scholarly journals RTEF-1 Inhibits Vascular Smooth Muscle Cell Calcification through Regulating Wnt/β-Catenin Signaling Pathway

2021 ◽  
Author(s):  
Jingjing Cong ◽  
Bei Cheng ◽  
Jinyu Liu ◽  
Ping He
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Osteogenic Differentiation ◽  
Signaling Pathway ◽  
Glycogen Synthase ◽  
Critical Role ◽  
Transcriptional Enhancer ◽  
Alizarin Red ◽  
Gsk 3Β ◽  
Enhancer Factor

AbstractVascular calcification (VC) is highly prevailing in cardiovascular disease, diabetes mellitus, and chronic kidney disease and, when present, is associated with cardiovascular events and mortality. The osteogenic differentiation of vascular smooth muscle cells (VSMCs) is regarded as the foundation for mediating VC. Related transcriptional enhancer factor (RTEF-1), also named as transcriptional enhanced associate domain (TEAD) 4 or transcriptional enhancer factor-3 (TEF-3), is a nuclear transcriptional factor with a potent effect on cardiovascular diseases, apart from its oncogenic role in the canonical Hippo pathway. However, the role and mechanism of RTEF-1 in VC, particularly in calcification of VSMCs, are poorly understood. Our results showed that RTEF-1 was reduced in calcified VSMCs. RTEF-1 significantly ameliorated β-glycerophosphate (β-GP)-induced VSMCs calcification, as detected by alizarin red staining and calcium content assay. Also, RTEF-1 reduced alkaline phosphatase (ALP) activity and decreased expressions of osteoblast markers such as Osteocalcin and Runt-related transcription factor-2 (Runx2), but increased expression of contractile protein, including SM α-actin (α-SMA). Additionally, RTEF-1 inhibited β-GP-activated Wnt/β-catenin pathway which plays a critical role in calcification and osteogenic differentiation of VSMCs. Specifically, RTEF-1 reduced the levels of Wnt3a, p-β-catenin (Ser675), glycogen synthase kinase-3β (GSK-3β), and p-GSK-3β (Ser9), but increased the levels of p-β-catenin (Ser33/37). Also, RTEF-1 increased the ratio of p-β-catenin (Ser33/37) to β-catenin proteins and decreased the ratio of p-GSK-3β (Ser9) to GSK-3β protein. LiCl, a Wnt/β-catenin signaling activator, was observed to reverse the protective effect of RTEF-1 overexpression on VSMCs calcification induced by β-GP. Accordingly, Dickkopf-1 (Dkk1), a Wnt antagonist, attenuated the role of RTEF-1 deficiency in β-GP-induced VSMCs calcification. Taken together, we concluded that RTEF-1 ameliorated β-GP-induced calcification and osteoblastic differentiation of VSMCs by inhibiting Wnt/β-catenin signaling pathway.

scholarly journals The Role and Mechanism ofα-Klotho in the Calcification of Rat Aortic Vascular Smooth Muscle Cells

2015 ◽  
Vol 2015 ◽  
pp. 1-7 ◽  
Author(s):  
Tianlei Chen ◽  
Huijuan Mao ◽  
Cheng Chen ◽  
Lin Wu ◽  
Ningning Wang ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Signaling Pathway ◽  
Vascular Smooth Muscle Cells ◽  
Calcium Content ◽  
Alizarin Red S ◽  
Alizarin Red ◽  
Calcium Deposits

Objective. To investigate the role and possible mechanism ofα-Klotho in the calcification and the osteogenic transition of cultured VSMCs.Methods. VSMCs were culturedin vitroand divided into 5 groups, each using a different medium: (1) control; (2)β-GP; (3)β-GP + Klotho; (4)β-GP + LiCl; (5)β-GP + Klotho + LiCl. Calcium deposits were visualized using Alizarin Red S staining. The calcium concentrations were determined by the o-cresolphthalein complexone method. BMP2, Runx2 andβ-catenin levels were estimated by western blotting, and the level ofα-SMA was determined by using immunofluorescence at day 12.Results.β-GP induced an increase in the expression of BMP2, Runx2, andβ-catenin. The calcium content increased, and the expression ofα-SMA decreased. Alizarin Red S staining was positive under the high phosphorus conditions. BMP2, Runx2, andβ-catenin levels and the calcium content decreased when the cells were cultured with rmKlotho; however, the levels of each were upregulated after treatment with the LiCl.Conclusions. Klotho can ameliorate the calcification and osteogenic transition of VSMCs induced byβ-GP. The mechanism of Klotho in preventing calcification in VSMCs may be partially mediated by the inhibition of the Wnt/β-catenin signaling pathway.

scholarly journals Downregulation of miR-542-3p promotes osteogenic transition of vascular smooth muscle cells in the aging rat by targeting BMP7

2019 ◽  
Vol 13 (1) ◽  
Author(s):  
Huan Liu ◽  
Hongwei Wang ◽  
Sijin Yang ◽  
Dehui Qian
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Osteogenic Differentiation ◽  
Close Association ◽  
Luciferase Reporter ◽  
Calcium Deposition ◽  
Alizarin Red ◽  
Qrt Pcr ◽  
Rna Transfection ◽  
Aging Rat

Abstract Background Aging is believed to have a close association with cardiovascular diseases, resulting in various pathological alterations in blood vessels, including vascular cell phenotypic shifts. In aging vessels, the microRNA(miRNA)-mediated mechanism regulating the vascular smooth muscle cell (VSMC) phenotype remains unclarified. MiRNA microarray was used to compare the expressions of miRNAs in VSMCs from old rats (oVSMCs) and young rats (yVSMCs). Quantitative reverse transcription real-time PCR (qRT-PCR) and small RNA transfection were used to explore the miR-542-3p expression in oVSMCs and yVSMCs in vitro. Calcification induction of yVSMCs was conducted by the treatment of β-glycerophosphate (β-GP). Alizarin red staining was used to detect calcium deposition. Western blot and qRT-PCR were used to investigate the expression of the smooth muscle markers, smooth muscle 22α (SM22α) and calponin, and the osteogenic markers, osteopontin (OPN), and runt-related transcription factor 2 (Runx2). Lentivirus was used to overexpress miR-542-3p and bone morphogenetic protein 7 (BMP7) in yVMSCs. Luciferase reporter assay was conducted to identify the target of miR-542-3p. Results Compared with yVSMCs, 28 downregulated and 34 upregulated miRNAs were identified in oVSMCs. It was confirmed by qRT-PCR that oVSMC expressed four times lower miR-542-3p than yVSMCs. Overexpressing miR-542-3p in yVSMCs suppressed the osteogenic differentiation induced by β-GP. Moreover, miR-542-3p targets BMP7 and overexpressing BMP7 in miR-542-3p–expressing yVSMCs reverses miR-542-3p’s inhibition of osteogenic differentiation. Conclusions miR-542-3p regulates osteogenic differentiation of VSMCs through targeting BMP7, suggesting that the downregulation of miR-542-3p in oVSMCs plays a crucial role in osteogenic transition in the aging rat.

Insulin-like growth-factor-1-induced PKB signaling and Egr-1 expression is inhibited by curcumin in A-10 vascular smooth muscle cells

2013 ◽  
Vol 91 (3) ◽  
pp. 241-247 ◽  
Author(s):  
Viktoria Youreva ◽  
Georgia Kapakos ◽  
Ashok K. Srivastava
Keyword(s):  
Smooth Muscle ◽  
Growth Factor ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Glycogen Synthase ◽  
Muscle Cells ◽  
Insulin Like Growth Factor ◽  
Dose Dependent Fashion ◽  
Gsk 3Β

Insulin-like growth factor 1 (IGF-1) is a mitogenic factor that stimulates the signaling pathways responsible for inducing hypertrophic and proliferative responses in vascular smooth muscle cells (VSMC). We have previously demonstrated that IGF-1 receptor (IGF-1R) plays a key role in transducing the hypertrophic and proliferative responses of angiotensin II (Ang-II) and endothelin-1 (ET-1). Curcumin, a polyphenolic compound derived from the spice turmeric is known to possess antiproliferative properties and exerts vasculoprotective effects. However, the ability of curcumin to modulate IGF-1-induced signaling responses in VSMC remains to be investigated. In this study, we determined the effect of curcumin on IGF-1-induced phosphorylation of protein kinase B (PKB), glycogen synthase kinase-3β (GSK-3β), and IGF-1R in VSMC. Curcumin inhibited IGF-1-induced phosphorylation of PKB and GSK-3β as well as the IGF-1R β subunit in a dose-dependent fashion. In addition, IGF-1-induced expression of early growth response protein 1 (Egr-1) which plays a pathogenic role in vascular dysfunctions, was also attenuated by curcumin. In conclusion, these results indicate that curcumin is a potent inhibitor of key components of the IGF-1-induced mitogenic and proliferative signaling system in VSMC, and suggest that curcumin-induced attenuation of these signaling components may constitute a potential mechanism for its vasculoprotective effects.

scholarly journals RhoA/Rho kinase (ROCK) alters fetuin-A uptake and regulates calcification in bovine vascular smooth muscle cells (BVSMC)

2010 ◽  
Vol 299 (3) ◽  
pp. F674-F680 ◽  
Author(s):  
Neal X. Chen ◽  
Xianming Chen ◽  
Kalisha D. O'Neill ◽  
Simon J. Atkinson ◽  
Sharon M. Moe
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Signaling Pathway ◽  
Rho Gtpase ◽  
Critical Role ◽  
Negative Regulator ◽  
Rock Inhibitor ◽  
Uptake Inhibition ◽  
Fetuin A ◽  
Alp Activity

RhoA/Rho kinases (ROCK) play a critical role in vascular smooth muscle cell (VSMC) actin cytoskeleton organization, differentiation, and function and are implicated in the pathogenesis of cardiovascular disease. We have previously determined that an important step in the regulation of calcification is fetuin-A endocytosis, a process that is dependent on changes in the cytoskeleton, which, in turn, is known to be affected by the RhoA/ROCK signaling pathway. In the present study, bovine VSMC (BVSMC) were treated with the ROCK inhibitor Y-27632 or transfected with ROCK small interfering (si) RNA to knock down ROCK expression. Both conditions resulted in reduced actin stress fibers and increased Cy5-labeled fetuin-A uptake. Inhibition of ROCK by Y-27632 or siRNA also significantly increased BVSMC alkaline phosphatase (ALP) activity and calcification of BVSMC and rat aorta organ cultures. Cells were then incubated in calcification media in the presence or absence of Y-27632 and matrix vesicles (MV) isolated by collagenase digestion. These MV, isolated from BVSMC incubated with Y-27632, had increased ALP activity and increased ability of MV to subsequently calcify collagen by 66%. In contrast, activation of RhoA, which is upstream of ROCK, by transfecting plasmids encoding the dominant active Rho GTPase mutant (Rho-L63) led to decreased fetuin-A uptake and reduced calcification in BVSMC. These results demonstrate that the RhoA/ROCK signaling pathway is an important negative regulator of vascular calcification.

scholarly journals Globular adiponectin inhibits osteoblastic differentiation of vascular smooth muscle cells through the PI3K/AKT and Wnt/β-catenin pathway

2021 ◽  
Author(s):  
Yun Zhou ◽  
Li-Long Wei ◽  
Rui-Ping Zhang ◽  
Cheng-Wu Han ◽  
Yongtong Cao
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells ◽  
Osteoblastic Differentiation ◽  
Alizarin Red S ◽  
Regulation Mechanism ◽  
Alizarin Red ◽  
Globular Adiponectin

AbstractLipid metabolism is closely related to the improvement of vascular calcification (VC) in chronic kidney disease (CKD). Globular adiponectin (gAd) has been reported to be involved in the development of VC in CKD, but the detailed regulatory role remains unclear. The present study is aimed to investigate the biological function and the underlying regulation mechanism of gAd in the process of VC during CKD. Vascular smooth muscle cells (VSMCs) calcification was determined by Alizarin Red S staining. Protein signaling related with VC was tested by western blotting. The expression and intracellular localization of runt-related transcription factor 2 (Runx2) was detected by immunofluorescence and uraemic rat with VC was established by a two-step nephrectomy. Combined with the results of Alizarin Red S staining, we discovered that β-glycerophosphate (β-Gp)-induced the osteoblastic differentiation of VSMCs was significantly reversed by gAd treatment. Along with the VSMCs calcification and the increase of Runx2 in β-Gp-exposed VSMCs, the activities of protein kinase B (AKT) and Wnt/β-catenin pathway were enhanced, but that were counteracted by the exposure of gAd in rat and human VSMCs. After administration with agonists of the Wnt (SKL2001) and AKT (SC79), there appeared more osteoblastic differentiation and higher expression of Runx2 in gAd-treated VSMCs, but showing lower impact in the presence of SC79 than that in the presence of SKL2001. In the in vivo experiments, intravenous injection of gAd also significantly inhibited VC and Runx2 level in uraemic rat in a dose-dependent manner, possibly through regulating Wnt/β-catenin pathway. This study demonstrates that gAd ameliorates osteoblastic differentiation of VSMCs possibly by blocking PI3K/AKT and Wnt/β-catenin signaling transduction. The findings provide an important foundation for gAd in treating VC in kidney diseases.

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