Caveolin-1-deficient aortic smooth muscle cells show cell autonomous abnormalities in proliferation, migration, and endothelin-based signal transduction

We previously showed that ablation of caveolin-1 (Cav-1) gene expression in mice promotes neointimal hyperplasia in vivo, a phenomenon normally characterized by smooth muscle cell (SMC) migration and proliferation. Whether these defects are cell autonomous, i.e., due to loss of Cav-1 within SMCs or loss of Cav-1 expression in other adjacent cell types in vivo, remains unknown. Cav-1 has been shown to associate with receptors for many vasoactive factors on the SMC surface. Therefore, Cav-1 might be an important regulator of SMC proliferation, migration, and signal transduction. To mechanistically dissect the role of Cav-1 in SMC signaling, we isolated SMCs from the aortas (AoSMCs) of Cav-1-deficient (Cav-1(-/-)) mice and characterized these cells with respect to their proliferation, migration, and Ca(2+) response to an important vasoactive factor, endothelin-1 (ET-1). 5-Bromo-2'-deoxyuridine incorporation and a wound-healing assay showed an increase in proliferation and migration rates in Cav-1(-/-) compared with wild-type (Cav-1(+/+)) AoSMCs. Cav-1(-/-) AoSMCs demonstrated upregulation of phosphorylated ERK1/2, cyclin D1, and proliferating cell nuclear antigen and reduced expression of the cyclin-dependent kinase inhibitor p27(Kip1). The Ca(2+) response was examined in the presence of ET-1 and assessed by confocal microscopy with the Ca(2+)-sensitive fluorescent probe fluo 3. When treated with ET-1, Cav-1(-/-) AoSMCs exhibited a faster and larger increase in free intracellular Ca(2+) than Cav-1(+/+) cells. The ET-1-induced response in Cav-1(-/-) cells was mediated by the ET(B) receptor, as shown using the ET(B) receptor antagonist BQ-788 and the ET(A) receptor antagonist BQ-123. In Cav-1(-/-) cells, ET(A) receptor expression was reduced and ET(B) receptor expression was upregulated. Therefore, Cav-1 ablation increased the ET-1-induced Ca(2+) response in SMCs by altering the type and expression level of the ET receptor (i.e., receptor isoform switching). These data suggest a novel regulatory role for Cav-1 in SMCs with respect to their proliferation, migration, and Ca(2+)-mediated signaling.     

Vanin-1 pantetheinase drives smooth muscle cell activation in post-arterial injury neointimal hyperplasia

The pantetheinase vanin-1 generates cysteamine, which inhibits reduced glutathione (GSH) synthesis. Vanin-1 promotes inflammation and tissue injury partly by inducing oxidative stress, and partly by peroxisome proliferator-activated receptor gamma (PPARγ) expression. Vascular smooth muscle cells (SMCs) contribute to neointimal hyperplasia in response to injury, by multiple mechanisms including modulation of oxidative stress and PPARγ. Therefore, we tested the hypothesis that vanin-1 drives SMC activation and neointimal hyperplasia. We studied reactive oxygen species (ROS) generation and functional responses to platelet-derived growth factor (PDGF) and the pro-oxidant diamide in cultured mouse aortic SMCs, and also assessed neointima formation after carotid artery ligation in vanin-1 deficiency. Vnn1(-/-) SMCs demonstrated decreased oxidative stress, proliferation, migration, and matrix metalloproteinase 9 (MMP-9) activity in response to PDGF and/or diamide, with the effects on proliferation linked, in these studies, to both increased GSH levels and PPARγ expression. Vnn1(-/-) mice displayed markedly decreased neointima formation in response to carotid artery ligation, including decreased intima:media ratio and cross-sectional area of the neointima. We conclude that vanin-1, via dual modulation of GSH and PPARγ, critically regulates the activation of cultured SMCs and development of neointimal hyperplasia in response to carotid artery ligation. Vanin-1 is a novel potential therapeutic target for neointimal hyperplasia following revascularization.     

Xenobiotic pregnane X receptor promotes neointimal formation in balloon-injured rat carotid arteries

Pregnane X receptor (PXR) is a member of nuclear receptor superfamily and responsible for the detoxification of xenobiotics. Recent studies demonstrated that PXR was also expressed in the vasculature and protected the vessels from endogenous and exogenous insults, thus representing a novel gatekeeper in vascular defense. In this study, we examined the potential function of PXR in the neointimal formation following vascular injury. In the rat carotid artery after balloon injury, overexpression of a constitutively active PXR increased the intima-to-media ratio in the injured region. PXR increased cell proliferation and migration in cultured rat aortic smooth muscle cells (SMCs) by inducing the expressions of cyclins (cyclin A, D1, and E) and cyclin-dependent kinase 2. In addition, PXR increased the phosphorylation and activation of extracellular-signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK). Inactivation of ERK1/2 and p38 MAPK pathways using selective inhibitors (U0126 and SB203580) abrogated PXR-induced SMC proliferation and migration. Furthermore, cigarette smoke particles (CSP) activated PXR in SMCs. Knockdown of PXR by small interfering RNA suppressed the cell proliferation, migration, and activation of the MAPK pathways by CSP. These findings suggested a novel role for PXR in promoting SMC proliferation and migration, and neointimal hyperplasia. Therefore, PXR may be a potential therapeutic target for vascular disease related to xenobiotics such as cigarette smoking and other environmental pollutants.     

Increase of smooth muscle cell migration and of intimal hyperplasia in mice lacking the alpha/beta hydrolase domain containing 2 gene

Multiple steps, including the migration of vascular smooth muscle cells (SMCs), are involved in the pathogenesis of atherosclerosis. To discover genes which are involved in these steps, we screened mutant mouse lines established by the exchangeable gene trap method utilizing X-gal staining during their embryonic development. One of these lines showed strong reporter gene expression in the vitelline vessels of yolk sacs at embryonic day (E) 12.5. The trap vector was inserted into the fifth intron of alpha/beta hydrolase domain containing 2 (Abhd2) gene which was shown to be expressed in vascular and non-vascular SMCs of adult mice. Although homozygous mutant mice were apparently normal, enhanced SMC migration in the explants SMCs culture and marked intimal hyperplasia after cuff placement were observed in homozygous mice in comparison with wild-type mice. Our results show that Abhd2 is involved in SMC migration and neointimal thickening on vascular SMCs.     

MCP-1 deficiency is associated with reduced intimal hyperplasia after arterial injury

Monocyte chemoattractant protein (MCP)-1 is abundant in smooth muscle cells (SMC) and macrophages of atherosclerotic plaques and in the injured arterial wall. MCP-1 and its receptor, CCR2, are important mediators of macrophage accumulation and atherosclerotic plaque progression. We have recently reported that CCR2(-/-) mice have a approximately 60% decrease in intimal hyperplasia and medial DNA synthesis in response to femoral arterial injury. We have now examined the response to femoral arterial injury in MCP-1(-/-) mice. MCP-1 deficiency was associated with a approximately 30% reduction in intimal hyperplasia at 4 weeks and was not associated with diminished medial DNA synthesis. Despite inducing tissue factor in SMC culture, MCP-1 deficiency was not associated with a decrease in neointimal tissue factor after injury. These data suggest that MCP-1 and CCR2 deficiencies have distinct effects on arterial injury. The effects of MCP-1 on intimal hyperplasia may be mediated largely through SMC migration.     

Combined loss of INK4a and caveolin-1 synergistically enhances cell proliferation and oncogene-induced tumorigenesis: role of INK4a/CAV-1 in mammary epithelial cell hyperplasia

Tumorigenesis is a multistep process that involves a series of genetic changes or "multiple hits," leading to alterations in signaling, proliferation, immortalization, and transformation. Many of the molecular factors that govern tumor initiation and progression remain unknown. Here, we evaluate the transformation suppressor potential of caveolin-1 (Cav-1) and its ability to cooperate with a well established tumor suppressor, the INK4a locus. To study the effects of loss of caveolin-1 on cellular transformation, we established immortalized primary mouse embryonic fibroblasts (MEFs) expressing and lacking caveolin-1 by interbreeding Cav-1 (+/+) and Cav-1 (-/-) mice with INK4a (-/-) mice. Analysis of these cells reveals that loss of caveolin-1 confers a significant growth advantage, as measured via cellular proliferation and cell cycle analysis. Loss of caveolin-1 in the INK4a (-/-) genetic background results in constitutive hyperactivation of the p42/44 MAP kinase cascade, decreased expression of p21(Cip1), as well as cyclin D1 and PCNA overexpression, consistent with their hyperproliferative phenotype. Importantly, in cells lacking Cav-1 expression, transformation by activated oncogenes (H-Ras(G12V) or v-Src) results in increased tumor growth in vivo (up to >40-fold). Finally, INK4a (-/-)/Cav-1 (-/-) mice demonstrate disturbed mammary epithelial ductal morphology, with hyperplasia, increased side-branching, and fibrosis. Our results provide important new evidence for the transformation suppressor properties of Cav-1 and the first molecular genetic evidence that Cav-1 cooperates with a tumor suppressor, namely the INK4a genetic locus.     

Carotid artery ligation induced intimal thickening and proliferation is unaffected by ageing

Following interventions to treat atherosclerosis, such as coronary artery bypass graft surgery, restenosis occurs in approximately 40% of patients. Identification of proteins regulating intimal thickening could represent targets to prevent restenosis. Our group previously demonstrated that in a murine model of vascular occlusion, Wnt4 protein expression and β-catenin signalling was upregulated which promoted vascular smooth muscle cell (VSMC) proliferation and intimal thickening. In this study, the effect of age on VSMC proliferation, intimal hyperplasia and Wnt4 expression was investigated. In vitro proliferation of VSMCs isolated from young (2 month) or old (18–20 month) C57BL6/J mice was assessed by immunocytochemistry for EdU incorporation. As previously reported, 400 ng/mL recombinant Wnt4 protein increased proliferation of VSMCs from young mice. However, this response was absent in VSMCs from old mice. As our group previously reported reduced intimal hyperplasia in Wnt4^+/? mice compared to wildtype controls, we hypothesised that impaired Wnt4 signalling with age may result in reduced neointimal formation. To investigate this, carotid artery ligation was performed in young and old mice and neointimal area was assessed 21 days later. Surprisingly, neointimal area and percentage lumen occlusion were not significantly affected by age. Furthermore, neointimal cell density and proliferation were also unchanged. These data suggest that although Wnt4-mediated proliferation was impaired with age in primary VSMCs, carotid artery ligation induced neointimal formation and proliferation were unchanged in old mice. These results imply that Wnt4-mediated proliferation is unaffected by age in vivo, suggesting that therapeutic Wnt4 inhibition could inhibit restenosis in patients of all ages.     

Proliferation of neointimal smooth muscle cells after arterial injury. Dependence on interactions between fibroblast growth factor receptor-2 and fibroblast growth factor-9

The growth factor signaling mechanisms responsible for neointimal smooth muscle cell (SMC) proliferation and accumulation, a characteristic feature of many vascular pathologies that can lead to restenosis after angioplasty, remain to be identified. Here, we examined the contribution of fibroblast growth factor receptors (FGFRs) 2 and 3 as well as novel fibroblast growth factors (FGFs) to such proliferation. Balloon catheter injury to the rat carotid artery stimulated the expression of two distinctly spliced FGFR-2 isoforms, differing only by the presence or absence of the acidic box, and two distinctly spliced FGFR-3 isoforms containing the acidic box and differing only by the presence of either the IIIb or IIIc exon. Post-injury arterial administration of recombinant adenoviruses expressing dominant negative mutant forms of these FGFRs were used to assess the roles of the endogenous FGFR isoforms in neointimal SMC proliferation. Dominant negative FGFR-2 containing the acidic box inhibited such proliferation by 40%, whereas the dominant negative FGFR-3 forms had little effect. Expression of FGF-9, known to be capable of binding to all four neointimal FGFR-2/-3 isoforms, was abundant within the neointima. FGF-9 markedly stimulated both the proliferation of neointimal SMCs and the activation of extracellular signal-related kinases 1/2, effects which were abrogated by the administration of antisense FGF-9 oligonucleotides to injured arteries and the expression of the dominant negative FGFR-2 adenovirus in cultured neointimal SMCs. These studies demonstrate that, although multiple FGFRs are induced in neointimal SMCs following arterial injury, specific interactions between distinctly spliced FGFR-2 isoforms and FGF-9 contribute to the proliferation of these SMCs.     

Host bone-marrow cells are a source of donor intimal smooth- muscle-like cells in murine aortic transplant arteriopathy

Long-term solid-organ allografts typically develop diffuse arterial intimal lesions (graft arterial disease; GAD), consisting of smooth-muscle cells (SMC), extracellular matrix and admixed mononuclear leukocytes. GAD eventually culminates in vascular stenosis and ischemic graft failure. Although the exact mechanisms are unknown, chronic low-level alloresponses likely induce inflammatory cells and/or dysfunctional vascular wall cells to secrete growth factors that promote SMC intimal recruitment, proliferation and matrix synthesis. Although prior work demonstrated that the endothelium and medial SMCs lining GAD lesions in cardiac allografts are donor-derived, the intimal SMC origin could not be determined. They are generally presumed to originate from the donor media, leading to interventions that target donor medial SMC proliferation, with limited efficacy. However, other reports indicate that allograft vessels may contain host-derived endothelium and SMCs (refs. 8,9). Moreover, subpopulations of bone-marrow and circulating cells can differentiate into endothelium, and implanted synthetic vascular grafts are seeded by host SMCs and endothelium. Here we used murine aortic transplants to formally identify the source of SMCs in GAD lesions. Allografts in beta-galactosidase transgenic recipients showed that intimal SMCs derived almost exclusively from host cells. Bone-marrow transplantation of beta-galactosidase--expressing cells into aortic allograft recipients demonstrated that intimal cells included those of marrow origin. Thus, smooth-muscle--like cells in GAD lesions can originate from circulating bone--marrow-derived precursors.     

The kinase inhibitor fasudil (HA-1077) reduces intimal hyperplasia through inhibiting migration and enhancing cell loss of vascular smooth muscle cells.

Smooth muscle cell (SMC) migration plays an important role in restenosis after angioplasty. Myosin phosphorylation is necessary for cell migration. Fasudil is an inhibitor of protein kinases, including myosin light chain kinase and Rho associated kinase, thereby inhibiting myosin phosphorylation, and it has been clinically used to prevent vasospasm following subarachnoid hemorrage. Based on these findings, we examined the anti-migrative action of fasudil. In SMC (SM-3), fasudil (1-100 microM) inhibited SMC migration in a dose-dependent manner (p < 0.001). Fasudil suppressed actin stress fiber formation dose dependently. In rabbit carotid artery, fasudil (10 mg/kg/day) markedly reduced intimal hyperplasia 14 days following balloon injury. Cell kinetic study showed that fasudil did not affect proliferation but enhanced cell loss in the media after injury. We concluded that fasudil reduced neointimal formation after balloon injury through both inhibiting migration and enhancing cell loss of medial SMC.     

IL-10 inhibits vascular smooth muscle cell activation in vitro and in vivo

The anti-inflammatory cytokine IL-10 inhibits intimal hyperplasia after stent implantation via a powerful inactivation of monocytes. We tested the hypothesis that IL-10 may also inhibit vascular smooth muscle cell (SMC) activation via the inhibition of the NF-kappaB/I-kappaB system. The IL-10 receptor was detected in rat SMCs in vitro and in vivo. In LPS-stimulated rat SMCs, 1 ng/ml recombinant murine IL-10 (mIL-10) reduced I-kappaBalpha and I-kappaBbeta degradation, NF-kappaB activation, as well as the expression of the NF-kappaB-dependent gene IL-6 by 32%, 31%, 75%, and 19%, respectively (P < 0.05 for all). Similar results were obtained in vivo 6 h and 4 days after balloon abrasion of the rat aorta, a model in which intimal hyperplasia results essentially from SMC activation. Moreover, mIL-10 reduced SMC proliferation and migration in vitro (by 60% for both, P < 0.0001), resulting in reduced SMC proliferation and intimal growth 14 days after balloon abrasion of the rat aorta (by 76% and 75%, respectively; P < 0.005). In conclusion, mIL-10 has a direct inhibitory effect on SMCs in vitro and in vivo. This effect is mediated in part by NF-kappaB inactivation and may participate in the overall protective effect of IL-10 on postangioplasty restenosis.     

Noncanonical Matrix Metalloprotease-1-Protease-activated Receptor-1 Signaling Triggers Vascular Smooth Muscle Cell Dedifferentiation and Arterial Stenosis

Vascular injury that results in proliferation and dedifferentiation of vascular smooth muscle cells (SMCs) is an important contributor to restenosis following percutaneous coronary interventions or plaque rupture. Protease-activated receptor-1 (PAR1) has been shown to play a role in vascular repair processes; however, little is known regarding its function or the relative roles of the upstream proteases thrombin and matrix metalloprotease-1 (MMP-1) in triggering PAR1-mediated arterial restenosis. The goal of this study was to determine whether noncanonical MMP-1 signaling through PAR1 would contribute to aberrant vascular repair processes in models of arterial injury. A mouse carotid arterial wire injury model was used for studies of neointima hyperplasia and arterial stenosis. The mice were treated post-injury for 21 days with a small molecule inhibitor of MMP-1 or a direct thrombin inhibitor and compared with vehicle control. Intimal and medial hyperplasia was significantly inhibited by 2.8-fold after daily treatment with the small molecule MMP-1 inhibitor, an effect that was lost in PAR1-deficient mice. Conversely, chronic inhibition of thrombin showed no benefit in suppressing the development of arterial stenosis. Thrombin-PAR1 signaling resulted in a supercontractile, differentiated phenotype in SMCs. Noncanonical MMP-1-PAR1 signaling resulted in the opposite effect and led to a dedifferentiated phenotype via a different G protein pathway. MMP-1-PAR1 significantly stimulated hyperplasia and migration of SMCs, and resulted in down-regulation of SMC contractile genes. These studies provide a new mechanism for the development of vascular intimal hyperplasia and suggest a novel therapeutic strategy to suppress restenosis by targeting noncanonical MMP-1-PAR1 signaling in vascular SMCs.     

Expression of adhesion molecule T-cadherin is increased during neointima formation in experimental restenosis

Phenotypic modulation, migration and proliferation of vascular smooth muscle cells (SMCs) are major events in restenosis after percutaneous transluminal angioplasty. Surface cell adhesion molecules, essential to morphogenesis and maintenance of adult tissue architecture, are likely to be involved, but little is known about cell adhesion molecules expressed on SMCs. T-cadherin is a glycosyl phosphatidylinositol-anchored member of the cadherin superfamily of adhesion molecules. Although highly expressed in vascular and cardiac tissues, its function in these tissues is unknown. We previously reported increased expression of T-cadherin in intimal SMCs in atherosclerotic lesions and proposed a role for T-cadherin in phenotype control. Here we performed immunohistochemical analysis of spatial and temporal changes in vascular T-cadherin expression following balloon catheterisation of the rat carotid artery. T-cadherin expression in SMCs markedly increases in the media early (1-4 days) after injury, and later (day 7-28) in forming neointima, especially in its preluminal area. Staining for monocyte/macrophage antigen ED-1, proliferating cell nuclear antigen and smooth muscle alpha-actin revealed that spatial and temporal changes in T-cadherin level coincided with the peak in cell migration and proliferation activity during neointima formation. In colchicine-treated cultures of rat aortic SMCs T-cadherin expression is increased in dividing M-phase cells but decreased in non-dividing cells. Together the data support an association between T-cadherin expression and SMC phenotype.     

Increased Expression of Phosphorylated Polo-Like Kinase 1 and Histone in Bypass Vein Graft and Coronary Arteries following Angioplasty

Interventional procedures, including percutaneous transluminal coronary angioplasty (PTCA) and coronary artery bypass surgery (CABG) to re-vascularize occluded coronary arteries, injure the vascular wall and cause endothelial denudation and medial vascular smooth muscle cell (VSMCs) metaplasia. Proliferation of the phenotypically altered SMCs is the key event in the pathogenesis of intimal hyperplasia (IH). Several kinases and phosphatases regulate cell cycle in SMC proliferation. It is our hypothesis that increased expression and activity of polo-like kinase-1 (PLK1) in SMCs, following PTCA and CABG, contributes to greater SMC proliferation in the injured than uninjured blood vessels. Using immunofluorescence (IF), we assessed the expression of PLK1 and phosphorylated-PLK1 (pPLK1) in post-PTCA coronary arteries, and superficial epigastric vein grafts (SEV) and compared it with those in the corresponding uninjured vessels. We also compared the expressions of mitotic marker phospho-histone, synthetic-SMC marker, contractile SMC marker, IFN-γ and phosphorylated STAT-3 in the post-PTCA arteries, SEV-grafts, and the uninjured vessels. Immunostaining demonstrated an increase in the number of cells expressing PLK1 and pPLK1 in the neointima of post PTCA-coronary arteries and SEV-grafts compared to their uninjured counterparts. VSMCs in the neointima showed an increased expression of phospho-histone, synthetic and contractile SMC markers, IFN-γ and phosphorylated STAT-3. However, VSMCs of uninjured coronaries and SEV had no significant expression of the aforementioned proteins. These data suggest that PLK1 might play a critical role in VSMC mitosis in hyperplastic intima of the injured vessels. Thus, novel therapies to inhibit PLK1 could be developed to inhibit the mitogenesis of VSMCs and control neointimal hyperplasia.     

Caveolin-1 influences vascular protease activity and is a potential stabilizing factor in human atherosclerotic disease

Caveolin-1 (Cav-1) is a regulatory protein of the arterial wall, but its role in human atherosclerosis remains unknown. We have studied the relationships between Cav-1 abundance, atherosclerotic plaque characteristics and clinical manisfestations of atherosclerotic disease.We determined Cav-1 expression by western blotting in atherosclerotic plaques harvested from 378 subjects that underwent carotid endarterectomy. Cav-1 levels were significantly lower in carotid plaques than non-atherosclerotic vascular specimens. Low Cav-1 expression was associated with features of plaque instability such as large lipid core, thrombus formation, macrophage infiltration, high IL-6, IL-8 levels and elevated MMP-9 activity. Clinically, a down-regulation of Cav-1 was observed in plaques obtained from men, patients with a history of myocardial infarction and restenotic lesions. Cav-1 levels above the median were associated with absence of new vascular events within 30 days after surgery [0% vs. 4%] and a trend towards lower incidence of new cardiovascular events during longer follow-up. Consistent with these clinical data, Cav-1 null mice revealed elevated intimal hyperplasia response following arterial injury that was significantly attenuated after MMP inhibition. Recombinant peptides mimicking Cav-1 scaffolding domain (Cavtratin) reduced gelatinase activity in cultured porcine arteries and impaired MMP-9 activity and COX-2 in LPS-challenged macrophages. Administration of Cavtratin strongly impaired flow-induced expansive remodeling in mice. This is the first study that identifies Cav-1 as a novel potential stabilizing factor in human atherosclerosis. Our findings support the hypothesis that local down-regulation of Cav-1 in atherosclerotic lesions contributes to plaque formation and/or instability accelerating the occurrence of adverse clinical outcomes. Therefore, given the large number of patients studied, we believe that Cav-1 may be considered as a novel target in the prevention of human atherosclerotic disease and the loss of Cav-1 may be a novel biomarker of vulnerable plaque with prognostic value.