STAT4 deficiency protects against neointima formation following arterial injury in mice.

Signal transducer and activator of transcription 4 (STAT4) has been associated with susceptibility to autoimmune diseases. Intriguingly, we previously reported that STAT4 might play a critical role in vascular smooth muscle cell (VSMC) proliferation. The present study therefore investigated the impact of STAT4 on VSMC migration, apoptosis and neointimal hyperplasia postinjury, as well as the underlying mechanisms. Guide-wire injury was associated with development of intimal neointima, STAT4 and phosphorylated STAT4 (p-STAT4) expressions were apparently up-regulated in the injured arteries. Neointima was greatly blocked in STAT4 knockout (KO) mice compared with wild type (WT) mice. A marked loss of inflammatory cells was identified in the vasculature postinjury in STAT4 KO mice. VSMC apoptosis was enhanced in the vasculature postinjury in STAT4 KO mice compared with WT mice. Cultured primary STAT4 KO VSMCs displayed reduced migration in comparison with WT controls. Mechanically, the deletion of STAT4 potently decreased the level of MCP-1, and its downstream targets MMP1 and MMP2. The effect of STAT4 on VSMC apoptosis was mainly mediated by the activation of the mitochondrial apoptotic pathway, as manifested by increased cytochrome c release and the activation of caspase-3. STAT4 therefore represents a promising molecular target to limit restenosis after artery intervention.

Lv L ，Meng Q ，Ye M ，Wang P ，Xue G ... -  《-》

Zinc finger protein 191 deficiency attenuates vascular smooth muscle cell proliferation, migration, and intimal hyperplasia after endovascular arterial injury.

Restenosis engenders surgical vascular intervention failure. Zinc finger protein 191 (ZFP191) is a novel member of the SCAN domain family of Krüppel-like zinc finger transcription factors. Previous work reveals that ZFP191 is a pleiotropic factor that plays important roles in hematopoiesis, brain development, and tumor growth. Here, we sought to determine whether intimal hyperplasia was affected by the activity of ZFP191 and to investigate the molecular mechanisms that may underpin the process. Intimal hyperplasia was induced by guidewire injury in mouse femoral arteries. The arteries were harvested for morphometric assessment and determination of ZFP191 expression. Next, ZFP191 knockdown in cultured mouse aortic vascular smooth muscle cells (VSMCs) was achieved by lentiviral transduction of short-hairpin RNA. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, [(3)H]thymidine incorporation assay, scratch assay, and transwell migration assay were used to evaluate the effects of ZFP191 knockdown on VSMC growth and migration. In addition, β-catenin, c-myc, cyclin D1, matrix metalloproteinase (MMP) 9, MMP2, and MMP7 were measured by Western blotting in the absence of ZFP191 in vitro and in vivo. Zymography was used to evaluate MMP activity in cell culture-conditioned media. Lastly, artery injury was performed in wild-type (WT) and heterozygous ZFP191 knockout (KO) mice, and morphometric analysis of the arteries was determined. Guidewire injury was associated with development of intimal hyperplasia, and ZFP191 expression was enhanced by 51% in the injured arteries. Cultured primary VSMCs transfected with lentiviral shZFP191 displayed reduced proliferation and migration compared with controls. Mechanically, ZFP191 knockdown potently decreased the level of β-catenin and its downstream targets c-myc and cyclin D1. ZFP191 knockdown downregulated the expression of MMP9, MMP2, and MMP7, and zymography confirmed that ZFP191 knockdown reduced the activity of MMPs. Consistent with the in vitro data, elevated expression of β-catenin, c-myc, cyclin D1, MMP9, MMP2, and MMP7 accompanied upregulation of ZFP191 after injury in the femoral arteries of mice, and these levels were downregulated in ZFP191 KO vessels. Finally, intimal hyperplasia was greatly blocked in heterozygous ZFP191 KO mice compared with WT mice (intima/media ratio, 0.124 vs 0.412; P < .05). ZFP191 played an essential role in aggressive proliferation and migration of VSMCs, which in turn facilitated intimal hyperplasia. Our findings offer the first genetic evidence of ZFP191 as a potential therapeutic target to prevent restenosis.

Lv L ，Zhang J ，Wang P ，Meng Q ，Liang W ，Zhang L ... -  《-》

Syndecan-4 deficiency limits neointimal formation after vascular injury by regulating vascular smooth muscle cell proliferation and vascular progenitor cell mobilization.

Ikesue M ，Matsui Y ，Ohta D ，Danzaki K ，Ito K ，Kanayama M ，Kurotaki D ，Morimoto J ，Kojima T ，Tsutsui H ，Uede T ... -  《-》

Increased neointimal formation in cystathionine gamma-lyase deficient mice: role of hydrogen sulfide in α5β1-integrin and matrix metalloproteinase-2 expression in smooth muscle cells.

The physiological and pathological roles of hydrogen sulfide (H(2)S) in the regulation of cardiovacular functions have been recognized. Vascular smooth muscle cells (SMCs) express cystathionine gamma-lyase (CSE) and produce significant amount of H(2)S. Although growing evidence demonstated the anti-atherosclerotic effect of H(2)S, less is known about the contribution of the endogenous CSE/H(2)S pathway to the development of vascular remodeling. This study investigated the roles of the CSE/H(2)S pathway on SMC migration and neoimtimal formation by using CSE knockout (KO) mice. SMCs and aortic explants isolated from CSE KO mice exhibited more migration and outgrowth compared with that from wild-type (WT) mice, and exogenously applied NaHS (a H(2)S donor) at 100 μM significantly inhibited SMC migration and outgrowth. SMCs became more elongated and spread in the absence of CSE, and fibronectin significantly stimulated adhesion and migration of SMCs from CSE KO mice (KO-SMCs) in comparison with SMCs from WT mice (WT-SMCs). The expressions of α5- and β1-integrins were significantly higher in KO-SMCs, and functional blocking of α5β1-integrin effectively abrogated KO-SMC migration. CSE deficiency also enhanced matrix metalloproteinase-2 (MMP-2) expression, and the selective blocking of MMP-2 decreased KO-SMC migration. NaHS treatment decreased both the expressions of α5- and β1-integrins and MMP-2. We further found that the expressions of α5- and β1-integrins as well as MMP-2, were stimulated by fibronectin, and that the blockage of α5β1-integrin reduced but overexpression of α5β1-integrin induced MMP-2 expression in both WT-SMCs and KO-SMCs. We also noticed that CSE deficiency in mice led to increased neointima formation in carotid arteries 4 weeks after ligation, which were attenuated by NaHS administration. In conclusion, inhibition of SMC migration by H(2)S may be a novel target for the treatment of vascular occlusive disorder.

Yang G ，Li H ，Tang G ，Wu L ，Zhao K ，Cao Q ，Xu C ，Wang R ... -  《-》

Regulator of G-Protein Signaling 5 Prevents Smooth Muscle Cell Proliferation and Attenuates Neointima Formation.

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. 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. 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.

Daniel JM ，Prock A ，Dutzmann J ，Sonnenschein K ，Thum T ，Bauersachs J ，Sedding DG ... -  《-》