Ferulic Acid Alleviates Atherosclerotic Plaques by Inhibiting VSMC Proliferation Through the NO/p21 Signaling pathway

The benefits and risks of inhibiting the proliferation and migration of vascular smooth muscle cells (VSMCs) in atherosclerosis (AS) remain a subject of debate. In this study, we investigated the effect of ferulic acid (FA) on the proliferation and migration of VSMCs induced by platelet-derived growth factor (PDGF) and the associated mechanism and used ApoE-/- mice to study whether the effect of FA on VSMC proliferation and migration is beneficial in alleviating AS plaques. It was found that FA not only reduced blood lipid levels but also promoted the production of nitric oxide (NO) by MOVAS cells through the endothelial nitric oxide synthase (eNOS) pathway, inhibited the migration and proliferation of VSMCs induced by PDGF, promoted the expression of p21 in VSMCs, and exerted a therapeutic effect against AS.

High Glucose-Induced Vascular Smooth Muscle Cell Proliferation and Migration are Regulated by the miR-34a-Notch1 Pathway

High glucose(HG)-induced excessive proliferation and migration of the media vascular smooth muscle cell(VSMC) are the main pathological characteristics in diabetes related vascular injuries. Previous studies have shown that microRNA-34a (miR-34a) is involved in cancer metastasis, proliferation and invasion and plays an essential role in cardiovascular disease. However, little is known about the regulating role miR-34a in HG-induced proliferation and migration of VSMC. Here we demonstrated that miR-34a was downregulated at different timepoints under HG stimulation. Then, HG induced proliferation and migration was found to be impaired by miR-34a overexpression using transwell, CCK8 and RT-qPCR assays. Furthermore, the HG-induced depression of “contractile” VSMC-specific markers were reversed by the overexpression of miR-34a. Moreover, we confirmed that miR-34a regulated HG-induced VSMC proliferation and migration through its target gene, Notch1, which has been shown to be associated with cell proliferation and migration in previous studies. Taken together, we propose that the miR34a-Notch1 axis plays an important role in regulating HG-induced VSMC proliferation and migration.

M6A methylation-mediated elevation of SM22α inhibits the proliferation and migration of vascular smooth muscle cells and ameliorates intimal hyperplasia in type 2 diabetes mellitus

Abnormal proliferation of vascular smooth muscle cells (VSMCs) induced by insulin resistance facilitates intimal hyperplasia of type 2 diabetes mellitus (T2DM) and N6-methyladenosine (m6A) methylation modification mediates the VSMC proliferation. This study aimed to reveal the m6A methylation modification regulatory mechanism. In this study, m6A demethylase FTO was elevated in insulin-treated VSMCs and T2DM mice with intimal injury. Functionally, FTO knockdown elevated m6A methylation level and further restrained VSMC proliferation and migration induced by insulin. Mechanistically, FTO knockdown elevated Smooth muscle 22 alpha (SM22α) expression and m6A-binding protein IGF2BP2 enhanced SM22α mRNA stability by recognizing and binding to m6A methylation modified mRNA. In vivo studies confirmed that the elevated m6A modification level of SM22α mRNA mitigated intimal hyperplasia in T2DM mice. Conclusively, m6A methylation-mediated elevation of SM22α restrained VSMC proliferation and migration and ameliorated intimal hyperplasia in T2DM.

17β-Estradiol Inhibits Proliferation and Oxidative Stress in Vascular Smooth Muscle Cells by Upregulating BHLHE40 Expression

Background: Intimal hyperplasia is a major complication of restenosis after angioplasty. The abnormal proliferation and oxidative stress of vascular smooth muscle cells (VSMCs) are the basic pathological feature of neointimal hyperplasia. 17β-Estradiol can inhibit VSMCs proliferation and inflammation. However, it is still unclear whether and how 17β-Estradiol affects intimal hyperplasia.Methods: The neointima hyperplasia was observed by hematoxylin/eosin staining. The expression of PCNA, cyclin D1, NOX1, NOX4 and p47phox in neointima hyperplasia tissues and VSMCs was determined by qRT-PCR and Western blotting. MTS assay, cell counting and EdU staining were performed to detect cells proliferation. The oxidative stress was assessed by ROS staining.Results: 17β-Estradiol suppressed carotid artery ligation-induced intimal hyperplasia, which is accompanied by an increase of BHLHE40 level. Furthermore, loss- and gain-of-function experiments revealed that BHLHE40 knockdown promotes, whereas BHLHE40 overexpression inhibits TNF-α-induced VSMC proliferation and oxidative stress. 17β-Estradiol inhibited TNF-α-induced VSMC proliferation and oxidative stress by promoting BHLHE40 expression, thereby suppressing MAPK signaling pathways. In addition, enforcing the expression of BHLHE40 leads to amelioration of intimal hyperplasia.Conclusions: Our study demonstrates that 17β-Estradiol inhibits proliferation and oxidative stress in vivo and in vitro by promotion of BHLHE40 expression.

Essential role of Orai1 and SARAF in vascular remodeling

Orai1 and STIM1, molecular components of store-operated calcium entry (SOCE), have been associated with vascular smooth muscle cell (VSMC) proliferation in vascular remodeling. Nevertheless, the role of SARAF (SOCE-associated regulatory factor), a regulatory protein involved in STIM1 inhibition, in vascular remodeling has not been examined. The aim of this study is to examine the role of SARAF and Orai1 in VSMC proliferation and neointima formation after balloon injury of rat carotid arteries. Experiments were conducted in an animal model of rat carotid angioplasty to characterize neointima formation. VSMC isolated from rat coronary arteries was also used to examine cell proliferation. The formation of neointima after balloon injury of rat carotid arteries was confirmed by hematoxylin and eosin staining of tissue sections up to 3 wk after surgery. Injured arteries showed significantly higher expression of SARAF, STIM1, and Orai1 compared with control tissues, corroborating the presence of these regulatory proteins in the neointima layer. Proximity ligation and coimmunoprecipitation assays revealed that SARAF interacts with Orai1 in the neointima. Furthermore, selective silencing of SARAF and Orai1 by small interfering RNA (siRNA) inhibited IGF-1–induced VSMC proliferation. Our data suggest that SARAF interacts with Orai1 to modulate SOCE and VSMC proliferation after vascular injury.

Identification of a Novel Gene Correlated With Vascular Smooth Muscle Cells Proliferation and Migration in Chronic Thromboembolic Pulmonary Hypertension

Objective: Chronic thromboembolic pulmonary hypertension (CTEPH) is characterized by thrombofibrotic obstruction of the proximal pulmonary arteries, which result in vascular remodeling of the distal pulmonary artery. While the cellular and molecular mechanisms underlying CTEPH pathogenesis remain incompletely understood, recent evidence implicates vascular remodeling. Here, we identify the molecular mechanisms that contribute to vascular remodeling in CTEPH.Methods: Microarray data (GSE130391) for patients with CTEPH and healthy controls were downloaded from the Gene Expression Omnibus (GEO) and screened for differentially expressed genes (DEGs). DEGs were functionally annotated using Gene Ontology (GO) functional analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. A protein–protein interaction (PPI) network was constructed to identify hub genes. Finally, pulmonary artery samples were harvested from patients with CTEPH (n = 10) and from controls (n = 10) and primary vascular smooth muscle cells (VSMCs) were cultured. Effects of the proto-oncogene FOS on VSMC proliferation and migration were assessed using expression and knockdown studies.Results: We detected a total of 292 DEGs, including 151 upregulated and 141 downregulated genes. GO analysis revealed enrichment of DEGs in biological processes of signal transduction, response to lipopolysaccharide, signal transduction, and myeloid dendritic cell differentiation. Molecular function analysis revealed enrichment in tumor necrosis factor (TNF)-activated receptor activity, transcriptional activator activity, and protein homodimerization activity. The expression of TNF-α and its receptor (sTNFR1 and sTNFR2) were significantly higher in CTEPH group, compared with control group. KEGG pathway analysis revealed enrichment in salmonella infection, pathways in cancer, osteoclast differentiation, and cytokine-cytokine receptor interaction. Hub genes in the PPI included FOS, suggesting an important role for this gene in vascular remodeling in CTEPH. Primary VSMCs derived from patients with CTEPH showed increased FOS expression and high proliferation and migration, which was attenuated by FOS inhibition. In control VSMCs, TNF-α treatment increased proliferation and migration, which FOS inhibition likewise attenuated.Conclusion: TNF-α drives CTEPH pathogenesis by promoting VSMC proliferation and migration via increased FOS expression. These results advance our understanding of the molecular mechanisms of vascular remodeling in CTEPH, and may inform the development of new therapeutic targets.

MIA SH3 Domain ER Export Factor 3 Deficiency Prevents Neointimal Formation by Restoring BAT-Like PVAT and Decreasing VSMC Proliferation and Migration

Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) and excessive accumulation of dysfunctional PVAT are hallmarks of pathogenesis after angioplasty. Recent genome-wide association studies reveal that single-nucleotide polymorphism (SNP) in MIA3 is associated with atherosclerosis-relevant VSMC phenotypes. However, the role of MIA3 in the vascular remodeling response to injury remains unknown. Here, we found that expression of MIA3 is increased in proliferative VSMCs and knockdown of MIA3 reduces VSMCs proliferation, migration, and inflammation, whereas MIA3 overexpression promoted VSMC migration and proliferation. Moreover, knockdown of MIA3 ameliorates femoral artery wire injury-induced neointimal hyperplasia and increases brown-like perivascular adipocytes. Collectively, the data suggest that MIA3 deficiency prevents neointimal formation by decreasing VSMC proliferation, migration, and inflammation and maintaining BAT-like perivascular adipocytes in PVAT during injury-induced vascular remodeling, which provide a potential therapeutic target for preventing neointimal hyperplasia in proliferative vascular diseases.

Inhibition of Vascular Smooth Muscle and Cancer Cell Proliferation by New VEGFR Inhibitors and Their Immunomodulator Effect: Design, Synthesis, and Biological Evaluation

Abnormal vascular smooth muscle cell (VSMC) proliferation has an important role in the pathogenesis of both atherosclerosis restenosis and hypertension. Vascular endothelial growth factor (VEGF) has been shown to stimulate VSMC proliferation. In addition, angiogenesis is one of the hallmarks of cancerous growth. VEGF is the key modulator for the initial stages of angiogenesis that acts through the endothelial-specific receptor tyrosine kinases (VEGFRs). VEGFR-2 blockage is a good approach for suppression of angiogenesis. In order to discover novel VEGFR-2 TK inhibitors, we have designed and synthesized three new series of pyridine-containing compounds. The new compounds were all screened against a panel of three cell lines (HepG-2, HCT-116, and MCF-7). Promising results encouraged us to additionally evaluate the most active members for their in vitro VEGFR-2 inhibitory effect. Compound 7a, which is the most potent candidate, revealed a significant increase in caspase-3 level by 7.80-fold when compared to the control. In addition, Bax and Bcl-2 concentration levels showed an increase in the proapoptotic protein Bax (261.4 Pg/ml) and a decrease of the antiapoptotic protein Bcl-2 (1.25 Pg/ml) compared to the untreated cells. Furthermore, compound 7a arrested the cell cycle in the G2/M phase with induction of apoptosis. The immunomodulatory effect of compound 7a, the most active member, showed a reduction in TNF-α by 87%. Also, compound 7a caused a potent inhibitory effect on smooth muscle proliferation. Docking studies were also performed to get better insights into the possible binding mode of the target compounds with VEGFR-2 active sites.

TET2 modulates proliferation and migration of vascular smooth muscle cells via LEMD2/NOX1/NOX4 signaling pathway

Background Ten-eleven translocation 2 (TET2), a widely reported DNA hydroxymethylation enzyme, is involved in active DNA demethylation. TET2 may play an critical role in numerous cellular processes by regulating the level of DNA hydroxymethylation and altering gene expression. TET2 expression was proved to be down-regulated in aorta of spontaneously hypertensive rats (SHR) compared to Wistar-Kyoto rats (WKY) as well as in VSMCs subjected to15% cyclic stretch compared to 5% cyclic stretch. However, whether TET2 regulates vascular smooth muscle cells (VSMCs) proliferation and migration and its underlying mechanisms remains unclear. Purpose The present study aims to investigate whether TET2 affects VSMC proliferation and migration and its possible underlying mechanisms. Methods TET2 knockdown rat VSMC was established using crispr/Cas9 method. Different expression genes were identified by next generation sequencing (NGS) between TET2 knockdown VSMC and control VSMCs while differentially hydroxymethylated promoter region were identified by hydroxymethylcytosine DNA immunoprecipitation (hMeDIP) sequencing. Expression of mRNA and proteins was detected by qRT-PCR and western blot respectively. The proliferation and migration of VSMCs were assessed by EdU assay, flowcytometry assay, and wound healing assay. LEMD2 overexpression and knockdown stable VSMC lines were established through Lentiviral infection. Results The EdU assay and wound healing assay show that knockdown of TET2 enhanced proliferation and migration of VSMCs. NGS identified 123 differentially expressed genes (91 up-regulated and 32 down-regulated) between TET2 knockdown VSMCs and control VSMCs while hMeDIP sequencing identified 166 genes with differentially hydroxymethylated promoter region (68 up-regulated and 98 down-regulated) between TET2 knockdown VSMCs and control VSMCs. Through comparing these sequencing results, we identified a gene named as LEMD2 simultaneously present in both sequencing results. Expression of LEMD2 at mRNA and protein level was significantly increased in TET2 knockdown VSMCs compared to control VSMCs. Then we successfully established LEMD2 overexpression and knockdown stable VSMC lines and found that overexpression of LEMD2 enhanced proliferation and migration of VSMCs while knockdown of LEMD2 inhibited proliferation and migration of VSMCs. Furthermore, overexpression of LEMD2 up-regulated the expression of NOX1 and down-regulated the expression of NOX4 while knockdown of LEMD2 show the opposite effect. Conclusion The present study confirmed that TET2 modulates VSMC proliferation and migration via LEMD2/NOX1/NOX4. The ROS level may be involved in VSMC proliferation and migration. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National natural science foundation of China

Expression and Functional Analysis of lncRNAs Involved in Platelet-Derived Growth Factor-BB-Induced Proliferation of Human Aortic Smooth Muscle Cells

Abnormal proliferation of vascular smooth muscle cells (VSMCs) is a common feature of many vascular remodeling diseases. Because long non-coding RNAs (lncRNAs) play a critical role in cardiovascular diseases, we analyzed the key lncRNAs that regulate VSMC proliferation. Microarray analysis identified 2,643 differentially expressed lncRNAs (DELs) and 3,720 differentially expressed coding genes (DEGs) between fetal bovine serum (FBS) starvation-induced quiescent human aortic smooth muscle cells (HASMCs) and platelet-derived growth factor-BB (PDGF-BB)-stimulated proliferative HASMCs. Gene Ontology and pathway analyses of the identified DEGs and DELs demonstrated that many lncRNAs were enriched in pathways related to cell proliferation. One of the upregulated lncRNAs in proliferative HASMC was HIF1A anti-sense RNA 2 (HIF1A-AS2). HIF1A-AS2 suppression decreased HASMC proliferation via the miR-30e-5p/CCND2 mRNA axis. We have thus identified key DELs and DEGs involved in the regulation of PDGF-BB induced HASMC proliferation. Moreover, HIF1A-AS2 promotes HASMC proliferation, suggesting its potential involvement in VSMC proliferative vascular diseases.