Key Words: peroxisome proliferator-activated receptor Ⅲ atherosclerosis Ⅲ diabetes mellitus Ⅲ vascular biology T he peroxisome proliferator-activated receptor ␥ (PPAR␥) belongs to the nuclear receptor family of ligand-dependent transcription factors. 1 PPAR␥ plays an important role in glucose homeostasis and is pharmacologically targeted by the class of insulin-sensitizing drugs named thiazolidinediones or glitazones. 1,2 In addition to its critical metabolic function, glitazone-activated PPAR␥ exhibits potent anti-inflammatory and vascular protective effects by directly affecting gene expression in monocytes/macrophages, T lymphocytes, endothelial cells, and vascular smooth muscle cells (VSMCs). 3,4 PPAR␥-mediated gene regulation comprises several distinct mechanisms, including ligand-dependent transactivation, ligand-independent repression, and ligand-dependent transrepression. 4 For its anti-inflammatory actions in macrophages, inhibition of gene expression by ligand-dependent transrepression has been identified as a key molecular process. 5 However, the molecular mechanisms underlying Original received July 26, 2011; revision received December 14, 2011; accepted December 16, 2011. In November 2011, the average time from submission to first decision for all original research papers submitted to Circulation Research was 15 days.From the Center for Cardiovascular Research, Institute of Pharmacology, Campus Charité Mitte (M.B., V.B., I.N.B., L.H., H.W., K.K., T.U., A.F.-L., U.K.), and Department of Endocrinology, Diabetes, and Nutrition (J.S.), Charité Universitätsmedizin Berlin, Berlin, Germany; Department of Cardiology, Giessen University (A.P., D.S.), Giessen, Germany; Department of Clinical and Experimental Medicine G. Salvatore, University of Catanzaro Magna Graecia (F.P., A.B.), Catanzaro, Italy; German Heart Institute Berlin, Department of Cardiology (P.S.), Berlin, Germany; and Instituto di Endocrinologia ed Oncologia Sperimentale del CNR Gaetano Salvatore, Università di Napoli Federico II (A.F.), Napoli, Italy.* Matrix metalloproteinase-9 (MMP-9) and endothelin-1 (ET-1) are PPAR␥ target genes in vascular cells involved in the development of atherosclerosis and have been characterized as important mediators of the vascular protective actions of PPAR␥. 6 -8 PPAR␥ activation by glitazones results in marked inhibition of MMP-9 mRNA/protein expression and its gelatinolytic activity in VSMCs, which indicates MMP-9 as a potential candidate gene for ligand-dependent transrepression in these cells. 8 High-mobility group (HMG) proteins are chromatinbinding proteins that consist of the 3 family members HMGA, HMGB, and HMGN. 9 HMG proteins act as architectural elements that affect various DNA-dependent processes in the context of chromatin. 9 Via DNA-protein or protein-protein interactions, HMG proteins regulate gene transcription and influence multiple biological processes, including cell growth, proliferation, differentiation, and death. 9 The present study aimed to characterize the molecular process of ligand-de...

Objective-Regulator of G-protein signaling 5 (RGS5) is abundantly expressed in vascular smooth muscle cells (SMCs) and inhibits G-protein signaling by enhancing the guanosine triphosphate-hydrolyzing activity of Gα-subunits. In the present study, we investigated the effects of RGS5 on vascular SMC function in vitro and neointima formation after wire-induced injury in mice and determined the underlying mechanisms. Approach and Results-We found a robust expression of RGS5 in native arteries of C57BL/6 mice and a highly significant downregulation within neointimal lesions 10 and 21 days after vascular injury as assessed by quantitative polymerase chain reaction, immunoblotting, and immunohistochemistry. In vitro, RGS5 was found significantly downregulated after mitogenic stimulation of human coronary artery SMCs. To restore RGS5 levels, SMCs were transduced with adenoviral vectors encoding wild-type RGS5 or a nondegradable mutant. RGS5-WT and, even more prominently, the C2A-RGS5 mutant prevented SMC proliferation and migration. In contrast, the siRNA-mediated knockdown of RGS5 significantly augmented SMC proliferation. Following overexpression of RGS5, fluorescence-activated cell sorting analysis of propidium iodide-stained cells indicated cell cycle arrest in G0/G1 phase. Mechanistically, inhibition of the phosphorylation of the extracellular signal-regulated kinase 1/2 and mitogen-activated protein kinase downstream signaling was shown to be responsible for the anti-proliferative effect of RGS5. Following wire-induced injury of the femoral artery in C57BL/6 mice, adenoviral-mediated overexpression of RGS5-WT or C2A-RGS5 significantly reduced SMC proliferation and neointima formation in vivo. Conclusions-Downregulation of RGS5 is an important prerequisite for SMC proliferation in vitro and in vivo.Therefore, reconstitution of RGS5 levels represents a promising therapeutic option to prevent vascular remodeling processes.

No abstract

Introduction: Regulator of G-protein signaling 5 (RGS5) is abundantly and specifically expressed in vascular smooth muscle cells (SMCs) and inhibits G-protein signaling by enhancing the GTP-hydrolyzing activity of Gα-subunits. Hypothesis: In the present study, we aimed to investigate the effects of RGS5 on vascular SMC function in vitro and neointima formation in vivo and to determine the underlying molecular mechanisms. Methods: Expression of RGS5 was assessed in vitro and in vivo by qPCR, immunoblotting and immunohistochemistry. Proliferation and migration of human coronary artery SMC were assessed in vitro following the adenoviral-mediated over expression of wild type (WT) RGS5, of a non-degradable mutant (C2A-RGS5) or siRNA-mediated knock down. In vivo, the proliferation and neointima formation were assessed in a wire-mediated injury model of the femoral artery in C57BL/6J mice. Results: We found a robust expression of RGS5 in native arteries of C57BL/6 mice and a significant down-regulation within neointimal lesions 10 and 21 days after vascular injury as assessed by qPCR, immunohistochemistry and immunoblotting. In vitro, RGS5 was found significantly down-regulated after mitogenic stimulation of SMCs. Overexpression of RGS5 or, even more prominently, the C2A-RGS5 mutant prevented SMC proliferation and migration. In contrast, the siRNA-mediated knockdown of RGS5 significantly augmented SMC proliferation. Following overexpression of RGS5, FACS analysis of propidium iodide-stained cells indicated cell cycle arrest in G0/G1 phase. Mechanistically, inhibition of ERK1/2 phosphorylation and MAPK downstream signaling was found to be responsible for the anti-proliferative effect since over expression of a constitutive active form of MEK reversed the growth-inhibitory effect of of RGS5. Following wire-induced injury, adenoviral-mediated overexpression of RGS5 or C2A-RGS5 significantly decreased ERK phosphorylation, SMC proliferation and neointima formation in vivo (n=8; p<0.001). Conclusions: Down-regulation of RGS5 is an important prerequisite for SMC proliferation and neointima Formation. Therefore, reconstitution of RGS5 levels might represent a promising therapeutic option to prevent vascular remodeling processes.