Accelerated Restitution of Endothelial Integrity and Endothelium-Dependent Function After phVEGF165 Gene Transfer
Background Delinquent reendothelialization (rET) has been shown to have a permissive, if not facilitatory, impact on smooth muscle cell proliferation. This inverse relation has been attributed to certain functions of the endothelium, including barrier regulation of permeability, thrombogenicity, and leukocyte adherence, as well as production of growth-inhibitory molecules. Accordingly, the present investigation was designed to test the hypothesis that an endothelial cell (EC) mitogen could serve as the basis for a novel gene therapy strategy designed to facilitate EC regeneration, reduce neointimal thickening, and promote recovery of EC dysfunction after balloon injury.
Methods and Results New Zealand White rabbits underwent simultaneous balloon injury and gene transfer of one femoral artery with phVEGF165, encoding the 165–amino acid isoform of vascular endothelial growth factor (VEGF), or pGSVLacZ. In each animal transfected with phVEGF165 or pGSVLacZ, the contralateral femoral artery was also subjected to balloon injury but not to gene transfer. For pGSVLacZ, rET remained incomplete at 4 weeks after transfection; in contrast, phVEGF165 produced prompt rET, which was 95% complete by 1 week. Furthermore, rET in the contralateral, balloon-injured, nontransfected limb of the VEGF group was similarly accelerated. Consequently, intimal thickening was diminished, thrombotic occlusion was less frequent, and recovery of EC-dependent vasomotor reactivity was accelerated in VEGF transfectants compared with control animals. A similar benefit was observed for the contralateral, balloon-injured, nontransfected limb.
Conclusions Catheter-mediated, site-specific arterial gene transfer of phVEGF165 can accelerate rET at local and remote sites, leading to inhibition of neointimal thickening, reduction in thrombogenicity, and restoration of endothelium-dependent vasomotor reactivity. These findings support the notion that gene transfer encoding for an EC-specific mitogen may be useful for preventing the complications, including restenosis, of balloon angioplasty.
Multiple strategies have been used clinically and/or experimentally to inhibit recurrent intimal thickening that occurs after balloon angioplasty, (ie, restenosis).1 2 These strategies have included antiproliferative strategies designed to interfere with smooth muscle cell proliferation, antiplatelet or anticoagulant therapies designed to preempt development of a platelet-fibrin scaffold, anti-inflammatory drugs, spasmolytic drug therapy, and lipid-lowering agents. It is conceivable that the limitations that have been recognized to many of these approaches may relate in part to belated rET because none of these strategies include a specific proactive effect on EC repaving of the balloon-injured arterial segment.
Delinquent rET has been shown to have a permissive, if not facilitatory, impact on smooth muscle cell proliferation.3 4 5 6 This inverse relation has been attributed to certain functions of the endothelium; these functions include barrier regulation of permeability, thrombogenicity, and leukocyte adherence, as well as production of growth-inhibitory molecules.7
The capability of certain cytokines to serve as mitogens for ECs in vitro suggests that such growth-stimulatory molecules might be exploited to accelerate rET consequent to balloon injury or plaque rupture. VEGF, known also as vascular permeability factor and vasculotropin, has been shown to be an EC-specific mitogen in vitro8 9 10 and an angiogenic growth factor in vivo.11 12 13 14 More recently, VEGF has been shown to modulate qualitative aspects of EC biology.15 16 VEGF is distinguished from most other angiogenic cytokines by the presence of a secretion signal at its amino terminus17 that allows VEGF to be naturally secreted by intact cells; this feature has led to its investigation in a strategy of arterial gene therapy for therapeutic angiogenesis.18 Accordingly, the present investigation was designed to test the hypothesis that an EC mitogen could serve as the basis for a novel gene therapy strategy designed to facilitate EC regeneration, reduce neointimal thickening, and promote recovery of EC dysfunction after balloon injury.
Male New Zealand White rabbits (3.5 to 4.0 kg) were used for all experiments according to protocols approved by St Elizabeth's Institutional Animal Care and Use Committee. In one femoral artery, 32 rabbits underwent simultaneous balloon injury and transfection with phVEGF165 (VEGF-Tf), while the contralateral femoral artery underwent identical balloon injury but no gene transfer (VEGF-nTf) (Fig 1⇓). Another group of 32 rabbits underwent simultaneous balloon injury and transfection of one femoral artery with β-Gal (LacZ-Tf), whereas in the contralateral femoral artery, the rabbits underwent identical angioplasty balloon injury but no gene transfer (LacZ-nTf). The angiographic and histological consequences of phVEGF165 versus pGSVLacZ gene transfer were systematically studied in these 64 animals at the following time points: 3 days (n=4 each group), 5 days (n=4 each), 1 week (n=8 each), 2 weeks (n=8 each), and 4 weeks (n=8 each).
Three additional groups of rabbits were used to study specific issues regarding arterial gene transfer. A total of 24 rabbits were transfected with phVEGF165 and with 3 normal noninstrumented rabbits were used to determine the time course of gene expression at the mRNA level at 0 days (before angioplasty); 36 hours; 3 and 5 days; and 1, 2, 3, 4, and 6 weeks (3 animals at each time point). Six rabbits were transfected with phVEGF165 or pGSVLacZ and were studied with an ELISA to allow evaluation of gene expression at the protein level. Finally, 4 rabbits were transfected with pGSVLacZ and killed 5 days later for assessment of transfection efficiency.
The mammalial expression vector used in these experiments contains a cytomegalovirus promoter and the cDNA for recombinant human VEGF165.18 The plasmid pGSVLacZ (courtesy of Dr Claire Bonnerot) containing a nuclear targeted β-Gal sequence coupled to the simian virus 40 early promoter19 was used for all control transfections as previously described.20
Percutaneous Arterial Gene Transfer and Balloon Angioplasty In Vivo
Access to the right common carotid artery was obtained via a small midline incision in the neck. In each rabbit, a 2.0-mm-diameter, 2.0-cm-long, Hydrogel-coated balloon catheter (Boston Scientific) was used to perform balloon angioplasty and arterial gene transfer.21 The angioplasty balloon was prepared ex vivo by first advancing the deflated balloon through a 5F Teflon protective sheath (Boston Scientific), which was designed to minimize contact between blood and the balloon on introduction of the catheter into the carotid artery. To apply plasmid DNA to the exterior Hydrogel polymer coating the angioplasty balloon, the balloon was inflated to 3 atm pressure. A conventional pipette was then used to apply 400 μg of phVEGF165 to the 20-μm-thick layer of Hydrogel polymer coating the external surface of the inflated balloon. The plasmid DNA was applied at a concentration of 10 μg/μL. The balloon was next deflated, withdrawn into the protective sheath, and reinflated to 3 atm within the sheath to minimize blood flow across the balloon surface. The sheath and angioplasty catheter were then introduced via the right common carotid artery and advanced to the lower abdominal aorta with the use of a 0.014-in guide wire under fluoroscopic guidance. The balloon catheter was then deflated and advanced into one femoral artery, where it was positioned with the use of angiographic landmarks. Balloon inflation was then performed three times for 1 minute each at 4 atm. After the final deflation, the balloon and sheath were withdrawn. An identical protocol was used to transfect the femoral artery of control animals with the plasmid pGSVLacZ containing a nuclear targeted β-Gal sequence.
For each rabbit, after completion of transfection of one femoral artery (with phVEGF165 or pGSVLacZ), the contralateral femoral artery underwent balloon injury (but no transfection) using a new 2.0-mm-diameter, 2.0-cm-long Hydrogel-coated angioplasty balloon. Balloon injury, with or without gene transfer, was performed by one person who was blind to the treatment.
In Vivo Vasomotor Reactivity
Vasomotor reactivity of the arterial segment subjected to balloon angioplasty and arterial gene transfer was evaluated on the day on which the animals were killed. A 3F end-hole infusion catheter (Target Therapeutics) was inserted into the left carotid artery and advanced to the origin of transfected iliac artery with the use of a 0.018-in guide wire under fluoroscopic guidance. This catheter was used for both infusion of vasoactive drugs and selective angiography of the femoral artery. Angiography was performed immediately after drug administration with 1 mL of nonionic contrast media.
To assess endothelium-dependent vasomotor reactivity, ACh and 5-HT were delivered from a constant infusion pump (1 mL/min) via the 3F catheter at dosages of 0.15, 1.5, and 15 μg·kg−1·min−1, each for 2 minutes. Five minutes was allowed to elapse between each dose of agent to reestablish basal blood flow conditions. After administrations of ACh and 5-HT were completed, a 2-minute intra-arterial infusion of NP (1.5 μg·kg−1·min−1) was administered to assess endothelium-independent vasomotor reactivity. Finally, an identical protocol was used to evaluate the contralateral injured but nontransfected femoral artery.
The angiographic luminal diameter of the femoral artery before and after drug infusion, before and after gene transfer, was determined using an automated edge-detection system as previously described.22 23 The average angiographic luminal diameter and the minimum luminal diameter were then determined for the defined 20-mm-long segment of each transfected as well as nontransfected balloon-injured artery.
Ex Vivo Animal Examination
Thirty minutes before the animals were killed, they received an intravenous injection of 6 mL of 0.5% Evans' blue dye (Sigma Chemical Co)3 delivered via the ear vein to identify the remaining nonendothelialized area. A cannula was inserted into the lower abdominal aorta and used to perfuse a total of 100 mL of 0.9% saline solution with 10 U/mL heparin in situ, followed by 100 mL of 100% methanol. The baseline angiogram recorded before balloon injury and the pilot radiographic recording of the angioplasty balloon were used to identify the arterial segment to be harvested. The initially injured 2-cm-long segment of femoral artery was then dissected free and incised longitudinally. The harvested arterial segment was inspected for the presence of occlusive thrombus; if present, this finding was noted, and such segments were excluded from subsequent analyses. Segments with nonocclusive thrombus were also noted but not excluded from further study. The arterial segment was then pinned to a corkboard, further fixed in 100% methanol, and photographed using a dissecting microscope in preparation for planimetric analysis of rET (see below). Tissues were further fixed by immersion in 100% methanol, embedded on longitudinal edge in paraffin, and cut into 5-μm sections on slides coated with 3-aminopropyl-triethoxy-silane.
Planimetric Analysis of rET
Planimetric analysis was performed using the photograph of the harvested arterial segment taken through the dissecting microscope. The area of the intimal surface that was stained blue after the application of Evans' blue dye was interpreted as identifying the portion of the arterial segment that remained endothelium deficient; these macroscopic analyses were confirmed through immunostaining of light microscopic sections with an EC-specific marker (vide infra). A computerized sketching program (MacMeasure) interfaced with a digitizing board was used by one person who was blinded to the treatment protocol to outline the Evans' blue–positive and –negative areas. Specifically, the extent of endothelialized area was calculated as a percentage of the total intimal area encompassed within the 2-cm length of artery.
Evaluation of Intimal Hyperplasia
Morphometric analysis of intimal thickness was performed on longitudinally cut light microscopic arterial sections, which is consistent with the requirement that each vessel be opened initially in a longitudinal fashion to perform gross morphometric analysis of endothelial integrity with Evans' blue dye. Longitudinal histological sections obtained from the 20-mm length of injured artery and stained with an elastic tissue trichrome stain were projected onto the digitizing board, and the area of the intima and media were measured by a technician who was blinded to treatment regimen with use of the computerized sketching program discussed above.
The thickness of the native media of the artery wall is variable, reflecting in part the dimensions (diameter) of the individual rabbit femoral artery. Accordingly, the thickness of the media was used to index the extent of neointimal thickening and thus is stated as the I/M. The average circumference of each arterial sample was 2.3 mm, permitting two longitudinal sections to be cut at ≈500-μm intervals for morphometric examination; the I/M ratio was calculated as the average of measurements performed on these two sections. (Analysis of step-sections [n=20] cut from the paraffin-embedded blocks of control and treated animals selected at random was used to validate results obtained from the two principal sections.)
Evaluation of Proliferative Activity in Injured Artery
Proliferative activity in the injured arterial segment harvested at the time of death was evaluated by immunostaining analysis for PCNA as previously described.24 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% BSA/PBS. Negative controls were incubated with MOPC-21, a purified nonspecific mouse monoclonal antibody (Sigma). Bound primary antibody was detected using an avidin/biotin/immunoperoxidase method (Signet). Sections were lightly counterstained with hematoxylin and mounted in aqueous mounting medium. ECs were identified by immunostaining with BSI lectin (5 μg/mL, Vector).25 For lectin staining, sections pretreated with 0.3% hydrogen peroxidase were incubated with 5 μg/mL biotinylated lectin (Vector) overnight at 4°C. After washing, slides were incubated with peroxidase-conjugated streptavidin (BioGenex) for 1 hour; 3-amino-9-ethylcarbazole (AEC, 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 one person who was blinded to the treatment regimen and was determined as the number of positive cells in the intima per length of the 20-mm-long longitudinal section (ie, cells/mm). PCNA staining of the rabbit ileum served as the positive control for this study.
Analysis of Gene Expression
Expression of phVEGF165 was evaluated using reverse-transcription polymerase chain reaction (RT-PCR). Amplification was performed for 35 cycles of 94°C for 20 seconds, 55°C for 20 seconds, and 72°C for 20 seconds, ending with 5 minutes at 72°C. A pair of oligonucleotide primers (22 mer) was designed to amplify a 258-bp sequence from the mRNA of human VEGF. To ensure specificity and avoid amplification of endogenous rabbit VEGF, each primer was selected from a region that is not conserved among different species. Sequences of primers used were 5′-GAGGGCAGAATCATCACGAAGT-3′ (sense) and 5′-TCCTATGTGCTGGCCTTGGTGA-3′ (antisense). Preliminary experiments showed RT-PCR product only when cultured rabbit smooth muscle cells were transfected with a plasmid containing the cDNA for human VEGF. Cultures of normal (nontransfected) or β-Gal–transfected rabbit smooth muscle cells showed no amplification product.
To assess the efficiency of in vivo arterial gene transfer in this model, LacZ-Tf arteries were harvested at day 5, and β-Gal activity was determined through incubation with X-Gal (5-bromo-4-chrolo-3-indolyl-β-d-galactoside chromogen) (Sigma) as previously described.21 After staining with X-Gal solution, tissues were paraffin embedded, sectioned, and counterstained with nuclear fast red. Nuclear localized β-Gal expression of the plasmid pGSVLacZ could not result from endogenous β-Gal activity; accordingly, histochemical identification of β-Gal within the cell nucleus was interpreted as evidence for successful gene transfer and gene expression.
Serum Concentration of VEGF
Serum levels of VEGF were measured with a commercially available ELISA (R&D Systems) with use of a monoclonal antibody for human recombinant VEGF165 precoated onto the 96-well microtiter plate. After incubation of each serum sample (100 μL) pipetted into the well, unbound proteins were washed away. Bound antigen was then detected with an enzyme (peroxidase)-linked second anti-VEGF antibody (goat anti-VEGF polyclonal antibody), which sandwiched the VEGF immobilized during incubation. After a wash to remove unbound antibody, a substrate (hydrogen peroxide) and chromogen (tetramethylbenzidine) were added to the wells, and the developed color was read at 450 nm with a microplate reader. Freshly diluted solutions of human recombinant VEGF165 protein were used as standards. The sensitivity of this assay is 9.0 pg/mL, and no significant cross-reactivities to several cytokines, including platelet-derived growth factor, fibroblast growth factor, and epidermal growth factor, have been noted.
All results are expressed as mean±SEM. Statistical significance was evaluated using unpaired Student's t test or Mann-Whitney U test for comparisons between two means or contingency table analysis for comparisons of frequency. A value of P<.05 was considered statistically significant.
Planimetric analysis performed with Evans' blue dye staining disclosed near-complete rET of the 20-mm-long segment of balloon-injured rabbit femoral artery by 7 days among VEGF-transfected arteries, whereas the extent of rET in LacZ-transfected arteries was <50% complete at 7 days and remained nearly 20% incomplete at 4 weeks (Fig 2A and 2C).⇓ In 10 VEGF-Tf arteries, rET at 4 weeks was 100% complete, a finding that was not observed in any of the LacZ-Tf arteries. rET was accelerated not only in arteries transfected with phVEGF165 (VEGF-Tf) but also in the contralateral balloon-injured, nontransfected arteries (VEGF-nTf) (Fig 2B and 2C).⇓
Previous investigations of rET have demonstrated that restoration of anatomic integrity and recovery of physiological function do not proceed simultaneously.26 27 28 Accordingly, we determined the vasomotor response to endothelium-dependent agonists with the use of quantitative angiography. Consistent with previous studies of the balloon-injured rabbit iliac artery reported by Weidinger et al,28 control rabbits transfected with LacZ demonstrated persistent impairment in vasomotor response to endothelium-dependent agents at 4 weeks after injury (Table⇓). In contrast, arteries transfected with phVEGF165 disclosed recovery of near-normal endothelium-dependent vasoreactivity within 1 week (Fig 3⇓). A similar benefit was observed for the contralateral, balloon-injured, nontransfected limb (Table⇓).
Angiographic Luminal Diameter Narrowing
The impact of accelerated rET on angiographic luminal diameter narrowing was evaluated through quantitative assessment of percent luminal diameter narrowing performed before and at serial time points after gene transfer (Fig 4⇓A). LacZ-Tf arteries developed progressive narrowing, ultimately to 55.2±5.9%. In contrast, luminal diameter narrowing was markedly reduced in VEGF-Tf arteries to a statistically significant degree at 1, 2, and 4 weeks after gene transfer. Similar results were observed for LacZ-nTf versus VEGF-nTf arteries. Representative angiographic findings are illustrated in Fig 4B⇓.
The impact of accelerated rET on neointimal thickening was evaluated with light microscopic examination as the I/M in longitudinally cut sections (Fig 5⇓). LacZ-Tf arteries showed progressive neointimal thickening through 4 weeks (1 week, 0.11±0.02; 2 weeks, 0.59±0.04; and 4 weeks, 0.69±0.10). In contrast, VEGF-Tf arteries disclosed significantly less intimal thickening, including regression of intimal thickening between weeks 2 and 4 (1 week, 0.06±0.02; 2 weeks, 0.20±0.05, P<.01; and 4 weeks, 0.14±0.02, P<.01). Furthermore, the I/M of the contralateral balloon-injured artery in the VEGF gene transfer group (VEGF-nTf) developed less intimal thickening and displayed no progression over 2 to 4 weeks compared with the contralateral artery of the LacZ controls (LacZ-nTf: 1 week, 0.05±0.03; 2 weeks, 0.54±0.06; and 4 weeks, 0.84±0.09; VEGF-nTF: 1 week, 0.09±0.02; 2 weeks, 0.29±0.08, P<.05; and 4 weeks, 0.31±0.09, P<.01).
The reduction in neointimal thickening observed with phVEGF165-accelerated rET was associated with a concomitant reduction in proliferative activity in both the media and neointima (Fig 6⇓A). Cellular proliferation in the media of LacZ-Tf reached a peak at 3 days after injury and then decreased gradually over the remaining 4 weeks (3 days, 32.5±4.7 PCNA-positive cells/mm; 5 days, 27.1±2.9; 1 week, 10.5±2.0; 2 weeks, 6.2±2.7; and 4 weeks, 0.3±0.2). Proliferative activity in the media of VEGF-Tf also peaked at 3 days after gene transfer (24.7±6.8 cells/mm) but then decreased rapidly (5 days, 12.2±4.2 cells/mm; 1 week, 2.6±1.0; 2 weeks, 1.6±0.5; and 4 weeks, 0.1±0.0). Selective immunostaining of adjacent sections established that proliferating cells were predominantly smooth muscle cells.
Temporal differences in proliferative activity were also observed for the neointima of LacZ- versus VEGF-transfected animals. Neointimal proliferation in LacZ-Tf arteries continued to crescendo to 2 weeks after transfection (3 days, 0.2±0.1 cells/mm; 5 days, 4.1±1.9; 1 week, 8.6±2.0; 2 weeks, 10.7±2.0; and 4 weeks, 2.3±0.9). For VEGF-Tf, neointimal proliferative activity peaked at 5 days and then fell precipitously (3 days, 2.4±0.6 cells/mm; 5 days, 7.0±3.4; 1 week, 0.8±0.3; 2 weeks, 2.0±0.7; and 4 weeks, 0.1±0.1).
PCNA-positive cells were identified as ECs through the use of positive BSI lectin staining of apparently identical cells located along the luminal border of adjacent (serially cut) histological sections. Proliferative activity of ECs was constant throughout the 4 weeks after balloon injury in LacZ-Tf arteries. In contrast, EC proliferation in VEGF-Tf arteries demonstrated a prominent peak at 3 days and was then reduced by 1 week after transfection (Fig 6B).⇑
Thromboresistance constitutes an important consideration in the immediate postangioplasty setting. This is particularly true when angioplasty is performed for acute myocardial infarction, in which case the risk of subacute reocclusion is increased. In the present study, phVEGF165-accelerated rET led to augmented thromboresistance. Specifically, thrombotic occlusion developed less frequently in animals transfected with phVEGF165 (2 of 64; 3.1%) than in those transfected with Lac-Z (14 of 64; 21.9%) (P<.01). Nonocclusive (mural) thrombus was also less frequent among VEGF-transfected animals: excluding the animals in which occlusive thrombus was observed, 9 of 60 VEGF-transfectants (15%) versus 18 of 50 LacZ-transfected (36%) animals (P<.05) had mural thrombus in the transfected artery at necropsy.
VEGF gene expression was assessed by RT-PCR in three normal, noninstrumented rabbits (0 days, or before angioplasty) and in rabbits transfected with phVEGF165 after intervals of 36 hours; 3 and 5 days; and 1, 2, 3, 4, and 6 weeks (n=3 each). Gene expression was observed as early as 36 hours after transfection (data not shown) and persisted for 2 weeks before diminishing at 3 weeks (Fig 7⇓A). No evidence of similar gene expression was observed among the noninstrumented (day 0) rabbits (data not shown).
RT-PCR was also performed on sections from other organs; no mRNA expression of VEGF at any site remote from the VEGF-transfected arterial segment was found (Fig 7B)⇑, including the contralateral balloon-injured, nontransfected arterial site of animals transfected with phVEGF165. RT-PCR for mRNA encoding β-actin documented similar loading conditions in all cases (data not shown).
Four rabbits were transfected with LacZ and killed 5 days later; quantitative examination comprising all cells in selected light microscopic fields indicated that transfection efficiency was <0.1%, which is consistent with previous reports.18 23
Serum Concentration of VEGF
Blood samples obtained immediately before and at 5 days after gene transfer from the central ear artery of six rabbits were analyzed with an ELISA for VEGF to determine whether expression and secretion of VEGF165 after arterial gene transfer was sufficient to increase serum concentration of the protein in systemic blood and thus provide a potential explanation for accelerated rET of the nontransfected balloon-injured site in the contralateral limb. Five days after transfection, VEGF level in VEGF-Tf arteries (n=3) increased by >15 pg/mL (day 0, 19.5±2.5; day 5, 34.8±2.6, Fig 8⇓), whereas that in LacZ-Tf arteries (n=3) remained unchanged (day 0, 18.3±1.7; day 5, 14.8±5.5) (P<.05).
Previous experiments performed in a rat carotid artery model of balloon injury have established in a systematic manner the impact of delinquent rET on intimal proliferation. Clowes et al,3 Bjorkerud and Bondjers,4 Fishman et al,5 and Haudenschild and Schwartz6 all observed an inverse relation between EC integrity and smooth muscle cell proliferation. 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 through neointimal thickening.29
Conversely, application of EC mitogens to a freshly injured arterial segment has been shown previously to exert favorable reparatory effects. Lindner et al,30 for example, established clear evidence for the mitogenic effect of basic fibroblast growth factor on EC replication in the rat carotid artery model of balloon injury; total EC regrowth was achieved through the administration of basic fibroblast growth factor in dosages of 12 μg twice weekly for ≤8 weeks. In this particular case, concurrent effects on smooth muscle cell proliferation were not discussed. In the case of acidic fibroblast growth factor, which is also a known mitogen for vascular smooth muscle cells in vitro,31 low doses (<1% of the doses of basic fibroblast growth factor used in the mentioned 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 rET, the extent to which endothelial repair per se was completed was not discussed in this report.32 More recently, we demonstrated in the same animal model that a 30-minute incubation of VEGF recombinant protein accelerated rET, reduced intimal proliferation, and likewise reduced intimal thickening.33
In contrast to these previous three studies, the present investigation represents the first successful attempt to achieve accelerated rET using local, percutaneous catheter delivery. In this sense, the strategy used was technically analogous to, and might therefore be conveniently coupled to, standard balloon angioplasty. The continuous local expression of a transgene overexpressing an EC mitogen for a period of ≥2 weeks constitutes the equivalent of a local depot effect and is consistent with previous suggestions that such an agent “should be most potent for the first 4-7 days after the procedure.”34 Subsequent analysis of earlier time points (<3 days) may permit more disclosure of the precise determination of the temporal relation between the onset of gene expression and rET.
Blood samples drawn from the central ear artery documented a nearly twofold increase in circulating levels of VEGF protein at 5 days after gene transfer. In the absence of pharmacokinetic data regarding the removal of VEGF protein from circulating blood and binding to heparin sulfate moieties of the cells or extracellular matrix, the precise significance of this data remains uncertain. Taken at face value, the picogram levels might be interpreted to suggest a more profound effect of VEGF on EC migration versus proliferation synergism of VEGF with other EC mitogens is also plausible. In any case, results of the ELISA analysis in combination with accelerated rET at sites remote from gene transfer have implications for the use of recombinant protein as well. Clinical studies of VEGF recombinant protein are not possible because the protein is not available for human application. Should protein therapy become an option, however, it will be intriguing to see whether the slow-release depot aspect of gene therapy proves to be superior in terms of bioactivity and/or safety to bolus and/or continuous administration of the protein use of recombinant protein as well.
Previous investigations of rET in a variety of animal models have demonstrated that restoration of anatomic integrity and recovery of physiological function do not proceed simultaneously.26 27 28 35 Weidinger et al,28 for example, found “a persistent and generalized loss of endothelium-dependent relaxation after balloon injury, despite regrowth of endothelium”; evidence of dysfunction persisted for ≥4 weeks. Similarly, in a study of the “acute” phase of arterial injury, Shimokawa et al27 reported evidence of impaired endothelial dysfunction for ≥4 weeks after injury, whereas analyses of the “chronic” response have disclosed dysfunction persisting beyond 8 weeks.26 Accordingly, we attempted to characterize the functional behavior of VEGF-induced rET in three ways. First, we determined the vasomotor response to endothelium-dependent agonists. Consistent with previous studies of vasomotor reactivity in the balloon-injured rabbit iliac artery,28 control rabbits transfected with pGSVLacZ demonstrated persistent impairment in response to ACh and 5-HT at 4 weeks after injury. In contrast, arteries transfected with phVEGF165 disclosed recovery of near-normal endothelium-dependent response within 1 week. Parenthetically, the physiological response among VEGF-Tf rabbits at 4 weeks can be assumed to represent the response of restored endothelium in the absence of ongoing VEGF165 secretion because analyses that involved the use of RT-PCR in the present and previous18 studies have consistently disclosed no evidence of transgene expression beyond 3 weeks.
The reduced proliferative activity among smooth muscle cells and associated reduction in intimal thickening observed in arterial segments subjacent to sites of VEGF gene transfer constitute a second index of expeditiously restored EC function. The precise mechanism by which the neoendothelium modulates intimal hyperplasia remains to be clarified (vide infra), but presumably it reflects prompt restoration of antiproliferative features characteristic of healthy ECs.
Third, VEGF-induced rET appeared to result in improved thromboresistance, a function of particular importance in the postangioplasty setting. In fact, given the current trend favoring increased application of angioplasty in the treatment of acute myocardial infarction, VEGF gene transfer for the purpose of stabilizing acutely ruptured plaque may have additional merit.
The extent to which these findings may be extrapolated to human subjects is complicated by the multiple variables that determine the magnitude, rapidity, and quality of EC regeneration; these include vessel size, axial distribution of EC disruption, presence of side branches (which may serve as a reservoir of ECs), associated lipid disorders (known to accelerate EC turnover,27 ) and, presumably, species-dependent characteristics. With regard to the latter, limited data are available in humans. Davies et al36 harvested the coronary arteries from explanted hearts of 6 transplant recipients and found that the severity and extent of EC defects varied in direct proportion to the severity and extent of intimal disease in an individual patient. Schwarz et al37 found foci of but not complete rET in only 6 of 16 carotid specimens (38%) from patients with recurrent carotid artery narrowing after an initial endarterectomy. Perhaps most relevant to the present study, Gravanis and Roubin38 found no EC regeneration among 11 patients who died ≤1 month after coronary angioplasty. The present findings can only be regarded as proof of principle for the concept that catheter-mediated, mitogen-accelerated rET can be achieved experimentally through arterial gene transfer and thereby inhibit neointimal thickening in an animal model. Further studies are clearly required to establish the potential clinical usefulness of this strategy.
The reduction in intimal thickness and smooth muscle cell proliferation achieved indirectly in the VEGF-Tf animals compares favorably with recent gene therapy strategies that have used genes encoding for growth regulatory proteins39 40 41 42 to more directly inhibit proliferative activity. Such equivalent benefit is noteworthy given the fact that the VEGF-Tf animals received naked plasmid DNA delivered from the Hydrogel polymer coating of an angioplasty balloon, whereas the previously cited efforts involved the use of adjunctive, including viral, vectors.
The efficient rET demonstrated in the VEGF-Tf rabbits contrasts sharply with the low transfection efficiency inferred from the LacZ-Tf animals and is in large part attributable to the fact that VEGF includes a signal sequence that permits it to be naturally secreted.17 Previous work from our laboratory43 44 and others45 46 has established that low-efficiency transfection with genes encoding for secreted proteins may achieve biologically meaningful effects not realized by transfection with genes encoding for proteins that remain intracellular. Adenoviral vectors may thus be required to generate thymidine kinase39 40 42 or a hypophosphorylated form of the retinoblastoma gene product in a critical proportion of smooth muscle cells, whereas many fewer cells made to overexpress VEGF may yield equipotent effects. How direct cytotoxic/cytostatic strategies described by previous investigators compare with the indirect cytostatic effect of VEGF for modifying associated increases in extracellular matrix production that contribute to intimal thickening47 remains to be determined.
The design of the present experiments permitted a unique opportunity to test the effect of local (ie, site-specific) gene transfer (evidence of gene expression was not observed at remote sites) on rET of nontransfected, balloon-injured remote arterial sites in the contralateral limb. It is intriguing to note that rET and intimal thickening at these contralateral sites were affected in a manner similar to the specific sites selected for arterial gene transfer. We attribute the prompt restoration of endothelium-dependent vasomotor reactivity and absence of thrombotic occlusion at these remote sites to the secretion feature of the gene product, in this case presumably circulating to the contralateral limb. This interpretation is supported by ELISA analysis, which documented increased circulating VEGF levels in VEGF transfectants but not animals transfected with LacZ.
Finally, further study is required to clarify the mechanisms responsible for the favorable impact of EC regeneration on intimal thickening and vasomotor tone observed in the VEGF-Tf animals. Previous work by Peters et al48 suggested that the modulating effects of VEGF on ECs may extend beyond that of a mitogen, a notion that is consistent with the first functional activity attributed to VEGF, enhancement of vascular permeability.49 Ku et al15 showed that VEGF may influence constitutive production of NO in regenerated ECs, and we recently documented VEGF-induced reduction in systemic blood pressure after intracoronary as well as intravenous administration of the recombinant protein; this effect may be obviated or reversed by preadministration or post hoc treatment, respectively, with inhibitors of NO synthase (J.M. Isner, unpublished observations). Similar effects of VEGF may be inferred from the experimental observations that intra-arterial administration of the recombinant protein to an ischemic rabbit hindlimb promotes prompt restoration of endothelium-dependent flow,16 and the administration of the protein to hypercholesterolemic rabbits modulates endothelial dysfunction of conduit and resistance vessels.50
Selected Abbreviations and Acronyms
|BSI||=||Bandeiraera simplicifolia I|
|PCNA||=||proliferating cell nuclear antigen|
|PCR||=||polymerase chain reaction|
|VEGF||=||vascular endothelial growth factor|
This work was supported in part by grants HL-40518 and HL-53354 and an Academic Award in Vascular Medicine (HL-02824) from the National Institutes of Health. We are grateful to N. Ferrara for providing us with phVEGF165, to C Bonnerot for pGSVLacZ, to J. Barry for Hydrogel-polymer coated balloon catheters, and to K. Walsh for helpful discussions regarding the work.
- Received March 20, 1996.
- Revision received July 25, 1996.
- Accepted July 31, 1996.
- Copyright © 1996 by American Heart Association
Ross R. Atherosclerosis: a defense mechanism gone awry. Am J Pathol.. 1993;143:985-1002.
Keck PJ, Hauser SD, Krivi G, Sanzo K, Warren T, Feder J, Connolly DT. Vascular permeability factor, an endothelial cell mitogen related to PDGF. Science.. 1989;246:1309-1312.
Connolly DT, Hewelman DM, Nelson R, Olander JV, Eppley BL, Delfino JJ, Siegel RN, Leimgruber RS, Feder J. Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis. J Clin Invest.. 1989;84:1470-1478.
Leung DW, Cachianes G, Kuang WJ, Goeddel DV, Ferrara N. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science.. 1989;246:1306-1309.
Takeshita S, Zheng LP, Brogi E, Kearney M, Pu LQ, Bunting S, Ferrara N, Symes JF, Isner JM. Therapeutic angiogenesis: a single intra-arterial bolus of vascular endothelial growth factor augments revascularization in a rabbit ischemic hindlimb model. J Clin Invest.. 1994;93:662-670.
Banai S, Jaklitsch MT, Shou M, Lazarous DF, Scheinowitz M, Biro S, Epstein SE, Unger EF. Angiogenic-induced enhancement of collateral blood flow to ischemic myocardium by vascular endothelial growth factor in dogs. Circulation.. 1994;89:2183-2189.
Ku DD, Zaleski JK, Liu S, Brock TA. Vascular endothelial growth factor induces EDRF-dependent relaxation in coronary arteries. Am J Physiol.. 1993;265:H586-H592.
Bauters C, Asahara T, Zheng LP, Takeshita S, Bunting S, Ferrara N, Symes JF, Isner JM. Recovery of disturbed endothelium-dependent flow in the collateral-perfused rabbit ischemic hindlimb following administration of vascular endothelial growth factor. Circulation.. 1995;91:2802-2809.
Tischer E, Mitchell R, Hartmann T, Silva M, Gospodarowicz D, Fiddes J, Abraham J. The human gene for vascular endothelial growth factor: multiple protein forms are encoded through alternative exon splicing. J Biol Chem.. 1991;266:11947-11954.
Isner JM, Walsh K, Symes JF, Piezcek A, Takeshita S, Lowry J, Rosenfield K, Weir L, Brogi E, Jurayj D. Arterial gene therapy for therapeutic angiogenesis in patients with peripheral artery disease. Circulation.. 1995;91:2687-2692.
Bonnerot C, Rocancourt D, Briand P, Grimber G. A beta-galactosidase hybrid protein targeted to nuclei as a marker for developmental studies. Proc Natl Acad Sci U S A.. 1987;84:6795-6799.
Takeshita S, Gal D, Leclerc G, Pickering JG, Riessen R, Weir L, Isner JM. Increased gene expression after liposome-mediated arterial gene transfer associated with intimal smooth muscle cell proliferation following vascular injury. J Clin Invest.. 1994;93:652-661.
LeFree HT, Simon SB, Mancini BJ, Vogel RA. Digital radiographic assessment of coronary arterial geometric diameter and videodesitometric cross-sectional area. Proc SPIE.. 1986;626:334-341.
Mancini CBJ, Simon SB, McGillem MJ, LeFree MT, Friedman HZ, Vogel RA. Automated quantitative arteriography: morphologic and physiologic validation in vivo of a rapid digital angiographic method. Circulation.. 1987;75:452-460.
Pickering JG, Weir L, Jekanowski J, Kearney MA, Isner JM. Proliferative activity in peripheral and coronary atherosclerotic plaque among patients undergoing percutaneous revascularization. J Clin Invest.. 1993;91:1469-1480.
Shimokawa H, Vanhoutte P. Dietary cod-liver oil improves endothelium-dependent responses in hypercholesterolemic and atherosclerotic porcine coronary arteries. Circulation.. 1988;78:1421-1430.
Shimokawa H, Aarhus LL, Vanhoutte PM. Porcine arteries with regenerated endothelium have a reduced endothelium-dependent responsiveness to aggregating platelets and serotonin. Circ Res.. 1987;61:256-270.
Weidinger FF, McLenachan JM, Cybulsky MI, Bordon JB, Rennke HG, Hollenberg NK, Fallon JT, Ganz P, Cooke JP. Persistent dysfunction of regenerated endothelium after balloon angioplasty of rabbit iliac artery. Circulation.. 1990;81:1667-1679.
Koos RD, Goldman WF. Hypoxia stimulates vascular endothelial growth/permeability factor expression by rat vascular smooth muscle cells. Abstracts of the Scientific Conference on the Molecular Cellular Biolory of the Vascular Wall, Boston, Mass, October 15-17, 1993.
Lindner V, Majack RA, Reidy MA. Basic fibroblast growth factor stimulates endothelial regrowth and proliferation in denuded arteries. J Clin Invest.. 1990;85:2004-2008.
Winkles JA, Gay CG. Serum, phorbol ester, and polypeptide mitogens increase class 1 and 2 heparin binding (acidic and basic fibroblast growth factor) gene expression in human vascular smooth muscle cells. Cell Growth Differ.. 1991;2:531-540.
Bjornsson TD, Dryjski M, Tluczek J, Mennie R, Ronan J, Mellin TN, Thomas KA. Acidic fibroblast growth factor promotes vascular repair. Proc Natl Acad Sci U S A.. 1991;88:8651-8655.
Asahara T, Bauters C, Pastore CJ, Kearney M, Rossow S, Bunting S, Ferrara N, Symes JF, Isner JM. Local delivery of vascular endothelial growth factor accelerates reendothelialization and attenuates intimal hyperplasia in balloon-injured rat carotid artery. Circulation.. 1995;91:2793-2801.
Steele PM, Chesebro JH, Stanton AW, Holmes DR, Dewanjee MK, Badimon L, Fuster V. Balloon angioplasty: natural history of the pathophysiological response to injury in a pig model. Circ Res.. 1985;57:105-112.
Tanaka H, Sukhona GK, Swanson SJ, Clinton SK, Ganz P, Cybulsky MI, Libby P. Sustained activation of vascular cells and leukocytes in the rabbit aorta after balloon injury. Circulation.. 1993;33:1788-1803.
Davies MJ, Woolf N, Rowles PM, Pepper J. Morphology of the endothelium over atherosclerotic plaques in human coronary arteries. Br Heart J.. 1988;60:459-464.
Chang MW, Barr E, Jonathan S, Jiang YQ, Nabel GJ, Nabel EG, Parmacek MS, Leiden JM. Cytostatic gene therapy for vascular proliferative disorders with a constitutively active form of the retinoblastoma gene product. Science.. 1995;267:518-522.
Guzman RJ, Hirschowitz EA, Brody SL, Crystal RG, Epstein SE, Finkel T. In vivo suppression of injury-induced vascular smooth muscle cell accumulation using adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene. Proc Natl Acad Sci U S A.. 1994;91:10732-10736.
Heiko E, Leyen VD, Gibbons GH, Morishita R, Lewis NP, Zhang L, Nakajima M, Kaneda Y, Cooke JP, Dzau VJ. Gene therapy inhibiting neointimal vascular lesion: in vivo transfer of endothelial cell nitric oxide synthase gene. Proc Natl Acad Sci U S A.. 1995;92:1137-1141.
Ohno T, Gordon D, San H, Pompili VJ, Imperiale MJ, Nabel GJ, Nabel EG. Gene therapy for vascular smooth muscle cell proliferation after arterial injury. Science.. 1994;265:781-784.
Losordo DW, Pickering JG, Takeshita S, Leclerc G, Gal D, Weir L, Kearney M, Jekanowski J, Isner JM. Use of the rabbit ear artery to serially assess foreign protein secretion after site specific arterial gene transfer in vivo: evidence that anatomic identification of successful gene transfer may underestimate the potential magnitude of transgene expression. Circulation.. 1994;89:785-792.
Nabel EG, Shum L, Pompili VJ, Yang ZY, San H, Shu HB, Liptay S, Gold L, Gordon D, Derynck R, Nabel GJ. Direct gene transfer of transforming growth factor beta-1 into arteries stimulates fibrocellular hyperplasia. Proc Natl Acad Sci U S A.. 1993;90:10759-10763.
Nabel EG, Yang Z, Liptay S, San H, Gordon D, Haudenschild CC, Nabel GJ. Recombinant platelet-derived growth factor B gene expression in porcine arteries induces intimal hyperplasia in vivo. J Clin Invest.. 1993;91:1822-1829.
Peters KG, De Vries C, Williams LT. Vascular endothelial growth factor receptor expression during embryogenesis and tissue repair suggests a role in endothelial differentiation and blood vessel growth. Proc Natl Acad Sci U S A.. 1993;90:8915-8919.
Connolly DR, Olander JV, Heuvelman D, Nelson R, Monsell R, Siegel N, Haymore BL, Leimgruber R, Feder J. Human vascular permeability factor: isolation from U937 cells. J Biol Chem.. 1989;264:20017-20024.
Asahara T, Bauters C, Wu T, Chen D, Zhen LP, Kearney M, Rossow S, Bunting S, Ferrara N, Symes JF, Isner JM. Restoration of endothelial function in hypercholesterolemic rabbit by intermittent administration of vascular endothelial growth factor. J Am Coll Cardiol.. 1995;25:366A.