PERIVASCULAR ADIPOSE TISSUE AND VESSEL
CONTRACTILITY IN HEALTH AND OBESITY
A thesis submitted to the University of Manchester for the degree of
Doctor of Philosophy (PhD)
in the faculty of Medical and Human Sciences
2013
Reza Aghamohammadzadeh
School of medicine
Institute of Cardiovascular Sciences
List of Contents
Title page.............................................................................................................1
List of contents....................................................................................................2-3
Abstract...............................................................................................................4
Declaration..........................................................................................................5
Contribution.......................................................................................................5
Copyright statement...........................................................................................6
Acknowledgements.............................................................................................7
Dedication............................................................................................................7
Abbreviations......................................................................................................8-9
The Author (about the candidate)....................................................................10
Publications related to this thesis......................................................................11-12
Abstracts and conference presentations...........................................................13-15
Chapter 1: Introduction.....................................................................................16-90
Perivascular adipose tissue from human systemic and coronary vessels: the emergence of a
new pharmacotherapeutic target. Aghamohammadzadeh et al, Br J Pharmacol. 2012
Feb;165(3):670-82. (PMID: 21564083)
Obesity-related hypertension: epidemiology, pathophysiology, treatments, and the
contribution of perivascular adipose tissue.Aghamohammadzadeh et al, Ann Med. 2012
Jun;44 Suppl 1:S74-84. (PMID: 22713152)
Chapter 2: Materials and Methods....................................................................91-102
2
Chapter 3: ...........................................................................................................103-133
Effects of bariatric surgery on human small artery function: evidence for reduction in
perivascular adipocyte inflammation, and the restoration of normal anticontractile activity
despite persistent obesity. Aghamohammadzadeh et al, J Am Coll Cardiol. 2013 Jul
9;62(2):128-35. (PMID: 23665100)
Chapter 4:............................................................................................................134-163
Obesity-induced damage to perivascular adipose tissue anticontractile function is nitric
oxide dependent, independent of the endothelium and reversible. Aghamohammadzadeh et
al, Submission pending
Chapter 5:............................................................................................................164-189
High Density Lipoprotein Antioxidant Function is Impaired and Adipose Tissue
Inflammation is More pronounced in Obese Patients with Obstructive Sleep Apnea.
Yadav, France, Aghamohammadzadeh et al, Submission pending
Chapter 6: Discussion.........................................................................................190-193
WORD COUNT: 39824
3
Abstract
PERIVASCULAR ADIPOSE TISSUE AND VESSEL CONTRACTILITY IN
HEALTH AND OBESITY
Reza Aghamohammadzadeh (PhD candidate, Faculty of Medical and Human Sciences),
September 2013
White adipocytes surround almost all blood vessels in the human body. It was thought
previously that these cells merely provide mechanical support for the adjacent small
vessels and are little more than fat storage units. Recent studies have identified these cells
as metabolic and vasoactive engines that produce and secrete molecules that can affect the
function of their adjacent small vessels. The adipocytes and a number of other cell types
(including inflammatory cells) surrounding the vessels are collectively termed the
PeriVascular Adipose Tissue (PVAT). Work from our group has shown previously that, in
health, PVAT conveys a vasorelaxant effect on adjacent small arteries and that this effect
is not observed in obesity thus the vessels must exist at an elevated level of basal tone. It is
plausible that increased basal vessel constriction can explain the elevated blood pressure
amongst the obese population and a better understanding of the obesity-induced PVAT
damage may lead to clues to a new approach in the treatment of the condition which
burdens its sufferers with a greater cardivascular risk profile.
In this thesis we have studied individuals with morbid obesity at baseline and six months
following surgery and observed that PVAT function following dramatic weight loss
restores the PVAT vasorelaxant effect close to that observed in lean patients. Moreover,
we have concluded that inflammation plays a significant role in this process and indeed
using protocols with antioxidant enzymes we were able to restore the damaged PVAT
function at baseline. We have have shown also that in health, PVAT vasorelaxant function
is independent of the endothelium, and that obesity-induced PVAT damage and its
reversal following weight loss and ex-vivo anti-oxidant treatment are both independent of
the endothelium and at least in part due to nitric oxide bioavailability. Finally, we have
observed that in sleep apnoea, which often coexists with morbid obesity and hypertension,
there is a greater degree of PVAT inflammation.
4
Declaration
No portion of the work referred to in the thesis has been submitted in support of an
application for another degree or qualification of this or any other university.
Contribution
Chapter 3: I was involved at all stages of this project. I helped write the ethics application
and its amendments, I recruited the patients and personally performed all the biopsies and
harvested the tissue. I dissected all the samples, was involved with the design of the
protocols and performed wire myography in each case. I preserved the samples for
immunohistochemistry which was performed by Dr Jeziorska. I was involved with all the
data analysis for immunohistochemistry and personally performed all the analysis for
myography data. The protocols for measurement of lipids and circulating biomarkers were
primarily performed by Phil Pemberton and our collaborator in the lipid study group. I
performed all the analysis on this data and wrote the manuscript.
Chapter 4: The human biopsies were performed by Dr Greenstein, however I performed
all the analysis. The Proteomics protocols were outsourced to Oxford Genomics and I
performed all the analysis on the data. All the animal protocols were designed, performed,
and analysed by me. I have written the manuscript.
Chapter 5: I was involved with the ethics application, patient recruitment and biopsies. I
was involved with tissue preparation and analysis of the data as well as preparing the
manuscript. Dr Maria Jeziorska performed the immuhistochemistry protocols. All the lipid
oxidation laboratory protocols were performed by Rahul Yada, Salam Hama and Yifen
Liu.
5
COPYRIGHT STATEMENT
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certain copyright or related rights in it (the “Copyright”) and s/he has given The
University of Manchester certain rights to use such Copyright, including for administrative
purposes.
ii. Copies of this thesis, either in full or in extracts and whether in hard or electronic copy,
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iii. The ownership of certain Copyright, patents, designs, trade marks and other intellectual
property (the “Intellectual Property”) and any reproductions of copyright works in the
thesis, for example graphs and tables (“Reproductions”), which may be described in this
thesis, may not be owned by the author and may be owned by third parties. Such
Intellectual Property and Reproductions cannot and must not be made available for use
without the prior written permission of the owner(s) of the relevant Intellectual Property
and/or Reproductions.
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commercialisation of this thesis, the Copyright and any Intellectual Property and/or
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http://documents.manchester.ac.uk/DocuInfo.aspx?DocID=487), in any relevant Thesis
restriction declarations deposited in the University Library, The University Library’s
6
regulations (see http://www.manchester.ac.uk/library/aboutus/regulations) and in The
University’s policy on Presentation of Theses
ACKNOWLEDGEMENTS
I am very grateful to all the study participants who voluntarily and without remuneration
underwent gluteal fat biopsies and provided blood samples for the purposes of the
protocols detailed in this thesis.
The nursing and administrative staff at the Manchester NIHR/ Wellcome Trust Clinical
Research Facility provided much needed support in coordinating patient scheduling and
collating demographic data.
I would like to thank my supervisor Professor Anthony Heagerty for his invaluable
support, guidance and mentorship throughout the whole process, as well my advisor
Professor Ryaz Malik who remained approachable, helpful and available in times of need.
I would like to thank Dr Adam Greenstein for his help and support throughout the steep
learning curve and for providing much needed technical guidance with the biopsies and
laboratory protocols.
Lastly, I would like to thank all members of the Heagerty laboratory for providing
technical assistance with research methodology in the laboratory and for being great
friends throughout the process.
DEDICATION
This thesis is dedicated to my father Dr Hossein Aghamohammadzadeh, mother Akram
Akhtari and brother Dr Soheil Aghamohammadzadeh who provided me with support, both
7
emotionally and scientifically, and for putting up with my endless moans and rants during
the three year process.
LIST OF THE MOST FREQUENTLY USED ABBREVIATIONS
AdipoR1
Adiponectin Receptor 1
ADRF
Adventitium Derived Relaxing Factor
BMI
Body Mass Index
BP
Blood Pressure
CAD
Coronary Artery Disease
HDL
High Density Lipoprotein
hsCRP
High sensitivity C-Reactive Protein
ICAM-1
Intercellular Adhesion Molecule-1
IL-6
Interleukin-6
K+
Potassium
L-NAME
NG-nitro-L-Arginine Methyl Ester
LDL
Low Density Lipoprotein
MCP-1
Monocyte Chemotactic Protein-1
MS/ MetS
Metabolic Syndrome
8
PPAR
Peroxisome proliferator-activated receptor
PVAT
Perivascular Adipose Tissue
NA
Noradrenaline
NE
Norepinephrine
NO
Nitric Oxide
OSA
Obstructive Sleep Apnoea
ROS
Reactive Oxygen Species
TNF
Tumour Necrosis Factor
VCAM-1
Vascular Cell Adhesion Molecule-1
WHO
World Health Organisation
9
THE AUTHOR
Reza Aghamohammadzadeh graduated from the University of Manchester with an
Honours degree in Medicine in 2006. He was appointed to an Academic Foundation
Training Programme in Manchester as a junior doctor. He was awarded his full
membership of the Royal College of Physicians in 2009 and subsequently took time out of
his clinical training programme to pursue research towards this thesis. In 2009, he was
awarded a Clinical Research Fellowship from NIHR Manchester Biomedical Research
Centre to start work on this thesis and in 2010 he was awarded futher personal funding in
the form of a Clinical Research Training Fellowship from the British Heart Foundation to
support this body of work. In 2012 he was awarded an NIHR Manchester Biomedical
Research Research Centre International Scholar award to pursue a 6 month research
project at Harvard Medical School (Boston, USA) in the field of vascular biology in
relation to pulmonary hypertension. This work was carried out during his thesis pending
year. Currently, he is an NIHR Academic Clinical Fellow in Cardiology and an honorary
research fellow at the University of Manchester and continues to pursue his research
endeavours alongside his cardiology traininig.
10
RELATED PUBLICATIONS

The Effects of Bariatric Surgery on Human Small Artery Function: Evidence for
Reduction in Perivascular Adipocyte Inflammation, and the Restoration of Normal
Anticontractile Activity Despite Persistent Obesity. Reza Aghamohammadzadeh, AS
Greenstein, R Yadav, M Jeziorska, S Hama, F Soltani, PW Pemberton, B Ammori, RA
Malik, H Soran, AM Heagerty. J Am Coll Cardiol. 2013 Jul 9;62(2): pp. 128-35.
(PMID:23665100)

Obesity-related hypertension: epidemiology, pathophysiology, new treatments and the
contribution of perivascular adipose tissue. R Aghamohammadzadeh, AM Heagerty.
Annals of Medicine Ann Med. 2012 Jun;44 Suppl 1:S74-84. (PMID: 22713152)

Perivascular adipose tissue from human systemic and coronary vessels: The emergence of
a new pharmacotherapeutic target. Aghamohammadzadeh R, Withers SB, Lynch FM,
Greenstein AS, Malik RA, Heagerty AM. Br J Pharmacol. 2012 Feb;165(3):670-82.
(PMID: 21564083)

Obesity, diabetes and atrial fibrillation; epidemiology, mechanisms and
interventions.Asghar O, Alam U, Hayat SA, Aghamohammadzadeh R, Heagerty AM,
Malik RA. Curr Cardiol Rev. 2012 Nov;8(4):253-64. (PMID: 22920475)

Mutation in the beta adducin subunit causes tissue-specific damage to myogenic tone.
Sonoyama K, Greenstein AS, Micheletti R, Ferrari P, Schiavone A,
Aghamohammadzadeh R, et al. J Hypertens 29(3): 466-474, (PMID: 21150638)
Book Chapters

Small artery structure and function in hypertension. Anthony M Heagerty, Sarah B
Withers, Ashley S Izzard, Adam S Greenstein, Reza Aghamohammadzadeh (2013).
11
Manual of Hypertension of the European Society of Hypertension 2 nd edition. Published
by Informa

Vani Subbarao, David Lowe, Reza Aghamohammadzadeh, Robert J. Wilkinson (2011).
Endothelial Dysfunction in HIV, HIV Infection in the Era of Highly Active Antiretroviral
Treatment and Some of Its Associated Complications, Elaheh Aghdassi (Ed.), ISBN: 978953-307-701-7, InTech
Conference proceedings

Effects of bariatric surgery on human small artery function: evidence for reduction in
perivascular adipocyte inflammation, and restoration of normal anticontractile activity
despite persistent obesity. The Lancet, Volume 381, Issue , Page S18, 27 February 2013
doi:10.1016/S0140-6736(13)60458-4

Alterations in subcutaneous small artery wall structure correlate with diastolic parameters
in patients with metabolic syndrome. K Khavandi, M Luckie, R Aghamohammadzadeh, A
Khavandi, A Mastan, A Greenstein, R Malik, R Khattar, AM Heagerty. J Am Coll
Cardiol, 2011; 57:1545, doi:10.1016/S0735-1097(11)61545-3

Protection from development of obesity in high fat diet rats is associated with preservation
of the anticontractile function of perivascular adipose tissue. R Aghamohammadzadeh*,
PH Park, AS Greenstein, AM Heagerty. Heart 2010;96:e17
doi:10.1136/hrt.2010.205781.29
12
ABSTRACTS AND CONFERENCE PRESENTATIONS
1. Raptor activation by aldosterone promotes apoptosis resistance in pulmonary artery
smooth muscle cells to modulate adverse pulmonary vascular remodelling in pulmonary
arterial hypertension. Reza Aghamohammadzadeh, Anthony Heagerty, Joseph Loscalzo,
Bradley A. Maron, Jane A. Leopold. Experimental Biology, Boston April 2013 (Oral and
poster presentation)
2. Aldosterone activates autophagy to increase fibroblast collagen synthesis and vascular
stiffness. S Torquato, BA Maron, R Aghamohammadzadeh, J Loscalzo, JA Leopold.
Experimental Biology, Boston April 2013 (Poster presentation)
3. Damage to the anticontractile property of perivascular adipose tissue in morbidly obese
individuals is rescued by weight loss following bariatric surgery. R
Aghamohammadzadeh*, A Greenstein, R Yadav, S Hama, P Pemberton, H Soran, B
Ammori, AM Heagerty. European Society of Hypertension, London April 2012 (Oral
presentation)
4. Perivascular adipose tissue (PVAT) nitric oxide bioavailability is reduced in obesity and
contributes to the loss of PVAT anticontractile function. R Aghamohammadzadeh*, F.
Soltani, A Greenstein, V Cunha, A Khanna, AM Heagerty. European Society of
Hypertension, London April 2012 (Poster presentation)
5. The obesity-induced perivascular adipose tissue damage can be rescued by both free
radical scavengers and weight loss following bariatric surgery. R Aghamohammadzadeh*,
Adam Greenstein, R Yadav, S Hama, P Pemberton, H Soran, B Ammori, AM Heagerty.
Manchester Medical Society (section of medicine) April 2012 (Oral presentation)
6. Damage to the anticontractile capacity of perivascular adipose tissue in obesity is in
keeping with reduced nitric oxide bioavailability and strongly correlates with blood
pressure elevation. R Aghamohammadzadeh*, B Park, AS Greenstein, AM Heagerty.
13
HBPR council of AHA scientific sessions, Orlando, September 2011 (Poster
presentation)
7. The effect of accelerated weight gain on blood pressure, intrinsic heart rate and the
function of the cardiac conduction system. R Aghamohammadzadeh*, O Monfredi, A
Mastan, , M Boyett, AM Heagerty. European Society of Hypertension, Milan June 2011
(Oral presentation)
8. Free radical scavengers rescue obesity-induced damage to the anticontractile property of
perivascular adipose tissue in rat and human small arteries. R Aghamohammadzadeh*, AS
Greenstein, AM Heagerty. European Society of Hypertension, Milan June 2011 (Oral
presentation)
9. A study of Vitamin D levels and circulatory risk factors in primary care. A Mastan*, A
Bukhari, R Aghamohammadzadeh, AM Heagerty. European Society of Hypertension,
Milan June 2011- Winner of best poster award
10. There is reduced nitric oxide production in perivascular adipose tissue (PVAT) of dietinduced obese rats, and the loss of PVAT function is endothelium-independent and is
rescued by superoxide dismutase and catalase. R Aghamohammadzadeh*, AS Greenstein,
A Mastan, AM Heagerty. International Symposium on Resistance Arteries, Skørping,
Denmark May 2011- Winner of best poster award
11. Damage to the anticontractile capacity of perivascular adipose tissue strongly correlates
with elevation of blood pressure: Results from an environmental model of obesity, R
Aghamohammadzadeh*, PH Park, AS Greenstein, AM Heagerty. International Society of
Hypertension, Vancouver Sep 2010 (poster) Selected as highest ranking abstract
12. Alterations in subcutaneous small artery wall structure correlate with diastolic parameters
in patients with metabolic syndrome. K Khavandi*, M Luckie, R Aghamohammadzadeh,
14
A Khavandi, A Mastan, A Greenstein, R Malik, R Khattar, AM Heagerty. American
College of Cardiology April 2011 (Poster presentation)
13. Protection from development of obesity in high fat diet rats is associated with preservation
of the anticontractile function of perivascular adipose tissue. R Aghamohammadzadeh*,
PH Park, AS Greenstein, AM Heagerty. British Cardiovascular Society, Manchester 2010
(Poster presentation)
14. Changes in function and structure of high-density lipoprotein in obese patients compared
with healthy controls. Salam Hama, H Soran*, Y Liu, V Charlton-Menys, R
Aghamohammadzadeh, M France, R Yadav, M Jeziorska, B Ammori, P Durrington.
European Atherosclerosis Society 2011 and Heart UK 2011 (Poster presentation)
15
CHAPTER 1: Introduction
Perivascular adipose tissue
from human systemic and coronary vessels:
The emergence of a new pharmacotherapeutic target
Running title: Signals from PVAT
Aghamohammadzadeh R, Withers S, Lynch F, Greenstein A, Malik R, Heagerty A
Corresponding author:
Professor A. M. Heagerty
Cardiovascular Research Group
Core Technology Facility (3rd Floor)
University of Manchester
46 Grafton Street
Manchester M13 9NT
Email: [email protected]
16
Summary
Fat cells or adipocytes are distributed ubiquitously throughout the body and
are often regarded purely as energy stores.
clear
of
that
these
metabolically
adipocytes
active
are
engine
substances
with
However, recently it has become
rooms
producing
large
numbers
both
endocrine
and
paracrine
actions. White adipocytes surround almost every blood vessel in the human body and are
collectively termed PeriVascular Adipose Tissue (PVAT). It is now well recognised that
PVAT not only provides mechanical support for any blood vessels it invests, but also
secretes vasoactive and metabolically essential cytokines known as adipokines, which
regulate vascular function. The emergence of obesity as a major challenge to our
healthcare systems has contributed to the growing interest in adipocyte dysfunction with a
view to discovering new pharmacotherapeutic agents to help rescue compromised PVAT
function. Very few PVAT studies have been carried out on human tissue. This review will
discuss these and the hypotheses generated from such research, as well as highlight the
most significant and clinically relevant animal studies showing the most pharmacological
promise.
Keywords
Perivascular adipose tissue; PVAT; adipocytes; adipokines; obesity; metabolic syndrome;
adiponectin; leptin; epicardial adipose tissue; coronary vessels
17
Abbreviations
PVAT: perivascular adipose tissue; MetS: metabolic syndrome; BMI: body mass index;
NO: nitric oxide; LNAME: NG-nitro-L-arginine methyl ester; CAD: coronary artery
disease; TNF: tumour necrosis factor; IL-6: interleukin-6; PPAR: peroxisome proliferatoractivated receptor; ROS: reactive oxygen species; ADRF: adventitium derived relaxing
factor; VCAM-1: vascular cell adhesion molecule-1; ICAM-1: intercellular adhesion
molecule-1; MCP-1: monocyte chemotactic protein-1
Introduction: the clinical problem
Obesity, defined as a body mass index (BMI) of greater or equal to 30kg/m2 is a major
problem in acculturated and developing societies. It often co-exists with a number of
other diseases including hypertension, dyslipidaemia and insulin resistance.
Such a
constellation has been labelled the metabolic syndrome (MetS). The international obesity
taskforce (IOTF) estimates that approximately 1billion adults are currently overweight
(BMI 25-29.9 Kg/m²), and of these 475 million are obese (≥30kg/m2) (IOTF, 2010).
The enormity of this epidemic highlights the need for novel approaches to obesity
management and a furthering of our knowledge of the mechanisms responsible for the
consequences of being overweight.
A number of reports has indicated that the distribution of fat around the body determines
not only the obese phenotype but its consequences. For example, intra-abdominal and
visceral fat depots have been linked with an increased cardiometabolic risk and the
mortality associated with obesity (Fox et al., 2007; Gesta et al., 2007). The total amount
of internal fat rises with increasing subcutaneous adipocity, but even individuals classed as
18
thin may have more visceral fat than some obese individuals. In addition increased
gluteofemoral fat mass has been negatively linked to levels of inflammatory cytokines and
positively linked to raised concentrations of adipokines resulting in decreased metabolic
and cardiovascular risk (Manolopoulos et al., 2010).
Our current understanding of this problem focuses on our ancestors and the fact that fat
was the energy store developed in times of plenty which could then be burned during
famine. Therefore genes which predisposed to obesity would confer survival benefits and
such individuals would live long enough to reproduce. Several reviews have suggested
that it is the breakdown of this system that is responsible for the contemporary problems
associated with obesity, where susceptible individuals no longer have periods of famine
and are instead constantly over-eating readily available high energy foods (Diamond,
2003; Neel, 1962). Whilst in hibernating mammals, short-term obesity and insulin
resistance have the beneficial effect of directing glucose to the brain, only man has
developed chronic obesity with its associated cardiovascular morbidity and mortality
(Scott et al., 2006).
Yudkin et al. (2005) originally postulated that perivascular adipose tissue might hold the
key to linking obesity with the development of metabolic syndrome and diabetes as a
result of an adverse influence upon the vasculature (Yudkin et al., 2005). In health, PVAT
could produce adipokines that profoundly influence metabolism and the control of local
vascular tone via vasocrine actions. It was suggested that the loss of such substances
would result in a change in vascular function and development of insulin resistance. These
authors suggested that the effect of circulating insulin on NO-mediated vasodilatation,
19
which is of paramount importance in modulating the postprandial increase in nutritive
flow, could be challenged by the paracrine action of adipokines released from local fat
stores in obesity. They further highlighted the role inflammation may play and suggested
elevated levels of TNF-α in obesity could disrupt the crosstalk between fat and blood
vessels.
In this review we intend to focus on the vasoactive properties of perivascular adipose
tissue as mediated by adipokines.
What are the principle adipokines released from adipocytes?
The recent interest in adipose tissue as a dynamic endocrine organ has resulted in a
number of studies examining the shared, and in cases distinct, properties of different fat
depots. Perivascular adipose tissue (PVAT) surrounds subcutaneous small arteries,
coronary vessels (peri-coronary and epicardial fat), aorta and systemic vessels and secretes
a number of important adipokines (Figure1).
Amongst the adipokines, leptin and adiponectin have been subject of recent reviews
(Kadowaki et al., 2005; Kadowaki et al., 2006; Ren, 2004; Sweeney, 2010). Here, we
review briefly the most significant findings on leptin, adiponectin and adrenomedullin. A
more comprehensive list of adipokines and their roles with regards to the metabolic
syndrome has been published recently (Deng et al.).
20
Leptin
In rats leptin has a direct vasodilator effect acting via an endothelium and nitric oxide
dependent mechanism (Lembo et al., 2000). However, it has an endothelium-independent
vasodilator effect on segments of human subclavian vein and internal mammary arteries
harvested during surgery, thus highlighting the need for careful selection of vascular tissue
in order to design clinically relevant studies (Momin et al., 2006).
The role of leptin as an indirect vasoconstrictor has also been studied. Fruhbeck et al have
shown that leptin has a sympathoexcitatory effect leading to a rise in BP in Wistar rats
(Fruhbeck, 1999). They demonstrated a significant rise in both systolic and diastolic BP
when rats were given an intravenous bolus of the nitric oxide inhibitor NG-nitro-Larginine methyl ester (L-NAME) followed by leptin, demonstrating the role NO plays in
facilitating the vasorelaxant effects of leptin. In the same experiment they showed that
leptin administration, following post ganglionic blockade using chlorisondamine, resulted
in a reduction in BP, which was abolished by NO inhibition.
Weight gain is often associated with increased insulin resistance and resultant
hyperinsulinaemia. Interestingly, Vecchione et al have demonstrated that insulin
potentiates leptin-induced NO release and even at physiological levels, insulin enhances
the vasodilator effects of leptin (Vecchione et al., 2003).
That leptin, a proinflammatory adipokine, possesses vasodilator properties, and that
obesity is associated with leptin resistance, implies its role in obesity is not understood
fully. In MetS leptin levels are increased within the epicardial fat adjacent to the coronary
21
vessels (Payne et al., 2010). In dogs, hyperleptinaemia has been associated with
endothelial dysfunction (Knudson et al., 2005), and work on a swine model of MetS has
shown that leptin exacerbates endothelial dysfunction via a protein kinase C – beta
dependent pathway (Payne et al., 2009).
It is unclear whether elevated levels of leptin in epicardial fat are of benefit to the adjacent
coronary vessels, or whether they may contribute to endothelial dysfunction in human
arteries.
Adiponectin
Adiponectin is a 30kDa protein made up of 244 amino acids. It is the most abundant
adipokine (Dridi et al., 2009; Liu et al., 2010) and exists in two forms, full length, or a
smaller globular fragment.(Dridi et al., 2009; Kadowaki et al., 2005; Kadowaki et al.,
2006). The full length form acts via the R2 receptor and the globular form via R1
(Yamauchi et al., 2003a). In human adipocytes, the expression of Adipo R1 is ~15 fold
higher than that of Adipo R2 (Rasmussen et al., 2006). Circulating adiponectin levels are
reduced in obesity (Sowers, 2008), diabetes (Kadowaki et al., 2006), and there is a down
regulation of adiponectin receptors in the adipose tissue of obese individuals (Rasmussen
et al., 2006). Given its anti-diabetic (Berg et al., 2001; Davis et al., 2008; Maeda et al.,
2002; Pajvani et al., 2003; Yamauchi et al., 2003b; Yamauchi et al., 2001), antiatherosclerotic (Han et al., 2009; Yamauchi et al., 2003b) and vasodilator (Fesus et al.,
2007; Greenstein et al., 2009) properties, adiponectin is believed to be the link between
obesity and metabolic syndrome. Its mechanism of action has yet to be determined fully.
22
In the liver, full-length adiponectin activates adenosine monophosphate-activated protein
kinase (AMPK) and the trimeric form is known to activate AMPK in adipose tissue and
muscle. Activation of AMPK leads to phosphorylation of Acetyl-Coenzyme-A
Carboxylase (ACC) which results in fatty acid ß-oxidation and inhibition of triacylglycerol
and fatty acid synthesis (Liu et al., 2010); (Kadowaki et al., 2005).
Deng et al. have demonstrated that adiponectin improves endothelial dysfunction by
increasing nitric oxide (NO) production via phosphorylation of endothelial NO in the aorta
of high-fat-fed obese Sprague-Dawley rats (Deng et al., 2010). Moreover, in adiponectin
receptor-KO mice, there is a significant attenuation of endothelium-dependent
vasodilatation in response to ACh (Ouchi et al., 2003). Of relevance, adiponectin
suppresses both basal and oxidised-LDL induced superoxide generation in bovine
endothelial cells (Motoshima et al., 2004), and also suppresses excess ROS production
under high-glucose conditions via a cAMP/PKA-dependent pathway (Ouedraogo et al.,
2006). Given the elevated levels of oxidised-LDL (Holvoet et al., 2008) and plasma
glucose in patients with metabolic syndrome, the concomitant reduction in adiponectin
levels may explain partly the endothelial dysfunction observed by our group (Greenstein et
al., 2009) in the cohort of patients with MetS.
Incubation of healthy human vessels with a blocking peptide for the adiponectin R1
receptor almost abolished completely the anticontractile effect of PVAT in response to
cumulative noradrenaline doses (Greenstein et al., 2009). Our unpublished proteomic
analysis of adipose tissue from obese patients shows a significant reduction in adiponectin
levels as compared with lean individuals. Therefore we suspect that the absence of the
23
anticontractile function of PVAT in obesity is accounted for by a reduction in adiponectin
levels in PVAT at least in part. It has been suggested that the high levels of adiponectin in
some disease states may be a compensatory response to the development of ‘adiponectin
resistance’ (i.e. a dysfunction in the adiponectin signalling pathway) (Sam et al., 2009).
Clearly, further research is required to describe the exact mechanisms of action of
adiponectin in health and to explain how these pathways become affected in disease.
Adrenomedullin
Amongst the adipokines, adrenomedullin (AM) has received the least attention in recently
published literature. It is a 52 amino acid peptide first isolated from a sample of human
phaeochromocytoma, but later shown to be synthesised by adrenal, heart, and vascular
endothelial and smooth muscle cells as well as white adipocytes of both rodents and
humans (Fukai et al., 2005; Silaghi et al., 2007). Its dose-dependent vasodilator effect on
the rat mesenteric vessels was first reported in 1993 (Nuki et al., 1993). Its direct and
potent vasodilatory action on blood vessels would suggest that it plays an important role in
the control of vessel tone. Human studies have shown that intravenous infusion of the
peptide leads to significant vasodilatation of pulmonary vessels providing a potential
therapeutic strategy for pulmonary hypertension (Nagaya et al., 2003; Nagaya et al.,
2000). Also there is evidence of AM-induced human coronary (Terata et al., 2000) and
skeletal artery (Nakamura et al., 1997) vasodilatation via a nitric oxide dependent
pathway.
24
The exact role of adrenomedullin in obesity needs further evaluation, but we know that
AM reduces levels of reactive oxygen species (ROS) in vascular smooth muscle cells
(Yoshimoto et al., 2005), and AM knockout mice express higher levels of ROS
(Shimosawa et al., 2003).
Catecholamine stimulation of beta3 adrenoceptors on adipocytes results in lipolysis and
release of stored adipokines (Robidoux et al., 2006). Adrenomedullin inhibits lipolysis via
a nitric oxide dependent mechanism (Harmancey et al., 2005) which in theory, would
affect vessel tone indirectly by blocking the release of vasoactive adipokines such as
adiponectin (Figure 2). Gettys et al have reported that stimulation of the beta3-adrenergic
receptor on white rat adipocytes by the selective agonist CL316,243 leads to the inhibition
of the release of leptin (Gettys et al., 1996). The balance between the potential lipolysisinduced release of adiponectin and the inhibition of leptin release and their relevance to
vascular tone needs further clarification.
Epicardial adipose tissue from patients with CAD displays higher levels of adrenomedullin
than those without CAD. Given its vasorelaxant properties, one would assume this may
serve as a protective mechanism for the diseased coronary vessels (Shibasaki et al., 2010).
Plasma AM concentrations are also elevated in disease states, which may provide further
protection against oxidative stress and vasoconstrictors. Despite its antioxidant and
vasorelaxant properties, it is possible that elevated AM levels in disease states may
actually contribute to the vascular dysfunction.
25
PVAT and control of local vascular tone
Perivascular adipose tissue function has been assessed in canine, swine and rodent models
and demonstrated different functional and structural properties of PVAT which vary both
between species and anatomical site. Examples of structural differences include the fact
that PVAT from rat aorta comprises smaller adipocytes compared with mesenteric vessels
(Galvez-Prieto et al., 2008). Whilst the murine thoracic aorta is surrounded by brown
adipocytes, peri-abdominal fat is comprised of white adipocytes (Police et al., 2009).
From a functional perspective, coronary artery PVAT in healthy dogs attenuates
acetylcholine induced relaxation (marker of endothelial function) (Payne et al., 2009;
Payne et al., 2008), but does not affect bradykinin-mediated dilatation in healthy pig
arteries (Payne et al., 2010).
Three studies have shown that healthy human PVAT exerts an anticontractile effect on
adjacent vessels. Rodent (both mouse and rat) mesenteric and aortic vascular beds have
been the most frequently studied models of PVAT function. Data from these models have
matched closely the data obtained from limited human studies.
In 1991, Soltis and Cassis were the first to report that vessels with intact PVAT were less
responsive to noradrenaline than naked vessels (Soltis et al., 1991). Later studies used
solution transfer protocols to demonstrate the existence of a transferrable adventitiumderived relaxing factor (ADRF) (Gao et al., 2005b; Greenstein et al., 2009; Lohn et al.,
2002; Malinowski et al., 2008). The solution transfer experiments involved using a small
volume of solution from a tissue bath with PVAT, adding it to a pre-constricted vessel and
26
measuring the vascular response. These suggest that the observed anticontractile property
of PVAT is not merely a consequence of it acting as an obstacle to diffusion for
vasoconstrictors, but as a dynamic tissue which secretes adipokines with anticontractile
properties.
There are likely to be a number of substances which account for the anticontractile effects
of PVAT. Indeed, both endothelium dependent and endothelium independent mechanisms
have been demonstrated. Protocols using rat mesenteric vessels have shown that both
PVAT and exogenous adiponectin exert anticontractile effects on preconstricted small
arteries (Greenstein et al., 2009); the effects of PVAT have been corroborated by
experiments on rat aorta and its surrounding PVAT, which mostly consists of brown
adipocytes (Gao et al., 2007; Lohn et al., 2002). Gao has shown that the endothelium
independent mechanism involves hydrogen peroxide (H2O2) and subsequent activation of
soluble guanylyl cyclase. At low concentrations (10-100µM) of H2O2, mesenteric vessels
preconstricted with phenylephrine undergo further constriction, but higher concentrations
of H2O2 (0.3-1mM) result in a biphasic response, with an early constriction followed by
relaxation. Hydrogen peroxide released from both adipocytes and macrophages can act via
the raf/MEK/ERK pathway and influence the phosphorylation of contractile apparatus in
vascular smooth myocytes (Figure 2).
Interestingly, exposure of rat mesenteric arteries to electric field stimulation (EFS) leads to
a contractile response via stimulation of α1-adrenoceptors by the perivascular sympathetic
nerves. PVAT enhances this contractile response by stimulating superoxide generation and
activation of tyrosine kinase and MAPK/ERK pathway (Gao et al., 2006) (Figure 2). More
27
recently it has been shown that Angiotensin II derived from adipocytes potentiates the
contractile response to EFS (Lu et al.), thereby highlighting the role of the reninangiotensin system within PVAT with regards to local vascular tone modulation.
Physiological release and action of ADRF: Possible role of the potassium channel
The exact mechanisms by which adipocytes exert their effects on adjacent arteries are not
well understood. Figure 2 shows a number of the potential mechanisms involved.
Downstream in the PVAT anticontractile pathway are vascular potassium (K) channels,
which occupy a central position in the maintenance and regulation of vascular tone. Early
experiments on potassium channels have identified roles for a number of K channels. In
rat mesentery the anticontractile effect is attenuated by blockade of the delayed rectifier K
channel (KV) (Verlohren et al., 2004). Studies of rat aorta suggest roles for adenosine
triphosphate (ATP)-sensitive K channels (KATP) (Lohn et al., 2002) as well as small and
intermediate conductance calcium-sensitive potassium channels (SKCa and IKCa) (Gao et
al., 2007). In human internal mammary artery, the relaxing factor appears to work via
large conductance calcium-sensitive K channels (BKCa) (Gao et al., 2005b).
More recent experiments have highlighted the pivotal role of the BKCa channel in
mediating the PVAT effect. Work from our group supports the role of the BKCa channel in
the vasodilator function of PVAT. Pharmacological inhibition of the channel using
paxilline or using mesenteric arteries from BKCa knockout mice (Slo-/- ,
-/-
) results in
a significant impairment of the response (Lynch et al., 2010b). This highlights the role of
BKCa channels in facilitating the ADRF effect, but further work is needed to clarify
28
whether BKCa channels are also present on adipocytes as well as the vascular myocytes.
Additionally the removal of endothelium or inhibition of NOS can significantly attenuate
the response in these mouse models (Lynch et al., 2010a).
Elegant micro-electrode studies of de-endothelialised rat mesenteric arteries have shown
that in non-constricted vessels the hyperpolarisation to exogenous adiponectin is inhibited
by selective blockade of BKCa (Egner et al., 2010). This group has reported also that
stimulation of ß3 adrenoceptors releases a factor which indirectly activates myocyte BK Ca
channels. They have suggested that this factor is adiponectin working via myocyte
adipoR1 receptors to activate AMPK. These protocols were performed in the absence of
endothelium. However, there are BKCa channels present on the endothelium (Hughes et
al., 2010) and stimulation of these channels by circulating ADRF could result in
hyperpolarisation of endothelial cells and subsequent hyperpolarisation of vascular
myocytes via the myoendothelial junction, thus leading to their relaxation (Figure 2).
Obesity is a state of adrenergic overdrive with increased circulating noradrenaline levels
(Prezio et al., 1964) and an overactive sympathetic nervous system (Lambert et al.)
releasing noradrenaline at nerve terminals which are present in PVAT. Noradrenaline can
bind to ß3 adrenoceptors, although with lower affinity than to ß1 and ß2 adrenoceptors. In
obesity one would expect that, overstimulation of the ß3 adrenoceptor by noradrenaline
would result in an increase in adiponectin release from adipocytes thus lowering vessel
tone and enhancing metabolic homeostasis. However in obesity, there are reduced blood
and adipocyte adiponectin levels (Asayama et al., 2003; Kern et al., 2003) as well as
downregulation of adiponectin receptors (Kadowaki et al., 2005). Further research is
29
required to explain the reduced levels of adiponectin and its receptors. There may be an
increase in breakdown of adiponectin secondary to oxidative stress and inflammation or a
reduction in its production due to obesity-induced damage to the intracellular mechanisms
involved in production of the protein. We shall discuss further evidence for inflammationinduced PVAT damage later in this review.
PVAT and human vessels
Harvesting human arteries for PVAT studies is fraught with difficulty, which may explain
the limited number of published studies. The most accessible human blood vessel is the
internal thoracic artery, obtained during coronary artery bypass graft operations (Gao et
al., 2005b; Malinowski et al., 2008) and small arteries from gluteal fat biopsies
(Greenstein et al., 2009). Both are clinically relevant vascular beds. However, the
atraumatic harvest of saphenous vein grafts, via the ‘no-touch’ technique has been shown
to result in vessels with intact PVAT and immunohistochemical evidence of the potent
vasodilator nitric oxide (Dashwood et al., 2007; Dashwood et al., 2009). Venous PVAT
has been shown to exert an anticontractile effect on adjacent tissue by releasing Ang-(1-7)
which activates Kv channels and relaxes vascular myocetes through eNOS release (Lu et
al., 2011) .
Gao et al first studied the anticontractile properties of human PVAT using segments of
human internal thoracic artery. It was shown that human PVAT exerted an anticontractile
effect on its adjacent vessel upon exposure to contractile agents including U46619, which
is a thromboxane A2/prostaglandin H2 receptor antagonist (Gao et al., 2005b). This is a
30
significant finding because thromboxane A2 and its stable metabolite thromboxane B2
(TxB2) are known to be potent vasoconstrictors and the levels of TxB 2 are known to be
increased during cardiopulmonary bypass (Davies et al., 1980). Perioperative spasm of the
internal thoracic artery is a commonly encountered issue (He et al., 1994), therefore
leaving the perivascular fat intact may help counteract the vasoconstricting effects of the
increased plasma thromboxane levels. In this study, the anticontractile effect of PVAT was
shown to be due to a transferrable relaxing factor (adipose derived relaxing factor or
ADRF) that activated BKCa channels.
Malinowski et al also used segments of human internal thoracic artery and investigated
whether it is perivascular adipose tissue per se, or adipose tissue in general, that is capable
of exerting an anticontractile effect (Malinowski et al., 2008). Following a coronary artery
bypass operation, the in situ graft (taken from the internal thoracic artery) heals in close
proximity to pleural tissue. Dose responses to serotonin were constructed for skeletonised
vessel segments with and without incubation with pleural fat. The study showed that,
despite the presence of white adipocytes in pleural fat, there was no anticontractile effect.
The study also demonstrated clearly that adipokines released by PVAT are able to affect
the blood vessel even if the fat tissue has been loosely added to the tissue bath and is not in
direct contact with the artery.
In order to assess whether ADRF acts via the endothelium, Malinowski et al. incubated
their vessels with inhibitors of nitric oxide synthase and cyclo-oxygenase (COX). It is well
known that nitric oxide and prostacyclin produced in the endothelium have vasodilatory
effects. This study showed that inhibition of NO and COX did not abolish the
31
anticontractile effect of PVAT. This does not mean that PVAT function is endotheliumindependent; rather that it can exert its effect independently of the NO and COX pathways.
The most recent study of human PVAT has been carried out on small arteries harvested
from adipose tissue samples obtained by gluteal biopsies in patients with metabolic
syndrome (MetS). Figure 3A demonstrates the anticontractile function of PVAT in healthy
individuals whilst figure 3B shows that PVAT did not exhibit an anticontractile function in
the group with MetS (Greenstein et al., 2009). Incubating healthy PVAT intact human
vessels with a fragment of the human type 1 adiponectin receptor completely abolished
PVAT anticontractile function, thus identifying adiponectin as an adipose-derived relaxing
factor (ADRF) in human PVAT. Also the presence of nitric oxide in human PVAT was
demonstrated by incubating PVAT intact healthy vessels with L-NMMA which resulted in
attenuation of the anticontractile property of PVAT. This study will be discussed further in
the next section.
PVAT, obesity and inflammation
The effect of obesity on PVAT function was first reported in 2005. It was shown that,
prenatal exposure of rats to nicotine caused postnatal obesity and an increased amount of
PVAT. However, the rise in PVAT volume which was associated with weight gain
resulted in a reduction in the anticontractile function of PVAT surrounding the aorta. It
was thought that this may be due to a change in the nature of PVAT or a reduction in
secretion of relaxation factor(s) from PVAT(Gao et al., 2005a).
32
Often obesity is associated with hyperglycaemia and hyperinsulinaemia in the context of
the metabolic syndrome. The role of circulating insulin on vascular tone has been well
documented (Eringa et al., 2004), but its effect on PVAT mediated vascular responses has
not been established. However, both acute and chronic hyperglycaemia enhance the PVAT
anticontractile effect (Lee et al., 2009b). This highlights the complex nature of the
metabolic syndrome and the difficulty of controlling for variables in ex-vivo experiments.
Adipocytes undergo significant hypertrophy in obesity. The cross-sectional area of
adipocytes in obese individuals with metabolic syndrome is up to 1000 μm2 larger than
that of healthy individuals (Greenstein et al., 2009). Given that the diffusion limit of
oxygen is thought to be around 100μm (Hosogai et al., 2007), the hypertrophied
adipocytes are likely to be subjected to a decreased oxygen tension. In obese individuals,
there is no increase in blood supply to match the increase in adipocyte size, and the
postprandial increase of blood flow that occurs in lean subjects is also absent in obesity
(Bulow et al., 1987; Coppack et al., 1992). This implies that hypertrophied adipocytes
exist in a state of relative hypoxia. This has been confirmed by pimonidazole staining (a 2nitroimidazole which is activated at low oxygen concentrations) of adipocytes from obese
mice (Hosogai et al., 2007), as well as measurements of partial pressures of oxygen in
abdominal subcutaneous adipose tissue of obese humans (Pasarica et al., 2009). In the
context of hypoxia, vasa vasorum in the adipose tissue surrounding vessels play an
important role in providing oxygen to the hypoxic PVAT. Recent work has shown that
PVAT induces the formation of vasa vasorum (Manka et al. 2010) which would be
beneficial in obesity.
33
Hypoxia reduces PVAT anticontractile effects in rat mesenteric arteries (Greenstein et al.,
2009), however in aorta the opposite occurs, namely an enhancement of PVAT function
(Maenhaut et al., 2010). Clearly, these regional differences merit further evaluation.
From a therapeutic perspective, experimental hypoxia-induced damage to PVAT function
can be rescued using free radical scavengers (Greenstein et al., 2009) and Eplerenone
(Withers et al., 2011). Future clinical trials can explore the possibility of using such drugs
in the treatment of patients with obesity.
The chronic low-grade inflammatory state which develops as a consequence of hypoxia in
obesity is marked by an increase in blood levels of reactive oxygen species and proinflammatory cytokines such as CRP, IL-6 and TNF-α (Trayhurn et al., 2008). Incubation
with TNF reduces the anticontractile effect of PVAT in vessels harvested from healthy
individuals, suggesting that the higher levels of the inflammatory cytokine in obesity may
partly account for the reduced anticontractile function of PVAT. Similar results were
shown in healthy rat tissue where incubation with TNF and IL6 resulted in a reduction of
the anticontractile function of PVAT (Greenstein et al., 2009).
Further human studies have shown a link between high levels of IL6 and increased risk of
CAD (Pai et al., 2004). Epicardial adipose tissue IL6 mRNA levels have been shown to be
higher in CAD than non-CAD patients, with higher levels correlating with greater degrees
of angiographically defined vascular injury (Eiras et al., 2008). The upregulation of
inflammatory cytokines in the adipose tissue suggest a role for oxidative stress as a
mechanism for damage to PVAT function. Proteomic analysis of epicardial adipose tissue
34
and subcutaneous adipose tissue obtained from CAD patients has demonstrated higher
levels of reactive oxygen species (ROS) in epicardial adipose tissue, and mRNA analysis
has revealed lower levels of the antioxidant enzyme catalase (Salgado-Somoza et al.).
The role of the macrophage
Increased macrophage numbers in adipose tissue of obese animals (Rausch et al., 2008)
and humans (Weisberg et al., 2003) also support the hypoxia theory, as hypoxic cells
secrete chemokines to attract macrophages (Pasarica et al., 2009). Further data from our
group suggest that when PVAT from mouse vessels is rendered hypoxic, the presence and
activation of macrophages is the key modulator of increased vascular contractility.
Moreover, following the conditional ablation of macrophages, hypoxic insult has no effect
on contractility of the vessels (Figure 4), further implicating a role for macrophages in
mediating the response to inflammatory stimuli (Withers et al., 2011).
The levels of monocyte chemotactic protein-1 (MCP-1) are raised in plasma and adipose
tissue of both genetically obese and diet-induced obese mice. In MCP-1 KO diet-induced
obese mice, there is a significant reduction in numbers of macrophages in the adipose
tissue as well as improved insulin sensitivity (Kanda et al., 2006). Paradoxically however,
while it is likely that activated macrophages in obese patients contribute to PVAT damage,
in health, perivascular macrophages enable structural remodelling of the small artery wall
in response to flow. Bakker et al have shown that in mouse mesenteric arteries,
inactivation of macrophages by clodronate reduces flow-induced remodelling (Bakker et
35
al., 2006) which is observed in arteries from diabetic animals (Belin de Chantemele et al.,
2009).
We propose that reduced levels of adiponectin in obesity enhances macrophage effects on
PVAT function as adiponectin suppresses macrophage phagocytosis and TNF-α
production (Yokota et al., 2000). Moreover, it has been shown that hypoxia and ROS
decrease adiponectin production from 3T3-L1 adipocytes (Chen et al., 2006).
A major process in the development of atherosclerosis is the recruitment of leucocytes to
the endothelium and their subsequent migration into the subendothelial space. VCAM-1
and ICAM-1 are two of the adhesion molecules expressed on vascular endothelial cells
and are responsible for facilitating the recruitment of the white blood cells. In patients with
coronary heart disease and carotid artery atherosclerosis, there are increased levels of
intercellular adhesion molecule-1 (ICAM-1) (Hwang et al., 1997). Resistin is produced by
adipocytes and is also expressed in macrophages which are present in larger numbers in
the PVAT of obese animals and humans. It has been shown that resistin induces the
expression of ICAM-1 and VCAM-1 in human endothelial cells and that adiponectin can
block this effect of resistin (Kawanami et al., 2004). PPARγ agonists have been shown to
reduce the expression of resistin mRNA in macrophages (Patel et al., 2003) whilst
increasing plasma levels of adiponectin (Long et al., 2010). Ouchi et al have also reported
that adiponectin can dose-dependently inhibit the expression of VCAM-1 and ICAM-1 in
human aortic endothelial cells (Ouchi et al., 1999).
36
The RAS within PVAT
The Renin- Angiotensin System (RAS) is an important regulator of blood pressure and
vascular function. Circulating angiotensinogen is cleaved by renin to produce angiotensin
1(Ang I). Angiotensin converting enzyme ACE) converts this to Ang II (Engeli et al.,
1999) which binds to two receptors, AT1 and AT2. Adipocytes secrete products of the
renin-angiotensin system (RAS) and the functional significance of this has been reviewed
in depth (Engeli et al., 2003; Gorzelniak et al., 2002). RAS products such as ACE,
angiotensinogen and AT 1 and AT2 receptors have been identified on human and rodent
adipocytes (Cassis, 2000; Cassis et al., 1988; Crandall et al., 1999; Harp et al., 1995;
Schling et al., 1999). However it is becoming apparent that the adipocyte RAS system
influences tissues beyond its local environment and that it plays a role in the development
of vascular diseases such as obesity and hypertension. Much is known about adipose RAS
however this section will focus on the RAS component of perivascular adipose tissue.
There appears to be differential distribution of components of the RAS system expressed
in both white and brown perivascular adipose tissue (Campbell et al., 1995; Campbell et
al., 1993; Cassis et al., 1988; Engeli et al., 1999; Galvez et al., 2006). Angiotensinogen
and Ang I expression is similar between white and brown tissue (Galvez et al., 2006);
however Ang II expression appears to vary between brown and white perivascular
adipocytes (Engeli et al., 1999; Giacchetti et al., 2002; Jonsson et al., 1994; Schling et al.,
1999). There is conflicting evidence of renin expression in perivascular brown or white
adipose tissue with some groups reporting no detectable levels of the protein and others
reporting its presence (Engeli et al., 1999; Galvez-Prieto et al., 2008; Giacchetti et al.,
2002)
37
There is a growing body of evidence to suggest that elements of adipocyte-derived RAS
play vital roles in the normal and pathogenic responses of blood vessels. Ang1-7 has
recently been identified as an important PVAT diffusible RAS product (Lee et al., 2009a).
It is released by PVAT and acts on the endothelium to stimulate the release of nitric oxide
which in turn hyperpolarises smooth muscle cells through KCa channels. Ang 1-7 is
thought to counterbalance the contractile influence of Ang II (Ferrario et al., 2005;
Reudelhuber, 2006; Yagil et al., 2003).
Increased expression of Ang II and decreased
expression of Ang 1-7 has been reported in hypertensive models. Indeed treatment of
hypertensive models with ACE inhibitors lowers Ang II and increases Ang 1-7 (Yagil et
al., 2003). Ang I antagonism using Losartan restored PVAT induced anticontractility in
fructose fed rats (Huang et al.). Ang II type 1 receptor antagonism using olmesartan can
reduce blood pressure and increase adiponectin levels in a rat model of metabolic
syndrome (Mizukawa et al., 2009). Lu et al have recently shown that PVAT-induced
anticontractility is impaired in spontaneously hypertensive rats and that the anticontractile
response could be inhibited with an Ang 1-7 antagonist in normotensive rats (Lu et al.).
Interestingly Ang 1-7 is also thought to be vital for the PVAT-induced anticontractile
response observed in venous rings(Lu et al.).
Epicardial adipose tissue and its clinical correlates
Given its proximity to the coronary vessels, epicardial and pericoronary fat present an
attractive therapeutic target for coronary artery disease and the treatment of its consequent
symptoms. Epicardial adipose tissue thickness correlates well with waist circumference,
visceral adipose tissue mass, fasting insulin and diastolic blood pressure (Iacobellis et al.,
38
2003a; Iacobellis et al., 2003b) and has been shown to be significantly greater in patients
with MetS than those without (Iacobellis et al., 2008).
In man, Chatterjee et al have shown that PVAT surrounding coronary vessels is made up
of smaller, more irregularly shaped adipocytes which exhibit a reduced differentiation
state as compared with visceral and subcutaneous fat depots. They also report that PVAT
from the coronary vessels secretes lower levels of the anti-inflammatroy cytokine
adiponectin and higher levels of proinflamatory cytokines IL-8 and IL-6 as compared with
subcutaneous and visceral adipocytes (Chatterjee et al., 2009). Moreover, exposure to IL6
has been linked with a reduction in adiponectin production by human adipocytes (Simons
et al., 2007). There is a high level of macrophage infiltration in the epicardial fat tissue of
CAD patients (Baker et al., 2006); indeed there is a greater pro-inflammatory profile of
the epicardial adipose tissue as compared with the subcutaneous adipose tissue of
individuals with CAD (Mazurek et al., 2003). Also it has been shown that peri-coronary
adipose tissue contains higher levels of monocyte chemotactic protein-1 (MCP-1) as
compared with visceral and subcutaneous tissue (Chatterjee et al., 2009).
There are lower adiponectin mRNA levels in the epicardial adipose tissue of patients with
coronary artery disease as compared with samples from those undergoing thoracic
operations for non-CAD related indications (Eiras et al., 2008; Iacobellis et al., 2009;
Iacobellis et al., 2005). Lower levels of adiponectin in epicardial tissue have also been
associated with hypertension (Ohashi et al., 2006; Teijeira-Fernandez et al., 2008) and
increased risk of myocardial infarction (Pischon et al., 2004).
39
Greif et al (Greif et al., 2009) have studied the relationship between epicardial adipose
tissue and coronary atherosclerosis in 286 consecutive patients with an intermediate pretest
likelihood for CAD using dual-source multi-slice CT coronary angiography. Interestingly,
they found no correlation between BMI and coronary atherosclerosis, but those with
atherosclerotic lesions were found to have higher volumes of pericardial adipose tissue
(226+/-92 cm3) than those without lesions (134 +/-56 cm3; P<0.001). Those with larger
volumes of pericardial adipose tissue had lower levels of plasma adiponectin and HDL,
but higher levels of the pro-inflammatory cytokine TNF and highly-sensitive CRP.
A recent comprehensive post-mortem study on 41 human cadavers carried out by
Spiroglou et al. has shown that adiponectin is present in both peri-aortic and peri-coronary
human fat depots and its levels inversely correlate with the degree of atherosclerosis
(Spiroglou et al., 2010). In animal models of vascular injury, adiponectin knock-out mice
exhibit more severe neointimal thickening and increased proliferation of vascular smooth
muscle cells than wild type mice (Matsuda et al., 2002).
Conclusions and perspectives
In this review we have discussed clinically significant PVAT studies and highlighted a
number of potential mechanisms via which PVAT may exert its anticontractile effect in
health and how these can become disordered in obesity (figure 2).
Our current understanding of the intricate nature of interactions between adipocytes,
vascular myocytes and endothelial cells is not sufficient to explain precisely the role
adipokines play and the receptors and signalling pathways involved in facilitating their
40
actions. We can conclude that in response to vasoconstrictor challenges, adipocytes release
adipokines with anticontractile effects on the smooth myocytes of adjacent vessels. The
mechanism of action of the ADRF(s) may involve direct action on potassium channels of
vascular myocytes. Circulating ADRF(s) can also work via potassium channels on the
endothelium and there is also the possibility that the potassium channels present on white
adipocytes may be involved in facilitating the ADRF action. ADRF action on its receptors
can also lead to nitric oxide release from adipocytes and endothelial cells. We have also
discussed the contribution of macrophages and ROS in compromising the anticontractile
effect of PVAT in hypoxia.
It is clear that animal studies have contributed significantly to our current understanding of
the mechanism of action of PVAT. However the studies have been conducted using
different vascular beds in different animal models using different pharmacological
approaches. Also it has become apparent that PVAT property varies amongst species and
vascular beds, and not all animal studies are clinically relevant thus highlighting the
paramount importance of research using human tissue.
Obesity and its associated co-morbidities pose a huge threat to public health and to our
increasingly fragile economies. Clearly, prevention of obesity by effective educational
programmes needs to be coupled with treatments for those currently suffering from the
consequences of the disorder. Currently, the most radical and arguably the only effective
intervention in treatment of obesity is bariatric surgery which is available only to a limited
number of patients worldwide. The recent appreciation of the contribution of perivascular
adipose tissue to metabolic homeostasis and control of local vessel tone, as well as its
41
involvement in disease states such as obesity, hypertension and atherosclerosis has
generated great interest in the possibility of reversing the PVAT damage and rescuing its
pre-morbid properties. This field of research will no doubt continue to provide new
challenges and opportunities in the years to come.
Acknowledgements and funding
Our group is supported by the Manchester Wellcome Trust Clinical Research Facility and
Dr Aghamohammadzadeh is a British Heart Foundation and NIHR Manchester
Biomedical Research Centre Clinical Research Fellow
42
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Figure 1
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Figure 1: PVAT is the source of a number of vasoactive and metabolically
significant adipokines. IL: Interleukin; PAI-1: Plasminogen Activator Inhibitor1; ROS: Reactive Oxygen Species; TNF- α: Tumour Necrosis Factor-α
54
Figure2
55
Figure 2: Potential mechanisms via which perivascular adipocytes, vascular
smooth muscle cells and endothelial cells interact. Dotted lines represent unproven
pathways.
ADRF: adventitium derived relaxing factor, AMPK: AMP-activated protein kinase, Ang
II: angiotensin II, BKca: large conductance calcium-activated potassium channel, H2O2:
hydrogen peroxide, ERK: extracellular signal-regulated kinases, 5-HT: 5hydroxytryptamine (serotonin), GTP: guanosine triphosphate, cG0MP: cyclic guanosine
monophosphate, IP3: inositol triphosphate, IRAG: IP3 receptor associated cGMP kinase
substrate, IKca: intermediate conductance calcium- activated potassium channel, Kv:
voltage-gated potassium channel, KATP: ATP- sensitive potassium channel, L-Arg: LArgine, NA: noradrenaline, NO: nitric oxide, NOS: nitric oxide synthase, O2.-: superoxide
anion, ONOO: peroxynitrite, PCS: prostacyclin pathway, PHE: phenylephrine,
PGH2:prostaglandin H2, PGI2: prostaglandin I2 (prostacyclin), PKG: protein kinase G, R:
receptor, sGC: soluble guanylate cyclise, SKca: small conductance SOD: superoxide
dismutase, SR: sarcoplasmic reticulum, calcium-activated potassium channel, TNF:
tumour necrosis factor, VSMC: vascular smooth muscle cell.
56
Figure 3
A
% constriction of KPSS
250
200
No PVAT
150
PVAT
100
50
0
-9
-8
-7
-6
-5
Log [NA] (M)
% constriction of KPSS
250
B
200
No PVAT
150
PVAT
100
50
0
-9
-8
-7
-6
Log [NA] (M)
57
-5
Figure 3: Effect of obesity and the metabolic syndrome on anticontractile capacity of
PVAT on small arteries from subcutaneous gluteal fat *=p<0.01 (Greenstein et al., 2009)
A: In healthy control participants PVAT exerts a significant anticontractile effect
(p=0.009, Multiple ANOVA) when compared with contractility of arteries without PVAT
(n=10).
B: In patients with obesity and metabolic syndrome, presence of PVAT has no effect on
contractility. (n=10).
58
Figure 4
59
Figure 4: The presence of macrophages is the key modulator of increased vascular
contractility in vessels with hypoxic PVAT (Withers et al., 2011)
A: When PVAT from mouse vessels is rendered hypoxic, there is increased
sensitivity of the vessel to cumulative doses of noradrenaline
B: In CD11b–diphtheria toxin (DT) receptor (DTR) transgenic mice (DT
administration selectively kills monocytes/macrophages), hypoxia has no effect on
vascular contractility
60
Obesity-related hypertension: epidemiology, pathophysiology, treatments and the
contribution of perivascular adipose tissue
Running title: Obesity-related hypertension
Dr Reza Aghamohammadzadeh and Professor Anthony M. Heagerty
Cardiovascular Research Group, University of Manchester, UK
Corresponding author:
Anthony M. Heagerty,
Cardiovascular Research Group,
Core Technology Facility (3rd floor),
University of Manchester,
46 Grafton Street,
Manchester,
M13 9NT
Email: [email protected]
61
Abstract
The advent of the obesity epidemic has highlighted the need to re-assess more closely the
pathophysiology of obesity-related hypertension with the aim of identifying new therapies.
In this article, we review the role of the renin-angiotensin-aldosterone system, sympathetic
nervous system and inflammation in relation to the pathophysiology of this condition. We
also discuss the potential role of the perivascular adipose tissue in the context of obesityrelated hypertension.
Key words
Adiponectin, bariatric surgery, hypertension, inflammation, obesity, oxidative stress,
perivascular adipose tissue, sympathetic nervous system
Key messages
1) Obesity-related hypertension is a major and growing public health concern.
2) The pathophysiology of obesity-related hypertension is complex and involves reninangiotensin-aldosterone system, sympathetic nervous system, oxidative stress and
adipokine dysregulation.
3) Perivascular adipose tissue damage in obesity may be a contributing factor to the
development of hypertension in obesity and rescuing its function may offer therapeutic
potential.
62
Abbreviations
ACE: Angiotensin converting enzyme
ADRF: Adipose derived relaxing factor
AMPK: 5' adenosine monophosphate-activated protein kinase
BMI: Body mass index
BP: Blood pressure
CNS: Central nervous system
DEXA: Dual-energy x-ray absorptiometry
eNOS: Endothelial nitric oxide synthase
G6PD: Glucose-6-phosphate dehydrogenase
IRS: Insulin receptor substrate
MCP-1: Monocyte chemotactic protein-1
MetS: Metabolic syndrome
MR: Mineralocorticoid receptor
NADPH: Nicotinamide adenine dinucleotide phosphate
NO: Nitric oxide
NOS: Nitric oxide synthase
OSA: Obstructive sleep apnoea
PAME: Palmitic acid methyl ester
63
PVAT: Perivascular adipose tissue
RAAS: Renin-angiotensin-aldosterone system
ROS: Reactive oxygen species
SHR: Spontaneously hypertensive rat
SNS: Sympathetic nervous system
WC: Waist circumference
Trends in obesity and hypertension
The obesity epidemic has emerged as a major challenge to our increasingly stressed
healthcare systems. Globally, obesity has more than doubled in the past 30 years. In 2008
there were 1 billion overweight and 500 million obese adults worldwide (1). In England
nearly a quarter and in the US close to a third of all adults has been classified as obese (2,
3). Public health initiatives and education have had an impact on diet in England as less
saturated and trans fats were consumed in 2008/09 compared to 10 years ago, but despite
this, 38% of adults had a raised waist circumference in 2009 as compared to 23% in 1993,
and those with a raised waist circumference were at least twice as likely to have high
blood pressure (2).
Whilst it is widely accepted that obesity and its associated disorders are major risk factors
for cardiovascular events, a recent study has reported that BMI, waist circumference and
waist-to-hip ratio do not improve cardiovascular disease risk prediction substantially when
information regarding other aspects of the medical history such as blood pressure, lipid
profile and a history of diabetes are available (4). However this does not understate the
64
importance of obesity and metabolic syndrome as predictors of future cardiovascular
events. The heterogeneity of body fat distribution has prompted the American Heart
Association to stress the importance of measuring waist circumference (WC) as well as
BMI. In the case of those with disproportionately high WC for a given BMI, assessment of
further cardiometabolic risk factors is encouraged (5). A more streamlined assessment of
body composition is fraught with difficulties as there are no internationally agreed cut-offs
for measurements such as waist circumference for a given BMI, or DEXA and bioelectric
impedance values (5).
Hypertension affects nearly one third of the US population (6), and globally around 40%
of adults had raised BP in 2008 (7). The co-occurrence of obesity and hypertension has
focused minds on understanding the pathophysiology of obesity-related hypertension.
Data from the Framingham Heart Study implicate obesity as a contributory factor in 6070% of essential hypertension (8) and obese individuals have a 3.5 fold increase in the
likelihood of suffering from hypertension (9).
According to a 2011 National Health Service survey in England, high blood pressure was
recorded in 48% of men and 46% of women in the obese group, compared with around
30% of those in the overweight and 15% of those in the normal weight category (2).
In this review, we aim to discuss briefly the pathophysiology of obesity-related
hypertension and highlight the role of perivascular adipose tissue in this context.
What causes hypertension in obesity?
There are many mechanisms via which obesity can lead to hypertension (Figure 1).
Gosmanov et al have reported the acute effects of high fat ingestion by normotensive
65
obese individuals (10). Their work suggested that both bolus oral ingestion and the
intravenous infusion of fat result in a significant rise in systolic blood pressure, attenuated
endothelial function (assessed by flow-mediated dilatation), increased oxidative stress
markers, and activation of the sympathetic nervous system as measured by heart rate
variability.
Clearly, obesity is a chronic disorder with a complex aetiology. Genetic causes of obesity
are rare and the majority of cases encountered in clinics is a consequence of indulgence in
readily available and calorie-rich food which provides a large amount of fat as well as a
significant proportion of the recommended daily allowance of salt. High salt diets
accelerate the development of hypertension in diet-induced obese rats without raising the
ceiling of the systolic blood pressure beyond that observed in diet-induced obese rats fed a
low salt diet (11). This effect may be propagated by an increase in oxidative stress levels
in the vasculature as the same study also demonstrated a significant increase in superoxide
levels within aortic rings of high-fat and high-salt diet fed animals.
There are numerous facilitators of obesity-related hypertension. These include the reninangiotensin-aldosterone system (RAAS), the over-active sympathetic nervous system,
metabolic dysregulation including hyperinsulinaemia, adipokine imbalance, and
potentially PVAT damage, defined as a disturbance in the normal metabolic and
vasoactive function of the adipocytes surrounding blood vessels (Figure 1). There is
currently no direct evidence to suggest that a loss of PVAT anticontractile function leads
to systemic hypertension, but the theory behind such a proposition will be discussed in this
review.
66
The contribution of the renal system is significant and manifold; these include sodium
retention and impaired pressure natriuresis (12). The kidneys contribute to the disease
process only to fall victim to its detrimental effects as end organ damage ensues in the
form of chronic kidney disease, thus leading to a vicious cycle.
There are three major factors that highlight the involvement of the renal system in obesityrelated hypertension: increased activity of the renal sympathetic nervous system (SNS),
activation of the RAAS and physical compression of the kidneys by intrarenal fat
deposition and extracellular matrix modifications (13).
Obesity has a differential effect on local SNS activity. A significant increase in renal
sympathetic activity is observed in obese individuals, but compared with lean
normotensive subjects the obese normotensive individuals exhibit suppression of their
cardiac sympathetic activity, whilst the hypertensive obese individuals show an increased
sympathetic activity in both cardiac and renal nerves (14). The degree of renal sympathetic
stimulation in obesity is similar in both normotensive and hypertensive cohorts, thus
emphasising the significance of other contributory factors such as the suppression of
cardiac sympathetic drive in the normotensive individuals (15). A lower cardiac
sympathetic tone in obesity can be viewed as a protective factor, and it may be it is overactivity that tips the balance in favour of the development of hypertension, but it is not
clear which factors dictate the differential activity of local SNS. Also there is evidence of
central stimulation of the SNS by reactive oxygen species in obesity. There are raised
levels of oxidative stress markers such as the superoxide anion and animal studies suggest
67
that NADPH oxidase-dependent oxidative stress in the brain may be a cause of increased
sympathetic tone leading to hypertension in high fat fed animals (16).
In obesity, the full compliment of the RAAS components is raised. In part this is as a
consequence of the intrinsic RAAS system within the adipocyte and includes ACE,
angiotensin type 1 and type 2 receptors. In addition adipocytes secrete angiotensinogen,
the levels of which are raised in obesity (17), as well as renin; though the source of the
adipocyte renin activity remains controversial (18). Raised plasma aldosterone levels also
seen in obesity correlate with the degree of visceral adiposity and waist: hip ratio (19-21).
The elevated aldosterone levels will not only contribute to increased blood volume by
increasing sodium reabsorption, but also lead to the generation of reactive oxygen species
(ROS) (Figure 2). Aldosterone activates NADPH oxidase thus increasing ROS levels
leading to oxidative posttranslational changes to guanylyl cyclase rendering it NOinsensitive (22). The generated ROS also react with nitric oxide to reduce its
bioavailability by forming molecules such as peroxynitrite, thus contributing to endothelial
dysfunction. There is also evidence of a vicious cycle with ROS stimulating the
mineralocorticoid receptor (MR) (23), thereby theoretically contributing to further
elevations in ROS levels. Aldosterone also decreases endothelial glucose-6-phosphate
dehydrogenase (G6PD) activity. G6PD is a cytosolic enzyme and the main source of
intracellular NADPH which functions to limit ROS activity (24). There are two
aldosterone receptor antagonists in clinical use; Spironolactone is a nonselective
aldosterone receptor antagonist, whereas Eplerenone is a selective aldosterone receptor
antagonist which has a lower degree of cross reactivity with sex-steroid hormones and a
longer half life than Spironolactone (25). Spironolactone leads to increased expression of
G6PD and its activity, as well as elevated NADPH levels leading to diminished ROS
68
generation in aortas of aldosterone-treated mice (24). Eplerenone, a mineralocorticoid
receptor antagonist, attenuates hypertension associated with diet-induced obesity in dogs,
despite only modestly elevated levels of plasma aldosterone. It is not clear to what extent
the blood pressure reduction is a consequence of reductions in blood volume and cardiac
output secondary to reduced sodium reabsorption, or due to a reduction in sympathetic
activity through the direct CNS effect of aldosterone (26, 27).
Sodium reabsorption in the kidneys is a major contributor to disease progression. The
raised Angiotensin II levels in obesity raise blood pressure by direct vasoconstrictor action
and also by increasing sodium reabsorption either via direct action on the kidneys or by
stimulation of aldosterone release. Insulin also acts on renal tubules to increase salt
reabsorption leading to retention of water and blood volume expansion (28, 29). Insulin
resistance in obesity leads to a state of hyperinsulinaemia and there is a suggestion that
renal tubular cells may possess a different insulin receptor substrate (IRS) to that of
adipocytes and skeletal muscle cells (IRS1), thus renal tubular cells may well remain
sensitive to the increased levels of plasma insulin in obesity via IRS1-independent
stimulation (30) .
Another important contributing factor in the pathophysiology of obesity related
hypertension is vessel stiffness. Obesity has long been associated with arterial stiffness
and increased pulse wave velocity independently of age, blood pressure and ethnicity.
However, the association is stronger for waist circumference and visceral adiposity, rather
than global obesity as measured by BMI (31). Obesity is a complex multifaceted disorder
and dysregulation of any number of factors can affect vascular stiffness. Leptin has been
69
linked with impairment of arterial distensibility and its raised levels in obesity may be a
significant contributing factor in arterial stiffness (32). Hyperinsulinaemia is another
contributor to vascular stiffness. In lean individuals, insulin reduces central arterial
stiffness before it exerts its slow vasodilatory effect on peripheral small arteries. In obese
individuals, its effect on arterial stiffness is severely blunted; this attenuation correlates
with the degree of obesity (33).
Inflammation, oxidative stress and monocyte recruitment all play their part in initiating
endothelial dysfunction in obesity. There is also disruption to the fine balance between the
vasoconstrictor action of endothelin-1 and the vasodilator effect of NO in endothelial cells.
In health, insulin activates phosphoinositide 3-kinase leading to increased NO production
secondary to eNOS phosphorylation(29). Postprandial physiological surge in insulin
concentrations leads to dilatation of precapillay arterioles, thus improving blood flow and
delivery of nutrients to tissues; a process known as nutritive flow (34). In obesity, NO
mediated vasorelaxation is impaired, leading to vasoconstriction via unopposed
endothelin-1 action (29, 34). Reduced endothelial nitric oxide bioavailability in obesity is
a significant consequence of the reactions between free radicals and the vasodilator gas.
Reactive oxygen species such as the superoxide anion react with nitric oxide to produce
peroxynitrite and deplete endothelial NO levels (figure 2). The role of nitric oxide in
vessel tone modulation and its fate in inflammatory diseases have been extensively
reviewed by Jin and Loscalzo (35).
There is a close correlation between obesity, Obstructive sleep apnoea (OSA) and
hypertension. OSA has been identified as both a cause and a consequence of a number of
70
the physiological and metabolic sequelae of obesity. A prospective study of 709
individuals with a follow up period of four years has reported a dose-response relationship
between sleep-disordered breathing and hypertension, independent of confounding factors
(36). There is a significant degree of OSA in about 40% of obese individuals and nearly
70% of OSA individuals suffer from obesity (37). Fat deposition around the upper airway
in obesity is thought to be the most significant contributor to the development of OSA in
obesity. Almost half of all hypertensive patients suffer from sleep apnoea and half of all
sleep apnoea patients are hypertensive (19). There are a number of potential mechanisms
linking OSA with hypertension, including endothelial dysfunction, CNS stimulation,
oxidative stress and inflammation (37). It is hypothesised that the most significant causal
factor is the elevated oxidative stress levels initiated by intermittent hypoxia, coupled with
hyperleptinaemia (38) with its direct stimulatory effects on the sympathetic nervous
system. There is also the suggestion that elevated levels of aldosterone exist in OSA and
that this correlates with severity of OSA. Clearly, elevated aldosterone levels would lead
to blood pressure elevations and there is a suggestion that it may even worsen upper
airway resistance by contributing to pharyngeal oedema via fluid retention (39).
PVAT function and damage in relation to obesity-related hypertension
Adipocytes surround almost every blood vessel in the body and secrete a number of
adipokines with metabolic and vasoactive properties. These predominantly white
adipocytes form the perivascular adipose tissue or PVAT (Figure 2).
71
Healthy PVAT exerts an anticontractile effect on adjacent small arteries when subject to
vasoconstrictors (40). The exact mechanism via which PVAT has this effect has been the
subject of many recent publications and yet remains controversial. Experimental protocols
have identified both endothelium- dependent and –independent (41) mechanisms, and a
number of molecules have been implicated which will be briefly discussed here. It is
important to highlight that white and brown adipocytes have slightly different secretion
profiles (42), but the anticontractile property of PVAT has been documented in both
tissues (43, 44).
Adiponectin is the most abundant adipokine with a significant vasorelaxant effect on small
arteries and is able to reverse endothelial dysfunction in diet-induced obese rats via the
AMPK-eNOS pathway (45). Clinical studies have shown its levels to be low in
hypertension and to improve with antihypertensive treatment (46). It has been shown that
adiponectin secreted from murine PVAT modulates the tone of the adjacent vessel by
functioning as an adipose tissue derived relaxant factor (47). Moreover, data from our
group have clearly demonstrated that adiponectin receptor type-1 blockade abolishes
PVAT anticontractile effect on adjacent small arteries from healthy human tissue (48);
clearly demonstrating that adiponectin is also an ADRF in humans. Recently we have
reviewed in detail the properties of this adipokine and its role as an ADRF (49).
Apart from adiponectin, PVAT secretes a number of other molecules with vasorelaxant
properties; these include Angiotensin 1-7 (Ang 1-7), nitric oxide (NO), leptin, hydrogen
sulphide and palmitic acid methyl ester (PAME).
72
Angiotensin 1-7 is secreted from PVAT and exerts its anticontractile effects by
stimulating the release of endothelial NO thus activating calcium dependent potassium
channels (43) in arteries, and voltage dependent potassium channels in veins (50). In
support of this finding, Ang 1-7 receptor antagonists have been shown to attenuate PVAT
anticontractile function (51). Ang 1-7 is also able to function via AT2 and Mas receptors
to decrease the nerve stimulated overflow of noradrenaline(52). This may be of particular
therapeutic interest as sympathetic over-activity contributes to pathophysiology of obesityrelated hypertension. Recently, an oral preparation of Ang 1-7 has been prepared to asses
its cardioprotective effects in infarcted rats (53); the effect of this product on small vessel
tone remains to be assessed.
In health, white adipose tissue (54) and PVAT itself (55) are known to be sources of Nitric
Oxide (NO). Insulin (56) and leptin (57) have been shown to stimulate NO production in
adipocytes and, in theory, the elevated levels of leptin and insulin in obesity should
enhance NO levels in PVAT. In early diet-induced obesity, there is evidence of improved
NO bioavailability in mesenteric PVAT of rats (55), but factors such as elevated
superoxide levels in chronic obesity would lead to a reduction in NO bioavailability via
mechanisms already discussed in this review.
Leptin is secreted from white adipocytes and its plasma levels are increased in obesity. It
acts centrally on the hypothalamus to reduce appetite and also to increase SNS activity
(58), and locally, it has a direct endothelial nitric oxide dependent vasorelaxant effect in
health. It is proposed that leptin plays a major role in pathophysiology of obesity-related
hypertension. Leptin deficient ob/ob mice develop severe obesity, but remain
73
normotensive (59). In the vasculature, leptin stimulates the release of endothelial NO, so
an acute rise in leptin levels does not significantly affect BP despite SNS activation.
However, in obesity, its plasma levels are chronically raised, and endothelial dysfunction
means a reduction in NO bioavailability (60), thus the vasopressor effects of leptin
become more apparent.
Hydrogen sulphide and palmitic acid methyl ester (PAME) are the most recent additions to
the list of potential ADRFs. Hydrogen sulphide is thought to function via KCNQ, and at
least partly contribute to the PVAT anticontractile effect (61); whilst PAME functions via
Kv channels, independent of nitric oxide and endothelium (62) and its release is calciumdependent. PAME is implicated in the pathophysiology of hypertension. There is a
reduction in its release from PVAT of 20 week old SHR as compared with prehypertensive SHR and normotensive Wistar Kyoto Rats, and exogenously applied PAME
has a reduced vasorelaxant effect on de-endothelialised aortic rings of SHR as compared
with its significant vasorelaxant effect on preconstricted vessels from pre-hypertensive
SHR and normotensive rats (62).
There may be more than one substance released from PVAT that satisfies all the criteria
for ADRF. We have shown that adiponectin is the ADRF from human subcutaneous
PVAT (48). The identity of the relaxant factor may vary according to species and site of
the PVAT compartments being studied.
In obesity, the anticontractile function of PVAT is attenuated or lost completely.
74
A number of explanations and theories exist as to the cause of this loss of function.
Amongst the most likely are the effects of oxidative stress and inflammation, as well as
adipokine dysregulation and increased sympathetic nervous system action.
We have shown that incubation of healthy PVAT with TNF-α and IL-6 leads to significant
attenuation of PVAT anticontractile function similar to that observed in the obese
phenotype (48). Macrophages secrete a number of inflammatory cytokines including TNFα, IL-6 and free radicals such as the superoxide anion. Following experimental hypoxia,
which approximates obesity-induced PVAT damage, macrophage recruitment and
activation in adipose tissue is an essential step leading to the loss of PVAT anticontractile
function (63). Interestingly, the PVAT surrounding rat thoracic aorta expresses brown
adipose tissue genes and appears to resist inflammation and macrophage infiltration in
diet-induced obesity (64).
The contribution of aldosterone to obesity-related hypertension has been discussed
previously in this article. It has been shown that aldosterone increases the expression of
TNF-α from macrophages. Moreover, activation of the mineralocorticoid receptor results
in generation of reactive oxygen species (ROS), and blockade of the receptor, using
Eplerenone, leads to a reduction of ROS and increased levels of adiponectin in obese and
diabetic mice (65). The superoxide anions generated by macrophages and MR stimulation
also contribute to PVAT damage. Work by Gao et al has shown that superoxide generated
by NADPH oxidase in response to electric field stimulation enhances the contractile
response of adjacent small arteries (66). It has been shown that Candesartan (angiotensin
II type 1 receptor antagonist) reduces this PVAT-mediated potentiation of EFS-induced
75
contractile response, thus providing another potential explanation for the increased
vascular resistance in obesity where there is both increased sympathetic nerve activity and
increased angiotensin II levels (67).
In view of the significant role macrophages play in PVAT damage, their recruitment from
blood to the perivascular adipose tissue is of paramount importance. Monocyte
chemotactic protein-1 (MCP-1) levels are increased in adipose tissue and plasma of
genetically obese and diet-induced obese mice (68), as well in obese humans (69). Insulin
increases the secretion of MCP-1 in insulin resistant 3T3-L1 adipocytes and in ob/ob mice
(70), thus the hyperinsulinaemic state in obesity leads to macrophage recruitment in PVAT
and subsequent release of cytokines which attenuate its anticontractile function.
Fractalkine (CX3CL1) is a recently identified chemokine secreted from adipocytes that
promotes monocyte adhesion to human adipocytes (71). Its levels are increased in
inflammatory states such as diabetes, HIV and rheumatoid arthritis as well as in obesity
(72). There is also direct evidence for involvement of fractalkine in hypertension. The
expression of CX3CL1 receptor gene in blood leukocytes from patients with arterial
hypertension has been shown to be significantly increased (73). The increased levels of
MCP-1 and fractalkine in obesity can be considered as facilitators for the initiation of the
macrophage-induced loss of PVAT anticontractile function.
Adipose tissue depots have unique inflammatory profiles. PVAT from murine aortic arch
expresses lower levels of adipocyte-associated genes when compared to subcutaneous and
visceral fat and this becomes even more pronounced after 2 weeks of high fat feeding
whilst proinflammatory genes become upregulated (74). Visceral adipose tissue exhibits a
76
more inflammatory profile with a higher macrophage content than subcutaneous fat (75).
This may somewhat explain the stronger link between central obesity and hypertension
than between BMI and raised blood pressure (76).
Treatments
There is no robust guidance with regards to the use of antihypertensive drugs in the
treatment of obesity related hypertension and we will not cover the topic in this review.
An important way to tackle the growing problem of obesity-related hypertension is to treat
obesity using therapies that result in sustained weight loss. Weight reduction in obese
hypertensive patients does indeed lead to an improvement in blood pressure (77).
Diet and exercise are the first line in both prevention and treatment of obesity. Studies
have shown that the combination of intense exercise and moderate caloric restriction can
lead to major reductions in markers of inflammation such as high sensitivity C-reactive
protein as well as improving insulin sensitivity and enhancing adiponectin levels (78).
Exercise and weight loss can also individually help lower BP. A study of 133 overweight
and sedentary individuals with either unmedicated high normal BP or stage 1 to 2
hypertension has reported a reduction of 7 mm Hg in systolic BP of those assigned to
behavioural weight management strategies and a 4 mm Hg reduction in systolic BP of
those who took up aerobic exercise 3 to 4 times per week over the 6 month study period
(79); thus clearly demonstrating that simple lifestyle measures should not be ignored.
77
Apart from antihypertensive treatment and lifestyle modifications, there are a number of
therapeutic interventions that have either been shown to be effective or show promise in
the treatment of obesity-related hypertension.
Bariatric surgery was first performed in 1953 and with the advent of the obesity epidemic
the procedure has become increasingly popular (80). In the US the number of procedures
has risen from 16,200 in 1992 to 220,000 in 2008 (81).
Lifestyle changes including diet and exercise are often the first line in the treatment of
obesity. However, for the morbidly obese, bariatric surgery is able to achieve a
significantly greater degree of sustained weight loss. The remission rate of hypertension is
also significantly higher after bariatric surgery than after lifestyle changes (82). A one year
follow up of 88 hypertensive obese patients who underwent gastric banding has shown
that 59% were normotensive and 33% had reduced BP with a reduction in doses of
medication (83). A recent systematic review of literature has shown that out of 16,867
patients, 49% had hypertension before the operation and 68% of these cases were either
improved or resolved completely after an average follow up period of 34 months (80).
A review of 18 studies looking at bariatric surgery outcomes has shown that blood
adiponectin levels increased by nearly 70% post gastric bypass and by around 36% post
gastric banding procedures. The greatest increase was achieved after losing 35% of the
original body weight. They also demonstrated a strong correlation between percentage
increase in adiponectin levels and percentage decrease in BMI (84). This is not true of
weight loss by liposuction where despite a 10% weight reduction, no improvements in
adiponectin or insulin resistance have been noted (85). This is likely due to the differing
78
qualities of adipose tissue depots with visceral fat exhibiting a more inflammatory profile
as compared with subcutaneous adipose tissue (86).
Bariatric surgery has also been shown to improve the inflammatory profile of obese
individuals (87) . In subcutaneous adipose tissue, the expression of IL-6 and TNFα mRNA
decreases significantly and expression of adiponectin and its receptors increase after
dramatic weight loss post surgery (88). The significant degree of weight loss, together
with improvements in adipokine and inflammatory cytokine profile, as well as resolution
or improvement in diabetes status (89) make this an invaluable procedure in those
suffering from morbid obesity and its sequelae.
The over-activity of the renal SNS has already been discussed in this review. In 1995,
Kassab et al had shown that renal denervation leads to a reduction in sodium retention and
lower blood pressures in dogs fed a high-fat diet (90). More recently, the Symplicity
HTN-2 has shown that catheter-based renal denervation can reduce BP by 32/12 mmHg in
treatment resistant hypertension (91), and has already been performed in the private sector
in England in November 2010 (92).
The oxidative stress-induced damage to the endothelium and PVAT in obesity would
suggest that antioxidants should serve as suitable therapeutic agents to reverse this damage
and possibly lower blood pressure in obesity. Indeed, administration of desmethyltirilazad
(Lazaroid), a potent antioxidant, to SHR rats for three weeks has been shown to
79
significantly ameliorate blood pressure in these animals whilst having no effect in control
animals (93).
We have already described that MR activation by aldosterone results in the generation of
reactive oxygen species. Data from our group have also shown that MR blockade using
Eplerenone is able to reduce macrophage activation and rescue aldosterone-induced and
hypoxia-induced PVAT damage (63).
Well designed and large scale clinical trials are required to assess the potential
antihypertensive actions of traditional antioxidants such as vitamin C or scavengers of
ROS such as superoxide dismutase. It has been recently proposed that prevention of ROS
generation using NADPH oxidase (NOX1 and NOX 2) inhibitors may be a better way of
tackling the oxidative stress problem rather than attempting to scavenge the free radicals
once they have been generated (94).
Vitamin D is known to function as a negative modulator of RAAS by suppressing renin
transcription via a vitamin D receptor (VDR) - mediated mechanism. It has been shown
that VDR-null mice have increased renin and angiotensin II levels and develop
hypertension and cardiac hypertrophy (95), thus vitamin D analogues can potentially serve
as antihypertensive agents in the context of obesity. However, vitamin D is also implicated
in energy expenditure and adipocyte biology. Compared with WT animals, VDR-null
transgenic mice, fed a high fat diet remain lean and exhibit smaller adipocytes and lower
leptin and triglyceride levels, as well as higher energy expenditure and oxygen
consumption leading to lower fat mass (96). These findings are a result of global ablation
of the vitamin D receptor and do not identify the organ system or tissues responsible for
the effects. A recent publication has shown that over-expression of VDR in adipocytes
80
leads to suppression of lipolysis, reduction in fatty acid beta-oxidation and thermogenesis
and overall reduction in energy expenditure leading to obesity (97).
The current evidence suggests that use of vitamin D analogues to treat obesity-related
hypertension would be fraught with difficulties. Vitamin D is a highly fat-soluble
compound and is readily deposited within adipose tissue leading to lower plasma levels of
the vitamin. This leads to attenuation in the negative regulation of renin expression.
Higher levels of vitamin D deposited in adipocytes would also lead to reduced lipolysis
and energy expenditure and contribute to further weight gain (98).
Conclusion
Obesity-related hypertension has a complex aetiology and pathophysiology. Development
of new therapies to treat this disorder requires a better understanding of the physiological
dysfunction and the intricate interplay between obesity and hypertension. Rescuing the
damaged PVAT function in obesity may be a new target for treatment of hypertension
associated with obesity.
Sources of funding
Dr Aghamohammadzadeh is a British Heart Foundation and NIHR Manchester
Biomedical Research Centre clinical research fellow. Work within Professor Heagerty’s
group is supported by Wellcome Trust Clinical Research Facility.
81
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Figure 1
Figure 1: The complex pathophysiology of obesity- related hypertension
OSA: Obstructive sleep apnoea, RAAS: renin-angiotensin-aldosterone system, SNS:
sympathetic nervous system.
89
Figure 2
Figure 2: Perivascular adipose tissue as relevant to vessel tone and adipokines implicated
in obesity-related hypertension
eNOS: endothelial nitric oxide synthase, Kv: voltage gated potassium channel,
CNQ: A member of the Kv family, MR: mineralocorticoid receptor, NO: nitric oxide,
O2.- : superoxide anion, ONOO-: peroxynitrite, PAME: palmitic acid methyl ester,
VSMC: vascular smooth muscle cell
90
CHAPTER 2: METHODS AND MATERIALS
Methods
Introduction
Aim
The overarching aim of this thesis was to use wire myography to study the effects of
perivascular adipose tissue on vessel contractility in obesity.
The major focus was to ascertain how PVAT anticontractile function is altered in obesity,
are the changes endothelium-dependent and can they be reversed following weight loss
surgery. In addition, further protocols using immunohisotchemistry and proteomics
contributed to findings from our functional protocols. These are detailed in the methods
sections of the corresponding papers.
Methods
The chapter contains details of the methods used in this thesis. Tissue collection and wire
myography is described in detail in the first part of the introduction. In the second part, the
clinical assessment and examination procedures for patients are described, and finally,
statistical methods used to analyse data are described in section three.
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Tissue collection
There are two arms to this thesis (animal and human) and they will be discussed in
separate sections.
Animal protocols
The animal studies were carried out on murine mesenteric arteries. I have attended the
Home Office animal handling course and have been trained in schedule 1 sacrifice of
rodents.
The animals were sacrificed according to the aforementioned regulations and a midline
laparotomy incision was used to visualise the intestines and mesenteric bed.
The whole mesenteric bed was removed, placed in a petri dish containing chilled PSS, and
prepared for dissection using micro-dissection tweezers and scissors.
Figure 1: Dissected rat mesentery1
1. Small intestine - covered in a thin layer of visceral peritoneum
2. Cecum (Caecum), 3. Mesentery, 4. Small intestine
92
Human protocols
Study participants were approached at the Bariatric surgery outpatient clinic at Salford
Royal Hospital (Manchester, UK). New patients attending the clinic were invited to take
part in the study. On the day of the clinic I explained the study in detail and those willing
to take part in the study were contacted after a few days (minimum 48 hours in accordance
with our local ethics guidelines) to arrange a visit to the Wellcome Trust Clinical Research
Facility (WTCRF) where further assessments including the gluteal fat biopsy were
performed. The same patients were invited back 6 months following weight loss surgery to
undergo the same set of investigations.
In each case, the patient was prepared, lying on their front, on a bed. The area extending
from their lower back to their upper thighs was exposed. The area between the natal cleft
and the outer aspect of the buttock was identified. The area was cleaned with Betadine and
then a sterile field delineated using drapes. Local anaesthesia was achieved by infiltration
of 2% Lignocaine. An elliptical incision was made measuring 2cm in length and just less
than 1cm in width at its widest point. A sample of skin, subcutaneous tissue and fat was
removed (Figure 2). The wound was closed using mattress sutures and dressed using a
waterproof dressing.
93
Skin
Adipose
tissue
Figure 2: Human fat biopsy sample
The sample was immediately immersed in chilled PSS and processed into 3 sections:
a) small samples of the adipose tissue were snap frozen by immersion in liquid nitrogen
b) a cross-section of tissue was cut vertically (skin down to the deepest adipose tissue),
processed and stored for immunohistochemistry.
c) the remaining part of the biopsy sample was dissected using micro-dissection
equipment to identify arteries for myography (figure 3).
Figure 3: Examples of human arteries and surrounding adipose tissue
94
Isolation of arteries
In the case of the rat mesenteric bed, there is always a vein running alongside the artery
and the first step is the correct identification of the artery using its distinct characteristics
which only become apparent with practice.
In the human samples, there may be arteries without a corresponding vein, but the issue of
the correct identification of vessel is again of paramount importance.
In the humans samples, the sample stored in PSS, was transferred to the laboratory and
pinned out on a dissection dish. Arteries were identified and dissected out under a light
microscope as demonstrated in the diagram below.
Artery for
wire
myography
(250-400μm
in diameter)
Figure 4: Dissection of an artery from a gluteal fat biopsy
95
Wire Myography
Preparation of arteries
Arteries of internal diameter 250-400μm and approximately 6mm in length were identified
by blunt dissection whilst the tissue was placed in ice cold PSS and oxygenated.
Perivascular adipose tissue (PVAT) was removed from a 3mm section using dissection
scissors, whilst PVAT was left intact in the adjacent 3mm segment of artery, thus
harvesting paired vessel rings both with and without PVAT intact. Care was taken to avoid
contact of dissection equipment (scissors and tweezers) with the adventitia or the
endothelium of segments.
Mounting and Normalisation
The arteries were mounted in two separate baths of a multi-chamber myograph (Danish
Myotechnology, DMT) and set up as illustrated by figure 5.
A
B
Figure 5 A: Vessel mounted onto wire myograph with perivascular adipose tissue
removed. B: Vessel mounted onto wire myograph with perivascular adipose tissue
intact.
96
The baths were filled with 6ml of PSS solution and the length of the mounted vessel was
manually measured using eye piece calibration. Baths were oxygenated (95% O 2 and 5%
CO2) via an attachment to the myograph and the bath solution was warmed to 37°C. At
this point, the vessel was left to equilibrate for one hour before a normalisation procedure
was performed. This procedure enables arteries mounted in a wire myograph to be
stretched to such an extent that the transmural stretch approximates to 90mmHg and
therefore reproducing a similar environment to that present in vivo, thereby providing a
standard and clinically relevant degree of tension on each vessel segment.
Figure 6: The technique of wire myography. Taken from Mulvany et al (Physiological
Reviews 1990)2.
Assessment of contractility
Each vessel was stimulated with KPSS three times prior to commencing dose response
studies with Noradrenaline. To do this, all 6ml of the PSS in the myograph bath was
aspirated and replaced with 6ml of KPSS. Once stable contraction was achieved, the KPSS
was replaced with PSS and the vessel left to relax to its baseline tension. In addition to
97
establishing arterial viability, the contraction in response to KPSS established a baseline
force in contraction to standardise dose response contractions across different
preparations. Thus, the data presented for agonist induced vascular contractility in this
thesis is as a percentage of the KPSS constriction in keeping with previously published
literature.
Cumulative dose response curves were constructed using agonist-induced contractions
elicited by the addition of increasing concentrations of Noradrenaline to the myograph
bath (10-8 to 3X10-5M). Successive doses were only added once the previous contraction
had reached a stable tension.
On analysis, all data regarding tension generated, whether by KPSS or agonist induced
were taken from the highest, stable point of contraction. All data are expressed as mean ±
standard deviation (SD).
98
Concentrations used to construct dose response curves:
Stock solution: Noradrenaline (Sigma-Aldrich, Dorset, United Kingdom)
10-2M:
0.0319g in 10ml of PSS
Volume (µL)
Noradrenaline
Final bath
stock solution
concentration
(M)
(M)
10-5
6
10-8
10-5
12
3 x 10-8
10-4
4.2
10-7
10-4
12
3 x 10-7
10-4
12
5 x 10-7
10-3
3
10-6
10-3
6
2 x 10-6
10-3
6
3 x 10-6
10-3
12
5 x 10-6
10-2
3
10-5
10-2
6
2 x 10-5
10-2
6
3 x 10-5
Table 1: Doses used for noradrenaline dose response curves
99
Krebs solution: Physiological Saline Solution (PSS)
g/L
mmol/L
NaCl
6.9
118
KCl
0.25
3.4
MgSo4
0.3
1.2
CaCl2.2H20
0.147
1
NaHCO3
2.1
25
Glucose
1.98
11
KH2PO4
0.163
1.2
Table 2: PSS
Modified Krebs solution: Depolarising (high potassium) kerbs solution
g/L
mmol/L
NaCl
3.588
61.4
KCl
4.473
60
MgSo4
0.3
1.2
CaCl2.2H20
0.147
1
NaHCO3
2.1
25
Glucose
1.98
11
KH2PO4
0.163
1.2
Table 3: KPSS
100
Clinical studies
Ethical considerations
Ethical approval for the study was initially granted in July 2009 (REC reference number
09/H1011/22) with further amendments granted during the study period to accommodate
the changing requirements for specific protocols. Study participants were recruited from
outpatient clinics at Salford Royal Infirmary and all assessments and procedures were
performed at the Manchester Wellcome Trust Clinical Research Facility.
Clinical Procedures
Anthropomorphic measurements
All examinations were performed by nurses at the Wellcome Trust Clinical Research
Facility. These included height, weight, waist and hip circumference, skin fold thickness
(scapula, triceps and biceps).
Blood pressure
Blood pressure was measured after 15 minutes of rest by a semiautomatic machine
(OMRON 75). Three readings were taken with the last of the three recorded as final
pressure.
Bioimpedance
Bioelectrical impedance analysis is a means of measuring body composition by measuring
movement of an electrical current through the body. Fat within the body allows virtually
no electricity to pass through, while electricity passes quite easily through water, much of
which is found in muscles. The degree of difficulty with which electricity passes through a
substance is known as the electrical resistance and thus the percentage of body fat and
101
water can be calculated by measurement of this resistance. For the purposes of the studies
in this thesis, bioimpedance was measured using a Tanita Body Composition Analyser
(Model: BC-418 MA).
Blood tests
Protocols were performed both at the Clinical Research Facility, University of Manchester
and at the specialist laboratories at Manchester Royal Infirmary
Statistical analysis
Data are presented as mean ±SD. The data were assessed for normality in each case and
the appropriate t-test (parametric/non-parametric) was used to assess statistical
significance. In order to assess the difference between vessels with intact PVAT and
vessels without PVAT, a 2-way Analysis of Variance (ANOVA) test was used (doseresponse experiments). The software Graphpad Prism5 (version 5.03) is used to analyse
the data.
References
1. Perkis M. Rat mesentry. Available from: faculty.orangecoastcollege.edu/mperkins/zooreview/rat-abdomen/rat-abdomen5.html
2. Mulvany MJ, Aalkjaer C. Structure and function of small arteries. Physiological Reviews.
Oct 1990;70(4):921-961
102
CHAPTER 3
The Effects of Bariatric Surgery on Human Small Artery Function: Evidence for
Reduction in Perivascular Adipocyte Inflammation, and the Restoration of Normal
Anticontractile Activity Despite Persistent Obesity
Short title: Obesity and Perivascular Adipose Tissue
Reza Aghamohammadzadeh MBChB (Honours) MRCP *†; Adam S. Greenstein MBChB
MRCP PhD*†; Rahul Yadav MBBS MRCP *†, Maria Jeziorska MD PhD *; Salam Hama,
MBChB MSc*†; Fardad Soltani, BSc†; Phil W. Pemberton, MSc†; Basil Ammori MD
FRCS§, Rayaz A. Malik FRCP PhD*†, Handrean Soran MD FRCP *†, Anthony M.
Heagerty, MD FMedSci*†.
*: Cardiovascular Research Group, University of Manchester, UK
†: Manchester Wellcome Trust Clinical Research Facility, UK
‡: Department of Clinical Biochemistry, Manchester Royal Infirmary, UK
§: Salford Royal NHS Foundation Trust, Manchester, UK
This study was funded by two Clinical Research Fellowships awarded to Dr R
Aghamohammadzadeh (British Heart Foundation (FS/10/42/ 28372), and NIHR
Manchester Biomedical Research Centre) and the support of Wellcome Trust Clinical
Research Facility
Address for correspondence:
Dr Reza Aghamohammadzadeh, MB ChB (Honours), MRCP (London)
Cardiovascular Research Group, Core Technology Facility (3rd floor), 46 Grafton Street,
Manchester, M13 9NT, United Kingdom; [email protected]
103
Abstract
Objectives: The aim of this study was to investigate the effects of bariatric surgery on
small artery function and the mechanisms underlying this.
Background: In lean healthy humans, perivascular adipose tissue (PVAT) exerts an
anticontractile effect on adjacent small arteries, but this is lost in obesity-associated
conditions such as the Metabolic Syndrome and Type II Diabetes where there is evidence
of adipocyte inflammation and increased oxidative stress.
Methods: Segments of small subcutaneous artery and perivascular fat were harvested
from severely obese individuals before (n = 20) and 6 months after bariatric surgery (n =
15). Small artery contractile function was examined in-vitro using wire myography and
perivascular adipose tissue (PVAT) morphology was assessed using
immunohistochemistry.
Results: The anticontractile activity of PVAT was lost in obese patients before surgery
when compared with healthy volunteers, and was restored 6 months after bariatric surgery.
In-vitro protocols using superoxide dismutase and catalase rescued PVAT anticontractile
function in tissue from obese individuals prior to surgery. The improvement in
anticontractile function following surgery was accompanied by improvements in insulin
sensitivity, serum glycaemic indices, inflammatory cytokines, adipokine profile, and
systolic blood pressure together with increased PVAT adiponectin and nitric oxide
bioavailability and reduced macrophage infiltration and inflammation. These changes
were observed despite the patients remaining severely obese.
Conclusions: Bariatric surgery and its attendant improvements in weight, blood pressure,
inflammation and metabolism collectively reverse the obesity-induced alteration to PVAT
104
anticontractile function. This reversal is attributable to reductions in local adipose
inflammation and oxidative stress with improved adiponectin and nitric oxide
bioavailability.
Introduction
Obesity has become a global public heath challenge affecting almost half a billion adults 1
and an estimated 40 million children2. Large artery disease is very common in obese
patients , and manifests clinically as myocardial infarction, stroke and hypertension3.
Small artery disease is also common in obesity and contributes to the development of
hypertension and microvascular disease due to changes in peripheral resistance and local
autoregulation4. Historically, the small artery dysfunction in obesity has been attributed to
damage to the endothelium5, most notably to generation and release of nitric oxide 6 .
However, more recently appreciation has grown for an additional mechanism by which
vascular damage occurs in obesity; the function of fat surrounding arteries, or Perivascular
Adipose Tissue (PVAT). PVAT surrounds the majority of blood vessels in the body and in
addition to adipocytes contains inflammatory cells, stem cells, and microvasculature. In
health, PVAT modulates the contractile tone of adjacent small arteries by secreting
vasodilatory molecules. These adipose derived vasodilators act independently of the
endothelium and include adiponectin7, nitric oxide7 , hydrogen sulphide8 and palmitic
acid methyl ester9. In 2009, we performed the first human small artery study of PVAT and
showed that subcutaneous gluteal PVAT from lean healthy individuals reduced adrenergic
constriction in adjacent arteries, an ‘anticontractile’ effect. However, in patients with
metabolic syndrome the vasodilatory effect of the PVAT was entirely lost, due to dual
processes of adipose tissue hypoxia and inflammation, both of which are established
105
sequelae of obesity in fat depots7. More recently, we have seen that macrophage
activation in adipose tissue contributes to the attenuation in PVAT anticontractile effect 10.
Bariatric surgery has been performed for nearly 60 years and is established as the most
effective clinical intervention to achieve both significant and sustained weight-loss in
severely obese individuals. There are three types of bariatric surgical procedures:
restrictive, malabsorptive and combined operations 11. Gastric bypass surgery is a
combination of restriction and malabsorption and has been shown to achieve a
significantly higher degree of weight loss than restrictive bariatric surgery12.
Bariatric surgery also dramatically improves cardiovascular risk profiles in obese patients
and reduces overall mortality13, 14. The mechanisms which underlie these cardiovascular
improvements remain unclear, however. In the present study we investigated the effects
of bariatric surgery achieved by the gastric bypass method on the vasodilatory properties
of PVAT. We report amelioration of inflammation in PVAT with complete restoration of
anticontractile activity as a consequence of improved adiponectin and nitric oxide
bioavailability, despite persisting obesity.
106
Methods:
Study population
Patients with severe obesity (BMI > 35) who were awaiting gastric bypass surgery (n=15)
and lean healthy volunteers (n=7) were recruited following full informed written consent
in accord with Local Research Ethics Committee approval. All participants provided a
fasting venous blood sample for the measurement of inflammatory markers and
adipokines.
The study participants also provided gluteal subcutaneous fat samples (1.5 x 1.5 x 1.5 cm)
by undergoing a surgical biopsy under local anaesthesia 7. The sample was immediately
processed in three sections. One part was stored for immunohistology, the second was
snap frozen for estimation of NO levels, and the remainder was used to harvest small
subcutaneous arteries using micro-dissection.
Blood pressure was recorded as a mean of three recordings measured using a
semiautomated machine (OMRON 705CP, White Medical, Clifton-Upon-Dunsmore,
United Kingdom) whilst the participants were seated at rest for 15 minutes.
Further measurements including body mass index (BMI) and waist circumference were
also recorded.
The obese patients were invited to return for a follow-up assessment, including a biopsy,
six months after bariatric surgery.
107
Biochemical analyses
High-sensitivity CRP (hs-CRP) was measured in serum by an in-house, antibody sandwich
ELISA technique using anti-human CRP antibodies, calibrators and controls from Abcam
(Cambridge, UK). Il-6, TNFα, adiponectin, leptin and resistin were measured in serum,
and E-selectin in plasma, all using DuoSet® ELISA development kits from R&D Systems
(Abingdon, UK).
Wire Myography
One section of the gluteal fat biopsy was placed in chilled physiological saline solution
(PSS, composition in mmol/L: NaCl 118, KCl 3.4, MgSO4 1.2, CaCl2 1, NaHCO3 25,
Glucose 11, KH2PO4 1.2) and oxygenated. The dissection dish was placed on ice during
microdissection to preserve the integrity of the tissue whilst arterial segments 250-350 μm
in diameter were harvested, one segment with PVAT intact and the adjacent segment
devoid of PVAT. Both segments came from the same artery.
Endothelium denuded vessels were mounted on 40 μm wires and studied using wire
myography (Danish MyoTech, Aarhus, Denmark).
The mounted vessel segments were oxygenated and maintained at a temperature of 37°C
before vessel diameter and wall tension was normalised as previously described 7, 15.
Vessels were challenged with a 60mM KPSS solution to establish viability and baseline
constriction.
108
Each vessel segment was stimulated with cumulative doses of norepinephrine (SigmaAldrich, Dorset, UK) at the following doses: 10-9, 10-8, 3 X 10-8, 10-7, 3 X 10-7, 5 X 10-7,
10-6, 2 X 10-6, 3 X 10-6, 5 X 10-6, 10-5, 2 X 10-5, 3 X 10-5 mol/L. Contractile responses to
norepinephrine are presented as a percentage of KPSS constriction, consistent with
published literature 7, 16-18.
Pharmacological Assessment
Pharmacological protocols were applied to study the effect of PVAT on adjacent small
arteries. In each case two segments of the same artery were prepared with and without
PVAT attached as previously described7.
The role of oxidative stress was evaluated by incubation of samples from pre-operative
patients with superoxide dismutase and catalase (superoxide dismutase, Sigma-Aldrich,
100u/ml incubation period 45 minutes and Catalase, Sigma-Aldrich, 100u/ml incubation
period 45 minutes; n=4).
Further protocols assessed the contribution of nitric oxide and adiponectin to PVAT
function in samples taken from patients after weight loss. Arteries with PVAT were
incubated with blocking peptide for adiponectin receptor 1 (5μg/ml, 1.6 X 10-4 mol/L,
Enzo Life Sciences; 45 minutes; n=7) and L-NMMA (5 X 10-5 mol/L, Sigma; 45 minutes;
n=4).
109
Nitric oxide assay
Five paired pre-operative and post-operative frozen PVAT samples were homogenised by
suspending the tissue in 400μl of lysis buffer (50 mmol/l Tris base, 150 mmol/l NaCl, 2
mmol/l EDTA, 2 mmol/l EGTA, 40 mmol/l β-glycerophosphate, 50 mmol/l NaF, 10
mmol/l sodium pyrophosphate, 10% glycerol, 1% Triton X-100 (pH 7.4)) and protease
inhibitor (Complete Mini EDTA-Free; Roche Diagnostics) on ice, and utilizing a Dounce
(glass/glass) tissue grinder set (Sigma-Aldrich, Dorset, UK). Upon complete
homogenisation, the solution was incubated at 4°C for 10 minutes then centrifuged for 10
minutes at 16,000g. The resulting supernatant was then removed for application of the
assay.
Total nitrate/nitrite concentration was measured with a nitric oxide colorimetric assay kit
according to the manufacturer’s instructions (Abcam, Cambridge, UK).
Immunohistochemistry
Macrophage markers and TNF-α.
Immediately after dissection, tissue from each gluteal biopsy consisting of skin and
subcutaneous fat was placed in 4% paraformaldehyde in phosphate buffered saline for 24
hours and subsequently processed to paraffin wax blocks. Consecutive 5μm sections were
de-waxed, rehydrated and immunostained for two macrophage markers: CD68 [KP1],
(Dako, Glostrup, Denmark; dilution 1:100), and CD68 [PGM1] (Biocare Medical,
Concord, CA; dilution 1:100) and the pro-inflammatory cytokine TNF-α (Abcam,
Cambridge, UK; dilution 1: 100). Antigen retrieval was performed followed by blocking
of endogenous peroxidase and nonspecific protein binding with Dako blocking solutions.
110
Tissue sections were incubated with primary antibodies for 18 hours followed by antirabbit/anti-mouse EnVision-HRP (Dako) and finally by Vector SG chromogen kit (Vector
Laboratories, Burlingame, CA), used to disclose the presence of the macrophages and
TNF-α. Colour images were captured with a Go-3 QImaging camera (QImaging Corp.,
Vancouver, Canada) mounted on a Leitz Diaplan microscope. Cells stained with
macrophage markers present in adipose tissue were counted and the results expressed as
cells/mm2. For assessment of TNF-α quantitative analysis of immunostaining was
obtained converting colour images to greyscale and using a macro subroutine in an
ImagePro version 6.2 image analysis programme (MediaCybernetics UK, Marlow, UK).
The extent of staining was expressed as a percentage of the entire area photographed.
Adipocyte size was quantified on microphotographs obtained on Zeiss Axio Imager M2
microscope equipped with AxioCam camera and AxioVision Rel. 4.8 programme by freehand tracing the margins of 100 consecutive cells (on average) per case to avoid selection
bias (total: 3000 cells).
Immunohistochemistry for Adiponectin receptor 1 (AdipoR1)
Five µm deparaffinised formalin-fixed tissue sections were microwaved in citrate buffer
pH 6.0, treated with 0.3% H2O2, blocked with Protein Block (Dako), and incubated with
one of the anti-AdipoR1 antibodies, goat polyclonal to AdipoR1 (abcam), or a rabbit
monoclonal to AdipoR1 (Epitomics), followed by anti-rabbit/anti-mouse EnVision-HRP
(Dako) and Vector SG chromogen kit (Vector Laboratories, Burlingame, CA). The results
of immunostaining were compared and the monoclonal rabbit antibody was selected for
further use. Slides were examined and images captured on a Zeiss Axio Imager M2
microscope.
111
Statistical Analysis
The statistical presentation includes paired and unpaired tests. In the case of ordinal tests,
medians and quartiles are used and for parametric tests means and standard deviation (SD)
is used. Cumulative concentration-response curves were constructed using data obtained
by wire myography and analysed using a two-way ANOVA and a Bonferroni post hoc test
for each dose. A P value of less than 0.05 was considered statistically significant.
Analyses were performed using the GraphPad Prism 5 software.
Results
Study design and participants
Six months after surgery and weight loss, the patients had a significantly lower waist
circumference (n = 15; P < 0.0001), body mass index (P < 0.0001) and systolic blood
pressure (P = 0.0025) (Table1).
Post-operatively there was a significant reduction in insulin (P = 0.0042), fasting glucose
(P = 0.0312), HbA1c (P < 0.0071) and an improvement in pancreatic ß-cell function
(HOMA-B; P < 0.01) and insulin resistance (HOMA-IR; P = 0.0023). After surgery serum
levels of adiponectin increased significantly (P = 0.0197), leptin decreased (P = 0.0014)
but resistin levels did not change significantly (P = 0.0796).
Weight-loss surgery restores PVAT anticontractile function
112
In severely obese patients before surgery and weight-loss, PVAT did not significantly alter
the norepinephrine-induced contractility of the small arteries in comparison to
skeletonised segments of the same vessels (n = 15, P = 0.96; Figure 1A).
In samples taken from patients 6 months following surgery and weight-loss, PVAT had a
significant anticontractile effect on vessels when compared to vessels without PVAT (n =
15, P< 0.01; Figure 1B). The vessels with intact PVAT following surgery had a very
similar response to cumulative doses of norepinephrine as the PVAT-intact vessels
harvested from healthy lean volunteers (P = 0.52, n = 7, Figure 1B). The maximal degree
of constriction is lower in PVAT-intact vessels in healthy and post-surgery samples as
compared with skeletonised vessels, but the degree of constriction of skeletonized vessels
to NE is similar in segments from pre and post surgery (Supplementary figure 1).
The anticontractile property of PVAT after weight loss is abolished by adiponectin
blockade
Samples taken from patients six months following surgery were incubated with blocking
peptide for adiponectin receptor 1. This had no effect on segments without PVAT, but in
vessels with PVAT there was a significant increase in vessel contractility (P < 0.0001, n=
7; Figure 2A).
Free radical scavengers can rescue PVAT anticontractile function in obesity
In patients with severe obesity, the presence of perivascular adipose tissue had no effect on
vessel contractility in comparison with vessel segments devoid of PVAT. Incubation of the
vessels with the free radical scavengers superoxide dismutase and catalase resulted in a
113
shift in the curve to resemble that of vessels with intact PVAT taken from healthy
individuals (P <0.001, n = 7, Figure 2B)
Increased NO bioavailability after surgery
There was a significant increase in nitric oxide in PVAT of severely obese patients 6
months after surgery (1.016 vs 1.196 nmol/μl, P = 0.029, n = 5).
In post surgery samples, de-endothelialised vessel segments were incubated with LNMMA. The incubation had no effect on vessels without PVAT, but in vessels with
PVAT, there was a significant increase in contractility to cumulative doses of
norepinephrine (P < 0.001, n = 4, Figure 2C).
Reduced PVAT inflammation, adipocyte size and adipoR1 receptors following
bariatric surgery
In order to quantify the inflammation, the perivascular adipose tissue was stained for both
CD68 staining macrophages (Figure 3) and the cytokine TNF-α (Figure 4A-C).
There was a significant reduction in the CD68 [KP1] staining macrophage numbers
following surgery (7.3 ± 1.1 vs. 4.1 ± 0.7, P = 0.0067, n =14; Figure 4C&D), but the
change in CD68 [PGM1] staining was not statistically changed. There was a significant
reduction in the percentage of adipose tissue area staining for TNF-α (1.41 ± 0.34 vs. 0.68
± 0.09, P < 0.05; Figure 4A-C) following surgery.
114
The average adipocyte area was 7672 ± 369.6 μm2 pre-surgery and 3955 ± 207.5 μm2 (P
< 0.0001) post-surgery (Figure 4D). The change in average adipocyte area correlated with
the change in BMI after surgery (R2 = 0.356, P = 0.0243, n = 14; Supplementary Figure 2).
There was a significant reduction in circulating markers of inflammation including highsensitivity C-reactive protein (P < 0.001), IL-6 (P = 0.013), MCP-1 (283.1 vs 258.3pg/ml;
P =0.013) and the adhesion molecule E-selectin (12.7 vs 7.47 ng/ml; P<0.001). However,
there was no significant change in circulating TNF-alpha levels (P = 0.519) (Table1).
Discussion
The present study investigated the effect of bariatric surgery on the vasodilatory properties
of PVAT. We designed the study following our earlier observation that in severely obese
patients, whilst there is an accumulation of adipose tissue, there is also a paradoxical
inhibition of the beneficial PVAT vasodilation 7. There are now three main findings
presented here. First, bariatric surgery reverses the obesity-induced damage to PVAT
anticontractile function. Second, the functional recovery of the PVAT is independent of
the endothelium. Third, bariatric surgery restores the function of PVAT by reducing
adipose inflammation and increasing local adiponectin and nitric oxide bioavailability.
The observations advance our understanding of the mechanisms by which obesity disrupts
the vasodilating function of adipose tissue and how this pathology may be reversed by
clinical intervention.
115
The cardiovascular complications of obesity are undoubtedly amongst the most pressing
issues facing clinicians today19. To date, the most common recommended intervention has
been the encouragement of dietary modification. Without doubt, effective and sustained
dieting does lead to significant weight loss, but unfortunately the weight loss is not
sustained20. Furthermore, the impact of dietary modification on cardiovascular outcomes is
unclear13, 21. For patients with severe obesity, the most effective method of achieving
significant and sustained weight reduction is by weight-reducing surgery14. In addition, the
weight loss following surgery also significantly correlates with improvements to blood
pressure22, 23 , left ventricular mass 24, exercise capacity25 and glucose metabolism26
. Changes to weight in patients is associated with profound modulation of the cytokine
and inflammatory profiles of adipose tissue. In this regard, it is now established that in
obese patients, adipose tissue undergoes dual processes of hypoxia and inflammation,
leading to a reduction in the secretion of adipocytokines such as adiponectin. The hypoxia
is thought to be due to adipocyte hypertrophy27 twinned with reductions to capillary
density and angiogenic capacity28. This results in up-regulation of hypoxia inducible
factor-1 (HIF-1) and inflammatory differentiation of macrophages which subsequently
secrete Tumour Necrosis Factor alpha (TNF) 29-31. Differentiated macrophages in obesity
also release superoxide anions which further contribute to local vascular dysfunction by
diminishing availability of nitric oxide32.
The functional damage we observe in PVAT and its recovery following bariatric surgery
are entirely consistent with this hypoxia/inflammation in fat hypothesis. Thus, in our
previous studies we observed adipocyte hypertrophy and increased TNF staining in
116
adipose tissue associated with loss of PVAT vasodilatory function. We now show that
following bariatric surgery there is restoration of PVAT vasodilatory capacity to a degree
which is similar to that observed in healthy non-obese participants. It is interesting to note
that this functional restoration of the PVAT occurs even though the patients are still
severely obese. The improvement in PVAT function after surgery is associated with
smaller adipocytes (in the context of a dramatic weight loss), a reduction in PVAT TNF
and increased local adipose tissue nitric oxide bioavailability. Our findings are consistent
with other studies which have shown significant reductions in macrophage numbers as
well as MCP-1 and HIF-1α in the stromal vascular fraction of white adipose tissue
following bariatric surgery33. Also, importantly, we show that the functional vasodilatory
improvement in the subcutaneous fat is due to restitution of PVAT derived adiponectin
and NO bioavailability.
The functional improvement of PVAT following bariatric surgery was independent of the
vascular endothelium as all arteries in this study were denuded of endothelium. Also
notably, there were no differences in constriction of arteries devoid of fat to
Norepinephrine before and after surgery. Taken together, these observations indicate that
the improvement in adipose-vascular coupling after surgery was due to improvements in
the vasodilating capacity of the PVAT rather than changes to isolated arterial contractility
or endothelial function.
There are a number of limitations to this study which need to be considered in parallel
with the findings. With respect to our patient selection, we did not monitor the level of
exercise performed by the participants at baseline or following surgery. At six months
117
after surgery, the patients had lost a significant amount of weight and were presumably
more mobile. As such, we acknowledge that the improvement in PVAT function may be
as a consequence of increased levels of mobility. Second, it would have been preferential
to recruit participants who were losing weight as part of a calorie controlled diet in order
to compare changes to PVAT function due to this intervention. This was not possible
within the limitations of our faculty, but we hope to perform this study in the future.
Similarly, a drawback of this study is that we were not able to perform a 6 month followup biopsy on severely obese patients who received no intervention. However, we assume
that there would have been no change in PVAT function over this time. We have studied
subcutaneous small arteries under the assumption that all small arteries and the
surrounding PVAT share similar properties. A separate consideration is that we have
detected changes only to subcutaneous gluteal PVAT following surgery. We did not study
the function of PVAT from other anatomical sites (i.e. mesenteric or skeletal). As such, we
acknowledge that changes to subcutaneous vascular function may not be reflected in other
vascular territories, but previous studies have shown similar remodeling profiles of human
cerebral and mesenteric arteries to those seen in arteries taken from gluteal subcutaneous
biopsies. Finally, it should be noted that norepinephrine was the only contractile agent
used. We acknowledge that it would have been preferable to study a variety of
vasoconstrictors. However, it should be noted that studies from other groups have shown
that PVAT antagonizes contraction to U4661934, Angiotensin II35, 36, Phenylephrine35, 37
and serotonin35, 36.
Despite these limitations however, we believe that we present convincing evidence that
PVAT induced vasodilation of small arteries can be fully restored following bariatric
surgery. Furthermore, this restoration of function is due to a reduction in inflammation
118
within the adipose tissue which leads to increased adiponectin secretion. The data offer an
insight into one mechanism by which bariatric surgery improves vascular function. We
anticipate that further work will harness this observation to improve therapeutic options
for the severely obese patient.
Acknowledgements
We thank all the study participants who took part in this study for their time and for
providing the invaluable gluteal fat samples as a gift without any remuneration.
We would also like to thank Aisha Meskiri, Simon Forman and Louisa Nelson for their
help with the sample preparation for immunohistochemical analysis of the tissue, and
Mahmoud Abudaka for assisting with patient recruitment.
Funding Sources
Dr Reza Aghamohammadzadeh is a British Heart Foundation and NIHR Manchester
Biomedical Research Centre Clinical Research Fellow.
The Manchester Wellcome Trust Clinical Research Facility supported this study.
Disclosures
The authors declare no conflict of interest.
119
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122
Figure legends
Figure 1: Background and Hypothesis
A: In obesity there is an increase in PVAT levels of inflammatory cells and cytokines as
well as increase in levels of leptin
B: We hypothesize that following bariatric surgery, there will be an increase in
adiponectin levels and nitric oxide bioavailability in PVAT and reduction this
inflammation which would result in restoration of PVAT anticontractile function.
Figure 2: The effect of perivascular adipose tissue on small artery tone before and after
surgery
A: In pre-surgery patients, the presence of PVAT did not affect vessel contractility as
compared with skeletonised segments of the same vessel (n = 20, P = 0.95)
B: In post-surgery patients, the presence of PVAT had a significant anticontractile effect
on the small artery contractility (n = 15, P <0.01).
123
Figure 3: Pharmacological protocols on PVAT pre-surgery and post-surgery
A: Blocking peptide for PAb to adiponectin receptor 1 increases vessel contractility to
norepinephrine (n = 7, P <0.0001).
B: Incubation with SOD and catalase rescues the PVAT anticontractile effect in samples
taken from pre-surgery patients (n = 7, P < 0.001).
C: Post-surgery, inhibition of nitric oxide synthase by incubation with L-NMMA leads to
increased contractility of vessel segments with intact PVAT (n = 4, P < 0.001)
Figure 4: Subcutaneous adipose tissue before and after surgery. Inflammatory infiltrates
composed mostly of macrophages were present either in wreath-like arrangements (A), in
groups (B), or were scattered. Staining for macrophage marker (CD68 (KP1) (C) allowed
for performing cell counts. There was a significant reduction in the number of CD-68
staining macrophages post-surgery (n =14, 7.3 ± 1.1 vs 4.1 ± 0.7, P < 0.01) (D). Scale bar
= 20 µm.
Figure 5: Assessment of TNF-α staining and adipocyte size pre- and postsurgeryImmunostaining for TNF-α showed that adipocytes themselves were positive for
this cytokine, to a higher extent pre-surgery (A) than post-surgery (B). Note positive
staining of microvessels in the pre-operative biopsy. There is a significant reduction
in the percentage of adipose tissue area that stains for TNF-α pre-surgery versus postsurgery (1.41 ± 0.34 vs 0.68 ± 0.09, P < 0.05; (C)). The average adipocyte area was 7672
369.6 μm2 pre-surgery and 3955 ± 207.5 μm2 (P < 0.0001) post-surgery (D). Scale bar =
20 µm.
124
Healthy
Age
(Years)
Waist
(cm)
BMI
(kg/m2)
Systolic BP
(mmHg)
hs-CRP
(mg/l)
IL-6
(pg/ml)
TNFα
(pg/ml)
Adiponectin
(mg/l)
Leptin
(ng/ml)
Resistin
(ng/ml)
E-selectin
(ng/ml)
Insulin
(mU/l)
Glucose
(mmol/l)
HbA1C
(%)
HOMA- B
(%)
HOMA- IR
(%)
Presurgery
51 (11.5)
Post surgery
P*
53.2 (6.73)
92 (11.6)
140.9 (14.85)
115.5 (13.3)
<0.0001
25.4 (2.68)
51.58 (7.47)
37.96 (7.175)
<0.0001
135 (17.2)
146.1 (28.8)
132.7 (22.15)
0.0025
1.33 (1.09)
6.321 (2.444)
2.747 (2.282)
0.0027
2.07 (0.811)
2.83 (1.755)
0.644 (0.774)
0.0052
4.83 (1.75)
35.14 (40.3)
27.14 (37.3)
0.12
2.19 (0.633)
1.845 (0.699)
2.41 (0.828)
0.011
9.67 (7.76)
59.9 (17.7)
24.91 (10.42)
0.0014
9.26 (3.05)
15.78 (6.823)
13.55 (4.605)
0.0796
131 (5.06)
12.53 (5.581)
8.753 (3.374)
0.0033
7.18 (4.11)
27.83 (16.17)
14.83 (10.12)
0.004
5.08 (0.377)
6.825 (3.585)
5.858 (2.355)
0.0312
5.74 (0.689)
7 (2.119)
6.33 (1.603)
0.0071
90.2 (26.9)
243 (175.7)
169.9 (143.7)
0.01
0.933 (0.557)
7.838 (4.213)
3.86 (2.683)
0.0023
Table1: Patient demographics and biomarker profile
Mean (SD) is indicated. P values compare the matched baseline and post-surgery values,
and were determined using paired t-tests.
125
Figures
% c o n s t r ic t io n o f K P S S
1A
250
N o P V A T (P re -s u rg e ry )
200
PVAT
(P re -s u rg e ry )
150
100
50
0
-9
-8
-7
-6
-5
-4
L o g [N E ] (M )
%constriction of KPSS
1B
250
*
No PVAT (Post-surgery)
PVAT
(Post-surgery)
Healthy PVAT
200
150
100
50
0
-9
-8
-7
-6
-5
Log [NE] (M)
126
-4
2A
% K P S S c o n s t r ic t io n
250
*
200
P V A T ( P o s t- s u r g e r y ) +
A d ip o n e c tin R 1 b lo c k e r
150
P V A T ( P o s t- s u r g e r y )
100
50
0
-9
-8
-7
-6
-5
-4
L o g [N E ] (M )
2B
% K P S S c o n s t r ic t io n
250
P V A T (P re -s u rg e ry )
*
200
P V A T (P re -s u rg e ry ) +
S O D & C a ta la s e
150
100
50
0
-9
-8
-7
-6
-5
-4
L o g [N E ] (M )
% c o n s t r ic t io n o f K P S S
2C
*
250
200
P V A T ( P o s t- s u r g e r y ) +
L -N M M A
150
P V A T ( P o s t- s u r g e r y )
100
50
0
-9
-8
-7
-6
-5
L o g [N E ] (M )
127
-4
3
A
A
B
B
D
2
No. of macrophages/mm
C
10
8
*
6
4
2
0
Pre-surgery
128
Post-surgery
A
Fat stained for TNF-/frame (%)
4
B
C
2.0
1.5
*
1.0
0.5
0.0
Pre-surgery
Post-surgery
D
Adipocyte area m2
10000
8000
*
6000
4000
2000
0
Pre-surgery
Post-surgery
5A
Decreased
Obesity
Adiponectin
Increased
Leptin
TNF α
Nitric oxide
bioavailability
Free radical
scavengers
MCP-1
Oxidative
stress
Macrophages
129
5B
130
Supplementary materials
Supplemental figures
Figure 1
%constriction of KPSS
A
250
No PVAT (pre-surgery)
No PVAT (post-surgery)
200
150
100
50
0
-9
-8
-7
-6
-5
-4
Log [NE] (M)
%constriction of KPSS
B
250
PVAT (post- surgery)
PVAT (pre-surgery)
200
*
150
100
50
0
-9
-8
-7
-6
-5
Log [NE] (M)
131
-4
Supplementary figure 1: Comparison of vessel constriction pre- and post-surgery
A: There is no difference in constriction of skeletonised to NE before and after surgery (P
= 0.07, n=15)
B: There is a significant difference in constriction of vessels with intact PVAT before and
after surgery (P < 0.0001, n=15)
132
Figure 2
*
Change in BMI (kg/m2)
18
16
14
12
10
8
0
2000
4000
6000
8000
Change in adipocyte size (m2)
Supplementary figure 2: The change in average adipocyte area post-surgery as compared
with baseline correlates with the change in BMI post-surgery (n = 14, R2 = 0.356, P =
0.024)
133
CHAPTER 4
Obesity-induced damage to perivascular adipose tissue anticontractile function is
nitric oxide dependent, independent of the endothelium and reversible
Short title: PVAT damage in obesity
Reza Aghamohammadzadeh MBChB (Honours) MRCP *†; Adam S. Greenstein MBChB
MRCP PhD*†; Richard Unwin MSC¥ ; Pemberton, MSc¥; Anthony M. Heagerty, MD
FMedSci*†.
*: Cardiovascular Research Group, University of Manchester, UK
†: Manchester Wellcome Trust Clinical Research Facility, UK
¥: Manchester Royal Infirmary, UK
Funding: R. Aghamohammadzadeh was funded by a British Heart Foundation Clinical
Research Training Fellowship (FS/10/42/28372).
Corresponding author:
Dr Reza Aghamohammadzadeh
Institute of Cardiovascular Sciences,
The University of Manchester,
Core Technology Facility (3rd floor),
46 Grafton Street,
Manchester, M13 9NT
[email protected]
134
Abstract
Objective
White adipocytes are the main constituents of perivascular adipose tissue (PVAT) which
surrounds most vessels in the human body. PVAT is the source of a number of molecules
with vasoactive and metabolic properties. In health, PVAT exhibits a vasodilating effect
on its adjacent vessel; however, this effect is not observed in obesity. The aim of this study
was to investigate whether the obesity-induced damage to PVAT function is endotheliumdependent and assess the role of inflammation and nitric-oxide (NO) in obesity-induced
PVAT damage.
Methods
Gluteal fat biopsies were performed on 10 obese and 10 control individuals. Adipose
tissue was immediately snap frozen and stored for proteomic analysis. Small arteries were
isolated from the biopsy samples and their function was assessed using wire myography
(n=7 for obese and control). Sprague Dawley rats were fed a high fat diet (obese; n=16) or
normal chow (control; n=6) and PVAT function was assessed using wire myography.
Skeletonised vessel segments as well as segments with intact PVAT were prepared with
both intact endothelium and as endothelium-denuded segments. Vessel segments were
incubated with L-NNA (10-4M, 45 minutes) or SOD (100 U/ml, 45 minutes) and Catalase
(100 U/ml, 45 minutes), to assess NO bioavailability and the contribution of
inflammatory-mediated damage to PVAT function respectively.
135
Results
Human protocols: In the control samples, presence of PVAT results in a significant
attenuation in vessel contractility to cumulative doses of NE, but this effect is not observed
in samples from obese individuals. Superoxide dismutase [Cu-Zn] (SOD1; fold change 2.4182, P = 0.018), peroxiredoxin-1 (PRDX1; fold change -2.15, P = 0.048), and
adiponectin (fold change -2.1; P = 0.018) were present in lower abundances in obese
PVAT compared with controls.
Animal protocols: Obese rats developed significantly higher systolic (127 ± 3 mmHg vs
144 ± 3 mmHg; P<0.01) and diastolic (100 ± 5 mmHg vs 118 ± 3 mmHg; P <0.01) blood
pressures (BP) as compared with controls. There were significant correlations between
weight and both systolic (r = 0.5044, P = 0.0167) and diastolic (r = 0.5329, P = 0.0107)
blood pressures. There was a significant correlation between weight and damage to PVAT
function (r = 0.5059, P=0.0163), and PVAT function correlated with both systolic (r =
0.5324, P = 0.0108) and diastolic (r = 0.4722, P = 0.0265) blood pressures.
In healthy animals, there was preservation of PVAT anticontractile function after removal
of the endothelium. In the obese animals, PVAT did not affect vessel contractility and
presence of endothelium did not affect this. In endothelium-denuded vessels, incubation
with L-NNA attenuated PVAT anticontractile function in control vessels (P < 0.0001), but
had no effect on vessel segments from the obese (P = 0.4). Incubation with SOD and
Catalase lead to an attenuation in PVAT-intact vessel contractility in the presence (P <
0.0001) and after removal of the endothelium (P <0.001).
136
Conclusions
In keeping with our prior observations, obese PVAT has no effect on vessel contractility.
The obesity-induced PVAT damage is independent of the endothelium, and in part, due to
a reduction in NO bioavailability within PVAT.
There are significantly lower levels of SOD, Peroxiredoxin 1 and adiponectin in obese
human PVAT as compared with healthy controls, and incubation with free radical
scavengers restores PVAT function in obese animals, independently of endothelial
presence.
Key words
Obesity
Vascular tone
Perivascular adipose tissue
Contractility
Endothelium
Nitric oxide
137
Abbreviations
PVAT: Perivascular adipose tissue
BP: Blood pressure
NE: Norepinephrine
SOD: Superoxide dismutase
CAT: Catalase
L-NNA: L-NG-Nitroarginine; NG-nitro-L-Arginine
NO: Nitric oxide
138
Introduction
Obesity has a profound effect on cardiovascular risk profile. Specifically with regards to
hypertension, weight gain is associated with a rise in BP whilst weight loss is associated
with a reduction in BP. Understanding this relationship has been substantially advanced by
studies into the vasoactive properties of adipose tissue surrounding blood vessels, known
as Perivascular Adipose Tissue (PVAT). Soltis and Cassis were the first to show that
PVAT antagonised arterial vasoconstriction in 19911. More recently, the vasorelaxant
effect of PVAT has been demonstrated in both resistance and conduit arteries, and appears
to utilise multiple physiological mechanisms. Thus, both endothelial independent and
endothelial dependent pathways are present and nitric oxide, hydrogen sulphide,
angiotensin 1-7, adiponectin and insulin have all been implicated in the vasorelaxant
effect. We have shown that in subcutaneous human tissue from healthy participants,
adiponectin release from PVAT acting via nitric oxide was the predominant mediator of
the anticontractile effect on small arteries. We also observed that in obese patients with
metabolic syndrome, there was complete loss of the vasoactive properties of PVAT. In
this follow-on study, we have used a proteomic approach to examine, for the first time,
molecular changes to subcutaneous PVAT in obese patients compared with healthy
participants; and interpret these in tandem with the functional properties of the adipose
tissue. We have used the findings to advance an existing hypothesis to account for damage
to PVAT function in obesity and validated this in an animal model of diet-induced obesity.
Overall, the project provides an insight into the changes of human adipose tissue which
develop in obesity and how these affect the vasoactive function of PVAT.
139
Materials and Methods
Study population
10 patients with obesity and 10 control participants gave full written informed consent and
participated in the study, which was approved by the Local Research Ethics Committee.
Fasting venous blood samples were taken to assess glycaemic and lipid profiles and
inflammatory markers. Blood pressure was measured sitting, after 15 minutes of rest, by a
semiautomatic machine (OMRON 705CP, White Medical) with a mean of 3 readings
recorded. Anthropometric measurements and bioimpedance were also measured.
Gluteal fat biopsy
A subcutaneous gluteal fat biopsy was obtained from each subject, under local
anaesthesia, allowing tissue (2x1.5x1.5cm) to be harvested. Using microdissection, a small
section of perivascular adipose tissue was dissected from the tissue and placed into liquid
nitrogen for proteomic analysis. The remainder of the biopsy was then placed in
physiological saline solution (PSS) with a composition (mmol/L) of: NaCl 119, KCl 4.7,
NaHCO3 25, KH2PO4 1.17, MgSO4 1.17, EDTA 0.026, CaCl2 1.6 and glucose 5.5.
Biochemical analysis of serum
Highly sensitive-CRP, leptin, IL-6, TNF and adiponectin were assayed by an in-house
ELISA technique (hs-CRP:Abcam: remainder:R&D Systems). Insulin levels were
measured using a radiolabelling technique described previously 2. The homeostasis model
140
assessment (HOMA) 3 was used to estimate beta cell function (HOMA-B) and peripheral
insulin sensitivity (HOMA-S).
Wire Myography
Viable arterial segments were obtained from 7 control participants and 7 obese patients
with metabolic syndrome. An arterial segment with an internal diameter of 250-350μm
was dissected from each biopsy. One segment was cleaned of PVAT, whilst on the
adjacent segment PVAT was left intact. Both arteries were mounted on 40μm wires in a
wire myograph (Danish MyoTech). After an initial 30 minute incubation, vessel wall
tension and diameter were normalized in a standardised procedure 4 and stabilised for 1
hour.
Arteries were first challenged with 60mM KPSS to establish a baseline contractile
response. A cumulative dose response to Norepinephrine was then constructed. Each
vessel was stimulated by addition of Norepinephrine (Sigma) at the following
concentrations: 10-9, 3x10-9, 10-8, 3x10-8, 10-7, 3x10-7, 10-6, 3x10-6, 10-5 mol/L. Contractile
responses to Norepinephrine are expressed as a percentage of KPSS contraction,
consistent with other studies 5-8.
Proteomic analysis
Gluteal perivascular adipose tissue fat samples from the 10 healthy and 10 of the obese
patients were immediately immersed in liquid nitrogen and stored at -80°C for analysis by
Oxford Biomarker Services (Oxford BioTherapeutics Ltd). Protein levels were compared
using a label free mass spectrometric approach. Given our previously published data
141
highlighting the significance of adiponectin in PVAT anticontractile function9, we
established a targeted quantification method for adiponectin in these samples using
Selected Reaction Monitoring (SRM) mass spectrometry to determine the relative levels of
two adiponectin peptides: GDIGETGVPGAEGPR and IFYNQQNHYDGSTGK
(Supplemental methods)
Animal model development
Animals and Diets
Four week old male Sprague Dawley rats were purchased from Charles River (UK). The
rats were housed in groups of three per cage (changed to two per cage after two months) in
a standard experimental animal laboratory, illuminated from 6.30 a.m. to 6.30 p.m., at a
temperature of 22±1°C. The protocol was approved by the Animal Experimentation
Committee of the Medical Faculty of University of Manchester. The animals had free
access to food and water during the experiment.
After a 1-week acclimatization period, the rats were divided into 2 groups receiving either
a high-fat diet (Western RD; 4.24 kcal/g (AFE); 35% of energy derived from fat; Special
Diets Services n = 16) or standard laboratory chow (AFE: 3.29 kcal/g; n = 6) for 3 months.
Body weight and blood pressure was monitored once a week and the consumption of feed
was monitored daily using a standard laboratory table scale. At the time of sacrifice, the
animals were weighed, blood pressure was recorded (LE5002 Storage Pressure Meter
from Panlab Harvard Apparatus) and blood glucose was analysed using a portable glucose
monitoring system (Ascensia® CONTOUR® blood glucose monitoring system, Bayer
HealthCare).
142
Vessel preparation and myography
The mesenteric bed was immediately removed after animal sacrifice and placed in chilled
PSS (detailed above) for transfer to the laboratory. Isolated mesenteric vessels with
average internal diameter of 250 μm were dissected and prepared as segments with intact
perivascular (PVAT) and segments without PVAT. The vessels were studied using a multi
wire myograph system (Danish Myo Technology; Model 610 M, version 2.2) and
analysed on ADIinstruments Chart™ 5 (version 5.5.1). The vessels were bathed in PSS (as
above), maintained at 37°C and gassed with a mixture of (95% air, 5% CO2). The vessels
were normalised and their viability was checked by addition of PSS containing 60mM KCl
following which concentration-response curves to NE (Sigma-Aldrich A0937) were
constructed. To assess the effect of free radical scavengers on the obese PVAT, vessels
from the obese cohort were incubated with superoxide dismutase (100 U/ml) and catalase
(100 U/ml) for 45 minutes, following which another norepinephrine concentrationresponse curve was constructed. Similarly, vessels were incubated with L-NNA (10-4M,
45minutes) to assess NO bioavailability within PVAT.
Results
Patient details and PVAT function
The obese individuals had significantly greater waist circumference, body mass index,
HDL, total cholesterol, HbA1C, leptin, insulin, glucose and HOMA-S (P<0.05). However,
triglycerides, blood pressure, resistin, TNF-alpha, IL-6, adiponectin and HOMA-B were
not significantly different (table 1). As we have demonstrated previously 9, 10, in healthy
143
participants the presence of perivascular adipose tissue lead to an attenuation in
contractility of adjacent small arteries (P < 0.05; figure 1A) while in obesity this PVAT
effect was not observed (figure 1B).
Animal characteristics, blood pressure, NO and PVAT function
At the time of sacrifice, the high fat fed group weighed significantly more than the control
group (511 ± 13g vs 622 ± 14g ; P <0.001), and developed both systolic (127 ± 3 mmHg
vs 144 ± 3 mmHg; P<0.01) and diastolic (100 ± 5 mmHg vs 118 ± 3 mmHg; P <0.01)
blood pressures that were significantly raised as compared to controls. Glucose was not
significantly different (P = 0.66). PVAT from the control group attenuated vessel
contractility to NE (P < 0.0001), but no such effect was observed in the obese group (P =
0.191). There were significant correlations between weight and both systolic (r = 0.5044,
P = 0.0167; figure 2A) and diastolic (r = 0.5329, P = 0.0107; figure 2B) blood pressures
of the rats at the time of sacrifice.
In order to quantify the PVAT anticontractile effect, at a given concentration of
norepinephrine, constriction of the vessel segment with PVAT was compared to the
constriction of vessel segment without PVAT, and this value was presented as a
percentage calculation (constriction of vessel with PVAT / constriction of vessel without
PVAT * 100). There was also a significant correlation between weight and damage to
PVAT function (r = 0.5059, P=0.0163; figure 2C). The damage to PVAT function also
correlated with both systolic (r = 0.5324, P = 0.0108; figure 2D) and diastolic (r = 0.4722,
P = 0.0265; supplementary figure 1) blood pressures at the time of sacrifice.
144
Incubation of PVAT-intact endothelium-denuded vessels with the NO synthase inhibitor
L-NNA resulted in a significant increase in vessel contractility in vessels segments from
healthy animals (figure 3A) , however, no such phenomenon was observed in obese PVAT
(figure 3B).
Downregulation of Superoxide Dismutase, Peroxiredoxin 1 and adiponectin in
human PVAT
Proteomic analysis revealed 932 peaks present in over half of the samples analysed and
these were used for quantification. Following statistical analysis, 67 peptides were found
to be significantly different in these tissues. Removal of peptides emanating from bloodderived proteins left 47 significant peptides (table2). Levels of the free radical scavengers
superoxide dismutase [Cu-Zn] (SOD1; fold change -2.4182, P = 0.018) and
peroxiredoxin-1 (PRDX1; fold change -2.15, P = 0.048) were found to be significantly
lower in the obese versus lean volunteers. Adiponectin (two peptides:
GDIGETGVPGAEGPR and IFYNQQNHYDGSTGK) levels in PVAT were lower in
obese patients compared with the lean volunteers with an average fold change of 2.1 (P <
0.05; supplementary figure 2).
PVAT anticontractile function in obesity is damaged independently of the
endothelium and incubation with free radical scavengers restores PVAT function in
obese animals
145
Cumulative dose response protocols with norepinephrine were performed both with intact
endothelium and after removal of endothelium. Vessel segments from healthy animals
constricted significantly less as compared with segments from obese animals, both when
the endothelium was present (P < 0.05, n=8; figure 4A) and in endothelium denuded
vessels (P < 0.05, n=8; figure 4B).
Incubation with SOD and catalase resulted in attenuation of vessel constriction to a similar
degree whether the endothelium was intact (figure 5A) or removed (figure 5B).
Discussion
The present study investigated the effects of obesity on PVAT function and assessed the
contribution of nitric oxide and endothelium in this process. We have shown previously
that in healthy individuals PVAT exerts a vasorelaxant effect on its adjacent small vessel
and that this is not observed in obesity9. Moreover, we have shown that this vasorelaxant
effect is restored following significant weight loss10. We now present four novel findings
here. First, using proteomic analysis, we show that levels of adiponectin and the free
radical scavengers SOD and peroxiredoxin-1 are significantly lower in the PVAT from
obese individuals as compared with healthy controls. Second, in healthy animals, there
was preservation of PVAT anticontractile capacity after removal of the endothelium,
confirming observations by Gao et al6. The obesity-induced damage to PVAT function is
independent of the endothelium and the PVAT vasorelaxant effect is restored using free
radical scavenger independent of the endothelium. Third, NO from PVAT is an important
endothelium-independent contributor to PVAT vasorelaxant function in healthy tissue, but
146
not observed in obesity. Fourth, PVAT function correlates with systemic blood pressure
and obesity-induced damage to PVAT vasorelaxant quality correlates with a rise in BP.
In keeping with increased inflammation in adipose tissue, our proteomic data show that
levels of the free radical scavengers superoxide dismutase [Cu-Zn] and peroxiredoxin-1
are significantly lower in the obese patients as compared with the healthy lean volunteers.
Macrophages present within PVAT contribute to the inflammatory environment by
secreting superoxide anions. Superoxide dismutase converts the highly volatile superoxide
anion into hydrogen peroxide which is then broken down into oxygen and water with the
help of catalase, glutathione peroxidise (GTP) and peroxiredoxin (PRX) enzymes.
Peroxiredoxin-1 resides within the cytoplasm and is the most abundant of the Prxs 11. The
catalytic efficiency of Prxs is less than that of glucothione peroxidase and catalase 12, but a
reduction in their levels may lead to a possible reduction in the rate of H 2O2 catabolism
and its accumulation in cells with its consequent effects on vascular tone 12. In the present
study, we have shown that in obesity we can rescue the PVAT vasorelaxant effect by
incubating the obese PVAT with the free radical scavengers superoxide dismutase and
catalase, and that this is not endothelium-dependent.
Gil-Ortega et al have shown previously that after 8 weeks on a high fat diet, mouse
mesenteric PVAT possesses greater NO bioavailability as compared with controls, and
have described this as an adaptive overproduction of the vasorelaxant molecule in early
diet-induced obesity13. In keeping with their findings, we confirm that, in health, PVAT
vasorelaxant function is partly dependent on NO present within the PVAT itself,
independent of the endothelium. However, after 3 months on the high fat diet, our
147
functional data show a significant reduction in NO bioavailability within PVAT, thus
suggesting that the proposed adaptive overproduction of NO is no longer observed once
the animals remain on the diet for longer.
Finally, we have shown for the first time, that damage to PVAT vasorelaxant function
correlates with a significant rise in BP and this remains to be explored further using human
protocols.
Conflict of Interest/Disclosure
There are no conflicts of interest to declare.
148
References
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muscle responsiveness. Clin Exp Hypertens A. 1991;13(2):277-296.
2. Yates AP, Laing I. Age-related increase in haemoglobin A1c and fasting plasma
glucose is accompanied by a decrease in beta cell function without change in insulin
sensitivity: evidence from a cross-sectional study of hospital personnel. Diabet Med.
2002;19(3):254-258.
3. Levy JC, Matthews DR, Hermans MP. Correct homeostasis model assessment
(HOMA) evaluation uses the computer program. Diabetes Care. 1998;21(12):2191-2192.
4. Mulvany MJ, Halpern W. Contractile properties of small arterial resistance vessels in
spontaneously hypertensive and normotensive rats. Circ Res. 1977;41(1):19-26.
5. Dubrovska G, Verlohren S, Luft FC, Gollasch M. Mechanisms of ADRF release from
rat aortic adventitial adipose tissue. Am J Physiol Heart Circ Physiol. 2004;286(3):H11071113.
6. Gao YJ, Lu C, Su LY, Sharma AM, Lee RM. Modulation of vascular function by
perivascular adipose tissue: the role of endothelium and hydrogen peroxide. Br J
Pharmacol. 2007;151(3):323-331.
7. Malinowski M, Deja MA, Golba KS, Roleder T, Biernat J, Wos S. Perivascular tissue
of internal thoracic artery releases potent nitric oxide and prostacyclin-independent
anticontractile factor. Eur J Cardiothorac Surg. 2008;33(2):225-231.
8. Verlohren S, Dubrovska G, Tsang SY, Essin K, Luft FC, Huang Y, Gollasch M.
Visceral periadventitial adipose tissue regulates arterial tone of mesenteric arteries.
Hypertension. 2004;44(3):271-276.
9. Greenstein AS, Khavandi K, Withers SB, Sonoyama K, Clancy O, Jeziorska M, Laing
I, Yates AP, Pemberton PW, Malik RA, Heagerty AM. Local inflammation and hypoxia
abolish the protective anticontractile properties of perivascular fat in obese patients.
Circulation. 2009;119(12):1661-1670.
10. Aghamohammadzadeh R, Greenstein AS, Yadav R, Jeziorska M, Hama S, Soltani F,
Pemberton PW, Ammori B, Malik RA, Soran H, Heagerty AM. The Effects of Bariatric
Surgery on Human Small Artery Function: Evidence for Reduction in Perivascular
Adipocyte Inflammation, and the Restoration of Normal Anticontractile Activity Despite
Persistent Obesity. J Am Coll Cardiol.
11. Immenschuh S, Baumgart-Vogt E. Peroxiredoxins, oxidative stress, and cell
proliferation. Antioxid Redox Signal. 2005;7(5-6):768-777.
12. Ardanaz N, Pagano PJ. Hydrogen peroxide as a paracrine vascular mediator:
regulation and signaling leading to dysfunction. Exp Biol Med (Maywood).
2006;231(3):237-251.
13. Gil-Ortega M, Stucchi P, Guzman-Ruiz R, Cano V, Arribas S, Gonzalez MC, RuizGayo M, Fernandez-Alfonso MS, Somoza B. Adaptative Nitric Oxide Overproduction in
Perivascular Adipose Tissue during Early Diet-Induced Obesity. Endocrinology. 2010.
149
Table 1
Control
Patients with
Participants
obesity
P
Age
51± 3.6
52 ± 4
0.95
Waist (mm)
920 ± 37
1140 ± 70
<0.001
BMI (kg/m2)
23.9 ± 0.7
35.5
<0.001
Triglyceride
1.6 ± 0.06
0.52 ± 0.03
0.64
HDL (mmol/L)
1.4 ± 0.06
1.2 ± 0.07
0.031
Glucose
5.1 ± 0.1
5.9 ± 0.3
0.013
134 ± 5
132 ± 3
0.63
82 ± 2
80 ± 4
0.67
Chol/HDL ratio
4 ± 0.2
4.2 ± 0.3
0.85
total chol
5.91 ± 0.31
4.79 ± 0.21
<0.01
HbA1c (%)
5.7 ± 0.2
6.4 ± 0.1
0.027
Fat (Kg)
19.7 ± 1.8
38 ± 2.7
< 0.001
Impedance
567 ± 22
493 ± 18
0.02
leptin (ug/l)
9.67 ± 2.5
40.4 ± 4.9
< 0.001
resistin (ug/l)
9.2 ± 1.2
7.8 ± 0.9
0.35
TNF (pg/ml)
4.8 ± 0.7
4.6 ± 1.6
0.93
IL-6 (pg/ml)
2.1 ± 0.3
2.6 ± 0.8
0.63
adiponectin
2.1 ± 0.3
2.6 ± 0.8
0.48
Insulin (mU/L)
7.2 ± 1.7
18.3 ± 2.7
<0.01
HOMA B
90.15 ± 10.9
118.8 ± 15.5
0.198
HOMA S
132.4 ± 24.7
49.9 ± 8.3
<0.01
(mmol/L)
(mmol/L)
Systolic BP
(mmHg)
Diastolic BP
(mmHg)
(mmol/L)
(mg/L)
150
Table 2
Protein Name
P<0.05
DERPC
Parathymosin
Fibrinogen beta chain precursor
Histone H2B.c (H2B/c) * Peptide sequence 1
Fibrinogen gamma chain precursor.
Serum albumin precursor
Hemoglobin subunit delta * Peptide sequence 1
annexin A2 isoform 1 *Peptide sequence 1
Alpha 2 globin variant * Peptide sequence 1
Sorting nexin-13
annexin A2 isoform 1 *Peptide sequence 2
Haptoglobin precursor
Histone H2B.c (H2B/c) * Peptide sequence 2
Hemoglobin subunit delta * Peptide sequence 2
Protein S100-A6
Lumican precursor
Serum albumin precursor * Peptide sequence 1
Serum albumin precursor * Peptide sequence 2
Hemoglobin beta chain * Peptide sequence 1
Superoxide dismutase
annexin A2 isoform 1 * Peptide sequence 3
Alpha 2 globin variant * Peptide sequence 2
Vimentin * Peptide sequence 1
Hemoglobin beta chain * Peptide sequence 2
Collagen alpha 2(I) chain precursor * Peptide sequence 1
Profilin-1 (Profilin I)
Peroxiredoxin-1
Collagen alpha 2(I) chain precursor * Peptide sequence 2
Ubiquitin
Adiponectin
Vimentin * Peptide sequence 2
151
0.047619
0.035714
0.04798
0.015873
0.029304
0.024242
0.034279
0.019048
0.019048
0.047619
0.015873
0.012121
0.005051
0.049073
0.015873
0.015873
0.018385
0.035714
0.043421
0.017677
0.015873
0.015873
0.0043
0.030393
0.011988
0.034965
0.04798
0.031219
0.017316
0.0184
0.025974
Fold
change
-9.585
-7.182
-5.0104
-4.949
-4.6283
-4.468
-4.286
-4.024
-4.024
-3.827
-3.739
-3.122
-2.933
-2.886
-2.835
-2.6968
-2.666
-2.469
-2.466
-2.4182
-2.416
-2.416
-2.3734
-2.238
-2.198
-2.1584
-2.15
-2.143
-2.1031
-2.1
-2.028
Figure Legends
Figure 1
In healthy participants the presence of PVAT lead to an attenuation in contractility of
adjacent small arteries (P < 0.05, n=7; figure 1A) while in obesity, the presence of PVAT
did not affect vessel contractility (n=7; figure 1B).
Figure 2
There were significant correlations between weight and both systolic (r = 0.5044, P =
0.0167; figure 2A) and diastolic (r = 0.5329, P = 0.0107; figure 2B) BP. There was a
significant correlation between weight and damage to PVAT function (r = 0.5059,
P=0.0163; figure 2C). The damage to PVAT function also correlated with systolic BP (r =
0.5324, P = 0.0108; figure 2D).
Figure 3
Incubation of PVAT-intact endothelium-denuded vessels with the NO synthase inhibitor
L-NNA resulted in a significant increase in vessel contractility in healthy vessels (figure
3A) , however, no such phenomenon was observed in obese vessels (figure 3B).
Figure 4
Vessel segments from healthy animals constricted significantly less as compared with
segments from obese animals, both when the endothelium was present (P < 0.05, n=8;
figure 4A) and in endothelium denuded vessels (P < 0.05, n= 8; figure 4B).
152
Figure 5
Incubation with SOD and catalase resulted in attenuation of vessel constriction to a similar
degree whether the endothelium was intact (figure 5A) or removed (figure 5B).
153
Figures
Figure 1
A
%constriction of KPSS
250
*
200
* *
150
100
50
0
-10
-9
-8
-7
-6
-5
LogLog
[NE][NA]
(M) (M)
Healthy: P(-)E(+)
Healthy: P(+)E(+)
B
%constriction of KPSS
250
200
150
100
50
0
-10
-9
-8
-7
-6
-5
LogLog
[NE] [NA]
(M) (M)
Obese: P(-)E(+)
Obese: P(+)E(+)
154
Figure 2
A
Systolic BP (mmHg)
175
150
125
100
400
500
600
700
800
700
800
Weight (g)
B
Diastolic BP (mmHg)
150
100
400
500
600
Weight (g)
155
C
PVAT / No PVAT
constriction (%)
200
150
100
50
0
400
500
600
700
Weight (g)
D
Systolic BP (mmHg)
175
150
125
100
0
50
100
150
200
PVAT/ No PVAT constriction (%)
156
250
800
Figure 3
% KPSS constriction
A
P(+)E(-) Healthy
P(+)E(-) Healthy + LNNA
200
150
100
50
0
-7.0
-6.5
-6.0
-5.5
-5.0
-4.5
Log [NE]
(M)(M)
Log
[NA]
% KPSS constriction
B
200
P(+)E(-) Obese
P(+)E(-) Obese + LNNA
150
100
50
0
-7.0
-6.5
-6.0
-5.5
-5.0
Log
[NA](M)
(M)
Log [NE]
157
-4.5
Figure 4
A
% KPSS constriction
*
*
200
PVAT(+)E(+) Healthy
PVAT(+)E(+) Obese
150
100
50
0
-7.0
-6.5
-6.0
-5.5
-5.0
-4.5
Log
[NA]
Log [NE]
(M)(M)
% KPSS constriction
B
*
200
*
PVAT(+)E(-) Healthy
PVAT(+)E(-) Obese
150
100
50
0
-7.0
-6.5
-6.0
-5.5
-5.0
Log
[NA](M)
(M)
Log [NE]
158
-4.5
Figure 5
% KPSS constriction
A
200
150
100
50
0
-7.0
-6.5
-6.0
-5.5
-5.0
-4.5
LogLog
[NE][NA]
(M) (M)
Obese: P(+)E(+)
Obese: P(+)E(+) with SOD+CAT
Healthy: P(+)E(+)
% KPSS constriction
B
200
150
100
50
0
-7.0
-6.5
-6.0
-5.5
-5.0
-4.5
Log
Log
[NE][NA]
(M) (M)
Obese: P(+)E(-)
Obese: P(+)E(-) with SOD+CAT
Healthy: P(+)E(-)
159
Supplementary figures
Figure 1
Diastolic BP (mmHg)
150
125
100
75
0
50
100
150
200
PVAT/ No PVAT constriction (%)
Figure 2
160
250
Supplemental methods
Comparative profiling by label-free MS
Frozen tissue was resuspended in a minimal volume of lysis buffer containing 8M Urea,
2M Thiourea, 4% (w/v) CHAPS, 65mM DTT. Lysate was clarified at 10,000rpm in a
microfuge for 3 mins to remove particulates, and protein lysate was collected taking care
to avoid the lipid layer in top of the sample. Protein was diluted 1:10, protein
concentration determined using a Bradford assay, then further diluted to a final
concentration of 1mg ml-1. Fifty μg of protein was then digested by addition of 50μL
200mM ammonium bicarbonate and 25μL of 0.5% (w/v) Rapigest (Waters). Proteins
were reduced by addition of 10μL 75mM DTT and incubation at 80°C for 15 mins.
Samples were cooled and alkylated using 10μL 150mM iodoacetamide in the dark for 30
mins. Trypsin was added in 50mM ammonium bicarbonate to a final enzyme:substrate
ratio of 1:25, and incubated at 37°C overnight. Following digestions, peptides were
cleaned up using a 96-well strong anion exchange plate (Bio-Rad) following the
manufacturers instructions.
For each sample, an amount corresponding to 0.15µg of total protein was injected onto an
HPLC Chip (High Capacity Loading, 150mm 300 Å C18 chip with 160 nL trap column;
Agilent Technologies). Peptides were eluted using a gradient of 5-35% acetonitrile over 65
minutes at 300 nL min-1. Eluted peptides were detected using a 6510 Q-ToF mass
spectrometer, with MS1 data collected in profile mode and MS2 data in centroid..
Mass, retention time and area of each peak in each sample were extracted using
MassHunter (Agilent) and were analysed using GeneSpring (Agilent). Peaks of interest
were identified by tandem MS using a targeted method where peptides were ordered by
retention time and assigned sequentially to one of five target lists. LC-MS/MS methods
161
were built using these lists as MS/MS target lists. MS/MS data were searched against
mouse and human protein databases using SpectrumMill software (Agilent).
Selected Reaction Monitoring (SRM) determination of Adiponectin levels
Adiponectin levels were determined by measuring the levels of two peptides in adipose
tissue digests prepared as above. Peptide standards GDIGETGVPGAEGPR and
IFYNQQNHYDGSTGK were synthesized with stable isotope labels on their terminal
residues, using 13C6,15N4-Arg and 13C6,15N2-Lys respectively. A pool of aliquots from all
samples in the study was used to optimise SRM transitions and calculate appropriate
spiking volumes (GDIGETGVPGAEGPR at 1 fmol µl-1, IFYNQQNHYDGSTGK at 55.5
fmol µl-1). Digested samples were spiked and 4µL analysed using a Tempo
chromatography system (AB Sciex) coupled to a 4000 QTrap hybrid triple
quadropole/linear ion trap MS (AB Sciex) in random order. Peptides were separated using
a PepMap 100-C18 150mm,75µm i.d. column using a gradient of water with 0.1% formic
acid and an increasing concentration of acetonitrile from 5% to 40% over 20 minutes at
300 nL min-1. Data was acquired using MRM transitions detailed in Supplementary Data
Table 1. Data was extracted by manually integrating peaks to ensure veracity. Values were
normalised to overall protein content using the total peak area from the label free
quantification experiments. Data was then log transformed prior to statistical analysis.
162
Peptide
GDIGETGVP
GAEGPR
GDIGETGVP
GAEGPR
IFYNQQNH
YDGSTGK
IFYNQQNH
YDGSTGK
Charge
state (z)
Transition
(endogenous)
Transition
(labelled)
Dwell
time (ms)
2
706.34/683.35
711.34/693.35
300
Collision
energy
(eV)
41
2
706.34/1126.55
711.34/1136.55
300
41
15.3
3
591.27/449.24
593.94/457.24
300
30
14.7
3
591.27/727.33
593.94/735.33
300
30
14.7
Table 1. SRM transitions for quantification of adiponectin
163
Retention
time
(mins)
15.3
CHAPTER 5
Adipose Tissue Inflammation is More Pronounced and High Density Lipoprotein
Antioxidant Function is Impaired in Obese Patients with Obstructive Sleep Apnea
Authors:
Rahul Yadav1,2, Reza Aghamohammadzadeh1, Michael France1,
2, 3
, Yifen Liu1,
Salam Hama1, See Kwok1, 2, Jonathan Schofield1,2, Peter Turkington4, Akheel A.
Syed4, Rayaz Malik1, Adam Greenstein1, Paul Durrington1, Basil Ammori5, Maria
Jeziorska1, Handrean Soran1,2.
Institutions: 1Cardiovascular Research Group, Core Technologies Facility, University of
Manchester; UK; 2Cardiovascular Trials Unit and 3Department of Clinical Biochemistry,
Central Manchester University Hospitals NHS Foundation Trust, Manchester, UK;
4
Department of Medicine, and 5Surgery, Salford Royal NHS Foundation Trust, Salford,
UK.
Corresponding author:
Dr Handrean Soran MSc, MD, FRCP
Consultant Physician & Endocrinologist and Lead Cardiovascular Trials Unit, University
Department of Medicine, Central Manchester University Hospitals NHS Foundation Trust,
Manchester, M13 9WL, UK.
E-mail: [email protected]; [email protected]
Tel: 0044 (0) 161 276 4066/4348, Fax: 0044 (0) 161 276 3630.
164
Abstract
Background: Obstructive sleep apnea (OSA) is a common disorder in morbidly obese
patients and is associated with oxidative stress and development of cardiovascular disease.
Tumor necrosis factor α (TNFα), a mediator of vascular inflammation and paraoxonase 1
(PON1), an antioxidant enzyme associated with HDL may influence each other’s role
inversely but their relation has not been studied in OSA. Moreover, the relation between
obesity, inflammation (TNFα in particular) and PVAT function has been studied.
However, PVAT function in relation to OSA has not been assessed
Methods: Matched 41 morbidly obese patients were divided into two groups based on the
severity of apnea (high or low apnea-hypo-apnea index (AHI) groups) and on presence of
OSA (“OSA” and “no OSA or nOSA” group). HDL’s in vitro antioxidant function, serum
PON1 activity, TNFα and intercellular adhesion molecule 1 (ICAM-1) were assessed. In a
subset of 19 patients we immunostained gluteal subcutaneous adipose tissue (SAT) for
TNFα expression, macrophages and measured adipocyte size. PVAT function was
assessed in OSA (n= 5) verus no OSA (n= 5).
Results: The in vitro HDL lipid peroxide levels were higher and serum PON1 activity was
lower in the high AHI group versus the low AHI group (p<0.05 & p<0.0001) and in the
OSA group versus the “nOSA” group (p=0.005 & p<0.05). Serum TNFα and ICAM1
levels and TNFα expression in SAT increased with the severity of OSA. PON1 inversely
correlated with AHI, whereas TNFα expression in SAT directly correlated with AHI.
Presence of PVAT had no significant effect on vessel activity in OSA or nOSA groups
(p>0.05)
Conclusion: Serum PON1 activity and HDL’s capacity to protect itself from in vitro
oxidation in morbidly obese patients are impaired with the presence and severity of OSA.
The differences in serum TNFα, ICAM-1 and TNFα expression in SAT suggest enhanced
165
inflammation due to OSA.
Reduced PON1 activity in these patients could be a
mechanism for HDL and endothelial dysfunction.
Despite lower background
inflammation, there is no difference in PVAT anticontractile function in morbidly obese
individuals with no OSA.
166
Introduction
Early in atherogenesis, monocytes adhere to arterial endothelium [1, 2] via adhesion
molecules such as intercellular adhesion molecule 1 (ICAM-1) [3, 4]. Mast cells,
neutrophils, macrophages and adipose tissue release pro-inflammatory cytokines such as
tumor necrosis factor α (TNFα) that induce expression of adhesion molecules in
endothelium and recruitment of leukocytes, which is essential for the pathogenesis of
vascular inflammatory diseases [5]. Products of phospholipid peroxidation originating
from oxidized low density lipoprotein (oxLDL) may propagate vascular inflammation by
promoting increased production of TNFα and ICAM-1 [6]. Paraoxonase 1 (PON1)
associated with high density lipoprotein (HDL) has anti-oxidant and anti-inflammatory
potential mainly by protecting phospholipids on the surface of HDL [7] and LDL from
peroxidation [8, 9]. These protective properties depend on the peroxidase and esterase
activity of PON1 allowing the detoxification of oxidized molecules such as phospholipids
and lipid hydroperoxides safely [10, 11].
HDL cholesterol (HDL-C) is inversely associated with ICAM-1 concentration in
individuals with low plasma HDL-C [12]. HDL, incubated with human umbilical vein
endothelial cells (HUVEC) before and after stimulation with TNFα, inhibits TNFα
induced expression of ICAM-1 [13]. Jurek et al have examined the ability of HDL from
dialysis patients to suppress the expression of adhesion molecules in endothelial cells and
in monocytes and to inhibit the uptake of oxidized LDL by macrophages. This study
showed that a decreased ability of HDL to suppress expression of adhesion molecules in
endothelial cells was a potential risk factor for atherosclerosis development. This could be
due to oxidative modification of HDL and the significantly reduced PON1 activity in these
patients [14]. In vitro studies have demonstrated that purified PON1 had an antiinflammatory effect in the presence of HDL, causing a 3- fold reduction in ICAM-1 levels.
167
PON1 also significantly decreased TNFα and oxidized phospholipid induced ICAM-1
expression [15]. This indicates PON1 associated with HDL is required for its anti-oxidant
and anti-inflammatory functions. Apolipoprotein M (apoM) has also been proposed to
assist HDL in its anti-oxidant and vasculo-protective functions [16, 17].
Perivascular adipose tissue conveys an anticontractile effect on adjacent small vessels, but
this effect is not observed in obesity [30, 38]. This loss of function is attributed, in part, to
the elevated inflammatory state in obesity. Given the heightened inflammatory state in
OSA verus nOSA, it would be intuitive to compare PVAT function in these two groups.
In summary, there is evidence that oxidatively modified HDL has low PON1 activity and
is unable to remove lipid peroxides originating from oxidized LDL, from the site of
atheroma formation. This could lead to activation of monocytes and macrophages which
release TNFα, promoting recruitment of monocytes from the circulation by inducing
expression of adhesion molecules on the endothelial cell surface. This may initiate the
process of atheroma formation and propagate a vicious circle as it has been suggested that
TNFα also influences PON1 and HDL anti-oxidant function [18].
Many studies have reported an independent correlation between the presence and severity
of obstructive sleep apnea (OSA) and cardiovascular diseases [19] like hypertension [20],
coronary artery disease [21, 22] and stroke [23]. The chronic state of intermittent hypoxia
due to OSA can lead to increased oxygen free radical stress [24] and endothelial
dysfunction [25]. This process may propagate atherogenesis. Lipid peroxidation occurs in
patients with OSA [26]. There is evidence that in patients with OSA, the HDL is
dysfunctional [27] and the PON1 activity is reduced [28, 29]. The mechanisms that link
oxidatively modified HDL with increased atherogenesis in these patients remain unclear.
Furthermore, there is evidence that adipocytes influence local vascular tone but this
168
capacity is lost in obesity by the development of adipocyte hypertrophy, leading to
hypoxia, inflammation, and oxidative stress [30].
In this study we showed that patients with OSA may have dysfunctional HDL due to low
PON1 activity and ApoM component of HDL. Dysfunctional HDL is less able to protect
itself from in vitro oxidation and has reduced ability to protect the endothelium. We also
showed adipose tissue inflammation increases with severity of OSA and may influence
anti oxidant function of HDL.
169
Methods
Subjects
Forty one patients with body mass index (BMI) >40 kg/m2 were recruited from Salford
Royal Hospital NHS Foundation Trust (Salford, UK) obesity management clinic. All
patients underwent overnight respiratory variable polysomnography to determine the
apnea/hypo apnea index (AHI). The diagnosis of OSA was established if the AHI was
≥5/hour. Patients were divided into two groups based on the presence or absence of OSA
(“OSA” and “nOSA” group) and were also divided into two groups around the median
AHI (high or low AHI groups) in order to assess the effect of OSA and its severity on
antioxidant function of HDL. We recruited morbidly obese patients without OSA (nOSA)
that matched the OSA group for age, gender and BMI in order to avoid any bias.
Isolation of lipoproteins
Blood samples were collected between 9:00 and 11:00 after participants had fasted from
22:00 hours the previous day. Serum and EDTA-plasma were isolated by centrifugation at
2000 × g for 15 min at 4°C within 2 hours of collection and were maintained at that
temperature until further use. Serum was used for total cholesterol (TC), triglycerides
(TG) and HDL-C assays and EDTA-plasma for apolipoprotein B100 (apoB).
HDL (density range 1.063-1.21g/mL) was isolated by density gradient ultracentrifugation
without the addition of EDTA in a Beckman preparative M8-55M ultracentrifuge with a
50.4Ti fixed angle rotor at speeds of 34,000 rpm (144,361 × g) for 22 h [31, 32]. The
isolated HDL fraction was dialyzed against Tris buffered saline (TBS) overnight at 4 °C.
HDL protein concentration was determined using bicinchoninic acid [33].
In vitro studies
Susceptibility of HDL to oxidation by copper in vitro was assessed immediately after
isolation by incubating 0.25 mg protein/mL of HDL fraction with 5 µl copper for 3 hours
170
at 37ºC in a Gallenkamp Economy Size 1 Incubator (Gallenkamp, Leicester, UK). Lipid
peroxide (LPO) production was measured by spectrophotometry at 365 nm at baseline and
3 hours [34]. Group comparisons were made between OSA and ‘nOSA’ controls and
between OSA patients with AHI above and below the median.
Other laboratory analyses
Cholesterol and triglycerides (TG) were determined using cholesterol oxidase phenol 4aminoantipyrine peroxidase (CHOD-PAP) and glycerol phosphate oxidase phenol 4aminoantipyrine peroxidase (GPO-PAP) methods respectively, using reagents from ABX
Horiba-UK, Northampton, UK. HDL-C was measured by a direct second-generation
homogeneous method (Roche Diagnostics, Burgess Hill, UK). Low density lipoprotein
cholesterol (LDL-C) was estimated using Friedewald Formula. Apolipoprotein B100
(apoB) and apolipoprotein A1 (apo A1) were assayed immunoturbidimetrically. A Cobas
Mira auto-analyzer (ABX Horiba-UK, Northampton, UK) was employed for all these
assays. Serum PON1 was determined by a semi-automated micro-titre plate method using
paraoxon (O,O-Diethyl O-(4-nitrophenyl) phosphate) as a substrate and read by
spectrophotometer at 405 nm [35].
Small dense LDL apoB (sdLDL) (density range 1.044 – 1.063g/mL) was isolated from
plasma adjusted to density of 1.044g/mL and at 100,000 rpm (435,680 x g) for 5h at 4C
using a Beckman Optima TLX bench top ultracentrifuge fitted with TLA 120.2 fixed
angle rotor (Beckman Coulter UK, High Wycombe, UK). Glycated haemoglobin (HbA1c)
and fasting blood glucose were measured using the standard laboratory methods of the
Department of Clinical Biochemistry, Central Manchester University Hospitals NHS
Foundation Trust. Insulin was determined in plasma using Mercodia ELISA kits from
Diagenics Ltd, Milton Keynes, UK. Homeostatic model assessment was used to assess
beta-cell function (HOMA-β) and insulin resistance HOMA-IR using formulas (HOMA-β)
171
= (20 X insulin (mU/l))/(glucose (mmol/l) – 3.5) and HOMA-IR = (insulin (mU/l) X
glucose (mmol/l))/22.5 [36]. TNFα and adiponectin were measured in serum, and ICAM-1
in plasma all using DuoSet® ELISA development kits from R&D Systems (Abingdon,
UK). Apo M was assayed in serum using Bluegene E01A0522 kits from Hölzel
Diagnostika Handels GmbH, Köln, Germany.
Subcutaneous adipose tissue biopsies and immunohistochemistry
Gluteal subcutaneous adipose tissue (SAT) biopsies were obtained from a subset of 19
individuals (13 with OSA and 6 without OSA) in order to assess the effect of apnea on
adipose tissue inflammation. Tissues were routinely processed to paraffin wax blocks. The
adipose tissue inflammation was assessed on 5µm tissue sections immunostained for
TNFα and the presence of macrophages. Immunostaining and adipocyte size were
assessed using quantitative image analysis.
Adipocyte size was quantified on microphotographs captured on Zeiss Axio Imager M2
microscope equipped with AxioCam camera and AxioVision Rel. 4.8 programme by freehand tracing the margins of 100 consecutive cells (on average) per case to avoid selection
bias. Colour images were captured on the Leitz Diaplan microscope equipped with a
digital G0-3 Q Imaging camera. Cells staining with the CD68 (KP1) macrophage marker
were counted and the results expressesd as cells/mm2. Colour microphotographs of TNFα
immunostained adipose tissue were converted to greyscale assessed using a macro
subroutine in an ImagePro version 6.2 image analysis programme (MediaCybernetics UK,
Marlow, UK) and the results were expressed as a percentage of the entire area
photographed.
172
Assessment of PVAT function
Small subcutaneous arteries measuring 250-400µm internal diameter were dissected from
gluteal fat samples from 5 individuals with and 5 without OSA and vessels contractility
was assessed using wire myography as previously described [30]. In brief, segments of
vessel measuring approximately 6mm were isolated from each sample, and split into 2
segments. One segment was prepared with the perivascular adipose tissue intact, and the
adjacent segment with PVAT removed. Vessel contractility to noradrenaline was assessed
by constructing concentration-response curves in order to measure the effect of PVAT on
contraction of adjacent vessels.
Statistical analyses
The distribution or normality of data was determined using Kolmogorov-Smirnov test,
D’Agastino and Pearson omnibus normality test and Shapiro-Wilk normality test on
GraphPad Prism version 5.03 (GraphPad Software Inc., California, USA). Statistical
significance of differences between groups was determined using the Student’s t-test (for
parametric data) or Mann Whitney test (for non parametric data), taking p< 0.05 as
statistically significant. Statistical significance of correlations was determined using
Spearman’s test, taking p< 0.05 as statistically significant. Results were expressed as mean
± standard deviation (SD) for parametric data and median (interquartile range) for nonparametric data. SPSS statistical software version 16.0 (SPSS, Illinois, USA) was used for
all comparisons and correlations. We did not use the multiple regression models to study
the strength of relationships between various parameters in this study due to the relatively
small number of patients.
Vessel contractility to NA and PVAT function was analaysed using two-way ANOVA
analysis performed by GraphPad Prism v5.03, as above.
173
Results
Low and high AHI groups: demographics, lipid profile, HDL in vitro antioxidant
function and factors affecting vascular inflammation
The two groups were well matched for age, gender, smoking history, body mass index
(BMI), waist circumference, blood pressure, equivalent rosuvastatin dose and prevalence
of diabetes, hypertension and cardiovascular disease (Table 1). There was no significant
difference in the number of patients on insulin or insulin sensitizers like metformin and
glitazones. There were no significant differences between the high and low AHI groups in
TC, TG, HDL-C, LDL-C , apoB, apoA1 , sdLDL apoB , HbA1c , HOMA β and HOMAIR respectively (Table 1).
LPO concentration at 3 hours on incubating HDL with copper was higher in the high AHI
group at 30 (14 – 43) nmol/mL compared to the low AHI group at 17 (6 – 31) nmol/mL;
(p<0.05) (Figure 1). PON1 activity was significantly lower in the high AHI group
compared with the low AHI group (101 ± 64 vs. 186 ± 68 nmol/mL/min; p<0.0001).
ApoA1 and apoM were lower in the high AHI group compared with the low AHI group
but the difference was not statistically significant (121 vs. 129 mg/dl; p=0.3; 31 vs. 37
mg/l, p=0.2 respectively) (Table 2). TNFα and ICAM-1 were significantly higher in the
high AHI group compared with the low AHI group {87 vs. 15.5 pg/mL, p<0.05; 267 vs.
218 ng/mL, p<0.0001 respectively). There was no significant difference in the levels of
adiponectin between the two groups (high AHI vs. low AHI; 2.1 vs. 2.0 mg/l, p=0.5)
(Table 2).
Subcutaneous adipose tissue (SAT) data (table 3)
To assess the effect of severity of apnea on adipose tissue inflammation, the subset of 19
patients from whom SAT biopsies were obtained, were divided into two groups based on
median AHI. There was no significant difference in the adipocyte size and the number of
174
cells staining with CD68 (KP1) marker for macrophages in the two groups (7592 vs. 6139
µm2; p=0.12 and 7.3 vs. 6.6 per mm2 respectively). On immunostaining the adipose tissue,
the expression of TNFα as a percentage of the area examined when measured at the same
grey pixel range in both groups revealed significantly greater staining in the high AHI
group as compared with low AHI group (0.8 vs. 0.6; p=0.05) (figure 3).
Obese OSA and “nOSA” groups: demographics, lipid profile, HDL in vitro antioxidant
function and factors affecting vascular inflammation
The two groups were matched for age, gender, smoking history, BMI, waist
circumference, blood pressure, equivalent Rosuvastatin dose and prevalence of diabetes,
hypertension and cardiovascular disease. There was no significant difference in the
number of patients on insulin or insulin sensitizing drugs. The AHI scores were
significantly higher in the OSA group compared to the nOSA group (13.5/hour vs.
3.8/hour; p<0.0001). There was no significant difference between the two groups in TC,
TG, HDL-C, LDL-C, apoB, apoA1, HbA1c, HOMA β and HOMA-IR. SdLDL was
significantly higher in OSA group compared to nOSA group (18.3 vs. 11.7 mg/dl, p<0.05).
The in vitro LPO concentrations at 3 hours on incubating HDL with copper in OSA group
were significantly higher at 31 (15 – 38) nmol/mL compared with the nOSA group at 9 (2
– 19) nmol/mL; (p= 0.005) (Figure 2). PON1 activity and apoM were significantly lower
in the OSA group compared with the nOSA group {127 vs. 187 nmol/mL/min; p<0.05 and
31 vs. 49 mg/l, p<0.05 respectively}. ApoA1 did not differ significantly between the OSA
and nOSA group (123 vs. 128 mg/dl). PON1 activity was inversely correlated to AHI in
the OSA group (Spearman’s correlation coefficient = -0.41, p=0.03). TNFα and ICAM-1
were significantly higher in the OSA group than in the nOSA group (66 vs. 15 pg/mL;
p<0.05 and 255 vs. 163 ng/mL, p<0.0001 respectively). There was no significant
175
difference in the levels of adiponectin between the two groups (OSA vs. nOSA; 2.0 vs. 2.1
mg/l).
Effect of PVAT on vessel contractility
Vessel segments with intact PVAT constricted to a similar degree as compared with
segments without PVAT (p>0.05, figure 4). This was true in vessels from both OSA and
nOSA groups.
176
Discussion
In this study of morbidly obese patients with or without OSA we found impaired HDL
antioxidant function, lower PON1 activity and apoM concentration associated with
presence of OSA and increasing severity of OSA. The changes in serum TNFα and
ICAM1 blood levels and TNFα expression in SAT suggest enhanced inflammation due to
OSA and increasing AHI. Reduced PON1 activity in these patients, associated with
enhanced oxidative stress and increased TNFα expression could be a mechanism for HDL
and endothelial dysfunction. However, PVAT did not affect vessel contractility in either
group.
We have shown that HDL’s in vitro antioxidant capacity is reduced in morbidly obese
patients with OSA compared with matched patients without OSA. This function of HDL
diminishes with increasing severity of OSA. Our results confirm findings from previous
studies that the presence and increasing severity of OSA leads to reduction in PON1
activity [28, 29]. However, the previous studies were not matched for basic characteristics
like BMI, gender and age. We recruited age, gender and BMI matched patients in order to
avoid bias.
Others have demonstrated HDL dysfunctionality in OSA patients by its inability to
prevent in vitro LDL oxidation or a lower PON1 activity in the OSA group. The degree of
dysfunctionality of HDL and levels of oxidised LDL were directly related to AHI (which
reflects the severity of OSA) [27]. We have shown that lower PON1 activity contributes to
HDL dysfunction in these patients. PON1 was lower in the OSA and the high AHI groups
compared to the obese nOSA and low AHI groups respectively. For the first time we have
reported that the serum PON1 activity has an inverse relation to AHI. These results
suggest that PON1activity is increasingly affected by the oxidative stress associated with
the presence and increasing severity of OSA. As PON1 is known to play an important role
177
in HDL’s antioxidant function, it is likely that the diminished PON1 activity due to OSA
contributed to HDL’s reduced antioxidant function.
ApoM and apoA1also play a role in HDL’s anti-oxidant function through distinct
pathways. We show that apoM concentrations are lower in the OSA and high AHI groups,
possibly contributing to the diminished anti-oxidant function of HDL. ApoA1 did not
differ significantly between the two groups. Similar result was obtained in a previous
study [27], suggesting that ApoA1concenteration may not be affected by the excessive
oxidative stress in sleep apnea. OSA does not affect the levels of adiponectin and insulin
sensitivity, consistent with a recent study showing an association between adiponectin and
obesity but not OSA [37].
Obesity is a state of chronic inflammation where adipocytes hypertrophy faster than the
rate of angiogenesis to oxygenate the cells, hence they exist in a state of relative hypoxia
which leads to inflammatory differentiation of macrophages and secretion of TNF-α [38].
In animal studies it has been shown that in conditions that mimic OSA, there is a 2.5 to 3
fold increase in TNFα secretion by adipose tissue [39].
In the present study, we have shown greater levels of the circulating inflammatory
biomarker TNFα in the OSA group as compared with the nOSA group, as well as higher
levels in the high AHI group compared with low AHI. In keeping with this, adipose tissue
staining for TNFα increased with severity of OSA, thus suggesting that within the obese
population, higher AHI and OSA result in a more inflammatory state as compared with
low AHI and without OSA. Adipose tissue macrophage numbers increase in obesity and
macrophages are responsible for most of the TNFα within adipose tissue [40]. In this study
we observed much stronger staining for TNFα around the macrophages with increasing
AHI. There was no statistical difference in adipocyte size and macrophage number
between the two groups. However, this may be due to the small numbers of patients from
178
whom gluteal SAT biopsy was obtained, which is a limitation of this study. Whilst CD68
is a widely used marker for macrophages [41] a more specific stain for activated
macrophages may help explain this anomaly in macrophage numbers.
We have shown previously that, in health, PVAT exerts an anticontractile effect on
adjacent small vessels and that in obesity this effect is not observed [30]. Moreover, we
have shown that following weight loss surgery, PVAT exerts an anticontractile effect on
its adjacent small vessels, in keeping with observations of a reduction in macrophage
numbers and TNFα staining in PVAT and a reduction in adipocyte size [38]. Thus given a
higher grade of background inflammation in OSA versus nOSA, we assessed PVAT
function in the two groups and did not observe a difference in PVAT function between the
two groups. This may be explained by the fact that despite the difference in OSA status,
both groups are morbidly obese and adipocyte size in the two groups are similar and
significantly larger than those in healthy individuals [38]. Also, there was not a significant
difference in macrophage numbers between the two groups and whilst TNFα, secreted by
macrophages has vasoactive effects [30], there may well be other inflammatory factors
secreted by macrophages which result in increased vessel contractility contributing to the
loss of PVAT anticontractile function that remain to be assessed. One limitation of this
study is the small number of vessels studied.
We report a relation between PON1 activity and TNFα in OSA patients, which may be
causative. Adenovirus based over-expression of human PON1 in apolipoprotein E knockout mice is associated with reduced TNFα levels [42]. PON1 activity is correlated to
serum TNFα levels in atheromatous patients with rheumatoid arthritis [43] and TNFα
antagonist therapy leads to enhanced PON1 levels, heightened anti-oxidant capacity of
HDL and a reduced inflammatory status [18]. Alternatively, a study showing low PON1
179
activity in HDL obtained from coronary disease patients suggests that reduced PON1
activity may lead to increased action of TNFα. This could result in activation of
endothelial protein kinase C beta 2 (PKC β2) pathways and loss of endothelium’s ability
to resist TNFα mediated expression of adhesion molecules [44].
Moreover, low PON1 activity may be one of the initial triggers for development of OSA.
Reduced PON1 activity leads to accumulation of lipid peroxides and increased oxidative
stress [8]. Free radicals can cause disruption of the blood brain barrier and reach the brain
where they can damage the neurons [45]. It would be of interest to study the antioxidant
capacity of HDL in CSF where it is the only lipoprotein present [46].
Finally we conclude that serum PON1 activity and capacity of HDL to protect itself from
in vitro oxidation in morbidly obese patients are impaired with the presence and increasing
severity of OSA. The differences in serum TNFα, ICAM-1 and TNFα expression in SAT
suggest enhanced inflammation due to OSA. Reduced PON1 activity in these patients
could be a mechanism for HDL and endothelial dysfunction. Further studies are needed to
elucidate the interaction between PON1, adipose tissue inflammation and TNFα in this
important group of patients.
180
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183
Table 1
High AHI group
(n=20)
21.3 (13.5 – 45.7)
Low AHI group
(n=21)
4.3 (3.7 – 6.8) ***
49 ± 10
45 ± 9
15/85
20/80
Smokers (%)
30
20
Hypertension (%)
65
50
Cardiovascular disease (%)
25
10
Type 2 diabetes (%)
30
30
Body Mass Index (kg/m2)
52 ± 6
50 ± 8
Waist circumference (cm)
143 ± 14
140 ± 18
Systolic BP (mmHg)
139 ± 23
139 ± 19
Diastolic BP (mmHg)
76 ± 13
75 ± 10
40
40
Total cholesterol (mmol/l)
4.8 (4.5 – 5.5)
5.3 (4.7 – 5.7)
Triglycerides (mmol/l)
1.7 (1.2 – 2.0)
1.6 (1.1 – 2.1)
HDL-C (mmol/l)
1.3 (1.1 – 1.5)
1.3 (1.2 – 1.5)
LDL-C (mmol/l)
3.2 ± 1.3
3.2 ± 0.8
90 (81 – 106)
102 (89 – 123)
121 (113 – 136)
129 (122 – 138)
17.3 (10.9 – 31.4)
16.5 (11.2 – 22.6)
HbA1c (%)
6.2 (5.7 – 6.7)
6.0 (5.6 – 6.9)
HOMA – IR
6.6 (4.3 – 10.7)
6.9 (4.0 – 10.2)
HOMA – B
198.6 (95.5 –
390.4)
5
176.6 (104.7 – 299.9)
Apnea
hypo-apnea
(episodes/hr)
index
Age (years)
Gender Male/Female (%)
Statin use (%)
Apolipoprotein B100 (mg/dl)
Apolipoprotein A1 (mg/dl)
Sd LDL apoB (mg/dl)
Equivalent rosuvastatin doses in
5
patients on statins (mg/day)
Table 3. Characteristics of low AHI and high AHI groups. Values are mean ± SD or
median (interquartile range). Statistical analyses – T test for parametric and Mann
Whitney for non-parametric data. ***p<0.0001. HDL-C – high density lipoprotein
cholesterol, LDL-C – low density lipoprotein cholesterol, sdLDL apoB – small dense LDL
apolipoprotein B100, HbA1c – glycated hemoglobin, HOMA-IR – homeostatic model of
assessment – insulin resistance, HOMAβ – homeostatic model of assessment – insulin
sensitivity
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Figure 1
Figure 1. This figure demonstrates lipid peroxides (LPO) generated at 3 hours on
incubating HDL with copper ions. LPO levels in HDL from high AHI group were
significantly higher as compared to LPO generated in HDL from low AHI group at the end
of 3 hours (p<0.05). Statistical analyses – Mann Whitney test used as data was nonparametric. LPO – lipid peroxides.
185
Figure 2
Figure 2. This figure demonstrates lipid peroxides (LPO) generated at 3 hours on
incubating HDL with copper ions. LPO levels in HDL from obese OSA group were
significantly higher as compared to LPO generated in HDL from obese no OSA group at
the end of 3 hours (p = 0.005). Statistical analyses – Mann Whitney test used as data was
non-parametric. LPO – lipid peroxides
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Table 2
Apolipoprotein A1 (mg/dL)
Paraoxonase1 activity
(nmol/mL/min)
Apolipoprotein M (mg/L)
TNFα (pg/mL)
ICAM-1 (ng/mL)
Adiponectin (mg/L)
High AHI group
(n=20)
121 (113 – 136)
101 ± 64
Low AHI group
(n=21)
129 (122 – 138)
186 ± 68 ***
31 (25 – 35)
87.2 (12.4 – 133.8)
267 (237 – 280)
2.1 ± 0.8
37 (26 – 50)
15.5 (7.2 – 38.2) *
218 (157 – 245) **
2.0 ± 0.7
Table 2. Apolipoproteins and enzymes associated with HDL that promote HDL’s antioxidant capacity (for both groups). This table also shows TNFα, ICAM-1 and adiponectin
values in both groups. Values are mean ± SD or median (interquartile range). Statistical
analyses – T test for parametric and Mann Whitney for non-parametric data. Values are
mean ± SD or median (interquartile range). * p<0.05, **p=0.01*** p<0.0001. TNFα –
tumor necrosis factor alpha, IL6 – interleukin 6, ICAM-1 – intercellular adhesion
molecule 1.
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Table 3
Adipocyte size (µm2)
SAT data for high
AHI (n=10)
7592 (7034 – 7971)
SAT data for low
AHI (n=9)
6139 (4743 – 7331)
Macrophages (per mm2)
7.3 (5.6 – 7.7)
6.6 (5.0 – 12.9)
TNFα expression
0.8 (0.6 – 1.3)
0.6 (0.2 – 0.8)*
Table 3. Gluteal subcutaneous adipose tissue (SAT) data from a subset of patients (n=19).
Divided into two groups based on median AHI. Statistical analyses – Mann Whitney for
non-parametric data. *p=0.05. TNFα expressed as a percentage of the area examined when
measured at the same grey pixel range. Difference in adipocyte size and macrophage
numbers between the two groups was not statistically significant.
Figure 3
Figure 3. Immunostaining for TNFα in adipose tissue of high AHI (A) and low AHI (B)
patient. Panel A shows more intense expression of TNFα comparing to B. (Bar = 100 m)
188
Figure 4
% constrction of KCl
A
250
PVAT (No OSA)
No PVAT (No OSA)
200
150
100
50
0
-8 -7.5 -7 -6.5-6.3 -6 -5.7-5.5-5.3 -5 -4.7-4.5
Log [NA] (M)
% constrction of KCl
B
250
No PVAT (OSA)
PVAT (OSA)
200
150
100
50
0
-8 -7.5 -7 -6.5-6.3 -6 -5.7-5.5-5.3 -5 -4.7-4.5
Log [NA] (M)
% constrction of KCl
C
250
PVAT (No OSA)
PVAT (OSA)
200
150
100
50
0
-8 -7.5 -7 -6.5-6.3 -6 -5.7-5.5-5.3 -5 -4.7-4.5
Log [NA] (M)
Figure 4: Presence of PVAT did not significantly alter vessel constriction in morbidly
obese individuals without OSA (A) or with OSA (B) (P>0.05, n=5). Comparison of vessel
contractility of segments with intact PVAT from those with OSA or No OSA (P>0.05,
n=5)
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Discussion
The overarching aims of the protocols comprising this thesis were to assess the effects of
obesity on perivascular adipose tissue function, the contribution of inflammation in this
process and whether the alterations in function could be reversed following weight loss
surgery.
The principal observations were derived from the functional assessment of vessel
segments harvested from human and rodent samples and studied using wire myography.
Further collaborative and complimentary data were collated using immunohistochemistry,
proteomics and lipid studies.
The main findings from each chapter are summarised here.
Effects of obesity and weight loss surgery on PVAT function
This study assessed the effect of weight loss (bariatric) surgery on the vasodilatory
properties of PVAT. We confirmed that in severely obese patients, whilst there is an
increase in adipose tissue volume, the beneficial PVAT anticontractile effect is abolished
1
. There are three novel findings presented here. First, bariatric surgery achieves a
dramatic degree of weight loss within only six months and reverses the obesity-induced
damage to PVAT anticontractile function. Second, the functional recovery of PVAT is
independent of the endothelium. This was confirmed by solely studying vessels denuded
of endothelium, thus eliminating obesity-induced endothelial damage as a potential
confounding factor. Third, bariatric surgery restores the function of PVAT by reducing
adipose tissue inflammation and increasing local adiponectin and nitric oxide
bioavailability. These findings advance our understanding of the mechanisms by which
190
obesity disrupts the vasodilating function of adipose tissue and confirm the relevance of
enhanced adiponectin levels and nitric oxide bioavailability in the reversibility of PVAT
anticontractile function following bariatric surgery. Future work needs to elucidate
whether other factors including a change in the metabolic status following surgery plays a
role.
Contribution of inflammation and obesity-induced endothelial damage to PVAT
function
This study investigated the effects of obesity on PVAT function and assessed the contribution of
nitric oxide and endothelium in more detail. We have shown previously that PVAT vasorelaxant
effect is restored following significant weight loss 2. Here we observed four novel findings. First,
using proteomic analysis, we show that levels of adiponectin and the free radical scavengers SOD
and peroxiredoxin-1 are significantly lower in the PVAT from obese individuals as compared with
healthy controls. We have reported previously that adiponectin secreted from PVAT has a major
vasorelaxant effect on adjacent small arteries1 and the data presented in this study confirms that
PVAT adiponectin levels are lower in the obese as compared with the healthy. Moreover, our
proteomic data show that levels of the free radical scavengers are significantly lower in the obese
patients as compared with the healthy lean volunteers. Macrophages present within PVAT
contribute to the inflammatory environment by secreting superoxide anions. The accumulation of
macrophages in the obese PVAT 2 means a higher level of background superoxide levels; a
reduction in free radical scavenger enzymes contributes to accumulation of inflammatory
mediators such as hydrogen peroxide which has adverse vasoactive effects on adjacent vessels 3.
Second, in healthy animals, there was preservation of PVAT anticontractile capacity after removal
of the endothelium, thus confirming observations by Gao et al4 that PVAT effect is not dependent
on endothelial status. Third, Nitric oxide from PVAT is an important endothelium-independent
191
contributor to PVAT vasorelaxant function in healthy tissue which is not observed in obesity.
Fourth, PVAT function correlates with systemic blood pressure and obesity-induced damage to
PVAT vasorelaxant quality correlates with a rise in BP.
Assessment of the effects of sleep apnoea in obesity on PVAT function
Obstructive sleep apnoea affects a large proportion of morbidly obese individuals and
contributes to elevated cardiovascular risk factors. This has been attributed to increased
prevalence of hypertension secondary to high circulating aldosterone and leptin levels, as
well as increased oxidative stress and sympathetic nervous system overstimulation5, 6.
Given the significant contribution of OSA to cardiovascular risk and its link to
hypertension, this study assessed whether there was a difference in inflammation within
the PVAT and if this translated to a difference in PVAT function between those with and
without OSA. We observed higher levels of circulating and adipose tissue TNFα in those
with OSA, however there was no significant difference in adipocyte size or macrophage
numbers within the PVAT in those with high apnoeic scores as compared to those with
low AHI scores. Similarly, no difference in circulating adiponectin was observed
depending on the severity of apnoea. PVAT did not exert an effect on vessel contractility
in either group and this can be explained by the fact that despite a lower level of TNFα in
serum and adipose tissue in those without OSA, the individuals remain obese with high
numbers of adipose tissue macrophages, low adiponectin levels and large adipocytes, thus
eliminating the possibility of any discernible PVAT effect between the two groups using
wire myography. A larger sample size may prove beneficial to assess this further.
192
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