MiPEP31 inhibits the vascular smooth muscle cell proliferation via cooperation with transcription factor Trps1

Nesfatin-1 functions as a switch for phenotype transformation and proliferation of VSMCs in hypertensive vascular remodeling

USP10 exacerbates neointima formation by stabilizing Skp2 protein in vascular smooth muscle cells

ALDH1A3 Regulations of Matricellular Proteins Promote Vascular Smooth Muscle Cell Proliferation.

The intermediate conductance calcium-activated potassium channel KCa3.1 contributes to a variety of cell activation processes in pathologies such as inflammation, carcinogenesis, and vascular remodeling. We examined the electrophysiological and transcriptional mechanisms by which KCa3.1 regulates vascular smooth muscle cell (VSMC) proliferation. Platelet-derived growth factor-BB (PDGF)-induced proliferation of human coronary artery VSMCs was attenuated by lowering intracellular Ca(2+) concentration ([Ca(2+)]i) and was enhanced by elevating [Ca(2+)]i. KCa3.1 blockade or knockdown inhibited proliferation by suppressing the rise in [Ca(2+)]i and attenuating the expression of phosphorylated cAMP-response element-binding protein (CREB), c-Fos, and neuron-derived orphan receptor-1 (NOR-1). This antiproliferative effect was abolished by elevating [Ca(2+)]i. KCa3.1 overexpression induced VSMC proliferation, and potentiated PDGF-induced proliferation, by inducing CREB phosphorylation, c-Fos, and NOR-1. Pharmacological stimulation of KCa3.1 unexpectedly suppressed proliferation by abolishing the expression and activity of KCa3.1 and PDGF β-receptors and inhibiting the rise in [Ca(2+)]i. The stimulation also attenuated the levels of phosphorylated CREB, c-Fos, and cyclin expression. After KCa3.1 blockade, the characteristic round shape of VSMCs expressing high l-caldesmon and low calponin-1 (dedifferentiation state) was maintained, whereas KCa3.1 stimulation induced a spindle-shaped cellular appearance, with low l-caldesmon and high calponin-1. In conclusion, KCa3.1 plays an important role in VSMC proliferation via controlling Ca(2+)-dependent signaling pathways, and its modulation may therefore constitute a new therapeutic target for cell proliferative diseases such as atherosclerosis.

Cyclic nucleotide phosphodiesterase 3 (PDE3) is an important regulator of cyclic adenosine monophosphate (cAMP) signaling within the cardiovascular system. In this study, we examined the role of PDE3A and PDE3B isoforms in regulation of growth of cultured vascular smooth muscle cells (VSMCs) and the mechanisms by which they may affect signaling pathways that mediate mitogen-induced VSMC proliferation. Serum- and PDGF-induced DNA synthesis in VSMCs grown from aortas of PDE3A-deficient (3A-KO) mice was markedly less than that in VSMCs from PDE3A wild type (3A-WT) and PDE3B-deficient (3B-KO) mice. The reduced growth response was accompanied by significantly less phosphorylation of extracellular signal-regulated kinase (ERK) in 3A-KO VSMCs, most likely due to a combination of greater site-specific inhibitory phosphorylation of Raf-1(Ser-²⁵⁹) by protein kinase A (PKA) and enhanced dephosphorylation of ERKs due to elevated mitogen-activated protein kinase phosphatase 1 (MKP-1). Furthermore, 3A-KO VSMCs, compared with 3A-WT, exhibited higher basal PKA activity and cAMP response element-binding protein (CREB) phosphorylation, higher levels of p53 and p53 phosphorylation, and elevated p21 protein together with lower levels of Cyclin-D1 and retinoblastoma (Rb) protein and Rb phosphorylation. Adenoviral overexpression of inactive CREB partially restored growth effects of serum in 3A-KO VSMCs. In contrast, exposure of 3A-WT VSMCs to VP16 CREB (active CREB) was associated with inhibition of serum-induced DNA synthesis similar to that in untreated 3A-KO VSMCs. Transfection of 3A-KO VSMCs with p53 siRNA reduced p21 and MKP-1 levels and completely restored growth without affecting amounts of Cyclin-D1 and Rb phosphorylation. We conclude that PDE3A regulates VSMC growth via two complementary pathways, i.e. PKA-catalyzed inhibitory phosphorylation of Raf-1 with resulting inhibition of MAPK signaling and PKA/CREB-mediated induction of p21, leading to G₀/G₁ cell cycle arrest, as well as by increased accumulation of p53, which induces MKP-1, p21, and WIP1, leading to inhibition of G₁ to S cell cycle progression.

Expression and Suppressive Effects of Interleukin-19 on Vascular Smooth Muscle Cell Pathophysiology and Development of Intimal Hyperplasia

Proliferation and oxidative stress of vascular smooth muscle cells (VSMCs) contribute to vascular remodeling in hypertension and several major vascular diseases. B-cell lymphoma 6 (BCL6) functions as a transcriptional repressor. The present study is designed to determine the roles of BCL6 in VSMC proliferation and oxidative stress and underlying mechanism. Angiotensin (Ang) II was used to induce VSMC proliferation and oxidative stress in human VSMCs. Effects of BCL6 overexpression and knockdown were, respectively, investigated in Ang II-treated human VSMCs. Therapeutical effects of BCL6 overexpression on vascular remodeling, oxidative stress, and proliferation were determined in the aorta of spontaneously hypertensive rats (SHR). Ang II reduced BCL6 expression in human VSMCs. BCL6 overexpression attenuated while BCL6 knockdown enhanced the Ang II-induced upregulation of NADPH oxidase 4 (NOX4), production of reactive oxygen species (ROS), and proliferation of VSMCs. BCL6 expression was downregulated in SHR. BCL6 overexpression in SHR reduced NOX4 expression, ROS production, and proliferation of the aortic media of SHR. Moreover, BCL6 overexpression attenuated vascular remodeling and hypertension in SHR. However, BCL6 overexpression had no significant effects on NOX2 expression in human VSMCs or in SHR. We conclude that BCL6 attenuates proliferation and oxidative stress of VSMCs in hypertension.

Background/Aims: Angiotensin (Ang) II plays vital roles in vascular inflammation and remodeling in hypertension. Phenotypic transformation of vascular smooth muscle cells (VSMCs) is a major initiating factor for vascular remodeling. The present study was designed to determine the roles of NLRP3 inflammasome activation in Ang II-induced VSMC phenotypic transformation and vascular remodeling in hypertension. Methods: Primary VSMCs from the aorta of NLRP3 knockout (NLRP3^-/-) mice and wild-type (WT) mice were treated with Ang II for 24 h. Subcutaneous infusion of Ang II via osmotic minipump for 2 weeks was used to induce vascular remodeling and hypertension in WT and NLRP3^-/- mice. Results: NLRP3 gene deletion attenuates Ang II-induced NLRP3 inflammasome activation, phenotypic transformation from a contractile phenotype to a synthetic phenotype and proliferation in primary mice VSMCs. Ang II-induced hypertension and vascular remodeling in WT mice were attenuated in NLRP3^-/- mice. Furthermore, Ang II-induced NLRP3 inflammasome activation, phenotypic transformation and proliferating cell nuclear antigen (PCNA) upregulation were inhibited in the media of aorta of NLRP3^-/- mice. Conclusions: NLRP3 inflammasome activation contributes to Ang II-induced VSMC phenotypic transformation and proliferation as well as vascular remodeling and hypertension.

Inflammation is involved in pathogenesis of hypertension. NLRP3 inflammasome activation is a powerful mediator of inflammatory response via caspase-1 activation. The present study was designed to determine the roles and mechanisms of NLRP3 inflammasome in phenotypic modulation and proliferation of vascular smooth muscle cells (VSMCs) in hypertension. Experiments were conducted in spontaneously hypertensive rats (SHR) and primary aortic VSMCs. NLRP3 inflammasome activation was observed in the media of aorta in SHR and in the VSMCs from SHR. Knockdown of NLRP3 inhibited inflammasome activation, VSMC phenotypic transformation and proliferation in SHR-derived VSMCs. Increased NFκB activation, histone acetylation and histone acetyltransferase expression were observed in SHR-derived VSMCs and in media of aorta in SHR. Chromatin immunoprecipitation analysis revealed the increased histone acetylation, p65-NFκB and Pol II occupancy at the NLRP3 promoter in vivo and in vitro. Inhibition of NFκB with BAY11-7082 or inhibition of histone acetyltransferase with curcumin prevented the NLRP3 inflammasome activation, VSMC phenotype switching and proliferation in VSMCs from SHR. Moreover, curcumin repressed NFκB activation. Silencing of NLRP3 gene ameliorated hypertension, vascular remodeling, NLRP3 inflammasome activation and phenotype switching in the aorta of SHR. These results indicate that NLRP3 inflammasome activation response to histone acetylation and NFκB activation contributes to VSMC phenotype switching and proliferation and vascular remodeling in hypertension.

Proliferation of vascular smooth muscle cells (VSMCs) greatly contributes to vascular remodeling in hypertension. This study is to determine the roles and mechanisms of miR-135a-5p intervention in attenuating VSMC proliferation and vascular remodeling in spontaneously hypertensive rats (SHRs). MiR-135a-5p level was raised, while fibronectin type III domain-containing 5 (FNDC5) mRNA and protein expressions were reduced in VSMCs of SHRs compared with those of Wistar-Kyoto rats (WKYs). Enhanced VSMC proliferation in SHRs was inhibited by miR-135a-5p knockdown or miR-135a-5p inhibitor, but exacerbated by miR-135a-5p mimic. VSMCs of SHRs showed reduced myofilaments, increased or even damaged mitochondria, increased and dilated endoplasmic reticulum, which were attenuated by miR-135a-5p inhibitor. Dual-luciferase reporter assay shows that FNDC5 was a target gene of miR-135a-5p. Knockdown or inhibition of miR-135a-5p prevented the FNDC5 downregulation in VSMCs of SHRs, while miR-135a-5p mimic inhibited FNDC5 expressions in VSMCs of both WKYs and SHRs. FNDC5 knockdown had no significant effects on VSMC proliferation of WKYs, but aggravated VSMC proliferation of SHRs. Exogenous FNDC5 or FNDC5 overexpression attenuated VSMC proliferation of SHRs, and prevented miR-135a-5p mimic-induced enhancement of VSMC proliferation of SHR. MiR-135a-5p knockdown in SHRs attenuated hypertension, normalized FNDC5 expressions and inhibitedvascular smooth muscle proliferation, and alleviated vascular remodeling. These results indicate that miR-135a-5p promotes while FNDC5 inhibits VSMC proliferation in SHRs. Silencing of miR-135a-5p attenuates VSMC proliferation and vascular remodeling in SHRs via disinhibition of FNDC5 transcription. Either inhibition of miR-135a-5p or upregulation of FNDC5 may be a therapeutically strategy in attenuating vascular remodeling and hypertension.

MDM2: A Novel Mineralocorticoid-Responsive Gene Involved in Aldosterone-Induced Human Vascular Structural Remodeling

Introduction: Induction of heme oxygenase 1 (HO-1) ameliorates the development of neointimal hyperplasia, which normally involves oxidative stress and vascular smooth muscle cell (VSMC) proliferation [1]. Bilirubin and biliverdin, generated during heme catabolism by HO-1, are potent anti-oxidants that play a role in maintenance of homeostasis [2]. Based on the clinical observation that healthy individuals with higher levels of bilirubin develop less arteriosclerosis3, we hypothesized that the bile pigments would inhibit VSMC proliferation in vitro and neointima formation in vivo. Methods: Balloon injury (BI) was performed in the left common carotid artery of congenitally hyperbilirubinemic Gunn rats. The artery was harvested two weeks after BI, and neointima formation was assessed by quantifying intima/media ratio and luminal cross-sectional area narrowing (LCAN). In vitro, proliferation of primary vascular smooth muscle cells (VSMC) was measured by [3H]-thymi-dine incorporation in the presence or absence of bilirubin/biliverdin. Cell cycle progression was monitored by DNA content analysis; apoptosis by the Annexin V binding assay. Protein levels were detected by western blot. Immunocytochemistry was performed using primary antibodies directed against cdk2 and phospho-p38 in growth stimulated primary VSMCs, in the presence or absence of bilirubin/biliverdin. To further delineate the pathways of bilirubin/biliverdin action on VSMC proliferation, we used VSMCs derived from p53 KO, p21 KO and HO-1 KO mice. Results: BI mediated neointima formation was significantly reduced in hyperbilirubinemic Gunn rats (serum bilirubin levels: 12.0 ± 2.5 mg/dl) as compared to control Wistar rats (LCAN 0.18 vs. 0.44; p<0.05). Systemic administration of biliverdin, the precursor of bilirubin, to normal rats also suppressed neointima proliferation (LCAN 0.29 vs. 0.43; p<0.05). In vitro, bilirubin inhibited VSMC proliferation by arresting VSMC cell cycle progression at the G0/G1 phase, while it did not increase apoptosis (p=0.2816). Inhibition of cell cycle progression was mediated by reduced phosphorylation of p38 MAPK and c-Jun NH2-terminal kinase 1/2 (JNK 1/2), down-regulation of cdk2, cyclins D1, E and A resulting in hypophosphorylation of pRb as demonstrated in nuclear extracts of cultured cells. Further the effect of bilirubin on VSMC proliferation was dependent on p53, but independent of p21 and HO-1. Conclusions: Our studies implicate bilirubin as the molecule to explain the observed associations between higher levels of plasma bilirubin in humans and a lesser incidence of atherosclerosis-related diseases and suggest the future use of bilirubin/biliverdin as a therapeutic to prevent restenosis, chronic rejection and atherosclerosis.

The Role of Estrogen Receptor α and β in Regulating Vascular Smooth Muscle Cell Proliferation is Based on Sex

Qingda granule inhibits angiotensin Ⅱ induced VSMCs proliferation through MAPK and PI3K/AKT pathways

O386* Aims: Induction of heme oxygenase 1 (HO-1) ameliorates the development of chronic allograft arteriosclerosis, which normally involves oxidative stress and vascular smooth muscle cell (VSMC) proliferation. Bilirubin and biliverdin, generated during heme catabolism by HO-1, are potent antioxidants that play a role in maintenance of homeostasis. Based on the clinical observation that healthy individuals with higher levels of bilirubin develop less arteriosclerosis, we hypothesized that the bile pigments would inhibit VSMC proliferation in vitro and neointima formation in vivo. We used balloon injury (BI) as a model of the neointimal proliferation seen in transplant arteriosclerosis. Methods: BI was performed in the left common carotid artery of congenitally hyperbilirubinemic Gunn rats and congenic wild-type Wistar rats. The artery was harvested two weeks after BI, and neointima formation was assessed by quantifying intima/media ratio and luminal cross-sectional area narrowing (LCAN). In vitro, proliferation of primary vascular smooth muscle cells (VSMC) was measured by 3H-thymidine incorporation in the presence or absence of bilirubin/ biliverdin. Cell cycle progression was monitored by DNA content analysis; apoptosis by the Annexin V binding assay. Protein levels were detected by western blot. Immuncytochemistry was performed using antibody directed against the retinoblastoma tumor suppressor protein (pRb). Results: BI mediated neointima formation was significantly reduced in hyperbilirubinemic Gunn rats (serum bilirubin levels: 12.0 ± 2.5 mg/dl) as compared to control Wistar rats (LCAN 0.18 vs. 0.44; p < 0.05). Systemic administration of biliverdin, the precursor of bilirubin, to normal rats also suppressed neointima proliferation (LCAN 0.29 vs. 0.43; p < 0.05). In vitro, bilirubin inhibited VSMC proliferation in a dose-dependent manner. Bilirubin (200 μM) arrested VSMC cell cycle progression at the G0/G1 phase, while it did not increase apoptosis (p=0.2816). Inhibition of cell cycle progression was mediated by reduced phosphorylation of p38 MAPK and c-Jun NH2-terminal kinase 1/2 (JNK 1/2), down-regulation of cdk2, cyclins D1, E and A, and the resulting decreased phosphorylation of pRb. In nuclear extracts, lower total levels of pRb were found in bilirubin treated VSMCs, consistent with the findings that a significant portion of pRb was kept in the cytosol after bilirubin treatment as assessed by immunocytochemistry. Further, bilirubin treatment reduced expression levels of the transcription factor YY1, a zinc finger DNA-binding transcription factor known to regulate the expression of genes with important functions during cell growth and differentiation. The effects of bilirubin were independent of HO-1 expression, as demonstrated in HO-1−/− VSMCs. Conclusions: Biliverdin and bilirubin suppress both BI induced neointima formation in vivo and VSMC proliferation in vitro. Our data suggest that bilirubin treatment inhibits VSMC growth by impaired activation of p38 and JNK 1/2, resulting in two events. Firstly, the inhibition of pRb phosphorylation impedes the release of transcription factors such as YY1 that are important for VSMC growth. Secondly, there is reduced expression of the transcription factor YY1 itself, resulting in a suppressed capacity to induce DNA synthesis and transcriptional activity. Thus, administration of the bile pigments bilirubin and biliverdin may have a beneficial effect on VSMC proliferation in chronic allograft arteriopathy and may become a potent strategy for the treatment of chronic rejection.