The Ran GTPase-activating protein (RanGAP1) is critically involved in smooth muscle cell differentiation, proliferation and migration following vascular injury: implications for neointima formation and restenosis.

Differentiation and dedifferentiation, accompanied by proliferation play a pivotal role for the phenotypic development of vascular proliferative diseases (VPD), such as restenosis. Increasing evidence points to an essential role of regulated nucleoporin expression in the choice between differentiation and proliferation. However, whether components of the Ran GTPase cycle, which is of pivotal importance for both nucleocytoplasmic transport and for mitotic progression, are subject to similar regulation in VPD is currently unknown. Here, we show that differentiation of human coronary artery smooth muscle cell (CASMC) to a contractile phenotype by stepwise serum depletion leads to significant reduction of RanGAP1 protein levels. The inverse event, dedifferentiation of cells, was assessed in the rat carotid artery balloon injury model, a well-accepted model for neointima formation and restenosis. As revealed by temporospatial analysis of RanGAP1 expression, neointima formation in rat carotid arteries was associated with a significant upregulation of RanGAP1 expression at 3 and 7 days after balloon injury. Of note, neointimal cells located at the luminal surface revealed persistent RanGAP1 expression, as opposed to cells in deeper layers of the neointima where RanGAP1 expression was less or not detectable at all. To gain first evidence for a direct influence of RanGAP1 levels on differentiation, we reduced RanGAP1 in human coronary artery smooth muscle cells by siRNA. Indeed, downregulation of the essential RanGAP1 protein by 50% induced a differentiated, spindle-like smooth muscle cell phenotype, accompanied by an upregulation of the differentiation marker desmin. Reduction of RanGAP1 levels also resulted in a reduction of mitogen induced cellular migration and proliferation as well as a significant upregulation of the cyclin-dependent kinase inhibitor p27KIP1, without evidence for cellular necrosis. These findings suggest that RanGAP1 plays a critical role in smooth muscle cell differentiation, migration and proliferation in vitro and in vivo. Appropriate modulation of RanGAP1 expression may thus be a strategy to modulate VPD development such as restenosis.

Local statin therapy differentially interferes with smooth muscle and endothelial cell proliferation and reduces neointima on a drug-eluting stent platform.

OBJECTIVE: Therapeutic strategies to provide local inhibition of mitogen mediated proliferation and migration of human coronary artery smooth muscle cells (CASMC) by means of drug-eluting stents have been shown to enable effective limitation of neointimal hyperplasia. However, currently available drug-eluting stents utilize compounds that may also adversely affect endothelial regrowth, thus possibly precipitating subsequent cardiac events. Accordingly, identification of compounds that differentially inhibit smooth muscle and endothelial cell migration and proliferation could be of substantial clinical usefulness. METHODS AND RESULTS: In addition to lipid lowering, statins are known to display auxiliary pleiotropic activities. The purpose of this study was to evaluate the effect of local administration of cerivastatin on proliferation, migration and cytotoxicity of CASMC as well as coronary artery endothelial cells (CAEC) and to evaluate the effect of cerivastatin-coated stents on the inhibition of neointima formation as well as endothelial regrowth within the stented vessel. Cerivastatin displayed a differential effect on CASMC as compared to CAEC with regard to proliferation and migration; both were more profoundly inhibited in CASMC. Appreciable cytotoxicity and pro-apoptotic effects were low in both cell lines at therapeutic concentrations. Cerivastatin-elution led to significant inhibition of neointima formation in the rat carotid stent model, endothelial coverage of in-stent vascular tissue was similar with control and cerivastatin-eluting stents. CONCLUSIONS: As proof of principle, our study provides evidence that local application of a HMG-CoA reductase inhibitor on a drug-eluting stent platform can efficiently limit neointima formation. Consequently, these compounds warrant further clinical evaluation to confirm this finding. Our data further suggest that the anti-restenotic effect of local statin administration might be associated with a more protective interaction with the endothelium than that observed with compounds currently employed on drug-eluting stents.

Vascular remodeling in mice lacking the cytoplasmic domain of tissue factor.

Tissue factor (TF), the cell surface receptor for the serine protease FVIIa supports cell migration by interaction with the cytoskeleton. Intracellular signaling pathways dependent on the cytoplasmic domain of TF modify cell migration and may alter vascular remodeling. Vascular remodeling was analyzed in a femoral artery injury and a blood flow cessation model in mice with a targeted deletion of the 18 carboxy-terminal intracellular amino acids of TF (TF(Deltact/Deltact)) and compared with TF wild-type mice (TF(wt/wt)). Morphometric analysis revealed a decrease in the intima/media ratio after vascular injury in arteries from TF(Deltact/Deltact) compared with TF(wt/wt) mice (femoral artery injury: 2.4+/-0.3 TF(wt/wt) versus 0.6+/-0.3 TF(Deltact/Deltact), n=9 to 10, P=0.002; carotis ligation: 0.45+0.11 TF(wt/wt) versus 0.22+0.03 TF(Deltact/Deltact), n=12 to 14, P=0.09). This was caused by an increase in the media by 54% (P=0.04) in the femoral artery model and by 32% (P=0.03) after carotis ligation and was associated with an increased number of proliferating cells. Isolated aortic smooth muscle cells (SMCs) of TF(wt/wt) mice showed an increased migratory response toward the TF ligand active site-inhibited FVIIa that was abolished in TF(Deltact/Deltact) SMC. In contrast, the unstimulated proliferation rate was increased in TF(Deltact/Deltact) SMC compared with TF(wt/wt) SMCs. Thus, retention of SMCs attributable to a migratory defect and increased proliferation results in thickening of the media and in decrease in neointima formation after arterial injury. TF cytoplasmic domain signaling alters vascular remodeling and, thereby, may play a role in the development of restenosis, atherosclerotic disease, and neovascularization.

Targeting 2A protease by RNA interference attenuates coxsackieviral cytopathogenicity and promotes survival in highly susceptible mice.

BACKGROUND: Enteroviridae such as coxsackievirus B3 (CVB3) are important infectious agents involved in viral heart disease, hepatitis, and pancreatitis, but no specific antiviral therapy is available. METHODS AND RESULTS: The aim of the present study was to evaluate the impact of RNA interference on viral replication, cytopathogenicity, and survival. Small interfering RNA (siRNA) molecules were designed against the viral 2A region (siRNA-2A), which is considered to be highly conserved and essential for both virus maturation and host cytopathogenicity. siRNA-2A exhibited a significant protective effect on cell viability mediated by marked inhibition of CVB3 gene expression and viral replication. In highly susceptible type I interferon receptor-knockout mice, siRNA-2A led to significant reduction of viral tissue titers, attenuated tissue damage, and prolonged survival. Repeated siRNA-2A transfection was associated with a further improvement of survival. Various control siRNA molecules had no protective effect in vitro or in vivo. CONCLUSIONS: RNA interference directed against the 2A protease encoding genomic region effectively confers intracellular immunity toward CVB3-mediated cell injury and improves survival, suggesting a potential role for RNA interference for future treatment options targeting enteroviral diseases.

Inhibition of neointima formation by a novel drug-eluting stent system that allows for dose-adjustable, multiple, and on-site stent coating.

OBJECTIVE: The risk of in-stent restenosis can be considerably reduced by stents eluting cytostatic compounds. We created a novel drug-eluting stent system that includes several new features in the rapidly evolving field of stent-based drug delivery. METHODS AND RESULTS: The aim of the present study was the preclinical evaluation of a stent-coating system permitting individual, on-site coating of stents with a unique microporous surface allowing for individualizable, dose-adjustable, and multiple coatings with identical or various compounds, designated ISAR (individualizable drug-eluting stent system to abrogate restenosis). Stents were coated with 0.75% rapamycin solution, and high-performance liquid chromatography (HPLC)-based determination of drug release profile indicated drug release for >21 days. Rapamycin-eluting microporous (REMP) stents implanted in porcine coronary arteries were safe. To determine the efficacy of REMP stents, this novel drug-eluting stent platform was compared with the standard sirolimus-eluting stent. At 30 days, in-stent neointima formation in porcine coronary arteries was similar in both groups, yielding a significant decrease of neointimal area and injury-dependent neointimal thickness compared with bare-metal stents. CONCLUSIONS: The ISAR drug-eluting stent platform as a novel concept for stent coating allows for a safe, effective, on-site stent coating process, thus justifying further clinical evaluation to decrease in-stent restenosis in humans.

Local cyclin-dependent kinase inhibition by flavopiridol inhibits coronary artery smooth muscle cell proliferation and migration: Implications for the applicability on drug-eluting stents to prevent neointima formation following vascular injury.

In-stent restenosis is a hyperproliferative disease which can be successfully treated by drug-eluting stents releasing compounds that exhibit cell-cycle inhibitory properties to inhibit coronary smooth muscle cell (CASMC) proliferation and migration, resembling the key pathomechanisms of in-stent restenosis. Cyclin-dependent kinases (CDK) are key regulators of the eukaryotic cell cycle. CDK activity may be blocked by novel compounds such as flavopiridol. Therefore, CDK inhibitors are attractive drugs to be used for the local prevention of in-stent restenosis. In this study, we demonstrate that flavopiridol leads to potent inhibition of CASMC proliferation and migration. Molecular effects on cell-cycle regulatory mechanisms and distribution were evaluated by post-transcriptional assessment of distinct cyclins and cyclin-dependent kinase inhibitor (CKI) levels and flow cytometry. Cellular necrosis and apoptosis was assessed in CASMC and coronary endothelial cells. Flavopiridol induced a potent antiproliferative effect by cell-cycle inhibition in G1 and G2/M and led to increased protein levels of CKIs p21cip1 and p27kip1 as well as p53 in CASMC. Hyperphosphorylation of retinoblastoma protein was abrogated and mitogen-mediated smooth muscle cell migration significantly reduced. No accelerated cytotoxicity or increased apoptosis was detectable. Flavopiridol-coated stents, implanted in rat carotid arteries, led to significant decrease of neointima formation. As proof of principle, our results demonstrate that stents eluting CDK inhibitors such as flavopiridol effectively inhibit neointima formation. Therefore, this new class of therapeutics may be suitable for further clinical investigations on drug-eluting stents to prevent in-stent restenosis.