(Circulation. 2000;102:III-370.)
© 2000 American Heart Association, Inc.
Myocardial Protection and Vascular Biology |
Hypercholesterolemia Inhibits Angiogenesis in Response to Hindlimb Ischemia
Nitric Oxide–Dependent Mechanism
From The Cardiovascular Research Institute and Departments of Internal Medicine III and Radiology, Kurume University School of Medicine, Kurume, Japan.
Correspondence to Toyoaki Murohara, MD, PhD, The Cardiovascular Research Institute, Kurume University School of Medicine, 67 Asahi-machi, Kurume, 830-0011 Japan. E-mail toyom{at}med.kurume-u.ac.jp
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Abstract |
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Background—Endothelium-derived nitric oxide (EDNO) plays an important role in the regulation of angiogenesis, whereas hypercholesterolemia (HC) impairs EDNO release. We examined the hypothesis that HC may inhibit ischemia-induced angiogenesis by inhibition of EDNO in a rat model of unilateral hindlimb ischemia and that oral L-arginine supplementation, a substrate for NO synthase, may prevent HC-related impairment of angiogenesis.
Methods and Results—Male Sprague-Dawley rats were fed (A) standard diet (control), (B) 2% high-cholesterol diet (HC group), or (C) high-cholesterol diet with oral L-arginine (2.25% in drinking water) (HC+L-arg group). At 2 weeks of the dietary intervention, unilateral limb ischemia was surgically induced in all animals. Dietary HC groups (B and C) revealed elevated total and LDL cholesterol levels compared with control animals. Laser Doppler blood flow analyses showed significant decreases in the ischemic/normal limb blood flow ratio in the HC group compared with controls (P<0.05) when followed up until 4 weeks after surgery. Selective angiography and immunohistochemical analyses in the ischemic limb at postoperative day 14 revealed significantly lower angiographic scores (P<0.01) and capillary densities (P<0.01) in the HC group than controls, which were associated with decreased tissue contents of NOx and cGMP. Oral L-arginine supplementation (HC+L-arg) significantly improved all parameters of the laser Doppler blood perfusion ratio, angiographic scores, and capillary densities (P<0.01 versus HC group), which were accompanied by significant elevations in serum L-arginine levels and tissue NOx and cGMP contents.
Conclusions—Collateral vessel formation and angiogenesis in response to hindlimb ischemia were significantly attenuated in rats with dietary HC. The mechanism may be related to the reduced NO bioactivity in the ischemic tissues. Augmentation of the tissue NO activity by oral L-arginine supplementation restored the impaired angiogenesis in HC.
Key Words: peripheral vascular disease • hypercholesterolemia • angiogenesis • nitric oxide • endothelium
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Introduction |
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There are increasing numbers of patients with peripheral arterial occlusive diseases. Promotion of collateral vessel formation in such patients is an emerging therapeutic strategy to minimize tissue necrosis associated with severe ischemia, which is called therapeutic angiogenesis.1 Collateral vessels initially respond to various cytokines and vasoactive substances to grow into ischemic foci, in which activation, migration, and proliferation of vascular endothelial cells (ECs) play essential roles.2 We and others recently showed that endothelium-derived nitric oxide (EDNO) is a critical modulator for angiogenesis.3 4 5 For example, angiogenesis induced by a potent angiogenic cytokine, vascular endothelial growth factor, was attenuated by inhibitors of NO synthase (NOS).3 4 More recently, we showed that angiogenesis and collateral vessel formation after operatively induced hindlimb ischemia were severely impaired in mice lacking the gene for the endothelial constitutive NOS (ecNOS).5
Hypercholesterolemia (HC) is an established coronary risk factor that induces EC dysfunction. Studies demonstrated that endothelium-dependent vasodilator response (ie, EDNO release) is impaired in experimental HC animals and in humans with HC.6 7 On the basis of this background, we hypothesized that in vivo angiogenesis in ischemic tissues may be impaired because of reduced EDNO bioactivity in the HC state and that angiogenesis may be augmented by promoting EDNO synthesis by dietary L-arginine supplementation, a substrate for NOS. However, these issues have not been addressed previously. Accordingly, we tested the effects of dietary HC on angiogenesis and collateral vessel formation in a rat model of acute hindlimb ischemia in vivo. We also examined the hypothesis that dietary supplementation of L-arginine, a substrate for NOS and an antiatherogenic molecule,8 may have beneficial effects on the ischemia-induced angiogenesis in this rat model.
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Methods |
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Animal Model
All animal protocols were approved by the Institutional Animal Care and Use Committee. A rat model of hindlimb ischemia was induced by a previously described method9 with slight modification. Male Sprague-Dawley rats (250 to 300 g) were anesthetized with intraperitoneal pentobarbital (50 mg/kg). After skin incision, the entire femoral artery and vein were excised after all of their major branches were tied. Consequently, the blood flow to the ischemic lower limb became completely dependent on collateral vessels issuing from the internal iliac artery and its branches.
Study Protocol
Rats (n=53) were divided into 3 groups (Figure 1
). Rats in the control group (n=18) were
fed a standard diet throughout the experiment. Rats in the HC group
(n=18) were fed a 2% high-cholesterol diet, and rats in
the HC+L-arg group (n=17) were fed an HC diet and given oral
L-arginine in the drinking water (2.25%). At 2 weeks of
the dietary modification, all rats were subjected to unilateral
hindlimb ischemia as described above. On the day of surgery,
rats in the HC+L-arg group started to receive L-arginine
until the end of the protocol. The oral dose of L-arginine
was chosen according to a previous study,10 and this
regimen of L-arginine administration effectively increased
serum levels of L-arginine and tissue contents of stable NO
metabolites [nitrite
(NO2-)+nitrate
(NO3-);
NOx] in rats.10 Before surgery and
at postoperative days 7, 14, and 28, a tail-cuff method was applied for
systemic blood pressure measurements in the conscious state.
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Laser Doppler Blood Perfusion Analysis
We measured the ischemic/normal hindlimb blood perfusion
ratio using a laser Doppler perfusion image (LDPI) analyzer
(moorLDI, Moor Instrument) as described previously.5 In
this method, a color-coded image representing blood flow
distribution is displayed. Low or no blood perfusion is displayed as
dark blue, and the highest perfusion level is displayed as red to white
colors. At 7 predetermined time points (before and immediately after
surgery and at postoperative days 3, 7, 14, 21, and 28) (Figure 1
), we performed 2 consecutive LDPI scans over the same region
of interest. After 2 scans, the average perfusion values of the
ischemic and nonischemic (normal) hindlimbs were
computed from histograms of the colored pixels. To minimize variations
due to ambient light, calculated blood perfusion (relative units) was
expressed as the ischemic (left)/normal (right) limb blood
perfusion ratio.5
Angiographic Score
At postoperative day 14, under pentobarbital
anesthesia (50 mg/kg), a 22-gauge soft-tip catheter was
inserted through the abdominal aorta of control (n=6), HC (n=6), and
HC+L-arg (n=5) rats. Both hindlimbs were perfused with 10 mL of saline
containing heparin (10 U/mL). Postmortem angiography was then performed
by injection of 3 mL of contrast medium through the catheter inserted
into the abdominal aorta. X-ray films were recorded by mammography
(Senographe 500T, General Electric), and the extent of collateral
vessels was calculated by the angiographic score. For calculation of
angiographic score, a composite of 2-mm2 grids
printed on a transparent sheet was placed over the thigh area of each
film. The score was determined by calculating intersections crossed by
opacified arteries divided by the total number of grid intersections
within the area of interest.
Immunohistochemistry and Determination of the Capillary
Density
Six animals in each group were euthanized at postoperative day
14 with an overdose of sodium pentobarbital. Ischemic (left)
and nonischemic (right) hindlimb skeletal muscles were
harvested and fixed in methanol. Tissues were embedded in paraffin, and
5-µm-thick histological sections were prepared. We
used a monoclonal antibody (MAb) directed against von
Willebrand factor (vWF) (DAKO) as a marker for ECs because this
molecule is constitutively expressed on all ECs and its expression does
not depend on either phenotypic changes or activation states of ECs.
The vWF-bound MAb was detected by an avidin-biotin-peroxidase method
from a commercially available kit (Vector Laboratories). Positively
stained ECs were counted, and the capillary densities of both
ischemic and nonischemic limb muscles were
analyzed for specific evidence of the vascularity at the
microcirculation. Ten different microscopic fields from 
3 different
sections from each animal were counted, and capillary density was
expressed as number of capillaries/field (x400).
Measurements of Tissue cGMP Contents
Four tissue blocks (1 to 1.2 g) from the thigh adductor
muscle were harvested from the ischemic hindlimb of 6 rats in
each group at postoperative day 14. The tissue samples were weighed,
snap-frozen in liquid nitrogen, and stored at -80°C. The assay for
tissue cGMP contents was performed as previously
described.5 Tissues were homogenized in a
10-fold volume of 6% trichloroacetic acid and centrifuged at
2000g for 15 minutes. The supernatant was collected and
washed 4 times with water-saturated diethyl ether. The samples were
then frozen in liquid nitrogen and lyophilized. The lyophylate was
resuspended in 1 mL of 0.05 mol/L sodium acetate buffer (pH 5.8).
Measurements of cGMP were performed with a cGMP enzyme immunoassay kit
(Biotrak, Amersham). Values for cGMP were standardized by tissue wet
weight (grams).
Biochemical Analyses
Serum levels of total, LDL, and HDL cholesterols and
of triglyceride were determined enzymatically with
commercially available kits (Boehringer Diagnostica
and Wako Chemicals). Serum L-arginine levels, tissue
contents of NOx (stable metabolites of NO), and
asymmetrical dimethyl arginine (ADMA), an endogenous NOS
inhibitor, were also measured by high-performance
liquid chromatography as described
previously.11 12
Statistical Analysis
Results are all expressed as mean±SEM. Comparisons were
performed by use of ANOVA followed by Fischer’s test for comparisons
between any 2 groups. Statistical significance was assumed at
P<0.05.
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Results |
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Table 1
summarizes body
weight, heart rate, and systolic and diastolic
blood pressures of each group measured before surgery and at
postoperative days 7, 14, and 28. Mean body weights of rats in the HC
and HC+L-arg groups were heavier than those of control animals at days
7, 14, and 28. However, there were no significant differences in body
weight between the HC and HC+L-arg groups at any time points. Thus, the
HC diet increased body weight but supplemental oral
L-arginine did not affect the body weight in the HC state.
There were no differences in heart rate or systolic and
diastolic blood pressures among the 3 experimental groups
at any time points (Table 1).
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Serum Lipid Levels
Table 2 summarizes serum lipid
levels of each group examined at postoperative day 14 (ie, 28 days
after dietary modification). Serum total and LDL
cholesterol levels were significantly but modestly
increased in the HC and HC+L-arg groups compared with controls. Serum
HDL cholesterol levels decreased significantly in the HC
and HC+L-arg groups compared with the controls. Serum
triglyceride levels did not differ among the 3 groups.
There were no significant differences in total, LDL, and HDL
cholesterol levels between HC and HC+L-arg groups,
indicating that supplemental oral L-arginine did not affect
the serum lipid profiles in the HC state.
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Laser Doppler Analysis for Hindlimb Blood
Perfusion
Before surgery, the left/right hindlimb blood perfusion ratio was
1.0 in all groups. Immediately after operative induction of the left
hindlimb ischemia, the ratios of ischemic/normal blood
perfusion markedly decreased, ranging from 0.37 to 0.41. These ratios
immediately after induction of unilateral limb ischemia did not
differ among the 3 groups, indicating that the severity of the
ischemia created was comparable among the 3 groups.
Representative images of the hindlimb blood perfusion
before and immediately after surgery and at postoperative days 7, 14,
and 28 are shown in Figure 2A. LDPI
analyses disclosed progressive recovery of the blood perfusion
within 28 days after surgery in controls (Figure 2B). However,
the blood perfusion ratios in the HC group were significantly smaller
than those of controls at postoperative days 7, 14, 21, and 28. In
contrast, the blood perfusion ratio improved significantly in the
HC+L-arg group compared with the HC group at days 7, 14, 21, and 28
(Figure 2B).
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Angiographic Score
To further examine whether the altered blood perfusion detected by
the LDPI is associated with changes in collateral vessel formation, we
performed iliac angiography. Figure 3A
shows representative angiograms taken at postoperative
day 14. There are numerous collateral vessels in the ischemic
thigh muscle area in a rat from the control group. However, there are
fewer collateral vessels issuing from the internal iliac artery in a
rat from the HC group. Oral L-arginine supplementation,
however, markedly increased the number of collaterals.
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Collateral vessels in the medial thigh area were quantitatively
assessed by calculation of the angiographic score. The angiographic
score was significantly lower in the HC group than in controls.
However, the angiographic score was restored to a level comparable to
that of the control group by oral L-arginine
supplementation (Figure 3B).
Immunohistochemical Identification of ECs and Capillary
Density
To examine whether the changes in the hindlimb blood perfusion (by
LDPI) and collateral vessel formation (by angiographic score) are
associated with changes in capillary EC formation at the
microcirculation level, we measured capillary densities in
histological sections harvested from ischemic
and nonischemic hindlimbs at postoperative day 14.
Immunohistochemical staining by an anti-vWF MAb identified capillary
ECs in the skeletal muscle tissues. Representative
photomicrographs of histological sections are shown in
Figure 4A. The number of capillary ECs in
the ischemic limb of an HC rat is decreased compared with a
control rat, which was restored by oral L-arginine
supplementation.
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Quantitative analyses counting the vWF-positive capillary ECs
under light microscopy (x400) revealed that the capillary densities
were significantly lower in the HC group than controls in the
ischemic limb (P<0.001) at postoperative day 14
(Figure 4B). However, capillary densities were restored in the
HC+L-arg group (P<0.001 versus HC group). In the
contralateral nonischemic right limb, capillary densities were
not different among the 3 groups (Figure 4B). Therefore, oral
L-arginine increased ischemia-induced
angiogenesis without affecting capillary densities of
nonischemic tissues.
Serum Levels of L-Arginine and Tissue Contents of
NOx and cGMP
The serum L-arginine levels at postoperative day 14
were not significantly different between the control and HC groups.
Oral L-arginine supplementation significantly increased
that level (Table 3). To evaluate whether
HC and L-arginine supplementation altered NO formation in
the ischemic tissues, we analyzed the contents of
NOx and cGMP in the ischemic hindlimb
tissues. Both tissue NOx and cGMP contents were
significantly lower in the HC rats than controls (Table 3). Oral
L-arginine supplementation, however, significantly restored
both tissue NOx and cGMP contents
(P<0.05) compared with the HC group (Table 3).
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Tissue Contents of ADMA in the Ischemic Hindlimb
Accumulation of ADMA, an endogenous
inhibitor of NOS, in vascular tissue has been implicated as
a risk factor for atherogenesis. We therefore measured tissue contents
of ADMA in the ischemic hindlimb. Tissue ADMA levels were
significantly greater in the HC group than the control group. Oral
L-arginine supplementation restored tissue ADMA contents
(P<0.05) to a level similar to that of the control group
(Figure 5).
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Discussion |
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Major findings of the present study are that (1) ischemia-induced angiogenesis was impaired in rats with dietary HC; (2) the HC-related impairment of angiogenesis was associated with decreased NO and cGMP productions in the ischemic hindlimb tissues; and (3) oral L-arginine supplementation, a substrate for NOS, rescued the HC-related impairment of ischemia-induced angiogenesis, accompanied by improved NO/cGMP production in the ischemic tissues. Therefore, HC impairs angiogenesis and collateral vessel formation in response to regional tissue ischemia, possibly through decreased tissue NO bioactivity. HC, however, does not preclude augmentation of collateral vessel development by means of oral L-arginine supplementation, a precursor of NO.
Rat Model of HC
In the present study, rats with HC revealed mild increases (by
37%) in total cholesterol levels compared with control
rats (Table 2). One may speculate that animals with such modest
HC may not be a proper model to test the effects of HC in vivo.
However, serum LDL cholesterol levels were almost doubled
in HC rats compared with controls in the present study (Table 2). Moreover, previous studies showed that rat models of HC
elicit impaired vascular functions. For example, Nunnari et
al13 showed that dietary HC resulted in significant
deposition of oil red O–positive lipids on the rat
arterial wall. We previously showed that HC attenuated
endothelium-dependent relaxation to acetylcholine in
the isolated rat aorta ex vivo, and HC enhanced
leukocyte-endothelium interactions in the mesenteric
microvasculature in vivo.14 Furthermore, alterations in
microvascular reactivity to vasoactive substances have been identified
in a rat model of HC before any histological changes of
atherosclerosis.15 Taken together, the rat
model of HC, despite the modest increase in serum
cholesterol levels, exhibits vascular dysfunction
regardless of morphological atherosclerotic changes and thus seems to
be a proper model to analyze the effects of HC on angiogenesis
in vivo.
HC Impaired Ischemia-Induced Angiogenesis in Vivo
HC is one of the established risk factors for atherosclerotic
vascular diseases. A recent report by van Belle et al16
showed that angiogenesis in an ischemic limb was severely
impaired in Watanabe heritable hyperlipidemic (WHHL)
rabbits. The WHHL rabbit is a genetic model of HC, however, and has
extremely high concentrations of serum cholesterol levels
(ie, 600 to 900 mg/dL). Therefore, the WHHL rabbit may mimic familial
HC but may not represent the modest HC commonly observed in
humans. In this regard, the effects of diet-induced modest HC on in
vivo angiogenesis were little known.
In the present study, angiogenesis in response to hindlimb
ischemia was significantly attenuated by diet-induced modest HC
in rats. There are several possible mechanisms for the impaired
angiogenesis in the HC state. First, HC-related EC dysfunction and
decreased EDNO formation may account for the impaired angiogenesis,
because proliferation and migration of ECs are essential processes for
angiogenesis. HC impairs EDNO formation not only in large conduit
arteries but also in microvessels.7 17 We and
others3 4 5 previously reported that EDNO is an important
regulator for angiogenesis both in vitro and in vivo. EDNO maintains
arterial EC integrity18 and expression of
integrin
vß3,19 a
key adhesion molecule for angiogenesis, and promotes EC
migration.19 Moreover, angiogenesis in response to tissue
ischemia was severely impaired in ecNOS-deficient
mice.5 Therefore, reduced EDNO formation in the HC state
most likely accounts for the impaired angiogenesis. Consistent
with this hypothesis, tissue contents of both
NOx, stable metabolites of NO, and cGMP, a
biological product of the L-arginine/NO pathway, were
all significantly reduced in the HC rats compared with control rats in
the present study. Second, HC has been shown to increase vascular
superoxide anion production.20 Superoxide anions
degrade NO, which results in further reduction of EDNO
bioactivity.20 Third, HC itself might directly inhibit EC
proliferation and/or migration. A recent study demonstrated that
oxidized LDL and lysophosphatidylcholine, major atherogenic molecules
in the arterial wall, can inhibit EC
migration.21 Furthermore, Chen et al22 showed
that capillary plexus formations from explants of human and rabbit
coronary arteries were impaired by HC ex vivo. Taken together,
EC dysfunction and decreased EDNO bioactivity in HC seem to be
responsible for the impaired angiogenesis in the HC state.
L-Arginine Rescued the HC-Related Impairment of
Angiogenesis
To further address the above issues, we examined the effects of
oral supplementation of L-arginine, a substrate for NO
synthase, on angiogenesis in response to hindlimb ischemia in
rats with HC. In previous studies, L-arginine has been
shown to improve endothelium-dependent relaxation in
humans and experimental animals with HC without changing systemic
hemodynamics.7 17 Oral
L-arginine improved EC function not only in large conduit
arteries but also in microvessels.7 17
In the present study, oral L-arginine supplementation
rescued the impaired angiogenesis in rats with HC. The improved
angiogenesis by L-arginine was documented by the increased
ischemic/normal blood perfusion ratio by the LDPI
analysis, increased angiographic score, and increased capillary
density compared with the HC group without L-arginine.
Moreover, oral L-arginine supplementation significantly
increased the contents of NOx and cGMP in the
ischemic tissues (Table 3). L-Arginine did
not alter the serum levels of total, LDL, and HDL
cholesterols and triglyceride, indicating that
the effects of L-arginine on angiogenesis were not due to
the changes in serum lipid profiles. Because oral
L-arginine did not affect systemic blood pressure, the
beneficial effects of L-arginine on angiogenesis and
collateral vessel formation probably are not due to blood pressure
changes. On the basis of these findings, oral L-arginine
administration seems to have improved ischemia-induced
angiogenesis in HC rats, possibly by augmenting the NO bioactivity in
the ischemic tissues.
The precise mechanisms by which L-arginine improved tissue NO activity and angiogenesis in the ischemic limb are still enigmatic. Previous studies23 24 have documented that regenerating ECs in ischemic tissues are generally dysfunctional. It is possible that the availability of L-arginine by ecNOS in regenerating ECs may be impaired.5 23 Alternatively, endogenous antagonists of NOS, such as ADMA, may accumulate in regenerating ECs.24 In the present study, HC significantly elevated tissue contents of ADMA. Therefore, elevated ADMA may in part account for the decreased tissue NOx contents and impaired angiogenesis in the ischemic limb in the HC state. Administration of L-arginine might have favorably changed the enzymatic kinetics of ecNOS by competitive inhibition of endogenous antagonists of NOS, such as ADMA.25
Study Limitations
In our rat model of hindlimb ischemia, angiogenesis in the
HC rats kept up with the extent of angiogenesis seen in the control
animals in the chronic phase (at postoperative day 35 and thereafter).
Thus, we could not determine the effects of L-arginine on
angiogenesis in the chronic phase. In this sense, our rat model of limb
ischemia is an acute model of ischemia and therefore
may not mimic chronic arterial occlusive diseases or
critical limb ischemia observed in humans. Nevertheless,
supplemental oral L-arginine significantly improved
angiogenesis at postoperative days 7 through 28, which is a critical
period for endothelial regeneration in the
ischemic tissues in these animal models.5 9
We started administration of oral L-arginine on the day of surgery in the HC+L-arg group in the present study. At postoperative day 3, the HC+L-arg group tended to have better blood perfusion than the HC group despite there being no significant difference between them. It then may be clinically relevant to examine the effects of oral L-arginine starting at a later time point (several days) after surgery on angiogenesis. This issue is now under investigation in our laboratory.
Conclusions and Clinical Implications
Our findings provide evidence that HC impairs angiogenesis in
response to regional tissue ischemia, presumably by the
decreased activity of the L-arginine/NO pathway in the
ischemic tissues. Therefore, augmentation of
endogenous NO bioactivity (eg, by means of
L-arginine, tetrahydrobiopterin, or ecNOS gene transfer)
may deserve further consideration as a therapeutic strategy for
patients with peripheral arterial occlusive
diseases associated with lipid disorders, who often have impaired
endothelial functions and decreased EDNO release.
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Acknowledgments |
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This study was supported by a grant (No. 10770336) from the Ministry of Education, Science, Sports, and Culture of Japan (Dr Murohara) and by the Japan Heart Foundation (Dr Murohara). We thank K. Kimura, A. Shimizu, K. Moriyama, and M. Tsuru for technical assistance.
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References |
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1. Isner JM, Pieczek A, Schainfeld R, et al. Clinical evidence of angiogenesis after arterial gene transfer of phVEGF165 in patient with ischaemic limb. Lancet. 1996;348:370–374.
2.
Folkman J. Angiogenesis in cancer, vascular,
rheumatoid and other disease. Nat Med. 1995;1:27–30.
3.
Ziche M, Morbidelli L, Choudhuri R, et al. Nitric
oxide synthase lies downstream from vascular
endothelial growth factor-induced but not basic
fibroblast growth factor-induced angiogenesis. J Clin
Invest. 1997;99:2625–2634.
4.
Papapetropoulos A, Garcia-Cardena G, Madri JA, et al.
Nitric oxide production contributes to the angiogenic
properties of vascular endothelial growth factor in
human endothelial cells. J Clin Invest. 1997;100:3131–9.
5.
Murohara T, Asahara T, Silver M, et al. Nitric oxide
synthase modulates angiogenesis in response to tissue ischemia.
J Clin Invest. 1998;101:2567–2578.
6.
Cohen RA, Zitnay KM, Haudenschild CC, et al. Loss of
selective endothelial cell vasoactive functions caused
by hypercholesterolemia in pig coronary
artery. Circ Res. 1988;63:903–910.
7.
Creager MA, Gallagher SJ, Girerd XJ, et al. L-Arginine
improves endothelium-dependent vasodilation in
hypercholesterolemic humans. J Clin
Invest. 1992;90:1248–1253.
8.
Cooke JP, Singer AH, Tsao P, et al. Antiatherogenic
effects of L-arginine in the hypercholesterolemic
rabbit. J Clin Invest. 1992;90:1168–1172.
9.
Yang HT, Ogilvie RW, Terjung RL. Heparin increases
exercise-induced collateral blood flow in rats with femoral artery
ligation. Circ Res. 1995;76:448–456.
10.
Matsuoka H, Nakata M, Kohno K, et al. Chronic
L-arginine administration attenuates cardiac
hypertrophy in spontaneously hypertensive rats.
Hypertension. 1996;27:14–18.
11.
Green LC, Wagner DA, Glogowski J, et al.
Analysis of nitrate, nitrite, and
[15N]nitrate in biological fluids. Anal
Biochem. 1982;126:131–138.
12.
Park KS, Lee HW, Hong SY, et al. Determination of
methylated amino acids in human serum by high-performance
liquid chromatography. J Chromatogr. 1988;440:225–230.
13.
Nunnari JJ, Zand T, Joris I, et al. Quantitation of oil
red O staining of the aorta in hypercholesterolemic
rats. Exp Mol Pathol. 1989;51:1–8.
14.
Gauthier TW, Scalia R, Murohara T, et al. Nitric oxide
protects against leukocyte-endothelium interactions in
the early stages of hypercholesterolemia.
Arterioscler Thromb Vasc Biol. 1995;15:1652–1659.
15.
Schuschke DA, Joshua IG, Miller FN. Comparison of early
microcirculatory and aortic changes in
hypercholesterolemic rats. Arterioscler
Thromb. 1991;11:154–160.
16.
van Belle E, Rivard A, Chen D, et al.
Hypercholesterolemia attenuates angiogenesis
but does not preclude augmentation by angiogenic cytokines.
Circulation. 1997;96:2667–2674.
17.
Cooke JP, Andon NA, Girerd XJ, et al. Arginine restores
cholinergic relaxation of hypercholesterolemic rabbit
thoracic aorta. Circulation. 1991;83:1057–1062.
18.
Tsurumi Y, Murohara T, Krasinski K, et al. Reciprocal
relation between VEGF and NO in the regulation of
endothelial integrity. Nat Med. 1997;3:879–886.
19.
Murohara T, Witzenbichler B, Spyridopoulos I, et al.
Role of endothelial nitric oxide synthase in
endothelial cell migration. Arterioscler Thromb
Vasc Biol. 1999;19:1156–1161.
20.
Ohara Y, Peterson TE, Harrison DG.
Hypercholesterolemia increases
endothelium superoxide anion production.
J Clin Invest. 1991;91:2546–2551.
21.
Murugesan G, Fox PL. Role of lysophosphatidylcholine in
the inhibition of endothelial cell motility by oxidized
low density lipoprotein. J Clin Invest. 1996;97:2736–2744.
22.
Chen CH, Cartwright J Jr, Li Z, et al.
Inhibitory effects of
hypercholesterolemia and ox-LDL on
angiogenesis-like endothelial growth in rabbit aortic
explants: essential role of basic fibroblast growth factor.
Arterioscler Thromb Vasc Biol. 1997;17:1303–1312.
23.
Azuma H, Sato J, Hamasaki H, et al. Accumulation of
endogenous inhibitors for nitric oxide
synthesis and decreased content of L-arginine in regenerated
endothelial cells. Br J Pharmacol. 1995;115:1001–1004.
24.
Böger RH, Bode-Böger SM, Szuba A, et al.
Asymmetric dimethylarginine (ADMA): a novel risk factor for
endothelial dysfunction: its role in
hypercholesterolemia. Circulation. 1998;98:1842–1847.
25.
Miyazaki H, Matsuoka H, Cooke JP, et al.
Endogenous nitric oxide synthase inhibitor: a
novel marker of atherosclerosis.
Circulation. 1999;99:1141–1146.
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