Coronary Microvascular Endothelial Cell Redox State in
Left Ventricular Hypertrophy
The Role of Angiotensin II
Derek Lang, Salah I. Mosfer, Alison Shakesby, Francis Donaldson, Malcolm J. Lewis
Downloaded from http://circres.ahajournals.org/ by guest on July 31, 2017
Abstract—Left ventricular hypertrophy (LVH) is associated with elevated plasma angiotensin II (Ang II) levels and
endothelial dysfunction. The relationship between Ang II and endothelial dysfunction remains unknown, however, but
it may involve an alteration in endothelial cell redox state. We therefore investigated the effect of Ang II on
NADH/NADPH oxidase–mediated superoxide anion (O2⫺) production by cultured guinea pig coronary microvascular
endothelial cells (CMVEs) and CMVEs freshly isolated from a guinea pig, pressure-overload model of LVH. Lucigenin
chemiluminescence was used to measure O2⫺ production in the particulate fraction of CMVE lysates. In cultured cells,
incubation with Ang II (0.1 nmol/L to 1 ␮mol/L for 18 hours) resulted in significant (P⬍0.01) increases in both NADHand NADPH-dependent O2⫺ production, with a peak effect at 1 nmol/L. The latter was significantly (P⬍0.01) inhibited
by the AT1 receptor antagonist losartan (1 ␮mol/L for 18 hours). In contrast, the O2⫺ response to Ang II (0.1 nmol/L
to 1 ␮mol/L for 18 hours) was largely unaffected by concomitant exposure to the AT2 antagonist PD 123319 (1 ␮mol/L).
In freshly isolated CMVEs from nonoperated animals, NADH- and NADPH-dependent O2⫺ production was not different
from that in sham-operated animals but was significantly (P⬍0.05) elevated in the aortic-banded animals. Plasma Ang
II levels were significantly (P⬍0.001) elevated in the aortic-banded (1.25⫾0.12 ␮g/L, n⫽12) compared with
sham-operated animals (0.63⫾0.06 ␮g/L, n⫽12). These data suggest that the endothelial dysfunction associated with
LVH may be due, at least in part, to the Ang II–induced upregulation of NADH/NADPH oxidase-dependent
O2⫺ production. (Circ Res. 2000;86:463-469.)
Key Words: NADH/NADPH oxidase 䡲 coronary microvascular endothelium 䡲 angiotensin II
䡲 left ventricular hypertrophy 䡲 superoxide anion
R
aised circulating levels of angiotensin II (Ang II) are
associated with a number of pathological states, such as
hypertension,1 congestive heart failure,2 and nitrate tolerance.3 Although the vasoactive effects of Ang II have been
well described for ⬎40 years,4 recent evidence suggests that
this peptide has many other effects on the cardiovascular
system. For example, Ang II has been shown to promote
vascular hypertrophy,5,6 stimulate the Janus kinase/signal
transducers and activators of transcription pathway,7 activate
the gene transcription of proto-oncogenes including c-fos,8 –10
induce cardiac hypertrophy,11–13 and modulate cardiac
remodeling.14
Another important action of Ang II is the induction of
superoxide anion (O2⫺) production by vascular smooth muscle cells via the stimulation of the NADH/NADPH oxidase
system.15 It is known that these oxygen free radicals are
intimately involved in the inactivation of endotheliumderived nitric oxide (NO).16 –18 The question remains, however, whether Ang II has a similar effect on this free
radical– generating enzyme system in endothelial cells
themselves.
It is becoming increasingly apparent that in disease states
affecting the cardiovascular system in which reduced NO
activity has been demonstrated, such as hypercholesterolemia,19 diabetes,20 smoking-related disorders,21 reperfusion
injury,22 hypertension,23 and possibly Alzheimer’s disease,24
that this inactivation of NO is associated with increased O2⫺
production. The general view is now emerging that it is the
balance between the release of NO and O2⫺ that ultimately
determines the level of NO activity. It is highly likely that an
imbalance in this delicate system will contribute to the
concomitant alterations in vascular tone seen in atherosclerosis and hypertension.
In all of these diseases, there are obviously many “candidate” cell types that could be involved in the overproduction
of O2⫺, eg, vascular smooth muscle cells.15 More recently,
vascular smooth muscle cells from animal models of hypertension,25 coronary artery endothelial cells,26 and rabbit aorta
Received October 18, 1999; accepted November 24, 1999.
From the Cardiovascular Sciences Research Group, Sir Geraint Evans Wales Heart Research Institute, Department of Pharmacology, Therapeutics, and
Toxicology, University of Wales College of Medicine, Cardiff, Wales.
Correspondence to D. Lang, Department of Pharmacology, Therapeutics, and Toxicology, University of Wales College of Medicine, Heath Park,
Cardiff, CF14 4XN UK. E-mail [email protected]
© 2000 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
463
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Circulation Research
March 3, 2000
adventitial tissues27,28 have also been shown to possess the
O2⫺-generating NADH/NADPH oxidase system, thus providing a potential source of O2⫺.
Given what is known about the marked effect that coronary
microvascular endothelium– derived NO has on cardiac function,29 the generation of O2⫺ by these cells may have an
important role in the pathogenesis of left ventricular hypertrophy (LVH) and could contribute significantly to the
endothelial dysfunction associated with this and other cardiovascular diseases.
The aim of this study, therefore, was to investigate the
relationship between Ang II and NADH/NADPH oxidase–
mediated O2⫺ production, firstly in vitro and secondly ex vivo
in an experimental model of LVH. We used cultured guinea
pig coronary microvascular endothelial cells (CMVEs) for
the in vitro part of this study and freshly isolated CMVEs
from a pressure-overload model of LVH in the guinea pig for
the ex vivo experiments.
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Materials and Methods
Aortic Banding
Pressure-overload LVH was induced in Dunkin Hartley guinea pigs
(200 to 250 g), as previously described.30 All experiments involving
aortic-banded and sham-operated animals were carried out 6 weeks
after operation when optimum LVH had developed. Control animals
for these experiments were matched for both age and weight, but
with no surgical intervention.
Isolation and Characterization of Coronary
Microvascular Endothelium
Guinea pig CMVEs were isolated and characterized as described
previously.31
NADH/NADPH Oxidase Assay
NADH/NADPH oxidase activity was measured essentially as described previously.15 The protein content of an aliquot of the
appropriate cellular fraction was assayed as described previously.32
A custom-built luminometer (see Reference 33) was used to detect
changes in chemiluminescence, and the output (in volts) was then
displayed on a Macintosh computer via a Maclab apparatus. The
integral for the first 10 minutes of the reaction represents the total
O2⫺ produced over this time and was normalized to fraction protein
content, and data were then expressed as volts⫻seconds/mg protein
(V.s/mg protein in Figures).
Even though CMVEs were isolated and cultured under identical
conditions, both basal (control) and Ang II–stimulated NADH- and
NADPH-dependent responses showed considerable interbatch variability. Each separate experiment was therefore performed on cells
from a single culture preparation.
Measurement of Plasma Ang II Levels
Blood (5 mL) from anesthetized guinea pigs (control, sham-operated,
and aortic-banded) was collected by cardiac puncture into prechilled
tubes containing 1.52 mg/mL EDTA and 500 kallikrein inhibitor
units/mL aprotinin immediately before removal of the heart for
CMVE isolation. It was then centrifuged at 1200g for 15 minutes at
4°C and the resulting platelet-free plasma stored in fresh plastic
tubes at ⫺70°C. After extraction, Ang II concentrations were
measured as described by a commercially available radioimmunoassay kit (Nichols Institute Diagnostics Ltd).
Ferricytochrome c Reduction
It has recently become apparent that lucigenin, under certain circumstances, can generate its own O2⫺,34,35 and its suitability for the
detection of these free radicals has been questioned.36 Therefore, to
validate our findings and confirm that the O2⫺ production measured
in this study is via NADH/NADPH oxidase activity and not from
lucigenin itself, ferricytochrome c reduction was also used.
Cultured CMVE lysates were prepared as above. O2⫺ production
was then measured using a reaction buffer of the following composition (in mmol/L): phosphate buffer (pH 7.4) 10, NaCl 100, KCl 4,
MgCl2 1.3, glucose 2, CaCl2 1, and ferricytochrome c 0.07. An
aliquot of the appropriate fraction was added to the reaction buffer in
the presence and absence of either NADH or NADPH (both
1 mmol/L) and allowed to incubate for 30 minutes at 37°C. These
additions were repeated in the presence of superoxide dismutase
(SOD, 350 U/mL). Tiron produced a nonspecific color change when
added to the reaction buffer, so it could not be used in these
experiments. The reduction of ferricytochrome c in the supernatant
was measured at 550 nm by a standard spectrophotometer.
Statistics
All data are expressed as mean⫾SEM and are compared by ANOVA
followed by either the Dunnett or Student-Newmann-Keul multiplerange test where appropriate. Significant differences are identified at
the P⬍0.05 level.
An expanded Materials and Methods section is available online at
http://www.circresaha.org.
Results
Characterization of Guinea Pig CMVEs
The endothelial nature of the freshly isolated and cultured
CMVEs used in this study was confirmed as described
previously.31 They selectively took up fluorescently labeled,
acetylated LDL, stained positively with the microvascular
endothelium-specific fluorescently labeled lectin from Lycopersicon esculentum, quickly formed tubes (as compared with
a slower rate for large-vessel endothelial cells) when grown
on Matrigel, stained negatively for smooth muscle ␣-actin,
and grew normally in D-valine– containing culture medium
(data not shown).
NADH/NADPH Oxidase–Dependent O2ⴚ
Production in Cultured CMVEs
The cytosolic fraction of the cultured (first-passage) cells
(and the freshly isolated cells) failed to produce a chemiluminescent response, either in the absence or presence of
NADH or NADPH (both 1 mmol/L). All of the following
data describe the responses of the particulate fraction of both
cell types.
Incubation of cultured cells with Ang II (0.1 nmol/L to
1 ␮mol/L) for 6 hours had no effect on control levels of O2⫺
production (data not shown). However, incubation with Ang
II for 18 hours resulted in significant (P⬍0.01) increases in
both NADH and NADPH oxidase–mediated O2⫺ production,
with the peak effect occurring at an Ang II concentration of
1 nmol/L (Figure 1). In a separate experiment, the NADH and
NADPH oxidase–mediated increases in O2⫺ production in the
presence of Ang II (1 nmol/L for 18 hours) were completely
inhibited (P⬍0.01) by the AT1 receptor antagonist losartan
(1 ␮mol/L for 18 hours) (Figure 2). In a further experiment,
cultured CMVEs were incubated for 18 hours with Ang II
(0.1 nmol/L to 1 ␮mol/L) in the absence or presence of the
AT2 antagonist PD 123319 (1 ␮mol/L). NADH and NADPH
oxidase–mediated increases in O2⫺ production in the presence
of Ang II were largely unaffected by this antagonist, although
Lang et al
LVH, Ang II, and Endothelial Superoxide
465
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Figure 1. Cultured guinea pig CMVE (particulate fraction) NADH
(1 mmol/L) (open columns)– or NADPH (1 mmol/L) (filled columns)– dependent O2⫺ production in the presence of either culture medium alone (control, C) or Ang II (0.1 nmol/L to 1 ␮mol/L
for 18 hours). **P⬍0.01 compared with control (all nⱖ3).
a significant (P⬍0.05) inhibition of the O2⫺ response to 10
nmol/L Ang II was observed (Figure 3).
The production of O2⫺ by both control and Ang II (1 nmol/L
for 18 hours)–stimulated cells was unaffected by preincubation
(10 minutes at 37°C) of the appropriate particulate fraction with
the NO synthase (NOS) inhibitor NG-nitro-L-arginine methyl
ester (L-NAME, 5 ␮mol/L), the xanthine oxidase inhibitor
oxypurinol (1 mmol/L), or the cycloxygenase inhibitor indomethacin (10 ␮mol/L) (Figure 4). In a separate experiment, O2⫺
production by both control and Ang II (1 nmo/L)–stimulated
CMVEs was significantly (P⬍0.001) inhibited by ⬎90% in the
presence of the specific O2⫺ scavenger tiron37,38 (10 mmol/L)
(Figure 5).
Figure 3. Cultured guinea pig CMVE (particulate fraction) NADH
(1 mmol/L) (A)– or NADPH (1 mmol/L) (B)– dependent O2⫺ production in the presence of either Ang II alone (0.1 nmol/L to
1 ␮mol/L for 18 hours, open columns) or Ang II and PD 123319
(1 ␮mol/L, both for 18 hours, filled columns). Controls (C) represent incubations with either culture medium alone (open column)
or PD 123319 alone (both for 18 hours, filled column). **P⬍0.01,
*P⬍0.05 compared with appropriate control; ⫹P⬍0.05 compared with Ang II alone at appropriate concentration (all nⱖ4).
No chemiluminescent response was demonstrated by any
CMVE fraction in the absence of NADH or NADPH.
Development of LVH
To assess the development of LVH, combined left/right
ventricle–to– body weight (LRV/BW) ratios were measured
in a sample (age-matched) population of control, shamoperated, and aortic-banded animals at a time point that
coincided with CMVE isolation from the rest of the animals
in the study. Combined LRV/BW ratios are shown in the
Table and demonstrate a significant (P⬍0.01) 25% increase
in the aortic-banded animals compared with the shamoperated group. There was no difference in LRV/BW ratio
between control and sham-operated animals.
Figure 2. Cultured guinea pig CMVE (particulate fraction) NADH
(1 mmol/L) (open columns)– or NADPH (1 mmol/L) (filled columns)– dependent O2⫺ production in the presence of culture
medium alone (CONTROL), Ang II alone (1 nmol/L for 18 hours),
or Ang II (1 nmol/L) and losartan (LOS 1 ␮mol/L, both for 18
hours). *P⬍0.05 compared with control; ⫹⫹P⬍0.01 compared
with Ang II alone (all nⱖ3).
Plasma Ang II Concentrations
Plasma Ang II levels were unaltered in the sham-operated
animals compared with controls (no operation) (0.63⫾0.06
versus 0.68⫾0.06 ␮g/L, n⫽12 and 10, respectively), but were
significantly (P⬍0.001) elevated in the aortic-banded animals
(1.25⫾0.12 ␮g/L, n⫽12).
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March 3, 2000
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Figure 5. Effect of the O2⫺ scavenger tiron (100 mmol/L) on
control and Ang II (1 nmol/L for 18 hours)–stimulated NADH
(1 mmol/L, open columns)– or NADPH (1 mmol/L, filled columns)– dependent O2⫺ production in cultured guinea pig CMVEs
(particulate fraction). **P⬍0.01, ***P⬍0.001 compared with control; ⫹⫹⫹P⬍0.001 compared with Ang II (all nⱖ4).
Figure 4. Effect of the NOS inhibitor L-NAME (5 ␮mol/L), the
xanthine oxidase inhibitor oxypurinol (OXYP, 1 mmol/L), or the
cycloxygenase inhibitor indomethacin (INDO, 10 ␮mol/L) (all preincubated for 10 minutes at 37°C) on control (A) and Ang II (1
nmol/L for 18 hours)–stimulated (B) NADH (1 mmol/L, open columns)– or NADPH (1 mmol/L, filled columns)– dependent O2⫺
production in cultured guinea pig CMVEs (particulate fraction)
(all nⱖ4).
NADH/NADPH Oxidase–Dependent O2ⴚ
Production in Freshly Isolated CMVEs
NADH and NADPH oxidase activity in CMVEs from control
animals (no operation) was not different from that in shamoperated animals. However, this activity was significantly
(P⬍0.05) elevated in the aortic-banded animals (Figure 6).
Again, O2⫺ production in CMVE lysates from both the
sham-operated and aortic-banded animals was unaffected by
preincubation of the lysate (10 minutes at 37°C) with
L-NAME (5 ␮mol/L), oxypurinol (1 mmol/L), or indomethacin (10 ␮mol/L) (data not shown).
Ferricytochrome c Reduction
NADH- and NADPH-dependent reduction in ferricytochrome c absorbance measured at 550 nm was significantly
(P⬍0.001) greater in cultured CMVE lysates (particulate
fraction) from Ang II–treated (1 nmol/L for 18 hours)
compared with control (Figure 7) CMVEs. The presence of
SOD (350 U/mL) caused a significant (P⬍0.05) inhibition in
absorbance seen with both NADH and NADPH.
Discussion
The microvascular endothelial nature of the cells isolated
from the perfused hearts was confirmed by techniques de-
scribed previously.31 Fixing the pericardial surface of each
heart with 70% ethanol (vol/vol) ensured that endothelial
cells from the large epicardial vessels were not part of the cell
population used in the present study. However, it is not
possible to completely exclude endocardial endothelial cells
from our cultures/cell isolates. Because the vast majority of
endothelial cells in heart tissue are contained in small vessels
and the capillary bed, the statistical average population of
cardiac endothelial cells is predominantly microvascular in
origin,39 with CMVEs vastly outnumbering endocardial endothelial cells. We can therefore be confident that the cells
under investigation in the present study were principally
CMVEs.
The data presented in this study demonstrate that Ang II
upregulates an oxygen free radical– generating enzyme system in cultured guinea pig CMVEs in a time- and
concentration-dependent manner. We have also shown that in
this pressure-overload model of LVH, upregulation of this
oxidase system is associated with increased plasma levels of
Ang II. This study also demonstrates that this enzyme(s) is
membrane bound and is activated to a greater extent by
NADH than by NADPH.
Previous studies investigating the influence of Ang II on
NADH-/NADPH-dependent O2⫺ production have shown this
Guinea Pig Combined LRV/BW Ratios
Group
Control
LRV/BW Ratio
n
0.0065⫾0.0002
15
Sham-operated
0.0065⫾0.0001
15
Aortic-banded
0.0081⫾0.00021*
15
LRV/BW ratios were measured in a sample (age-matched) population of
guinea pigs, at a time point that coincided with CMVE isolation from the
remaining animals in the study.
*P⬍0.01 vs sham-operated guinea pigs.
Lang et al
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Figure 6. Effect of aortic banding–induced LVH on freshly isolated guinea pig CMVEs (particulate fraction) NADH (1 mmol/L,
open columns)– or NADPH (1 mmol/L, filled columns)– dependent O2⫺ production. **P⬍0.01, *P⬍0.05 compared with sham
(all nⱖ5).
effect to be concentration dependent. The present findings,
however, clearly demonstrate an inverse effect of Ang II
concentration on O2⫺ generation by CMVEs. The pattern of
activation is similar to that demonstrated in neutrophils in
Figure 7. Changes in NADH (A) (1 mmol/L)– and NADPH (B)
(1 mmol/L)– dependent ferricytochrome c absorbance by cultured guinea pig CMVEs (particulate fraction) in the presence of
either culture medium alone (control) or Ang II (1 nmol/L for 18
hours), with these incubations being repeated in the absence
(open columns) and presence (filled columns) of SOD (350
U/mL). ***P⬍0.001 compared with control; *P⬍0.05 compared
with the absence of SOD (all nⱖ8).
LVH, Ang II, and Endothelial Superoxide
467
response to Ang II.40 Chemotactic migration in these leukocytes is increased at concentrations up to 0.1 nmol/L but
inhibited at those of 10 nmol/L and above. The mechanism of
this effect is unclear, and a direct comparison between these
2 cell types is clearly difficult. Nevertheless, it is perhaps not
surprising that because both leukocytes and endothelial cells
are frequently involved in cell-cell interactions, they should
both respond in a similar way to vasoactive agents such as
Ang II.
The inverse effect of Ang II concentrations outlined above
may be explained by Ang II coinducing an inhibitory process
at the same time as upregulating O2⫺ generation. For instance,
an inhibitory role for AT2s on AT1-mediated responses41,42
may exist. Such a role seems unlikely in the present study,
because the responses to the highest concentrations of Ang II
were not potentiated by the AT2 antagonist PD 123319.
Indeed, the inhibitory effect of PD 123319 on the NADHdependent response to 10 nmol/L Ang II and the general
pattern of O2⫺ generation in the presence of this antagonist
suggests that the AT2 may be involved in Ang II– dependent
oxygen free radical production by this cell type. However, a
direct and nonspecific inhibitory effect of PD 123319 on the
AT1 receptor cannot be ruled out.
It is also possible that at the higher concentrations of Ang
II, the AT1s become desensitized. Furthermore, Ang II has
been shown to stimulate the synthesis of both the endothelial43 and inducible44 isoforms of NOS. Clearly, increased NO
production in the presence of the higher concentrations of
Ang II may result in decreased detection of O2⫺ anions,
because the latter will combine readily with NO to form
peroxynitrite. The upregulation, at least of endothelial NOS,
is unlikely in the present study, because these CMVEs lose
the ability to express both endothelial NOS protein and
mRNA after culture.31 Increased expression of inducible
NOS, however, cannot be excluded. Resolution of the exact
mechanism(s) involved would, however, require further extensive experimentation outside the scope of the present
study.
Plasma levels of Ang II provide an approximation only of
concentrations in the region of the endothelial cells in the
microvasculature. Levels of Ang II in the control and shamoperated animals were ⬇0.65 nmol/L. These were elevated to
⬇1.3 nmol/L in the LVH animals and were accompanied by
a 2-fold increase in NADH-dependent oxidase activity and a
1.5-fold increase in NADPH-dependent oxidase activity. A
small increase in plasma Ang II is therefore associated with a
significant increase in O2⫺ production. In the cultured cells,
an increase in the Ang II concentration from 0.1 to 1 nmol/L
again produced a 2-fold increase in NADH-dependent oxidase activity, but an almost 4-fold increase in NADPHdependent oxidase activity. Thus, the data from the in vitro
experiments closely approximated that from the ex vivo
experiments.
The development of LVH is likely to be multifactorial,
with the oxidant stress generated in this condition likely to
have many determinants. Superoxide anions are probably the
most important oxygen free radical generated in vivo, and it
is highly likely that they are derived from more than one
source, not just from the Ang II–sensitive NADH/NADPH
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March 3, 2000
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oxidase system. One such example is xanthine oxidase, an
enzyme that resides within endothelial cells but also exists in
the bloodstream of patients in some clinical settings.45 Moreover, leakage of electrons from the electron transport system
within mitochondria,46 the cycloxygenase pathway,45 and the
auto-oxidation of catecholamines47 are all potential sources of
oxygen free radicals. Indeed, evidence suggests that in
cerebral arterioles, an acute increase in blood pressure can
cause excessive activation of arachidonic acid production via
the cycloxygenase pathway with the subsequent overproduction of O2⫺ by endothelial cells.48 Even the activation of NOS,
albeit in the presence of suboptimal concentrations of the
substrate L-arginine or the cofactor tetrahydrobiopterin, can
lead to the production of O2⫺ from molecular oxygen.49,50
Furthermore, Ang II itself has been shown to induce the
release of NO in resistance vascular beds51 in certain pathophysiological conditions.52 As mentioned above, we have
previously demonstrated31 that the cultured CMVEs do not
possess NOS activity; the ability to express this enzyme,
although present in freshly isolated cells, is lost in culture.
This finding provides further evidence that in the in vitro
experiments, NOS is an unlikely source of the O2⫺. We have
therefore demonstrated that neither NOS, cycloxygenase, nor
xanthine oxidase is involved in O2⫺ production by the CMVE
lysates. However, care must be taken in extrapolating from
the in vitro to the in vivo situation, and it is quite feasible that
the aforementioned enzymatic pathways could be involved in
the generation of reactive oxygen species in the intact animal.
As indicated earlier, recent evidence suggests that lucigenin,
under certain circumstances, can be a source of O2⫺,34,35 and
questions have arisen as to its suitability for the detection of
these free radicals.36 To validate our findings and confirm that
the O2⫺ production measured in this study was via NADH/
NADPH oxidase activity and not from lucigenin itself, ferricytochrome c reduction was used in some experiments. As the data
indicate, the particulate fraction from cultured cells also produces a reduction in ferricytochrome c in the presence of either
NADH or NADPH. This reduction was significantly greater in
lysates from cells exposed to Ang II compared with controls, an
observation that mirrors the data obtained using lucigenin.
Furthermore, the NADH-dependent effect is the dominant response using both techniques. The ferricytochrome c reduction
is almost completely inhibited by SOD, indicating that NADH/
NADPH oxidase is the likely source of O2⫺. SOD was used in
these experiments instead of tiron, because the latter produced a
nonspecific color change on addition to the reaction buffer, thus
interfering with the assay.
The data presented in this study clearly demonstrate a role for
Ang II in the generation of oxidant stress and endothelial
dysfunction in vitro. How such changes contribute to the
development of the endothelial dysfunction associated with
LVH must remain a matter for speculation. Several studies have
shown impairment of endothelial function in LVH in both
animals and humans,53–55 the mechanism being attributed to a
reduction in the expression of the constitutive NOS gene in
aortic endothelial cells in one of the studies.55 Clearly, the
increased inactivation of NO by O2⫺ provides an additional
mechanism for the endothelial dysfunction and reduction in
coronary reserve seen in LVH.56,57
Endothelial dysfunction is an early event in many diseases
affecting the cardiovascular system. Recently, Ang II has been
shown to contribute to this dysfunction via the activation of
endothelial cell suicide pathways leading to apoptosis,58 although this effect does not seem to be mediated directly by Ang
II–induced O2⫺ production. NO has, however, been demonstrated to have an inhibitory effect on this process,58 suggesting
that the inactivation of NO by O2⫺ could lead to endothelial cells
being driven toward Ang II–induced apoptosis. This observation
provides another possible mechanism whereby Ang II may be
involved in the development of endothelial dysfunction.
NO and its intracellular second messenger cGMP are
inhibitors of cardiac myocyte and fibroblast growth.59 Reduced NO activity or downregulation of NOS in CMVEs may
therefore also contribute to increased growth of cardiac
myocytes and/or fibroblasts characteristic of LVH.
In summary, the results of the present study demonstrate that
Ang II upregulates an oxygen free radical– generating enzyme
system in cultured guinea pig CMVEs and, furthermore, that in
this model of LVH, upregulation of this oxidase in CMVEs is
associated with increased plasma levels of Ang II. This study
also demonstrates that the NADH/NADPH enzyme is membrane bound and is activated to a greater extent by NADH than
NADPH as previously described for other cell types. Ang
II–induced oxidative stress leading to the inactivation of NO and
to endothelial cell injury is likely, therefore, to contribute
significantly to the endothelial dysfunction associated with LVH
and to play an important role in disease progression. However, it
must be noted that the development of LVH is likely to be
multifactorial and that Ang II may not be the only determinant in
the generation of oxidant stress.
Acknowledgments
This work was supported by funding from the UK Medical Research
Council; The British Council in Sana’a; and the University of
Sana’a, Yemen.
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Coronary Microvascular Endothelial Cell Redox State in Left Ventricular Hypertrophy:
The Role of Angiotensin II
Derek Lang, Salah I. Mosfer, Alison Shakesby, Francis Donaldson and Malcolm J. Lewis
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Circ Res. 2000;86:463-469
doi: 10.1161/01.RES.86.4.463
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