(Circulation. 1997;96:2667-2674.)
© 1997 American Heart Association, Inc.
Articles |
Hypercholesterolemia Attenuates Angiogenesis but Does Not Preclude Augmentation by Angiogenic Cytokines
From Hopital Cardiologique (E. Van B., C.B.), Lille, France; Genentech, Inc (S.B., N.F.), South San Francisco, Calif; and Departments of Surgery (J.F.S.) and Medicine (Cardiology) (A.R., D.C., M.S., J.M.I.), St Elizabeth's Medical Center, Tufts University School of Medicine, Boston, Mass.
Correspondence to Jeffrey M. Isner, MD, St Elizabeth's Medical Center, 736 Cambridge St, Boston, MA 02135. E-mail VeJeff{at}AOL.COM
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Background The impact of hyperlipidemia on collateral vessel development in vivo remains enigmatic. We sought to determine the anatomic extent and functional capacity of the collateral bed that develops in response to limb ischemia in a well characterized animal model of spontaneous hypercholesterolemia, the Watanabe heritable hyperlipidemic (WHHL) rabbit. We further characterized the impact of exogenous angiogenic cytokine administration on collateral vessel development and function in the same animal model.
Methods and Results Weight-matched 6-month-old male homozygous WHHL (n=9) and normal New Zealand White (NZW) (n=9) rabbits underwent surgical resection of one femoral artery. Ten days later, the ischemic hindlimb was evaluated for collateral vessel formation, blood flow, and tissue damage. Collateral vasculature was less extensive among WHHL than NZW, as indicated by a significant reduction in angiographic score (0.19±0.02 versus 0.35±0.03, P<.001) and capillary density (46.4±4.1 versus 78.9±4.6/mm2, P<.0002). This was associated with a reduction in calf blood pressure index (9.5±3.5% versus 32.8±2.8%, P<.0001), arterial blood flow (7.5±0.6 versus 13.6±0.7 mL/min, P<.0001), and muscle perfusion index (40.1±3.2% versus 65.9±2.0%, P<.0001) and an increase in muscle necrosis (48.16±5.41% versus 25.90±3.83% negative 2,3,5-triphenyltetrazolium chloride staining, P<.004). Treatment of WHHL rabbits (n=9) with recombinant human vascular endothelial growth factor produced a statistically significant improvement in all functional as well as anatomic indices of collateral development.
Conclusions Collateral vessel development associated with hindlimb ischemia in vivo is severely attenuated in an animal model of spontaneous hypercholesterolemia but nevertheless may be augmented by administration of angiogenic cytokines.
Key Words: angiogenesis • atherosclerosis • growth factors • hypercholesterolemia
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Introduction |
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Angiogenesis, the development of new blood vessels, depends on the activation, migration, and proliferation of ECs1 under the regulatory control of growth factors and hypoxia.2 Administration of EC mitogens to promote collateral vessel growth, a strategy known as "therapeutic angiogenesis," has been proposed for the treatment of patients with peripheral and/or myocardial ischemia.3 This concept has been validated in several animal models of limb or myocardial ischemia.4 5 6 7 These studies, however, have been universally performed in normal healthy animals. Consequently, two important issues remain currently unresolved. First, what is the impact of hyperlipidemia on endogenous collateral development in vivo? Second, is exogenous administration of angiogenic growth factors effective in hyperlipidemic animals?
We sought to address these issues in WHHL rabbits,8 a strain of rabbits in which a single gene mutation leads to markedly reduced LDL receptor expression.9 As a result, these animals develop increased plasma levels of total (predominantly LDL) cholesterol and a pattern of atherosclerosis similar to that seen in familial hypercholesterolemia.10 11 12 13 Collateral vessel formation was evaluated in vivo 10 days after the induction of ischemia with or without exogenous administration of an EC-specific growth factor, rhVEGF. We observed that collateral vessel development associated with hindlimb ischemia was severely attenuated but could be nevertheless successfully augmented by angiogenic cytokine administration.
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Methods |
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Animal Model and Study Design
All protocols were approved by St Elizabeth's Institutional Animal Care and Use Committee. Weight-matched (3.0- to 3.5-kg) 6-month-old male homozygous WHHL (Camm Research) and NZW (Pine Acre Rabbitry) rabbits were used for the experiments. Representative angiograms performed in nonoperated NZW and WHHL rabbits are illustrated in Fig 1
. Surgical procedures were performed
with the animals under anesthesia with a mixture of
ketamine (50 mg/kg) and acepromazine (0.8 mg/kg)
after premedication with xylazine (2 mg/). Animals underwent
surgical removal of one femoral artery according to a previously
described technique.7 14 Excision of the femoral artery
results in occlusion of the external iliac artery; blood flow to the
ischemic limb is consequently dependent on collateral vessels
originating from the internal iliac artery.
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Two sets of pilot experiments were performed. The first was designed to document the severity of angiographic, hemodynamic, and blood flow deficits measured 1 day after surgical creation of unilateral hindlimb ischemia in WHHL rabbits (n=5) (see "Methods" for a description of these analyses). Angiography disclosed angiographic scores of 0.19 to 0.24; blood pressure in the ischemic limb was 0 mm Hg in all rabbits, and blood flow in the ischemic limb was reduced to 5.19±0.62 mm Hg. These findings established evidence of severely compromised hindlimb perfusion in WHHL rabbits.
A second set of pilot experiments were performed to evaluate the ability of WHHL rabbits to tolerate unilateral hindlimb ischemia past day 1. These experiments disclosed reproducible limb necrosis in WHHL rabbits (in the absence of exogenous cytokine therapy) if these animals were permitted to survive beyond 10 days after surgery. (In contrast, limb necrosis has not been observed as a consequence of this surgery in NZW rabbits.) In conjunction with our animal care and use committee, we therefore designed the current protocol to perform functional and anatomic analyses of the animals at 10 days after surgery, after which all animals were electively killed.
At 1 day after surgery (day 1), WHHL rabbits were randomly assigned to receive daily injections of rhVEGF (500 µg in 0.1% PBS; WHHLvegf, n=9; Genentech) or vehicle alone (WHHLctrl, n=9) from day 1 to day 6. NZW rabbits (n=9) received daily injections of vehicle alone. Although previous studies15 have indicated that this dose of rhVEGF leads to a transient reduction in mean arterial pressure (20.5±1.4%), this was typically tolerated by hypercholesterolemic as well as normal NZW rabbits without sequela.
Plasma levels of cholesterol, triglycerides, and CK were evaluated before surgery and 10 days later. Collateral vessel formation, blood flow, and the extent of tissue damage were evaluated at day 10 as described below.
After surgery, all animals were closely monitored. Analgesia (levorphanol tartrate [60 mg/kg]; Roche Laboratories) was administered subcutaneously as required for evidence of discomfort throughout the duration of the experiment. Prophylactic antibiotics (sulfamethoxazole [15 mg/kg] and trimethoprim [3 mg/kg]; Elkins-Sinn) were also administered subcutaneously for a total of 5 days after surgery.
Lower Limb Calf Blood Pressure Index
Calf blood pressure was measured noninvasively in both hindlimbs
using a Doppler flowmeter (model 1050; Parks Medical Electronics)
and a cuff connected to a pressure manometer.7 14 The calf
blood pressure index was defined for each rabbit as the ratio of
systolic pressure in the ischemic limb to
systolic pressure in the normal limb (x100).
Angiography and Doppler Guide Wire Measurements
Angiography and intra-arterial Doppler guide
wire measurement of blood flow velocity were performed on day 10 as
previously described.7 14
A 3F infusion catheter (Tracker-18TM; Target Therapeutics) was used for angiography and drug infusion, and an 0.018-in Doppler guide wire (Cardiometrics) was used for the measurement of flow velocity. The catheter and wire were introduced into the right common carotid artery through a small cutdown and advanced through the aorta to the proximal segment of the common iliac artery (catheter) and the proximal segment of the ipsilateral external or internal iliac artery (Doppler wire).
Measurements were performed in the external and internal iliac arteries in the nonischemic limb and in the internal iliac artery in the ischemic limb. In addition to measurements performed at rest, endothelium-dependent and -independent flows were measured in each vessel after intra-arterial administration of acetylcholine chloride and nitroprusside, respectively (Sigma Chemical Co) over 2 minutes via a constant infusion pump (1 mL/min). Each drug was administered at a dose of 1.5 µg · min-1 · kg-1.
Doppler-Derived Blood Flow in Nonischemic and
Ischemic Hindlimbs
Angiographic luminal diameter was measured in each vessel
at the site of Doppler sample volume recording, at rest and
after drug infusion, with the use of an automated edge-detection system
(Quantum 2000I; QCS) as previously described.7
Doppler-derived flow was calculated as
QD=(
d2/4)(0.5xAPV), where QD is
Doppler-derived time average flow, d is vessel diameter, and APV is
time average of the spectral peak velocity.16 Mean
velocity was estimated as 0.5xAPV by assuming a time-averaged
parabolic velocity profile across the vessel. Doppler-derived flow
calculated in this fashion has been previously validated in
vivo.16 Blood flow in the nonischemic hindlimb was
calculated as the sum of the flows recorded in the external and
internal iliac arteries. In the ischemic limb, flow measured in
the internal iliac artery represented the flow to the
entire hindlimb.
Angiographic Analysis of Collateral Vessels
Angiographic analysis of collateral vessels supplying
the ischemic limb was performed using angiograms recorded 4
seconds after the injection of contrast media into the internal iliac
artery. A grid overlay composed of 2.5-mm-diameter circles arranged in
rows spaced 5 mm apart was placed over the angiogram at the level
of the medial thigh. The number of contrast-opacified arteries crossing
over circles as well as the total number of circles encompassing the
medial thigh area were counted in a single blinded fashion. An
angiographic score was calculated as the ratio of circles crossed by
opacified arteries divided by the total number of circles in the
ischemic thigh. This angiographic score reflects vascular
density in the medial thigh.14 17
Measurement of Muscle Perfusion
Perfusion of hindlimb skeletal muscles was evaluated using
15-µm-diameter colored polystyrene
microspheres.14 17 After completion of the
invasive measurements described above, two sets of 3x106
Dye-Trak colored microspheres (Triton Technology) were injected
through a 3F Teflon catheter into the left ventricle. The first
injection was performed under resting conditions, and the second was
performed during stimulation with an infusion of nitroprusside (1.5
µg · min-1 ·
kg-1) in the lower aorta. Each time, a
reference blood sample was withdrawn using a syringe pump to collect
microspheres at a constant rate of 1.2 mL/min through a
peripheral artery (central ear artery). The animals were
then killed, and 14 tissue samples (weight, 
2 g each) were retrieved
from seven different muscles (tensor fascia latae, vastus lateralis,
vastus medialis, adductor, semimembranosus, gastrocnemius, tibialis
cranialis) in each hindlimb (ischemic and nonischemic).
Samples from the right and left kidneys were also collected and used as
controls to determine homogeneity of the blood content of
microspheres. After tissue and blood sample digestion using
potassium hydroxide, sphere-filtering extraction, and dimethylformamide
dye removal, each sample was analyzed with a conventional
spectrophotometer (model 8452A; Hewlett Packard).14 17 On
the basis of the OD measurements, muscle perfusion expressed in mL
· min-1 · 100
g-1 was calculated with the following
equation: Perfusion of Muscle Sample x (mL ·
min-1 ·
g-1)=(OD of Tissue Sample
x/OD of Reference Blood Sample)x[Withdrawal Rate of
Reference Blood Sample (mL/min)/Weight of Tissue Sample x
(g)].
Muscle perfusion in each hindlimb was expressed as the mean of 14 samples. Perfusion in the ischemic hindlimb was also expressed as percent of perfusion in the nonischemic hindlimb, or "muscle perfusion index."
Capillary Density
Collateral vessel formation was further examined by measuring
the number of capillaries in light microscopic sections taken from
ischemic and nonischemic hindlimbs.14
Tissue specimens were obtained from the adductor muscle and
semimembranosus muscle of both hindlimbs of each animal at the time
they were killed (day 10). These two muscles were chosen because they
are the two major muscles of the medial thigh. Samples were embedded in
O.C.T. Compound (Miles) and snap-frozen in liquid nitrogen. Frozen
sections (5 µm in thickness) were stained for alkaline
phosphatase using an indoxyl-tetrazolium method to detect capillary ECs
and then counterstained with eosin.18 Because method it is
based on histochemical staining for an enzyme within capillary
endothelium, it shows all capillaries present
within the tissue, regardless of the extent of tissue perfusion.
Capillaries were counted under a 20x objective to determine capillary
density (mean number of capillaries/mm2). A total of 20
different fields from the two muscles were randomly selected, and the
number of capillaries were counted. To ensure that analysis of
capillary density was not overestimated due to muscle atrophy,
capillary density was also evaluated as a function of the number of
muscle fibers in the histological section
(capillary-to-myocyte ratio).
TTC Staining
The application of the oxidation-reduction indicator TTC stains
tissue with normal levels of dehydrogenase red, whereas
ischemic and infarcted tissue remained unstained due to loss of
the enzyme. TTC staining was performed immediately after the animals
were killed.19 The remaining portions of the tensor fascia
latae, vastus lateralis, vastus medialis, adductor, semimembranosus,
gastrocnemius, and tibialis cranialis that were not used for the
microsphere evaluation were further sectioned and incubated in
TTC for 30 min at 37°C. Percent unstained/total muscle weight was
used as an index of tissue damage. The healthy limb was used as a
control for the quality of the staining (absence of unstained muscles
in the healthy limb).
Pathological Analysis of Atherosclerotic Lesions
The presence and extent of atherosclerotic lesions were
evaluated in tissue samples retrieved from the lower aorta, right and
left iliac arteries, and the remaining femoral artery of each rabbit
(six sections from each vessel for a total of 24 sections per animal).
Tissues were fixed by immersion in 100% methanol, embedded in
paraffin, stained by hematoxylin and eosin or elastic-trichrome, and
examined by light microscopy.
Statistical Analysis
Results are expressed as mean±SEM. Comparisons were performed
using ANOVA followed by Scheffé's procedure or, for categorical
data, contingency table analysis. Statistical significance was
assumed at P<.05.
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Results |
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Lipid Levels and Atherosclerotic Lesions
Both WHHLctrl and WHHLvegf rabbits had higher levels of total plasma cholesterol than NZW rabbits (WHHLctrl, 686±96; WHHLvegf, 846±115; NZW, 34±3 mg/dL; P<.0001). Similar results were observed for triglyceride levels (WHHLctrl, 527±65; WHHLvegf, 405±60; NZW, 86±6 mg/dL; P<.0001). No significant differences were observed between the values at day 0 and day 10 or between the two WHHL groups.
Atherosclerotic lesions were identified in 18 of 18 WHHL rabbits. Among 432 tissue sections retrieved from WHHL rabbits, atherosclerotic lesions were observed in 102 (24%). All lesions were focal, raised, and eccentric, and none resulted in significant luminal compromise, which is consistent with what has been described previously in WHHL rabbits of this age.11 The distribution, number, and anatomic extent of lesions were not different for WHHLctrl (47 of 216, 22%) versus WHHLvegf (55 of 216, 25%) rabbits (P=.36). No atherosclerotic lesions were observed in any of the tissue sections (n=216) retrieved from NZW rabbits.
Survival Rate
Two of 9 WHHLctrl (8.22%) rabbits died before
completion of the follow-up period (day 7 and day 8, respectively). In
both cases, examination of the ischemic limb disclosed
extensive necrosis. No other cause of death was found. No deaths
occurred among NZW or WHHLvegf rabbits
(
2=4.32; P=. 11). The 2 rabbits who
died were excluded from the subsequent analyses.
Angiography
In WHHLctrl, collateral artery development in
the medial thigh was attenuated compared with that observed for NZW
(Fig 2). The administration of rhVEGF,
however, markedly improved angiographically visible collateral vessels
in WHHL rabbits. An analysis of angiographic score
quantitatively confirmed the reduced collateral vessel network in
WHHLctrl rabbits compared with NZW and WHHLvegf
rabbits (WHHLctrl, 0.19±0.02; NZW, 0.35±0.03;
WHHLvegf, 0.47±0.04; P<.001; Fig 3A). The angiographic score for
WHHLvegf was significantly higher than that measured for
NZW rabbits (P=.04).
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Capillary Density
In normal limb muscles, capillary density was not significantly
different among the three groups (P=NS). In contrast, in the
ischemic limb muscles (Fig 2
), capillary density in sections of
muscle harvested from WHHLctrl was less than half of that
found in sections from NZW but was markedly increased among
WHHLvegf (WHHLctrl, 46.4±4.1; NZW, 78.9±4.6;
WHHLvegf, 98.7±4.4/mm2; P<.0002;
Fig 3). As was the case for angiographically detected vessels,
capillary density was also higher for WHHLvegf than for NZW
(P=.009). An analysis of capillary-to-myocyte ratio
yielded similar results (WHHLctrl, 0.14±0.01; NZW,
0.23±0.02; WHHLvegf, 0.28±0.01; P<.0004).
Hemodynamic Measurements
At day 10 after surgery, the blood pressure index was lower among
WHHLctrl than NZW but improved significantly in response to
rhVEGF (WHHLctrl, 9.5±3.5%; WHHLvegf,
46.4±4.6%; NZW, 32.8±2.8%; P<.0001).
Resting blood flow and endothelium-independent blood
flow (nitroprusside-induced) were similar in the normal limb of
WHHLctrl, NZW, and WHHLvegf rabbits
(P=NS; Fig 4A). In contrast,
endothelium-dependent blood flow
(acetylcholine-induced) was lower in WHHLctrl than in NZW
rabbits but increased significantly in response to rhVEGF
(WHHLctrl, 35.9±2.7; WHHLvegf, 44.9±3.0; NZW,
48.0±3.2 mL/min; P<.05; Fig 4A).
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In the ischemic limb, the resting (WHHLctrl,
7.5±0.6; WHHLvegf, 15.2±0.9; NZW, 13.6±0.7 mL/min;
P<.0001), endothelium-dependent
(WHHLctrl, 8.6±0.7; WHHLvegf, 25.8±1.7; NZW,
21.8±1.3 mL/min; P<.0001), and
endothelium-independent (WHHLctrl,
14.0±0.8; WHHLvegf, 30.2±1.8; NZW, 27.6±1.4
mL/min; P<.0001) blood flows were lower for
WHHLctrl than for NZW, and all improved to a statistically
significant degree after the administration of rhVEGF (Fig 4A). In
particular, endothelium-dependent flow was specifically
reduced in WHHLctrl as demonstrated by the adjustment to
endothelium-independent stimulated blood flow; the
latter may be considered an index for the size of the vascular bed.
Indeed, a reduction in the endothelium-dependent
flow/endothelium-independent flow ratio was observed
for WHHLctrl and improved with rhVEGF
(WHHLctrl, 0.61±0.03; WHHLvegf, 0.86±0.03;
NZW, 0.79±0.04; P<.002).
Systemic hemodynamic changes accompanying
administration of vasoactive agents are summarized in the
Table.
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Muscle Perfusion
As was the case for measurements performed with the
intra-arterial Doppler guide wire, muscle perfusion of
the normal limb measured at rest and after nitroprusside provocation
did not differ among WHHLctrl, NZW, and
WHHLvegf (P=NS).
In the ischemic hindlimb (Fig 4B), however, muscle perfusion at
rest was significantly lower in WHHLctrl than in NZW but
improved significantly after rhVEGF (WHHLctrl, 2.99±0.33;
NZW, 4.83±0.36; WHHLvegf, 5.58±0.46 mL ·
min-1 · 100
g-1; P<.004). Similar findings
were recorded in the ischemic hindlimb in response to
nitroprusside (WHHLctrl, 4.97±0.74; NZW, 9.63±1.06;
WHHLvegf, 11.32±0.92 mL ·
min-1 · 100
g-1; P<.002). The muscle perfusion
index was also lower in WHHLctrl than in the two other
groups at rest (WHHLctrl, 40.09±3.19%; NZW,
65.87±2.03%; WHHLvegf, 68.32±3.01%;
P<.0001) and after nitroprusside provocation
(WHHLctrl, 21.83±1.80%; NZW, 43.65±5.08%;
WHHLvegf, 49.62±5.96%; P=.001).
Tissue Damage
CK levels did not differ among groups before surgery
(P=NS). At day 10, however, the CK level for the
WHHLctrl group (10 714±1629 U/L) was still more than
fivefold that of NZW (2114±325 U/L) and WHHL treated with rhVEGF
(1630±475 U/L); P<.0002). Similarly, the percentage of TTC
negative (ischemic plus infarcted) muscles was significantly
higher for WHHLctrl (48.16±5.41%) than for NZW
(25.90±3.83%) or WHHLvegf (29.11±2.90%;
P<.004; Fig 5).
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Discussion |
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The present study establishes that in hypercholesterolemic rabbits, the anatomic extent of collateral vessel development in response to ischemia is both markedly reduced and associated with more severe tissue damage compared with that observed in control animals. The biological significance of these findings is underscored by the results of pilot experiments performed in our laboratory in which surgically induced limb ischemia in WHHL rabbits predictably resulted in severe limb necrosis beyond 10 days after surgery, necessitating the death of the animals. This contrasts with the results of similar surgery performed in NZW rabbits, in which gross evidence of vascular insufficiency is rare.7 20
The impact of spontaneous hypercholesterolemia on evidentiary findings of angiogenesis was both quantitative and qualitative. Quantitatively, the size of the vascular bed, including muscle capillaries and angiographically visible collateral vessels, was numerically reduced, as was endothelium-independent blood flow in the WHHL ischemic hindlimb. Qualitatively, endothelium-dependent responses in the collateralized ischemic hindlimb were more severely altered in hypercholesterolemic than in normal rabbits. We have previously demonstrated in a similar model, but using nonatherosclerotic animals, that endothelium-dependent responses are impaired in the collateral-dependent arterial bed.21 In WHHL rabbits, the reduction in endothelium-dependent flow was more severe than would be anticipated based on the anatomic reduction in the size of the vascular bed. Indeed, in the ischemic limb, the ratio of endothelium-dependent to endothelium-independent blood flow was lower in untreated WHHL than NZW rabbits. Whether these results are unique to inherited versus acquired hypercholesterolemia remains to be determined.
These results were not, however, due to baseline differences in the size of the vascular bed because in the "healthy" limb, no differences existed among groups with regard to resting or endothelium-independent blood flow or capillary density. Moreover, the findings cannot be attributed to stenotic or occlusive disease in the stem arteries that otherwise constitute the source of collateral vessel development in hindlimb ischemia. We intentionally used 6-month-old WHHL rabbits because previous pathological analyses11 have failed to disclose advanced lesions in WHHL rabbits at this age, a feature that was confirmed by the absence of obstructive lesions in 432 tissue sections we examined. Although the duration of follow-up (10 days) was too brief to evaluate the effects of rhVEGF on the extent of atherosclerotic lesion development, evidence that VEGF may inhibit neointimal thickening by expediting endothelial repair22 23 suggests longer-term studies to address this issue may be warranted.
The administration of rhVEGF markedly enhanced anatomic and functional indices of collateral vessel development in WHHL rabbits. Although all animals in the current protocol were electively killed at 10 days after surgery to permit concurrent anatomic analyses, subsequent experiments have shown that the regimen of rhVEGF administration we used permits indefinite integrity of the ischemic limb, including its neovasculature (E. Van Belle, unpublished data). These in vivo findings should not be interpreted as unique to VEGF. Chen et al,24 for example, have previously shown that basic fibroblast growth factor reverses atherosclerotic impairment of capillary-like microtubules that develop in vitro from explants of human coronary arteries.
Postnatal angiogenesis is considered to involve migration, proliferation, and remodeling of fully differentiated ECs derived from preexisting parent vessels.1 2 25 An increase in the number of blood vessels visualized angiographically in vivo and capillary density measured at necropsy were interpreted as evidence of augmented angiogenesis. Previous work performed in our laboratory using 5-bromodeoxyuridine immunostaining26 established that enhanced neovascularity after rhVEGF administration to NZW rabbits with unilateral hindlimb ischemia resulted from increased EC proliferation. The precise mechanisms responsible for secondary smooth muscle cell investment of endothelial neovessels remain to be defined, but they may involve elaboration by ECs of smooth muscle cell chemoattractants. Work in our laboratory, for example, has shown that VEGF stimulates release of platelet-derived growth factor-BB from activated ECs (E. Brogi and J. Isner, unpublished data).
The current in vivo and previous in vitro24 27 observations suggest that EC loss and/or dysfunction constitutes the basis for angiogenesis that is attenuated due to hyperlipidemia and/or atherosclerosis. Kolodgie et al,10 for example, previously documented focal EC disruption in similarly aged WHHL rabbits. Endothelial dysfunction, a well documented feature of atherosclerosis, was observed in the "healthy" limb of WHHL rabbits in the current study, as indicated by the reduction in endothelium-dependent flow.
Several different mechanisms may account for the reduced capacity of WHHL ECs to form new blood vessels. ECs have been previously shown to represent a contingency source of VEGF synthesis under hypoxic conditions.28 VEGF receptor expression is typically upregulated in a paracrine fashion by ischemic myocytes.29 Both of these responses may be potentially impaired in WHHL rabbits. EC migratory activity, a fundamental step in angiogenesis, appears impaired in atherosclerosis,24 possibly due to the effect of oxidized LDL components on the promigratory activity of endogenous angiogenic cytokines.27 Furthermore, the response to certain agonists requiring receptor binding and signal transduction to release intracellular calcium may be impaired in dysfunctional ECs.30 31 Such an impairment in transmembrane signaling could alter the responsiveness of ECs to EC mitogens.
The favorable impact of supplemental rhVEGF on collateral development in WHHL rabbits may be interpreted to imply that hypercholesterolemia leads to a relative diminution in endogenous EC mitogens required to stimulate angiogenesis. In this regard, our findings are consistent with the seminal work of Chen et al24 cited above. Whether this involves an absolute reduction in the synthesis and/or secretion of angiogenic cytokines, a relative increase in cytokine requirements due to endothelial dysfunction, or upregulation of angiogenesis inhibitors32 remains to be determined.
From a clinical standpoint, these results suggest that native collateral vessel formation may be inherently limited in the setting of hyperlipidemia and/or atherosclerosis but that its consequences at the tissue level may be at least in part reversible. In particular, the favorable effect of angiogenic cytokines on endothelial function demonstrated previously in nonatherosclerotic animal models with VEGF21 and basic fibroblast growth factor33 was reproduced in the WHHL rabbit. Thus, hyperlipidemia and/or atherosclerosis may not preclude successful application of therapeutic angiogenesis in patients with limb and possibly myocardial ischemia.
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Received February 26, 1997; revision received May 8, 1997; accepted May 15, 1997.
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