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Circulation. 1995;91:2793-2801

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(Circulation. 1995;91:2793-2801.)
© 1995 American Heart Association, Inc.

Articles

Local Delivery of Vascular Endothelial Growth Factor Accelerates Reendothelialization and Attenuates Intimal Hyperplasia in Balloon-Injured Rat Carotid Artery

Takayuki Asahara, MD; Christophe Bauters, MD; Christopher Pastore, BS; Marianne Kearney, BS; Susan Rossow, BS; Stuart Bunting, PhD; Napoleone Ferrara, MD; James F. Symes, MD; Jeffrey M. Isner, MD

From the Departments of Medicine (Cardiology) (T.A., C.B., C.P., M.K., S.R., S.B., N.F., J.M.I), Surgery (Cardiovascular) (J.F.S.), and Biomedical Research (T.A., C.B., C.P., M.K., S.R., S.B., N.F., J.M.I.), St Elizabeth's Medical Center, Tufts University School of Medicine, Boston, Mass; and the Department of Cardiovascular Research (S.B., N.F.), Genentech, Inc, South San Francisco, Calif.

Correspondence to Jeffrey M. Isner, MD, St Elizabeth's Medical Center, 736 Cambridge St, Boston, MA 02135.


*    Abstract
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 *Abstract
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Background Most strategies designed to reduce restenosis by the use of pharmacological or biological reagents involve direct inhibition of vascular smooth muscle cell (SMC) proliferation. Alternatively, SMC proliferation might be indirectly inhibited if reendothelialization could be specifically facilitated at sites of balloon-induced arterial injury. Accordingly, we investigated the hypothesis that application of an endothelial cell (EC)-specific mitogen to a freshly denuded intimal surface could accelerate reendothelialization and thereby attenuate intimal hyperplasia.

Methods and Results The left carotid artery of 31 Sprague-Dawley rats was subjected to balloon injury, after which 16 rats were treated with a 30-minute incubation with 100 µg of vascular endothelial growth factor (VEGF), an EC-specific mitogen. Control animals (n=15) received a 30-minute incubation with 0.9% saline. At 2 weeks after balloon injury, carotid artery reendothelialization was markedly superior in the VEGF-treated group compared with the control group (14.59±1.12 versus 7.96±0.51 mm2, P<.0005). The extent of reendothelialization measured at 4 weeks after balloon injury remained superior for arteries treated with VEGF (18.04±0.90 mm2) versus saline (13.42±0.84 mm2, P<.005). Neointimal thickening was correspondingly attenuated to a statistically significant degree in arteries treated with VEGF versus the control group at both the 2-week and 4-week time points. Immunostaining for proliferating cell nuclear antigen (PCNA) disclosed a threefold increase in PCNA-positive cells in the neointima of control arteries versus VEGF-treated arteries at 2 weeks after injury.

Conclusions Application of VEGF, an EC-specific growth regulatory molecule, may be effectively used in vivo to promote reendothelialization and thereby indirectly attenuate neointimal thickening due to SMC proliferation.


Key Words: growth factors • endothelium • neointima • stenosis


*    Introduction
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 up arrowAbstract
 *Introduction
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Vascular endothelial growth factor (VEGF) is unique among angiogenic growth factors by virtue of the fact that its high-affinity binding sites (Flt-11 and Flk-1/KDR2 3 ) are present on endothelial cells (ECs) but not on other cell types; consequently, the mitogenic effects of VEGF are limited to ECs.4 5 This contrasts, for example, with acidic and basic fibroblast growth factors (aFGF and bFGF), both of which are mitogenic for smooth muscle cells (SMCs)6 7 and fibroblasts in addition to ECs. Recent studies from our laboratory have documented that VEGF administered as a single intra-arterial bolus of 500 to 1000 µg is sufficiently potent to augment collateral artery development and thereby ameliorate hemodynamic8 and physiological9 deficits in a rabbit model of hindlimb ischemia. The extent to which this is likely related to the potency of VEGF to serve as an EC mitogen is suggested by studies documenting the effects of VEGF on EC proliferation in vitro10 and, more recently, in vivo.11 In the present study, we sought to exploit the EC-specific mitogenic properties of VEGF for another purpose, ie, to expedite reendothelialization of a freshly injured artery. Specifically, we investigated the hypothesis that a single, direct application of VEGF to the intimal surface of a balloon-injured artery could accelerate reendothelialization and reduce neointimal thickening.


*    Methods
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 *Methods
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Study Protocol
All surgical procedures were performed by one trained operator. A total of 31 male 2- to 3-month-old Sprague-Dawley rats (weight, 450 to 600 g) were used. All rats were anesthetized with an intraperitoneal injection of pentobarbital sodium solution (60 mg/kg) (Abbott). The method of balloon denudation was adapted from the technique established by Reidy et al.12 The bifurcation of the left common carotid artery was exposed, and blood flow to the site of surgical manipulation was temporarily interrupted by lifting up the left common, internal, and external carotid arteries with surgical ligatures. A 2F Fogarty catheter (Baxter Edwards) was introduced through an arteriotomy in the external carotid artery and advanced to the proximal edge of the omohyoid muscle. To produce deendothelialization injury of the left common carotid artery, we inflated the balloon with saline and withdrew it three times from just under the proximal edge of the omohyoid muscle to the carotid bifurcation. Immediately after removing the balloon catheter, we introduced an infusion cannula into the external carotid artery to administer treatment or control solution into the temporarily isolated segment of left common carotid artery.

The VEGF-treated group of 16 rats received a 30-minute incubation with 100 µg VEGF (Genentech, Inc) in 0.15-mL solution of 0.1% albumin; this solution was shown to contain no detectable lipopolysaccharide. This dosage was arbitrarily selected based in part on previous demonstrations that 500 µg (approximately 1.25 mg/kg) administered as an intra-arterial bolus induced enhanced angiogenesis in a rabbit model of hindlimb ischemia.8 9 The control group of 15 rats received a 30-minute incubation with 0.9% saline solution. After the 30-minute incubation period, which was performed at room temperature, the ligatures applied to the left common and internal carotid arteries were released; the external carotid artery was permanently ligated after the infusion cannula was withdrawn. The choice of agent to be administered was decided in a random manner, and the operator was blinded to the content of treatment solution.

After surgery, the rats were allowed to recuperate, housed in a US Department of Agriculture–approved facility with free access to rat chow (Agway) and water, and exposed to a 12-hour light/dark cycle. The experimental protocol for this project was approved by the St Elizabeth's Medical Center Institutional Animal Care and Use Committee and complied with the "Guide for the Care and Use of Laboratory Animals" (NIH publication No. 86-23, revised 1985).

Rats were killed 2 weeks after injury (2-week VEGF group [n=7], 2-week control group [n=7]) or 4 weeks after injury (4-week VEGF group [n=9], 4-week control group [n=8]).

Necropsy Examination
Thirty minutes before they were killed, all rats received an intravenous injection via the tail vein of 0.5 mL 0.5% Evans blue dye (Sigma Chemical Co)13 to allow us to microscopically identify the remaining denuded area. With the rats under general anesthesia with intraperitoneal sodium pentobarbital solution (60 mg/kg), a cannula inserted into the left ventricle was used to perfuse phosphate-buffered saline (PBS) in situ at a pressure of 90 mm Hg until the effluent ran clear via bilateral jugular venous vents; this was followed by 5 minutes of fixation with 100% methanol. The same anatomic landmarks used to apply the initial balloon injury were used to identify the arterial segment to be harvested. Specifically, the initially denuded segment of the left common carotid artery, from just under the proximal edge of the omohyoid muscle to the carotid bifurcation, was dissected free and incised longitudinally. The harvested segment of carotid artery was pinned to a cork board, fixed in 100% methanol, and photographed with a dissecting microscope (STEMI SR, Zeiss) in preparation for planimetric analysis of the reendothelialized area. Tissues were then fixed by immersion in 100% methanol, embedded on the longitudinal edge in paraffin, and cut into 5-µm sections onto silane-coated slides.

Planimetric Analysis of Reendothelialized Arterial Segment
Planimetric analysis of the photograph of the harvested arterial segment stained with Evans blue dye to identify the remaining endothelium-denuded site was performed with a computerized sketching program (MACMEASURE, version 1.9, National Institute of Mental Health) on a digitizing board (Summagraphics) by one analyst, who was blinded to the treatment regimen. The initially denuded area was defined as the total surface area of the harvested arterial segment. The length of the harvested segment corresponded to the total length of the injured segment; in each case, this length was similarly defined proximally by the carotid bifurcation and distally by the edge of the omohyoid muscle. The reendothelialized area was defined macroscopically as the area that was not stained with Evans blue dye.

Evaluation of Intimal Hyperplasia
Because the thickness of the native media of the artery wall is variable and reflects the size (diameter) of the rat carotid artery, it was used to index the area of neointima resulting from balloon injury.14 Accordingly, neointimal thickening was assessed in terms of the intima area–to–media area ratio (I/M) measured from longitudinal sections of hematoxylin and eosin– or elastic-trichrome–stained arterial sections by one analyst, who was blinded to the treatment regimen. The histological sections were projected onto the digitizing board, and the areas of the intima and media were measured with the computerized sketching program by a technician who was blinded to the treatment regimen.

Evaluation of Proliferative Activity in Injured Artery
Proliferative activity in the injured arterial segment harvested when the rats were killed was evaluated by histochemical analysis for proliferating cell nuclear antigen (PCNA), as previously described.15 Endogenous peroxide activity was blocked with 3.0% hydrogen peroxide in PBS. Nonspecific protein binding was blocked with 10% normal horse serum. Sections were incubated overnight at 4°C with a mouse monoclonal antibody against PCNA (clone PC10, Signet) at a dilution of 1:40 in 1% bovine serum albumin/PBS. Negative control sections were incubated with MOPC-21, a purified nonspecific mouse monoclonal antibody (Sigma). Bound primary antibody was detected using an avidin-biotin-immunoperoxidase method according to supplier's guidelines (Elite Avidin-Biotin Detection System, Signet). Sections were lightly counterstained with hematoxylin. Sections were finally mounted in aqueous mounting medium. Regenerated endothelium was identified by immunostaining with Bandeiraea simplicifolia I lectin (5 µg/mL; Vector Laboratories). Additional staining for Bandeiraea simplifolia I lectin B416 was performed on adjacent sections to identify ECs. For lectin staining, sections pretreated with 0.3% hydrogen peroxidase were incubated with 5 µg/mL of biotinylated lectin (Vector) overnight at 4°C. After washing, slides were incubated with peroxidase-conjugated streptavidin (BioGenex Laboratories) for 1 hour; 3-amino-9-ethylcarbazole (Signet) was then applied as a substrate for the enzyme, resulting in a brown reaction product.

The extent of proliferative activity for each longitudinal section was measured by the number of positive cells in the intima per length in millimeters of the longitudinal section (ie, cells per millimeter). PCNA staining of the rat ileum served as the positive control. Reendothelialized intima was identified as area covered with a lining of cells staining positive for lectin. Nonendothelialized area was defined as an area of intima not covered by a lining of lectin-positive cells. These examinations were performed by one analyst, who was blinded to the treatment regimen.

Scanning Electron Microscopy
Tissue specimens from 4-week VEGF-treated and 4-week control animals were studied using scanning electron microscopy to evaluate the extent of reendothelialization of the harvested arterial segment. Harvested arteries were fixed overnight at 4°C in 2.5% glutaraldehyde in 0.13 mol/L cacodylate buffer, pH 7.4. After being rinsed twice in buffer, specimens were postfixed in 1% ostium tetroxide at 4°C for 1.5 hours, rinsed three times in cacodylate buffer, and dehydrated through graded concentrations of ethanol. The specimens were critical point–dried from 100% ethanol with liquid CO2. After the specimens were dried, they were bisected to expose the luminal surface, mounted on stubs, and sputter-coated with gold. The samples were viewed and photographed at 10 kV with a scanning electron microscope (model ISI-DS130, International Scientific Instrument). Photomicrographs were taken at x200 and x700 magnifications.

Statistical Analysis
Results were expressed as mean±SEM. Differences between groups were evaluated using two-tailed, unpaired Student's t test. Differences were considered significant at P<.05.


*    Results
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*Results
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Total Area of Initial Balloon Injury
Digitized planimetric analysis of the initially deendothelialized area included the total intimal surface area of the balloon-injured carotid artery retrieved when the animals were killed. As indicated, for both treatment and control groups, the length of the excised segment of artery was determined by the identical anatomic landmarks used to direct the initial balloon injury: from just under the proximal edge of the omohyoid muscle, craniad, to the arteriotomy site, caudad. Planimetric analysis disclosed no statistically significant difference between the total area of initial balloon injury (mm2) in the VEGF-treated group (17.6±0.6) and that of the control group (18.3±0.7) (P=NS) (Fig 1Down).



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  		Figure 1. Bar graph showing planimetric analysis of extent of reendothelialization. Injured area was similar for animals in control group and in vascular endothelial growth factor (VEGF)-treated group. Reendothelialized area, indicated macroscopically by absence of blue dye stain, was more extensive in VEGF-treated animals than in control animals, at both 2 weeks and 4 weeks after injury.

Extent of Reendothelialization
Acceleration of reendothelialization was observed in the VEGF-treated group versus the control group at 2 weeks after balloon injury. The reendothelialized area (in mm2) in the 2-week control group was 8.0±0.5; in contrast, the reendothelialized area in the 2-week VEGF-treated group was 14.6±1.1 (P<.0005) (Fig 1Up). Representative examples of the macroscopic appearance of Evans blue dye–stained segments from control and VEGF-treated animals are shown in Fig 2Down. Expressed as a percentage of the total area that was initially deendothelialized, the reendothelialized area in the control group was 44.0±3.6%; in contrast, the percentage of reendothelialized area in the VEGF-treated group was nearly twice as great (80.7±4.9%) (P<.0001) (Fig 3Down).



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  		Figure 2. Representative macroscopic photographs of harvested arterial segments at 2 weeks (top) and 4 weeks (bottom); at both time points, extent of reendothelialization of vascular endothelial growth factor (VEGF)-treated segments exceeds that observed for control segments. Evans blue dye was injected 1 hour before the animals were killed. Nonendothelialized dye is clearly defined by blue staining, whereas reendothelialized area appears white.



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  		Figure 3. Scatterplots of extent of reendothelialization calculated as percentage of area initially injured. Percentage of reendothelialization in 2-week vascular endothelial growth factor (VEGF) group (80.7±4.9) is nearly twice that of the 2-week control group (44.00±3.56, P<.0001); note absence of any overlap for results at this time point. At 4 weeks, percentage reendothelialization for VEGF-treated group (mean±SEM, 94.9±1.7) is still more than that of control animals (75.6±6.0, P<.005).

The extent of reendothelialization measured at 4 weeks after balloon injury remained superior for arteries treated with VEGF versus arteries of control animals. The reendothelialized area (in mm2) in the 4-week control group was 13.4±0.8; in contrast, the reendothelialized area in the 4-week VEGF-treated group was 18.0±0.9 (P<.005) (Fig 1Up). Although reendothelialization in the control group at 4 weeks extended to only 75.6±6.0% of the total area of initial balloon injury, reendothelialization at 4 weeks was nearly complete (94.9±1.7%) among the VEGF-treated group (P<.005) (Fig 3Up).

Scanning electron microscopy disclosed a regular and smooth-surfaced endothelial lining in the 4-week VEGF group, similar to the endothelial lining of a normal, uninjured rat carotid artery. The intimal surface of segments harvested from the 4-week control group, however, demonstrated an irregular endothelial lining, leaving exposed subendothelial tissue (Fig 4Down).



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  		Figure 4. Scanning electron photomicrographs of intimal surface of left common carotid artery of uninjured animals (left), of balloon-injured, untreated 4-week control animals (center), and of balloon-injured, 4-week vascular endothelial growth factor (VEGF)-treated animals (right). Top, original magnification of each x200; bottom, each x700. Reendothelialized segment of the VEGF-treated artery appears similar to that of the untreated, uninjured normal artery. In contrast, examination of the control artery discloses exposed subendothelium.

Neointimal Thickening
A highly significant reduction in neointimal thickening was observed in arteries treated with VEGF versus the control group at both 2 and 4 weeks. For the 2-week control group, I/M was 1.23±0.07; in contrast, I/M for VEGF-treated balloon-injured arterial segments was 0.81±0.11 (P<.01) (Figs 5Down and 6Down).



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  		Figure 5. Plot of ratio of intimal area to medial area (I/M ratio) at 2 and 4 weeks after injury. I/M ratio (1.23±0.07) is significantly higher among 2-week control animals than 2-week animals treated with vascular endothelial growth factor (VEGF) (0.81±0.11, P<.01). Intimal thickening progressed in control group during subsequent 2 weeks; in contrast, intimal thickening essentially ceased in VEGF-treated group. Consequently, I/M ratio at 4 weeks was 1.60±0.12 for control animals vs 0.86±0.05 for VEGF-treated group (P<.0001).



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  		Figure 6. Representative photomicrographs demonstrating intimal area–to–medial area ratio (of balloon-injured arterial segment) at 2 weeks (top) and 4 weeks (bottom) for control animals (left) and vascular endothelial growth factor–treated animals (right). VEGF indicates vascular endothelial growth factor; IEM, internal elastic membrane.

This difference between the two groups was more profound at the 4-week time point. In the control group at 4 weeks, I/M increased further to 1.60±0.12; in contrast, I/M among the arteries treated with VEGF remained similar at 4 weeks (0.86±0.05) to that measured at 2 weeks and significantly less than that of the 4-week control group (P<.0001) (Figs 5Up and 6Up).

Proliferative Activity
Proliferative activity in the balloon-injured arteries of both groups was evaluated at 2 weeks and again at 4 weeks. For the 2-week control group, proliferative activity was significantly higher than that of the VEGF-treated group. At this time point, proliferative activity was limited predominantly to the neointima. PCNA-positive cells were identified more frequently in the neointima of the 2-week control group (22±4.5 mm-1) than in the neointima of the 2-week VEGF-treated group (8±3 mm-1) (P<.05) (Fig 7Down). Furthermore, most of the PCNA-positive cells identified at this time point were localized to the nonendothelialized segment of neointima; this was the case for both control (96.9±1.5%) and VEGF-treated (97.2±2.0%) animals. When analysis of proliferative activity was limited to the nonendothelialized segment of neointima, PCNA-positive cells were identified as frequently in the 2-week VEGF-treated group (36±12 mm-1) as in the 2-week control group (34±6 mm-1) (P=NS) (Fig 8Down). Shown in Fig 9Down is a representative illustration of the fact that PCNA-positive cells were far less frequent in segments of the artery in which the endothelium had been reestablished than in those in which the endothelium remained absent; furthermore, Fig 9Down shows that positive immunostaining for PCNA was commonly observed as well in ECs at the leading edge of repair.



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  		Figure 7. Bar graph of proliferative activity, assessed by immunostaining for proliferating cell nuclear antigen (PCNA). Nonendothelialized area was identified as area devoid of superficial layer of lectin-positive cells. PCNA-positive cells were identified approximately three times more frequently in the total length of neointima of the 2-week control animals (22±5 mm-1) vs the neointima of the 2-week vascular endothelial growth factor (VEGF)-treated group (8±3 mm-1) (P<.05). Furthermore, most of the PCNA-positive cells identified at this time were localized to the nonendothelialized segment of neointima; this was the case for both the control animals (96.9±1.5%) and the VEGF-treated animals (97.22±2.00%).



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  		Figure 8. Bar graph of analysis of proliferative activity limited to the nonendothelialized segment of neointima at 2 weeks after injury. When analysis of proliferative activity was limited to the nonendothelialized segment of neointima, proliferating cell nuclear antigen (PCNA)-positive cells were identified as frequently in the 2-week vascular endothelial growth factor (VEGF)-treated group (36±12 mm-1) as in the 2-week control group (35±6 mm-1) (P=NS).



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  		Figure 9. Representative photomicrographs demonstrating proliferating cell nuclear antigen (PCNA) immunostaining in control (top) and vascular endothelial growth factor (VEGF)-treated (bottom) arteries at 2 weeks. Lectin-stained sections are on left; PCNA immunostaining sections are on right. PCNA-positive cells were far less frequent in those segments of artery in which endothelium (identified by lectin immunostain) had been reestablished than in those in which endothelium remained deficient.

At 4 weeks after injury, the frequency of PCNA-positive cells was reduced in both control (0±0 mm-1) and treated (1±1 mm-1) animals, and the difference between these two groups at 4 weeks was insignificant (P=NS). If analysis of proliferative activity at 4 weeks was limited to the nonendothelialized neointima, PCNA-positive cells were as frequent in the narrow zone of residual nonendothelialized neointima of the VEGF-treated group (5±4 mm-1) as in the 4-week control group (5±4 mm-1) (P=NS).


*    Discussion
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*Discussion
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Previous studies carried out in a variety of animal species have repeatedly shown that extensive endothelial denudation of the arterial wall leads to neointimal thickening.17 Because it is difficult to achieve extensive endothelial denudation without injuring the underlying media, it has been difficult to establish with certainty that the loss of endothelial integrity per se constitutes the sole and/or primary basis for neointimal thickening. In fact, in certain animal models, neointimal thickening can be reproducibly created by electrode stimulation18 or other forms of injury19 20 applied exclusively to the adventitial aspect of the vessel wall. Nevertheless, at least four studies13 21 22 23 in the rat model of arterial balloon injury have clearly established an inverse relation between endothelial integrity and SMC proliferation. Moreover, elaboration of extracellular matrix, an important if not dominant contributor to the development of both primary and restenotic lesions in humans,24 is likewise augmented after endothelial disruption.25 26

These experimental studies have been cited to support the notion that certain functions of the endothelium—including barrier regulation of permeability, thrombogenicity, and leukocyte adherence, as well as production of growth-inhibitory molecules—are critical to prevention of luminal narrowing by neointimal thickening.27 This concept has stimulated efforts to preserve intact the endothelium of native veins used for bypass surgery,28 29 accelerate reendothelialization after balloon-induced arterial injury,30 31 and facilitate endothelialization of prosthetic conduits32 33 34 35 or endovascular stents.36 37

In the present study, local delivery of VEGF to the site of balloon injury resulted in expeditious reendothelialization. By 2 weeks after injury, reendothelialization of balloon-injured arterial segments in the VEGF-treated group was 80% complete; this finding represented profound acceleration of repair in comparison to identically injured arteries in the control group, in which reendothelialization remained limited to only 44% of the balloon-injured arterial segment. Even at 4 weeks after injury, natural mechanisms of repair among the control animals failed to match the extent of reendothelialization observed in response to a single application of VEGF: Although reendothelialization was nearly complete in the VEGF-treated group, the injured arterial segment of control animals remained approximately 24% nonendothelialized.

This rapid regeneration of ECs appeared to contribute to suppression of intimal hyperplasia. Animals in the VEGF-treated group showed less intimal thickening than those in the control group at 2 weeks after injury; moreover, the modest extent of neointimal thickening observed among the VEGF-treated animals remained essentially unchanged during the subsequent 2 weeks (weeks 2 to 4 after injury), whereas neointimal thickening in the control group continued to progress. The notion that this observed differential in neointimal thickening truly represents a differential degree of intimal hyperplasia is supported by the results of PCNA immunostaining: The frequency of PCNA-positive cells in the neointima of control animals at 2 weeks was nearly threefold that observed among animals treated with VEGF. The inverse relation between reendothelialization and neointimal SMC proliferation was graphically demonstrated by the fact that >96% of PCNA-positive cells in the neointima of both groups were observed at neointimal sites devoid of endothelium. Neointimal thickening thus developed in association with delayed reendothelialization. Conversely, the observations from this series of animal experiments suggest that if reendothelialization can be accelerated after arterial injury, then associated neointimal thickening may be correspondingly reduced.

In the present study, we did not attempt to measure the extent to which a potential reduction in extracellular matrix may have contributed to reduced intimal thickening in VEGF-treated arteries. This would not be an unexpected finding, given that the inhibitory effect of VEGF on SMC accumulation would likely translate into reduced SMC synthesis of matrix proteoglycans.38 Furthermore, the potential for regenerating ECs to contribute directly via elaboration of mediators capable of matrix degradation cannot be excluded.

The facilitatory effect of VEGF on reendothelialization constitutes a novel therapeutic application for this growth-regulatory molecule. Previous studies from our laboratory established that VEGF administered intra-arterially8 9 or intravenously39 as a single bolus of 500 to 1000 µg was sufficiently potent to accomplish therapeutic angiogenesis in a rabbit model of hindlimb ischemia; more recently, a similarly efficacious effect was demonstrated in a canine40 model of chronic coronary artery occlusion. Both applications—on arterial reendothelialization as well as collateral artery development—derive at least in part from the documented ability of VEGF to stimulate EC proliferation.10 Adjunctive studies using bromodeoxyuridine labeling have quantified the extent to which EC proliferation stimulated by VEGF contributes to the development of collateral vessels in the rabbit hindlimb model.11

Potentially contributory as well, however, and beyond its mitogenic effects, is the potential for VEGF to modulate qualitative aspects of EC function.41 Ku et al,42 for example, demonstrated that direct application of VEGF to isolated segments of canine coronary arteries induces endothelium-dependent relaxation. Because the response was diminished by pretreatment with the tyrosine kinase inhibitor genistein, Ku et al further inferred that arterial relaxation was in this case mediated by VEGF receptor–induced tyrosine phosphorylation of phospholipase C-{gamma}1. The finding by Peters et al41 of the fms-like tyrosine kinase receptor (Flt) in the endothelium of mature (quiescent) endothelium of adult organs was likewise interpreted as evidence for the concept that VEGF may be important for the maintenance and repair of the endothelium.

These quantitative and qualitative effects of VEGF are limited to ECs by virtue of the exclusive disposition of high-affinity binding sites to this cell type. (Interaction of VEGF with lower-affinity binding sites has been shown to induce mononuclear phagocyte chemotaxis.43 44 ) This cell specificity represents a potential advantage of VEGF in contrast to other EC mitogens that have been studied for their ability to promote angiogenesis as well as endothelial repair. In vivo studies performed in the rat carotid artery model of balloon injury, for example, established clear evidence for the mitogenic effect of bFGF on EC replication in vivo and further demonstrated that total EC regrowth could be achieved by administration of bFGF in doses of 12 µg twice weekly for up to 8 weeks.45 In this particular case, concurrent effects on SMC proliferation were not discussed. Previous work from this same group of investigators,46 however, has documented the mitogenic effects of bFGF on SMC proliferation in vivo, including the demonstration that specific antibodies to bFGF inhibit SMC proliferation.47 In the case of aFGF—also a known mitogen for vascular SMCs in vitro48 —low doses (<1% of the doses of bFGF used in the aforementioned studies) administered to this same animal model were shown to have an inhibitory effect on neointimal thickening; although morphometric analyses suggested that the extent of neointimal thickening was inversely related to the extent of reendothelialization, the extent to which endothelial repair per se was completed was not discussed in this report.49

Finally, it is encouraging from a practical standpoint that in the present study, a single application of VEGF was sufficient to facilitate endothelial repair that was 80% complete by 2 weeks after injury. Provided that a similar magnitude of recombinant protein could be efficiently delivered in association with balloon angioplasty, the potential for VEGF to optimize the short-term as well as long-term consequences of this widely used intervention is intriguing. In contrast to most strategies that have been designed to reduce restenosis by directly inhibiting SMC proliferation, the present findings suggest that SMC proliferation might be indirectly inhibited by directly facilitating reendothelialization. In this regard, it will be important to verify that the qualitative characteristics of the neoendothelium demonstrated anatomically in the present study are satisfactory to reestablish those aspects of EC function believed to confer the antithrombotic, antiadhesive, and antiproliferative effects that limit neointimal thickening. Several experimental studies, for example, have suggested that regenerating ECs, particularly soon after disruption, are functionally impaired with regard to vasomotor reactivity of the traumatized vessel.50 51 52 53 Whether the favorable effects of reendothelialization on intimal thickening demonstrated in the present study indicate functional dissociation between effects on vasomotor tone versus SMC proliferation in regenerating ECs remains to be determined.


*    Acknowledgments
 
This work was supported in part by Academic Award in Vascular Medicine grant HL-02824 and grant HL-40518, both from the National Heart, Lung, and Blood Institute, National Institutes of Health. Dr Bauters was the recipient of the French Federation of Cardiology Research Fellowship 1993. We gratefully acknowledge Mickey Neely for assistance in the preparation of the manuscript.

Received October 31, 1994; revision received January 18, 1995; accepted January 22, 1995.


*    References
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 up arrowIntroduction
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*References
 

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