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Arterial calcification is a common cardiovascular disease that initiates from a process of osteoblastic differentiation of vascular smooth muscle cells (VSMCs). Accumulating evidence has demonstrated that microRNAs play an important role in regulating arterial calcification. miR-204 was significantly downregulated in calcified human renal arteries from patients with uremia; calcified arteries of mice, due to 5/6 nephrectomy with a high-phosphate diet (5/6 NTP); and in VSMCs induced by high phosphate concentration. The overexpression of miR-204 alleviated the osteoblastic differentiation of VSMCs. Bisulphite sequencing PCR revealed that CpG sites upstream of miR-204 DNA were hypermethylated in calcified VSMCs; in calcified arteries of mice, due to 5/6 NTP; and in calcified renal artery tissues from patients with uremia. Moreover, increased DNMT3a resulted in the hypermethylation of miR-204 in high phosphate concentration-induced VSMCs, whereas 5-aza-2'-deoxycytidine could restore the expression of miR-204 in high phosphate concentration-induced VSMCs. Moreover, we found that DNMT3a was the target of miR-204, and the methylation ratio of miR-204 was decreased significantly, meaning that the expression of miR-204 was restored when DNMT3a was knocked down by using DNMT3a small interfering RNA, resulting in abrogation of the effect of high phosphate concentration on VSMC calcification. The progress of arterial calcification is regulated by the miR-204/DNMT3a regulatory circuit.

Arterial calcification is a complex and active regulated process, which results from a process of osteoblastic differentiation of vascular smooth muscle cells (VSMCs). Leptin, the product of the ob gene, mainly regulates food intake and energy expenditure and recently has been considered to be correlated with the arterial calcification. However, the mechanisms of the effects of leptin on osteoblastic differentiation of VSMCs are unknown. We used calcifying vascular smooth muscle cells (CVSMCs) as a model to investigate the relationship between leptin and the osteoblastic differentiation of CVSMCs and the signaling pathways involved. Our experiments demonstrated that leptin could increase expression of receptor activator of nuclear factor-κB ligand (RANKL) and bone morphogenetic protein 4 (BMP4), as well as alkaline phosphatase (ALP) activity, runt-related transcription factor 2 expression, calcium deposition, and the formation of mineralized nodules in CVSMCs. Suppression of RANKL with small interfering RNA abolished the leptin-induced ALP activity and BMP4 expression in CVSMCs. Leptin could activate the ERK1/2 and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Furthermore, pretreatment with the ERK inhibitor PD98059 and the PI3K inhibitor LY294002 abolished leptin-induced RANKL expression and blocked the promotion of ALP activity of CVSMCs. Silencing of the leptin receptor OB-Rb with small interfering RNA abolished leptin-induced activation of ERK and Akt and the expression of RANKL and reversed the effects of leptin on ALP activity. Meanwhile, addition of Noggin (the BMP4 inhibitor) blunted the effect of leptin on ALP activity. These results show that leptin can promote osteoblastic differentiation of CVSMCs by the OB-Rb/ERK1/2/RANKL-BMP4 and OB-Rb/PI3K/Akt/RANKL-BMP4 pathways.

Aims: Medial artery calcification is a common macroangiopathy that initiates from a cell-regulated process similar to osteogenesis. Although the mechanisms governing this process remain unclear, epigenomic regulation by specific microRNAs might play a role in vascular smooth muscle cell (VSMC) calcification. In this study, we aimed to investigate whether miR-204 participates in the regulation of VSMC calcification., Methods and Results: We found that miR-204 was suppressed in mouse aortic VSMCs during β-glycerophosphate-induced calcification, whereas Runx2 protein levels were elevated. Overexpression of miR-204 by transfection of miR-204 mimics decreased Runx2 protein levels and alleviated β-glycerophosphate-induced osteoblastic differentiation of VSMCs, whereas miR-204 inhibition by transfection of miR-204 inhibitors significantly elevated Runx2 protein levels and enhanced osteoblastic differentiation of VSMCs, suggesting the role of miR-204 as an endogenous attenuator of Runx2 in VSMC calcification. Luciferase reporter assays revealed Runx2 as the direct target of miR-204 by overexpression of miR-204 on the wild-type or mutant 3'-UTR sequences of Runx2 in VSMCs. In vivo overexpression of miR-204 by injection of miR-204 agomirs in Kunming mice attenuated vitamin D3-induced medial artery calcification., Conclusion: Our study has shown that down-regulation of miR-204 may contribute to β-glycerophosphate-induced VSMC calcification through regulating Runx2. miR-204 represents an important new regulator of VSMC calcification and a potential therapeutic target in medial artery calcification.

Vascular calcification results from osteoblastic differentiation of vascular smooth muscle cells (VSMCs) and is a major risk factor for cardiovascular events. Ghrelin is a newly discovered bioactive peptide that acts as a natural endogenous ligand of the growth hormone secretagog receptor (GHSR). Several studies have identified the protective effects of ghrelin on the cardiovascular system, however research on the effects and mechanisms of ghrelin on vascular calcification is still quite rare. In this study, we determined the effect of ghrelin on osteoblastic differentiation of VSMCs and investigated the mechanism involved using the two universally accepted calcifying models of calcifying vascular smooth muscle cells (CVSMCs) and beta-glycerophosphate (beta-GP)-induced VSMCs. Our data demonstrated that ghrelin inhibits osteoblastic differentiation and mineralization of VSMCs due to decreased alkaline phosphatase (ALP) activity, Runx2 expression, bone morphogenetic protein-2 (BMP-2) expression and calcium content. Further study demonstrated that ghrelin exerted this suppression effect via an extracellular signal-related kinase (ERK)-dependent pathway and that the suppression effect of ghrelin was time dependent and dose dependent. Furthermore, inhibition of the growth hormone secretagog receptor (GHSR), the ghrelin receptor, by siRNA significantly reversed the activation of ERK by ghrelin. In conclusion, our study suggests that ghrelin may inhibit osteoblastic differentiation of VSMCs through the GHSR/ERK pathway.

Vascular calcification, which results from a process osteoblastic differentiation of vascular smooth muscle cells (VSMCs), is a major risk factor for cardiovascular morbidity and mortality. Apelin is a recently discovered peptide that is the endogenous ligand for the orphan G-protein-coupled receptor, APJ. Several studies have identified the protective effects of apelin on the cardiovascular system. However, the effects and mechanisms of apelin on the osteoblastic differentiation of VSMCs have not been elucidated. Using a culture of calcifying vascular smooth muscle cells (CVMSCs) as a model for the study of vascular calcification, the relationship between apelin and the osteoblastic differentiation of VSMCs and the signal pathway involved were investigated. Alkaline phosphatase (ALP) activity and osteocalcin secretion were examined in CVSMCs. The involved signal pathway was studied using the extracellular signal-regulated kinase (ERK) inhibitor, PD98059, the phosphatidylinositol 3-kinase (PI3-K) inhibitor, LY294002, and APJ siRNA. The results showed that apelin inhibited ALP activity, osteocalcin secretion, and the formation of mineralized nodules. APJ protein was detected in CVSMCs, and apelin activated ERK and AKT (a downstream effector of PI3-K). Suppression of APJ with siRNA abolished the apelin-induced activation of ERK and Akt. Furthermore, inhibition of APJ expression, and the activation of ERK or PI3-K, reversed the effects of apelin on ALP activity. These results showed that apelin inhibited the osteoblastic differentiation of CVSMCs through the APJ/ERK and APJ/PI3-K/AKT signaling pathway. Apelin appears to play a protective role against arterial calcification.

Several reports have shown that circulating insulin level is positively correlated with arterial calcification; however, the relationship between insulin and arterial calcification remains controversial and the mechanism involved is still unclear. We used calcifying vascular smooth muscle cells (CVSMCs), a specific subpopulation of vascular smooth muscle cells that could spontaneously express osteoblastic phenotype genes and form calcification nodules, to investigate the effect of insulin on osteoblastic differentiation of CVSMCs and the cell signals involved. Our experiments demonstrated that insulin could promote alkaline phosphatase (ALP) activity, osteocalcin expression and the formation of mineralized nodules in CVSMCs. Suppression of receptor activator of nuclear factor κB ligand (RANKL) with small interfering RNA (siRNA) abolished the insulin-induced ALP activity. Insulin induced the activation of extracellular signal-regulated kinase (ERK)1/2, mitogen-activated protein kinase (MAPK) and RAC-alpha serine/threonine-protein kinase (Akt). Furthermore, pretreatment of human osteoblasts with the ERK1/2 inhibitor PD98059, but not the phosphoinositide 3-kinase (PI3K) inhibitor, LY294002, or the Akt inhibitor, 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (HIMO), abolished the insulin-induced RANKL secretion and blocked the promoting effect of insulin on ALP activities of CVSMCs. Recombinant RANKL protein recovered the ALP activities decreased by RANKL siRNA in insulin-stimulated CVSMCs. These data demonstrated that insulin could promote osteoblastic differentiation of CVSMCs by increased RANKL expression through ERK1/2 activation, but not PI3K/Akt activation.