(Circulation. 1997;95:438-448.)
© 1997 American Heart Association, Inc.
Articles |
Stent Endothelialization
Time Course, Impact of Local Catheter Delivery, Feasibility of Recombinant Protein Administration, and Response to Cytokine Expedition
the Departments of Medicine (Cardiology) and Biomedical Research, St Elizabeth's Medical Center, Tufts University School of Medicine, Boston, Mass, and the Department of Pathology, University of Texas, San Antonio (F.O.T.).
Correspondence to Jeffrey M. Isner, MD, St Elizabeth's Medical Center, 736 Cambridge St, Boston, MA 02135. E-mail jisner{at}opal.tufts.edu
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Abstract |
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Background Because prior studies have established the critical role of the endothelium in preventing vascular thrombosis and intimal thickening, we designed a series of experiments to determine the feasibility of percutaneous local catheter delivery of recombinant protein to accelerate development of an intact endothelial monolayer after stent implantation.
Methods and Results Balloon injury followed by percutaneous delivery of a 15-mm-long, balloon-expandable metallic stent was performed in 64 rabbit external iliac arteries (baseline diameter, 2.67±0.07 mm). Planimetric time-course analysis disclosed <20% stent endothelialization at 4 days, <40% at 7 days, and near-complete endothelialization at 28 days. The reporter protein horseradish peroxidase and the endothelial cell–specific recombinant protein vascular endothelial growth factor (VEGF) were each effectively delivered from a local delivery catheter (channel balloon catheter, ChB) after stent implantation. Although local catheter delivery (of vehicle control) itself mildly retarded the extent of stent endothelialization (10.6±2.9%) versus no local delivery (25.5±6.6%, P=.045), local ChB delivery of 100 µg VEGF overcame this catheter effect: By day 7, stent endothelialization was nearly complete (91.8±3.8%) (P<.0001 versus no local delivery). Consequently, stent thrombus was reduced in the VEGF-treated group (mural thrombus, 5.3±3.7%) versus no local delivery (29.3±6.8%, P=.006). Occlusive thrombus was seen only in the absence of local VEGF administration.
Conclusions (1) Local delivery of recombinant protein to the arterial wall is feasible after stent implantation, and (2) local delivery of the endothelial cell mitogen VEGF accelerates stent endothelialization, reducing stent thrombosis. These results thus establish a novel means by which the safety and/or bioactivity of endovascular stents may be further enhanced.
Key Words: angioplasty • stents • endothelium • thrombosis • growth substances
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Introduction |
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Two landmark clinical trials1 2 have established stents as the first mechanical device to reduce restenosis after balloon angioplasty. Efforts to extend the potential clinical applications of coronary and peripheral vascular stents include strategies designed to further reduce the thrombogenic potential of metallic stents and/or inhibit intimal thickening within the stent, thus further reducing the incidence of restenosis. Both of these goals are considered particularly important if the use of stents is to be extended to smaller-diameter (<3.0 mm) and longer (infrainguinal) vascular segments.
The results of prior investigations have established the critical role of the endothelium in preventing intimal thickening and vascular thrombosis.3 4 5 We therefore considered that these two issues could be satisfactorily addressed in the case of endovascular prostheses by minimization of the time required for stents to develop an endothelial covering. To investigate this strategy, we first sought to determine the time course for stent endothelialization. We then investigated the possibility that the trauma of local catheter delivery could affect this process adversely. The third issue we addressed was the feasibility of performing local protein delivery through a deployed stent to the underlying arterial wall. Finally, we investigated local delivery of an endothelial cell–specific mitogen, rhVEGF165,4 to balloon-injured, stented arteries of New Zealand White rabbits as a means of accelerating stent endothelialization (StE).
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Methods |
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Animals
New Zealand White rabbits (weight, 5.0 to 5.5 kg) were used exclusively for these experiments, according to protocols approved by St Elizabeth's Institutional Animal Care and Use Committee. All animals received aspirin 50 mg/d for 7 days before the initial intervention and until they were killed. All procedures were performed under general anesthesia induced by intramuscular injection of ketamine (50 mg/kg) and acepromazine (0.2 mg/kg) after premedication with xylazine (10 mg/kg).
In Vivo Catheter Procedures
The EIA was used for all experiments. A 5F introducer sheath was positioned in the femoral artery under surgical exposure, after which nitroglycerin 0.25 mg and heparin 1000 USP units were administered intra-arterially. All catheters were subsequently introduced through this sheath and advanced to the EIA via a 0.014-in guidewire. Arterial injury was performed with a 3F Fogarty balloon catheter (Baxter Edwards).6 Stent implantation was performed with a 15-mm-long Palmaz-Schatz coronary stent (Johnson & Johnson Interventional Systems) over an angioplasty balloon catheter (SCIMED) and apposed to the vessel wall by high-pressure balloon inflation (10 atm) to achieve a 1.1:1.0 to 1.2:1.0 stent-to-artery ratio (see "Results"). After stent deployment, local catheter delivery was performed as described below.
Quantitative Angiography
Baseline angiograms of the EIAs and radiographs of the stents after implantation were obtained for each animal. Quantitative analysis was performed with a computerized analysis system (ImageComm) that has previously been validated.7 In each vessel, the stent-to-artery ratio was calculated as the ratio of the mean stent diameter to the mean baseline angiographic luminal diameter.
Percutaneous Local Catheter Delivery With the ChB
Local protein delivery was performed through a ChB (Boston Scientific). The ChB incorporates a conventional, 20-mm-long polyethylene teraphthalate balloon covered by a layer of 24 perforated channels, each with a single 100-µm hole through which drug is delivered via an independent lumen.8 9 The ChB was advanced into the EIA and inflated at nominal pressure (6 atm). Protein solution was instilled slowly through the infusion port of the ChB at low pressure (100 to 150 mm Hg). Infusion and incubation times were 5 and 10 minutes, respectively. The balloon was then deflated and the ChB removed.
Quantitative Analysis of StE as a Function of Time (Protocol A)
Because quantitative data regarding StE as a function of time are limited, we first sought to define the temporal sequence of StE. This experiment involved unilateral balloon injury of the EIA followed by stent implantation (protocol A, Fig 1A
). The animals were killed after stent deployment on days 0 (n=3), 4 (n=2), 7 (n=2), 14 (n=2), and 28 (n=3).
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Effect of Local Catheter Delivery on StE (Protocol B)
The impact on StE of additional injury that could potentially result from local catheter delivery (ie, inflation of the ChB and infusion of vehicle solution) has not been defined previously. Such putative injury could compromise the net benefit of any locally administered agent. To address this issue, 9 rabbits underwent balloon injury followed by stent implantation in both EIAs. In one stented EIA (randomly chosen), vehicle control was delivered locally; the contralateral stented EIAs of these 9 rabbits received no therapy and thus served as an internal control for the stented arterial site receiving local catheter delivery. This experiment (protocol B) is illustrated in Fig 1B
. The stent site that was completely untreated in these 9 animals was analyzed to determine the extent to which StE develops in the absence of additional balloon injury. On the basis of the results of the above time-course experiment, all 9 animals were killed at day 7.
Feasibility and Efficacy of Local Protein Delivery After Stent Implantation
To determine the feasibility of local protein delivery to the arterial wall immediately after stent deployment, a preliminary study was performed with local delivery of HRP (type II, 10 mg/mL, dissolved in PBS with 0.1% rabbit serum albumin, Sigma) as a reporter protein. The peroxidase reaction of HRP with the enzyme substrate 3,3'-diaminobenzidine produces a characteristic brown precipitate, which may be detected on sections of the artery wall and provides semiquantitative information (staining intensity is proportional to tissue concentration of HRP).10 HRP is a relevant reporter protein for the present experiments because the size of the HRP protein (44 kD)10 is similar to the size of VEGF (46 kD).11
Three animals underwent focal balloon injury of the EIA and stent implantation as described above, followed by local delivery of 0.5 mL HRP. Three additional animals underwent the same procedure without stent implantation and were used as positive controls. All 6 animals were killed within 1 to 2 hours after completion of the experiment, and three 5-mm-long arterial segments were retrieved from the site of balloon injury, placed immediately in OCT compound, frozen in liquid N2, and stored at -80°C. Two or 3 days later, 6-µm frozen sections were placed on gelatin-coated slides. Incubation of the slides with 3,3'-diaminobenzidine was carried out as described previously,12 after which the slides were passed through dehydration solutions to xylene, coverslipped, and allowed to dry. Reaction product was visualized as a dense brown precipitate on a colorless background of tissue. Remote vessels and vessels of uninstrumented animals were used as negative controls.
Local Delivery of rhVEGF165 After Stent Implantation (Protocol C)
The possibility that StE could be expedited after implantation has not been previously tested. We used rhVEGF165 (a generous gift from B. Keyt and S. Bunting, Genentech) expressed, refolded, and purified from transfected Escherichia coli as previously described13 to test this strategy. The dose of rhVEGF165, 100 µg, was the lowest dose previously found to be efficient for in vivo acceleration of reendothelialization after local protein administration.4
These 17 rabbits underwent bilateral EIA balloon injury and stent implantation. Local delivery of 100 µg of rhVEGF165 in PBS/0.1% albumin was performed as described above to one EIA (randomly chosen), and vehicle alone (PBS/0.1% albumin) was administered to the contralateral artery (protocol C, Fig 1C). These 17 rabbits were killed 4 (n=8) or 7 (n=9) days after the procedure.
Detection of rhVEGF165 After Local Delivery
To confirm the presence of locally delivered rhVEGF165 in the arterial wall, VEGF (100 µg, n=3) or saline (n=3) was delivered after stent implantation. Animals were killed 3 hours later, and arterial segments were retrieved from the site of balloon injury. VEGF protein was detected with a mouse monoclonal antibody to human VEGF (Sigma). Briefly, tissues were fixed in 100% methanol and embedded in paraffin, and 5-µm sections were cut. After deparaffinization, endogenous peroxide activity was blocked with 3% hydrogen peroxide. Sections were next preincubated with 10% horse serum for 20 minutes before incubation with the anti-VEGF antibody (20 µg/mL) overnight at 4°C. Bound primary antibody was detected with an avidin-biotin immunoperoxidase method according to the supplier's guidelines (Signet). Sections were lightly counterstained with Gill's hematoxylin.
Histological and Ultrastructural Analysis
Animals received heparin (2000 U) via the ear vein before they were killed. A cannula was inserted into the lower abdominal aorta to perfuse in situ 100 mL of 5% dextrose solution with 100 U/mL heparin, followed by 0.25% silver nitrate for 20 seconds. This was followed by 5% dextrose and then pressure-perfusion at 100 mm Hg for 2 hours with 10% buffered formalin.
To analyze StE and thrombosis, specimens were cut lengthwise and photomacrographed with a Zeiss Tessovar System 3. One half was used for morphometric studies of intimal surface endothelialization; the other half was submitted for scanning electron microscopy. Surface endothelialization was quantified with a Nikon Labophot compound microscope equipped with 2x to 10x objectives and a pair of 10x eyepieces. The visual field of the microscope was integrated into the LED-lit cursor of a standard digitizing pad through a drawing tube attachment with a x1.25 magnification factor. Measurements were carried out with Sigmascan morphometric software on an Intel/486-based personal computer system. Integration of the microscope with the computer via the digitizing tablet facilitated direct examination of the endothelial surface at x25 to x125.
The theoretical surface area for a 15-mm-long stent with a deployed diameter of 3.0 mm is 141 mm2. The half-stent used for planimetry was cut longitudinally, then opened along the longitudinal cut. Because the stent could not be completely flattened without jeopardizing tissue integrity, quantitative analysis was confined to the continuous longitudinal section of intima that remained flat enough to permit focussing of the microscope. The mean area of this longitudinal strip, located opposite the longitudinal stent incision and traversing the entire length of the stent, was 29 mm2.
To confirm the extent of StE and thrombosis, the remaining half of the specimen was processed for scanning electron microscopy by the modified Peldri II sublimation technique.14 Specimens were rinsed in phosphate buffer and dehydrated in graded ethanols. After dehydration, a 1:1 mixture of Peldri II and ethanol was used as a transition solvent. After a 30-minute incubation in a 37°C oven, specimens were transferred to straight Peldri II and reincubated in the oven for 30 minutes more. This was repeated twice. As the solidified specimens were brought to room temperature, the Peldri II began to solidify, and they were refrigerated for 20 minutes. The solidified specimens were sublimated with ice packs under vacuum to remove solidified Peldri II. The specimens were coated with gold/palladium and examined under the Jeol JSM 840A scanning electron microscope. Scanning electron photomicrographs (x40 to x500) were then obtained from the proximal, medial, and distal portions of each specimen.
Thrombotic Occlusion
Thrombotic stent occlusion precluded the quantitative analyses described above. Accordingly, such specimens were not included in these analyses, and the frequency of this event was analyzed separately (see "Results," "Thrombotic Occlusion").
Data Analysis
Values are expressed as mean±SEM. Differences between means were assessed by ANOVA and Scheffe's F test for multiple comparisons. Correlations were assessed by linear regression analysis. A value of P<.05 was considered to denote statistical significance.
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Results |
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Quantitative Angiographic Analysis
Mean baseline luminal diameter for all stents deployed in protocols A, B, and C (n=64) was 2.67±0.07 mm. Mean stent diameter was 3.04±0.04 mm, and mean stent-to-artery ratio was 1.15±0.04. No differences were observed among groups.
Quantitative Analysis of StE as a Function of Time (Protocol A)
One of 12 vessels, from an animal killed at day 28, was occluded by thrombus. At day 0, examination of the luminal surface disclosed no measurable endothelialization. By day 4, <20% of the stent was endothelialized. By day 7, StE was still <40% complete. By 4 weeks, StE in each of 3 stents studied was typically complete (Fig 2). Representative photomicrographs of StE are shown in Fig 3.
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Local Catheter Delivery Per Se May Compromise StE
The extent to which the trauma of local catheter delivery per se could retard StE was tested in 9 rabbits in which balloon injury and stent implantation were performed in both EIAs. Local ChB infusion of PBS vehicle solution was performed at one stent site, while the contralateral side was untreated. At day 7, percent StE, obtained by planimetric analysis of the silver-stained endothelial surface (see above), was lower at the stent site receiving local infusion of the vehicle solution (protocol B/day 7/PBS, 10.57±2.88%) compared with the contralateral, untreated stented artery (protocol B/day 7/no delivery, 25.54±6.68%, P=.045; Fig 4). Thus, the trauma of local delivery per se may further compound injury resulting from primary balloon angioplasty/stent implantation and thereby retard StE.
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P<.0001 vs contralateral; 
P=.045 vs contralateral.
Local Protein Delivery Is Feasible After Stent Implantation
Transmural peroxidase staining of the wall (Fig 5) was documented for all three stented vessels in which HRP was delivered to assess the feasibility of protein delivery after stent implantation. Intensity and extent of staining in stented vessels was similar to that observed in nonstented vessels. These results thus established that local delivery of a 44-kD protein immediately after stent deployment is feasible and is similar in magnitude to that achieved in nonstented vessels.
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These results were confirmed by the immunodetection of VEGF in the vessel wall of stented vessels after local delivery of 100 µg of rhVEGF165 (Fig 6).
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Local Infusion of rhVEGF165 Enhances StE
Because spontaneous endothelialization remained quite limited at days 4 and 7, these time points were specifically analyzed by planimetric analysis of the silver-stained endothelial surface for StE in animals receiving rhVEGF165. Local delivery of rhVEGF165 accelerated StE at day 4 compared with the contralateral side treated with vehicle alone (protocol C/day 4/VEGF, 48.34±10.31% of stent surface versus protocol C/day 4/PBS, 6.64±1.37%, P=.0013; Fig 4). By day 7, StE was virtually complete for sites treated with rhVEGF165 (protocol C/day 7/VEGF, 91.80±3.83%) versus vehicle alone (protocol C/day 7/PBS, 29.06±7.84%, P<.0001; Fig 4). Scanning electron microscopy (Figs 7 and 8) confirmed these findings and showed, in addition, qualitative changes in endothelial cell phenotype: endothelial cells seen by day 7 at the rhVEGF165 treatment sites were typically ellipsoid and organized parallel to axial blood flow (Fig 8); these changes have previously been associated with the functional recovery of endothelial function after balloon injury.15
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Analysis of the stented artery treated with vehicle alone (PBS sides of protocol B/day 7 and protocol C/day 7) was used to control for the possibility of accelerated StE that might have resulted from systemically circulating rhVEGF165 protein "leaking" from the site of local delivery in the treated animals described above. Indeed, by day 7, StE in EIAs receiving local infusion of vehicle alone was greater if the contralateral artery was infused with rhVEGF165 (protocol C/day 7/PBS, 29.06±7.84%) than if it was not (protocol B/day 7/PBS, 10.57±2.88%, P=.05; Fig 4). This result suggests that local infusion of rhVEGF165 may have an effect, albeit modest, on endothelialization of remote stent sites.
StE observed after local delivery of rhVEGF165 was also compared with that observed after stent deployment followed by no local delivery (ie, no further manipulation). Indeed, StE at day 7 in vessels receiving a local infusion of rhVEGF165 exceeded that observed in unmanipulated stent sites (protocol C/day 7/VEGF, 91.80±3.83% versus protocol B/day 7/no delivery, 25.54±6.58%, P=.0001; Fig 4).
Local Delivery of rhVEGF165 Reduces Stent Thrombus
Local delivery of rhVEGF165 decreased the stent surface covered with thrombus compared with the contralateral side receiving vehicle alone (protocol C/day 7/VEGF, 5.26±3.72% versus protocol C/day 7/PBS, 37.46±10.12%, P=.007; Fig 9). This finding was confirmed by scanning microscopy (Fig 8).
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Local infusion (with vehicle alone) per se was associated with more extensive stent thrombus compared with contralateral stented arteries that were not instrumented, although this did not achieve statistical significance (protocol B/day 7/PBS, 48.49±10.41% versus protocol B/day 7/no delivery, 29.34±6.82%, P=.13; Fig 9). Nevertheless, stent thrombus was still reduced in rhVEGF165-treated stents versus stents in which no further manipulation was performed (protocol C/day 7/VEGF, 5.26±3.72% versus protocol B/day 7/no delivery, 29.34±6.82%, P=.006; Fig 9). The systemic effect of VEGF did not appear to be significant with regard to this parameter (protocol C/day 7/PBS, 37.46±10.12% versus protocol B/day 7/PBS, 48.49±10.41%, P=.47; Fig 9).
In all these comparisons, the development of organized thrombus was dependent on the extent of the StE. Indeed, at day 7, a significant and inverse linear relationship could be found between StE and the extent of organized thrombus (r=.72, F=33.05, P=.0001). This is consistent with the notion that the antithrombogenic property of the rhVEGF165-accelerated stent neoendothelium is functionally intact.
Thrombotic Occlusion Was Not Observed After Administration of rhVEGF165
Although thrombotic occlusion was a rare event in these experiments (5/61, 8.21% of the stented vessels; animals killed within 1 to 2 hours after completion of the experiment are not included), it is noteworthy that thrombotic occlusion was not observed in vessels treated by local administration of rhVEGF165 (0/17, 0%). All cases of thrombotic occlusion were observed in control vessels (5/44, 11.36%) receiving either local vehicle infusion (3/26, 11.54%) or no local delivery (2/18, 11.11%), consistent with the frequency of thrombotic occlusion reported in previous animal models of stent deployment.16 17 18 Peak incidence of thrombotic occlusion was observed between days 4 and 7.
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Discussion |
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Development of restenosis after stent implantation has been attributed principally to smooth muscle cell proliferation,19 since stents are considered to neutralize geometric effects such as so-called "remodeling."20 21 22 23 Indeed, the successful application of intravascular radiation to limit thickening of stent neointima in a variety of animal models24 25 26 is based on the premise that actively proliferating cells have enhanced sensitivity to the lethal effects of ionizing radiation.26 Subacute stent thrombotic occlusion, a troublesome complication in early clinical trials,27 has been reduced in frequency with the advent of high-pressure balloon inflation28 and may be reduced further with the development of heparin-coated stents.16 29
Both of these pathological targets, intimal thickening and thrombosis, have been conceptually linked to the initial absence of an intact endothelial monolayer.30 This fundamental concept has in fact stimulated efforts to promptly establish a functional neoendothelium in vascular prostheses,31 32 33 34 including endovascular stents.35 36 37 38
Previous reports from several laboratories, including our own,4 5 39 40 have demonstrated that treatment of a balloon-injured artery with recombinant proteins mitogenic for endothelial cells results in expedited reendothelialization. The logical extension of these previous experiments is that such a strategy could be applied after stent implantation to increase the rate of endothelial coverage of the stented surface, preventing thrombus formation and intimal thickening.
To test this hypothesis, however, required successfully addressing a series of issues. First, demonstration that StE may be accelerated requires quantitative data regarding the time course of StE that occurs de novo. Although this issue has received comment in several previous pathological studies, there is a paucity of morphometric data available for comparison. In rabbit aorta, the process has been described as completed after 1 week41 ; for rabbit iliac arteries, it was observed that "after 1 week arteries began to endothelialize"24 ; and in dog and pig coronary models, endothelialization has been evaluated as incomplete42 or advanced16 after 1 week. Several animal studies have been interpreted to indicate that, independent of the implant site, by 4 weeks the process is completed.24 25 41 42 43 In a series of stents implanted in human venous bypass grafts, no significant endothelialization was observed in three specimens retrieved within 14 days after implantation.44 In the only published series of human coronary stents examined at necropsy, endothelialization was observed in one of three specimens retrieved 3 weeks after implantation.45
To acquire morphometric data regarding the time course of StE, we specifically evaluated arteries <3.0 mm in diameter. Arteries of this caliber are frequent potential targets for revascularization, and the notion that such arteries may not be as resistant to thrombosis and restenosis as arteries 
3.0 mm in diameter tested in the STRESS and BENESTENT trials1 2 suggests that arteries of this size might benefit most from a local delivery strategy designed to expedite StE. Our analysis indicates that complete StE in arteries <2.67±0.07 mm in diameter does, as suggested previously, require up to 4 weeks.
The second issue we addressed was the extent to which local catheter delivery could pose a liability for StE. Our findings disclosed that the delivery technique we used did in fact have a deleterious effect on StE. This may have been due to additional removal of remaining endothelial cells, on the edge or within the injured area, induced by the additional and prolonged balloon inflation (15 minutes) and/or by trauma associated with the impact of recombinant protein infusion itself. Alternatively, more protracted injury could render the residual luminal surface of the vessel wall less appropriate for initial adhesion and migration of endothelial cells. The compromising effect of local catheter delivery on StE was overcome when the ChB was used to administer rhVEGF165.
The third issue we studied was the feasibility of performing local protein delivery through a deployed stent to the underlying arterial wall. To the best of our knowledge, although the feasibility of protein delivery has been amply documented in elegant studies10 46 involving the bare arterial wall, no such analyses have been reported after stent implantation. Our results clearly show that local delivery of a 44-kD macromolecule (HRP as well as VEGF165) from a low-pressure infusion catheter (ChB) is feasible and, moreover, is not altered by the presence of a metallic stent in the vessel wall.
The above series of three experiments provided the requisite suitability for proceeding with the fourth experiment, namely, assessment of local delivery of a recombinant endothelial cell mitogen on the time course of StE. StE was indeed accelerated: by day 7, endothelialization was nearly complete (91.80±3.83%). Although this is encouraging, it must be noted that previous studies of spontaneous reendothelialization have shown quite explicitly that restoration of anatomic integrity and recovery of physiological function do not proceed simultaneously.47 48 These previous studies used endothelium-dependent vasoreactivity to evaluate endothelial function. Experimental work performed in animal models of balloon arterial injury and myocardial or limb ischemia has in fact suggested that treatment with endothelial cell mitogens may accelerate recovery of endothelium-dependent vasomotor responses in balloon-injured arteries40 49 and in arterial beds perfused via newly generated collaterals.50 51 In the present study, such an analysis was precluded by the presence of the stent itself. We did, however, observe a dramatic reduction in mural thrombus on the luminal surface of stents treated with VEGF165. This finding may well have important implications for preservation of stent patency in vessels <3.0 mm in diameter in the absence of anticoagulation therapy. Moreover, the reduction in stent thrombosis may constitute alternative evidence that VEGF promotes recovery of neoendothelial function at a rate equal to that of anatomic regrowth; this is consistent with other data52 53 54 55 suggesting that VEGF may have profound effects on certain aspects of endothelial cell biology separate from its mitogenic and migratory influences. In this respect, the apparent impact of VEGF165 treatment on endothelial cell morphology is intriguing: the spindle shape of the endothelial cells with their long axes oriented in the direction of blood flow (Fig 8) has been associated with functional recovery of endothelial cells.15
Analysis of stent sites receiving local infusion of vehicle alone disclosed enhanced endothelialization if the contralateral stent site was infused with rhVEGF165. This effect is likely related to a "leak" of recombinant protein from the ChB.46 That some systemic effect was observed is not entirely surprising, because we have previously described systemic effects on therapeutic angiogenesis after intravenous administration of 500 µg of rhVEGF165 in an animal model of hindlimb ischemia.56 The fact that StE was only 
30% complete at day 7, however, suggests that this systemic effect may be insufficient for accelerating endothelialization of a remote stent site.
Certain limitations of the present study must be acknowledged. First, it remains for subsequent studies to determine how the interpretations made here may be modified by the use of other endothelial cell mitogens, delivered from alternative delivery devices, to different arterial sites, diseased as well as normal, of other species. Perhaps even more important, the manner in which alternative stent designs, both structural and compositional, may alter the current findings deserves to be studied as well.
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Acknowledgments |
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This study was supported in part by an Academic Award in Vascular Medicine (HL-02824) and grant HL-40518, both from the National Heart, Lung, and Blood Institute, National Institutes of Health.
Received May 13, 1996; revision received August 15, 1996; accepted August 22, 1996.
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