Adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene inhibits vascular smooth muscle cell proliferation and neointima formation following balloon angioplasty of the rat carotid artery.

BACKGROUND: Vascular smooth muscle cell (VSMC) proliferation following arterial injury plays a critical role in a variety of vascular proliferative disorders, including atherosclerosis and restenosis after balloon angioplasty. In this study, we tested the hypothesis that localized arterial infection at the time of balloon angioplasty with an adenovirus (ADV-tk) encoding the herpes simplex virus thymidine kinase gene (HSV-tk), followed by systemic ganciclovir administration, can inhibit VSMC proliferation and neointima formation in a well-characterized model of arterial injury and restenosis. MATERIALS AND METHODS: The left carotid arteries of 31 male Sprague-Dawley rats were subjected to balloon angioplasty and immediately infected with 2 x 10(9) pfu of either ADV-tk or a control adenovirus that does not encode a recombinant protein (ADV-delta E1). Twenty-four hours after injury, animals from each experimental group were randomized to receive a course of systemic ganciclovir (ADV-tk/+GC, ADV delta E1/+GC) or saline (ADV-tk/-GC, ADV-delta E1/-GC). VSMC DNA synthesis was measured by 5'-bromodeoxuridine (BrdU) incorporation 2-4 days after balloon injury. The extent of restenosis, expressed as the neointima to media (I/M) area ratio was determined by digital planimetry 20 days after balloon injury in each of the four treatment groups. Immunohistochemistry using a mAb to von Willebrand factor (vWF) was used to determine the effects of ADV-tk infection and ganciclovir treatment on re-endothelialization of the carotid arteries 20 days following balloon angioplasty. RESULTS: Forty-one percent of the medial VSMCs in the ADV-tk/-GC arteries were labeled with BrdU 4 days after balloon injury. In contrast, ADV-tk infected animals that were treated with systemic ganciclovir (ADV-tk/+GC) displayed a 40% reduction in BrdU-staining medial VSMCs (p < 0.03). I/M area ratios of the three control groups were 1.17 +/- 0.18 (ADV-tk/-GC, n = 5), 1.15 +/- 0.10 (ADV-delta E1/+GC, n = 6), and 0.91 +/- 0.08 (ADV-delta E1/-GC, n = 6). These differences were not statistically significant (p > 0.05). In contrast, the ADV-tk/+GC animals (n = 6) displayed an I/M area ratio of 0.49 +/- 0.13 which was significantly lower than that seen in each of the three control groups (p < 0.02). None of the treated animals showed evidence of significant organ toxicity at autopsy. A regenerated endothelium was observed in the ADV-tk/+GC animals 20 days after balloon injury. CONCLUSIONS: Localized arterial infection with ADV-tk at the time of balloon angioplasty followed by systemic ganciclovir therapy reduces VSMC proliferation and neointimal expansion in the rat carotid artery injury model. Moreover, combined treatment with ADV-tk and systemic ganciclovir does not result in systemic toxicity and appears to selectively eliminate proliferating VSMCs, while preserving the capacity of the injured arterial segments to re-endothelialize within 3 weeks of injury. Taken together, these results support the feasibility of using this gene therapy approach for the treatment of human vascular proliferative disorders.     

Vascular gene transfer of the human inducible nitric oxide synthase: characterization of activity and effects on myointimal hyperplasia.

BACKGROUND: Nitric oxide (NO) has been shown to decrease myointimal hyperplasia in injured blood vessels. We hypothesize inducible No synthase (iNOS) gene transfer even at low efficiency will provide adequate local no production to achieve this goal. MATERIALS AND METHODS: A retroviral vector containing the human iNOS cDNA (DFGiNOS) was used to transfer the iNOS gene into vascular cells and isolated blood vessels to answer the following questions: can vascular endothelial and smooth muscle cells support iNOS activity and will low efficiency iNOS gene transfer suppress myointimal hyperplasia in injured porcine arteries? RESULTS: DFGiNOS-infected sheep pulmonary artery endothelial cells (SPAEC) expressed significant iNOS mRNA and protein, releasing nitrite levels of 155.0 +/- 10.7 nmol/mg protein/24 h vs. 5.5 +/- 1.1 by control cells. Transduced rat smooth muscle cells (RSMC) also expressed abundant iNOS mRNA and protein, but, in contrast to SPAEC, NO synthesis was dependent on exogenous tetrahydrobiopterin (BH4) (291.8 +/- 10.4 nmol nitrite/mg protein/24 hr with BH4, 37.7 +/- 2.6 without BH4). Only porcine arteries infected with DFGiNOS following balloon injury exhibited a 3-fold increase in total NO synthesis and a 15-fold increase in cGMP levels over control vessels in a BH4 dependent fashion, despite only a 1% gene transfer efficiency. Transfer of iNOS completely prevented the 53% increase in myointimal thickness induced by balloon catheter injury; the administration of a NOS inhibitor reversed this effect. CONCLUSIONS: These in vitro findings suggest that vascular iNOS gene transfer may be feasible. Furthermore, a low gene transfer efficiency may be sufficient to inhibit myointimal hyperplasia following arterial balloon injury, although a source of BH4 may be required.     

Adrenomedullin gene transfer induces neointimal apoptosis and inhibits neointimal hyperplasia in injured rat artery

Arterial wall injury leads to inflammatory reaction and release of growth factors that may mediate intimal regrowth. It is hypothesized that the neointimal cells may originate from adventitial myofibroblasts, medial smooth muscle cells, or differentiated bone marrow derived cells. Adrenomedullin (AM), an auto/paracrine cardiovascular peptide that is secreted from fibroblasts, endothelial cells, and vascular smooth muscle cells, may have a regulatory role in the intimal regeneration. In order to investigate the role of AM in neointimal growth, stimulation of stem cell migration, and apoptosis, we overexpressed AM with recombinant adenovirus in a rat arterial injury model. The intimae were significantly thinner in the arteries treated with AM adenovirus compared to the control group. Intima/media ratios were 0.48 +/- 0.18 and 1.01 +/- 0.20 (P < 0.05) in the AM group and the control group, respectively. In addition, a significantly higher apoptotic index of neointimal cells was seen in the AM gene transfer group compared to the control (2.78 +/- 0.5 vs. 0.57 +/- 0.20, P < 0.01). The neointimal cells stained positive for alpha-smooth muscle actin and negative for desmin suggesting possible myofibroblast origin. Very few c-Kit+ or MDR1+ cells were detected 2 weeks after the injury. We conclude that AM overexpression inhibits neointimal growth. The inhibition is associated with enhanced apoptosis of the neointimal cells which may be of myofibroblast origin.     

C-type natriuretic peptide for reduction of restenosis: gene transfer is superior over single peptide administration

BACKGROUND: Restenosis is still a significant clinical problem limiting the long-term therapeutic success following balloon dilation or stent implantation. New approaches are necessary inhibiting neointima formation and simultaneously promoting re-endothelialization. Therefore, long-term therapeutic effects of adventitial liposome-mediated C-type natriuretic protein (CNP) gene and CNP peptide applications in a porcine model for restenosis post-angioplasty were investigated. METHODS: For in vitro applications, primary cultures of porcine vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) were used. Gene transfer was performed with cationic lipid DOCSPER [1,3-dioleoyloxy-2-(N5-carbamoylspermine)propane]. In vivo treatment of pig femoral arteries was adventitial using a needle injection catheter following balloon angioplasty. Arteries were investigated by angiography, Evan's blue staining, histomorphometry, immunohistochemistry, PCR and RT-PCR. RESULTS: Using CNP gene transfer in vitro, 29.4+/-7.2% reduction of cell proliferation in VSMCs was observed. In ECs, the CNP gene did not compromise cellular growth. For the CNP peptide the optimal concentration was 1 mM with 50.7+/-11.3% reduction of VSMC proliferation and 12.1+/-5.3% enhancement of growth of ECs. Three weeks following application in vivo complete re-endothelialization was observed in all treated groups. At 3 months significant reduction of neointima formation was observed using CNP gene vs. CNP peptide (85.9+/-7.8% vs. 63.3+/-27.6% reduction, P<0.05) compared to control treatment. CONCLUSION: Periadventitial liposome-mediated CNP gene transfer in vivo resulted in a significant long-term reduction of neointima formation without compromising endothelial repair and was superior over single CNP peptide administration. Advantages of CNP are its physiological origin and simultaneous inhibition of VSMC proliferation and promotion of EC growth.     

Intramuscular gene transfer of CGRP inhibits neointimal hyperplasia after balloon injury in the rat abdominal aorta

CGRP is a well-known neuropeptide that has various protective effects on cardiovascular system. Our previous studies have shown that CGRP inhibits vascular smooth muscle cell (VSMC) proliferation in vitro. The present study aimed to explore the role of the CGRP in neointimal formation after balloon injury in the rat aortic wall and the underlying mechanism. Gene transfer of CGRP was performed with the use of intramuscular electroporation in a balloon-injured rat aorta model. Apoptosis in VSMCs was determined by electrophoresis assessment of DNA fragmentation and terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay. Overexpression of the CGRP gene significantly inhibited the neointimal formation after balloon injury compared with the mock transfer, as assessed by the intima-to-media ratio 14 days after balloon injury (29.2 +/- 3.7% vs. 52.7 +/- 5.4%; n = 9-12, P < 0.05). In addition, CGRP gene expression increased the number of apoptotic cells in the neointima in vivo 14 days after balloon injury. Similarly, the addition of bioactive CGRP and the nitric oxide donor induced similar apoptosis in cultured VSMCs. The antagonist of the CGRP(1) receptor and inhibitors of cAMP-PKA and nitric oxide blocked CGRP-mediated apoptosis. Furthermore, CGRP gene transfer increased inducible nitric oxide synthase and p53 but decreased PCNA and Bcl-2 protein levels in balloon-injured rat aorta. Our data demonstrated that CGRP potently inhibited neointimal thickening in the rat aorta, at least in part through its distinct effects on apoptosis and proliferation of VSMCs both in vivo and in vitro. Therefore, delivery of the CGRP gene may have therapeutic implications in limiting vascular restenosis.     

Antisense oligonucleotides to stromelysin mRNA inhibit injury-induced proliferation of arterial smooth muscle cells.

Smooth muscle cell migration and proliferation are important events in the formation of intimal lesions associated with atherosclerosis and restenosis following balloon angioplasty. To make this possible, the smooth muscle cell has to change from a contractile to an activated repair cell with capacity to synthesize DNA and extracellular matrix components. There is now considerable evidence that the extracellular matrix has important functions in modulating the phenotypic properties of smooth muscle cells, but less is known about the role of the matrix metalloproteinases. The present study investigates the role of stromelysin in the modulation of rat aortic smooth muscle cell morphology and function following mechanical injury in vitro and in vivo. Antisense mRNA oligonucleotides were used to investigate the role of stromelysin expression in injury-induced phenotypic modulation and the subsequent migration and proliferation of vascular smooth muscle cells. Cultured rat aortic smooth muscle cells and balloon-injured rat carotid arteries were used as experimental models. Light- and electron microscopy were used to follow changes in smooth muscle cell phenotype and lesion formation and incorporation of 3H-thymidine to detect DNA synthesis. Injury-induced DNA synthesis and migration in vitro were inhibited by 72% and 36%, respectively, by adding stromelysin antisense oligonucleotides to the medium prior to injury. In primary cultures, 67% of the smooth muscle cells treated with stromelysin antisense were retained in a contractile phenotype as judged by analysis of cell fine structure, compared to 15% untreated cells and 40% in cells treated with mismatched oligonucleotides. Examination of the carotid arteries one week after balloon injury likewise demonstrated a larger fraction of contractile cells in the inner parts of the media in vessels treated with antisense oligonucleotides compared to those treated with mismatched oligonucleotides. The neointima was also distinctly thinner in antisense-treated than in mismatched-treated and control arteries at this time. These findings indicate that stromelysin mRNA antisense oligonucleotides inhibited phenotypic modulation of rat arterial smooth muscle cells and so caused a decrease in migration and proliferation and neointima formation in response to vessel wall injury.     

Expression of T-cadherin in the rat carotid artery wall after balloon injury and in different rat organs

T-cadherin is an unusual glycosilphosphatidylinositol (GPI)-anchored member of the cadherin family of cell adhesion proteins. In contrast to classical cadherins, tissue distribution of T-cadherin so far remained unknown. We examined tissue distribution of T-cadherin in rats using Western blotting and immunohistochemical method. Our results show that T-cadherin is expressed in all types of muscles (cardiac, striated, and smooth muscles), in brain neurons, and spinal cord, in the vessel endothelium, at the apical pole of intestinal villar epithelium, in the basal layer of skin, and eosophagal epithelium. Blood-derived and lymphoid cells as well as connective tissue were T-cadherin-negative. The highest level of T-cadherin expression was revealed in the cardiovascular system. Although T-cadherin was detected in smooth muscle cells, its role in the intimal thickening and restenosis is not known. We examined T-cadherin expression within 1-28 days after balloon injury of rat left carotid arteries. T-cadherin expression was valued immunohistochemically with semiquantitative method. In uninjured arteries, T-cadherin was expressed in endothelial (vWF-positive) cells, and smooth muscle (alpha-actin-positive) cells (SMCs). After denudation of arterial wall, T-cadherin was present both in the media and neointima. We revealed dynamics of T-cadherin expression in the media of injured artery: an essential increase being registered at the stage of cell migration and proliferation in the media and neointima (1-7 days), followed by its decrease to the baseline level (10-28 days). The high upregulation of T-cadherin expression in the media and neointima during migration and proliferation of vascular cells after vessel injury enables us to suggest the involvement of T-cadherin in vessel remodeling after balloon catheter injury.     

LncRNA GAS5 regulates vascular smooth muscle cell cycle arrest and apoptosis via p53 pathway

Vascular remodeling is a pathological process following cardiovascular intervention. Vascular smooth muscle cells (VSMC) play a critical role in the vascular remodeling. Long noncoding RNAs (lncRNA) are a class of gene regulators functioning through various mechanisms in physiological and pathological conditions. By using cultured VSMC and rat carotid artery balloon injury model, we found that lncRNA growth arrest specific 5 (GAS5) serves as a negative regulator for VSMC survival in vascular remodeling. By manipulating GAS5 expression via adenoviral overexpression or short hairpin RNA knockdown, we found that GAS5 suppresses VSMC proliferation while promoting cell cycle arrest and inducing cell apoptosis. Mechanistically, GAS5 directly binds to p53 and p300, stabilizes p53-p300 interaction, and thus regulates VSMC cell survival via induction of p53-downstream target genes. Importantly, local delivery of GAS5 via adenoviral vector suppresses balloon injury-induced neointima formation along with an increased expression of p53 and apoptosis in neointimal SMCs. Our study demonstrated for the first time that GAS5 negatively impacts VSMC survival via activation the p53 pathway during vascular remodeling.     

Systemic tissue inhibitor of metalloproteinase-1 gene delivery reduces neointimal hyperplasia in balloon-injured rat carotid artery

Metalloproteinases (MMP)-2 and MMP-9 play a role in smooth muscle cell (SMC) migration from the media to the intima following arterial injury. Intravenous administration of adenovirus encoding tissue inhibitor of metalloproteinase-1 (TIMP-1) into balloon-injured rat arteries (3 x 10(11) viral particles/rat; n=7) resulted in a transient expression of TIMP-1 and a significant inhibition of neointima thickening within 16 days ( approximately 40% vs. control; P=0.012). Three days after injury, the number of intimal SMCs was decreased by approximately 98% in TIMP-1-treated rats. However, no alteration was seen in intimal SMC proliferation after 13 days of injury. Therefore, our results show that systemic gene transfer of TIMP-1 is a promising approach in early restenosis treatment.     

Regulation of vascular smooth muscle cell growth by aldose reductase

Aldose reductase (AR) is a broad-specificity aldo-keto reductase with wide species and tissue distribution. The enzyme has been implicated in the development of pleiotropic complications of long-term diabetes. However, the euglycemic function of the enzyme remains unclear. To examine its potential role in cell growth, changes in AR mRNA and protein were measured in human aortic smooth muscle cells exposed in culture to serum or thrombin. Stimulation by these mitogens led to an increase in the abundance of AR mRNA and protein. Furthermore, inhibition of the AR by tolrestat and sorbinil diminished DNA synthesis and cell proliferation in response to serum. Immunohistochemical staining with anti-AR antibodies revealed no significant expression of AR in the smooth muscle cells of rat carotid arteries. However, 10 and 21 days after balloon injury, intense staining was associated with the proliferating cells of the neointima. Treatment of these animals with 40 mg/kg/day sorbinil diminished the ratio of neointima to the media. Together, these observations suggest that, in vascular smooth muscle cells (VSMC), AR is a growth-responsive gene product and that inhibition of AR prevents VSMC growth and decreases intimal hyperplasia and restenosis.     

Regulation of vascular smooth muscle cell growth by aldose reductase

Aldose reductase (AR) is a broad-specificity aldo-keto reductase with wide species and tissue distribution. The enzyme has been implicated in the development of pleiotropic complications of long-term diabetes. However, the euglycemic function of the enzyme remains unclear. To examine its potential role in cell growth, changes in AR mRNA and protein were measured in human aortic smooth muscle cells exposed in culture to serum or thrombin. Stimulation by these mitogens led to an increase in the abundance of AR mRNA and protein. Furthermore, inhibition of the AR by tolrestat and sorbinil diminished DNA synthesis and cell proliferation in response to serum. Immunohistochemical staining with anti-AR antibodies revealed no significant expression of AR in the smooth muscle cells of rat carotid arteries. However, 10 and 21 days after balloon injury, intense staining was associated with the proliferating cells of the neointima. Treatment of these animals with 40 mg/kg/day sorbinil diminished the ratio of neointima to the media. Together, these observations suggest that, in vascular smooth muscle cells (VSMC), AR is a growth-responsive gene product and that inhibition of AR prevents VSMC growth and decreases intimal hyperplasia and restenosis.     

A selective somatostatin type-2 receptor agonist inhibits neointimal thickening and enhances endothelial cell growth and morphology following aortic balloon injury in the rabbit

Somatostatin analogs have been shown to inhibit vascular smooth muscle cell (VSMC) proliferation and attenuate neointimal thickening following experimental balloon catheter injury. In this study, the effects of a selective agonist for the somatostatin receptor subtype 2, PRL-2486, on neointimal thickening and endothelial cell regrowth 2 weeks following balloon catheterization of male New Zealand White rabbits were determined. Rabbits treated 2 days prior to and 2 weeks after catheter injury with 10 g/kg/day PRL-2486 (PRL-tx) had decreased I/M ratios (intimal area/medial area × 100; p < 0.05) but had no effect at lower (5 g/kg/day) or higher (20 g/kg/day) doses. PRL-tx had significantly decreased VSMC proliferation compared to untreated animals. PRL-tx increased endothelial regrowth by over 2-fold (p < 0.002) and improved endothelial cell morphology. Endothelial-dependent relaxation responses to acetylcholine were attenuated by catheter injury, and were not improved with PRL-tx. These data suggest that the PRL-2486-mediated inhibition of neointimal thickening exhibits a bell-shaped dose-response curve. This inhibition may be due in part to decreased VSMC proliferation, which may be a function of enhanced endothelial regrowth, but not the return of endothelium-dependent vascular function.     

Gene transfer of dominant negative Rho kinase suppresses neointimal formation after balloon injury in pigs

Restenosis after angioplasty still remains a major problem for which neointimal formation appears to play an important role. Recent studies in vitro suggested that Rho kinase, a target protein of Rho, is important in various cellular functions. We thus examined whether Rho kinase is involved in the restenotic changes after balloon injury. In vivo gene transfer was performed immediately after balloon injury in both sides of the porcine femoral arteries with adenoviral vector encoding either a dominant negative form of Rho kinase (AdDNRhoK) or beta-galactosidase (AdLacZ) as a control. One week after the transfer, immunohistochemistry confirmed the successful gene expression in the vessel wall, whereas 2 wk after the transfer, Western blotting showed the functional upregulation of Rho kinase at the AdLacZ site and its suppression at the AdDNRhoK site. Angiography showed the development of a stenotic lesion at the AdLacZ site where histological neointimal formation was noted, whereas those changes were significantly suppressed at the AdDNRhoK site. These results indicate that Rho kinase is involved in the pathogenesis of neointimal formation after balloon injury in vivo.     

Vascular smooth cell proliferation in perfusion culture of porcine carotid arteries

Objective of this study was to develop a novel in vitro artery culture system to study vascular smooth muscle cell (SMC) proliferation of porcine carotid arteries in response to injury, basic fibroblast growth factor (FGF2), and FGF2 conjugated with cytotoxin saporin (SAP). Perfusion-cultured porcine carotid arteries remained contractile in response to norepinephrine and relaxant to acetylcholine for up to 96 h. SMC proliferation of cultured arteries was detected by bromodeoxyuridine incorporation in both non-injured and balloon-injured arteries. In the inner layer of the vessel wall near the lumen, SMC proliferation were less than 10% in uninjured vessels, 66% in injured vessels, 80% in injured vessels with FGF2 treatment, and 5% in injured vessels with treatment of FGF2-SAP. Thus, the cultured porcine carotid arteries were viable; and the injury stimulated SMC proliferation, which was significantly enhanced by FGF2 and inhibited by FGF2-SAP.     

17-AAG对大鼠颈总动脉球囊损伤后内膜增生的影响及其作用机制

目的:研究17-丙烯胺-17去甲氧格尔德霉素(17-Allylamino-17-emethoxy-geldanamycin, 17-AAG)对球囊损伤后大鼠颈总动脉内膜增生的影响及可能作用机制。方法:将清洁级雄性SD大鼠36只按照随机数字法分为假手术组(Sham组)12只、球囊损伤组(Balloon injury, BI组)12只及17-AAG治疗组(17-AAG组)12只。采用2F Fogarty球囊建立大鼠颈总动脉球囊损伤组模型,17-AAG治疗组大鼠在建模后腹腔注射17-AGG(20 mg/kg 2d)。各组大鼠于球囊损伤3周后取损伤段颈总动脉,通过HE染色观察血管内膜形态学改变并评估内膜增生情况,免疫组化染色(Immunohistochemical staining,IHS)法检测血管壁增殖细胞核抗原(Proliferating cell nuclear antigen,PCNA)的表达,评估血管平滑肌细胞的增殖情况。流式细胞术检测血管平滑肌细胞的凋亡情况。结果:BI组、17-AAG组大鼠球囊损伤后颈总动脉内膜出现不同程度增生,内膜/中膜面积比(Intima area/Membrane area,I/M)均较Sham组显著升高(P0.05);17-AAG组的I/M较BI组明显下降(P0.05)。BI组、17-AAG组颈总动脉PCNA表达水平较Sham组明显升高(P0.05),较BI组显著降低(P0.05)。BI组、17-AAG组大鼠血管平滑肌细胞凋亡率较Sham组显著升高(P0.05);17-AAG组大鼠血管平滑肌细胞凋亡程度较BI组明显升高(P0.05)。结论:17-AAG对球囊损伤后颈总动脉内膜增生存在抑制作用,其机制可能是通过提高血管平滑肌细胞凋亡率影响其增殖程度。     

Telomeric restriction analysis of vascular smooth muscle cells following balloon angioplasty in rabbits

Intimal hyperplasia due to smooth muscle cell proliferation and migration has been reported to be responsible for the pathogenesis of atherosclerosis and restenosis, manifested following balloon angioplasty. In this study, we employed the balloon angioplasty model to study telomere length regulation in proliferating vascular smooth muscle cells. Our results showed that balloon angioplasty in iliac arteries resulted in intimal hyperplasia due to proliferation of the smooth muscle cells and small size telomeric restrictional fragments were evident in injured arteries.     

Novel heparanase-inhibiting antibody reduces neointima formation

Basic fibroblast growth factor (bFGF), stored bound to heparan sulfate proteoglycans in the extracellular matrix (ECM) of the arterial media, may initiate smooth muscle cell (SMC) proliferation after coronary intervention, thus contributing to restenosis. bFGF mobilization from ECM stores after injury may be induced by platelet degranulation products such as heparanase. Therapies aimed at the inhibition of bFGF release and activation may assist in prevention of restenosis. To test this theory, we first examined the mobilization and activation of bFGF in the arterial media by platelet-derived heparanase. Heparanase, locally delivered to the rat carotid artery, was found to release bFGF and induce substantial SMC proliferation in the absence of actual vascular injury. An antibody that neutralizes heparanase was then developed and evaluated in a rat carotid balloon injury model. Local delivery of anti-heparanase IgG was found to inhibit bFGF release by approximately 60% ( p < 0.001) at 4 d; this correlated with the significant reduction in neointima formation observed at 14 d (intimal area/medial area: control 1.3 +/- 0.3, anti-heparanase 0.35 +/- 0.12, p < 0.0001). Platelet-derived heparanase is therefore likely to be important in initiating events leading to restenosis via bFGF mobilization. Furthermore, heparanase neutralization may assist in the prevention of restenosis following vascular injury.     

Intimal hyperplasia following vascular injury is not inhibited by an antisense thrombin receptor oligodeoxynucleotide

Thrombin is a multifunctional serine protease with central functions in hemostasis, but demonstration of its role in the initiation and maintenance of cell proliferation which occurs following vascular injury is still lacking. To determine the role played by thrombin and its receptor in neointimal accumulation of smooth muscle cells in a rabbit carotid artery model, we have used an 18 mer antisense phosphorothioate oligonucleotide (ODN) directed against the translation initiation region of the human thrombin receptor gene. The antisense ODN inhibited in a dose-dependent manner thrombin- or thrombin receptor activating peptide-induced human aortic smooth muscle cell proliferation. The growth-inhibitory effect of thrombin receptor antisense ODN was preventable by an excess of sense oligomer and specific for thrombin. The suppression of growth was accompanied by a marked decrease of the level of thrombin receptor expression as evidenced by [¹²⁵l]-thrombin binding to smooth muscle cells. Under the same experimental conditions, the corresponding sense ODN was inactive. The effect of the antisense ODN on intimal smooth muscle hyperplasia in rabbit carotid arteries subjected to endothelial injury was then investigated. The topical application of the antisense (500 μg/artery) but not the sense ODN dissolved in F127 pluronic gel around the injured artery resulted, 2 weeks after the application, in a dramatic reduction of the expression of the thrombin receptor mRNA and protein levels as determined by in situ hybridization and immunohistochemistry. However, intimal smooth muscle cell accumulation as estimated by an intimal to medial cross-sectional area ratio was reduced only by 2.7% (vs. 10.3% for the sense ODN), whereas r-hirudin (200 μg/kg/day, sc), a potent direct thrombin inhibitor significantly reduced the formation of neointima in denuded carotid arteries (35.4% inhibition, P = 0.03). J. Cell. Physiol. 170:106–114, 1997. © 1997 Wiley-Liss, Inc.     

Activation of EphB2 and its ligands promotes vascular smooth muscle cell proliferation.

EphB2 and its ligands regulate interactions between endothelial and mesenchymal cells in developing arteries. In adult arteries, the relationship between smooth muscle cells and overlying intact endothelium is responsible for maintaining the health of the vessel. Heparin inhibits vascular smooth muscle cell growth in culture and intimal hyperplasia following endothelial denudation. Using gene microarrays, we identified the tyrosine kinase receptor EphB2 as being differentially expressed in response to continuous intravenous heparin administration in the rabbit model of arterial injury. EphB2 protein levels increased in cultured bovine vascular smooth muscle cells following serum stimulation and were decreased in a dose-dependent fashion by heparin. Fc chimeras of the binding domain of the EphB2 ligands blocked the formation of the EphB2 ligand-receptor complex and reduced growth of serum-stimulated vascular smooth muscle cells in a dose-dependent fashion. Activation of the ligand by an Fc chimera to EphB2 followed a parabolic dose-response growth curve, indicating growth stimulation until the chimera begins to compete with native receptors. Co-administration of EphB2/Fc chimera with heparin shifted the dose-response curve to the right. These data indicate a possible new route of Heparin's antiproliferative effect and a role of EphB2 and its ligands in vascular smooth muscle cell proliferation.     

Aging exacerbates negative remodeling and impairs endothelial regeneration after balloon injury

Many older patients, because of their high prevalence of coronary artery disease, are candidates for percutaneous coronary interventions (PCI), but the effects of vascular aging on restenosis after PCI are not yet well understood. Balloon injury to the right carotid artery was performed in adult and old rats. Vascular smooth muscle cell (VSMC) proliferation, apoptotic cell death, together with Akt induction, telomerase activity, p27kip1, and endothelial nitric oxide synthase (eNOS) expression was assessed in isolated arteries. Neointima hyperplasia and vascular remodeling along with endothelial cell regeneration were also measured after balloon injury. Arteries isolated from old rats exhibited a significant reduction of VSMC proliferation and an increase in apoptotic death after balloon injury when compared with adult rats. In the vascular wall of adult rats, balloon dilation induced Akt phosphorylation, and this was barely present in old rats. In arteries from old rats, Akt-modulated cell cycle check points like telomerase activity and p27kip1 expression were decreased and increased, respectively, compared with adults. After balloon injury, old rats showed a significant reduction of neointima formation and an increased vascular negative remodeling compared with adults. These results were coupled by a marked delay in endothelial regeneration in aged rats, partially mediated by a decreased eNOS expression and phosphorylation. Interestingly, chronic administration of L-arginine prevented negative remodeling and improved reendothelialization after balloon injury in aged animals. A decreased neointimal proliferation, an impaired endothelial regeneration, and an increase in vascular remodeling after balloon injury were observed in aged animals. The molecular mechanisms underlying these responses seem to be a reduced Akt and eNOS activity.