Downregulation of TRPC4 and TRPC5 Inhibits Smooth Muscle Cell Proliferation without Affecting Endothelial Cell Proliferation

Aims. The main treatment for coronary heart disease is percutaneous coronary intervention (PCI), and drug-eluting stents are designed to inhibit vascular smooth muscle cell (VSMCs) proliferation and migration causing restenosis by releasing pharmacological agents into the vessel wall. Once drug-eluting stents are deployed, these pharmacological agents exert many biological effects in the coronary circulation, not only inhibition of VSMCs but also extension to vascular endothelial cells (VECs). The purpose of this study was to explore target molecules that inhibit VSMCs proliferation without affecting VECs. Methods. mRNA and protein expressions of transient receptor potential channels (TRPCs) in cultured VSMCs and VECs were determined by western blotting and RT-qPCR. VSMCs and VECs proliferation was evaluated using CCK-8 assays and western blotting of proliferating cell nuclear antigen (PCNA). Calcium backfilling assays were performed to detect intracellular calcium ion concentration in cultured VSMCs and VECs. Results. The TRPC6 expression was more abundant in VECs than VSMCs, while TRPC4 and TRPC5 expressions were more abundant in VSMCs than VECs. Knockdown of TRPC4 or TRPC5 alone had no remarkable inhibitory effect on VSMC proliferation. Synergistic knockdown of TRPC4 and TRPC5 inhibited the proliferation of VSMCs, declined the expression of the PCNA, and reduced the intracellular calcium ion concentration but not VECs. Conclusion. These data suggest that concurrent inhibition of TRPC4 and TRPC5 inhibits VSMCs proliferation without affecting VECs, thus providing novel targets for developing pharmacological agents for drug-eluting stents.

Silence of Insulin-Like Growth Factor 2 mRNA-Binding Protein 1 Prevents Vascular Smooth Muscle Cells Proliferation via Nuclear Factor of Activated T Cells Isoform-3/Ca2+/Calmodulin Pathway

Increased proliferation of vascular smooth muscle cells (VSMCs) contributes to the pathogenesis of atherosclerosis (AS), and the insulin like growth factor 2 (IGF2) is involved in AS through effects on VSMCs growth and migration. The IGF2 mRNA-binding protein 1(IGF2BP1) is a secreted protein that can bind to IGF2 and regulate its localization, however, whether IGF2BP1 could regulate VSMCs proliferation remains to be elucidated. This study aimed to investigate the role of IGF2BP1 in VSMCs proliferation and uncover the potential mechanism. Primary human aortic VSMCs that transfected with or without shRNA-IGF2BP1 were stimulated by platelet-derived growth factor-BB (PDGF-BB), and then cell proliferation, intracellular Ca2+ level, cell apoptosis and the expression of IGF2BP1, calmodulin (CaM) and cell cycle-related proteins were detected. RNA pull down assay was used to determine the interaction between IGF2BP1 and nuclear factor of activated T cells isoform-3 (NFATc3). We found that PDGF-BB promoted cell proliferation and enhanced IGF2BP1 protein expression in a concentration-dependent manner. The 10 μg/L PDGF-BB significantly increased intracellular Ca2+ level, NFATc3, CaM and calcineurin A protein expression, TUNEL-positive cells, and the expression of cell cycle-related proteins cyclin D1/E1/B1. However, knockdown of IGF2BP1 significantly blunted all these effects induced by PDGF-BB. In addition, IGF2BP1 could bind to NFATc3 RNA. Collectively, knockdown of IGF2BP1 could inhibit PDGFBB- induced VSMCs proliferation via targeting NFATc3/Ca2+/calmodulin pathway and disturbing the effect of NFATc3/ on cell cycle.

Exogenous H2S Preventing PDC-E1 Translocation to Nucleus and Inhibiting Vascular Smooth Muscle Cell Proliferation in Diabetic State

BackgroundThe poliferation of vascular smooth muscle cells (VSMCs) is the main cause of diabetic vascular complications. Hydrogen sulfide (H2S), a gaseous molecule, is involved in modulating multiple physiological functions. H2S could inhibit VSMCs proliferation induced by hyperglycemia and hyperlipidemia, however, the mechanisms are unclear. ResultsOur results showed that H2S level was lower and expression of proliferative protein for PCNA and CyclinD1 was higher in db/db mice aorta and VSMC treated by glucose and palmitate, whereas, exogenous H2S decreased PCNA and CyclinD1 expression. We found that mitochondrial pyruvate dehydrogenase complex-E1 (PDC-E1) was significantly translocated to the nucleus of VSMCs with the treatment of high glucose and palmitate, and it increased the level of acetyl-CoA and histone acetylation (H3K9Ac). Exogenous H2S inhibited PDC-E1 translocation from mitochondria to nucleus, due to PDC-E1 being modified via S-sulfhydration. In addition, PDC-E1 was mutated at Cys101. Overexpression of PDC-E1 mutated at Cys101 enhanced histone acetylation (H3K9Ac) and VSMCs proliferation.ConclusionsThese findings suggested that H2S regulated PDC-E1 S-sulfhydration at Cys101 to prevent its translocation from mitochondria to nucleus and inhibit VSMCs proliferation in diabetic conditions.

miR-132-3p mediates Functions of Vascular Smooth Muscle Cells through targeting PTEN expression and ERK1/2 pathway

Background This study was aimed to investigate the functional role of the microRNA (miR)-132-3p in rat vascular smooth muscle cells (VSMCs) and the potential mechanisms in abdominal aortic aneurysm (AAA). Methods VSMCs were transfected with miR-132-3p mimics and inhibitors, and the effects of miR-132-3p on VSMCs proliferation, migration were assessed by CCK-8 assay and Boyden chamber cell invasion assay, respectively. miRNA targets were determined using bioinformatics and luciferase reporter assays. The protein expression of phenotype markers and related signaling pathways were detected by Western blot. Results Overexpression of miR-132-3p in VSMCs attenuated VSMCs proliferation and migration. Conversely, the opposite effect was obtained with the inhibition of miR-132-3p. We further demonstrated that miR-132 could significantly promote the expression of VSMCs marker genes ACTA2 and MYH1. Reporter assays and western blot validated that PTEN as a direct target of miR-132-3p in VSMCs. Besides, miR-132-3p overexpression could also promote the expression of ras and c-myc, and activate the phosphorylation levels of ERK1/2. Conclusions These results indicate that miR-132-3p is a critical regulator in maintaining normal functions of VSMCs through PTEN-ERK1/2 axis. Restoring expression of miR-132-3p may serve as a potential therapeutic approach for treatment of AAAs.

miR-223 Promotes Smooth Muscle Cell Proliferation and Atherosclerotic Plaque Formation by Inhibiting Phosphatase and Tensin Homolog (PTEN)/ Phosphatidylinositol 3-Kinase (PI3K) and Protein Kinase B (AKT) Signaling Pathway

Abnormal vascular smooth muscle cells (VSMCs) proliferation is the pathological basis of atherosclerosis (AS) pathogenesis. miR-223 is abnormally expressed in AS plaques and affects the proliferation of VSMCs, but the mechanism of miR-223 affecting the proliferation of VSMCs is unclear. Our study intends to investigate the mechanism of miR-223 in VSMCs proliferation and AS formation. Healthy SD rats and miR-223 knockout SD rats took high-fat diet to induce AS model. Oil red O staining was done to observe AS formation. miR-223 mimics/NC was transferred to VSMCs followed by analysis of miR-214 expression by real-time PCR, cell proliferation by CCK8 assay, phosphatase and tensin homolog gene (PTEN) level by Western blot detection. Compared with control group, after knocking out miR-223, the AS level was significantly decreased and PTEN expression was significantly elevated (P < 0 05). After transfection of miR-223 mimics into VSMCs, PTEN expression protein was significantly decreased and the number of cells was increased (P < 0 05). In addition, the luciferase signal of miR-223 mimics and pmirGLO-PTEN-3 UTR-wt co-transfection group was significantly reduced (P < 0 05). miR-223 promotes VSMCs proliferation and AS plaque formation by targeting PTEN/PI3K/Akt signaling pathway.

Abstract WMP32: Osteoprotegerin Attenuates the Growth of Intracranial Aneurysms Activating Collagen Biosynthesis and Vascular Smooth Muscle Cell Proliferation via Transforming Growth Factor-Beta1 Signaling

Introduction: The pathogenesis of intracranial aneurysms (IAs) are characterized by reduced extracellular matrix and decreased number of vascular smooth muscle cells (VSMCs) in cerebral vascular walls. It is previously reported that osteoprotegerin (OPG) increase collagen content and activate VSMCs proliferation via transforming growth factor-beta1 (TGF-beta1). We investigated whether to attenuate IAs growth through the effect of OPG on collagen expression and VSMCs proliferation in IAs model rats. Materials and Methods: IAs were surgically induced in 7-week-old male Sprague Dawley rats. 1 week after the operation, mouse recombinant OPG at 125 μg/mL or vehicle was continuously infused into the lateral ventricle via an osmotic pump. 5 weeks after the first operation, aneurysmal size, media thickness of IAs were measured in the both groups. The expression of type-I and type-III collagen, TGF-beta1 and phosphorylated Smad2/3 in IA walls were examined by immunohistochemistry and RT-PCR. Primary cell culture of mouse VSMCs were analyzed by RT-PCR and the cell proliferation assay with SB431542 (a selective TGF-β receptor I inhibitor). Results: In the OPG treatment group, size and media thickness ratio of IAs were significantly smaller (control vs. OPG: 42 ± 6.5 μm vs 66 ± 6.1 μm; P < 0.05) and higher (control vs. OPG: 55 ± 7 % vs 34 ± 3 %; P < 0.05) than in the vehicle-control group. In the immunohistochemistry, collagen and phosphorylated Smad2/3 were upregulated in IA walls in the OPG group. In the RT-PCR study, OPG treatment significantly upregulated expression of collagen and TGF-beta1 genes ( P < 0.05). In mouse VSMC cultures, OPG administration enhanced the expression of collagen and TGF-β1 genes. The cell proliferation assay revealed the OPG administration promoted VSMCs proliferation and SB431542 canceled this effect ( P < 0.05). Conclusions: Our results demonstrate that OPG has a suppressive effect on IAs growth through the activation of collagen biosynthesis and VSMC proliferation via TGF-beta1 signaling in IA walls. OPG may represent a novel therapeutic target of the medical treatment for IAs.

MicroRNA-183-5p acts as a potential diagnostic biomarker for atherosclerosis and regulates the growth of vascular smooth muscle cell

Background Atherosclerosis (AS) is a multifactorial chronic disease, and vascular smooth muscle cells (VSMCs) plays an important role in the pathology of AS. MicroRNAs regulate multiple cellular biological processes. This study aimed to investigate the clinical value of miR-183-5p in AS patients, and further explored the effects of miR-183-5p on the proliferation and migration of VSMCs. Methods qRT-PCR was used to test the level of miR-183-5p. The diagnostic value of miR-183-5p for AS patients was assessed by a receiver operating characteristic (ROC) analysis. Cell proliferation and migration were determined via CCK-8 and Transwell assay. Results MiR-183-5p was highly expressed in AS patients compared with the healthy group. Serum miR-183-5p expression was positively associated with CIMT and CRP in AS patients. The ROC analysis suggested that miR-183-5p had quality to be used as a biomarker with high specificity and sensitivity for AS detection. Overexpression of miR-183-5p promoted the proliferation and migration of VSMCs. Downregulation of miR-183-5p attenuated ox-LDL stimulated VSMCs proliferation and migration. Conclusion MiR-183-5p is highly expressed in AS patients, and downregulation of miR-183-5p attenuated ox-LDL stimulated VSMCs proliferation and migration. MiR-183-5p may be a key molecular for the diagnosis and treatment of AS in the future.