Heparan sulfate side chains have a critical role in the inhibitory effects of perlecan on vascular smooth muscle cell response to arterial injury.

Perlecan is a proteoglycan composed of a 470-kDa core protein linked to three heparan sulfate (HS) glycosaminoglycan chains. The intact proteoglycan inhibits the smooth muscle cell (SMC) response to vascular injury. Hspg2(Δ3/Δ3) (MΔ3/Δ3) mice produce a mutant perlecan lacking the HS side chains. The objective of this study was to determine differences between these two types of perlecan in modifying SMC activities to the arterial injury response, in order to define the specific role of the HS side chains. In vitro proliferative and migratory activities were compared in SMC isolated from MΔ3/Δ3 and wild-type mice. Proliferation of MΔ3/Δ3 SMC was 1.5× greater than in wild type (P < 0.001), increased by addition of growth factors, and showed a 42% greater migratory response than wild-type cells to PDGF-BB (P < 0.001). In MΔ3/Δ3 SMC adhesion to fibronectin, and collagen types I and IV was significantly greater than wild type. Addition of DRL-12582, an inducer of perlecan expression, decreased proliferation and migratory response to PDGF-BB stimulation in wild-type SMC compared with MΔ3/Δ3. In an in vivo carotid artery wire injury model, the medial thickness, medial area/lumen ratio, and macrophage infiltration were significantly increased in the MΔ3/Δ3 mice, indicating a prominent role of the HS side chain in limiting vascular injury response. Mutant perlecan that lacks HS side chains had a marked reduction in the inhibition of in vitro SMC function and the in vivo arterial response to injury, indicating the critical role of HS side chains in perlecan function in the vessel wall.

Inhibition of intimal hyperplasia after stenting by over-expression of p15: a member of the INK4 family of cyclin-dependent kinase inhibitors.

We evaluated the role of p15(Ink4), a member of the INK4 family of CDK inhibitors on vascular smooth muscle cells (VSMCs) proliferation, cell cycle progression and intimal hyperplasia after stenting. Aortic VSMCs transduced with either adenovirus encoding for p15(Ink4) or ß-galactosidase were assessed for DNA synthesis, cell cycle progression, and pRb phosphorylation. Rabbit carotid arteries were stented and treated with peri-adventitial delivery of saline or adenovirus encoding for p15(Ink4) or ß-galactosidase. p15(Ink4) transgene and protein expression were evaluated at 24 h and 72 h, respectively. In-stent cell proliferation was evaluated by BrdU at day 7. Histomorphometric analysis of in-stent intimal hyperplasia was performed at 10 weeks. Human p15(Ink4) DNA was detected in transduced VSMCs at 24h. p15(Ink4) over-expression reduced VSMCs DNA synthesis by 60%. Cell cycle progression was inhibited, with a 30% increase in G1 population accompanied by inhibition of pRb phosphorylation. Human p15(Ink4) transgene was identified in transduced stented arteries but not in control arteries. p15(Ink4) immunostaining was increased and cell proliferation significantly reduced by 50% in p15(Ink4) transduced arteries. Intimal cross-sectional area (CSA) of p15(Ink4)-treated group was significantly lower than the ß-gal treated and non-transduced groups (p=0.008). There were no differences in the intimal or medial inflammatory response between groups. p15(Ink4) over-expression blocks cell cycle progression leading to inhibition of VSMCs proliferation. Peri-adventitial delivery of p15(Ink4) significantly inhibits in-stent intimal hyperplasia.

In vitro differences between venous and arterial-derived smooth muscle cells: potential modulatory role of decorin.

OBJECTIVE: We analyzed the phenotypic and functional differences between venous and arterial smooth muscle cells (SMC) and the role of decorin in modulating these differences. METHODS AND RESULTS: SMC were isolated from the jugular veins and carotid arteries of male white New Zealand rabbits. Venous SMC demonstrated increased proliferation (2-fold, p<0.001), migration (1.7-fold, p<0.001), and collagen synthesis (4-fold, p<0.001), with decreased adhesion to collagen and fibronectin (1.2-fold, p<0.01) compared to arterial SMC. Higher levels of gelatinase activity (MMP-2 and MMP-9) and tissue inhibitor of metalloproteinase (TIMP) were also observed in venous SMC. Venous SMC demonstrated increased expression of SMemb and decreased expression of SM1--markers of a dedifferentiated and differentiated phenotype, respectively. Arterial SMC produced increased levels of the inhibitory proteoglycan, decorin, compared to venous SMC. Conditioned medium from arterial SMC (ASMC-CM) significantly decreased DNA synthesis, collagen synthesis, and gelatinase activity in venous SMC. Removal of decorin from ASMC-CM by immunoprecipitation significantly reversed the inhibitory effects of ASMC-CM on venous SMC proliferation and collagen synthesis but did not affect gelatinase activities. CONCLUSION: Venous SMC are more dedifferentiated and demonstrate increased proliferative and synthetic capacity than arterial SMC. Differential decorin expression between arterial and venous SMC contributes to these differences in biologic behavior. Venous SMC properties may contribute to accelerated atherosclerosis in venous bypass grafts.

Study of pomegranate (Punica granatum L.) peel extract containing anthocyanins on fatty streak formation in the renal arteries in hypercholesterolemic rabbits.

BACKGROUND: The influence of the supplementation of pomegranate peel extract containing anthocyanins on atherosclerotic plaque formation induced by hypercholesterolemia was investigated in renal arteries in rabbits. MATERIALS AND METHODS: After the determination of polyphenol and anthocyanin's content of P. granatum peel hydroalcoholic extract, 30 male rabbits were randomly divided into three groups. They were fed basic diet, hypercholesterolemic diet and hypercholesterolemic diet along with P. granatum peel extract (polyphenolic content for each rabbit 1 g/kg diet) for 2 month. Blood samples were collected at the begging, middle and end of the study in order to measure lipid concentration and oxidative and antioxidative status variables, and renal arteries were taken for the assessment of atherosclerotic plaques at the end of the study. RESULTS: The results reveal that P. granatum peel extract significantly increases serum antioxidant capacity in the extract recipient group in comparison with hypercholesterolemic control (P < 0.05). No significant differences are observed in total cholesterol, triglyceride, low-density lipoprotein, high-density lipoprotein, very low-density lipoprotein and in mean size of accumulated fatty streaks in renal arteries in the extract treatment group in comparison with hypercholesterolemic control (P > 0.05). CONCLUSION: The results of this study indicate that consumption of pomegranate peel extract containing anthocyanins (polyphenol content 1 g/kg diet) despite of a significant increase in serum antioxidant capacity cannot protect the kidneys from hypercholesterolemia-induced damages during the treatment period.

Collagenase plaque digestion for facilitating guide wire crossing in chronic total occlusions.

BACKGROUND: Chronic total occlusions (CTOs) are associated with significant angina, impaired left ventricular function, and worse long-term outcomes. Percutaneous coronary interventions in CTO are unsuccessful in up to 50% of cases, primarily because of inability to cross the lesion with a guide wire. Collagen is the predominant component of the atherosclerotic plaque. The objective of this study was to determine the efficacy and toxicity of local delivery of a collagen-degrading enzyme to facilitate guide wire crossing in CTO. METHODS AND RESULTS: Type IA collagenase (100 or 450 microg) or placebo was locally administered to 45 CTOs in a rabbit femoral artery model. Mean occlusion duration was 16+/-5 weeks. Attempts to cross the CTO (mean length, 28+/-9 mm) with conventional guide wires were assessed at 72 hours after treatment. An additional 3 arteries per group were assessed for collagenase effects at 24 hours after treatment. Successful guide wire crossings were significantly higher in collagenase-treated arteries (13 of 21, 62%) than in placebo-treated arteries (7 of 24, 29%) (P=0.028). No adverse effects on arterial structure were observed in collagenase-treated arteries. At 24 hours, collagenase-treated arteries demonstrated increased collagenase protein, gelatinase activity, and collagen fragments. CONCLUSIONS: Local delivery of collagenase can safely facilitate guide wire crossing of CTO. This novel approach could lead to higher percutaneous coronary intervention success rates in CTO.

The cardiac atria are chambers of active remodeling and dynamic collagen turnover during evolving heart failure.

OBJECTIVES: The role of atrial myocytes and extracellular matrix (ECM) changes in atrial chamber remodeling was studied in a canine model of heart failure (HF). BACKGROUND: Cardiac remodeling is a key process mediating the progression of HF. Studies of the structural mechanisms of cardiac remodeling have been limited to the left ventricle. The structural alterations associated with atrial chamber remodeling in evolving HF have not been studied. METHODS: Age- and weight-matched dogs were subjected to right ventricular pacing (240 beats/min) for one and three weeks to produce early and severe HF, respectively. Atrial tissues were assessed for myocyte and ECM changes. RESULTS: Right atrial and left atrial (LA) pressures were significantly increased in early and severe HF. The LA wall tension index was significantly increased at both HF stages by 116% and 443%, respectively. Atrial collagen synthesis and degradation were significantly increased in severe HF. Gelatinase activity was significantly increased at both early and severe stages of HF. Gelatin zymography showed increased matrix metalloproteinases (MMP)-9 with early HF and increased MMP-2 with severe HF. The LA wall tension index was significantly correlated with gelatinase activity and collagen synthesis. Although total atrial collagen content was not changed, disarray of collagen fibers was observed. Atrial myocyte hypertrophy without evidence of apoptosis was also present in severe HF. CONCLUSIONS: There is marked atrial chamber remodeling in canine pacing-induced HF, which is characterized by myocyte hypertrophy and dynamic collagen turnover. Atrial remodeling may contribute to the development of atrial arrhythmias and pulmonary hypertension and could offer a novel therapeutic target.

Arterial repair after stenting and the effects of GM6001, a matrix metalloproteinase inhibitor.

OBJECTIVES: This study compared the extracellular matrix (ECM) and cellular responses after stenting to balloon angioplasty (BA) and to determine the late effects of matrix metalloproteinase (MMP) inhibition on arterial repair after stenting. BACKGROUND: Although stenting is the predominant form of coronary intervention, there is limited understanding of the early and late arterial response. METHODS: In a double-injury rabbit model, adjacent iliac arteries in 87 animals received BA (3.0 mm diameter) or stenting (3.0 mm NIR). Rabbits were treated for 1 week postprocedure with either GM6001 (100 mg/kg per day), an MMP inhibitor or placebo and sacrificed at 1 week or at 10 weeks' postprocedure. Arteries were analyzed for morphometry, collagen content, gelatinase activity, cell proliferation and DNA content. RESULTS: Stented arteries had significant increases in collagen content (2-fold) at 10 weeks compared to BA-treated arteries. At one week, overall gelatinase activity was increased >2-fold in stented arteries, with both 72 kD and 92 kD gelatinase activity. Stented arteries also had increases in both intimal DNA content (1.5-fold) and absolute cell proliferation (4-fold). Compared to placebo, GM6001 significantly inhibited intimal hyperplasia and intimal collagen content, and it increased lumen area in stented arteries without effects on proliferation rates. CONCLUSIONS: Stenting causes a more vigorous ECM and MMP response than BA, which involves all layers of the vessel wall. Inhibition by MMP blocks in-stent intimal hyperplasia and offers a novel approach to prevent in-stent restenosis.

Human-grade purified collagenase for the treatment of experimental arterial chronic total occlusion.

PURPOSE: Chronic total occlusions (CTO) remain a major limitation of percutaneous interventions. Procedural failure is usually due to the inability to cross the lesion with a guide wire. We have previously shown that local administration of a laboratory-grade collagenase followed by a 72-h waiting period may facilitate guide-wire crossing. The aim of the present study was to evaluate the efficacy and toxicity of a human-grade purified collagenase, suitable for clinical use, in facilitating guide-wire crossing in a rabbit model of femoral artery CTO. METHODS AND RESULTS: A chronic total arterial occlusion was constructed in femoral arteries of New Zealand white rabbits. The local administration of purified collagenase solution (150 microg) via an over-the-wire balloon system was performed in 10 CTO. Guide-wire crossing was attempted after 24 h and was successful in all cases. Different doses (50-500 microg) were administered to an additional 17 rabbits to assess collagenase effects. Local subcutaneous bruising was observed at higher doses. Histological evaluation showed no damage to the arterial wall structure. Arterial extracts from collagenase-treated arteries showed increased MMP-2 and MMP-9 activities and higher levels of local MMP-1 and degraded collagen. CONCLUSIONS: Local administration of a human-grade purified collagenase degrades collagen in CTO and is highly effective for the facilitation of guide-wire crossing in CTO.

The role of perlecan in arterial injury and angiogenesis.

Perlecan is a large heparan sulfate proteoglycan (HSPG), which is a major component of the vessel wall. In relation to vascular biology, perlecan has been shown to be a potent inhibitor of smooth muscle cell (SMC) activity. In vivo experiments in animal models of arterial injury have shown that perlecan may inhibit thrombosis and intimal hyperplasia. On the other hand, perlecan has been shown to have opposing effects on endothelial cells (ECs), where it promotes in vitro and in vivo angiogenesis and plays an important role in mediating tumor growth. These diverse biological effects, or "the perlecan paradox", are discussed in this review paper. The properties of perlecan including inhibition of SMC activity and thrombosis while enhancing EC proliferation are ideal for the prevention of in-stent restenosis. Perlecan's pro-angiogenic effects may be used for the treatment of various ischemic diseases such as intractable coronary artery disease and peripheral vascular disease.

Effects of intravascular cryotherapy on vessel wall repair in a balloon-injured rabbit iliac artery model.

OBJECTIVE: Although the application of cold energy, cryotherapy, has been shown to cause selective damage to cellular components with preservation of matrix structure resulting in less fibrosis in a variety of tissues, the effects of intravascular cryotherapy on vessel wall repair after balloon angioplasty are unknown. We sought to characterize the effects of cryotherapy application on vessel wall repair after balloon angioplasty and study the relationship between collagen accumulation in the vessel wall and late lumen loss as assessed by serial intravascular ultrasound. METHODS: The immediate, early (72 h) and late (10 weeks) effects of three intravascular cryotherapy application time periods (60, 120 and 240 s) after iliac artery balloon angioplasty ('cryotherapy') were compared with balloon angioplasty alone ('control') in 59 rabbits. Arterial lumen area was measured by intravascular ultrasound immediately after the procedure, at 72 h and at 10 weeks. Collagen content was calculated separately for intima and media/adventitia layers and correlated with late lumen loss. RESULTS: Cryotherapy produced average vessel wall temperature of -26 degrees C (range, -20 to -45 degrees C) and resulted in significantly larger lumen cross-sectional area (CSA) immediately after application (5.74+/-1.18 vs. 4.14+/-0.75 mm(2), P=0.008) but was not different than control arteries at 10 weeks. At 72 h, there was extensive cell loss in the medial and adventitial layers accompanied by increased macrophage infiltration in cryotherapy treated arteries compared to control. At 10 weeks, intimal hyperplasia was increased 2-fold in cryotherapy treated arteries. Collagen content was increased 2-fold in the medial/adventitial layers, and nearly 3-fold in the intima of cryotherapy treated arteries. Collagen content in arterial intima (P=0.01) as well as media/adventitia (P=0.005) positively correlated with late lumen loss. Foci of chondro- and osseous metaplasia and calcification were evident at the medial-adventitial junction in cryotherapy treated arteries at 10 weeks. CONCLUSION: Intravascular cryotherapy induced early arterial wall cell loss and late intimal hyperplasia, vascular fibrosis and chondro- and osseous metaplastic changes with no late beneficial effects on lumen area compared to balloon angioplasty alone. Collagen accumulation in all three layers of the vessel wall contributes to the development of late inward remodeling after balloon angioplasty.

Vascular matrix remodeling in patients with bicuspid aortic valve malformations: implications for aortic dilatation.

BACKGROUND: Patients with bicuspid aortic valve malformations are at an increased risk of aortic dilatation, aneurysm formation, and dissection. Vascular tissues with deficient fibrillin-1 microfibrils release matrix metalloproteinases, enzymes that weaken the vessel wall by degrading elastic matrix components. In bicuspid aortic valve disease a deficiency of fibrillin-1 and increased matrix metalloproteinase matrix degradation might result in aortic degeneration and dilatation. METHODS: Samples of the pulmonary artery and aorta were obtained from surgical patients with bicuspid aortic valves (n = 21) and tricuspid aortic valves (n = 16). RESULTS: Fibrillin-1 content was reduced in bicuspid aortic valve aortas compared with that seen in tricuspid aortic valve aortas (P =.001), whereas the associated matrix components, elastin and collagen, were unchanged (P =.51 and P =.21). Reductions of aortic fibrillin-1 content were independent of valve function and patient age. Compared with tricuspid aortic valve aorta, matrix metalloproteinase 2 activity was increased more than 2-fold in bicuspid aortic valve aortas (P =.04) and correlated positively with aortic diameter (r = 0.74, P =.05). Matrix metalloproteinase 9 activity was not significantly different. Fibrillin-1 content was also reduced in the pulmonary arteries of patients with bicuspid aortic valves (P =.06), suggesting a systemic deficiency of fibrillin-1. Promatrix metalloproteinase 2 was increased (P =.04), reflecting an increased production of matrix metalloproteinase 2 in these fibrillin-1-deficient tissues, whereas active matrix metalloproteinase 2 and matrix metalloproteinase 9 species were unchanged, and correspondingly, the pulmonary arteries were not dilated. CONCLUSIONS: Deficient fibrillin-1 content in the vasculature of patients with bicuspid aortic valves might trigger matrix metalloproteinase production, leading to matrix disruption and dilatation. This process of vascular matrix remodeling in patients with bicuspid aortic valves offers novel therapeutic targets to prevent the aortic degeneration and dilatation characteristic of this disease.

Biochemical analysis of collagen and elastin synthesis in the balloon injured rat carotid artery.

INTRODUCTION: Balloon catheter denudation of the endothelium in the common carotid artery of the rat is routinely used as a model of neointimal lesion development. The response to endothelial denuding injury involves proliferation and migration of medial smooth muscle cells (SMCs), with the formation of a dramatically thickened neointima. While many studies have focused on the kinetics of the early proliferative and migration responses, much less attention has been paid to the pronounced accumulation of extracellular matrix that occurs as the neointima grows. The purpose of this study was to measure collagen and elastin content, and to assess collagen and elastin synthesis in injured rat carotid arteries. METHODS: Male Sprague-Dawley rats were subject to balloon catheter injury of the left carotid artery using a 2F embolectomy catheter. Rats were sacrificed at different time points after injury, and both carotids left (injured) and right (uninjured and control) were used for measurement of elastin and collagen synthesis (7 days) and content (7, 21 and 60 days). RESULTS: Elastin and collagen syntheses were significantly increased in the injured carotids at 7 days after injury. The increase in elastin synthesis was more dramatic (100% compared to control) than the increase in collagen synthesis (50% compared to control). Both elastin and collagen content were significantly increased by 21 days, and contents were further increased by 60 days in the injured carotid compared to uninjured controls. At 60 days, collagen content of the injured vessels was 2.09+/-0.01 mg/arterial segment compared to 1.32+/-0.08 mg/arterial segment in controls (P=.01). Elastin content of injured vessels was 2.40+/-0.43 mg/arterial segment compared to 1.19+/-0.25 mg/arterial segment in controls (P=.03). CONCLUSIONS: Collagen and elastin contents were significantly increased following injury of the rat carotid. This study provides for the first time a biochemical assessment of collagen and elastin in the balloon-injured rat carotid artery.

Poly(methylidene malonate 2.1.2) nanoparticles: a biocompatible polymer that enhances peri-adventitial adenoviral gene delivery.

Vascular gene therapy is currently limited by low and transient levels of gene transfection. The objectives of this study were to determine whether peri-adventitial delivery of adenovirus coupled to nanoparticles could improve transfection efficiency and duration. Adenovirus was absorbed to the surface of nanoparticles that were made from poly(methylidene malonate)2.1.2 (PMM2.1.2). These complexes were found to have good adhesive properties to both cultured vascular smooth muscle cells and to the luminal and adventitial layers of excised rabbit carotid arteries. Adenovirus encoding to beta-galactosidase coupled to PMM2.1.2 nanoparticles or adenovirus alone were delivered locally to the adventitia of rabbit carotid arteries. Transfection rate was assessed histologically by the percentage of beta-galactosidase positive cells in the vessel wall at 1 and at 2 weeks. There was significantly higher transfection rate when adenovirus was complexed with nanoparticles as compared to free adenovirus (At 1 week: 10+/-3.9% beta-gal positive cells vs. 2.4+/-0.3% and at 2 weeks: 4.3+/-4.1% vs. 0%, P<0.005 for all). This difference was present in both the medial and adventitial layers. In conclusion, adenoviral mediated gene therapy was significantly enhanced by adsorbing the virus to PMM2.1.2 nanoparticles. This delivery method may be a good therapeutic strategy for the treatment of various vascular diseases.

Unexpected calcification after direct intravascular injection of autologous bone marrow-derived cells in a chronic total occlusion model.

AIMS: Patients with symptomatic chronic total occlusions (CTO) remain a therapeutic challenge. Enhancement of intraluminal neovascularisation by pro-angiogenic therapies has been proposed as a new strategy to improve percutaneous revascularisation. The aim of this study was to investigate the effects of intraluminal injection of bone marrow-derived cells (BMC) into experimental CTO. METHODS AND RESULTS: CTO were created in the femoral arteries of 43 New Zealand White rabbits using the thrombin injection model. At 12 weeks following CTO creation, 33 rabbits were injected with either cultured BMC (n=19) or control DMEM alone (n=14) directly into the CTO. Ten rabbits were used for cell tracking (seven BMC and three control). BMC labelled with fluorescent Qdot® nanocrystals were identified in the CTO up to one week after injection. Animals were sacrificed at three to five weeks post-treatment and arterial samples were excised for micro-CT imaging and histologic morphometric analysis. There was a significant but modest increase in neovascularisation in BMC-treated arteries compared to controls (7.47±4.75% vs. 4.35±2.97%, p<0.05). However, unexpected intravascular calcification was only detected within the CTO in BMC cell treated arteries. Western blot for conditioned medium from BMC showed up-regulation of osteogenic proteins (BMP-2 and -7). CONCLUSIONS: Although direct delivery of BMC into CTO increases neovascularisation, undesirable vascular calcification will limit this therapeutic approach.

Statin therapy prevents expansive remodeling in venous bypass grafts.

BACKGROUND: Venous grafts (VG) have high failure rates by 10 years in aortocoronary bypass surgery. We have previously shown that expansive remodeling followed by increased LDL retention are early atherosclerotic changes in experimental VG placed in the arterial circulation. The objective of this study was to determine whether statin therapy prevents these expansive remodeling changes. METHODS AND RESULTS: Reversed jugular vein-to-common carotid artery interposition graft was constructed in 27 cholesterol-fed (0.5%) rabbits. Rabbits were randomized either to control or atorvastatin (5 mg/kg/day) groups, starting two weeks prior to vein graft implantation and continuing until sacrifice at 1 or 12 weeks post-surgery. Ultrasound measurements of arterial luminal cross-sectional area (CSA) were done at day 3 and at 4, 8 and 12 weeks post-surgery. Histomorphometric measurements were performed following sacrifice at 12 weeks. Atorvastatin treatment significantly decreased total plasma cholesterol levels at 4, 8 and 12 weeks (12 weeks: 6.7 ± 4.2 mmol/L versus control 38.7 ± 10.6 mmol/L, p<0.0002). Atorvastatin significantly reduced expansive remodeling at 4, 8 and 12 weeks (lumen CSA: 44.6 ± 6.6 mm(2) versus control 77.6 ± 10.7 mm(2), p<0.0001). Intimal CSA by histomorphometry was also significantly reduced by atorvastatin at 12 weeks (5.59 ± 2.19 mm(2) versus control 9.57 ± 2.43 mm(2), p<0.01). VG macrophage infiltration, MMP-2 activity and metalloelastase activity were reduced in the atorvastatin treated group. CONCLUSION: Atorvastatin inhibits both expansive remodeling and intimal hyperplasia in arterialized VG, likely through inhibition of macrophage infiltration and reduction of tissue proteolytic activity. The mechanism proposed above may be important for preventing VG atherosclerosis and late VG failure.

A perlecan-inducing compound significantly inhibits smooth muscle cell function and in-stent intimal hyperplasia: novel insights into the diverse biological effects of perlecan.

AIMS: Perlecan is the major heparan sulfate proteoglycan in the arterial wall. Previous studies have suggested that perlecan is a potent inhibitor of smooth muscle cell (SMC) activity. Therefore, perlecan overexpression may serve as a therapeutic modality to prevent in-stent restenosis (ISR). We have investigated a novel compound (RUS3108), identified in a SMC based screen to induce perlecan synthesis in SMC. The aims of this study were to assess the in vitro effects of RUS3108 and the effects of RUS3108-eluting stents in preventing ISR. METHODS AND RESULTS: Rabbit aortic SMC and bovine aortic endothelial cells (EC) were used in this study. Immunohistochemistry showed that RUS3108-treated SMC over-expressed perlecan indicating the drug effects. Furthermore, RUS3108 induced a SMC differentiated phenotype by SMembryonic staining. RUS3108 (1 microM) inhibited 3H-thymidine incorporation by >50%, which was completely reversed by a perlecan antibody. RUS3108 also inhibited SMC migration (Boyden chamber) and MMP-9 activity. In contrast, RUS3108 (100nM) modestly stimulated EC 3H-thymidine incorporation by 22% (p<0.02). In vivo, a total of 30 stents were deployed in rabbit iliac arteries as follows: 1) bare metal stents (n=10), 2) polymer onlycoated stents (n=10), and 3) polymer-coated stents containing RUS3108 (n=10). Rabbits were sacrificed at four weeks and stented segments were subjected to morphometric analysis. Intimal cross sectional area was significantly lower in the RUS3108-eluting stent group (0.31+ or -0.27 mm(2) versus 1.0 + or - 0.31 and 1.25 + or - 0.51 in the bare metal stents and polymer only coated stents groups, respectively, p<0.0001). CONCLUSIONS: RUS3108 is a novel perlecan-inducing compound, which is a potent inhibitor of SMC activity and a modest stimulator of EC proliferation. RUS3108-eluting stents may serve as an excellent modality for the prevention of ISR.

Expansive remodeling in venous bypass grafts: novel implications for vein graft disease.

OBJECTIVE: To date, intimal hyperplasia has been regarded as the principle mechanism responsible for subsequent vein graft disease. Lumen remodeling has not been previously considered as an additional mechanism. The objectives of this study were to determine changes in lumen remodeling in arterialized vein grafts, the accompanying cellular and extracellular matrix events contributing to remodeling, and the effects of a high cholesterol diet. METHODS AND RESULTS: Reversed jugular vein-to-common carotid artery interposition grafts were constructed in 70 normocholesterolemic and 11 hypercholesterolemic male New Zealand white rabbits. The lumen area initially remained unchanged between 1 and 4 weeks but significantly increased by 40% at 12 weeks. This phase of expansive positive remodeling was accompanied by significantly increased cell apoptosis, collagen synthesis (1.7-fold), collagen content (3.7-fold), gelatinase (MMP-2 and MMP-9) levels and decreased tissue inhibitor of metalloproteinase (TIMP) levels. Expansive remodeling temporally corresponded to high macrophage infiltration and increased low density lipoprotein (LDL) retention (fourfold) in the vein grafts. A high cholesterol diet stimulated early macrophage infiltration and increased MMP-12 (metalloelastase) levels, which was associated with earlier onset of expansive remodeling. CONCLUSION: Expansive lumenal remodeling is a novel mechanism of vein graft response to the arterial circulation, which is accelerated by a high cholesterol diet.

The cell cycle: a critical therapeutic target to prevent vascular proliferative disease.

Percutaneous coronary intervention is the preferred revascularization approach for most patients with coronary artery disease. However, this strategy is limited by renarrowing of the vessel by neointimal hyperplasia within the stent lumen (in-stent restenosis). Vascular smooth muscle cell proliferation is a major component in this healing process. This process is mediated by multiple cytokines and growth factors, which share a common pathway in inducing cell proliferation: the cell cycle. The cell cycle is highly regulated by numerous mechanisms ensuring orderly and coordinated cell division. The present review discusses current concepts related to regulation of the cell cycle and new therapeutic options that target aspects of the cell cycle.

Adventitial microvessel formation after coronary stenting and the effects of SU11218, a tyrosine kinase inhibitor.

OBJECTIVES: The aim of this study was to delineate the temporal profile of adventitial microvessel (Ad-MV) formation after stenting, its relationship to arterial wall hypoxia, and the effects of a tyrosine kinase inhibitor (TKI), SU11218, on Ad-MV and in-stent intimal hyperplasia (IH). BACKGROUND: Adventitial microvessels have been reported after arterial injury; however, the underlying stimulus for this response and its relationship to IH is unknown. METHODS: Coronary stenting was performed in 40 pigs randomized to SU11218 (n = 20) or placebo (n = 20). Vessel wall hypoxia was assessed by pimonidazole adducts and hypoxia-inducible factor (HIF)-1 alpha expression. Adventitial microvessels were quantified by three-dimensional microscopic computed tomography (3D micro CT). Intimal hyperplasia was measured by intravascular ultrasound (IVUS), 3D micro CT, and morphometry. The effects of SU11218 were assessed in vitro on smooth muscle cell (SMC) and endothelial cell (EC) functions and in vivo on Ad-MV and IH. RESULTS: Hypoxia was evident in the vessel wall at 48 h and persisted for four weeks. Adventitial microvessels increased significantly at one week (24 +/- 7 microvessels/segment) and four weeks (23 +/- 7 microvessels/segment) compared with uninjured arteries (16 +/- 2 microvessels/segment; p < 0.001) and correlated with IH (r = 0.77, p < 0.001). The TKI SU11218 inhibited platelet-derived growth factor receptor-beta phosphorylation, EC and SMC DNA synthesis, and migration in a dose-dependent manner in vitro and significantly inhibited Ad-MV (16 +/- 5 vs. 23 +/- 7 microvessels/segment in placebo, p < 0.001) and produced approximately 80% reduction in IH (0.52 +/- 0.51 mm2 vs. 2.47 +/- 1.66 mm2 in placebo, p < 0.001) at four weeks in vivo. CONCLUSIONS: Arterial stenting causes arterial wall hypoxia followed by Ad-MV formation. The TKI SU11218 inhibits both Ad-MV formation and IH and represents a promising therapeutic agent to prevent in-stent restenosis.

Cell transplantation preserves matrix homeostasis: a novel paracrine mechanism.

OBJECTIVES: Cell transplantation prevents chamber dilatation, but the underlying molecular mechanisms remain undefined. Structural cardiac remodeling involves matrix degradation from an imbalance of matrix metalloproteinases (MMP) relative to endogenous tissue inhibitors of metalloproteinases (TIMP). We aimed to determine the capacity of cell transplantation to alter extracellular matrix in the failing heart and, in so doing, identify novel paracrine molecular mediators underlying the beneficial effects of cell transplantation on chamber dilatation. METHODS: Smooth muscle cells were transplanted to the dilating left ventricle of cardiomyopathic hamsters (CTX, n = 15) compared with age-matched media-injected cardiomyopathic (CON, n = 15) and normal hamsters (n = 7). After 5 weeks, left ventricular volume was measured by computerized planimetry. Fibrillar collagen was examined by confocal microscopy. Matrix homeostasis was quantified by measuring MMP/TIMP expression/activity relative to myocardial collagen synthesis (14C-proline uptake). RESULTS: Left ventricular dilatation was attenuated in CTX hearts (P = .02). CTX restored perimysial collagen fiber content and architecture to normal levels. TIMP-2 and TIMP-3 expression were enhanced in CTX (TIMP-2, 195% +/- 42% of CON, P = .02; TIMP-3, 118% +/- 3% of CON, P = .002), and correspondingly, gelatinase MMP-2 activity was reduced (P < .05). The TIMP:MMP ratio was increased in CTX hearts (TIMP-2 to MMP-2, 410% +/- 134% of CON, P = .04, and TIMP-3 to MMP-9, 205% +/- 47% of CON, P = .03), reflecting a reduced capacity for matrix degradation. Collagen synthesis was equivalent (CTX vs CON), suggesting that restored matrix architecture was a function of attenuated matrix degradation. CONCLUSIONS: These data provide the first evidence that cell transplantation limits ventricular dilatation in the failing heart through a paracrine-mediated mechanism that preserves extracellular matrix homeostasis.