Micrornas And Apelin Regulate The Process Of Vascular Smooth Muscle Cells Differentiating Into Osteoblast-like Cells And Their Apoptosis

Part one Mechanisms of the effects of microRNA-204on the regulation of osteoblastic transdifferentiation of vascular smooth muscle cellsObjectiveMedial artery calcification is a common macroangiopathy that initiates from a cell-regulated process of osteoblastic transdifferentiation of vascular smooth muscle cells (VSMCs). Although the mechanisms governing this process remain unclear, epigenomic regulation by specific microRNAs might play a role in it. In this study, we aimed to investigate whether miR-204participates in regulating osteoblastic transdifferentiation of VSMCs.MethodsPrimary mouse VSMCs were treated with β-glycerophosphate to induce calcification. The expression of miR-204was tested by real-time quantitative PCR. Runx2protein levels were evaluated by western blot analysis. Overexpression of miR-204was obtained by transfection with miR-204mimics. Inhibition of miR-204was obtained by transfection with miR-204inhibitors. Runx2portein levels and osteoblastic differentiation markers were evaluated both before and after induction of VSMC calcification by P-glycerophosphate. Luciferase reporter assay was carried out to investigate if Runx2was the direct target of miR-204.ResultsWe found that miR-204was suppressed in mouse aortic VSMCs during β-glycerophosphate induced calcification, whereas Runx2protein levels were elevated. Overexpression of miR-204by transfection of miR-204mimics decreased Runx2protein levels and alleviated β-glycerophosphate induced osteoblastic differentiation of VSMCs while miR-204inhibition by transfection of miR-204inhibitors significantly elevated Runx2protein levels and enhanced osteoblastic transdifferentiation of VSMCs, suggesting miR-204's role as an endogenous attenuator of Runx2in osteoblastic transdifferentiation of VSMCs. Luciferase reporter assays revealed Runx2as the direct target of miR-204by overexpression of miR-204on wild-type or mutant3'-UTR sequences of Runx2in VSMCs.ConclusionOur study show that down-regulation of miR-204may contribute to β-glycerophosphate induced VSMC osteoblastic transdifferentiation through regulating Runx2. miR-204represents an important new regulator of osteoblastic transdifferentiation of VSMCs. ObjectiveArtery calcicification plays a critical role in the pathogenesis of numerous cardiovascular diseases. Recent evidence has suggested that microRNAs (miRNA) might play a role in vascular smooth muscle cell (VSMC) calcification. VSMC calcification initiates from an osteoblastic differentiation process. The aim of the present study is to expore novel miRNAs participating in regulating osteoblastic transdifferentiation of VSMCs.MethodsSmall RNAs were extracted from primary mouse VSMCs. After polyadenylation and ligation with a5'RNA adapter, the cDNAs were obtained by reverse transcription, amplified by PCR, inserted into pcDNA3.1TOPO cloning vectors, and sequenced. Novel miRNAs were detected by Bioinformatic analysis and verified by Northern blotting. Bioinformatic analysis was also carried out to detect the target of the novel miRNAs. Modulating the novel miRNA(miR-X) was achieved by transfection of pre-miR-X(gain-of-function) and miR-X inhibitors (loss-of-function). The effect of miR-X on osteoblastic differentiation markers and target gene expression was investigated after transfection. ResultsA novel miRNA (miR-X) was found preferentially expressed in VSMCs, and its expression level was decreased with the process of osteoblastic transdifferentiation of VSMCs. The levels of ALP activity, OC secretion, Runx2protein levels and mineralization were inhibited by transfection of pre-miR-X. Treatment with miR-X inhibitors attenuated the levels of ALP activity,OC secretion and Runx2protein levels induced by β-glycerophosphate. FosB is the target gene predicted by miRNA target prediction software tools. Overexpression of miR-X downregulated endogenous FosB protein. By contrast, no change in FosB mRNA levels was noted.ConclusionThe results suggested that the downregulation of miR-X may contribute to β-glycerophosphate induced osteoblastic transdifferentiation of VSMCs through regulating its target gene FosB. Thus, miR-X might play an important role in artery calcification. ObjectiveApoptosis of vascular smooth muscle cells (VSMCs) plays an important role in regulating vascular remodeling during cardiovascular diseases. Apelin is the endogenous ligand for the G-protein-coupled receptor APJ and plays an important role in the cardiovascular system. However, apelin's effect on apoptosis of VSMCs and its underlying mechanisms have not been elucidated. Using a culture of primary human VSMCs as a model for the study of apoptosis, the relationship between apelin and apoptosis of human VSMCs and the signal pathway involved were investigated.MethodsUsing western blot analysis, we investigated the expression of APJ in primary human VSMCs. To evaluate the possible role of apelin in VSMC apoptosis, we assessed its effect on apoptosis of human VSMCs by TUNEL and cell death ELISA essay. Western blot analysis was carried out to evaluate the expression of Bcl-2and Bax protein expression and the activation of MAPK and Akt signaling after apelin stimulati Expression of APJ was inhibited by small-interfering RNA (siRNA). Specific signaling inhibitors were used to block the activation of specific kinases.The activation of MAPK and Akt and the apoptosis of VSMCs was evaluated after the inhibition.ResultsWe confirmed the expression of APJ in VSMCs by western blot analysis. Apelin inhibited human VSMCs apoptosis induced by serum deprivation. Suppression of APJ with siRNA abolished the anti-apoptotic activity of apelin. Apelin increased Bcl-2protein expression, but decreased Bax protein expression. An increase in activation of ERK and Akt was shown after apelin stimulation. siRNA of APJ abolished the apelin-induced activation of extra-cellular signal-regulated protein kinase (ERK) and Akt. LY294002(a PI3-K inhibitor) blocked apelin-induced activation of Akt and abolished the apelin induced antiapoptotic activity.ConclusionOur study suggests that apelin suppresses serum deprivation-induced apoptosis of human VSMCs, and that the anti-apoptotic action is mediated through the APJ/PI3-K/Akt signaling pathways.