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 ... -  《-》

Epac1 Deficiency Attenuated Vascular Smooth Muscle Cell Migration and Neointimal Formation.

Vascular smooth muscle cell (SMC) migration causes neointima, which is related to vascular remodeling after mechanical injury and atherosclerosis development. We previously reported that an exchange protein activated by cAMP (Epac) 1 was upregulated in mouse arterial neointima and promoted SMC migration. In this study, we examined the molecular mechanisms of Epac1-induced SMC migration and the effect of Epac1 deficiency on vascular remodeling in vivo. Platelet-derived growth factor-BB promoted a 2-fold increase in SMC migration in a primary culture of aortic SMCs obtained from Epac1(+/+) mice (Epac1(+/+)-ASMCs), whereas there was only a 1.2-fold increase in Epac1(-/-)-ASMCs. The degree of platelet-derived growth factor-BB-induced increase in intracellular Ca(2+) was smaller in Fura2-labeled Epac1(-/-)-ASMCs than in Epac1(+/+)-ASMCs. In Epac1(+/+)-ASMCs, an Epac-selective cAMP analog or platelet-derived growth factor-BB increased lamellipodia accompanied by cofilin dephosphorylation, which is induced by Ca(2+) signaling, whereas these effects were rarely observed in Epac1(-/-)-ASMCs. Furthermore, 4 weeks after femoral artery injury, prominent neointima were formed in Epac1(+/+) mice, whereas neointima formation was significantly attenuated in Epac1(-/-) mice in which dephosphorylation of cofilin was inhibited. The chimeric mice generated by bone marrow cell transplantation from Epac1(+/+) into Epac1(-/-) mice and vice versa demonstrated that the genetic background of vascular tissues, including SMCs rather than of bone marrow-derived cells affected Epac1-mediated neointima formation. These data suggest that Epac1 deficiency attenuates neointima formation through, at least in part, inhibition of SMC migration, in which a decrease in Ca(2+) influx and a suppression of cofilin-mediated lamellipodia formation occur.

Kato Y ，Yokoyama U ，Yanai C ，Ishige R ，Kurotaki D ，Umemura M ，Fujita T ，Kubota T ，Okumura S ，Sata M ，Tamura T ，Ishikawa Y ... -  《-》

Redox-sensitive transcription factor Nrf2 regulates vascular smooth muscle cell migration and neointimal hyperplasia.

Ashino T ，Yamamoto M ，Yoshida T ，Numazawa S ... -  《-》

BET bromodomain-containing epigenetic reader proteins regulate vascular smooth muscle cell proliferation and neointima formation.

Recent studies revealed that the bromodomain and extra-terminal (BET) epigenetic reader proteins resemble key regulators in the underlying pathophysiology of cancer, diabetes, or cardiovascular disease. However, whether they also regulate vascular remodelling processes by direct effects on vascular cells is unknown. In this study, we investigated the effects of the BET proteins on human smooth muscle cell (SMC) function in vitro and neointima formation in response to vascular injury in vivo. Selective inhibition of BETs by the small molecule (+)-JQ1 dose-dependently reduced proliferation and migration of SMCs without apoptotic or toxic effects. Flow cytometric analysis revealed a cell cycle arrest in the G0/G1 phase in the presence of (+)-JQ1. Microarray- and pathway analyses revealed a substantial transcriptional regulation of gene sets controlled by the Forkhead box O (FOXO1)1-transcription factor. Silencing of the most significantly regulated FOXO1-dependent gene, CDKN1A, abolished the antiproliferative effects. Immunohistochemical colocalization, co-immunoprecipitation, and promoter-binding ELISA assay data confirmed that the BET protein BRD4 directly binds to FOXO1 and regulates FOXO1 transactivational capacity. In vivo, local application of (+)-JQ1 significantly attenuated SMC proliferation and neointimal lesion formation following wire-induced injury of the femoral artery in C57BL/6 mice. Inhibition of the BET-containing protein BRD4 after vascular injury by (+)-JQ1 restores FOXO1 transactivational activity, subsequent CDKN1A expression, cell cycle arrest and thus prevents SMC proliferation in vitro and neointima formation in vivo. Inhibition of BET epigenetic reader proteins might thus represent a promising therapeutic strategy to prevent adverse vascular remodelling.

Dutzmann J ，Haertlé M ，Daniel JM ，Kloss F ，Musmann RJ ，Kalies K ，Knöpp K ，Pilowski C ，Sirisko M ，Sieweke JT ，Bauersachs J ，Sedding DG ，Gegel S ... -  《-》

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 ... -  《-》