The Molecular Basis of Small Vessel
Constriction in Endothelin-1 Models and
Peripheral Arterial Disease
Kanchani Rajopadhyaya
Discipline of Physiology, School of Medical Science
The University of Adelaide
May 2013
A thesis submitted for the degree of Doctor of Philosophy
Table of contents
Thesis declaration
8
Acknowledgements
9
List of figures and tables
10
List of common abbreviations
12
Thesis abstract
14
A. Literature Review
16
A1. Peripheral artery disease
17
A1.1. Clinical characteristics
17
A1.2. Diagnosing PAD
18
A1.3. The prevalence of PAD
20
A1.4. Risk factors
21
A1.5. Treating PAD
23
A1.6. Large vs small vessel disease in the peripheral
circulation
26
A2. The pathophysiology of PAD
29
A2.1. Atherosclerosis
29
A2.2. Thrombosis
30
A2.3. Vasospasm and increased vascular tone
31
A3. The endothelium
35
A3.1. The role of the endothelium in regulating vascular
tone
35
A3.2. Endothelial dysfunction
36
A3.3. Nitric oxide
37
A3.4. Endothelial nitric oxide synthase
38
A3.5. Regulation of eNOS activity by Ca2+
40
A3.6. Regulation of eNOS activity by Ser1177
phosphorylation
41
A3.7. Non-NO endothelial-derived vasodilators
43
A4. Molecular mechanism underlying vascular smooth
muscle tone
46
A4.1. The contractile apparatus
46
A4.2. Regulation of contraction via myosin light chain
kinase
47
A4.3. Regulation of Ca2+ entry
47
-2-
A4.4. Regulation of Ca2+ sensitisation via myosin light
chain phosphatase
51
A4.5. PKC/CPI-17-mediated Ca2+ sensitisation
52
A4.6. RhoA/ROK-mediated Ca2+ sensitisation
54
A5. The physiological role and molecular basis of
important vasoconstrictors
58
A5.1. α1-adrenergic receptor activation
58
A5.2. Thromboxane A2
61
A5.3. Endothelin-1
63
A5.4. Serotonin
65
A6. Thesis aims
69
B. General Methods
70
B1. Materials
71
B1.1 Functional myography
71
B1.2. Mini-osmotic pump and jugular vein cannulation
71
B1.3. Sodium dodecyl sulphate polyacrylamide gel
electrophoresis
71
B1.4. Western blot analysis
72
B2. Functional vascular myography
73
B2.1. Background
73
B2.2. Animal tissue preparation
73
B2.3. Human tissue preparation
75
B2.4. Tissue integrity
75
B2.5. Resting tension
76
B2.6. Endothelial integrity
76
B2.7. Experimental protocol – single dose
77
B2.8. Experimental protocol – dose response
78
B2.9. Snap-freezing and tissue storage
78
B2.10. Data analysis
78
B3. Chronic endothelin-1 infusion model
80
B3.1. Background
80
B3.2. Preparation of mini-osmotic pumps
81
B3.3. Jugular vein cannulation and mini-pump implantation
82
B4. SDS-PAGE
84
B4.1. Background
84
-3-
B4.2. Sample preparation
84
B4.3. SDS-polyacrylamide gel preparation
85
B4.4. SDS-PAGE run
87
B4.5. Standardising volume loading
87
B4.6. Protein transfer to nitrocellulose membrane
88
B5. Western Blot Analysis
89
B5.1. Background
89
B5.2. Blocking
89
B5.3. Primary antibody
89
B5.4. Biotin-conjugated secondary IgG
90
B5.5. Streptavidin-conjugated 800nm fluorochrome
90
B5.6. Re-probe with monoclonal anti-MYPT and antieNOS
91
B5.7. Incubation with HRP-conjugated secondary IgG
91
B5.8. ECL detection
91
B5.9. Data analysis
92
C. PKC inhibition attenuated sustained endothelin1-mediated vasoconstriction while ROK
inhibition attenuated initial and sustained
endothelin-1-mediated vasoconstriction in large
and small arteries
93
C1. Introduction
94
C2. Methods
97
C2.1. Materials
97
C2.2. Functional vascular myography
97
C2.3. Data analysis
98
C3. Results
C3.1. Inhibition of PKC and ROK prior to ET-1 stimulation
attenuated the development and maintenance of
vasoconstriction in rat caudal artery
C3.2. Inhibition of PKC and ROK prior to ET-1 stimulation
attenuated the development and maintenance of
vasoconstriction in rat mesenteric arteries
C3.3. During the sustained phase of ET-1-mediated
contraction subsequent inhibition of PKC and ROK
attenuates vasoconstriction in rat caudal arteries
C4. Discussion
-4-
99
100
104
108
110
D. Chronic endothelin-1 infusion attenuated
thromboxane A2-mediated vasoconstriction in
second order rat mesenteric arteries
117
D1. Introduction
118
D2. Methods
121
D2.1. Materials
121
D2.2. Preparation of mini-osmotic pumps and jugular vein
cannulation
122
D2.3. Functional vascular myography
124
D2.4. SDS-PAGE and western blot analysis of eNOS and
myosin phosphatase
125
D2.5. Data analysis
127
D3. Results
D3.1. Chronic exposure to elevated ET-1 decreased the
sensitivity and maximum contractile response to
exogenously applied thromboxane A2 mimetic,
U46619
D3.2. The contractile response to exogenously applied
ET-1 is similar in rats chronically exposed to
elevated ET-1
D3.3. The contractile response to the exogenously applied
α1-adrenergic receptor agonist, phenylephrine and
serotonin are similar in rats chronically exposed to
elevated ET-1
D3.4. Chronic exposure to elevated ET-1 does not alter
the contractile response to KCl-mediated
depolarisation
D3.5. The abundance and activation state of eNOS is
similar in rats chronically exposed to ET-1
D3.6. The abundance and activation state of myosin
phosphatase is not significantly different in rats
chronically exposed to ET-1
D4. Discussion
E. Subcutaneous microvessels from patients
suffering from peripheral artery disease exhibit
enhanced serotonin and α 1-adrenergic receptor
mediated vasoconstriction
128
129
131
133
136
138
140
142
148
E1. Introduction
149
E2. Methods
153
E2.1. Materials
153
E2.2. Patient recruitment and tissue collection
154
-5-
E2.3. Functional vascular response
155
E2.4. SDS-Page and western blot analysis of eNOS,
myosin phosphatase and 5HT2A
156
E2.5. Data analysis
157
E3. Results
E3.1. PAD and age-matched non-PAD patients had similar
responses to KCl-mediated vasoconstriction
E3.2. The maximum vasoconstrictor response to serotonin
receptor activation is increased in patients with PAD
compared to age-matched patients asymptomatic
for PAD
E3.3. The maximum vasoconstrictor response to α1adrenergic receptor activation is increased in
patients with PAD compared to age-matched
patients asymptomatic for PAD
E3.4. Patients with and without PAD have similar
sensitivity and maximum vasoconstrictor responses
to thromboxane A2 receptor activation
E3.5. Patients with and without PAD have similar
sensitivity and maximum vasoconstrictor responses
to ET-1 receptor activation
E3.6. Patients with and without symptomatic PAD have
similar abundance and Ser1177 phosphorylation
state of eNOS in subcutaneous arteries
E3.7. Subcutaneous arteries from patients with and
without PAD have similar abundance and Thr855
MYPT-dependent activation state of myosin
phosphatase
E3.8. Subcutaneous arteries from patients suffering with
PAD have increased abundance of total 5HT2A
receptors compared to patients asymptomatic for
PAD
E4. Discussion
159
163
165
167
169
171
173
177
179
181
F. General Discussion
192
F1. Thesis discussion
193
F1.1. Thesis premise
193
F1.2. Summary of results
194
2+
F1.3. Is there a need to consider alternative to Ca
channel and multiple G-protein coupled receptor
blockade therapy?
196
F1.4. The influence of elevated ET-1 in vascular disease
198
F1.5. The pathophysiology of vascular reactivity in patients
with PAD
200
-6-
F1.6. Conclusions
202
References
204
-7-
Thesis Declaration
I certify that this work contains no material which has been accepted for the
award of any other degree or diploma in any university or other tertiary
institution and, to the best of my knowledge and belief, contains no material
previously published or written by another person, except where due reference
has been made in the text. In addition, I certify that no part of this work will, in
the future, be used in a submission for any other degree or diploma in any
university or other tertiary institution without the prior approval of the University
of Adelaide.
I give consent to this copy of my thesis, when deposited in the University
Library, being made available for loan and photocopying, subject to the
provisions of the Copyright Act 1968.
I also give permission for the digital version of my thesis to be made available
on the web, via the University’s digital research repository, the Library
catalogue and also through web search engines, unless permission has been
granted by the University to restrict access for a period of time.
Signed,
Kanchani Rajopadhyaya
-8-
Acknowledgements
I thank my primary supervisor Dr. David Wilson for his invaluable support and
guidance. I owe much of this thesis and my growth as an investigator to his
unique ability to encourage and motivate. Above all, I thank him for his patience
through this immense learning experience. His commitment to teaching and his
passion for research is inspirational and I am truly grateful for his belief in me.
I extend my sincere gratitude to my supervisors Prof. John Beltrame and Prof.
Robert Fitridge for their guidance and clinical perspective throughout my PhD
career. Together my supervisors hold an unfathomable wealth of knowledge
and I treasured their willingness to share this with me.
To my mother Laxmi Sharma, my brother Brajesh Rajopadhyaya, my partner
Kemuel Kitto and my close friends, particularly Janine Van Heer and Hien Le: I
am grateful for your love and encouragement at home. I am especially thankful
for your understanding during the challenging periods and for sharing with me
the joys of achievement and success.
To my colleagues and friends: Jessica Maddern, Scott Copley, Timothy
Spencer, Joanne Eng, Yann Chan, Amenah Jaghoori, Rachel Dreyer, Rachel
Jakobczak, Lauren Rimmer and Benjamin Reddi: I thank you for creating such
an enjoyable and supportive work environment. I was grateful for your
camaraderie through the many highs and lows and I especially thank you for
the countless laughs and crazy antics in the lab. I wish you all the very best in
your future endeavours.
-9-
List of figures and tables
Figure A1.
Figure A2.
Figure C1.
Figure C2.
Figure C3.
Figure C4.
Figure C5.
Figure D1.
Figure D2.
Figure D3.
Figure D4.
Figure D5.
Figure D6.
Figure D7.
Table E1.
Table E2.
Figure E1.
Molecular basis of eNOS-mediated nitric oxide
generation in the endothelium.
Molecular mechanisms involved in vascular smooth
muscle contraction.
In rat caudal artery, inhibition of ROK but not PKC
prior to ET-1 stimulation attenuated the initiation of
vasoconstriction.
In rat caudal artery inhibition of ROK but not PKC
prior to ET-1 stimulation attenuated the sustained
phase of vasoconstriction in rat caudal artery.
In small mesenteric arteries combined inhibition of
PKC and ROK prior to ET-1 stimulation attenuates
the initiation of contraction.
In small mesenteric arteries inhibition of PKC and
ROK prior to ET-1 stimulation attenuates sustained
contraction.
During the sustained phase of ET-1-mediated
contraction subsequent inhibition of PKC and ROK
attenuates contraction in rat caudal artery.
The sensitivity and maximum contractile responses
to the exogenously applied thromboxane mimetic,
U46619 are significantly decreased following
chronic exposure to elevated ET-1.
The contractile response to ET-1 receptor
activation is unchanged following chronic exposure
to elevated ET-1.
The contractile response to α1-adrenergic receptor
activation is not statistically different following
chronic exposure to elevated ET-1.
The contractile response to serotonin receptor
activation is not significantly different following
chronic exposure to elevated ET-1.
Rats chronically treated with elevated ET-1 have
similar maximum contractile responses to KClmediated depolarisation.
eNOS abundance and activation state is similar in
rats chronically treated with ET-1.
Myosin phosphatase abundance and activation
state is not significantly different in rats chronically
treated with ET-1.
Clinical information for PAD and non-PAD patients
In-patient medication on day of surgery
Figure E1. Patients with and without PAD had
similar high K+-mediated vasoconstriction.
- 10 -
45
57
102
103
106
107
109
130
132
134
135
137
139
141
161
162
164
Figure E2.
Figure E3.
Figure E4.
Figure E5.
Figure E6.
Figure E7.
Figure E8.
Figure E9.
The maximum subcutaneous arterial contractile
response to serotonin receptor activation is higher
in PAD vs non-PAD patients.
The maximum subcutaneous arterial contractile
response to α1-adrenergic receptor activation is
higher in PAD vs non-PAD patients.
Patients with and without PAD have similar
contractile responses to thromboxane A2 receptor
activation.
The sensitivity and maximum contractile responses
to ET-1 receptor activation is similar between
patients with and without PAD.
Subcutaneous arteries from PAD and non-PAD
patients have similar total eNOS and Ser1177
phosphorylation of eNOS.
Acetylcholine-mediated vasodilation was not
significantly different in 300-400µM subcutaneous
arteries from PAD and non-PAD patients.
Basal abundance and Thr855 MYPT-dependent
activation state of myosin phosphatase is similar in
patients with and without PAD.
Patients suffering with PAD have more 5HT2A
receptors in their subcutaneous arteries than nonPAD patients.
- 11 -
166
168
170
172
175
176
178
180
List of common abbreviations
5HT
ABI
ADMA
ADP
ANOVA
APS
ARB
ATP
BH4
cGMP
COX
CRP
CSFP
CT
DAG
DFP
DTT
EC
ECL
EDHF
EDIN
eNOS
ET-1
GDI
GDP
GEF
GF109203X
GTP
HRP
IgG
ILK
IMA
iNOS
IP3
L0
LAD
LC20
L-NAME
MLCK
MYPT
NO
nNOS
PAD
PDBu
PDE
PE
PGI2
5,hydroxytryptamine / serotonin
ankle brachial index
asymmetric dimethylarginine
adenosine diphosphate
analysis of variance
ammonium persulfate
angiotensin II receptor blocker
adenosine triphosphate
tetrahydrobiopterin
cyclic guanosine monophosphate
cyclooxygenase
c-reactive protein
coronary slow flow phenomenon
computer tomography
diacylglycerol
diisopropylfluorophosphate
dithiothreitol
effective concentration
enhanced chemiluminescent
endothelial-derived hyperpolarising factor
epidermal cell differentiation inhibitor
endothelial nitric oxide synthase
endothelin-1
guanine nucleotide dissociation inhibitor
guanosine diphosphate
guanine-nucleotide exchange factors
bisindolylmaleimide
guanosine triphosphate
horse radish peroxidase
immunoglobulin G
integrin-linked kinase
Internal mammary artery
inducible nitric oxide synthase
inositol triphosphate
optimum resting length
left anterior descending coronary artery
20kDa regulatory light chains of myosin
Ng-nitro-L-arginine methyl ester
myosin light chain kinase
myosin phosphatase targeting subunit
nitric oxide
neuronal nitric oxide synthase
peripheral artery disease
phorbol 12,13-dibutyrate
phosphodiesterase
phenylephrine
prostacyclin
- 12 -
PI3K
PIP2
PKA
PKB
PKC
PKG
ROK
SAH
SDS-PAGE
SR
SSRIs
TBS-T
TCA
TEMED
TPI
ZIPK
phosphatidylinositol 3-kinase
phosphatidylinositol 4,5-biphosphate
protein kinase A
protein kinase B
protein kinase C
protein kinase G
Rho-associated kinase
subarachnoid haemorrhage
sodium dodecyl sulphate-polyacrylamide gel
electrophoresis
sarcoplasmic reticulum
selective serotonin re-uptake inhibitors
Tris buffered saline – Tween 20
trichloroacetic acid
N,N,N’,N’-tetramethylethylenediamine
toe pressure index
zipper interacting protein kinase
- 13 -
Thesis Abstract
Peripheral artery disease (PAD) affects 20% of people over the age of 65 years
and the prevalence increases with age. Predominately affecting the lower limbs,
PAD causes chronic ischaemic leg pain, reduced quality of life, and increased
risk of death by heart attack and stroke. It is well established vascular disease
is a large vessel, atherothrombotic disorder, however the importance of
vasospasm and/or increased vascular tone is less well recognized, particularly
in the microvasculature. Current vasodilatory medical therapies have focused
on extracellular Ca2+ entry or specific receptor blockade. Targeting subcellular
enzymes to attenuate general agonist-mediated vasoconstriction has not yet
been implemented. Although specific agonists, such as endothelin-1 (ET-1),
have
been
implicated
in
PAD,
whether
chronic
exposure
to
these
vasoconstrictors causes altered receptor profiles of circulating hormones have
not been identified. The direct contractile responses of diseased human
microvessels to specific agonists also remain unclear.
We used 1) a rat model to identify the acute temporal activation of PKC and
ROK during rapid and sustained ET-1-mediated vasoconstriction 2) a rat model
of chronically elevated ET-1-meditated vasoconstriction to identify altered
receptor profiles to specific agonists and 3) human subcutaneous arteries from
patients with PAD to identify their functional and biochemical properties
compared to age-matched non-PAD patients.
We report that PKC and ROK inhibition in large caudal and small mesenteric rat
arteries are effective in attenuating acute ET-1-mediated vasoconstriction in
- 14 -
both the rapid and sustained phases of constriction. Chronically elevated ET-1
in a healthy rat model blunts the acute contractile response to the thromboxane
A2 mimetic, U46619 but does not change the vascular response to exogenously
added ET-1, the α1-adrenergic agonist, phenylephrine and serotonin receptor
activation. In human subcutaneous arteries we identified an increased
maximum contractile response to serotonergic and α1-adrenergic receptor
activation in PAD vs non-PAD patients, while vascular responses to K+mediated activation of voltage-gated Ca2+ channels, thromboxane A2, and ET-1
receptor
activation
were
unchanged.
Altered
vascular
reactivity
was
independent from the abundance and Ser1177-dependent and Thr855dependent activation state of eNOS and myosin phosphatase, respectively. We
identified, patients with PAD have more 5HT2A receptors than patients with no
symptomatic PAD, suggesting a possible mechanism for increased contractile
responses to serotonin receptor activation.
These data suggest 1) subcellular targets that block the inhibition of myosin
phosphatase may be valuable in attenuating the vasoconstrictor response to
several agonists, and provide additional benefit to specific receptor blockade, 2)
while elevated ET-1 is a strong marker of vascular disease, it may have less
direct impact on vascular reactivity, 3) decreased contractile responses to
thromboxane A2 following chronic ET-1 infusion is most likely caused by down
regulation of thromboxane A2 receptors, which could have important
implications for patients on antiplatelet agents, 4) blockade of enhanced
serotonin and α1-adrenergic vasoconstriction may be beneficial in improving
subcutaneous microvascular blood flow in patients with PAD
- 15 -
Section A
Literature Review
- 16 -
A1. Peripheral artery disease
Peripheral artery disease (PAD) describes disorders causing impaired blood
flow outside the coronary and cerebrovascular circulation. The underlying
pathogenesis of PAD involves interplay between 1) atheroma development 2)
thrombus formation and 3) vasospasm or increased vascular tone, leading to
declining blood flow and end organ dysfunction. Although PAD includes disease
of the aortic, mesenteric, renal and carotid vascular beds, the following review
will focus on the consequences of impaired blood flow in the lower extremities.
A1.1. Clinical characteristics
Intermittent claudication, defined as leg pain experienced during activity and
relieved by rest, is one of the first clinical presentations of impaired blood flow
to the lower limbs1. These symptoms can be compared to chest pain
experienced by patients with stable angina; where low-grade atherosclerosis of
the coronary arteries permits adequate blood flow to the myocardial tissue at
rest but cannot meet metabolic demand during activity. Without appropriate
management, previous findings have reported 63% of patients with low-grade
atherosclerosis will progress to complete stenosis of the affected blood vessel
within 5 years2. As the severity of PAD increases, patients suffer rest pain and
complain of interrupted sleep, difficulty performing simple activities and reduced
quality of life. During the advanced stages of PAD, critical tissue hypoperfusion
leads to the development of non-healing ulcers and tissue death; increasing the
risk of infection1. In patients with advanced PAD lower limb amputation is often
required to prevent systemic infection and risk of sepsis.
- 17 -
PAD commonly indicates the presence of systemic atherothrombosis and
increased vascular tone and frequently coexists with several co-morbidities
such as ischaemic heart disease and diabetes. Consequently, PAD is a strong
predictor of coronary and cerebrovascular events such as myocardial infarction
and stroke3, 4. In an elegant study from the Cleveland clinic, varying stages of
coronary artery atherosclerosis was discovered in 90% of patients undergoing
surgery for PAD. Of the 1000 patients assessed in the study, 27% had severe
stenosis of at least one coronary artery5. It is not surprising then, that the cause
of death in 40-60% of patients with lower-limb PAD is coronary artery related
complications5, 6. A further 10-20% of deaths occur from cerebrovascular
problems such as stroke and 20-30% from non-cardiovascular related causes
such as ischaemic limb-related infection and sepsis6.
A1.2. Diagnosing PAD
It is well recognized that early identification and treatment of lower-limb PAD is
important in determining the fate of both the patient and the affected limb. An
accurate history provides information about co-existing coronary and
cerebrovascular disease and intermittent claudication. Patient histories are
important in differentiating leg pain caused by lower limb ischaemia from other
aetiologies such as spinal stenosis, nerve root compression or foot/ankle
arthritis7; achieved by assessing the characteristics of the leg pain. For
example, cramping, sharp lancinating or aching pain and the effects of
exercise, rest and position in relieving the pain6, 8. Additionally, a physical
examination assesses palpation of the ulnar, radial, brachial, carotid, femoral,
popliteal, dorsalis pedis and tibial artery pulses to identify proximal obstructive
- 18 -
disease in these vessels. Furthermore, examination of skin colour and
temperature as well as reduced hair and nail growth in the feet can also
indicate decreased blood flow in the lower-limbs8.
It is important to note that patients with lower-limb PAD may be asymptomatic.
Population studies have shown that the fate of the limb is independent of
symptoms of intermittent claudication3, 9. In the early stages, leg pain is
dependent on the level of activity routinely carried out by the patient. Hence, in
sedentary patients the first presenting symptom is rest pain with developing
ulcers without a history of intermittent claudication. Using Doppler ultrasound, a
study which assessed 6880 patients found only 11% of patients with lower-limb
PAD were identified by physical examination and a history of intermittent
claudication as a diagnostic tool10.
Stenosis of large vessels can be directly examined through the use of computer
tomography (CT), angiography and ultrasound8. These are useful in
determining the severity of lower-limb PAD and directing appropriate therapy. In
addition, the ankle-brachial index (ABI) is a non-invasive and inexpensive tool
for detecting symptomatic and asymptomatic lower-limb PAD11,
12
. Using
Doppler flow, an ABI assesses the ratio of blood pressure at the ankles and the
brachial artery. A normal ABI is greater than one (1) while an ABI < 0.9
indicates decreased blood pressure in the legs, diagnostic of lower-limb PAD.
Although previous findings have shown that an ABI is 9 times more effective at
identifying PAD compared to physical examination alone, the ABI is frequently
used in conjunction with CT scans and toe pressures to improve diagnostic
- 19 -
accuracy in cases of patients with calcified atheroma. Calcification renders the
effected vessel incompressible and can provide a falsely high ABI13.
A1.3. The prevalence of PAD
The prevalence of PAD is dependent on both its definition and the diagnostic
techniques used. As previously discussed, some patients with PAD may be
asymptomatic or have uncommon symptoms; hence the true occurrence of
PAD may be greatly underestimated. When PAD was defined as an ABI < 0.9,
the Limburg PAOD study found that 6.9% of patients over 45 years had PAD
while only 22% of these patients showed symptoms of intermittent
claudication14. This was consistent with the Edinburgh Artery Study which found
that 30% of patients with asymptomatic PAD had complete occlusion of a major
blood vessel15.
As PAD is a progressive disease, it is well recognized that the prevalence of
PAD increases with age. The Rotterdam study analysed 7715 patients and
reported that while 19% of the general population had PAD, 16-25% of those
over 55 years had varying degrees of PAD and 60% of those aged over 85
years of age had moderate to severe PAD16. The PARTNERS study reported
20% of the general population had PAD which increased to 29% in those over
7017. Previous findings have reported the incidence of PAD increases with age
independent of sex and/or ethnicity18. Variability in diagnostic rates exists
between studies, presumably due to the patient population and diagnostic
techniques used.
- 20 -
The incidence of PAD in men vs women is controversial. The Framingham
study analysed 1554 males and 1759 females and found that the frequency of
intermittent claudication was greater in men than women19. This was consistent
with several studies including the TASCII report which documented an
increased prevalence of PAD in men. In addition, it was shown that men have a
greater rate of death by cardiovascular events than women with PAD6,
20
. In
contrast, recent population based studies are suggesting the risk of PAD in
women have been greatly underestimated and that the total occurrence of PAD
is higher in women than in men21,
22
. However, these studies also report the
incidence of PAD is similar or higher in men when screening those younger
than 79 years. The prevalence increases in women only when screening those
over 80 years.
A1.4. Risk factors
The risk factors for PAD are typical for general cardiovascular disease. In
addition to age, smoking is a major risk factor for the development of
atherosclerosis, thrombosis and chronic increases in vascular tone. Toxins and
free radicals generated by cigarette smoke damage the endothelial lining;
triggering an inflammatory response and decreasing the availability of
vasodilatory
nitric
oxide
(NO)23.
Additionally,
nicotine
causes
direct
vasoconstriction by either directly acting on vascular smooth muscle cells or by
releasing catecholamines from the adrenal glands, promoting vasospasm or
increased contractility of blood vessels24. The Framingham Heart Study
documented the risk of developing intermittent claudication doubled in smokers
compared to non-smokers, and that smoking cessation could reverse the
- 21 -
progression of PAD19,
25
. Previous findings have shown that hypertension is
more common in smokers compared to non-smokers and have suggested that
the risk of hypertension and atheroma development increases with the number
of cigarettes smoked per day26, 27.
Hypercholesterolemia, physical inactivity and obesity are also important risk
factors for PAD. Management of high lipid levels with several lipid-lowering
agents
including
atorvastatin,
simvastatin
and
bezafibrate
significantly
decreases the risk of adverse vascular events in patients with limb ischaemia28.
Low physical fitness has been reported to be independently associated with
increased levels of triglycerides, low-density lipoproteins and total cholesterol29.
It has been previously reported that obesity, indexed by Metropolitan Relative
Weight30 predicted 26 year incidence of vascular disease in 5209 men and
women independent of age, cholesterol, systolic blood pressure, smoking and
glucose tolerance31.
Hypertension and diabetes are additional major risk factors for PAD. The
Framingham Heart Study documented the risk of developing intermittent
claudication was 2-3 times greater in patients with blood pressures over
140/90mmHg19. The risk of developing intermittent claudication doubles if the
patient has diabetes as insulin resistance is associated with hyperglycaemia,
dyslipidaemia and hypertension which contribute to the development of
atherothrombosis and peripheral neuropathy6,
32
. The progression of PAD in
diabetic patients is markedly more aggressive than in non-diabetic patients,
reflected by a five (5) fold greater need for amputation in patients with co-
- 22 -
existing PAD and diabetes6. This is most likely due to an interplay between
ischaemia, peripheral neuropathy and decreased resistance to infection33.
Previous findings have suggested that insulin resistance in the absence of
diabetes still increases the risk of PAD by 40-50%34.
Coexisting risk factors increase the risk of PAD. The Basle study reported the
risk of PAD was 2.3% and 3.3% for patients who had one (1) and two (2) of the
following, respectively: smoking, diabetes, hypertension or obesity1, 2. The risk
of developing PAD doubled if any patients had three risk factors.
A1.5. Treating PAD
The treatment of PAD begins with lifestyle management such as smoking
cessation, decreasing cholesterol intake, and increasing daily activity. Smoking
cessation slows low-grade arterial stenosis progressing to severe stenosis and
development of critical limb ischaemia; reducing the risk of death by myocardial
infarction or cerebrovascular complications35. However it has previously been
reported that while smoking cessation decreases the progression of
atherosclerosis it does not improve maximal treadmill walking distance36. A
possible explanation may be that while subjects had ceased smoking, nicotinereplacement therapy was used to overcome difficulties associated with quitting.
Since the vasoconstrictor effects of nicotine had not been removed, blood flow
may still have been limited in the lower limbs. However the benefits of shortterm nicotine replacement therapy in facilitating compliance of smoking
cessation are still relevant in the management of PAD37,
38
. Increasing daily
physical activity comes second only to smoking cessation in the management
- 23 -
of PAD. It has been previously demonstrated a formal exercise program may be
as effective as surgical revascularisation procedures in improving symptoms of
claudication, increasing walking distance and improving quality of life in patients
suffering with PAD39, 40.
Compliance
necessitating
with
lifestyle
medical
management
management
of
strategies
is
commonly
hyperlipidaemia,
diabetes
low41
and
hypertension. Lipid lowering HMG-CoA reductase inhibitors (statins) have been
shown to decrease mortality in PAD patients; however whether this is due to a
systemic decrease in low-density lipoproteins, plaque stabilisation, reduced
inflammation or increased vasodilation is unclear42. Administration of
antiplatelet agents such as aspirin and clopidogrel are effective in decreasing
thrombus formation43. Blood glucose control is effective in managing
microvascular complications associated with diabetes41. However several trials
have shown insulin sensitizing therapy does not improve the risk of amputation
or death by vascular events in patients with PAD; which may be due to a lack of
improvement of large vessel disease following insulin therapy in patients with
type I and type II diabetes44, 45.
Vasodilator medical therapy includes Pentoxifylline, a competitive non-selective
phosphodiesterase inhibitor which has been shown to increase vasodilation and
decrease fibrin and platelet aggregation through a nitric oxide-dependent
mechanism36,
46
. Similarly, Cilostazol, a specific phosphodiesterase 3 (PDE3)
inhibitor with both antiplatelet and vasodilatory properties, has been shown to
improve walking distance, decrease rest pain and increase quality of life in
- 24 -
patients with PAD47. Naftidrofuryl, a serotonin 5HT2 receptor antagonist, has
been shown to increase pain-free walking distance in 26% of patients with
PAD48,
49
. Prostacyclin infusion has been shown to decrease rest pain and
accelerate healing of ischaemic ulcers50. However, side effects associated with
prostacyclin infusion include headaches, flushing, nausea and vomiting during
initial treatment. Unfortunately, the efficacy of many of these medical therapies
is limited in the treatment of microvascular dysfunction51,
52
. Often
pharmacotherapy fails to resolve clinical symptoms and surgical procedures are
required.
Surgical revascularisation procedures include angioplasty with or without,
stenting, endarterectomy and bypass grafts53. With recent advancement in
catheter design, it has been suggested that percutaneous arterial interventions
are also an effective alternative to open surgery. However whether
endovascular techniques have a better outcome compared to open repair
remains an open question1. A recent multi-centre study involving 881 patients
compared the benefits of endovascular vs open repair of abdominal aortic
aneurysms54. Lederle and colleagues reported endovascular repair and open
repair resulted in similar long-term survival while rupture following repair was
still a major concern.
Several studies have shown that 12-29% of patients experiencing rest pain and
tissue loss will require lower-limb amputation within three months of presenting
with these symptoms to prevent systemic infection and relieve rest pain6,
55
.
However, studies have shown that life expectancy of amputees is still
- 25 -
considerably reduced; 30% of patients die within 2 years of major amputation6.
In contrast, the need for amputation in patients with intermittent claudication is
relatively low. Only 2-7% of patients will require lower limb amputation after 5
years of symptom onset and 12% after 10 years56.
A1.6. Large vs small vessel disease in the peripheral circulation
Peripheral artery disease has predominately been considered a large vessel
disorder. However with the effective treatment of large vessel stenosis, the
important contribution of small vessel disease has become apparent; indicating
large and small vessel disease often co-exist. For example, surgical
revascularisation is ineffective in decreasing peripheral vascular resistance in
hypertensive PAD patients, probably due to an increase in microvascular
resistance57,
58
. Similarly, a low ABI, claudication and non-healing ulcers
frequently persist following mechanical opening of large vessels59 indicating an
impaired cutaneous circulation.
Criqui and colleagues assessed impaired blood flow in large and small vessels
in patients with PAD using several hemodynamic measures including ABI, postocclusive reactive hyperaemia, toe pressures and femoral and tibial pulse
decay60. It was reported that 16% of the 624 symptomatic patients screened
showed a low ABI and decreased toe pressures without evidence of large
vessel stenosis, indicating decreased small vessel blood flow60. However these
approaches exclusively assessed dynamic aspects of perfusion and/or large
vessel blood flow61,
62
. Anatomical obstruction was not directly examined and
- 26 -
hence these approaches can only provide an indirect description of small
vessel disease.
Previous findings have suggested the pathogenesis of large vessel PAD is
different from small vessel PAD63. Small vessel disease diagnosed with noninvasive Doppler Ultrasound and toe pressure index (TPI) has been reported to
be unrelated to some traditional risk factors for vascular disease; including
smoking and high density lipoproteins63-66 and while c-reactive protein (CRP) is
a biomarker of large vessel disease, there is no correlation between highsensitivity CRP and small vessel PAD progression67. Small vessel disease in
the peripheral circulation is largely associated with diabetes and high blood
pressure. Consistent with these studies, it has been reported that in contrast to
large vessel disease, small vessel disease occurs independently of age and
sex60.
These
studies
suggest
that
small
vessel
PAD
may
be
pathophysiologically different from large vessel disease. Small vessel perfusion
may be influenced less by atherothrombosis and is perhaps more dependent on
the vasoconstrictive effects of circulating hormones and/or altered profiles of Gprotein coupled receptors activated by specific vasoconstrictors.
Due to the difficulties associated with assessing small vessel disorders, the
current literature in the field is relatively sparse. Doppler ultrasound and ABI
directly measures large vessel blood flow but its ability to reliably characterise
small vessel flow is limited. Similarly, angiography cannot accurately resolve
small vessel dysfunction. For this reason the mechanism underlying increased
contractility of small vessels in patients with PAD is incompletely understood.
- 27 -
Importantly, improving current strategies to resolve intermittent claudication,
rest pain and/or non-healing ulcers can only be achieved with the effective
treatment of both large and small vessel disease.
- 28 -
A2. The pathophysiology of PAD
The following section will briefly review the contribution of atherosclerosis and
thrombosis in causing impaired blood flow and will focus on the importance of
vasospasm/increased vascular tone in contributing to reduced blood flow and
increased blood pressure. These concepts have traditionally been perceived as
separate entities; this section illustrates their interconnectivity and highlights
how one factor in this pathophysiological triad can predispose a patient to
another.
A2.1. Atherosclerosis
The presence of an encroaching atheroma coupled with a reduced ability to
under-go endothelial-dependent vasodilation can be a common cause of
myocardial infarction in patients with coronary artery disease. However,
atherosclerosis is more recently being considered a generalised disorder as it
frequently occurs in both the coronary and peripheral circulation68.
The development of an atheroma involves the activation and recruitment of
immune cells in response to damage to the endothelium. This is perceived to
be triggered by elevated levels of oxidised lipoproteins, free radicals and high
blood pressure69,
70
. The inflammatory process is dependent on specific
adhesion molecules, namely P-selectin and VCAM-1 which favour adhesion
and infiltration of lymphocytes and monocytes71. The inflammatory response
sets the stage for plaque formation as mononuclear leucocytes and
macrophages migrate from the blood to the intimal layer of the arterial wall and
- 29 -
assume a foamy histological appearance. These foam cells create the earliest
visible stage of atheroma development: the fatty streak. Although appropriate
lifestyle changes can reverse fatty streak formation, continued accumulation of
foam cells under the endothelial lining transforms the fatty streak into an
advanced plaque72. Smooth muscle migration and collagen deposition in the
lesion facilitates the formation of a fibrous matrix, creating a cap for the lipidrich core73. A stable developing atheroma will encroach into the lumen and
significantly reduce blood flow. It has become evident that while vascularrelated health issues manifest in older populations, the initiation of plaque
development often begins in early life; studies have shown atherosclerotic
lesions appearing in the aorta and coronary arteries of children under nine (9)
years74, 75, highlighting a need to establish healthy lifestyle practices at a young
age.
A2.2. Thrombosis
Although atheroma and thrombus have traditionally been considered separate
processes it has become clear that the pathogenic mechanisms underlying
atherosclerosis are common and necessary for thrombosis76, 77.
The final stage of atherogenesis is the formation of a complicated or an
unstable plaque. The blood and the lipid-rich atheromatous core are normally
separated by the fibrous cap, preventing the exposure of thrombogenic factors,
including von Willebrand factor, fibrillar collagens, fibronectin and laminin which
promote platelet recruitment, adhesion and contracture. Exposure of these
substances is dependent on the thickness and collagen/elastin content of the
- 30 -
fibrous cap78,
79
. Following adhesion, an important component of platelet
activation is the production and release of soluble agonists such as densegranule ADP and the prostanoid thromboxane A280. In summary, platelet
activation triggers thrombin generation, fibrin formation from fibrinogen and
cross-linking of fibrin monomers leading to thrombus formation81. The thrombus
can continue to enlarge gradually until it completely blocks the vessel or detach
from the atherosclerotic plaque and obstruct smaller vessels distally. A
detached thrombus frequently causes myocardial infarction, stroke, pulmonary
embolism and/or development and progression of lower-limb ischaemia80.
A2.3. Vasospasm and increased vascular tone
Inflammatory cells and platelets involved in the formation of atheroma and
thrombus release vasoactive factors, such as endothelin-1 (ET-1) by
macrophages82 and thromboxane A283 and serotonin by activated platelets84.
The catecholamines, adrenaline and noradrenaline are frequently elevated in
patients with vascular disease85, predisposing patients to undergo enhanced
vasoconstriction or vasospasm, an abnormal rapid vasoconstrictor response to
circulating or locally released hormones.
The consequence of vasospasm are best known in the coronary and
cerebrovascular circulation where constrictor responses may induce spasm of
the large epicardial arteries as in Prinzmetal variant angina86,
87
, or involve
microvascular dysfunction as occurs in the coronary slow flow phenomenon
(CSFP)88. Prinzmetal angina presents as transient bouts of chest pain at rest;
differing from stable angina which presents as chest pain during activity or
- 31 -
stress. In Prinzmetal variant angina, the epicardial arteries are hyper-reactive to
multiple
constrictor
agents
including
α1-agonists89,
serotonin90
and
acetylcholine91. It has been shown that many patients suffering from
vasospastic Prinzmetal angina often suffer from low-grade atherosclerosis in an
adjacent coronary artery92-94, illustrating the pathophysiology cannot be
attributed to one cause; atheroma, thrombus or vasospasm but the interplay of
all three.
In the cerebral circulation, vasospasm of cerebral arteries causes cerebral
ischaemia and is the most frequent cause of mortality in survivors of
aneurysmal subarachnoid haemorrhage (SAH). It occurs in up to 70% of
patients presenting with a SAH and significantly contributes to the overall 50%
mortality95. The pathogenesis of cerebral vasospasm is multi-factorial involving
oxyhaemoglobin, ET-1, arachidonic acid derivatives, lipid peroxides, neurogenic
dysregulation and other indirect effects of inflammatory atherothrombotic
processes96.
Hyper-contractility and increased vascular tone of the microvasculature is
important in the regulation of blood pressure. Although elevated blood pressure
isn’t a direct cause of mortality, overwhelming evidence exists of the influence
of blood pressure on cardiovascular events97-100. Large vessels consist of
proximal elastic-type arteries responsible for mass transport of blood and
oxygen. These conduit vessels buffer the large pressure gradient between
ventricular ejection and re-filling, but do not directly contribute to blood
pressure. Blood pressure is governed by cardiac output and total peripheral
- 32 -
resistance. Although the heart pumps blood into the systemic circulation, the
distribution depends on peripheral resistance. Small arteries (150-300µm) and
arterioles (<100µm) constitute 30 and 40% of total vascular resistance,
respectively101. In contrast to large conduit arteries, small arteries and arterioles
can dramatically alter their diameter in response to a plethora of stimuli
including circulating hormones and endothelial-derived vasoactive factors.
Arteries <300µm are capable of dilating up to 50% of their resting diameter,
decreasing peripheral resistance and increasing blood flow102. On the other
hand, they are also able to undergo strong contractions, sometimes closing
completely, increasing resistance and enabling tightly controlled regulation of
blood flow and intravascular pressure. This is important for adequate fluid
exchange, distribution of blood flow and structural conservation of delicate
capillaries102. In a pathological context, increased vasoconstriction of the
peripheral microvasculature can induce chronic ischaemia in the lower limbs,
evident by a failure to resolve high blood pressure, non-healing ulcers and
claudication despite the treatment of large vessel stenosis57-59. Raynaud’s
phenomenon is considered a peripheral microvascular vasospastic disorder
where episodes are precipitated by emotional stress or exposure to cold
stimuli103. Although a range of factors have been postulated to contribute to the
pathogenesis of Raynaud’s phenomenon, previous research has implicated a
heightened
sensitivity
activation103,
105
glyceryl
to
α2-adrenergic104
and/or
serotonergic
receptor
. L-type Ca2+ channel blockade, sublingual administration of
trinitrate
(Anginine®)
extracellular Ca2+ entry107,
108
and
PDE3
inhibitors106
which
decrease
and favour vasodilation, has been shown to
increase maximum walking distance in some patients with low to moderate
- 33 -
Raynaud’s disease. Similarly clinical symptoms in patients with limb-threatening
lower extremity ischaemia have partially resolved after infusion of the nonspecific tolazoline, an α-adrenergic inhibitor and agonist of histamine
receptors109.
- 34 -
A3. The endothelium
The endothelium is a single layer of specialised cells that line the lumen of
arteries. It is responsible for the release and activation of important vasoactive
agents crucial for vascular function. The following section will review the role of
the endothelium in health and disease.
A3.1. The role of the endothelium in regulating vascular tone
The endothelium influences vascular smooth muscle contraction and relaxation.
This was first shown by Furchgott in 1980, who’s elegant experiment
demonstrated rabbit aortic rings relaxed in the presence of acetylcholine but
this vasodilation was abolished following removal of the endothelium110. In
1987, the mediator of endothelium-dependent vasodilation was identified as
nitric oxide (NO)111,
112
. In the last three decades, our understanding of the
importance and complex nature of endothelial function has increased
dramatically. In response to altered blood pressure, shear stress, circulating
hormones and mechanical stimuli, the endothelium releases vasoconstrictors
such as ET-1113, and vasodilators such as nitric oxide111, 112, prostacyclin114 and
endothelial-derived hyperpolarising factor (EDHF)115,
116
. In addition, the
endothelium is, in part, involved in the degradation of specific hormones such
as serotonin and noradrenaline by housing monoamine oxidase, favouring
vasorelaxation117, 118. In contrast, the endothelium is one source of angiotensin
converting enzyme crucial for the conversion of inactive angiotensin I into the
active, potent vasoconstrictor, angiotensin II119, 120.
- 35 -
A3.2. Endothelial dysfunction
Adequate regulation of systemic blood flow requires an appropriate balance of
vasodilators and vasoconstrictors. Endothelial dysfunction is a multifactorial
disorder characterised by a reduction in endothelial-derived vasodilators, which
shifts this balance in favour of vasoconstriction; perceived to be a major
mediator of vascular disease. Clinically, endothelial dysfunction can be
assessed by measuring either the transient increase in brachial artery blood
flow following short-term ischaemia (reactive hyperaemia) or the increase in
blood flow following administration of acetylcholine121, 122. In a healthy patient,
intracoronary acetylcholine infusion dilates normal coronary arteries; this
vasodilator response is impaired in patients with atherothrombosis123. In the
complete absence of the endothelium, previous studies have reported
acetylcholine infusion induces vasoconstriction in patients with advanced
coronary stenosis, which has provided valuable insight into the generation of
coronary
artery
vasospasm91,
124
.
In
porcine
and
rabbit
models
of
hypercholesterolemia, a high cholesterol diet impaired endothelial-dependent
vasodilation in vitro before any hypercholesterolemia-induced structural
modifications were detected, indicating endothelial dysfunction occurs early in
the atherothrombotic process125,
endothelial
dysfunction
atherothrombosis;
for
is
126
. In humans, the increasing severity of
positively
example,
in
associated
patients
with
with
progressive
low-moderate
hypercholesterolemia, impaired endothelial-dependent vasodilation has been
reported in angiographically normal coronary arteries which gradually advances
to a complete loss of endothelium-mediated vasodilation in atherosclerotic
coronary arteries127.
- 36 -
A3.3. Nitric Oxide
NO is generated from L-arginine and oxygen by the enzyme, NO synthase
(figure A1)128-130. NO is lipophilic and readily diffuses into adjacent vascular
smooth muscle cells where it binds to soluble guanylate cyclase causing a
conformational change and subsequent activation of the enzyme131. Activated
guanylate cyclase generates cyclic guanosine monophosphate (cGMP) from
GMP, which in turn leads to the activation of protein kinase G (PKG)131 and
subsequent activation of Ca2+-dependent K+ channels132. This facilitates K+
efflux from vascular smooth muscle cells, hyperpolarisation of the plasma
membrane and as a consequence reduces the likelihood of voltage-gated Ca2+
channel activation. Inhibiting extracellular Ca2+ entry impairs Ca2+-calmodulin
dependent myosin light chain kinase (MLCK) from phosphorylating myosin,
favouring vasodilation.
In addition to its vasoactive role, NO has anti-inflammatory, anti-coagulation
and anti-proliferative properties. Reduced NO availability increases expression
of adhesion molecules, platelet activation, and smooth muscle proliferation133.
These promote inflammation of the vessel wall, and accelerate atheromatous
plaque growth, thrombus formation and vasoconstriction; significantly reducing
blood flow and tissue perfusion. Decreased NO availability has been attributed
but not limited to: a reduction in available L-arginine, an increase in NOscavenging reactive oxygen species (superoxide), and a decrease in the
abundance or expression of NO synthase134. Asymmetric dimethylarginine
(ADMA), an endogenous by-product of protein modification, has been shown to
decrease NO by inhibiting intracellular entry of L-arginine and by competitively
- 37 -
inhibiting NO synthase135. Clinical studies have found elevated ADMA levels in
patients with atherosclerosis and hypertension suggesting its role in promoting
endothelial dysfunction in these disorders136. In addition, the co-factor,
tetrahydrobiopterin (BH4) plays a crucial role in NO synthase activity and NO
production. A reduction in or oxidation of BH4 is associated with NO synthase
uncoupling and superoxide production137. Declining levels of BH4 was
perceived to be age related138. However recent studies have revealed that,
exercise can restore BH4 levels suggesting the associated decrease in BH4
levels is associated with reduced activity and not age per se. Consequently,
approaches to increase activity in the elderly are being examined as a strategy
to improve endothelial function139-141.
A3.4. Endothelial nitric oxide synthase
There are currently three known isoforms of nitric oxide synthase: neuronal
nitric oxide synthase (nNOS), inducible nitric oxide synthase (iNOS) and
endothelial nitric oxide synthase (eNOS). eNOS is a dimeric enzyme of 134kDa
responsible for NO production from L-arginine and oxygen in the endothelium142
but is also expressed in platelets143 and the myocardium144. eNOS is composed
of two identical monomers, where dimerization is dependent on the binding of
heme145. This then facilitates the binding of BH4 and formation of a stable
dimer. The functional activity of eNOS is dependent on its stabilization by BH4.
In the presence of low levels of BH4 or in the complete absence of the important
co-factor, eNOS cannot generate NO but instead produces the free radical,
superoxide which has important implications for increased oxidative stress and
decreased nitric oxide production during the early stages of atherogenesis137.
- 38 -
Several factors have been reported to up or down regulate basal expression
and activation of eNOS, including the circulating hormones: ET-1, bradykinin
and acetylcholine146-149, oxidised low-density lipoproteins150 and mechanical
stimuli151. Both the mRNA and protein expression of eNOS is increased in
response to shear stress152, 153. The effects hypoxia has on eNOS expression is
controversial; several studies have reported both an increase154,
155
and
decrease156-158 in mRNA half-life and protein expression, indicating hypoxiamediated regulation of eNOS may be dependent on the vascular bed and/or the
experimental approach.
Recent research has implicated eNOS dysfunction as a mediator of vascular
disease. Studies using eNOS knockout mouse models have reported increased
blood pressure, an impaired ability to undergo agonist-mediated vasodilation
and increased reactivity to specific vasoconstrictors in mice deficient of
eNOS159,
160
. In addition, atherosclerotic knockout mouse models have
demonstrated hypertension and increased acceleration of atherosclerotic
lesions in mice deficient of both apolipoprotein E and eNOS, indicating the
absence of eNOS promotes increased peripheral vascular resistance, elevated
blood pressure, smooth muscle proliferation and increased platelet reactivity
and aggregation161,
162
. Experiments using Ng-nitro-L-arginine methyl ester (L-
NAME)-induced inhibition163-166 of NO production causes hypertension,
increases leukocyte adherence167 and promotes platelet aggregation168,
important for the early stages and advancement of atherothrombosis.
- 39 -
In humans, studies have shown a decrease in eNOS expression in
atherosclerotic blood vessels which has been associated with hypertension,
atheroma development and hypercholesterolemia169. Additionally, there are
several known eNOS polymorphisms associated with an increased risk of
developing coronary artery disease, diabetes, and hypertension along with a
greater likelihood of suffering a myocardial infarction170-172, suggesting
endothelial dysfunction is linked to eNOS activity173.
Other isoforms of NO synthase include nNOS and iNOS. nNOS, as the name
suggests, is predominately found in neurons functioning as a neurotransmitter,
but is also present in skeletal, smooth and cardiac muscle174. Unlike eNOS and
nNOS, iNOS is Ca2+ independent and up-regulated in response to infection; the
free radicals being used to destroy invading organisms175; during chronic
inflammation this leads to oxidative damage of cellular proteins and lipids
contributing to cellular and organ dysfunction. While it has been shown that
nNOS is constitutively expressed, it is unclear whether iNOS is expressed at
rest129.
A3.5. Regulation of eNOS activity by Ca2+
As mentioned nitric oxide synthase isoforms have been identified and
characterised based on their primary location and whether their activation is
Ca2+-calmodulin dependent. eNOS is largely expressed in endothelial cells and
relative to nNOS and iNOS is most sensitive to changes in cytosolic Ca2+
concentrations. Previous studies have demonstrated acetylcholine and
bradykinin-mediated production of NO from endothelial cells and subsequent
- 40 -
vasodilation is abolished following the chelation of extracellular Ca2+ and in the
presence of inhibitory calmodulin binding peptides176,
177
. It is perceived that
Ca2+-bound calmodulin displaces an auto-inhibitory loop on eNOS which
enables NADPH-dependent transfer of electrons between specific domains on
eNOS, allowing the enzyme complex to catalyse the conversion of L-arginine
and oxygen to L-citrulline and vasodilatory NO178,
179
. Intracellular Ca2+ is
regulated by activation of ion channels on either the endothelial plasma
membrane or the sarcoplasmic reticulum. For example bradykinin or
acetylcholine-mediated activation of plasma membrane G-protein coupled
receptors activates 1) plasma membrane Ca2+ channels and 2) generates
inositol triphosphate (IP3) and subsequent release of Ca2+ from the
sarcoplasmic reticulum. The activation of stretch sensitive ion channels can
also lead to extracellular Ca2+ entry into the endothelial cell and activate
eNOS151, 180.
A3.6. Regulation of eNOS activity by Ser1177 phosphorylation
While it has been demonstrated that an increase in cytosolic Ca2+ is important
for eNOS activation, several studies have documented shear stress and
specific hormones (e.g. oestrogen, insulin) activate eNOS independent from
intracellular Ca2+ concentrations. eNOS can be phosphorylated on serine,
threonine and tyrosine residues179, 181. However, phosphorylation of Ser1177 is
the most thoroughly studied and is believed to be the predominant site of
positive regulation. Previous studies have reported phosphorylation of Ser1177
increases eNOS activity by 15 fold182. Several studies have shown this
phosphorylation is mediated by activation of phosphatidylinositol 3-kinase
- 41 -
(PI3K) and subsequent activation of protein kinase B (PKB)/Akt183-185. When
isolated human umbilical vein endothelial cells were incubated with a PKB
inhibitor, or transfected with a dominant negative (inactive) form of PKB/Akt,
phosphorylation of eNOS Ser1177 and nitric oxide production was abolished185.
Similarly mutation of eNOS Ser1177 to a non-phosphorylatable alanine residue
attenuated PKB/Akt-dependent phosphorylation of eNOS185.
Although Ser1177 phosphorylation-induced activation of eNOS is considered to
be Ca2+ independent, the chelation of Ca2+ using EGTA abolishes shear stress
mediated eNOS activity179,
185
. This indicates that although Ca2+ is necessary
for Ser1177 phosphorylation-dependent activation of eNOS, eNOS-mediated
generation of NO can occur at resting Ca2+ levels through this mechanism.
In contrast to Ser1177-dependent activation of eNOS, phosphorylation of the
Thr495 residue is associated with a decrease in enzyme activity and is
therefore a negative regulatory site of eNOS in vitro186,
187
. It is believed
phosphorylation of Thr495 impairs the binding of Ca2+-bound calmodulin. For
example, following stimulation with bradykinin, histamine or a Ca2+-ionophore,
Ca2+-calmodulin binding to eNOS is significantly increased when the Thr495
residue is not phosphorylated187. Decreased Thr495 phosphorylation has been
documented to increase eNOS activity by 10-20 fold above basal activation173,
179
. Further studies are required to examine the functional implications of eNOS
Thr495 phosphorylation in vivo. The regulation of eNOS activity and NO
generation is detailed in figure A1.
- 42 -
A3.7. Non-NO endothelial-derived vasodilators
Prostacyclin (PGI2), a member of the prostanoid family, is another important
endothelial-derived vasodilator114. In the endothelium, PGI2 production involves
a complex metabolic pathway initiated by phospholipase A2-mediated release
of arachidonic acid188. Cyclooxygenase (COX) is involved in the conversion of
arachidonic acid to specific prostanoids189. The type of prostanoid produced is
governed by the enzyme present in a given tissue type. In the endothelium,
COX-1 and COX-2 are involved in the production of prostacyclin190, which like
NO, causes hyperpolarisation of the vascular smooth muscle cell membrane
and consequently mediates vasodilation but also has anti-inflammatory, antiplatelet and anti-proliferative properties. A common outcome for patients on
COX-2 inhibitors is a decline in endothelial function191.
Interestingly, vasodilation occurs in the presence of NO and prostacyclin
blockade suggesting a third important endothelial-derived vasodilator. Like NO
and prostacyclin, this endothelium-dependent vasodilation also occurs through
activation of K+ channels and hyperpolarisation of the vascular smooth muscle
plasma membrane, consequently decreasing the activation of voltagedependent Ca2+ channels and reducing extracellular Ca2+ entry192. This
vasodilator has been so named endothelial-derived hyperpolarising factor
(EDHF) and has been shown to play an important role in resistance vessel
vasodilation and regulation of blood pressure. Several studies have nominated
5,6-epoxyeicosatrienoic acids193, C-type natriuretic peptide194 and hydrogen
peroxide as the putative EDHF195-197; perhaps with the strongest evidence in
- 43 -
favour of hydrogen peroxide195. Nevertheless its identity still remains an open
and important question.
- 44 -
Figure A1. Molecular basis of eNOS-mediated nitric oxide generation in
the endothelium. Endothelial nitric oxide synthase (eNOS) is composed of two
monomers and requires heme and BH4 for dimerisation and stabilisation,
respectively. Under normal physiological conditions, eNOS converts O2 and Larginine to L-citrulline and nitric oxide which hyperpolarises adjacent vascular
smooth muscle cells mediating vasodilation. Increased cytosolic Ca2+ by
plasma membrane Ca2+ channels and/or sarcoplasmic reticulum (SR) Ca2+
release enables Ca2+-bound calmodulin to bind to eNOS and as a consequence
increase eNOS activity. Ser1177 and Thr495 eNOS phosphorylation by a
phosphatidylinositol 3-kinase (PI3kinase), protein kinase B (PKB)-dependent
mechanism positively and negatively regulates eNOS activity, respectively.
Modified from Félétou et al., 2006198.
- 45 -
A4. Molecular mechanisms underlying vascular smooth
muscle tone
The ability to alter arterial lumen diameter is important for increased or
decreased blood flow through a specific blood vessel and subsequent perfusion
of downstream vascular beds. The following section will review the important
subcellular regulators governing vascular smooth muscle contraction and
relaxation.
A4.1. The contractile apparatus
The contractile apparatus of smooth muscle consists of thin actin filaments and
thick immobile myosin filaments. There are considerably more actin filaments
relative to myosin; 10-15 actin filaments can be detected for every myosin
filament199. Each actin filament is attached to structures called dense bodies
located in the membrane and the cytosol200. These structures are analogous to
the Z-lines of skeletal muscle and anchor the thin filaments. In response to
many stimuli, including nervous, endocrine or even changes to the surrounding
chemical environment, the thin actin filaments interact with the heads of thick
myosin filaments and draw the dense bodies towards each other causing the
smooth muscle cells to contract. The structure of the thick filaments of myosin
consists of multiple hexamers composed of 200kDa heavy chains and 20kDa
regulatory light chain subunits (LC20). Myosin LC20 forms the myosin heads and
contains both actin and Mg2+-ATP binding sites 201, 202.
- 46 -
A4.2. Regulation of contraction via myosin light chain kinase
Myosin LC20 plays a crucial role in vasoconstriction. When a vascular smooth
muscle cell is stimulated, the cytosolic concentration of Ca2+ increases and
binds to calmodulin. This induces a conformational change enabling calmodulin
to remove the auto-inhibitory domain from the inactive, actin-bound myosin light
chain kinase (MLCK); forming an active holoenzymatic complex. Active MLCK
catalyses the transfer of a phoshoryl group from Mg2+-bound adenosine
triphosphate (Mg2+-ATP) to a Ser19 residue of myosin LC20203-205. Myosin LC20
phosphorylation enables actin to interact with myosin and increase the
activation of myosin ATPase by up to 100 fold206. This promotes ATP hydrolysis
and increases the rate of actin-myosin cross-bridge cycling and as a
consequence mediates the development of force203, 205, 207. When Ca2+ efflux or
uptake of Ca2+ into the sarcoplasmic reticulum causes cytosolic Ca2+ to fall
below 1µM, calmodulin dissociates from MLCK causing the auto-inhibitory
domain to once again inactivate MLCK205,
208
. Dephosphorylation of myosin
LC20 by myosin phosphatase enables actin-myosin dissociation, favouring
vasorelaxation (detailed below).
A4.3. Regulation of Ca2+ entry
Ca2+ is an important second messenger involved in a plethora of cellular
functions including, cell growth, migration, proliferation and skeletal and smooth
muscle contraction209. Previous studies have shown increasing cytosolic Ca2+
concentrations consistent with the development of force in isolated rat aortic
preparations210. At rest, the cytosolic Ca2+ concentration in vascular smooth
muscle cells is maintained between 100-300nM which can be increased by up
- 47 -
to 10 fold upon stimulation. Ca2+ is tightly regulated by both extra- and
intracellular mechanisms as high cytosolic concentrations of Ca2+ can be
toxic209, 211. Long-term increases in cytosolic Ca2+ or increased reactivity to Ca2+
has been attributed to enhanced vascular tone, and increased vascular smooth
muscle proliferation and apoptosis. These consequences have important
implications for the development of increased peripheral vascular resistance,
hypertension and atherothrombosis212-214. Extracellular Ca2+ entry primarily
occurs through plasma membrane voltage-gated Ca2+ channels215. However it
is important to recognise several channels including the receptor-operated,
non-selective cation and store-operated Ca2+ channels are also involved in
extracellular Ca2+ influx216.
Although several types of voltage-gated Ca2+ channels have been identified,
the most clinically relevant are the L-type and T-type Ca2+ channels which are
composed for five (5) distinct subunits; α1, α2, β, γ and δ. The “long-acting” Ltype Ca2+ channels are characterised by a large conductance (23-25pS), high
activating potential (between -30mV and +10mV) and slow inactivation217-219.
The low voltage T-type or “transient” Ca2+ channels have a small conductance
(3.5-8pS), a low activating potential (between -50mV and 20mV) and rapid
inactivation (5-50 milliseconds)218-220. While T-type Ca2+ channels play an
important role in vascular smooth muscle contraction, they are known for their
significant pacemaker role in cardiomyocytes209. Recent evidence suggests
there is a greater abundance of T-type Ca2+ channels in small resistance
arteries compared to L-type Ca2+ channels, indicating an important role for Ttype Ca2+ channels in microvascular constriction221.
- 48 -
The importance of voltage-gated Ca2+ channels are evident by the documented
efficacy of voltage-gated Ca2+ channel antagonists, the primary medical therapy
for hypertension, acute coronary syndromes and PAD41, 222, 223. However, the Ltype Ca2+ channel is the sole target of clinically available Ca2+ channel blockers
and often do no provide 100% protection to patients suffering from vascular
disease. Combinatorial drug therapy with diuretics, ACE inhibitors, specific
agonist receptor blockers or anti-thrombotic agents is frequently required.
The activation or inhibition of other ion channels such as K+, Cl- and nonselective cation channels are also important regulators of extracellular Ca2+
entry. Potassium channels are the fundamental determinants of membrane
potential in vascular smooth muscle cells and thereby greatly influence the
activation of voltage-gated Ca2+ channels. Under normal physiological
conditions, the concentration of intracellular K+ (140mM) greatly exceeds
extracellular K+ (5mM) so the plasma membrane has a greater permeability
towards K+ compared to the other major ions, Na+, Cl-. Thus K+ distribution
across the smooth muscle cell membrane generates a resting membrane
potential of -60 to -40mV224,
225
. Increasing K+ efflux (e.g., by NO or EDHF-
mediated activation of cGMP and PKG) stabilises the plasma membrane at
more negative membrane potentials (hyperpolarisation), which reduces the
likelihood of opening voltage-gated Ca2+ channels, reducing Ca2+ influx and
favouring vasodilation132. Conversely inhibition of K+ efflux or promoting K+
influx (e.g., with high K+ buffers) causes membrane depolarisation and
promotes rapid Ca2+ influx through voltage-gated Ca2+ channels, favouring
vasoconstriction. Several types of K+ channels have been identified and these
- 49 -
include voltage-gated K+ channels, Ca2+-activated K+ channels and ATPsensitive K+ channels, among many, each with distinct tissue distributions and
patterns of regulation224-226.
Intracellular Ca2+ stored within the sarcoplasmic reticulum is approximately
10,000 times higher than resting cytosolic Ca2+ concentrations215, 227. This large
Ca2+ reservoir allows a robust Ca2+ release from the sarcoplasmic reticulum,
which is believed to play an important role in the initial-rapid phase of specific
agonist-mediated vasoconstriction. Dynamic control of Ca2+ release from the
sarcoplasmic reticulum occurs by two mechanisms: 1) via the ryanodinesensitive receptors and 2) via the inositol triphosphate (IP3) receptors227-229.
Previous studies have suggested intracellular and extracellular Ca2+ entry may
be coupled. A mechanism termed Ca2+ induced Ca2+ release describes a
process where small increases in cytosolic Ca2+ promotes its own release by
directly activating Ca2+ release from the sarcoplasmic reticulum and/or
activation of L-type Ca2+ channels leading to MLCK activation and
vasoconstriction230,
231
. The complexity of Ca2+ regulation in vascular smooth
muscle is detailed in figure A2.
Although increased cytosolic Ca2+ concentrations and subsequent activation of
MLCK has long been considered the primary mechanism underlying
vasoconstriction, studies have shown vascular smooth muscle cell contraction
can be initiated and maintained independent of cytosolic Ca2+ 232, 233. Although
low cytosolic Ca2+ concentrations and subsequent decreased Ca2+-bound
calmodulin enables auto-inhibition of MLCK, vasorelaxation cannot occur until
- 50 -
the phosphate groups have been removed from myosin LC20. Dephosphorylation of myosin LC20 requires the trimeric Ca2+ independent enzyme,
myosin phosphatase. When myosin LC20 is not phosphorylated, actomyosin
interaction is diminished and subsequent cross-bridge cycling cannot occur and
so the cell relaxes. Therefore, the ability of vascular smooth muscle cells to
contract or relax is dependent on the relative activity of MLCK and myosin
phosphatase, respectively203, 234.
A4.4. Regulation of Ca2+ sensitization via myosin light chain phosphatase
Several studies have reported an enhanced contractile response to fixed Ca2+
concentrations following ET-1 stimulation in α-toxin and β-escin permeabilised
rabbit mesenteric arteries235, 236. In 1994, Somlyo and Somlyo were one of the
first to provide the mechanism for increased LC20 phosphorylation at constant
cytosolic Ca2+ concentrations. They reported that when MLCK was inhibited, α1adrenergic receptor activation caused sensitivity of myosin LC20 to Ca2+ by the
inhibition of myosin phosphatase, favouring vasoconstriction237. This is
consistent with a greater force-Ca2+ ratio following noradrenaline, thromboxane
A2 and α1-adrenergic receptor activation of rat aorta and rabbit pulmonary
artery compared to K+-mediated membrane depolarisation where voltage-gated
Ca2+ channels are activated without influencing the activation state of myosin
phosphatase210, 238.
Myosin phosphatase is composed of three (3) distinct subunits: a type 1
phosphatase catalytic subunit of approximately 37kDa, a 130kDa regulatory
subunit and a 20kDa subunit of little known function. The regulatory subunit
- 51 -
anchors myosin phosphatase tightly to myosin and is so called the myosin
phosphatase targeting subunit (MYPT)239,
240
. Myosin phosphatase inhibition
has been credited to 1) direct inhibition of the catalytic subunit and 2) an
inhibitory phosphorylation of the regulatory MYPT subunit. In 1995, David
Hartshorne’s group showed that partially purified myosin-bound myosin
phosphatase from turkey and chicken gizzard could be phosphorylated at
MYPT Thr695 residues, which inhibited the ability of the enzyme to
dephosphorylate LC20241. Alternatively phosphorylation of MYPT Thr855 has
recently been reported to decrease myosin phosphatase activity following α1adrenergic and thromboxane A2 receptor activation242-246. Rho-associated
kinase (ROK) and protein kinase C (PKC) activation are well recognized Ca2+
sensitizing mechanisms247 (discussed below). However, it is also important to
recognize zipper interacting protein kinase (ZIPK) and integrin-linked kinase
(ILK) have also recently been shown to cause inhibitory phosphorylation of
myosin phosphatase in various vascular smooth muscle preparations248-250.
However, whether ILK and ZIPK-mediated inhibition of myosin phosphatase
occurs independently of ROK and PKC is yet to be identified.
A4.5. PKC/CPI-17-mediated Ca2+ sensitisation
Previous studies have indicated direct PKC activation by phorbol 12,13dibutyrate (PDbu) is consistent with LC20 phosphorylation and vascular smooth
muscle cell contraction independent of cytosolic Ca2+, indicating Ca2+
sensitization via myosin phosphatase inhibition243,
251-254
. PKC describes a
family of serine/threonine kinases, of which there are 11 known Ca2+ dependent
and Ca2+ independent isoforms. In the vasculature, PKCα, δ, ε, and ζ have
- 52 -
been identified as important regulators of vascular smooth muscle function255257
. Previous studies have shown G-protein coupled receptor activation,
specifically activation of Gq/11, by circulating vasoconstrictors (e.g. angiotensin
II, adrenaline and ET-1) activates phospholipase C which in turn cleaves
phosphatidylinositol 4,5-biphosphate (PIP2) to form diacylglycerol (DAG) and
IP3. IP3 releases Ca2+ from the sarcoplasmic reticulum causing Ca2+-calmodulin
dependent MLCK activation and vasoconstriction. This is coupled with DAGmediated activation of PKC and subsequent phosphorylation of the Thr38
residue of the 17kDa protein CPI-17247. When phosphorylated, CPI-17
activation increases by approximately 6000 fold at which point it becomes a
potent and direct inhibitor of the catalytic subunit of myosin phosphatase,
promoting a greater response to a given level of cytosolic Ca2+, favouring
vasoconstriction258, 259. Evidence indicates DAG also increases the activation of
non-selective cation channels, favouring membrane depolarisation and
subsequent opening of voltage-gated Ca2+ channels260; providing a possible
mechanism underlying Gq/11-mediated activation of voltage-gated Ca2+
channels by specific vasoconstrictors.
The importance of PKC/CPI-17 in modulating vascular smooth muscle
constriction may be tissue and agonist specific. For example, studies have
shown α1-adrenergic receptor activation causes CPI-17 phosphorylation in
intact rabbit femoral arteries, which can be abolished with PKC inhibitors242,
whereas thromboxane A2-mediated vasoconstriction in rat caudal arteries is
insensitive to PKC inhibitors243. Additionally, western blot analysis showed
abundant CPI-17 Thr38 phosphorylation following α1-adrenergic receptor
- 53 -
activation of rabbit femoral artery242 but no CPI-17 Thr38 phosphorylation
following thromboxane A2-mediated vasoconstriction in rat caudal artery243. In
contrast, PKC inhibition attenuated thromboxane A2-mediated vasoconstriction
in rat pulmonary artery261. Previous studies have suggested different isoforms
of PKC are present in different vascular beds and may be differentially activated
by specific agonists253,
255, 262
. Certainly it has been documented the relative
abundance of CPI-17 and myosin phosphatase is differentially expressed
between vascular beds254.
A4.6. RhoA/ROK-mediated Ca2+ sensitisation
Several other G-proteins, including G12/13 activate a signalling cascade involving
a small monomeric G-protein, RhoA. Experimental evidence has indicated
activation of RhoA is consistent with increased LC20 phosphorylation and
subsequent vascular smooth muscle contraction243,
245, 246, 263-266
. These data
indicate RhoA is involved in communicating an inhibitory signal to myosin
phosphatase following activation of plasma membrane G-protein coupled
receptors. Previous studies have shown ADP-ribosylation and consequent
inhibition of RhoA using the chimeric DC3B toxin and epidermal cell
differentiation inhibitor (EDIN) attenuated the tonic phase of α1-adrenergic
receptor activation and the increased sensitivity to Ca2+ following ET-1 and
guanosine triphosphate (GTP) stimulation of intact rabbit portal vein and rabbit
mesenteric artery helical strips267,
268
. Interestingly, ADP-ribosylation of RhoA
did not attenuate Ca2+ sensitivity following smooth muscle stimulation with
PDBu, a direct PKC activator; indicating the underlying mechanism of RhoA
and PKC-induced inhibition of myosin phosphatase activity are independent267.
- 54 -
At rest, the GTPase RhoA, exists largely in an inactive form in the cytosol,
bound to GDP and a guanine nucleotide dissociation inhibitor (GDI). Following
stimulation of receptors coupled to G13 G-proteins, the subsequent activation of
guanine-nucleotide exchange factors (GEFs) substitutes the nucleotide GDP
with GTP. The consequent displacement of the RhoA-bound GDI exposes an
isoprene moiety, which enables RhoA to translocate to and interact with the
plasma membrane203,
234
. Activated RhoA in turn activates a Rho-associated
serine/threonine kinase (ROK)269. Although the mechanism of RhoA-mediated
ROK activation is still unclear, activation of ZIPK and/or ILK have been
suggested as a possible intermediate pathway between RhoA and ROK248-250.
Unlike PKC/CPI-17 which directly inhibits the catalytic core of myosin
phosphatase, ROK activation has been shown to increase MYPT Thr855
phosphorylation242-244, 246, 270. This phosphorylation inhibits the ability of myosin
phosphatase to remove the phosphoryl groups from myosin LC20, presumably
by impairing the interaction between regulatory MYPT and catalytic subunits.
Although MYPT Thr695 phosphorylation has been previously shown to regulate
activity of purified myosin phosphatase241, cellular in vitro studies did not find an
agonist-mediated change in MYPT Thr695 phosphorylation nor inhibition of this
phosphorylation by the ROK inhibitors Y27632 and H1152; but did identify an
increase in Thr855 of regulatory MYPT, which was blocked by ROK
inhibition242-246 suggesting Thr855 is a ROK-mediated inhibitory site of MYPT.
Numerous studies have shown myosin phosphatase phosphorylation is slow in
onset and consistent with myosin LC20 phosphorylation during sustained
contraction.
This
suggests
that
RhoA/ROK
vasoconstriction242, 243.
- 55 -
is
important
for
tonic
ROK and PKC subcellular pathways have historically been considered
independent mechanisms of Ca2+ sensitization. However, over the last decade,
some studies have suggested cross-talk between ROK and PKC. For example,
in rabbit aortic preparations, ROK activation with a thromboxane A2, mimetic,
U46619 was consistent with CPI-17 Thr38 phosphorylation which was
abolished with the ROK inhibitor Y27632 but was insensitive to the general
PKC inhibitor GF109203X271, 272.
The physiological importance of ROK activity has been shown experimentally
with ROK inhibitors, Y27632 and H1152 which markedly reduced agonistmediated vasoconstriction. Western blot analysis of myosin phosphatase
phosphorylation has indicated basal RhoA/ROK activation242, 243. One must be
careful to interpret studies solely using ROK inhibitors in specific cellular
signalling pathways, i.e., blocking basal myosin phosphatase inhibition will lead
to enhanced dephosphorylation of myosin LC20 favouring general vasodilation
(or attenuation of contraction) to a variety of agents rather than a specific
response. Nevertheless, fasudil, a clinically available ROK inhibitor has been
shown to be effective in reducing peripheral vascular resistance, decreasing the
frequency of vasospastic variant angina273 and improving clinical outcomes
following acute ischaemic stroke274.
- 56 -
Figure A2. Molecular mechanisms involved in vascular smooth muscle
contraction. Increased cytosolic Ca2+ entry via voltage-gated (L- and T-type),
receptor operated (ROCC), and non-selective (NSCC) Ca2+ channels and/or
intracellular Ca2+ release via sarcoplasmic reticulum (SR) inositol triphosphate
(IP3) receptor, activates myosin light chain kinase (MLCK) leading to
phosphorylation
of
myosin
and
actomyosin
cross-bridge
cycling
and
contraction. Cytosolic Ca2+ extrusion occurs via the plasma membrane Ca2+
ATPase (PMCA) and SR uptake of Ca2+ via the sarco-endoplasmic reticulum
Ca2+ ATPase (SERCA). Myosin phosphatase (MLCP) dephosphorylates the
light chains of myosin, favouring relaxation. Activation of PKC/CPI-17 and
RhoA/ROK directly inhibits or causes an inhibitory phosphorylation of MLCP,
respectively, favouring vasoconstriction. Endothelial-derived, NO, PGI2 and
EDHF increases smooth muscle K+ extrusion and hyperpolarises the
membrane,
limiting
voltage
gated
Ca2+
channel
vasodilation. Modified from Wilson et al., 2011275.
- 57 -
activation,
favouring
A5. The physiological role and molecular basis of
important vasoconstrictors
Vascular tone can be modulated by both endogenous and clinically
administered vasoconstrictors. In this section, the molecular pathways involved
in α1-adrenergic (e.g. catecholamines), thromboxane A2, ET-1 and serotonergic
receptor activation will be reviewed.
In vivo, vasoactive agents rarely act in isolation; the physiological response to
any one vasoactive agent depends upon the local concentrations of other
vasoconstrictor and vasodilator substances. Of particular importance are
endothelium derived vasoactive substances and local neural input and these
influences need to be considered when interpreting the molecular mechanisms
discussed below. However, to simplify the discussion we shall consider each
vasoconstrictor agent individually, detailing how it acts through its specific Gprotein coupled receptor to effect vascular smooth muscle contraction.
A5.1. α1-adrenergic receptor activation
Oliver and Schafer first reported the vasoconstrictive properties of adrenal
gland extracts 127 years ago276. Catecholamines are water-soluble tyrosine
and phenylalanine derivatives, which include noradrenaline and adrenaline.
Production
of
these
catecholamines
from
the
adrenal
medulla
and
postganglionic neurons activate adrenergic receptors in the vasculature and the
heart playing a particularly important role in the regulation of heart rate and
peripheral vascular resistance. There are two classes of adrenergic receptors:
- 58 -
α-adrenergic and β-adrenergic receptors which are further sub-classified into
α1, α2, β1, β2 and β3-adrenergic receptors. Distribution of these receptors
throughout the cardiovascular, nervous and respiratory systems has differential
effects on specific organs. Briefly, noradrenaline and adrenaline stimulate
muscle tissue, liberate fatty acids, increase gluconeogenesis and regulate
specific endocrine secretions85. This section will largely focus on the molecular
basis underlying α1-adrenergic receptor activation in vascular smooth muscle
cells. Nevertheless it is important to recognize that β1-adrenergic receptors are
primarily cardiac and are both inotropic and chronotropic277. β2-adrenergic
receptors are found in the peripheral vasculature while β3–adrenergic receptors
exist in adipose tissue and some endothelial cells where receptor simulation
leads to the release of NO278.
Elevated levels of noradrenaline and adrenaline due to increased physical or
emotional stress have been associated with hypertension, coronary artery
disease, heart failure and PAD. The link between stress-induced catecholamine
release and vascular disease was first shown using monkeys by Manuck and
colleagues279. This study measured the acute heart rate response following
threatened capture and physical handling in monkeys on a moderately
atherogenic diet for 22 months. This threatening stimulus caused an acute
increase in heart rate, indicating a transient increase in catecholamine release.
The percentage increase in heart rate from baseline was used to group low
(61% increase) and high reactors to stress (88% increase). Following
termination, this study identified that high reactors to stress had significantly
- 59 -
greater atherosclerotic coronary and aortic arteries compared to the low reactor
experimental group279, implicating chronic stress as a mediator of vascular
disease.
In humans, it has been documented that the degree of increased blood
pressure following cold pressor testing (immersion of hands in ice-cold water for
60 seconds) was a strong predictor of cardiovascular disease. This study
reported the response to cold pressure testing was more strongly associated
with future cardiovascular events then standard risk factors examined in the
study, including hypercholesterolemia and physical inactivity280.
α1-adrenergic receptor blockers such as prazosin, decrease Ca2+ entry and
vasoconstriction mediated by noradrenaline and adrenaline and are one of
many
treatment
strategies
in
the
management
of
hypertension281.
Mechanistically, α1-adrenergic receptors are coupled to Gq/11 G-proteins on the
plasma membrane of vascular smooth muscle cells282. Activation of Gq/11protein leads to phospholipase-mediated cleavage of PIP2 to IP3 and DAG. IP3
binds to IP3 receptors on the sarcoplasmic reticulum causing Ca2+ release. DAG
can activate PKC/CPI-17-dependent inhibition of the catalytic core of myosin
phosphatase, favouring contraction. More recent experiments using rabbit
femoral artery have documented α1-adrenergic stimulation activates the G12/13
coupled RhoA/ROK and subsequent inhibitory phosphorylation of MYPT
Thr855 on myosin phosphatase242. α1-adrenergic receptor stimulation also
activates voltage-gated Ca2+ channels to augment extracellular Ca2+ entry283.
- 60 -
A5.2. Thromboxane A2
In 1969, Piper and Vane documented a compound being liberated in the
porcine pulmonary circulation during anaphylaxis, which was later identified as
a chemically unstable metabolite of prostaglandin endoperoxidase and named
thromboxane A2284,
285
. Thromboxane A2 is a potent vasoconstrictor and
activator of platelets286,
generated
by
the
287
and, like prostacyclin in the endothelium, is
conversion
of
arachidonic
acid
to
prostaglandin
endoperoxidase by COX-1 and COX-2284, 288, 289 which, in turn, is converted into
the intermediate, prostaglandin H2. In platelets, prostaglandin H2 is converted to
thromboxane A2 by thromboxane synthase290, 291. Thromboxane A2 generation
occurs in both the dense granules of platelets and vascular smooth muscle in
response to various stimuli286. It is well documented that thromboxane A2 has a
short half-life of 30 seconds before it is rapidly hydrolysed to near inactive but
stable thromboxane B2286, hence research investigating the structure and
physiological function of thromboxane A2 have had to employ stable
thromboxane analogues, such as U46619292-294.
The thromboxane A2 receptors, TP, are G-protein coupled receptors, of which
there are two human isoforms, namely TPα and TPβ295. Both isoforms express
similar ligand binding and have been shown to be coupled to both Gq/11- and
G12/13-proteins. Whilst TPα and TPβ both activate PLC they have an opposing
effect on adenylate cyclase: TPα stimulates whereas TPβ inhibits adenylate
cyclase activity296. The downstream signalling pathways mediating TP induced
contraction involve various protein kinases, including PKC, ROK and tyrosine
kinase. The equivocal nature of the signalling pathway is complicated by the
- 61 -
fact that there appears to be heterogeneity amongst vascular beds and that
some kinase activations are necessary and permissive, while others are directly
involved in the activation. For example, in rat pulmonary arterial smooth
muscle, thromboxane A2 has been reported to activate the atypical PKCζ,
which led to the inhibition of Kv channels261. This in turn caused membrane
depolarisation, opening of voltage-gated Ca2+ channels, MLCK activation and
vasoconstriction. In contrast, in rat caudal artery, thromboxane A2-mediated
contraction was insensitive to the broad-spectrum PKC inhibitor, GF109203X,
indexed by both the functional contractile response and the phosphorylation
status of CPI-17 Thr38297. Nevertheless, it is perceived that thromboxane A2induced contraction is mediated primarily through Ca2+ entry via voltage-gated
L-type Ca2+ channels and RhoA/ROK Ca2+ sensitisation pathways as inhibition
using a L-type Ca2+ channel blocker, nicardipine, successfully attenuated 80%
of thromboxane A2-mediated vasoconstriction243. Similarly, ROK inhibition
completely abolished the contractile response in intact vascular preparations243,
297
. Hence, this discrepancy may be a consequence of the heterogeneity of
vascular beds used in the studies.
As thromboxane A2 is a biologically potent compound, it has been implicated as
a mediator of thrombotic and vasospastic disorders298-300. Analysis of urinary
thromboxane B2 using gas chromatography revealed the biosynthesis of
thromboxane A2 is increased following exposure to a cold stimulus in patients
with vasospastic systemic sclerosis and Raynaud’s phenomenon; indicating
thromboxane A2 may be exacerbating vasospasm in these patients301. Whole
blood flow cytometry has previously shown platelet reactivity to be increased in
- 62 -
patients with stable coronary artery disease302. Consequently, antiplatelet
agents such as aspirin and clopidogrel have been shown to be beneficial for
patients with peripheral and coronary artery disease43. The clinical value of
thromboxane A2 receptor antagonists, such as terutroban, are also currently
being explored as therapeutic agents in the management of vascular
disease303,
304
, however the additional benefits of thromboxane A2 receptor
blockade over conventional aspirin/clopidogrel therapy are yet to be
established305.
A5.3. Endothelin-1
Furchgott’s Nobel Prize winning experiments illustrating the role of the
endothelium in mediating vasodilation110 initiated a global surge of research in
this field as investigators began to realise the importance of endotheliumdependent regulation of vascular function306. In 1985, Hickey and colleagues
discovered an endothelial-derived polypeptide vasoconstrictor which was later
isolated from porcine aortic endothelial cells by Yanagisawa’s group in 1988
and named endothelin (ET)113. ET is composed of 21-amino acid residues and
has been reported to have potent long lasting vasoconstrictor effects,
particularly in the microvascular circulation307. There are currently three (3)
known endogenous isoforms of ET: ET-1, ET-2 and ET-3 expressed in various
tissue and cell types308. ET-1 is the predominant ET derived from the
endothelium and will be the focus of the following section.
In response to various stimuli, including shear and mechanical stress309,
hypoxia310 and specific agonists311, ET-1 is generated from the precursor,
- 63 -
preproendothelin. Preproendothelin is converted into a biologically inactive
intermediate called big-ET. Endothelin-converting enzyme converts big-ET to
active ET-1312, 313. At present, there are three known ET-1 receptors, ETA, ETB
and non-mammalian ETC G-protein coupled receptors314-317. In the endothelium,
ETB receptor activation up-regulates vasodilatory NO via Ca2+ entry and PKBmediated activation of eNOS146,
147
. In vascular smooth muscle cells ETA
receptors are coupled to Gq/11, and G12/13 G-proteins while ETB receptors are
exclusively coupled to Gq/11 G-proteins317,
318
. Binding to the Gq/11 G-proteins
leads to the activation of PLCβ. This triggers IP3-mediated sarcoplasmic
reticulum Ca2+ release and direct PKC/CPI-17-mediated inhibition of the
catalytic core of myosin phosphatase. In addition, activation of the ETA-G13
signalling cascade activates the RhoA/ROK pathway, causing an inhibitory
phosphorylation
of
the
MYPT
Thr855,
favouring
vasoconstriction318.
Independent evidence suggests that ET-1 augments voltage-gated Ca2+
channel activation in smooth muscle cells221; however the exact mechanism is
yet to be established. Nevertheless, the following two mechanisms have been
proposed: ET-1 possibly evokes membrane depolarisation via 1) DAGmediated activation of non-selective cation channels319, 320 or 2) inhibition of Kv
channels via the atypical PKCζ261.
Clinical studies have identified elevated ET-1 plasma concentrations in patients
with vascular disease, implicating ET-1 as a mediator of hypertension,
atherosclerosis, chronic renal failure and coronary and peripheral artery
disease, among many disorders321, 322. Although ET-1 is primarily expressed in
the endothelium, it is also generated in the kidney323 and specific inflammatory
- 64 -
and smooth cells in atherosclerotic arteries82, indicating a role for ET-1 in
regulating renal perfusion and mediating atherogenesis. Previous findings have
reported patients with critical limb ischaemia and elevated serum ET-1 have a
greater likelihood of suffering from a major vascular event and have a greater
risk of death by myocardial infarction or stroke324. Reducing vascular reactivity
to ET-1 is hence of great clinical interest in the management of vascular
disease. Whether ET-1 increases blood pressure by directly mediating hypercontraction and increased vascular tone of small resistance arteries or by
altering renal hemodynamics and urinary and salt water excretion is still
unclear. Nevertheless, ETA and/or ETB receptor blockade is being explored as a
therapeutic strategy to reduce peripheral vascular resistance in patients with
coronary artery disease and pulmonary hypertension. Intra-arterial infusion of
BQ123, an ETA receptor antagonist has been shown to decrease hypercontractility in atherosclerotic coronary arteries325. Similarly, Bosentan, a
combined ETA/B receptor blocker has been shown to be effective in decreasing
digital ulcers326 in patients with PAD. Previous studies have also reported
Bosentan reduces pathological pulmonary arterial pressure and improves
exercise tolerance in patients with vascular disease327. ET-1 has largely been
studied in the coronary and pulmonary vasculature, however the direct
influence of ET-1 on peripheral arteries is unclear in health and disease.
A5.4. Serotonin
First identified and isolated by Rapport and colleagues in 1948328, 329, serotonin
or 5,hydroxytryptamine (5HT) is a monoamine, similar to adrenaline. In the
periphery, serotonin is namely produced in platelets but has also been found in
- 65 -
nerve endings and enterochromaffin cells. Although serotonin is an important
regulator of diverse functions, it is most recognised as a neurotransmitter,
facilitating many central nervous system functions, including pain perception,
mood, behaviour, appetite and sleeping patterns. However, 90-95% of
endogenous serotonin is stored in the periphery and as such, is involved in
gastrointestinal peristalsis, platelet aggregation, inflammation and regulation of
the microcirculation330.
The molecular basis of serotonin receptor mediated vasoconstriction is similar
to α1-adrenergic receptor activation but the specific isoforms of downstream
effector proteins vary depending on the vascular bed as does the relative
magnitude of the response. In the vascular system, serotonin causes
vasoconstriction by activating G-protein coupled receptors on vascular smooth
muscle cells. Currently there are seven (7) known subtypes of serotonin
receptors; vascular smooth muscle cells being predominately regulated by
5HT1B and 5HT2A subtypes, although 5HT7 receptors have also been identified
in the coronary vasculature331,
332
. On the endothelium, it has been proposed
that serotonin receptor activation increases cytosolic Ca2+ and induces eNOS
activation and the subsequent production of NO333. In vascular smooth muscle
cells, serotonin receptor stimulation activates both Gq/11 and G12/13 G-proteins in
addition to voltage-gated Ca2+ channels on the plasma membrane332,
334
. The
response to increased Ca2+ is augmented by inhibition of myosin phosphatase
through activation of PLC and subsequent activation of PKC/CPI-17335,
336
.
However it remains unclear which isoform of PKC is activated during serotoninmediated vasoconstriction. As described above, activation of the G12/13-protein
- 66 -
coupled receptor signalling cascade promotes vasoconstriction mediated by
RhoA/ROK-dependent inhibition of myosin phosphatase, further sensitizing the
smooth muscle to Ca2+ 337, 338.
The release of serotonin from platelets occurs during platelet aggregation,
thrombosis and inflammation and plays an important role in local hypercontractility. Like adrenaline and noradrenaline, the metabolism of serotonin is
dependent
on
membrane-bound
monoamine
oxidase118.
Hence,
the
pathophysiology of serotonin-mediated vasoconstriction is linked to a
decreased production of monoamine oxidase by endothelial cells coupled with
an increased release of serotonin from platelets. Intracoronary infusion of
serotonin in patients with stable and variant angina induces coronary artery
vasoconstriction at a dose that dilates normal arteries. Serotonin-mediated
vasoconstriction is significantly greater in small distal and collateral vessels in
these patients suggesting a role for serotonin in mediating acute myocardial
ischaemia and coronary microvascular dysfunction90. It has been suggested
paradoxical vasodilation and vasoconstriction to serotonin is due to the
presence of an intact endothelium, indicating that serotonin-mediated
vasodilation is dependent on a functional endothelium339. This is consistent with
the role of the endothelium in mediating atherogenesis and vasospasm.
Patients with peripheral vascular disorders have shown increased plasma
serotonin and increased reactivity to serotonin340 which is abolished in the
presence of non-selective serotonin antagonists, ketanserin and naftidrofuryl341.
Additionally, in animal models of hind limb perfusion, serotonin-receptor
antagonism has been shown to significantly improve blood flow and decrease
- 67 -
skeletal muscle damage342. However the direct influence of serotonin on
vascular constriction has not been identified in patients with PAD.
- 68 -
A6. Thesis aims
The overall objective of this thesis was to identify the intracellular signalling
mechanisms
of
microvascular
vasoconstriction
in
acute
and
chronic
experimental models of ET-1-mediated vasoconstriction and to identify whether
these molecular mechanisms could be translated to patients with PAD.
Specific aims:
1) To identify the temporal activation of PKC and ROK during acute ET-1mediated vasoconstriction in isolated large and small arteries.
2) To identify whether contractile responses to an α1-adrenergic agonist,
thromboxane A2, endothelin-1 and serotonin were altered following
chronic exposure to ET-1.
3) To identify whether the vasoconstrictor response to α1-adrenergic,
thromboxane A2, endothelin-1 and serotonergic receptor activation were
altered in human subcutaneous arteries (300-400µm) from patients with
and without diagnosed PAD and to identify whether these responses
were dependent on the abundance and activation of eNOS and myosin
phosphatase.
- 69 -
Section B.
General Methods
- 70 -
B1. Materials
B1.1. Functional myography
Materials used for functional vascular myography were purchased from the
following
suppliers:
HEPES
sodium
salt
((4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid), acetone, H1152 ((S)-(+)-2-Methyl-1-[(4-methyl5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine
dihydrochloride),
bisindolylmaleimide (GF109203X), acetylcholine, U46619, serotonin, and
phenylephrine were from Sigma-Aldrich, Australia. Endothelin-1 (ET-1) was
synthesized by Auspep, Australia. Sodium chloride, potassium chloride,
magnesium chloride, calcium chloride, potassium dihydrogen phosphate,
sodium bicarbonate, EDTA, D-glucose and trichloroacetic acid (TCA) were from
Merck & Co Inc., Australia. Sylgard® was from Dow Corning, Australia.
B1.2. Mini-osmotic pump and jugular vein cannulation
Materials used for mini-osmotic pump jugular vein catheterization were
purchased from the following suppliers: mini osmotic pumps (model 1002,
0.25µL/hr) were from ALZET® Durect, USA. Polyethylene tubes were from
Intramedic, Australia. Non-absorbable surgical sutures were from
©
Ethicon,
Inc., Australia. Ibuprofen was from Reckitt Benckiser, Australia.
B1.3. Sodium dodecyl sulphate polyacrylamide gel electrophoresis
Materials used for sodium dodecyl sulphate polyacrylamide gel electrophoresis
(SDS-PAGE) were purchased from the following suppliers: glycerol, sodium
dodecyl sulfate (SDS), bromophenol blue, diisopropylfluorophosphate (DFP),
ammonium
persulfate
(APS),
and
N,N,N’,N’-tetramethylethylenediamine
- 71 -
(TEMED) were from Sigma-Aldrich, Australia. Glycine and Tris were from
AMRESCO, USA. 30% Acrylamide/0.8% N,N’-methylenebisacrylamide solution,
Tween-20, and 0.2µm nitrocellulose membrane were from BioRad Laboratories,
Australia. Complete protease inhibitor cocktail™ was from Roche, Australia.
Pre-stained molecular weight markers (PageRuler™ prestained protein ladder
plus) were from Fermentas Life Sciences. Sucrose, dithiothreitol (DTT), acetic
acid, coomassie brilliant blue R-250, and ponceau-S were from Merck and Co
Inc., Australia.
B1.4. Western blot analysis
Materials used for western blot analyses were sourced from the following
suppliers: mouse monoclonal anti-eNOS was purchased from BD Biosciences,
USA. Mouse monoclonal anti-MYPT was made in house243,
249
. Rabbit
polyclonal anti- anti-P[Ser1177]eNOS and P[Thr855]MYPT were purchased
from Upstate/Millipore, USA. Mouse monoclonal anti-5HT2A was purchased
from Santa Cruz Biotechnology, USA. Anti-rabbit biotin-conjugated secondary
IgG, anti-mouse biotin-conjugated secondary IgG, streptavidin-conjugated
800nm fluorochrome and West Femto enhanced chemiluminescent (ECL)
reagents were from Pierce Thermo Fisher Scientific Australia Pty. Anti-mouse
horseradish peroxidase (HRP)-conjugated secondary IgG was from Santa Cruz
Biotechnology, USA. X-ray film and standard developing agents were
purchased from Afga, Belgium.
- 72 -
B2. Functional Vascular Myography
B2.1. Background
Wire myography (DMT Danish Myotechnologies, Denmark) is a technique that
enables the functional analysis of developed isometric tension from isolated
arteries <500µm in vitro. Prior to its development by Mulvany and Halpern in
1977, much of our understanding of the vasculature was derived from the aorta,
and specific conduit arteries343. This approach enables the examination of
contractile responses to various agonists and pharmacological blockers in the
absence of circulating vasoactive factors.
The apparatus consists of individual organ baths allowing for the study of
individual vessel segments. Within each organ bath, individual arteries are
mounted to clamps connected to a micrometer and a force transducer. The
micrometer allows for mechanical manipulation of vessel length and resting
tension to optimal resting length (L0) or equivalent to mean arterial pressure of
93mmHg, while the force transducer is connected to a PowerLab system and a
chart recorder (Chart 7, AD Instruments, Australia). Each organ bath is placed
on a common platform which is heated at a constant 37ºC344.
B2.2. Animal tissue preparation
All animal research was approved by the University of Adelaide and Queen
Elizabeth Hospital animal ethics committees. For rat experiments involving
acute ET-1 stimulation (section C) male Sprague Dawley rats (350-400g), were
killed using CO2 inhalation. The caudal artery and the mesenteric artery bed
was removed and placed in a Sylgard®-lined dissecting dish containing Ca2+- 73 -
free HEPES Tyrode buffer (containing in mM: 135.5 NaCl, 5.9 KCl, 1.2 MgCl2,
11.6 HEPES pH 7.4 and 11.6 glucose). Once isolated each artery was carefully
dissected from the surrounding adventitia and adipose tissue under a dissecting
microscope. Arteries were then cut into 2mm segments. Particular care was
taken not to stretch the blood vessel beyond 25% of its resting length to avoid
damage to the contractile apparatus. Following dissection two 40µm wires were
introduced through the lumen of each blood vessel with special care taken not
to damage the endothelium. Isolated artery segments were then placed into the
myograph organ baths containing Ca2+-free HEPES Tyrode buffer. Once
arterial segments were mounted, Ca2+-free HEPES Tyrode buffer was removed
with suction and replaced with normal Ca2+ containing HEPES Tyrode buffer
(containing in mM; 135.5 NaCl, 5.9 KCl, 1.2 MgCl2, 2.5 CaCl2, 11.6 HEPES pH
7.4 and 11.6 glucose) heated to 37ºC. Arterial segments were equilibrated in
normal HEPES Tyrode buffer for 20 minutes prior to initiating a specific
experimental protocol.
For experiments involving chronic ET-1 infusion (section D), Male Sprague
Dawley rats were anesthetised with isoflurane and killed by exsanguination
followed by removal of the heart and subsequent isolation of the mesenteric
arteries. Ca2+-free and normal HEPES Tyrode buffers were substituted with
KREBS solution (containing in mM: 118 NaCl, 1.18 KH2PO4, 25 NaHCO3, 1.05
MgCl2, 2.34 CaCl2, 0.01 EDTA and 5.56 glucose, pH7.4). Each organ bath was
constantly bubbled with carbogen (95% O2 and 5% CO2, BOC, Australia) to
maintain dissolved O2 levels and pH at 7.4. Direct comparisons have identified
no difference in contractile responses in the presence of HEPES Tyrode buffer
- 74 -
or KREBS based buffers. Tissue dissection and mounting was conducted as
described above.
B2.3. Human tissue preparation
All human research was approved by the University of Adelaide and Queen
Elizabeth Hospital human ethics committees. Patients with peripheral artery
disease (PAD) undergoing above or below knee amputations consented to
donate subcutaneous tissue from the proximal region of the amputated lower
limb. As the amputation must extend into viable tissue to enable healing of the
wound and subsequent fitting of prosthesis, the tissue collected from the
amputation site is from a non-ischemic area. Subcutaneous tissue from the
abdominal and inguinal region of patients undergoing open hernia repair was
collected as our control cohort. So called control patients had no symptomatic
PAD and had not been previously diagnosed with PAD. All patients provided
written consent to tissue donation prior to their surgery. Small blood vessels
(300-400µm in diameter) were isolated from the subcutaneous tissue and
placed in a Sylgard®-lined dissecting dish containing Ca2+-free HEPES Tyrode
buffer at room temperature. Once isolated vessels were dissected and mounted
as describe above in B2.2.
B2.4. Tissue integrity
The gold standard for measuring tissue integrity and consistent vessel size is
assessing contractile responses to high K+ stimulation. High K+ depolarises the
plasma membrane and activates voltage-gated Ca2+ channels causing a rapid
increase in cytosolic Ca2+ causing vasoconstriction. To determine tissue viability
- 75 -
following dissection and mounting, each vessel was stimulated with isotonic
87mM KCl HEPES Tyrode buffer (containing in mM; 54.4 NaCl, 87 KCl, 1.2
MgCl2, 2.5mM CaCl2, 11.6 HEPES pH 7.4 and 11.6 glucose) or KPSS for
experiments using KREBS (containing in mM: 217 KCl, 1.18 KH2PO4, 25
NaHCO3, 1.05 MgCl2, 2.34 CaCl2, 0.01 Na2EDTA and 5.56 glucose, pH7.4)
heated to 37ºC. Following 87mM KCl and KPSS stimulation, arterial segments
were relaxed with normal HEPES Tyrode buffer or KREBS, respectively. This
contraction/relaxation cycle was repeated three times at 20 minute intervals.
B2.5. Resting tension
In an isolated vessel preparation, optimal resting tension assumes the normal
blood pressure experienced by that vessel in the body. It is important that each
blood vessel is at its own optimal resting tension to allow for comparisons of
contractile responses344. For example, optimal resting tension of a caudal artery
is between 7-12mN while a mesenteric artery is set between 2-3mN. Optimal
resting tension for vessels was determined using a length tension curve. This
was conducted by applying increments of 0.5mN of tension and stimulating with
87mM KCl followed by relaxation. When beyond optimal resting tension,
developed tension declined and vessels were returned to optimal resting length.
The optimal resting tension for a given vessel diameter was then chosen from
this series of experiments.
B2.6. Endothelial integrity
The endothelium is well recognised as an important mediator of vascular tone.
To determine whether the endothelium was intact in these arterial preparations,
- 76 -
each vessel was constricted with a submaximal concentration of the α1adrenergic agonist, phenylephrine (2x10-6M) followed by acetylcholine (1x106
M)-induced vasorelaxation. The endothelium was considered intact if the
acetylcholine-mediated relaxation was greater than 80% of the response to
phenylephrine in rat vessels. However in human vessels, since patients had a
mean age of +70 years, endothelial integrity was simply reported and as
expected was impaired in both PAD and non-PAD groups (figure E7). Following
endothelial integrity testing, vessels were washed 3 times with normal HEPES
Tyrode buffer and equilibrated for 30 minutes.
B2.7. Experimental protocol – single dose
For experiments aimed to identify the molecular mechanisms of acute ET-1
actions (section C), arterial segments were stimulated with a single dose of ET1 (1x10-7M) before and after incubation with pharmacological blockers.
Following the final 87mM KCl stimulation, vessels were equilibrated for 20
minutes in normal HEPES Tyrode buffer before incubation with the specific Rho
kinase (ROK) inhibitor, H1152
(1x10-6M) and/or the broad spectrum protein
kinase C (PKC) inhibitor GF109203X (5x10-6M) for 15 minutes. Control vessels
were incubated in normal HEPES Tyrode buffer for an equal duration. Each
vessel was then stimulated with a maximum concentration of ET-1 (1x10-7M) for
30 seconds or 10 minutes. In post-treatment studies, the pharmacological
blockers were administered 5 minutes after ET-1 administration and the
experiment stopped at 10 minutes following ET-1 challenge.
- 77 -
B2.8. Experimental protocol – dose response
In sections D and E, the response to a specific agonist was assessed by
constructing a dose response curve. Incremental concentrations of each
agonist was directly added to the tissue bath and thoroughly mixed. At each
concentration, vessels reached a plateau response before the subsequent
concentration was added to the bath. The concentration range for each agonist
was as follows; serotonin and phenylephrine: 1x10-8M–3x10-4M, U46619: 1x109
M–3x10-6M, ET-1: 1x10-12M–3x10-7M. In an independent series of experiments
solvent concentrations were tested and found to have no impact on vascular
contraction; solvent concentrations were always less than 0.1%.
B2.9. Snap-freezing and tissue storage
Untreated and unmounted caudal, mesenteric (section D) and human
subcutaneous arteries (section E) were isolated in Ca2+-free HEPES Tyrode
buffer and snap-frozen by direct submersion in liquid nitrogen and stored at 80ºC.
B2.10. Data analysis
All myography recordings were in mN and analysed using Chart 7 (AD
Instruments, Australia). Net developed tension was determined by subtracting
the resting tension from the maximum tension recorded for a given treatment.
All data was presented as mean developed tension ± SEM. Single dose
experiments were statistically assessed using an analysis of variance (ANOVA)
with a Bonferroni’s post-hoc test. For dose response curve experiments a
sigmoidal curve of best fit was generated using four-parameter nonlinear
- 78 -
regression (GraphPad Prism 6). The Hillslope, EC50 and maximal efficacy was
calculated from these dose response curves. Data was statistically assessed
using a two-tailed, non-paired Students t-test where p<0.05 was considered
statistically significant.
- 79 -
B3. Chronic endothelin-1 infusion model
B3.1. Background
The mini-osmotic pump was developed in the 1970s by ALZA® Corporation for
the purpose of internal academic research and released to the global scientific
market in 1977 by ALZET®. Since its conception, mini-osmotic pumps have
emerged as a valuable research tool for constant and reliable delivery of
bioactive compounds in rats and mice345,
346
. The advantages of using mini-
osmotic pumps are three fold: 1) they provide a reliable and constant delivery
rate, 2) they allow for reproducible results, and 3) they are self-powered,
decreasing the need to excessively handle and stress laboratory animals.
The premise of its action is drug delivery by osmotic displacement which relies
on the osmotic difference between the pump and the body fluid of an animal.
Each mini-osmotic pump is composed of three concentric layers; the
impermeable core drug reservoir, the middle osmotic layer and the outer
semipermeable membrane which controls the rate of delivery. The drug
reservoir is filled with the bioactive solution and capped with a flow moderator.
When the mini-osmotic pump is implanted in subcutaneous tissue, the outer
membrane absorbs water and expands the osmotic layer. This compresses the
flexible core compartment, forcing the bioactive solution into the flow moderator
at a controlled and constant pre-determined rate. Attaching a catheter to the
flow moderator enables directed administration of the bioactive compound to
specific organs or tissue beds. For example, we chose to infuse ET-1 into the
jugular vein using a polyethylene catheter.
- 80 -
B3.2. Preparation of mini-osmotic pumps
ET-1 was commercially synthesized and lyophilized by Auspep, Australia. ET-1
(0.1mg/mL) was prepared in 0.9% Tris buffered saline and sterilised by
passage through a 0.2µm syringe filter. Following from the manufacturer’s
instructions347, the drug reservoir of the mini-osmotic pump (Model 1002,
0.25µL/hr, ALZET® Durect Co., USA) was then filled through the delivery portal
with 100µL of sterile ET-1 solution. This allowed for a constant delivery rate of
10ng/kg/min, a dose previously shown to increase blood pressure in rats348.
Osmotic mini-pumps were filled using a 1mL syringe attached to a 21-gauge
needle. Particular care was taken to ensure no air bubbles were introduced into
the drug reservoir. The cap and flow moderator was attached to ensure the
core drug compartment was sealed. In control pumps, the ET-1 solution was
substituted with sterile 0.9% Tris buffered saline.
The mini-osmotic pump catheter consisted of a 3.5cm segment of polyethylene
tubing (0.03 inches internal diameter. Intramedic, Australia). The polyethylene
catheters could not be autoclaved, so were sterilised with 70% ethanol followed
by rinsing with 0.9% sterile saline using a 1mL syringe and a 21-gauge needle.
One end of the polyethylene tube was cut at a 45º angle to facilitate insertion
into the jugular vein. Following from the manufacturer’s recommendations347 a
2mm piece of polyethylene tubing was fitted over the polyethylene catheter,
2cm from the end. This provided an anchoring collar stabilizing the sutures
holding the jugular vein catheter in place. The non-angled end of this catheter
was then fitted to the mini-osmotic pump flow moderator.
- 81 -
Each mini-osmotic pump has a “start-up” gradient during which the pumps
absorb fluid and equilibrates to body temperature before reaching its predetermined flow rate. Therefore it is crucial that each pump is first “primed” in
vitro before implantation. This is achieved by incubating the pumps in 0.9%
sterile saline at 37ºC for 4-6hrs. During this procedure, it is important the
catheter is not fully submerged to prevent saline from entering and diluting the
drug reservoir.
B3.3. Jugular vein cannulation and mini-pump implantation
Male Sprague Dawley rats (350-400g) were placed into a controlled chamber
infused with the gaseous anaesthetic, isoflurane, following removal from the
anaesthetic chamber. Rats were placed in the supine position on the surgical
table. Rats were determined unconscious by lack of pain reflexes in their fore
and hind limbs. A cone providing adjustable isoflurane flow was fitted over the
rat’s nose throughout the surgery. As isoflurane is a central nervous system
depressant it causes decreased blood pressure, reduced respiratory rate and
lowers body temperature349. An assistant monitored anaesthetic levels to
ensure rats remained in an unconscious state but also took particular care to
prevent isoflurane overdose by closely monitoring respiration rate and body
temperature. To maintain animals at 37ºC rats were placed on a heated mat
throughout the surgery.
Sterile technique was maintained during all surgical procedures. The neck was
shaved and skin bathed with 70% ethanol to provide a sterile field. A 1cm skin
incision was made superficial to and 2mm left of the jugular vein. Blunt
- 82 -
dissection revealed the jugular vein and was used to create a pouch in the
subcutaneous tissue to house the in-dwelling mini-osmotic pump. Using curved
forceps, two lengths of non-absorbable surgical sutures (4.0
©
Ethicon, Inc.,
Australia) were carefully threaded under the jugular vein and left untied, 1cm
apart. Haemostats held the sutures in position, while an 18-gauge needle was
used to puncture the outer aspect of the jugular vein. This provided an entry
point for the osmotic mini-pump catheter. The catheter was advanced into the
jugular vein until the anchoring collar reached the incision. To prevent
retrograde movement of the catheter, the two previously placed sutures were
positioned so they straddled the anchoring collar and were tied with triple box
knots. The mini-osmotic pump was then placed in the previously made
subcutaneous pouch and the initial incision closed with 4-5 separately tied triple
box knot sutures. The closed incision site was then cleaned with 70% ethanol
and the isoflurane nose cone was removed.
For pain management, once the animal had regained consciousness, 15mg/kg
ibuprofen (Reckitt Benckiser, Australia) was given per os using a plastic
Pasteur pipette. Throughout the seven (7) day ET-1 infusion protocol, each rat
was closely monitored for incision health, signs of distress and infection.
Following seven (7) days, the animals were again anesthetised with isoflurane
and sacrificed by exsanguination and removal of the heart. The mesenteric
arteries were removed and prepared for functional vascular myography and/or
snap frozen as described in B2.
- 83 -
B4. SDS-PAGE
B4.1. Background
Electrophoresis is the separation of molecules based on their rate of movement
through fluid under the influence of an electric field. In biological science,
protein electrophoresis is a valued and useful tool for the analysis of protein
abundance and activity in a sample. The ionic detergent used in SDS
polyacrylamide gel electrophoresis (SDS-PAGE) binds to proteins to provide a
near uniform negative charge. Consequently, when an electric field is applied,
most proteins migrate from the negative terminal to the positive terminal where
the rate of migration is largely dependent on molecular weight of the SDScoated proteins350-352.
B4.2. Sample preparation
Proteins from isolated vessel samples, snap frozen with 10% TCA/acetone or
liquid nitrogen were extracted in 2x SDS sample buffer (containing: 50mM TrisHCl pH 6.8, 1% SDS, 0.01% bromophenol blue, 20% sucrose, 10% complete
protease inhibitor cocktailTm, 10mM DTT and 10µM DFP). DFP is a protease
inhibitor, particularly important in preventing an unknown protease from
degrading myosin phosphatase. Since it is also a potent inhibitor of
acetylcholinesterases it is a dangerous neurotoxin353.
The volume of sample buffer added to each isolated arterial sample was
dependent on the source vessel. For example, all caudal arteries were
extracted in 200µL of 2x SDS sample buffer while ten (10) mesenteric arteries
- 84 -
from a single rat were collectively extracted in 300µL. Human samples were
extracted in 100-300µL of 2x SDS sample buffer. Extraction volume was
optimized in preliminary experiments and all samples were normalised to
myosin or total extracted protein per lane to account for differences in extraction
and volume loading. See section B4.5 below standardising sample loading.
Once all samples were in 2x sample buffer, they were kept for 5 minutes at
room temperature to inactivate DFP. All samples were then heated for 5
minutes at 95°C and then vortexed for 3 x 15 seconds at 5 minute intervals.
Samples were then loaded into SDS-polyacrylamide gels for protein separation.
B4.3. SDS-polyacrylamide gel preparation
SDS-polyacrylamide gels consist of polymerised acrylamide cross-linked with
N,N’-methylenebisacrylamide. This is normally a slow spontaneous process,
however the addition of the oxidising agent APS with the quaternary amine
TEMED, catalyses this reaction. SDS uniformly coats proteins with a net
negative charge, hence the separation of proteins is dependent on its molecular
weight and the size of pores formed by the polymerised acrylamide. Pore size
is dependent on the total amount of cross linker (%C) and acrylamide (%T). For
a fixed C/T ratio, pore size decreases with increasing concentration.
Conversely, reducing concentration increases pore size. For example, myosin
phosphatase and eNOS are 130kDa proteins and were analysed using 7.5%
polyacrylamide gels while 5HT2A (55kDa) was analysed using 12.5% gels; the
C/T ratio being 0.02%.
- 85 -
SDS-polyacrylamide gels consist of a bottom separating layer where the
density is adjusted to the desired %T. A relatively less dense stacking gel (5%)
was cast above the separating gel and its consistency is the same across all
gels. SDS-polyacrylamide gels were cast using large format glass plates and
1.5mm spacers (Protean® BioRad, Australia). Large format gels were employed
to enable 30 samples to be loaded onto a single gel, reducing inter-gel
variability. 7.5% separating gels were composed of the following: 7.3%
acrylamide, 0.2% bisacrylamide, 375mM Tris-HCl (pH 8.8), 0.1% SDS, 0.025%
TEMED and 0.025% APS. 12.5% separating gels were composed of the
following: 12.3% acrylamide, 0.2% bisacrylamide, 375mM Tris-HCl (pH 8.8),
0.1% SDS, 0.025% TEMED and 0.025% APS. High dissolved oxygen inhibits
the polymerization of SDS-polyacrylamide gels so this solution was de-gased
before the addition of APS and TEMED using a low pressure bell jar. This
acrylamide solution was slowly introduced into large format glass plates to
avoid generation of bubbles, using a 50mL syringe. A thin layer of watersaturated butanol was carefully overlayed across the separating gel to ensure a
uniform interface between the separating and stacking gel layers. Since butanol
is hygroscopic, to avoid dehydration of the top most layer of polyacrylamide,
butanol was water-saturated by mixing with an excess of deionised water. The
separating gel was left to polymerise at room temperature for 1 hour. The
water-saturated butanol was then removed and thoroughly rinsed with
deionised water. Residual water was carefully removed before the stacking gel
was cast. The stacking gel was composed of the following: 4.87% acrylamide,
0.13% bisacrylamide, 375mM Tris-HCl (ph 8.8), 0.1% SDS, 0.025% TEMED,
0.025% APS. Stacking gels were cast with two 1.5mm, 15 well combs.
- 86 -
B4.4. SDS-PAGE run
Polyacrylamide gels were placed into a large format BioRAD Protean® II
electrophoresis module. The linear range of protein to load was calculated for
each antibody in previous experiments and used to determine the volume
loaded for each sample270. The top and bottom chambers of the apparatus
were filled with SDS running buffer composed of: 25mM Tris-HCl, 192mM
glycine and 1% SDS and run at a constant 35mA per gel for 90 minutes.
Although this is a short run for a large format gel, it provides equivalent
separation to that afforded by the Protean® II mini format gels (BioRad
Laboratories, Australia).
B4.5. Standardising sample loading
Since most samples were subject to vascular myography, particular effort was
taken to dissect vessels of a consistent diameter and a uniform length. This
was validated by the similarity in K+-mediated vasoconstriction between
vessels. Since we were interested in assaying the targeting subunit of myosin
phosphatase (MYPT) on the relatively insoluble myofilament (in addition to
eNOS and 5HT2A receptors), we opted to solubilize vessels directly in SDSsample buffer. This precluded the use of traditional Bichionic acid or Bradford
protein assays. Consequently, we subjected an equal volume of each vascular
extract to SDS-PAGE followed by coomassie brilliant blue R250 staining and
subsequent de-staining with 10% acetic acid. Total protein per lane was
quantified using Odyssey V3 software (Li-Cor Biosciences, USA). Protein
concentrations were adjusted to equality by sample dilution. A second SDSPAGE was run and transferred for the analysis of specific proteins.
- 87 -
B4.6. Protein transfer to nitrocellulose membrane
Although proteins were separated on large format gels, the transfer of proteins
onto a nitrocellulose membrane was conducted in mini Protean® II transfer units
(BioRad Laboratories, Australia). Each gel was cut into multiple segments
(approximately 5x6cm) using a sharp knife and transferred on a single unit to
reduce variability imparted by differences in the transfer process. The transfer
buffer for eNOS and myosin phosphatase was composed of the following:
25mM Tris, 192mM glycine, 0.1% SDS and 20% methanol. Transfers for 5HT2A
were conducted in a similar buffer but in the absence of SDS. Samples
analysed for eNOS, myosin phosphatase and 5HT2A were transferred at a
constant 100V for 45, 30 and 60 minutes, respectively.
Following transfer, as a second confirmation of equal loading and transfer
efficacy, gels were stained using coomassie brilliant blue R-250 composed of
(0.2% coomassie blue, 10% acetic acid and 50% ethanol) and de-stained in
10% acetic acid. Even following transfer coomassie staining of high molecular
weight proteins, such as myosin, were still visible and were scanned using the
odyssey imager (Li-Cor Biosciences, USA). Following transfer, transfer efficacy
and equality of protein loading were confirmed by staining the nitrocellulose
membrane using ponceau-S solution (0.1% ponceau S and 5% glacial acetic
acid) for 5 minutes with gentle agitation followed by a rinse with deionized
water.
- 88 -
B5. Western Blot Analysis
B5.1. Background
Western blot analysis is a useful analytical tool which allows for the
identification and quantification of specific proteins following SDS-PAGE and
transfer. Antibodies specific to the protein of interest are used to quantify its
abundance in the sample354. In the current studies, ratiometric western blot
analysis using antibodies specific to both the total and phosphorylated protein
of interest were used to identify the phosphorylation and hence the activation
state of eNOS and myosin phosphatase. Total 5HT2A receptor abundance was
also quantified. A particular strength of multiplex western blot analyses is
afforded by the ability to use both anti mouse/rabbit monoclonal and polyclonal
primary antibodies as well as HRP and fluorochrome conjugated secondary
antibodies. This effectively allows for the analysis of multiple proteins from
samples transferred onto a single piece of nitrocellulose membrane.
B5.2. Blocking
Nitrocellulose membranes were blocked with 30mLs of blocking buffer (30mLs
50mM Tris 7.4, 150mM NaCl, 0.05% Tween-20, 5% non-fat milk powder) for 1
hour with gentle agitation, to avoid non-specific binding of primary and
secondary antibodies.
B5.3. Primary antibody
The primary antibody binds to the protein of interest. Nitrocellulose membranes
were incubated for 1 hour with a 1:1000 dilution of the primary antibody in Tris
buffered saline with Tween-20 (containing: 50mM Tris 7.4, 150mM NaCl, 0.05%
- 89 -
Tween-20) (TBS-T). Unbound primary antibody was then removed by one 30
second rinse followed by 3 x 5 minute washes with TBS-T.
B5.4. Biotin-conjugated secondary IgG
Individual western blots (nitrocellulose membranes) incubated with rabbit antiP[Ser1177]eNOS and rabbit anti-P[Thr855]MYPT, were incubated with antirabbit biotin-conjugated secondary IgG. Membranes treated with mouse anti5HT2A were incubated with anti-mouse biotin-conjugated secondary IgG. This is
a three (3) step antibody detection protocol; the biotin conjugated secondary
antibody increases the sensitivity of the assay in the presence of a streptavidin
conjugated fluorochrome. Membranes were incubated for 1 hour with 10mLs of
a 1:10,000 dilution of the secondary antibody in TBS-T. Unbound secondary
antibody was then removed by one 30 second rinse followed by 3 x 5 minute
washes with TBS-T.
B5.5. Streptavidin-conjugated 800nm fluorochrome
Streptavidin is a glycoprotein capable of binding biotinylated secondary
antibodies, allowing for significant signal amplification. Membranes were
incubated for 1 hour in 10mLs of a 1:10,000 dilution of streptavidin-conjugated
800nm fluorochrome in TBS-T with gentle agitation. Unbound streptavidinconjugated fluorochrome was then removed by one 30 second rinse followed
by 3 x 5 minute washes with TBS-T. Streptavidin was conjugated with a lightsensitive fluorochrome. As a consequence, all incubations and washes were
carried out in the dark. The membranes were left to dry and scanned using the
Odyssey imager.
- 90 -
B5.6. Re-probe with monoclonal anti-MYPT and anti-eNOS
Following analysis of each signal, membranes incubated with rabbit antiP[Ser1177]eNOS and anti-P[Thr855]MYPT were washed with TBS-T for 5
minutes before incubation for 1 hour with a 1:1000 dilution of mouse anti-eNOS
and anti-MYPT. This allowed for the analysis of both total and phosphorylated
eNOS and myosin phosphatase using the same sample and membrane as
previously documented243.
B5.7. Incubation with HRP-conjugated secondary IgG
Membranes incubated with mouse monocolonal anti-eNOS and anti-MYPT
were then incubated with anti-mouse HRP-conjugated secondary IgG.
Membranes were incubated for 1 hour with a 1:5000 dilution of the secondary
antibody in TBS-T with gentle agitation before 3 washes in TBS-T. Due to a
difference in both the species and the detection enzyme, the total protein signal
could be differentiated from the phosphorylated protein.
B5.8. ECL detection
Membranes incubated with HRP-conjugated secondary IgG were detected
using West Femto ECL reagents (Pierce Thermo Fisher Scientific Australia
Pty). The active substrate for HRP is created by mixing luminol with a stable
peroxide buffer in a 1:1 ratio. For each blot 50µL of each reagent was mixed
and then diluted with 400µL of TBS-T to allow for adequate dispersion of the
solution across the membrane. The excess ECL reagents were removed and
the membrane covered with conventional cling-film. Western blots were then
exposed to x-ray film (Afga, Belgium) in the dark and developed using standard
- 91 -
developing and fixing reagents (Afga, Belgium). Developed film was scanned
using a densiometric scanner (GS-710 BioRad, Australia)
B5.9. Data analysis
Integrated intensity (darkness of a given band) was determined using LiCor V3
software for all Odyssey scanned blots and BioRad Quantity One software was
used to analyse x-ray film-based results. In both cases, an analytical rectangle
was placed over the proteins of interest. The software provided the integrated
intensity within the area of each rectangle. The myosin bands and total protein
per lane of the coomassie stained gels were analysed in a similar manner to
determine differences in sample extraction or loading. For phosphorylation
analysis the integrated intensity was presented as phosphorylated / total
specific protein. All data are presented as mean integrated intensity ± SEM. A
two-tailed unpaired student’s t-test was used to compare treated and control
samples, where p<0.05 was considered statistically significant.
- 92 -
Section C.
PKC inhibition attenuated
sustained endothelin-1-mediated
vasoconstriction while ROK
inhibition attenuated initial and
sustained endothelin-1-mediated
vasoconstriction in large and small
arteries
- 93 -
C1. Introduction
Endothelin-1 (ET-1) is a potent 21-amino-acid peptide vasoconstrictor and an
important regulator of blood flow and pressure113. ET-1 also has mitogenic
properties and has consequently been postulated to contribute to intimal
thickening and progression of atheroma355, 356. Increased ET-1 mRNA has been
detected in large conduit arteries from patients with coronary and peripheral
atherosclerosis357,
exacerbating
358
. In this context it has been proposed that ET-1 may be
existing
physical
artery
stenosis
through
inappropriate
vasoconstriction. In addition, pulmonary and systemic hypertension, acute renal
failure and cerebral vasospasm are associated with increased circulating ET-1
and the plasma concentration has been shown to positively correlate with
disease severity359, 360. Hence pharmacological modulation of ET-1 action is an
attractive clinical strategy for the management of vascular disease in our aging
demographic.
ET-1 is principally released by the endothelium in response to shear stress and
infiltrating cells during inflammation82,
316
. ET-1 has dual vasoactive properties
and regulates vascular tone through activation of G-protein coupled receptors
ETA and ETB315,
316
. ETB receptors are located on both the endothelium and
vascular smooth muscle while in the vasculature ETA receptors are exclusive to
smooth muscle. ETB receptor activation on the endothelium increases
vasodilatory nitric oxide production by increasing endothelial nitric oxide
synthase
(eNOS)
activity
through
a
protein
kinase
B/AKt-dependent
mechanism146. In contrast ETA and ETB receptors on vascular smooth muscle
cells are coupled to Gq/11 and G13 G-proteins and cause vasoconstriction
- 94 -
through Ca2+ entry, myosin light chain kinase activation and protein kinase C
(PKC)/CPI-17 and RhoA/Rho kinase (ROK) dependent inhibition of myosin
phosphatase318.
ET-1-mediated vasoconstriction involves an initial transient Ca2+-dependent
phase
followed
by
a
Ca2+-independent
plateau
phase
of
sustained
contraction361. Evidence with other G-protein coupled receptors suggests
PKC/CPI-17, RhoA/ROK and myosin phosphatase also participate in both
rapid-transient and sustained-tonic phases of ET-1-mediated vasoconstriction.
For example, following α1-adrenergic stimulation of rabbit femoral artery,
PKC/CPI-17 mediated inhibition of myosin phosphatase is an initial event,
critical for rapid vasoconstriction but is less important for sustained
contraction242. In contrast, ROK-dependent phosphorylation of the targeting
subunit of myosin phosphatase, MYPT, is consistent with increased myosin
light chain phosphorylation and contraction during the sustained phase of α1adrenergic and thromboxane A2 receptor-mediated vasoconstriction242, 243.
Many current pharmacological strategies target the specific cellular receptor of
hormones known to be causing vasoconstriction. Often this may require
administration of several receptor blockers and therapeutically, the dosing and
management of side effects presents significant challenges. Alternatives to
such one-at-a-time therapy already exists; for example, L-type Ca2+ channel
blockers and nitrates limit extracellular Ca2+ entry and reduce vasoconstriction
caused by many circulating hormones. However the chronic use of L-type Ca2+
channel blockers and nitrates in the management of coronary microvascular
- 95 -
disorders362 and peripheral artery disease363,
364
(PAD) can be limited by the
development of tolerance or oedema. As we learn more about the basic cellular
mechanisms governing vascular smooth muscle contraction, specifically Ca2+dependent mechanisms of contraction and PKC/CPI-17 and RhoA/ROKdependent inhibition of myosin phosphatase, it is valuable to map the temporal
activation of these processes and investigate whether there may be any
therapeutic
value
in
blocking
Ca2+-sensitive
intracellular
pathways
independently or concurrently. Using functional vascular myography and
pharmacological inhibitors, this study aimed to identify the temporal sequence
of activation of PKC/CPI-17 and RhoA/ROK activation during the initial-transient
and sustained phases of ET-1-mediated vasoconstriction in isolated large rat
caudal artery and small mesenteric arteries. It was hypothesised that PKC and
ROK activation was important in the initial-rapid and sustained phases of ET-1mediated contraction, respectively. Arterial segments were incubated with
pharmacological blockers of PKC or ROK prior to and following ET-1
stimulation and the magnitude of contraction was determined at specific time
points (30 seconds and 10 minutes). Our results indicate PKC inhibition
attenuated the sustained ET-1-mediated vasoconstriction while ROK inhibition
attenuated both the initial and sustained ET-1-mediated vasoconstriction in
large and small arteries. These data indicate temporal activation of subcellular
signalling pathways is an important consideration when managing acute versus
chronic hyper-constriction and suggest targeting intracellular effectors may
provide additional benefit to existing L-type Ca2+ channel and specific receptor
blockade therapy.
- 96 -
C2. Methods
To
identify
the
molecular
basis
underlying
acute
ET-1-mediated
vasoconstriction, we used in vitro functional myography to stimulate isolated
caudal (500µm) and mesenteric (300µm) arterial segments from male Sprague
Dawley rats with ET-1 in the presence and absence of PKC and ROK inhibitors.
This study was approved by the University of Adelaide animal ethics committee.
C2.1. Materials
Materials used for functional vascular myography were purchased from the
following
suppliers:
HEPES
sodium
salt
((4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid), acetone, H1152 ((S)-(+)-2-Methyl-1-[(4-methyl5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine
dihydrochloride)
and
bisindolylmaleimide (GF109203X), were from Sigma-Aldrich, Australia. ET-1
was synthesized by Auspep, Australia. Sodium chloride, potassium chloride,
magnesium chloride, calcium chloride and D-glucose were from Merck & Co
Inc., Australia.
C2.2. Functional vascular myography
Male Sprague Dawley rats were killed using CO2 inhalation. The caudal
(500µm) and second order mesenteric (300µm) arteries were dissected in Ca2+free HEPES Tyrode buffer (containing in mM: 135.5 NaCl, 5.9 KCl, 1.2 MgCl2,
11.6 HEPES pH 7.4 and 11.6 glucose). The adventitia was carefully removed
without excessive manipulation of the artery. The caudal and mesenteric
arteries were cut into 2mm segments and mounted on a Mulvany wire
myograph (DMT Danish Myotechnologies, Denmark). Particular care was taken
- 97 -
not to damage the endothelium. A length tension curve using 87mM KCl
stimulation was generated to determine the optimal resting tension for caudal
and mesenteric arteries (10mN and 2mN, respectively). Arterial segments were
equilibrated for 20 minutes in normal HEPES-Tyrode buffer (containing in mM;
135.5 NaCl, 5.9 KCl, 1.2 MgCl2, 2.5 CaCl2 11.6 HEPES pH 7.4 and 11.6
glucose) and set to 10mN or 2mN. Vessels were then stimulated three (3) times
with 87mM KCl HEPES-Tyrode buffer (containing in mM; 54.4 NaCl, 87 KCl,
1.2 MgCl2, 2.5mM CaCl2, 11.6 HEPES pH 7.4 and 11.6 glucose) and washed
with normal HEPES Tyrode buffer at 20 minute intervals. Each vessel was then
incubated for 15 minutes with either normal HEPES Tyrode buffer (control), the
specific ROK inhibitor (1x10-6M), H1152 and/or the general PKC inhibitor,
GF109203X (5x10-6M). These concentrations have previously been shown to
maximally inhibit ROK and all isoforms of PKC243. Arterial segments were then
stimulated with a maximum concentration of ET-1 (1x10-7M) for 30 seconds or
10 minutes. In post-treatment studies, pharmacological blockers were
administered after 5 minutes of ET-1 stimulation and the experiment stopped
after a further 5 minutes (a total of 10 minutes of ET-1-mediated stimulation).
C2.3. Data Analysis
The vascular responses were expressed as maximum developed tension
(GraphPad Prism 6). All data is presented as mean ± SEM and assessed using
a one-way analysis of variance (ANOVA) with a Bonferroni’s post-hoc test;
p<0.05 was considered statistically significant.
- 98 -
C3. Results
Functional data was collected from 30 second and 10 minute time points.
Likewise tissue was snap frozen at equivalent time points. Functional data at 10
minutes was not available from tissue frozen following 30 seconds of ET-1
stimulation and hence the unbalanced sample size for the initial and sustained
phases of ET-1-mediated vasoconstriction. This approach was undertaken to
biochemically assess the temporal activation of PKC and ROK (data not
presented herein).
- 99 -
C3.1. Inhibition of PKC and ROK prior to ET-1 stimulation attenuated the
development and maintenance of vasoconstriction in rat caudal artery.
In the context of α1-adrenergic receptor activation, previous research has
suggested PKC is rapidly activated while ROK-mediated inhibition of myosin
phosphatase is important during sustained contraction in large femoral
arteries242. To identify whether PKC and ROK are involved in the initial and
sustained phases of ET-1-mediated contraction in large arteries, isolated rat
caudal artery segments (500µm) were incubated with the PKC and/or ROK
inhibitor, GF109203X (5x10-6M) and H1152 (1x10-6M), respectively, before
arterial segments were contracted with ET-1 (1x10-7M). Figure C1 shows that
the broad spectrum PKC inhibitor did not cause a significantly different
contractile response to control ET-1 stimulation in the initial phase of
contraction (4.29mN ± 0.62 and 4.71mN ± 0.60, respectively. p>0.05, n = 8-18).
While ROK and combined ROK/PKC inhibition caused a significant decrease in
the initial ET-1-mediated contractile response (30 seconds); (ROK inhibition:
1.57mN ± 0.25 and ROK/PKC inhibition: 0.69mN ± 0.18 vs control: 4.71mN ±
0.60 and PKC inhibition: 4.29mN ± 0.62. p<0.05, n = 8-18). The combination of
PKC and ROK inhibition did not further attenuate contraction compared to ROK
inhibition alone (p>0.05, n = 8-12). In contrast, during the sustained phase of
rat caudal artery contraction, pre-treatment with the broad spectrum PKC
inhibitor, GF109203X, attenuated ET-1-mediated contraction (figure C2. PKC
inhibition: 12.42mN ± 1.2 vs control: 16.18mN ± 1.31, p<0.05, n = 6-12).
Treatment with the ROK inhibitor, H1152, and combined ROK/PKC inhibition
caused a much greater attenuation of the sustained phase of ET-1-dependent
vasoconstriction in rat caudal arteries compared to PKC inhibition alone and
- 100 -
control (figure C2. ROK inhibition: 1.90mN ± 0.53, ROK/PKC inhibition: 1.11mN
± 0.33 vs, PKC inhibition: 12.42mN ± 1.2 and control: 16.18mN ± 1.31 p<0.05,
n = 6-12). The combination of PKC and ROK inhibition did not further attenuate
sustained ET-1-mediated vasoconstriction compared to ROK inhibition alone in
rat caudal arteries (p>0.05, n = 6-12).
- 101 -
Figure C1. In rat caudal artery, inhibition of ROK but not PKC prior to ET-1
stimulation attenuated the initiation of vasoconstriction. Rat caudal
arteries (500µm) were incubated for 15 minutes with the vehicle (n = 17) PKC
(n = 15) and/or the ROK inhibitor (n = 18), GF109203X (5x10-6M) and H1152
(1x10-6M), respectively, followed by a 30 second ET-1 stimulation (1x10-7M).
Inhibition of PKC prior to simulation did not significantly attenuate the response
to ET-1 in the initial 30 seconds of contraction (p>0.05). However, inhibition of
ROK and combined PKC/ROK inhibition (n = 8) significantly decreased the
contractile response to ET-1 compared to control and PKC inhibition alone
(*p<0.05). Combined PKC and ROK inhibition did not further attenuate ET-1mediated vasoconstriction compared to ROK inhibition alone (p>0.05).
- 102 -
Figure C2. In rat caudal artery inhibition of ROK but not PKC prior to ET-1
stimulation attenuated the sustained phase of vasoconstriction in rat
caudal artery. Rat caudal arteries (500µm) were incubated for 15 minutes with
the vehicle (n = 11) PKC (n = 9) and/or the ROK inhibitor (n = 12), GF109203X
(5x10-6M) and H1152 (1x10-6M), respectively, followed by a 10 minute ET-1
stimulation (1x10-7M). Inhibition of PKC prior to simulation significantly
attenuated sustained ET-1-mediated vasoconstriction (*p<0.05). However,
inhibition of ROK and combined PKC/ROK (n = 6) further attenuated the
contractile response to ET-1 compared to control and PKC inhibition alone
(**p<0.05). Combined PKC and ROK inhibition provided no further reduction in
developed tension than ROK inhibitor therapy alone (p>0.05).
- 103 -
C3.2. Inhibition of PKC and ROK prior to ET-1 stimulation attenuated the
development and maintenance of vasoconstriction in rat mesenteric
arteries.
Previous findings have suggested the importance of PKC-activated CPI-17 and
myosin phosphatase differs between large and small vessels254,
258, 365
.
However, it is not clear whether this manifests as differential functional
responses to specific agonists. To identify whether PKC and ROK are involved
in initial and sustained ET-1-mediated vasoconstriction in small arteries, second
order rat mesenteric arteries (300µm) were incubated with the PKC and ROK
inhibitor, GF109203X (5x10-6M) and H1152 (1x10-6M), respectively, followed by
ET-1 stimulation (1x10-7M). Figure C3 illustrates that neither PKC and ROK
inhibition alone did not attenuate the initiation of ET-1-mediated contraction
compared to control i.e. the contraction at 30 seconds following ET-1
stimulation (PKC inhibition: 6.30mN ± 0.93 and ROK inhibition: 7.09mN ± 0.82
vs control: 8.88mN ± 1.7. p>0.05, n = 4-5). However, combined PKC and ROK
inhibition significantly attenuated the initiation of contraction following ET-1
stimulation (PKC/ROK inhibition: 1.67mN ± 0.77 vs control: 8.88mN ± 1.7, PKC
inhibition: 6.30mN ± 0.93, and ROK inhibition: 7.09mN ± 0.82. p<0.05, n = 4-5).
In contrast, both PKC and ROK inhibitors GF109203X and H1152, respectively,
significantly attenuated the sustained phase of ET-1-mediated vasoconstriction
compared to control arteries (figure C4. PKC inhibition: 12.89mN ± 1.48 and
ROK inhibition: 12.22mN ± 2.56 vs control: 18.16mN ± 1.99. p<0.05, n = 4-5).
However combined PKC and ROK inhibition caused a greater attenuation of
sustained contraction compared to control and solely inhibiting PKC or ROK
(PKC/ROK inhibition: 2.60mN ± 0.43 vs control: 18.16mN ± 1.99, PKC
- 104 -
inhibition: 12.89mN ± 1.48 and ROK inhibition: 12.22mN ± 2.56. p<0.05, n = 45).
- 105 -
Figure C3. In small mesenteric arteries combined inhibition of PKC and
ROK prior to ET-1 stimulation attenuates the initiation of contraction. The
contractile response following 30 seconds of ET-1 (1x10-7M) stimulation in
isolated mesenteric arteries (300µm) incubated with the vehicle (n = 5) PKC (n
= 5) and/or the ROK inhibitor (n = 5), GF109203X (5x10-6M) and H1152 (1x106
M), respectively. PKC and ROK inhibition did not significantly alter ET-1-
mediated vasoconstriction in the initial phase of contraction (p>0.05). Combined
PKC and ROK inhibition (n = 4) significantly attenuated ET-1-mediated
vasoconstriction in the initial phase of contraction (*p<0.05).
- 106 -
Figure C4. In small mesenteric arteries inhibition of PKC and ROK prior to
ET-1 stimulation attenuates sustained contraction. The contractile response
following 10 minutes of ET-1 (1x10-7M) stimulation in isolated mesenteric
arteries (300µm) incubated with the vehicle (n = 5) PKC (n = 5) and/or the ROK
(n = 5) inhibitor, GF109203X (5x10-6M) and H1152 (1x10-6M), respectively.
PKC and ROK inhibition significantly attenuated sustained ET-1-mediated
vasoconstriction (*p<0.05). In the sustained phase of contraction combined
PKC and ROK inhibition (n = 4) significantly decreased the contractile response
to ET-1 and this inhibition was greater than PKC and ROK inhibition alone
(**p<0.05).
- 107 -
C3.3.
During
the
sustained
phase
of
ET-1-mediated
contraction
subsequent inhibition of PKC and ROK attenuates vasoconstriction in rat
caudal arteries.
Patients with symptoms of vasospasm unresponsive to traditional therapies of
nitrates, Ca2+ blockers and/or angiotensin II and α1-receptor blockers364, 366, 367
provide a major challenge for clinicians. In an effort to identify whether ET-1dependent vasoconstriction could be attenuated during the sustained phase
independent from traditional pharmacological therapy, we constricted rat caudal
arteries for 5 minutes with ET-1 (1x10-7M) prior to adding PKC or ROK inhibitors
GF109203X (5x10-6M) and H1152 (1x10-6M), respectively. Figure C5 illustrates
that while PKC inhibition significantly attenuated sustained contraction in an ET1 pre-constricted artery (PKC inhibition: 10.53mN ± 1.13 vs control: 12.33mN ±
1.41. p<0.05, n = 6), ROK inhibition causes a greater decrease in sustained
contraction following ET-1 stimulation (PKC inhibition: 10.53mN ± 1.13 vs
control: 12.33mN ± 1.41 and ROK inhibition: 2.38mN ± 0.38. p<0.05, n = 6).
- 108 -
Figure C5. During the sustained phase of ET-1-mediated contraction
subsequent inhibition of PKC and ROK attenuates contraction in rat
caudal artery. Rat caudal artery segments (500µm) were constricted with ET-1
(1x10-7M) for 5 minutes followed by administration of the vehicle (n = 6) PKC (n
= 6) or ROK inhibitor (n = 6), GF109203X (5x10-6M) and H1152 (1x10-6M),
respectively for a further 5 minutes. Inhibition of PKC during sustained ET-1
contraction significantly attenuated constriction in rat caudal arteries (*p<0.05, n
= 6). Subsequent ROK blockade also attenuated the sustained phase of ET-1mediated vasoconstriction in ET-1 pre-constricted rat caudal artery, but to a
greater extent compared to PKC inhibition (**p<0.05).
- 109 -
C4. Discussion
Experimental and clinical studies have suggested ET-1 release causes
enhanced vasoconstriction of atherosclerotic vessels, exacerbating obstruction
and causing further reductions in blood flow355, 357. ETA/B receptor blockade has
been shown to be effective in the healing of ulcers in patients with systemic
sclerosis and in reducing peripheral vascular resistance in patients with
pulmonary and systemic hypertension326, 368, 369. However with recent research
documenting myosin phosphatase as an important regulator of vascular smooth
muscle contraction234,
269
, it has become increasingly clear that downstream
effectors of ETA and ETB receptor signalling are incompletely understood. The
pathogenesis of vascular disease is complex, involving inflammation, atheroma
development, thrombus formation and enhanced vasoconstriction by several
circulating hormones1. The benefit of specific receptor blockers i.e. α1adrenergic370, angiotensin II371 and ETA/B receptor antagonists326, have been
documented. However, activation of specific G-protein coupled receptors
activate common subcellular pathways, such as extracellular Ca2+ entry shown
by the effectiveness of L-type Ca2+-channel blockers372. This warrants
investigating whether there is a therapeutic value in blocking Ca2+ sensitization
in ET-1-mediated vasoconstriction. As a first step, the current study aimed to
identify the involvement of specific downstream effectors of ETA/B receptor
activation during the initial-rapid and sustained phases of ET-1-mediated
vasoconstriction in an effort to investigate the potential of prophylactic treatment
vs treatment following initiation of contraction. Using functional vascular
myography and pharmacological blockade we identified PKC inhibition
- 110 -
attenuates sustained contraction while ROK inhibition attenuates initial and
sustained ET-1-mediated vasoconstriction in large rat caudal and small
mesenteric arteries.
In the context of α1-adrenergic receptor activation, recent evidence has
indicated Gq/11 G-proteins and PKC/CPI-17 is activated rapidly and is
responsible for the initial phase of contraction in rabbit femoral arteries242. In
contrast we identified PKC inhibition prior to ET-1 stimulation does not
attenuate contraction in the initial-rapid phase while ROK inhibition significantly
attenuated initial-rapid contraction in rat caudal arteries (figure C1). PKC and/or
ROK blockade prior to agonist stimulation relates to prophylactic treatment
aimed at preventing or reducing the likelihood of the onset of vasoconstriction.
This approach inhibits 1) any existing basal enzymatic activity and 2) blocks the
extent of agonist-mediated enzymatic activation prior to its activation. Although
the significant attenuation of contraction mediated by ROK inhibition implicates
its involvement, it is important to consider ROK inhibitors are most likely
removing basal myosin phosphatase inhibition allowing for increased relaxation
regardless of ETA-mediated activation of ROK. In contrast, direct inhibition of
the catalytic core of myosin phosphatase by CPI-17 is not present at rest in
vitro242,
243
and hence this may explain why there is no attenuation of the
initiation of contraction following PKC inhibition. In the context of ET-1-mediated
vasoconstriction in rat caudal artery preparations the robust initial-rapid
constriction is functionally PKC/CPI-17 independent. The initial phase of ET-1mediated vasoconstriction is most likely mediated by intra/extracellular Ca2+
release in large rat caudal arteries. Nevertheless, these data suggests that a
- 111 -
ROK inhibitor could be an effective prophylactic treatment to prevent the onset
of vasoconstriction whereas blocking PKC is not effective. The advantage is
that based on known cellular signalling in a large artery, one would anticipate
this would also attenuate α1-adrenergic and thromboxane A2-mediated
vasoconstriction242, 243.
Previous studies have shown G13 G-proteins and subsequent ROK activation
causes a slow but persistent inhibition of myosin phosphatase responsible for
sustained contraction while PKC activation is less important for sustained tonic
contraction242,
243
. In the current study, we identified PKC inhibition caused a
small but significant attenuation of sustained ET-1-mediated contraction. This
suggests that PKC activation and perhaps subsequent CPI-17-mediated
inhibition of myosin phosphatase is involved in the sustained phase of ET-1mediated contraction (figure C2). ROK inhibition and combined PKC/ROK
inhibition caused a greater inhibition of contraction in rat caudal arteries
compared to PKC inhibition alone. This indicates that while myosin
phosphatase is an important regulator of ET-1-mediated vascular tone, as
expected, for a multistep enzymatic process i.e. RhoA/ROK-mediated inhibition
of myosin phosphatase and subsequent accumulation of phosphorylated
myosin light chains, myosin phosphatase activation and inhibition takes time to
manifest functionally. This is consistent with rat caudal artery studies using
phorbol-dibutyrate a general PKC activator showing a slow, small contraction in
the absence of Ca2+. Similarly, thromboxane A2 receptor activation causes
ROK-mediated inhibition of myosin phosphatase resulting in a slow but
sustained contraction243. These data indicate that in the context of large vessel
- 112 -
diseases such as PAD, and coronary artery disease, prophylactic treatment
with ROK inhibitors may be beneficial in preventing both hyper-contractility and
increased vascular tone. These data are consistent with the current clinical use
of the ROK inhibitor, fasudil, which has been effectively used to treat cerebral
vasospasm following stroke274. Clinical studies have also reported the benefit of
ROK inhibitors in increasing vasodilation in patients with vasospastic angina273
and severe pulmonary hypertension373.
It is well documented that large and small vessels are heterogeneous and often
have different responses to agonist stimulation. For example, when examining
blood pressure in relation to vessel diameter, pressure decreases dramatically
in arteries under 150µm compared to large conduit arteries indicating a role for
small resistance arteries in blood pressure regulation101. Interestingly, it has
been suggested the importance of CPI-17 and myosin phosphatase differs
between large and small vessels254, 258, 365 which may have important functional
implications regarding temporal activation of PKC/CPI-17 and ROK in small
vessels. In the current study only combined inhibition of PKC and ROK
attenuated the initial phase of ET-1-mediated vasoconstriction in small
mesenteric arteries (figure C3). These data suggest that in small arteries, both
PKC and ROK are slow to activate and that myosin phosphatase
phosphorylation may have a lower basal level of tonic activation compared with
large arteries; perhaps illustrating ET-1-mediated vasoconstriction is vascular
bed specific. While neither PKC nor ROK inhibitors were able to affect maximal
ET-1-mediated vasoconstriction in the rapid-transient phase of contraction,
there was a clear attenuation of vasoconstriction with combined PKC and ROK
- 113 -
inhibition. This suggests the initial-rapid response is mediated largely by Ca2+
entry through voltage-gated Ca2+ channels consistent with significant
attenuation of ET-1-mediated vasoconstriction in the presence of nifedipine,
verapamil and efonidipine221.
PKC and ROK inhibition attenuated sustained ET-1-mediated vasoconstriction
in small mesenteric arteries (figure C4). In contrast to large arteries, our data
suggests that during sustained ET-1-mediated vasoconstriction PKC and ROKmediates Ca2+ entry or Ca2+ sensitization to a similar degree. These data are
consistent with previous western blot analyses documenting an increase in the
abundance of myosin phosphatase in large arteries254. Currently, vasodilator
therapies for patients with cardiac syndrome X374 and peripheral arterial
disease such as Raynaud’s phenomenon103 and Burger’s disease375 are limited
to
L-type
Ca2+-channel
blockers,
specific
receptor
antagonists,
phosphodiesterase inhibitors and nitrates. The current study suggests a
possible role for ROK and PKC to activate myosin phosphatase as additional
beneficial vasodilatory therapies for these vascular disorders. PKC is widely
involved in important processes including cell proliferation, differentiation,
gastric motility and respiration376,
377
; the inhibition of which can have severe
and adverse outcomes. However, clinical value may be achieved if therapy is
limited to acute use. Alternatively, once the specific isoforms of PKC are
identified in vascular smooth muscle cells, a more directed and specific
blockade of PKC in the vasculature might be beneficial in specifically effecting
vasodilation. Further studies with specific antibodies for the α, δ, ε ζ isoforms of
- 114 -
PKC in the presence and absence of Ca2+ are required to identify whether PKC
can exclusively be targeted in vascular smooth muscle.
Inhibiting PKC and ROK subsequent to ET-1 stimulation blocks 1) basal
enzyme activity or 2) continual agonist-mediated activation. It is important to
point out ET-1-mediated vasoconstriction has already reached maximum and
some of the tonic contraction is independent from Ca2+. This is perhaps the
most clinically pertinent setting in a patient that presents with acute vasospasm
such as cerebral, coronary and popliteal vasospasm where one wants to
attenuate an existing spasm. In the context of sustained ET-1-mediated
vasoconstriction in large arteries, subsequent PKC and ROK inhibition
attenuated existing vascular contraction (figure C5). This indicates that while
PKC is thought to be rapidly activated under α1-adrenergic stimulation, in the
context of ET-1-mediated vasoconstriction it remains active after ten (10)
minutes of stimulation. Alternatively, PKC may be affecting a different cellular
signalling mechanism which effects ET-1-dependent tone. Nevertheless, these
data indicate that a combination of both PKC and ROK inhibition may be
valuable in decreasing vascular spasm mediated by circulating hormone(s); a
possible alternative to multiple receptor blocker therapy.
In conclusion, inhibition of specific subcellular effectors governing vascular tone
may be an effective alternative to single or multiple receptor blockade therapy
and may be particularly valuable when one has not yet identified the cause of
enhanced vasoconstriction i.e. identified a specific culprit hormone causing
spasm or increased vascular tone. We identified that inhibiting PKC prior to ET- 115 -
1 stimulation decreases sustained contraction while ROK inhibition attenuated
both the initial and sustained ET-1-mediated vasoconstriction in large and small
arteries. Combined PKC and ROK inhibition may be useful in attenuating both
large and small vessel constriction.
- 116 -
Section D.
Chronic endothelin-1 infusion
attenuated thromboxane A2mediated vasoconstriction in
second order rat mesenteric
arteries
- 117 -
D1. Introduction
Endothelin-1 (ET-1) is a peptide vasoconstrictor predominately released by the
endothelium in response to shear stress but is also released by infiltrating
macrophages and leukocytes during inflammation82,
113
. ET-1 regulates
vascular smooth muscle contraction through activation of G-protein coupled
receptors, ETA and ETB314, 315. Activation of ET-1 receptors on the endothelium
increases Ca2+ entry and activation of endothelial nitric oxide synthase (eNOS),
favouring vasodilation146,
147, 378
. In contrast, in vascular smooth muscle cells
ET-1 receptor activation causes Ca2+ entry and activation of myosin light chain
kinase, coupled with protein kinase C (PKC) and Rho kinase (ROK) mediated
inhibition of myosin phosphatase, favouring vasoconstriction318, 379.
ET-1 is perceived as one of the most potent vasoconstrictors in the circulating
milieu. Enhanced ET-1-mediated vasoconstriction and an increase in ET-1
mRNA have been shown in the smooth muscle of atherosclerotic aorta, carotid
arteries and in muscle biopsies taken from patients with peripheral artery
disease (PAD)357,
380
. Consistent with these findings, immunohistochemical
analysis has documented the presence of ET-1 in high abundance in the intima
of atherosclerotic human coronary arteries381, suggesting a role for ET-1 in the
progression and reduced stability of atherosclerotic plaques and in contributing
to vasospasm or increased vascular tone in atherosclerotic blood vessels. ET-1
receptor blockade has been explored as a therapeutic strategy for reducing
pulmonary hypertension and overall peripheral vascular resistance in
hypertensive patients. The combined ETA/B receptor blocker, Bosentan, reduces
blood pressure in patients with both systemic and pulmonary hypertension327,
- 118 -
369
. Animal models of hind limb ischaemia and systemic hypertension have
demonstrated improved ischaemia-induced angiogenesis and flow dependent
dilation of resistance arteries following chronic blockade of ETA receptors382, 383.
In humans, administration of the selective ETA receptor antagonist, BQ-123 into
the femoral artery improves local tissue perfusion in patients with critical limb
ischaemia384. Similarly, intra-coronary administration of BQ-123, in patients
undergoing cardiac catheterization has been shown to induce significant
dilation of stenotic vessels and reduce the frequency of unstable angina325.
Elevated plasma ET-1 levels have been documented in patients with PAD,
coronary artery disease and hypertension357,
385
. Following subarachnoid
haemorrhage (SAH) patients showed elevated ET-1 concentrations in
cerebrospinal fluid and ET-1 is suggested to be a key player in inducing
cerebral artery vasospasm; the most common complication in survivors of
SAH386. In a hypoxic mesenteric vascular bed model, reverse-phase HPLC
quantification indicates that ET-1 may be released in high concentrations locally
during ischaemia387, which could have important implications for patients with
ischaemic heart disease and critical limb ischaemia. High circulating ET-1 in
patients with critical limb ischaemia324 has been suggested to be a predictor of
death. Femoral artery occlusion in experimental models of critical limb
ischaemia have also identified ET-1 is up-regulated in the ischaemic limb and
ET-1 receptor blockade reduces skeletal muscle injury by improving tissue
blood flow388. However, using a similar model of hind limb ischaemia in rats, our
group did not observe any functional differences in isolated femoral arteries
when challenged with either ET-1, serotonin, the α1-adrenergic agonist,
- 119 -
phenylephrine or the thromboxane A2 mimetic, U46619 (Maddern et al.,
unpublished). Hence it remains unclear whether enhanced vasoconstriction
associated with ET-1 is directly linked to elevated plasma ET-1.
To address this issue, the vascular consequences of direct, seven (7) day miniosmotic pump-mediated infusion of ET-1 has been investigated in healthy
Sprague Dawley rats. In addition to direct vasoactive properties, previous
studies have shown ET-1 potentiates the vascular response to other circulating
hormones including catecholamines and serotonin389,
390
. Consequently, our
approach included identification of vascular reactivity in isolated small
mesenteric arteries in response to exogenously applied vasoconstrictors
following seven (7) days of ET-1 infusion. We hypothesised that chronic
exposure to ET-1 would potentiate the contractile responses to acute ET-1,
thromboxane A2, serotonergic and α1-adrenergic receptor activation in isolated
mesenteric arteries. Interestingly, chronic ET-1 infusion blunted vascular
reactivity to the exogenously applied thromboxane A2 mimetic, U46619 but did
not influence the contractile responses to exogenously applied ET-1, α1adrenergic stimulus (phenylephrine) or serotonin. These responses were
independent from the abundance and Ser1177-dependent activation state of
eNOS and Thr855-dependent activation state of the targeting subunit of myosin
phosphatase, MYPT. These data indicates that while elevated ET-1 is a strong
marker of vascular disease, in an otherwise healthy organism circulating ET-1
alone has less impact on vascular reactivity.
- 120 -
D2. Methods
To identify whether chronic elevations in circulating ET-1 increased the vascular
responses to exogenously added ET-1, thromboxane A2 mimetic (U46619),
serotonin and the α1-agonist, phenylephrine, healthy male Sprague Dawley rats
(350g) were exposed to elevated circulating ET-1 (10ng/kg/min) for seven (7)
days using a mini-osmotic pump (ALZET®), jugular vein cannulation model
described previously348, 391. Rats were sacrificed and second order mesenteric
arteries (300µm) were collected for functional myography. To determine the
molecular
mechanism
underlying
altered
vascular
reactivity,
vascular
myography was coupled with western blot analysis to identify the abundance
and Ser1177- and Thr855-dependent activation state of eNOS and the MYPT
targeting subunit of myosin phosphatase, respectively. This study was
approved by the University of Adelaide and the Queen Elizabeth Hospital
animal ethics committees.
D2.1. Materials
Mini osmotic pumps (model 1002, 0.25µL/hr) were from ALZET® Durect, USA.
Polyethylene tubes were from Intramedic, Australia. Non-absorbable surgical
sutures were from ©Ethicon, Inc., Australia. Ibuprofen as Nurofen® was from
Reckitt Benckiser, Australia.
Materials used for functional vascular myography, sodium dodecyl sulphate
polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis
were purchased from the following suppliers: acetylcholine, U46619, serotonin,
- 121 -
phenylephrine, glycerol, sodium dodecyl sulfate (SDS), bromophenol blue,
diisopropylfluorophosphate (DFP), ammonium persulfate (APS), and N,N,N’,N’tetramethylethylenediamine (TEMED) were from Sigma-Aldrich, Australia. ET-1
was synthesized by Auspep, Australia. Sodium chloride, potassium chloride,
magnesium chloride, calcium chloride, potassium dihydrogen phosphate,
sodium bicarbonate, D-glucose, EDTA, sucrose, dithiothreitol (DTT), acetic
acid, coomassie brilliant blue R-250, and ponceau-S were from Merck & Co
Inc., Australia. Glycine and Tris were from AMRESCO, USA. Acrylamide / 0.8%
N,N’-methylenebisacrylamide (bisacrylamide) solution, Tween-20, and 0.2µm
nitrocellulose membrane were from BioRad Laboratories, Australia. Complete
protease inhibitor cocktail™ was from Roche, Australia. Pre-stained molecular
weight markers (PageRuler™ prestained protein ladder plus) were from
Fermentas Life Sciences. Mouse monoclonal anti-eNOS was purchased from
BD Biosciences, USA. Mouse monoclonal anti-MYPT was made in house243, 249.
Rabbit
polyclonal
anti-P[Ser1177]eNOS
and
anti-P[Thr855]MYPT,
were
purchased from Upstate/Millipore, USA. Anti-rabbit biotin-conjugated secondary
IgG, streptavidin conjugated 800nM Dylight fluorochrome and West Femto
enhanced chemiluminescent (ECL) reagents were from Pierce Thermo Fisher
Scientific, Australia Pty. Anti-mouse horseradish peroxidase (HRP)-conjugated
secondary IgG was from Santa Cruz Biotechnology, USA. X-ray film and
standard developing agents were purchased from Afga, Belgium.
D2.2. Preparation of mini-osmotic pumps and jugular vein cannulation
A detailed description of this protocol is included in Section B. General Methods
followed from the manufacturer’s instructions and previously published reports
- 122 -
using ET-1 infusion347, 348. In summary, 100µL of ET-1 (0.1mg/mL) prepared in
0.9% Tris buffered saline was filter-sterilised and injected into the drug reservoir
of the mini-osmotic pumps, providing a constant flow rate of 10ng/kg/min.
Control pumps were filled with 0.9% Tris buffered saline. The flow moderator
was attached to seal the core drug reservoir before attaching 3.5cm of 70%
ethanol-sterilized
polyethylene
tubing
(0.03
inches
internal
diameter.
Intramedic, Australia). The free end of this tube was cut at a 45º angle to
facilitate insertion into the jugular vein. An additional 2mm piece of polyethylene
tubing was placed over the original 3.5cm segment and secured 2cm from the
angled end of the catheter. This served as an anchoring collar for sutures
during
permanent
jugular
vein
cannulation.
Following
manufacturer’s
instructions the pumps were primed in sterile saline at 37ºC for 4-6 hours.
Following light general anaesthesia using isoflurane, male Sprague Dawley rats
were placed on the surgical table in the supine position and isoflurane was
administered via a nose cone throughout the surgery. Anaesthetic levels were
monitored throughout the surgery to prevent isoflurane overdose. The neck of
the rat was shaved and sterilised using 70% ethanol. Sterile technique was
used throughout the remaining procedure. A 1cm incision was made near the
midline of the neck and blunt dissection was used to expose the jugular vein.
Two sutures were placed under the vein. A small incision was made in the vein
using an 18-gauge needle to guide the catheter. The catheter was then inserted
into the vein and the two previously placed sutures were tied around the
catheter, straddling the 2mm collar to prevent retrograde movement of the
catheter. Blunt dissection was continued laterally from the jugular vein to create
- 123 -
a subcutaneous pouch to house the mini-osmotic pump. The incision was
closed using 3-5 individual triple box knot stiches. Rats were given ibuprofen for
pain management and closely monitored for signs of distress and infection.
Following seven (7) days of ET-1 infusion, rats were killed by exsanguination
under isoflurane-induced anaesthesia.
D2.3. Functional vascular myography
Second order mesenteric arteries (300µm) were isolated in ice-cold KREBS
(containing in mM: 118 NaCl, 1.18 KH2PO4, 25 NaHCO3, 1.05 MgCl2, 2.34
CaCl2, 0.01 EDTA and 5.56 glucose, pH7.4) and mounted on a wire myograph
(DMT Danish Myotechnologies, Denmark). Arteries were equilibrated for 20
minutes in KREBS pre-heated to 37ºC and set at an optimal resting tension of
2mN. A length tension curve was previously carried out to determine optimal
resting tension for second order rat mesenteric arteries. Organ baths were
constantly bubbled with carbogen (95% O2 and 5% CO2, BOC, Australia) to
maintain dissolved O2 and pH at 7.4. Artery segments were taken through a
series of three stimulations using high K+-mediated depolarisation with KPSS
(containing in mM: 217 KCl, 1.18 KH2PO4, 25 NaHCO3, 1.05 MgCl2, 2.34 CaCl2,
0.01 Na2EDTA and 5.56 glucose, pH7.4) followed by a 20 minute washout with
KREBS. Following the final stimulation with KPSS, vessels were incubated in
KREBS for 20 minutes and resting tension set to 2mN before incremental
doses of each agonist were added using the following concentration ranges:
serotonin and phenylephrine: 1x10-8M–3x10-4M, U46619: 1x10-9M–3x10-6M,
ET-1: 1x10-12M–3x10-7M. Once the maximum contractile response was reached
with each agonist, endothelial integrity was assessed on each arterial segment
- 124 -
by administering a sub-maximal concentration of the vasodilator, acetylcholine
(1x10-6M). All arterial segments returned to their baseline resting tension
indicating an intact endothelium.
C2.4. SDS-PAGE and western blot analysis of eNOS and myosin phosphatase
Second order mesenteric arteries (300µm in diameter) from saline and ET-1
infused rats were isolated in KREBS and immediately snap frozen in liquid
nitrogen. Ten (10) arteries of equal length from each rat were placed into a
single microcentrifuge tube (1.5ml). Samples were extracted directly in SDSPAGE buffer containing 50mM Tris/HCl, pH 6.8, 1% SDS, 40% sucrose, 10%
complete protease inhibitor cocktail™, 10mM DTT and 10µM DFP. Samples
were heated at 95°C for 5 minutes then vortexed for 3 x 15 seconds at 5 minute
intervals at room temperature. Slight variation in protein extraction was first
quantified by SDS-PAGE followed by staining with coomassie brilliant blue R250. Gels were then de-stained with 10% acetic acid. Total protein per lane was
quantified using Odyssey V3 software (Li-Cor Biosciences, USA). Protein
concentrations were adjusted to equality by sample dilution with SDS-PAGE
buffer. A detailed rationale for standardizing sample loading is included in
Section B4.5. A second SDS-PAGE was run using a 7.5% large format SDSpolyacrylamide gel using a BioRAD Protean® II Xi cell at 60mA for 90 minutes
in running buffer containing 25mM Tris, 192mM glycine and 1% SDS. Proteins
were transferred (using a single BioRad mini Protean® II transfer unit) onto
0.22µM nitrocellulose membrane at 100V for 45 minutes and 30 minutes for
eNOS and myosin phosphatase, respectively. Transfer buffer contained 25mM
Tris, 192mM glycine and 20% methanol and 0.1% SDS.
- 125 -
The nitrocellulose membranes were blocked for 60 minutes using 5% non-fat
dried milk in Tris buffered saline (20mM Tris, 150mM NaCl) and 0.05% Tween20 (TBS-T). Nitrocellulose membranes were incubated for 60 minutes with antiP[Ser1177]eNOS,
or
anti-P[Thr855]MYPT
(1:1000
dilution
in
TBS-T).
Membranes were then rinsed once and washed 3 x 5 minutes in TBS-T before
60 minute incubation in anti-rabbit biotin conjugated secondary IgG (1:10,000
dilution in TBS-T). Following another rinse and 3 x 5 minute washes in TBS-T,
membranes were incubated in streptavidin conjugated 800nM Dylight
fluorochrome for 60 minutes in the dark. Membranes were again rinsed and
washed 3 times for 5 minutes in TBS-T then dried overnight. Protein bands
were scanned using the Odyssey Imager and quantified using Odyssey V3
software (Li-Cor Biosciences, USA). Following a 5 minute wash in TBS-T,
membranes previously incubated with rabbit anti-P[Ser1177]eNOS, and rabbit
anti-P[Thr855]MYPT were next incubated for 60 minutes with mouse anti-eNOS
and mouse anti-MYPT, respectively (1:1000 dilution in TBS-T). These
membranes were washed 3 times for 5 minutes in TBS-T before 60 minute
incubation in horseradish peroxidase (HRP) conjugated goat anti-mouse
secondary IgG (1:5000 dilution in TBS-T). Following a 3 x 5 minute wash step,
antibody detection was achieved with West Femto ECL reagents and x-ray film.
Quantity One Software (BioRad Laboratories, Australia) was used to quantify
protein bands from x-ray film. This approach has the advantage of analysing
both total and phosphorylated Ser1177 eNOS and the total and phosphorylated
Thr855 MYPT subunit of myosin phosphatase on the same membrane,
decreasing inter-experimental variability. The dual rabbit/mouse labelling
technique has been previously validated243, 270.
- 126 -
D2.5. Data analysis
The vascular response to agonists was expressed as developed tension. A
sigmoidal curve of best fit was generated using four-parameter nonlinear
regression (GraphPad Prism 6). The Hillslope, EC50 and maximal response was
generated from these dose response curves. Total protein from western blot
analyses are presented as integrated intensity of anti-eNOS or anti-MYPT. The
phosphorylation
state
is
indexed
as
integrated
intensity
of
P[Ser1177]eNOS/Total eNOS and P[Thr855]MYPT/Total MYPT. All data are
presented as mean ± SEM and assessed using a two-tailed, non-paired
Students t-test; p<0.05 was considered statistically significant.
Although an ANOVA is frequently the statistical test of choice, a Students t-test
is an adequate statistical test when comparing treatment to control groups.
Multiple regression analysis (Hillslope) was used to identify whether a single
line of best fit could describe two (2) independent dose response curves (e.g.
control vs ET-1 treatment) and hence whether the two (2) curves were
significantly different.
- 127 -
D3. Results
Chronic agonist infusion using mini-osmotic pumps is an established model for
studying agonist actions in vivo. A dose of 10ng/kg/min has previously been
shown to increase blood pressure and alter haemodynamics in rat and guinea
pig models348, 392. The standard approaches for measuring blood pressure in rat
models is direct intra-arterial pressure through carotid artery cannulation or
caudal artery pressure using a tail-cuff393,
394
. Intra-arterial pressure
measurements require animals to be under anaesthesia; being a central
nervous depressant, anaesthetics naturally depress blood pressure. Alternately,
tail cuff measurements can be taken while the animal is conscious. However
researchers are then reliant on the animal being still and calm as any
movement or stress can also change blood pressure. With these limitations in
mind, we opted to validate our model through changes in vascular reactivity and
not alterations in blood pressure as has been done in previously published
iterations of this experimental model348, 391.
- 128 -
D3.1. Chronic exposure to elevated ET-1 decreased the sensitivity and
maximum contractile response to exogenously applied thromboxane A2
mimetic, U46619.
Thromboxane is released by platelets during activation and aggregation and is
an important mediator of local hyper-vasoconstriction of blood vessels during
inflammation
and
thrombus
formation80,
83
.
Patients
with
peripheral
microvascular vasospastic disorders have increased urinary metabolites of
thromboxane A2 (thromboxane B2) following a cold stimulus, indicating
thromboxane A2 may be contributing to vasospasm in these patients301. To
determine whether chronically elevated ET-1 alters the normal response to
acute thromboxane A2 receptor activation, male Sprague Dawley rats were
infused with ET-1 for seven (7) days followed by mesenteric artery isolation.
The half-life of thromboxane has previously been reported as 30 seconds,
hence for experimental purposes, the stable thromboxane mimetic, U46619
was used. Isolated second order mesenteric arteries (300µm) were stimulated
with incremental doses of U46619 (1x10-9M–3x10-6M). With the current sample
size, multiple regression analysis (Hillslope) identified no significant difference
in control vs ET-1 treated dose response curves (p>0.05, n = 6-8). However,
figure D1 illustrates a statistically significant increase in EC50 to thromboxane A2
receptor activation in rats chronically treated with ET-1 compared to rats treated
with saline (658.30nM ± 167.30 and 105.60nM ± 34.41, respectively. p<0.05, n
= 6-8). Additionally, the maximum efficacy of exogenously applied U46619 was
decreased in rats chronically treated with ET-1 compared to control rats (7.35 ±
2.02mN and 15.78mN ± 0.90, respectively. p<0.05, n = 6-8).
- 129 -
Figure D1. The sensitivity and maximum contractile responses to the
exogenously applied thromboxane mimetic, U46619 are significantly
decreased following chronic exposure to elevated ET-1. Dose response
curves of isolated second order mesenteric arteries (300µm) to the
exogenously applied thromboxane A2 mimetic, U46619, following saline
treatment (n = 8) or seven (7) day exposure to 10ng/kg/min ET-1 (n = 6) using a
mini-osmotic pump and jugular vein catheterization model in male Sprague
Dawley rats. Rats chronically exposed to intravenous ET-1, 10ng/kg/min for
seven (7) days (open circles) had a significantly higher EC50 and a lower
maximum contractile response to the thromboxane A2 mimetic U46619,
compared to control rats (closed circles). (*p<0.05)
- 130 -
D3.2. The contractile response to exogenously applied ET-1 is similar in
rats chronically exposed to elevated ET-1.
Elevated plasma levels of ET-1 and increased sensitivity to ET-1 has been
demonstrated in several cardiovascular disorders including atherosclerosis,
coronary microvascular dysfunction and pulmonary hypertension357,
380
. To
determine whether elevated ET-1 directly causes increased sensitivity to ET-1,
second order mesenteric arteries (300µm) isolated from male Sprague Dawley
rats infused with ET-1 for seven (7) days were stimulated with incremental
doses of ET-1 (1x10-12M–3x10-7M). Figure D2 shows the contractile response
to exogenously added ET-1 was similar in rats with elevated ET-1 (EC50 –
control: 13.59nM ± 3.46 and ET-1 treated: 11.33nM ± 3.33. Maximum response
– control: 16.80mN ± 1.91 and ET-1 treated: 15.97mN ± 1.30. p>0.05, n=7-9).
Similarly, multiple regression analysis (Hillslope) identified no significant
difference in control vs ET-1 treated dose response curves (p>0.05, n = 7-9).
- 131 -
Figure D2. The contractile response to ET-1 receptor activation is
unchanged following chronic exposure to elevated ET-1. Dose response
curves of isolated second order mesenteric arteries (300µm) to exogenously
applied ET-1 following saline treatment (n = 9) or seven (7) day exposure to
10ng/kg/min ET-1 (n = 7) using a mini-osmotic pump and jugular vein
catheterization model in male Sprague Dawley rats. Rats chronically exposed
to ET-1 (open circles) did not have a significantly different EC50 or maximum
contractile response to ET-1 receptor activation compared to control rats
(closed circles). The Hillslope was not significantly different between control vs
ET-1 treated dose response curves (p>0.05).
- 132 -
D3.3. The contractile response to the exogenously applied α 1-adrenergic
receptor agonist, phenylephrine and serotonin are similar in rats
chronically exposed to elevated ET-1.
Previous research has shown ET-1 potentiates the vascular response to
catecholamines and serotonin in acute, in vitro models389,
390
. To determine
whether chronically elevated ET-1 potentiates the contractile response to acute
α1-adrenergic and serotonin receptor activation, mesenteric arteries isolated
from male Sprague Dawley rats infused with ET-1 for seven (7) days were
stimulated
with
incremental
doses
of
α1-adrenergic
receptor
agonist,
phenylephrine and serotonin (1x10-8M–3x10-4M). Figures D3 and D4 show that
the EC50 and maximum contractile responses to exogenously added
phenylephrine and serotonin are not altered in rats chronically treated with ET-1
(phenylephrine EC50 – control: 4.86µM ± 2.09 and ET-1 treated: 2.56µM ± 0.43.
Phenylephrine maximum – control: 18.01mN ± 1.75 and ET-1 treated: 13.79mN
± 2.23. p >0.05, n = 7-8. Serotonin EC50 – control: 476.0nM ± 137.0 and ETtreated: 878.50nM ± 202.8. Serotonin maximum – control: 16.40mN ± 2.46 and
ET-1 treated 11.98mN ± 1.57. p>0.05, n = 6-7). With the current sample sizes,
multiple regression analysis (Hillslope) identified no significant difference in
control vs ET-1 treated dose response curves generated by either
phenylephrine (p>0.05, n = 7-8) or serotonin (p>0.05, n = 6-7)
activation.
- 133 -
receptor
Figure D3. The contractile response to α 1-adrenergic receptor activation
is not statistically different following chronic exposure to elevated ET-1.
Dose response curves of isolated second order mesenteric arteries (300µm)
exposed to exogenously applied phenylephrine (PE), a specific α1-adrenergic
receptor agonist, following saline treatment (n = 8) or seven (7) day exposure to
elevated ET-1 (n = 7) using a mini-osmotic pump and jugular vein
catheterization model in male Sprague Dawley rats. Rats chronically exposed
to ET-1 (open circles) did not have a significantly different EC50 or maximum
contractile response to α1-adrenergic receptor activation compared to control
rats (closed circles. p>0.05).
- 134 -
Figure D4. The contractile response to serotonin receptor activation is not
significantly different following chronic exposure to elevated ET-1. Dose
response curves of second order mesenteric arteries (300µm) exposed to
exogenously applied serotonin following saline treatment (n = 6) or seven (7)
day exposure to elevated ET-1 (n = 7) using a mini-osmotic pump and jugular
vein catheterization model in male Sprague Dawley rats. Rats chronically
exposed to ET-1 (open circles) do not have a significantly different EC50 or
maximum contractile response to serotonin receptor activation compared to
control rats (closed circles. p>0.05).
- 135 -
D3.4. Chronic exposure to elevated ET-1 does not alter the contractile
response to KCl-mediated depolarisation.
ET-1-mediated vasoconstriction causes Ca2+ entry through voltage-gated Ca2+
channels and the sarcoplasmic reticulum221, 318. Stimulation with high K+ causes
plasma membrane depolarisation and activates voltage-gated Ca2+ channels.
To determine whether the basal activation state and properties of voltage-gated
Ca2+ channel activation was altered following chronic elevation of ET-1, second
order mesenteric arteries (300µm) isolated from control or rats chronically
treated with ET-1 were stimulated with high K+ solution (KPSS). Figure D7
shows that the response to high K+ is not significantly different in rats treated
with ET-1 for seven (7) days compared to control rats (15.06mN ± 1.09 and
14.21mN ± 1.01, respectively. p>0.05 n = 7-9).
- 136 -
Figure D5. Rats chronically treated with elevated ET-1 have similar
maximum contractile responses to KCl-mediated depolarisation. Second
order mesenteric arteries (300µm) isolated from rats chronically treated with
elevated ET-1 (n = 7) using a jugular vein mini-pump catheterisation model
showed no difference in their maximum contractile response to KCl-mediated
depolarisation compared to control rats (n = 9), (p>0.05).
- 137 -
D3.5. The abundance and activation state of eNOS is similar in rats
chronically exposed to ET-1.
ET-1 has dual actions in that it regulates vascular tone both through increasing
endothelial cell eNOS activity, favouring vasodilation and in vascular smooth
muscle cells, causing Ca2+ entry and decreasing myosin phosphatase activity,
favouring vasoconstriction146, 318. To determine whether chronic exposure to ET1 results in physiological compensation by altering the Ser1177 activation state
or abundance of eNOS, second order mesenteric arteries (300µm) from rats
chronically treated with ET-1 were isolated and snap-frozen in liquid nitrogen.
SDS-PAGE and ratiometric western blot analysis of eNOS Ser1177 was used
to determine eNOS activation and abundance. Figure D6 illustrates that the
Ser1177-dependent activation state of eNOS was unchanged in rats treated
with ET-1 compared to control rats (219.17 ± 31.97 and 195.9 ± 37.89,
respectively. p>0.05, n = 5-7). Similarly the abundance of eNOS was
unchanged in rats treated with ET-1 compared to control rats (4.27 ± 0.36 and
4.53 ± 1.32, respectively. p>0.05, n = 5-7).
- 138 -
Figure D6. eNOS abundance and activation state is similar in rats
chronically treated with ET-1. Second order mesenteric arteries (300µm) from
rats treated with 10ng/kg/min ET-1 for seven (7) days were snap-frozen in liquid
nitrogen prior to protein extraction and SDS-PAGE. The activation state of
eNOS was indexed using ratiometric western blot analysis of P[Ser1177]eNOS
and total eNOS protein. There was no significant difference between the
Ser1177-dependent phosphorylation state (A, C) and total abundance of eNOS
(B, D) in rats chronically exposed to ET-1 (n = 5) compared to control rats (n =
7), (p>0.05).
- 139 -
D3.6. The abundance and activation state of myosin phosphatase is not
significantly different in rats chronically exposed to ET-1.
Activation of ETA receptors on vascular smooth muscle cells increases
RhoA/ROK-dependent inhibitory phosphorylation of Thr855 on the targeting
subunit of myosin phosphatase, MYPT318. To determine whether chronic
exposure to ET-1 alters the activation or abundance of myosin phosphatase,
second order mesenteric arteries (300µm) from rats chronically treated with ET1 were isolated and snap-frozen in liquid nitrogen. SDS-PAGE and ratiometric
western blot analysis of P[Thr855]MYPT/total MYPT was used to determine the
activation state and abundance of myosin phosphatase. Figure D7 shows that
the Thr855-dependent activation state of myosin phosphatase was unchanged
in rats treated with ET-1 compared to control rats (32.31 ± 9.14 and 29.45 ±
4.84, respectively. p>0.05, n = 5-7.) Similarly the abundance of myosin
phosphatase was unchanged in rats treated with ET-1 compared to control rats
(11.19 ± 1.96 and 9.15 ± 0.52, respectively. p>0.05, n = 5-7).
- 140 -
Figure D7. Myosin phosphatase abundance and activation state is not
significantly different in rats chronically treated with ET-1. Second order
mesenteric arteries (300µm) from rats treated with 10ng/kg/min ET-1 for seven
(7) days were snap-frozen in liquid nitrogen prior to protein extraction and SDSPAGE. The activation state of myosin phosphatase was indexed using
ratiometric western blot analysis of P[Thr855]MYPT and total myosin
phosphatase protein. There was no significant difference between the Thr855dependent phosphorylation state of myosin phosphatase (A, C) or total myosin
phosphatase (B, D) in rats chronically exposed to ET-1 (n = 5) compared to
control rats (n = 7), (p>0.05).
- 141 -
D4. Discussion
ET-1-mediated vasoconstriction is enhanced in several disease states including
PAD and ischaemic heart disease357,
380
. In addition, patients with
cardiovascular disease frequently present with elevated serum ET-1324, 357, 385.
ET-1-receptor blockade in vivo has been shown to significantly dilate coronary
arteries of patients with stable coronary artery disease395, and decrease systolic
blood pressure in patients with pulmonary and systemic hypertension327,
369
,
heart failure396, renal failure397 and cerebral artery vasospasm398. Chronic
infusion of ET-1 using mini-osmotic pump and jugular vein cannulation is a
validated experimental model and has previously been used to identify the role
of reactive oxygen species in mediating ET-1-induced hypertension348.
Assessment of haemodynamic changes identified increased mean arterial
pressure and renal vascular resistance and consequently, decreased renal
blood flow; this was abolished in the presence of a superoxide anion
scavenger. Acetylcholine and sodium nitroprusside induced vasodilation has
also been reported to be impaired through a nitric-oxide dependent mechanism
in rat pulmonary arteries following chronic ET-1 infusion391. However, these
studies have not addressed to what extent elevated ET-1 influences or
potentiate other vasoconstrictor responses once the ET-1 is removed.
Specifically, whether chronic ET-1 causes up or down regulation of ET-1 Gprotein coupled receptors or the receptors of other circulating hormones. We
have addressed this question by 1) chronically exposing healthy rats to
elevated ET-1, 2) isolating 300µm second order mesenteric arteries and
indexing the functional vascular response to high K+-mediated depolarisation,
the thromboxane A2 mimetic U46619, phenylephrine, serotonin and ET-1
- 142 -
receptor activation. We used biochemical western blot analysis of eNOS and
myosin phosphatase abundance with specific indices of their activation state, to
identify alternative points of regulation at the endothelial and vascular smooth
muscle levels, respectively.
We identified the vasoconstrictor response to thromboxane A2 receptor
activation was significantly decreased following seven (7) day infusion with ET1 in rats (figure D1). We speculate that down regulation of thromboxane A2
receptors may be contributing to reduced vascular reactivity to thromboxane A2
receptor activation for the following reasons: 1) ET-1 infusion has little influence
on ET-1, α1-adrenergic and serotonergic contractile responses (figures D2-4),
suggesting shared molecular pathways downstream of the thromboxane A2
receptor are not altered. 2) High K+-mediated depolarisation in control and ET-1
treated groups are similar (figure D5) suggesting there is no change in the
activation or abundance of voltage-gated Ca2+ channels. 3) Examination of
eNOS abundance and the Ser1177-dependent activation state (figure D6)
along with the unchanged sensitivity to ET-1, phenylephrine, and serotonin
suggest the mechanism of thromboxane A2 insensitivity is not endotheliummediated. 4) The finding that the abundance and Thr855-dependent activation
state of myosin phosphatase was not changed (figure D7) strongly suggests
that chronic ET-1 treatment neither up nor down regulates myosin phosphatase
or
its
Thr855
MYPT-dependent
activation
state.
Consequently,
since
thromboxane A2 is known to mediate contraction through L-type Ca2+ channels
and RhoA/ROK-dependent Ca2+ sensitivity243 and given that neither high K+dependent activation of voltage-gated Ca2+ channels nor myosin phosphatase
- 143 -
abundance or activation has been altered following chronic ET-1 treatment.
These data are consistent with the notion that chronic ET-1 alone results in the
down regulation of vascular smooth muscle thromboxane A2 receptors.
Although it remains controversial whether platelets are directly activated by ET1 there is increasing evidence that this may occur via a direct effect or indirectly
through the generation of free radicals, peroxynitrite399-401 or reduced nitric
oxide-mediated bioavailability which limits inhibition of platelet activation402. In
the short-term, elevated ET-1 may directly down regulate thromboxane A2
receptors or alternatively, cause an increased endogenous release of plateletderived thromboxane A2 and a plausible consequence of increasing circulating
thromboxane A2 may be long-term down regulation or internalisation of
thromboxane A2 receptors. This would compensate for increased thromboxane
A2-mediated vasoconstriction and over activation of platelets. Hence, a logical
next step would be to identify cell membrane thromboxane A2 receptor
expression and clotting times in animal models and perhaps patients with
significantly elevated serum ET-1. These data could have important
implications for patients on antiplatelet agents and/or anticoagulants; patients
with vascular disease and elevated serum ET-1 may be receiving more
antiplatelet agents than required, predisposing them to longer bleeding times
and perhaps haemolytic stroke.
Our study identified chronically elevated ET-1 does not change the vascular
response to exogenously added ET-1 (figure D2). Patients with vascular
disease frequently have concurrent elevations in many vasoactive molecules
- 144 -
such as catecholamines and angiotensin II. As our model exclusively elevated
plasma ET-1 in healthy rats, one possible explanation for normal ET-1
responses following chronic infusion may be that other circulating agonists are
contributing more to the increased vascular responses observed in patients
even though the ET-1 levels are elevated. For example, sub-pressor
concentrations of ET-1- have been shown to potentiate α1-adrenergic and
serotonin responses390. Experiments in cultured endothelial and smooth muscle
cells have shown that adrenaline, angiotensin II and vasopressin stimulates
local expression of ET-1 and may potentiate ET-1-mediated vasoconstriction403,
404
.
Consistent with these findings, infusion of angiotensin II in vivo using
osmotic mini-pumps have demonstrated significant increases in ET-1 in
vascular smooth muscle403,
404
. Importantly, these studies have demonstrated
ET-1 receptor blockade abrogates the hypertensive response to chronic
angiotensin II infusion. It is conceivable then an alternate explanation is that
chronically elevated ET-1 may be less involved in contributing to vascular
hyper-contractility and may contribute to elevated pressures more through renal
management of sodium and water balance348.
Isolated rat mesenteric arteries infused with threshold concentrations of ET-1
have shown enhanced pressor responses to noradrenaline389. Additionally,
acute organ bath preparations document sub-pressor concentrations of ET-1
potentiates vasoconstrictor responses to noradrenaline and serotonin in
isolated human internal mammary (IMA) and left anterior descending (LAD)
coronary arterial rings390. It is important to consider our experiments involved
removing arteries from a circulating milieu containing high levels of ET-1 to
- 145 -
enable one to assess vasoconstriction in response to individual pressors and
index chronic functional changes in receptor and cellular signalling. We
identified normal constrictor responses to exogenously added phenylephrine
and serotonin following seven (7) day infusion with ET-1 (figures D3 and D4).
Previous research has provided some valuable insights into acute ET-1
stimulation and subsequent adrenergic and serotonin receptor activation341, 389.
The current study does not exclude potentiation of adrenergic and serotonergic
receptor activation but suggests this occurs acutely, independent from changes
in receptor profile and, more simply, may be a reflection of circulating hormone
levels.
ETB receptor activation on the endothelium causes an increase in eNOS
activity, indexed by the phosphorylation of Ser1177146. In contrast in vascular
smooth muscle, ET-1 causes contraction through PKC and ROK dependent
inhibition of myosin phosphatase318. Our study identified the total abundance
and Ser1177-dependent activation state of eNOS is unchanged following
chronic exposure to ET-1 (figure D6). This indicates ET-1 differentially regulates
eNOS in an acute versus chronic context. Our study identified normal total
abundance and activation of myosin phosphatase in rats chronically treated
with ET-1 (figure D7). The activation state of myosin phosphatase was indexed
by the phosphorylation of myosin phosphatase Thr855, indicating no change in
the intracellular signalling pathway involving ROK. This is consistent with our
functional results showing no alteration in vascular reactivity to exogenously
added ET-1, the thromboxane A2 mimetic, U46619, phenylephrine and
serotonin. For example, if the influence of myosin phosphatase were altered
- 146 -
one would predict responses to all agonists would change. The findings that
eNOS and myosin phosphatase abundance and activation were not altered
suggest they are not physiological mediators to compensate for ET-1 induced
pressure.
In conclusion, chronic exposure to ET-1 decreases the sensitivity and maximum
contractile responses to thromboxane A2 receptor activation, likely as a
consequence of thromboxane A2 receptor down regulation. Chronic exposure to
elevated ET-1 does not alter the isolated vascular responses to exogenously
added ET-1, phenylephrine and serotonin, suggesting that enhanced ET-1
vasoconstriction in humans may be governed by the relative abundance of
circulating hormones and not changes in ET-1, α1-adrenergic or serotonergic
receptor profiles. Consistent with these functional data, our study found no
change in the fundamental regulators of vascular smooth muscle cell
contraction, i.e., voltage-gated Ca2+ channels, eNOS and myosin phosphatase
abundance and activation states. These results highlight the complexity of the
pathophysiological state in which multiple agonists are often responsible for
increased vascular reactivity frequently observed in patients with vascular
disease. This study also suggests the need to pose the question: is
thromboxane A2-mediated platelet reactivity reduced when ET-1 levels are
elevated? Further studies are aimed to explore the dynamic interplay of ET-1,
thromboxane A2 and platelet function and whether this alters sensitivity to
current antiplatelet agents.
- 147 -
Section E.
Subcutaneous microvessels from
patients suffering from peripheral
artery disease exhibit enhanced
serotonin and α 1-adrenergic
receptor mediated vasoconstriction
- 148 -
E1. Introduction
Peripheral artery disease (PAD) affects 20% of the population over 65 years
and is increasingly prevalent as the population ages16,
17
. Commonly, it is
caused by one or a combination of the following: atheroma, thrombosis and
vascular spasm. Modern angioplasty and stenting procedures are effective in
managing mild to moderate disease but in severe disease lower-limb
amputation is often required. An ankle brachial index (ABI) ≤ 0.9 is considered
diagnostic of PAD. In the early symptomatic stages of the disease, patients
experience claudication (leg pain) during activity but as the disease advances,
declining blood flow leads to ischaemic pain at rest. Without an improvement in
blood flow, consequences include: skin ulceration, tissue necrosis and infection,
potentially leading to sepsis, multiple organ system dysfunction and death1.
A variety of pathophysiological processes contribute to PAD but the most
common cause is perceived to be atherothrombosis. It is less well recognised
that this process can predispose patients to undergo vasospasm or
exaggerated vasoconstriction, further impairing blood flow. A common feature
of atheroma and thrombus formation is endothelial dysfunction and
inflammation of the vessel wall. Infiltrating macrophages and leukocytes release
the potent vasoconstrictor endothelin-182 (ET-1) while activated platelets
release thromboxane83 and serotonin84. In addition, endothelial dysfunction,
ischaemia and redox stress decrease the bioavailability of vasodilatory nitric
oxide and promote platelet activation, further potentiating vasoconstriction and
thrombus formation405,
406
. Cilostazol, a type three phosphodiesterase (PDE)
- 149 -
inhibitor, is an effective medical therapy for patients with PAD as it prevents
cAMP hydrolysis, and increases PKA activity, favouring activation of K+
channels and vasodilation106, 407. Additional medical therapies for patients with
PAD include lipid lowering agents and anticoagulants, such as statins and low
molecular weight heparin, respectively41.
Computer tomography and angiography studies suggest PAD predominately
affects large blood vessels supplying the legs. However, chronic increases in
vascular resistance in patients with atherosclerosis and hypertension indicate
hyper-contraction and/or impaired vasodilation of small resistance blood
vessels (10-300µm)408,
409
. Laser Doppler assessment has revealed reduced
resting skin blood flow in the legs of patients with moderate PAD and critical
limb ischaemia410, 411. Isolated vessel studies have shown thinning of the arterial
wall in subcutaneous vessels isolated from patients with severe PAD412.
Experienced
vascular
surgeons
often
find
large
vessel
mechanical
revascularisation procedures provide incomplete restoration of blood flow,
which fails to resolve non-healing skin ulcerations and regional subcutaneous
tissue death. This may indicate additional regional dysfunction of the
microcirculation.
Vascular tone in the microcirculation is controlled by two opposing enzyme
systems. Activation of Ca2+-calmodulin dependent myosin light chain kinase
(MLCK) leads to phosphorylation of myosin and interaction with actin. This
activation is opposed by dephosphorylation of myosin by myosin phosphatase.
The diversity of vascular responses are governed not only by the differential
- 150 -
receptor distribution but also differences in the spatio-temporal profile of
activation of voltage-gated Ca2+ channels, K+ channels, IP3 dependent
sarcoplasmic reticulum Ca2+ release and the extent of RhoA/ROK and PKC
dependent inhibition of myosin phosphatase234, 269. Agonists like angiotensin II,
noradrenaline,
serotonin
and
ET-1
activate
these
cellular
signalling
mechanisms, by activating plasma membrane G-protein coupled receptors to
increase vascular tone. Additionally, endothelial nitric oxide synthase (eNOS)
abundance and activation state can be reduced or increased depending on
disease progression and medical therapy185. For example endothelial
dysfunction is associated with reduced eNOS or impaired eNOS function while
statins have been shown to enhance eNOS abundance by stabilizing the
mRNA
and
activating
eNOS
through
protein
kinase
B-dependent
phosphorylation of Ser1177413.
This study aimed to characterise microvascular reactivity in patients suffering
from severe PAD and to identify the mechanisms responsible for the
heterogeneous responses to, serotonin, α1-adrenergic, thromboxane A2 and
ET-1 receptor activation. We hypothesised that vascular responses to
serotonin, α1-adrenergic, thromboxane A2 and ET-1 receptor activation would
be increased in PAD versus non-PAD patients independent of the activation
and abundance of eNOS and myosin phosphatase. Using wire myography and
western blot analysis, we have identified that the maximum contractile response
to serotonin and α1-adrenergic receptor but not thromboxane A2 or ET-1
activation is increased in patients with PAD. The enhanced vasoconstrictor
- 151 -
responses are independent from the abundance and activation state of eNOS
and myosin phosphatase, indexed by phosphorylation of eNOS Ser1177 and
the Thr855 targeting subunit of myosin phosphatase (MYPT), respectively. We
have identified an increased abundance of a specific serotonin receptor in
patients with PAD which suggests a possible mechanism for altered vascular
reactivity. These data suggest a therapeutic potential for agents that target
serotonin and α1-adrenergic mediated vasoconstriction.
- 152 -
E2. Methods
Subcutaneous arteries from the non-ischaemic proximal regions of legs of
patients undergoing elective lower limb amputations were collected. These
were compared to subcutaneous arteries isolated from the abdominal and
inguinal region of age-matched patients asymptomatic for PAD undergoing
open hernia repair. In vitro myography was used to determine vascular
contractility and this was coupled with biochemical analysis of the Ser1177dependent and Thr855 MYPT-dependent activity state of eNOS and myosin
phosphatase, respectively as well as total eNOS, myosin phosphatase and
serotonin 5HT2A receptor expression. This study was approved by the
University of Adelaide and Queen Elizabeth Hospital human ethics committees.
E2.1. Materials
Materials used for functional vascular myography, sodium dodecyl sulphate
polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis
were purchased from the following manufacturers: HEPES sodium salt ((4-(2hydroxyethyl)-1-piperazineethanesulfonic
acid),
acetylcholine,
U46619,
serotonin, phenylephrine, glycerol, sodium dodecyl sulfate (SDS), bromophenol
blue, diisopropylfluorophosphate (DFP), ammonium persulfate (APS), and
N,N,N’,N’-tetramethylethylenediamine (TEMED) were from Sigma-Aldrich,
Australia. ET-1 was synthesized by Auspep, Australia. Sodium chloride,
potassium chloride, magnesium chloride, calcium chloride, D-glucose, sucrose,
dithiothreitol (DTT), acetic acid, coomassie brilliant blue R-250, and ponceau-S
were from Merck & Co Inc., Australia. Glycine and Tris were from AMRESCO,
USA. 30% acrylamide/0.8% N,N’-methylenebisacrylamide solution, Tween-20,
- 153 -
and 0.2µm nitrocellulose membrane were from BioRad Laboratories, Australia.
Complete protease inhibitor cocktail™ was from Roche, Australia. Pre-stained
molecular weight markers (PageRuler™ prestained protein ladder plus) were
from Fermentas Life Sciences. Mouse monoclonal anti-eNOS was purchased
from BD Biosciences, USA. Mouse monoclonal anti-MYPT was made in
house243, 249. Rabbit polyclonal anti-P[Ser1177]eNOS and anti-P[Thr855]MYPT,
were purchased from Upstate/Millipore, USA. Mouse monoclonal anti-5HT2A
was purchased from Santa Cruz Biotechnology, USA. Anti-rabbit biotinconjugated secondary IgG, anti-mouse biotin-conjugated secondary IgG,
streptavidin conjugated 800nM Dylight fluorochrome and West Femto
enhanced chemiluminescent (ECL) reagents were from Pierce Thermo Fisher
Scientific Australia Pty. Anti-mouse horseradish peroxidase (HRP)-conjugated
secondary IgG was from Santa Cruz Biotechnology, USA. X-ray film and
standard developing agents were purchased from Afga, Belgium.
E2.2. Patient recruitment and tissue collection
Patients were recruited through the Queen Elizabeth Hospital vascular and
general surgery units. Patients diagnosed with severe PAD scheduled for
above or below knee amputations with an ABI <0.9 and/or with appropriate
computer tomography scans depicting significant large vessel stenosis and
critical limb ischaemia were included in the study. Subcutaneous arteries (300400µm) were collected from the non-ischaemic proximal region of amputated
limbs. The control cohort included age-matched patients undergoing open
hernia repair with no clinical diagnosis or symptoms of PAD. A donated sample
of subcutaneous tissue from the lower abdomen or inguinal region provided a
- 154 -
source of 300-400µm arteries. Similar anaesthetics were used in both
experimental groups.
E2.3. Functional vascular response
Subcutaneous arteries (300-400µm) were isolated in Ca2+-free HEPES Tyrode
buffer (containing in mM: 135.5 NaCl, 5.9 KCl, 1.2 MgCl2, 11.6 HEPES pH 7.4
and 11.6 glucose) and mounted on a wire myograph (DMT Danish
Myotechnologies, Denmark). Arteries were equilibrated for 20 minutes in
normal HEPES Tyrode buffer (containing in mM; 135.5 NaCl, 5.9 KCl, 1.2
MgCl2, 2.5 CaCl2 11.6 HEPES pH 7.4 and 11.6 glucose) and set at an optimal
resting tension of 2mN. A length tension curve was carried out to determine
optimal resting tension in small human arteries. Arterial segments were taken
through a series of three stimulations using isotonic 87mM KCl HEPES Tyrode
buffer in which an equimolar amount of NaCl was substituted with KCl, followed
by a 20 minute washout with normal HEPES Tyrode buffer. Following the third
87mM KCl stimulation, vessels were equilibrated in normal HEPES Tyrode
buffer for 20 minutes then constricted with a sub-maximal concentration of
phenylephrine (1x10-6M). Endothelial integrity was assessed with a submaximal concentration of acetylcholine (1x10-6M). Subsequently, vessels were
incubated for 30 minutes in normal HEPES Tyrode buffer. Incremental doses of
each agonist were added as follows; serotonin and phenylephrine: 1x10-8M–
3x10-4M, U46619: 1x10-9M–3x10-6M, ET-1: 1x10-12M–3x10-7M.
- 155 -
E2.4. SDS-PAGE and western blot analysis of eNOS, myosin phosphatase,
and 5HT2A
Untreated subcutaneous arteries (300-400µm) from both patient groups were
isolated in Ca2+ free HEPES Tyrode buffer and immediately snap frozen in
liquid nitrogen. Samples were extracted in SDS-PAGE buffer containing 50mM
Tris/HCl, pH 6.8, 1% SDS, 40% sucrose, 10% complete protease inhibitor
cocktail™, 10mM DTT and 10µM DFP. Samples were heated at 95°C for 5
minutes then vortexed for 3 x 15 seconds at 5 minute intervals at room
temperature. Although vessels dissected were 300-400µm x 2mm, slight
variation in protein extraction per vessel was quantified first by SDS-PAGE
followed by staining with coomassie brilliant blue R-250 and de-staining with
10% acetic acid. Total protein per lane was quantified using Odyssey V3
software (Li-Cor Biosciences). Protein concentrations were adjusted to equality
by sample dilution. A detailed rationale for using this approach to standardize
sample loading is included in Section B4.5. A second SDS-PAGE was run with
a 7.5% large format polyacrylamide gel using a BioRAD Protean ® II Xi cell at
35mA for 90 minutes in running buffer containing 25mM Tris, 192mM glycine
and 1% SDS. Following trimming of the large format gel, proteins were
transferred (using a single BioRad mini protean II transfer unit) onto a 0.22µM
nitrocellulose membrane at 100V for 45 minutes and 30 minutes for eNOS and
the MYPT targeting subunit of myosin phosphatase, respectively. Transfer
buffer contained 25mM Tris, 192mM glycine, 20% methanol and 0.1% SDS.
5HT2A receptors were transferred for 60 minutes at 100V using a similar buffer
but in the absence of SDS. Different transfer conditions were used to optimise
protein transfer for each protein of interest. The nitrocellulose membrane was
- 156 -
blocked for 60 minutes using 5% non-fat dried milk in Tris buffered saline
(20mM Tris, 150mM NaCl) and 0.05% Tween-20 (TBS-T). Nitrocellulose
membranes were incubated for 60 minutes with either anti-eNOS, antiP[Ser1177]eNOS, anti-MYPT, anti-P[Thr855]MYPT, or anti-5HT2A (1:1000
dilution in TBS-T) prior to being washed 3 x 5 minutes in TBS-T. Membranes
incubated with anti-MYPT and anti-eNOS were then incubated in horseradish
peroxidase (HRP) conjugated goat anti-mouse secondary IgG (1:5000 dilution
in TBS-T) for 60 minutes. Membranes were washed 3 x 5 minutes in TBS-T
before detection with West Femto ECL reagents (Pierce Thermofisher
Scientific) and X-ray film (Afga). Quantity One Software (BioRad) was used to
quantify protein bands from X-ray film. Membranes incubated with rabbit antiP[Ser1177]eNOS
and
anti-P[Thr855]MYPT
or
mouse
anti-5HT2A
were
incubated with anti-rabbit or anti-mouse biotin conjugated secondary IgG for 60
minutes. Membranes were washed 3 times with TBS-T for 5 minutes before
incubation in streptavidin conjugated 800nM Dylight fluorochrome (Li-Cor
biosciences) for 60 minutes in the dark. Protein bands were scanned using the
Odyssey Imager and Odyssey V3 software (Li-Cor Biosciences).
E2.5. Data analysis
Patient characteristics between the two patients groups were analysed using a
Chi-squared and Fisher’s exact test. The vascular response to agonists was
expressed as developed tension. A sigmoidal curve of best fit was generated
using four-parameter nonlinear regression (GraphPad Prism 6). The Hillslope,
EC50 and maximal contractile response were generated from these dose
response curves. Protein bands were exposed as either total or the ratio of
- 157 -
phosphorylated vs total protein. All data is presented as mean ± SEM and
assessed using a two-tailed, non-paired Students t-test; p<0.05 was considered
statistically significant. Although an ANOVA is frequently the statistical test of
choice, a Students t-test is an adequate statistical test when comparing
treatment to control groups. Multiple regression analysis (Hillslope) was used to
identify whether a single line of best fit could describe two (2) independent dose
response curves (PAD vs non-PAD) and hence whether the two (2) curves
were significantly different.
- 158 -
E3. Results
The challenge associated with analysing human tissue is delegating an
appropriate control and often the ideal control is unfeasible. For example,
patients suffering from lower-limb PAD frequently have several co-morbidities
and their medical therapy can be extensive. In this context, it would be
unsuitable to recruit young healthy patients with no history of cardiovascularrelated issues. Additionally, gaining regular access to remnant leg tissue from
elective surgeries is difficult and approaching patients for leg biopsies
specifically for the purposes of this study is ethically unjustifiable.
Open hernia repairs are regularly scheduled procedures which provide an
abundant source of remnant abdominal and inguinal subcutaneous arterial
tissue. Functional analyses from our laboratory compared subcutaneous
arteries from buttocks biopsies and the subcutaneous abdominal region from
patients with coronary artery disease and identified these arteries respond
similarly, indicating generalized microvascular dysfunction414. This rationalises
the use of subcutaneous abdominal and inguinal arteries from age-matched
patients as a reference for microvascular dysfunction in subcutaneous arteries
isolated from the lower-limb of patients with PAD. The reference cohort (nonPAD) consisted of age-matched patients (~70 years) undergoing open hernia
repair with no clinically diagnosed or angiographic evidence of PAD. Chisquared and Fisher’s exact statistical analysis showed there was a significantly
higher percentage of patients with renal dysfunction, type II diabetes, ischemic
heart disease and previous myocardial infarction in the PAD compared to the
non-PAD patient group (table E1, p<0.05). Similarly, a significantly higher
- 159 -
percentage of patients were on angiotensin II and β-adrenergic receptor
blockade therapy in the PAD compared to the non-PAD patient group (table E2,
p<0.05). Anaesthetics administered on the day of surgery were similar in both
patient groups.
- 160 -
Table E1. Clinical information for PAD and non-PAD patients
PAD
Non-PAD
73.78 ± 4.01
71.67 ± 3.48
p > 0.05
Male
10
*11
p < 0.05
Female
1
*11
p < 0.05
90.00 ± 2.72
*98.31 ± 2.45
p < 0.05
Type II diabetes
*55%
14%
p < 0.05
Hypertension
55%
23%
p > 0.05
Hypercholesterolemia
18%
23%
p > 0.05
Renal Dysfunction
*27%
0%
p < 0.05
Ischaemic heart disease
*27%
0%
p < 0.05
Myocardial infarction
*36%
0%
p < 0.05
Atrial fibrillation
9%
5%
p > 0.05
Congestive heart failure
9%
0%
p > 0.05
Smoker
18%
18%
p > 0.05
Age
Sex
Mean arterial pressure
Co-morbidities
- 161 -
Table E2. In-patient medication on day of surgery
Medication
PAD
Non-PAD
Ca2+ channel blockers
18%
5%
p > 0.05
Statins
46%
18%
p > 0.05
AngII receptor antagonists
*36%
5%
p < 0.05
ACE inhibitors
18%
14%
p > 0.05
Nitrates
18%
5%
p > 0.05
Antiplatelet Agents
9%
5%
p > 0.05
Aspirin
36%
18%
p > 0.05
α-adrenergic blockers
18%
5%
p > 0.05
β-adrenergic blockers
*45%
5%
p < 0.05
- 162 -
E3.1. PAD and age-matched non-PAD patients had similar responses to
KCl-mediated vasoconstriction.
Stimulation with 87mM KCl caused plasma membrane depolarisation and
activated voltage-gated Ca2+ channels. To identify whether vessels isolated
from the abdomen/inguinal region and lower-limbs respond similarly to voltagegated Ca2+ channel activation, isolated subcutaneous arteries (300-400µm)
from patients with and without PAD were stimulated with 87mM KCl. Data in
figure E1 identifies there was no statistical difference in the maximum response
to 87mM KCl-mediated depolarisation in vessels isolated from PAD and nonPAD patients (6.80mN ± 0.94 and 5.43mN ± 0.55, respectively. p>0.05, n = 9 18).
- 163 -
Figure E1. Patients with and without PAD had similar high K+-mediated
vasoconstriction. Subcutaneous arteries (300-400µm) from patients with PAD
(n = 9) and patients asymptomatic for PAD (n = 19) were stimulated with a high
K+ buffer (87mM KCl). Subcutaneous arteries from patients with PAD showed
no significant difference in the maximum response to high K+-mediated
vasoconstriction compared with arteries obtained from non-PAD patients
(p>0.05).
- 164 -
E3.2. The maximum vasoconstrictor response to serotonin receptor
activation is increased in patients with PAD compared to age-matched
patients asymptomatic for PAD.
Serotonin has been implicated in the cause of mood disorders, irritable bowel
syndrome and hypertension through modulation of cerebral vascular tone,
gastrointestinal motility and peripheral vascular tone84,
415
. To determine
whether serotonin-mediated vasoconstriction was altered in patients suffering
from PAD, isolated subcutaneous arteries (300-400µm) were stimulated with
incremental doses of serotonin (1x10-8M–3x10-4M). Figure E2 illustrates the
dose response in patients with and without symptomatic PAD. With the current
power, multiple regression analysis identified the dose response curves were
not significantly different (Hillslope: p>0.05, n = 9-13). The EC50 and hence
sensitivity was not significantly different between PAD and non-PAD patients
(0.36µM ± 0.15 and 1.24µM ± 0.54, respectively. p>0.05, n = 9-13). However,
there was a significant increase in the maximum response to serotonin in
isolated vessels from PAD vs non-PAD patients (10.62mN ± 2.14 and 3.19mN
± 1.09, respectively. p<0.05, n = 9-13).
- 165 -
Figure E2. The maximum subcutaneous arterial contractile response to
serotonin receptor activation is higher in PAD vs non-PAD patients. Dose
response curves (± SEM) to serotonin stimulation in subcutaneous arteries
(300-400µm) isolated from patients with PAD (closed circles, n = 9) and nonPAD patients (open circles, n = 13). Patients with PAD did not show a
significantly different Hillslope or EC50 compared to non-PAD patients (p>0.05).
The maximum response to serotonin receptor activation was significantly
greater in PAD vs non-PAD patients (*p<0.05).
- 166 -
E3.3. The maximum vasoconstrictor response to α 1-adrenergic receptor
activation is increased in patients with PAD compared to age-matched
patients asymptomatic for PAD.
The catecholamines, noradrenaline and adrenaline are elevated in many
cardiovascular disorders and increase peripheral vascular resistance85. α1adrenergic receptor blockers may be used to reduce vascular tone in the
periphery and reduce blood pressure in patients with cardiovascular disease281,
370
. In the current study, α1-adrenergic receptor blockers were administered to
patients in both experimental groups (table E2). To determine sensitivity to α1adrenergic receptor activation, a dose response (1x10-8M–3x10-4M) was
conducted in isolated human vessels with the specific α1-adrenergic receptor
agonist, phenylephrine. With the current power, multiple regression analysis
identified the dose response curves were not significantly different (figure E3.
Hillslope: p>0.05, n = 9-18). The EC50 and consequently sensitivity was not
significantly different between PAD and non-PAD patients (6.57µM ± 3.33 and
3.04µM ± 0.67, respectively, p>0.05, n = 9-18) while there was a significant
increase in the maximum response to α1-adrenergic receptor activation
(8.57mN ± 1.53 and 4.05mN ± 0.75, respectively. p<0.05, n = 9-18).
- 167 -
Figure E3. The maximum subcutaneous arterial contractile response to
α 1-adrenergic receptor activation is higher in PAD vs non-PAD patients.
Dose response curves (± SEM) to the specific α1-adrenergic receptor agonist,
phenylephrine (PE), in subcutaneous arteries (300-400µm) isolated from
patients with PAD (closed circles, n = 9) and non-PAD patients (open circles, n
= 18). Subcutaneous arteries from patients with PAD did not show a
significantly different Hillslope or EC50 compared to those from non-PAD
patients (p>0.05). The maximum contractile response to phenylephrine
stimulation was significantly greater in PAD compared with non-PAD patients
(*p<0.05).
- 168 -
E3.4. Patients with and without PAD have similar sensitivity and maximum
vasoconstrictor responses to thromboxane A2 receptor activation.
Thromboxane is a potent vasoconstrictor released by activated platelets and is
important in thrombus formation416. Previous findings have reported patients
with peripheral microvascular vasospasm have increased urinary metabolites of
thromboxane A2 following exposure to cold stimuli, indicating thromboxane A2
may be exacerbating vasospasm in these patients301. To identify whether
thromboxane-mediated vasoconstriction was altered in patients with PAD,
isolated subcutaneous arteries (300-400µm) were stimulated with incremental
doses of the stable thromboxane mimetic, U46619 (1x10-9M–3x10-6M). Figure
E4 shows that patients with and without PAD have similar functional responses
to thromboxane A2 receptor activation. Multiple regression analysis shows that
the dose response curves are not significantly different (Hillslope: p>0.05, n =
8-17). The EC50 and maximum response was not significantly different between
PAD and non-PAD patients (EC50: 31.56nM ± 10.25 and 25.65nM ± 6.47,
respectively. Maximum response: 10.55mN ± 1.34 and 9.07mN ± 1.71,
respectively. p>0.05, n = 8-17).
- 169 -
Figure E4. Patients with and without PAD have similar contractile
responses to thromboxane A2 receptor activation. Dose response curves to
the thromboxane A2 mimetic, U46619 in subcutaneous arteries (300-400µm)
isolated from PAD (closed circles, n = 8) and non-PAD patients (open circles, n
= 17). Patients with PAD did not have a significantly different Hillslope, EC50 or
maximum response to thromboxane A2 stimulation compared to patients with
no clinical diagnosis of PAD (p>0.05).
- 170 -
E3.5. Patients with and without PAD have similar sensitivity and maximum
vasoconstrictor responses to ET-1 receptor activation.
ET-1 is a potent peptide vasoconstrictor released by the endothelium and
infiltrating cells in response to sheer stress and during inflammation,
respectively82. To identify whether ET-1-mediated vasoconstriction was altered
in patients with PAD, isolated vessels were stimulated with incremental doses
of ET-1 (1x10-12M–3x10-7M). Data in figure E5 illustrates that the EC50 and
maximum response was not significantly different between PAD and non-PAD
patients (EC50: 17.03nM ± 8.12 and 8.15nM ± 1.56, respectively. Maximum
response: 10.89mN ± 2.64 and 7.11mN ± 1.12, respectively. p>0.05, n = 9-11).
With the current power, multiple regression analysis has identified the dose
response curves are not significantly different (Hillslope: p>0.05).
- 171 -
Figure E5. The sensitivity and maximum contractile responses to ET-1
receptor activation is similar between patients with and without PAD.
Representative dose response curves to ET-1 in subcutaneous arteries (300400µm) isolated from PAD (closed circles, n = 9) and non-PAD patients (open
circles, n = 11). Patients with PAD did not have a significantly different Hillslope,
EC50 or maximum response to ET-1 compared to non-PAD patients (p>0.05)
- 172 -
E3.6. Patients with and without symptomatic PAD have similar abundance
and Ser1177 phosphorylation state of eNOS in subcutaneous arteries.
Endothelial dysfunction commonly occurs in patients with hypercholesterolemia
and plaque burden, decreasing the bioavailability of vasodilatory nitric oxide405.
eNOS generates nitric oxide in the endothelium and one measure of its
activation can be indexed by examining its phosphorylation state at Ser1177,
which has been reported to enhance the activity of the enzyme up to 15
times185. Previous research has identified that statins, a class of agents
frequently used to decrease cholesterol biosynthesis, also increases the halflife of eNOS mRNA. This has been associated with increased eNOS protein
and improved nitric oxide generation. Additionally, experimental models have
indicated that statins have an ability to increase the activation state of eNOS by
blocking
ROK-dependent
inhibition
of
protein
kinase
B-dependent
phosphorylation of Ser1177413. As patients from both PAD and non-PAD
cohorts were hypercholesterolemic and consequently on statin therapy (table
E1 and E2), it was important to examine endothelial function in 300-400µm
arteries isolated from these patients and determine whether differential
endothelial function was responsible for altered vascular reactivity in PAD vs
non-PAD patients.
Endothelial function was assessed by two independent methods: 1) direct
biochemical analysis of the abundance and Ser1177 phosphorylation state of
eNOS 2) direct functional responses to acetylcholine-dependent vasodilation.
The abundance and activity of eNOS was determined in subcutaneous arteries
isolated from patients using SDS-PAGE and western blot analysis. Figure E6
- 173 -
shows that total eNOS was not significantly altered in PAD vs non-PAD patients
(PAD: 6.96 ± 2.49, non-PAD: 8.01 ± 2.89. p>0.05, n = 8). Similarly, there is no
significant difference between the level of Ser1177 phosphorylation of eNOS in
PAD vs non-PAD patients (PAD: 1.78 ± 0.05, non-PAD: 1.80 ± 0.56. p>0.05, n
= 7).
To identify whether patients with and without symptomatic PAD had different
functional responses to acetylcholine-mediated vasodilation, isolated vessels
were constricted with phenylephrine (1x10-6M) and challenged to relax with a
submaximal concentration of acetylcholine (1x10-6M); an established approach
in determining endothelial function in isolated vessels110. Figure E7 shows that
maximum acetylcholine-mediated relaxation was not significantly different
between PAD and non-PAD patient groups (48.29% ± 14.60 and 58.60% ±
9.11, respectively. p>0.05, n = 7-18). Furthermore, the rate of relaxation to
acetylcholine was not significantly different in PAD vs non-PAD patient groups
(data not shown).
- 174 -
Figure E6. Subcutaneous arteries from PAD and non-PAD patients have
similar total eNOS and Ser1177 phosphorylation of eNOS. Subcutaneous
arteries (300-400µM) from age-matched PAD and non-PAD patients were
snap-frozen with liquid nitrogen prior to protein extraction. Total eNOS and the
Ser1177 phosphorylation state of eNOS was determined using SDS-PAGE and
ratiometric western blot analysis. There was no significant difference between
Ser1177-dependent eNOS phosphorylation (A, C) or total eNOS (B, D) in PAD
and non-PAD patients (p>0.05, n = 8)
- 175 -
Figure E7. Acetylcholine-mediated vasodilation was not significantly
different in 300-400µM subcutaneous arteries from PAD and non-PAD
patients. Subcutaneous arteries isolated from PAD (n = 7) and non-PAD
patients (n = 18) were constricted with a submaximal dose of phenylephrine
(1x10-6M). Arteries were then challenged with acetylcholine (1x10-6M). The
maximum relaxation was presented as a percentage of the maximum response
to phenylephrine. Subcutaneous arteries from PAD and non-PAD patients had
no significant difference in acetylcholine-mediated vasodilation (p>0.05).
- 176 -
E3.7. Subcutaneous arteries from patients with and without PAD have
similar abundance and Thr855 MYPT-dependent activation state of
myosin phosphatase.
To more completely characterise the basal subcellular activation state of
myosin phosphatase, we assessed the quantity and activation state of myosin
phosphatase mediated by an inhibitory Thr855 phosphorylation of the targeting
subunit, MYPT in subcutaneous arteries. Total myosin phosphatase and the
ratio of phosphorylated Thr855 MYPT/total myosin phosphatase were
quantified. Figure E8 shows there was no significant difference in total myosin
phosphatase between patients with and without symptomatic PAD (4.53 ± 1.03
and 4.17 ± 0.96, respectively. p>0.05, n = 8). Similarly, the Thr855
phosphorylation of MYPT and hence the activation state of myosin
phosphatase was not significantly different in PAD vs non-PAD patients (PAD:
21.34 ± 5.17, non-PAD: 23.37 ± 4.03. p>0.05, n = 8).
- 177 -
Figure E8. Basal abundance and Thr855 MYPT-dependent activation state
of myosin phosphatase is similar in patients with and without PAD.
Subcutaneous arteries (300-400µM) from patients with PAD undergoing lower
limb amputation and from non-PAD patients were isolated and subject to SDSPAGE and western blot analysis. Total abundance of the MYPT targeting
subunit of myosin phosphatase and its phosphorylation state at Thr855 was
identified.
There
was
no
significant
difference
between
the
Thr855
phosphorylation state of MYPT (A, C) or total myosin phosphatase (B, D) in
patients with and without PAD (p>0.05, n = 8).
- 178 -
E3.8. Subcutaneous arteries from patients suffering with PAD have
increased abundance of total 5HT2A receptors compared to patients
asymptomatic for PAD.
To identify whether the increased maximum contractile responses to serotonin
were due to increased abundance of serotonin receptors we quantified total
5HT2A protein abundance in subcutaneous arteries from patients with and
without clinically diagnosed PAD using western blot analysis. Although there
are seven (7) classes of serotonin receptors, each with numerous subtypes,
5HT2A receptors have predominately been found in vascular smooth muscle
and have been suggested to be the principal regulators of serotonin-mediated
vascular smooth muscle contraction332. Western blot analysis identified that
patients with PAD have more 5HT2A receptors compared to patients
asymptomatic for PAD (Figure E9. 18.16 ± 2.71 and 9.57 ± 2.26, respectively.
p<0.05, n = 6).
- 179 -
Figure E9. Patients suffering with PAD have more 5HT2A receptors in their
subcutaneous arteries than non-PAD patients. Subcutaneous arteries (300400µM) from patients with and without PAD were isolated and subject to SDSPAGE/western blot analysis. Isolated arteries from patients with PAD have
significantly more 5HT2A receptors compared with non-PAD patients (*p<0.05, n
= 6).
- 180 -
E4. Discussion
This study identified that subcutaneous isolated arteries from patients with PAD
have more potent vasoconstrictor responses to serotonin and α1-receptor
activation than do subcutaneous arteries from patients asymptomatic for PAD.
In contrast, both groups of arteries constricted similarly to 87mM KCl-mediated
activation of voltage-gated Ca2+ channels, thromboxane A2 and ET-1 receptor
activation. Interestingly endothelial function, eNOS abundance and eNOS
activity based on Ser1177 phosphorylation was similar in vessels from both
patient groups. Consistent with the functional responses, the abundance and
activation
state
of
myosin
phosphatase,
governed
by
its
Thr855
phosphorylation state, was also similar in both patient groups. Quantification of
5HT2A receptors was increased in PAD vs non-PAD patients consistent with
increased vascular responses to serotonin receptor activation.
In the current study we used accessible abdominal/inguinal tissue from patients
undergoing open hernia repair to overcome the many challenges associated
with selecting an appropriate control to compare isolated arteries from nonischaemic areas of a leg from patients with severe PAD. Previous research
from our laboratory has indicated small subcutaneous arteries isolated from
buttocks biopsies and abdominal tissues reflect vascular responses observed in
the coronary microcirculation414. A conventional approach for evaluating
vascular responses in different vascular beds includes examining the vascular
response to high K+. High K+ depolarises the plasma membrane inducing
robust
voltage-gated
Ca2+
channel
activation.
- 181 -
Several
studies
have
documented the use of K+-mediated depolarisation as an internal normalising
construct for agonist responses221,
417
. Consistent with previous data from our
laboratory, we found no differences in the functional response to high K+dependent vasoconstriction between small subcutaneous arteries isolated from
ischaemic legs compared to subcutaneous abdominal/inguinal arteries isolated
from patients asymptomatic for PAD (figure E1). These data indicate that
voltage-gated Ca2+ channel activation is unchanged in subcutaneous leg
arteries isolated from patients with PAD. These data along with non-significant
differences in vascular responses to ET-1 and thromboxane A2 receptor
activation (figures E4 and E5) further supports our rationale for using human
abdominal and inguinal subcutaneous arteries as a comparator for the
assessment of agonist-mediated vascular responses in subcutaneous arteries
isolated from the leg.
Serotonin is stored and released by platelets and has been implicated in
several vascular disorders. Previous studies have identified patients with
various microvascular disorders have high and low serotonin concentrations in
plasma and platelets, respectively340,
418, 419
; possibly reflecting increased
release or reduced uptake of serotonin by platelets. Consistent with this notion,
isolated rat mesenteric arteries have shown increased contractile responses to
aspirates collected from patients with saphenous vein graft stenosis. These
contractile responses were abolished by ketanserin which has 5HT2A/2C, α1adrenergic and histaminergic blocking properties341, 417. In dog and rat models
of hind limb ischaemia, Doppler flow analysis identified oral and intravenous
administration of serotonin receptor antagonists significantly improve hind-limb
- 182 -
perfusion342,
420, 421
, supporting the emerging possibility that serotonin is an
important mediator of vasoconstriction in peripheral vascular disorders. In
contrast to previous findings, our human data did not show any differences in
sensitivity to exogenously added serotonin in isolated subcutaneous arteries
from patients with or without PAD (figure E2). However, given the
heterogeneous nature of our patient cohort and limited sample size, it is
conceivable we are under-powered to resolve altered sensitivity to serotonin
receptor activation. However, our study did identify a significant increase in the
maximum response to serotonin in isolated arteries from patients with PAD.
Although increased vascular responses to serotonin occurred outside the
normal circulating concentration range, it is conceivable that local serotonin
levels, at the point of release, may exceed physiological plasma concentrations
and thereby cause hyper-contractility in patients with PAD. Although serotonin
antagonists are recognized therapy to manage symptoms of claudication41
(large vessel disease), these data indicate the potential for serotonin receptor
antagonists to increase subcutaneous blood flow and improve healing of
cutaneous ulcers in patients with PAD. Although, several clinical studies have
reported contradicting efficacy of serotonin antagonists such as non-specific
ketanserin and the more selective 5HT2 receptor blocker, naftidrofuryl in
decreasing claudication and improving peripheral blood flow407,
422
. Serotonin
blockers in clinical studies are often administered intravenously, however no
indication has been made to drug concentrations in the critically ischaemic limb.
This raises the question whether reduced blood flow in the ischaemic leg
decreases the drug delivery time to the ischaemic tissue limiting immediate
benefit to the microvasculature. For example, serotonin receptor antagonists
- 183 -
have been shown to be effective in the treatment of Raynaud’s syndrome103, 105,
a rare microvascular disorder which occurs in the absence of large vessel
obstruction. It can be speculated then that the efficacy of intravenous
administration of serotonin receptor blockers may be dependent on the severity
of disease, and consequently the extent of restricted blood flow to the
periphery. Tissue analyses quantifying drug concentrations in the critically
ischaemic leg would provide valuable insights towards clinically implementing
serotonin
receptor
antagonists
for
the
management
of
microvascular
dysfunction in PAD.
Many patients suffering from cardiovascular disorders have elevated circulating
catecholamines85. Consequently, this has led to the development of clinically
useful α/β-adrenergic receptor blockers to manage hypertension281,
370
. Based
on patient profile data, tables E1 and E2 suggest that patients with PAD may
have experienced better management for hypertension. It is conceivable that
once arteries were removed from the patient and their circulating medication,
this benefit was lost leading to enhanced adrenergic vasoconstriction. It has
previously been shown, using wire and pressure myography, that the sensitivity
and the maximum response to noradrenaline and phenylephrine-mediated
vasoconstriction was increased in arteries within ischaemic skeletal muscle of
patients with critical limb ischaemia but decreased in ischaemic subcutaneous
arteries from the same patient423,
424
. In contrast, our study identified that the
maximum contractile response to α1-adrenergic receptor activation was
increased in subcutaneous arteries isolated from patients with advanced PAD
(figure E3). However, it is important to consider our study was designed to
- 184 -
explore vascular reactivity of non-ischaemic subcutaneous arteries from
patients with distal critical limb ischaemia in comparison to vascular responses
of subcutaneous arteries from age-matched patients asymptomatic of PAD; our
study did not compare proximal and distal regions of the same leg. The current
surgical approach is to access healthy tissue to enable efficient wound closure.
Our study is one of the first to report that there may be persistent vascular
dysfunction in the non-ischaemic subcutaneous arteries that may result in
patients experiencing delayed wound closure or development of skin
ulcerations in the remaining stump. Within the surgical community it is
recognized many patients undergoing below knee amputations often require
further debridement or a follow-up above knee amputation. It is conceivable that
therapeutic strategies to manage the abnormal vascular responses to serotonin
and α1- adrenergic stimuli may 1) improve wound closure, 2) resolve nonhealing ulcers and 3) reduce the risk of persistent ulcers and infections in the
remaining stump.
In contrast to serotonin and α1-adrenergic receptor activation, the contractile
responses to thromboxane A2 and ET-1 receptor activation was similar between
patients with and without clinically diagnosed PAD (figures E4 and E5).
Previous studies have documented increased platelet activation and upregulation of thromboxane A2, indexed by urinary thromboxane A2 metabolites,
in patients with PAD301,
425
. Additionally, increased vascular responses in
healthy rat mesenteric arteries to aspirate from patients with coronary artery
disease are shown to be abolished in the presence of a thromboxane A2
receptor antagonist417. Similarly it has been reported ET-1 predicts the risk of
- 185 -
mortality in patients with PAD324. Although these studies implicate thromboxane
and ET-1 as important mediators of vascular disease, direct thromboxane A2
and ET-1 receptor activation in human arteries has not previously been shown
in the context of PAD. Our data indicates that exogenous thromboxane A2 and
ET-1 receptor activation does not directly mediate hyper-constriction of human
subcutaneous small arteries. Nevertheless, it is conceivable its synergistic
action with other vasoconstrictors, such as angiotensin II and noradrenaline389,
403
may be important.
Doppler flow assessment of forearm blood flow in the presence and absence of
ischaemia has shown that endothelial-dependent vasodilation of subcutaneous
arteries is impaired in patients with peripheral and coronary artery disease426,
427
. To identify if endothelial dysfunction and decreased nitric oxide availability
was contributing to increased maximum vasoconstrictor responses to serotonin
and α1-adrenergic receptor activation, we identified the abundance of eNOS
and the activation state indexed by eNOS Ser1177 in 300-400µm
subcutaneous arteries. Interestingly, we found no significant differences in
eNOS abundance nor phosphorylation of eNOS Ser1177, an index of eNOS
activity, in PAD and non-PAD patients (figure E6). Importantly, this was
consistent
with
similar
functional
responses
to
acetylcholine-mediated
vasorelaxation in pre-constricted arteries from both PAD and non-PAD patient
groups (figure E7). It is important to point out that while no significant difference
between patient groups existed, acetylcholine-mediated vasodilation was not
normal (approximately 50-60% relaxation to phenylephrine vs 90% relaxation
as normal361). Importantly, our biochemical analysis used naïve, unmounted
- 186 -
arteries immediately snap-frozen following isolation allowing us to exclude
experimenter-induced mechanical endothelial damage. Several previous
studies have shown endothelial dysfunction and eNOS uncoupling caused by
reduced levels of the co-factor, tetrahydrobiopterin (BH4) which is associated
with age and/or reduced physical activity138, 139, 141; hence impaired responses
to acetylcholine-mediated vasorelaxation may well be appropriate for the +70
years age category of both patient groups. Although endothelial dysfunction
may still be present in these patients, it is not more prominent in patients with
PAD compared to those asymptomatic for PAD. Consequently, endothelial
function alone cannot explain specific enhanced vascular reactivity in patients
with PAD. It is important to point out that computer tomography scans or
intravascular ultrasound rarely show atheromatous plaque and calcified
vascular disease along the full length of large arteries, but rather in intermittent
and localised regions53, 428. Since, endothelial dysfunction occurs intermittently
along a vascular tree it is conceivable that our tissue sections predominately
contained regions of relatively healthy-aged endothelium.
Myosin phosphatase has attracted considerable interest regarding possible
mechanisms regulating central vascular disorders. However our data clearly
excludes the role of Thr855-dependent regulation of myosin phosphatase in
mediating microvascular dysfunction in PAD (figure E8). We saw no change in
myosin phosphatase abundance and activity, indexed by phosphorylation of
MYPT Thr855. This was consistent with normal functional responses to
thromboxane A2 and ET-1 receptor activation (figures E4 and E5). However like
eNOS abundance and activity, the mean age of +70 years in both groups (table
- 187 -
E1) begs the question whether myosin phosphatase abundance and activity is
different in young-healthy arteries compared to aged-diseased arteries;
implying the greatest risk factor associated with vascular disease may be age.
We identified subcutaneous arteries from patients with PAD have more 5HT2A
receptors and enhanced maximal responses to serotonin receptor activation
compared to subcutaneous arteries isolated from patients with no clinically
diagnosed PAD (figure E9). Although many studies have documented the
importance of serotonin in mediating large vessel disease340,
420, 421, 429
, our
study is the first to suggest a possible mechanism for increased vascular
reactivity to serotonin in small arteries. This novel finding could have important
implications to support the use of serotonin receptor antagonists in improving
subcutaneous blood flow and would healing in patients with PAD.
Comments on co-morbidities and patient medications
Compared with patients asymptomatic for PAD, the PAD cohort had an
increased frequency of type II diabetes mellitus, renal dysfunction, ischemic
heart disease and previous myocardial infarction (table 1). Vascular
complications associated with type II diabetes are well documented. Insulin
resistance and hyperglycemia contributes to endothelial dysfunction, reduced
nitric oxide release and increased vascular reactivity; consequently patients
with type II diabetes often have accelerated glaucoma, renal, coronary and
peripheral vascular disease430-432. Using isolated blood vessels in vitro, it is
reasonable to consider that the consequences of diabetes may alter vascular
reactivity to all agonists. However since we only observed increased contractile
- 188 -
responses specifically to serotonergic and α1-adrenergic receptor activation, it
is unlikely that increased incidence of diabetes in the PAD patient group
contributed to this response. The compensatory mechanisms associated with
renal dysfunction often include an increase in ET-1 and angiotensin II release
by the kidneys and vasopressin by the posterior pituitary. Similarly, a decreased
cardiac output in patients with ischemic heart disease and previous occurrence
of myocardial infarction may invoke compensatory catecholamine release by
the adrenal glands433,
434
. ET-1, angiotensin II and catecholamines have
previously been shown to potentiate the vascular response to ET-1,
serotonergic and α1-adrenergic receptor activation390,
404
. Although the current
study did not identify altered contractile responses to ET-1, it is possible that
co-morbidities contributed to altered vascular reactivity to serotonergic and α1adrenergic receptor activation in patients with PAD. Considering the comorbidities associated with PAD, it is not surprising that more patients with PAD
were on angiotensin II (ARB) and β-adrenergic receptor blockade therapy
compared to non-PAD patients (table 2). As a compensatory feedback
mechanism to ARB therapy, one would anticipate higher circulating angiotensin
II and as previously discussed this may have contributed to the altered vascular
reactivity reported in the current study. It is possible β-blockers would abolish βadrenergic receptor-mediated vasodilation in vascular smooth muscle cells and
may consequently contribute to increased contractile responses to α1adrenergic receptor activation; however one would have expected the βblockers to have been washed out in the in vitro vessel preparation.
Nevertheless, this does not preclude some form of long term feedback
- 189 -
compensation, which may have contributed to altered phenylephrine-mediated
vasoconstriction in the PAD patients. It is perhaps wise to consider that despite
and/or as a consequence of medical therapy, patients with PAD continue to
show increased vascular reactivity to specific agonists. This data warrants the
consideration of additional strategies to reduce microvascular vasoconstrictor
sensitivity to age matched control values. The current sample size does not
allow for the stratification of these co-morbidities and medical therapies. Future
studies will be directed at increasing the number of patients in PAD and nonPAD cohorts to enable a more through stratification and analysis of variance.
Conclusion
In conclusion our study has identified serotonin and α1-adrenergic receptor
agonists as mediators of increased microvascular constriction in patients with
PAD. We have excluded several proposed regulators of vascular tone and
identified an increased abundance of 5HT2A receptors as a possible mechanism
for hyper-contractility to serotonin. These data cast doubt on the wisdom and
vascular benefit of selective serotonin reuptake inhibitors frequently used to
treat depression in PAD patients. Future studies are aimed at quantification of
active 5HT2A and other serotonin receptor subtypes on the plasma membrane
using cell surface biotinylation and immunoprecipitation assays. Due to limited
tissue availability we were not able to analyse α1-adrenergic receptor
abundance in patients with and without PAD. However, the increased
vasoreactivity to α1-adrenergic receptor activation begs the question whether
phenylephrine-based over the counter cold medications and stress-induced
catecholamine release may contribute to reduced subcutaneous blood flow in
- 190 -
patients with PAD, favouring delayed ulcer healing and wound closure. Our
study supports the notion of α1-adrenergic and serotonin receptor blockade as
beneficial treatment strategies for PAD.
- 191 -
Section F
General Discussion
- 192 -
F1. Thesis discussion
F1.1. Thesis premise
As little as 20 years ago, it was predicted the treatment of high cholesterol and
hypertension through the use of lipid-lowering agents, diuretics and ACE
inhibitors would abolish death by cardiovascular complications over 10 years69.
However, even with significant advancements in therapy, vascular disease is
expected to remain a global killer due to an increasing prevalence of obesity
and diabetes in economically less developed and first world countries69,
435
.
Although vascular disease is perceived as an inflammatory atherothrombotic
disorder, the contribution of increased vascular tone and vasospasm is being
increasingly recognized. Large vessel atheroma and thrombosis can be
relatively well managed with lipid-lowering agents, antiplatelet agents,
angioplasty, stents and bypass6, 8, 41, 364. However, mechanical revascularisation
procedures and conventional medical therapies are limited in treating small
vessel disease, vasospasm and chronic increases in vascular tone51, 52, 57. Due
to a lack of appropriate animal models and the significant challenges
associated with the study of microvascular dysfunction in humans, the current
literature is sparse. This thesis aimed to identify the aetiologies and molecular
mechanisms underlying peripheral vascular reactivity in health and disease,
specifically in the microvasculature.
The inflammatory atherothrombotic process involves the release of a variety of
vasoconstrictors including endothelin-1 (ET-1), serotonin and thromboxane A2
from inflammatory cells, platelets and the vasculature which is often coupled
with a decreased release of endothelial-derived vasodilators82-84. Additionally,
- 193 -
patients with vascular disease often have elevated circulating noradrenaline
and adrenaline concentrations due to a vicious circle of increased pressure and
after load on the heart with necessary and compensatory catecholamine
release to increase cardiac output but also total peripheral resistance. As a
consequence, patients are predisposed to inappropriate vasoconstriction and/or
vasospasm, causing additional restrictions in blood flow. ET-1 has been
implicated in many vascular disease states including pulmonary hypertension,
vasospastic angina and digital vasospasm307 326, 360, 381. This thesis examined 1)
the molecular basis of acute ET-1 mediated vasoconstriction in rat caudal and
mesenteric arteries in vitro 2) the chronic influence of ET-1 on the expression of
specific G-protein coupled receptors in rat mesenteric arteries using an in vivo
ET-1 infusion model and 3) the molecular basis of vascular reactivity to
exogenously added ET-1, α1-adrenergic, serotonergic and thromboxane A2
receptor activation in human subcutaneous arteries from patients suffering with
peripheral artery disease (PAD) and age-matched patients asymptomatic for
PAD.
F1.2. Summary of results
To identify whether inhibition of specific subcellular effectors, specifically protein
kinase C (PKC) and Rho-associated kinase (ROK), common to several Gprotein coupled receptors is an effective alternative to multiple and specific
receptor blockade therapy242,
243, 253, 258, 338, 436
, we first examined their role in
regulating acute ET-1-mediated vasoconstriction in healthy rat caudal (500µm)
and mesenteric arteries (300µm). We identified that inhibiting PKC and ROK
prior to and following ET-1-mediated vasoconstriction attenuated the initial- 194 -
rapid Ca2+ dependent and sustained-tonic Ca2+ independent phases of ET-1mediated vasoconstriction in large conduit caudal arteries. Whereas in small
second order resistance mesenteric arteries, ROK and combined PKC/ROK
inhibition attenuated sustained ET-1-mediated vasoconstriction.
Although ET-1 up-regulation in patients with vascular disease has been well
documented322, 357, 359, 385 and the benefits of ETA/B receptor blockade has been
shown clinically and experimentally326,
327
, it is unclear whether the direct
influence of ET-1 on the microvasculature is solely caused by elevated
circulating ET-1. Following chronic exposure of young (9-10 weeks), healthy
Sprague Dawley rats to ET-1, we identified decreased contractile responses to
exogenously
added
thromboxane
A2,
while
ET-1,
α1-adrenergic
and
serotonergic receptor profiles remained unchanged in isolated rat mesenteric
arteries. Importantly, the decreased contractile response to thromboxane A2
receptor activation was independent of high K+-mediated activation of voltagegated Ca2+ channels, the abundance and Ser1177 eNOS, Thr855 MYPTdependent activation state of eNOS and myosin phosphatase, respectively.
In subcutaneous arteries collected from age-matched patients with and without
PAD, we identified increased maximum contractile responses to serotonergic
and α1-adrenergic receptor activation but no change in the contractile response
to ET-1 and thromboxane A2 receptor activation. Patient history, medications,
the vascular response to high K+-mediated activation of voltage-gated Ca2+
channels and the abundance and Ser1177 eNOS, Thr855 MYPT-dependent
activation state of eNOS and myosin phosphatase was similar between patients
- 195 -
groups. However, interestingly, western blot analysis identified more total 5HT2A
receptors in patients with PAD compared to non-PAD patients.
F1.3. Is there a need to consider alternatives to Ca2+ channel and multiple Gprotein coupled receptor blockade therapy?
Clinically, the management of vasospastic disorders, increased peripheral
vascular resistance and elevated blood pressure, is focused on reducing
agonist-mediated vasoconstriction through the individual or concomitant
blockade of G-protein coupled receptors, e.g., ET-1, angiotensin II, and α1adrenergic receptors326,
369-371
. Although a generalised strategy to reduce
agonist-mediated vasoconstriction also exists, these approaches almost
exclusively focus on reducing extracellular Ca2+ entry; for example, through the
use of Ca2+ channel blockers and nitrates363, 437. These therapies often do not
resolve small vessel dysfunction presumably because clinically available Ca2+
channel blockers exclusively target the L-type voltage-gated Ca2+ channel438,
439
, and neglect numerous additional modes of Ca2+ entry e.g., sarcoplasmic
Ca2+ release and activation of T-type voltage-gated Ca2+ channels. The T-type
Ca2+ channel has recently been suggested to be important for small vessel
vasoconstriction while the L-type Ca2+ channel is more abundant in large
vessels221. Additionally, it has been suggested vasodilators such as endothelialderived hyperpolarising factor (EDHF) may be more important than nitric oxide
in mediating small vessel dilation440, 441.
These data are consistent with the current dogma that heterogeneity amongst
both large and microvascular beds exists. In studies herein, PKC and ROK
- 196 -
were activated differently in large caudal arteries (500µm) and small mesenteric
arteries (300µm). It has been previously suggested PKC, ROK and myosin
phosphatase are activated differently in specific vascular beds254. These data
support the contention that there may well be therapeutic value in differentially
targeting ROK and PKC in large vessel disease and microvascular dysfunction.
Additionally, these data indicate ROK and PKC inhibition may be valuable for
either prophylactic treatment or treatment following initiation of contraction; this
may be particularly important for patients presenting with existing chest pain,
hemorrhagic stroke and peripheral ischaemic pain, which is not relieved by Ca2+
channel blockers or nitrate therapy.
Targeting common downstream effectors may be a particularly valuable first
line strategy if the aetiology of increased vascular contractility or vasospasm is
unknown. For example, essential hypertension, which accounts for 95% of all
hypertensive cases, is often idiopathic442. Similarly, patients with non-healing
ulcers are often already on L-type Ca2+ channel blockers and several receptor
antagonists41,
364
, implying there may be significant residual risk that remains
unidentified and unmanaged. Consistent with these observations, data herein
suggests chronic ET-1 infusion and direct examination of the contractile
properties of human subcutaneous arteries demonstrated responses to specific
circulating hormones were altered (e.g., thromboxane A2, an α1-adrenergic
agonist and serotonin). It is not surprising to find differences in healthy vessels
and those with a significant atherothrombotic plaque burden.
- 197 -
While ROK inhibitors are clinically available in Japan273, 373, 443, current research
in this field is increasingly favouring the international availability of ROK
inhibitors. In contrast, there is a limited availability of PKC inhibitors and
different PKC isoforms have been suggested to be regionally expressed and
activated by different agonists253, 255, 262. The specific identification of the PKC
isoforms involved in agonist-mediated vasoconstriction may enable the required
specificity to block PKC-mediated Ca2+ sensitization in vascular smooth muscle
and improve their clinical safety. Precedent for this approach currently exists in
clinical practice, i.e., it is well recognized that phosphodiesterase 5 (PDE5)
inhibitors are effective vasodilators in the penile and pulmonary circulations but
are relatively less effective in the coronary circulation444-446. While specific
PDE3 inhibitors have been shown to be effective treatment inotropes in
managing cardiac failure and cardiogenic shock as well an emerging treatment
options in the management of claudication and peripheral ischaemia447, 448.
F1.4. The influence of elevated ET-1 in vascular disease
Based on data presented herein one can speculate that chronic infusion of ET1 in a healthy animal model down-regulates thromboxane A2 receptors. As
thromboxane A2 is an important activator of platelets80, these data lead on to
the speculation that patients with elevated ET-1 may have decreased platelet
reactivity and consequently increased thromboxane A2-dependent bleeding and
clotting times relative to individuals without elevated ET-1. Several studies have
indicated increased thromboxane A2 up-regulation and enhanced platelet
reactivity in patients with vascular disease301,
302
. It is wise to point out that
vascular disease has manifold causes, nevertheless it may be worthwhile to
- 198 -
consider thromboxane A2-mediated vasoconstriction, platelet reactivity and
elevated ET-1 have not been specifically studied together. In human
subcutaneous arteries, we identified no change in thromboxane A2 receptor
activation between PAD and non-PAD patients, which may suggest that ET-1
levels are not elevated. Well-designed future studies should be established to
directly test this hypothesis. It is important to recognize, patients with vascular
disease often have elevations in multiple circulating vasoconstrictors and
decreased vasodilators85,
130, 360, 449
. Our PAD and non-PAD patients groups
were on medical therapy and had several co-morbidities. Furthermore, both
patient groups had a similar degree of endothelial dysfunction, appropriate for
their age group (+70 years). This was certainly not true for our healthy rat
model, in which one vasoconstrictor was exclusively elevated and each
mesenteric artery showed near complete (90%) acetylcholine-mediated
relaxation following stimulation with maximum concentrations of each agonist.
Hence the influence of ET-1 in the complex context of vascular disease
requires further investigation. Future studies will focus on quantifying
thromboxane A2 receptor expression and identifying platelet reactivity and
clotting times in animal models and perhaps in patients with PAD where ET-1 is
chronically elevated.
The similar responses to exogenously added ET-1 between saline and ET-1
infused groups indicated the increased response to ET-1 in patients may not be
due to elevated ET-1 per se or up-regulation of ET-1 receptors. This was
consistent with our human data which showed similar contractile responses to
exogenously added ET-1 in subcutaneous arteries from PAD and non-PAD
- 199 -
patients. This indicates that contractile responses to ET-1 typically observed in
patients with vascular disease may be due to potentiation by other circulating
vasoconstrictors and/or high concentrations of locally generated ET-1. These
data highlight a value in moving beyond the singular, step-wise approach
toward a multi-factorial, integrated approach to causality and perhaps
considering the evaluation of multiple risk factors concurrently.
F1.5. The pathophysiology of vascular reactivity in patients with PAD
Our study is the first to show that vessels from non-ischaemic regions of a
critically ischaemic lower limb still harbour abnormal constrictor responses to
serotonin and α1-adrenergic agonists. PAD and non-PAD patient groups were
age-matched, and hence had similar co-morbidities, were on similar medical
therapies
and
showed
a
similar
degree
of
endothelial
dysfunction.
Consequently, these factors could not explain the increased contractile
response to serotonergic and α1-adrenergic receptor activation. The finding that
patients with PAD had more serotonin 5HT2A receptors indicated a possible
mechanism for increased contractile responses to serotonin. Limited tissue
availability restricted our ability to quantify α1-adrenergic receptor expression;
nonetheless these data support the use of serotonin and α1-adrenergic receptor
blockers in the management of microvascular dysfunction in patients with PAD.
Decreasing the influence of α1-adrenergic and serotonergic agonists in the
cutaneous circulation has the potential to accelerate healing of ulcers and
reduce the risk of reoccurring infection and ischaemia-related complications in
patients with PAD. The inefficacy of clinically used serotonin antagonists may
be reflective of their relative non-specificity to serotonin receptors. For example,
- 200 -
ketanserin has both histaminergic, α1-adrenergic and dopamine receptor
blocking properties450-452; while naftidrofuryl is more selective for serotonin 5HT2
receptors453.
These data suggest one should consider a conservative or cautious approach
toward the use of specific antidepressants, namely serotonin re-uptake
inhibitors (SSRIs) as these may exacerbate clinical symptoms in patients with
PAD. In the coronary circulation, cardiac complications following administration
of SSRIs have been attributed to amplification of serotonin-induced
vasoconstriction in patients with endothelial dysfunction as a result of
increasing the availability of circulating serotonin454-456. However, it is important
to recognize several factors may be the cause of SSRI-induced cardiac
complications,
including
induction
of
arrhythmias
and
specific
drug
interactions454. Nevertheless, it is conceivable SSRIs may also be enhancing
serotonin-mediated vasoconstriction in the peripheral circulation. In a similar
context, phenylephrine-based cold medications may exacerbate α1-adrenergic
receptor activation in patients with PAD and contribute to a reduction in blood
flow to the cutaneous circulation.
In the clinical management of PAD, smoking cessation is one of the first
strategies to increase peripheral blood flow and tissue perfusion. It has been
shown chronic smokers have elevated blood pressure and an increased risk of
developing coronary and peripheral vascular disease23,
26
. Although cigarettes
(and nicotine-containing alternatives, e.g. tobacco) contain a myriad of harmful
compounds, nicotine is probably the most thoroughly studied. Nicotine causes
- 201 -
vasoconstriction by either directly acting on smooth muscle cells or by releasing
catecholamines from the adrenal glands457. Our data suggests that serotonin
and α1-adrenergic receptor activation may contribute to chronic elevations in
vascular tone in a similar manner to nicotine and hence may benefit from the
same rigor as encouragement of smoking cessation in the management for
PAD. This suggests that there is additional support and impetus to consider a
clinical need to decrease the release of serotonin from platelets during
inflammation or directly block serotonin and α1-adrenergic receptor activation in
an effort to improve perfusion through the microvasculature and salvage
critically ischaemic limbs.
Our study was limited by sample size, highlighting the difficulties associated
with a single centre study over the time course of PhD studies. Patient
recruitment is often a challenge, which illustrates the value of effective
teamwork. Future studies may benefit from larger multiple-centre studies to
broaden patient recruitment and clinical relevance.
F1.6. Conclusions
Although the burden of cardiovascular disease is well recognized, research in
this field largely focuses on the heart and coronary circulation. This thesis
emphasises the importance of the peripheral circulation and perhaps
importantly reminds us of the generalised nature of vascular disease. This
thesis suggests a need to better recognize and aggressively manage PAD to a
similar degree as coronary/cerebrovascular disease in an effort to increase
- 202 -
quality of life and decrease risk of death by cardiovascular-related
complications.
- 203 -
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