Taurine inhibits osteoblastic differentiation of vascular smooth muscle cells via the ERK pathway

Vascular calcification develops within atherosclerotic lesions and results from a process similar to osteogenesis. Taurine is a free β-amino acid and plays an important physiological role in mammals. We have recently demonstrated that vascular smooth muscle cells (VSMCs) express a functional taurine transporter. To evaluate the possible role of taurine in vascular calcification, we assessed its effects on osteoblastic differentiation of VSMCs in vitro. The results showed that taurine inhibited the β-glycerophosphate-induced osteoblastic differentiation of VSMCs as evidenced by both the decreasing alkaline phosphate (ALP) activity and expression of the core binding factor α1 (Cbfα1). Taurine also activated the extracellular signal-regulated protein kinase (ERK) pathway. Inhibition of ERK pathway reversed the effect of taurine on ALP activity and Cbfα1 expression. These results suggested that taurine inhibited osteoblastic differentiation of vascular cells via the ERK pathway.     

Adiponectin attenuates the osteoblastic differentiation of vascular smooth muscle cells through the AMPK/mTOR pathway

Vascular calcification is common in patients with peripheral artery diseases and coronary artery diseases. The osteoblastic differentiation of vascular smooth muscle cells (VSMCs) contributes significantly to vascular calcification. Adiponectin has been demonstrated to exert a protective effect in osteoblastic differentiation of VSMCs through regulating mTOR activity. However, the upstream and downstream signaling molecules of adiponectin-regulated mTOR signaling have not been identified in VSMCs with osteoblastic differentiation. In this study, the VSMC differentiation model was established by beta-glycerophosphate (β-GP) induction. The mineralization was identified by Alizarin Red S staining. Protein expression and phosphorylation were detected by Western blot or immunofluorescence. Adiponectin attenuated osteoblastic differentiation and mineralization of β-GP-treated VSMCs. Adiponectin inhibited osteoblastic differentiation of VSMCs through increasing the level of p-AMPKα. Pretreatment of VSMCs with AMPK inhibitor blocked while AMPK activator enhanced the effect of adiponectin on osteoblastic differentiation of VSMCs. Adiponectin upregulated TSC2 expression and downregulated mTOR and S6K1 phosphorylation in β-GP-treated VSMCs. Adiponectin treatment significantly attenuates the osteoblastic differentiation and calcification of VSMCs through modulation of AMPK–TSC2–mTOR–S6K1 signal pathway.     

Apelin attenuates the osteoblastic differentiation of vascular smooth muscle cells

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.     

Ghrelin attenuates the osteoblastic differentiation of vascular smooth muscle cells through the ERK pathway

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.     

Lanthanum suppresses osteoblastic differentiation via pertussis toxin‐sensitive G protein signaling in rat vascular smooth muscle cells

A major cellular event in vascular calcification is the phenotypic transformation of vascular smooth muscle cells (VSMCs) into osteoblast‐like cells. After demonstrating that lanthanum chloride (LaCl₃) suppresses hydrogen peroxide‐enhanced calcification in rat calcifying vascular cells (CVCs), here we report its effect on the osteoblastic differentiation of rat VSMCs, a process leading to the formation of CVCs. Cells were isolated from aortic media of male SD rats, and passages between three and eight were cultured in Dulbeccol's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS) and 10 mM β‐glycerophosphate (β‐GP) in the presence or absence of LaCl₃. Exposure of cells to LaCl₃ suppressed the β‐GP‐induced elevations in calcium deposition, alkaline phosphatase (ALP) activity, and Cbfa1/Runx2 expression, as well as the concomitant loss of SM α‐actin. Furthermore, LaCl₃ activated the phosphorylation of extracellular signal‐regulated kinase (ERK) and c‐Jun N‐terminal kinase (JNK), and the blockage of either pathway with a specific inhibitor abolished the effects of LaCl₃. In addition, pretreatment of the cells with pertussis toxin (PTx), an inhibitor of G protein‐mediated signaling pathway, repealed all the changes induced by LaCl₃. These findings demonstrate that LaCl₃ suppresses the β‐GP‐induced osteoblastic differentiation and calcification in rat VSMCs, and its effect is mediated by the activation of both ERK and JNK MAPK pathways via PTx‐sensitive G proteins. J. Cell. Biochem. 108: 1184–1191, 2009. © 2009 Wiley‐Liss, Inc.     

Taurine restores Axl/Gas6 expression in vascular smooth muscle cell calcification model

Our previous studies demonstrated that taurine inhibits osteoblastic differentiation of vascular smooth muscular cells (VSMCs) via the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway, but the underlying mechanism is not elucidated. The tyrosine kinase receptor Axl and its ligand growth arrest-specific protein 6 (Gas6) are expressed in VSMCs. Axl/Gas6 signaling system is known to inhibit VSMCs calcification. We herein showed that taurine partially restored Axl and Gas6 expression in β-glycerophosphate (β-GP)-induced VSMC calcification model. Taurine also induced activation of ERK, but not other two MAPKs including c-jun N-terminal Kinase (JNK) and p38 in VSMCs. Either knockdown of the taurine transporter (TAUT) or treatment with the ERK-specific inhibitor PD98059 blocked the activation of ERK by taurine and abolished taurine-induced Axl/Gas6 expression and calcium deposition reduction in β-GP-induced VSMC calcification model. These results demonstrate for the first time that taurine stimulates expression of Axl and Gas6 via TAUT/ERK signaling pathway in β-GP-induced VSMC calcification model.     

Superoxide production: A procalcifying cell signalling event in osteoblastic differentiation of vascular smooth muscle cells exposed to calcification media

Recent studies showed that hydrogen peroxide (H₂O₂) enhanced bone markers expression in vascular smooth muscle cells (VSMCs) implicated in osteoblastic differentiation. This study aimed at investigating the role of NAD(P)H oxidase in vascular calcification processes. A7r5 rat VSMCs were incubated with β-glycerophosphate (10 mm) or uremic serum to induce a diffuse mineralization. H₂O₂ production by VSMCs was determinated by chemiluminescence. NAD(P)H oxidase sub-unit (p22^phox), Cbfa-1, ERK phosphorylation and bone alkaline phosphatase (ALP) expressions were measured by Western blotting. VSMCs exhibited higher production of H₂O₂ and early expression of p22^phox with β-glycerophosphate or uremic serum within 24 h of treatment. β-glycerophosphate-induced oxidative stress was associated with Cbfa-1 expression followed by ALP expression and activity, meanwhile the VSMCs expressing ALP diffusely calcified their extracellular matrix. Interestingly, diphenyleneiodonium partly prevented the osteoblastic differentiation. Results from this model strongly suggest a major implication of vascular NAD(P)H oxidase in vascular calcification supported by VSMCs osteoblastic differentiation.     

Macrophages play a unique role in the plaque calcification by enhancing the osteogenic signals exerted by vascular smooth muscle cells

Vascular calcification is a major risk factor for the cardiovascular disease, yet its underlying molecular mechanisms remain to be elucidated. Recently, we identified that osteogenic signals via bone morphogenetic protein (BMP)-2 exerted by vascular smooth muscle cells (VSMCs) play a crucial role in the formation of atherosclerotic plaque calcification. Here we report a synergistic interaction between macrophages and VSMCs with respect to plaque calcification. Treatment with conditioned medium (CM) of macrophages dramatically enhanced BMP-2 expression in VSMCs, while it substantially reduced the expression of matrix Gla-protein (MGP) that inhibits the BMP-2 osteogenic signaling. As a result, macrophages significantly accelerated the osteoblastic differentiation of C2C12 cells induced by VSMC-CM. In contrast, macrophage-CM did not enhance the osteoblastic gene expressions in VSMCs, indicating that macrophages unlikely induced the osteoblastic trans-differentiation of VSMCs. We then examined the effect of recombinant TNF-α and IL-1β on the VSMC-derived osteogenic signals. Similar to the macrophage-CM, both cytokines enhanced BMP-2 expression and reduced MGP expression in VSMCs. Nevertheless, only the neutralization of TNF-α but not IL-1β attenuated the effect of macrophage-CM on the expression of these genes in VSMCs, due to the very low concentration of IL-1β in the macrophage-CM. On the other hand, VSMCs significantly enhanced IL-1β expression in macrophages, which might in turn accelerate the VSMC-mediated osteogenic signals. Together, we identified a unique role of macrophages in the formation of plaque calcification in coordination with VSMCs. This interaction between macrophages and VSMCs is a potential therapeutic target to treat and prevent the atherosclerotic plaque calcification.     

Phenotypic modulation of cultured vascular smooth muscle cells: a functional analysis focusing on MLC and ERK1/2 phosphorylation

Primary cultures of vascular smooth muscle cells (VSMCs) from rats offer a good model system to examine the molecular basis of mechanism of vascular contraction–relaxation. However, during pathological conditions such as atherosclerosis and hypertension, VSMCs characteristically exhibit phenotypic modulation, change from a quiescent contractile to a proliferative synthetic phenotype, which impairs this mechanism of vascular contraction–relaxation. Taking in account that Myosin light chain (MLC) and ERK1/2 directly participate in the process of vascular contraction, the aim of the current study was to analyze the involvement of MLC and ERK1/2 signaling during the process of VSMCs phenotypic modulation. Primary cultures of VSMCs from rat thoracic aortas were isolated and submitted to different number of passages or to freezing condition. Semi-quantitative RT-PCR was used to evaluate the mRNA levels of VSMCs differentiation markers, and western blot assays were used to determine the MLC and ERK1/2 phosphorylation levels during VSMCs phenotypic modulation. Also, immunocytochemical experiments were performed to evaluate morphological alterations occurred during the phenotypic modulation. Elevated number of passages (up to 4) as well as the freezing/thawing process induced a significant phenotypic modulation in VSMCs, which was accompanied by diminished MLC and ERK1/2 phosphorylation levels. Phosphorylation of MLC was suppressed completely by the treatment with a synthetic inhibitor of MEK-1, a direct upstream of ERK1/2, PD98059. These findings provide that ERK1/2-promoted MLC phosphorylation is impaired during VSMCs phenotypic modulation, suggesting that ERK1/2 signaling pathway may represent a potential target for understanding the pathogenesis of several vascular disease processes frequently associated to this condition.     

Selenium suppresses oxidative-stress-enhanced vascular smooth muscle cell calcification by inhibiting the activation of the PI3K/AKT and ERK signaling pathways and endoplasmic reticulum stress

Vascular calcification is a prominent feature of many diseases, including atherosclerosis, and it has emerged as a powerful predictor of cardiovascular morbidity and mortality. A number of studies have examined the association between selenium and risk of cardiovascular diseases, but little is known about the role of selenium in vascular calcification. To determine the role of selenium in regulating vascular calcification, we assessed the effect of sodium selenite on oxidative-stress-enhanced vascular smooth muscle cell (VSMC) calcification and the underlying mechanism. Oxidative stress induced by xanthine/xanthine oxidase increased apoptosis, as determined by Hoechst 33342 staining and annexin V/propidium iodide staining, and it enhanced osteoblastic differentiation and calcification of VSMCs, on the basis of alkaline phosphatase activity, the expression of Runx2 and type I collagen, and calcium deposition. These effects of oxidative stress were significantly inhibited by selenite. The following processes may explain the inhibitory effects of selenite: (1) selenite significantly suppressed oxidative stress, as evidenced by the decrease of the oxidative status of the cell and lipid peroxidation levels, as well as by the increase of the total protein thiol content and the activity of the antioxidant selenoenzyme glutathione peroxidase; (2) selenite significantly attenuated oxidative-stress-induced activation of the phosphatidylinositol 3-kinase/AKT and extracellular-signal-regulated kinase signaling pathways, resulting in decreased osteoblastic differentiation of VSMCs; (3) selenite significantly inhibited oxidative-stress-activated endoplasmic reticulum stress, thereby leading to decreased apoptosis. Our results suggest a potential role of selenium in the prevention of vascular calcification, which may provide more mechanistic insights into the relationship between selenium and cardiovascular diseases.     

Osteoprotegerin Inhibits Calcification of Vascular Smooth Muscle Cell via Down Regulation of the Notch1-RBP-Jκ/Msx2 Signaling Pathway

Objective

Vascular calcification is a common pathobiological process which occurs among the elder population and in patients with diabetes and chronic kidney disease. Osteoprotegerin, a secreted glycoprotein that regulates bone mass, has recently emerged as an important regulator of the development of vascular calcification. However, the mechanism is not fully understood. The purpose of this study is to explore novel signaling mechanisms of osteoprotegerin in the osteoblastic differentiation in rat aortic vascular smooth muscle cells (VSMCs).

Methods and Results

VSMCs were isolated from thoracic aorta of Sprague Dawley rats. Osteoblastic differentiation of VSMCs was induced by an osteogenic medium. We confirmed by Von Kossa staining and direct cellular calcium measurement that mineralization was significantly increased in VSMCs cultured in osteogenic medium; consistent with an enhanced alkaline phosphatase activity. This osteoblastic differentiation in VSMCs was significantly reduced by the addition of osteoprotegerin in a dose responsive manner. Moreover, we identified, by real-time qPCR and western blotting, that expression of Notch1 and RBP-Jκ were significantly up-regulated in VSMCs cultured in osteogenic medium at both the mRNA and protein levels, these effects were dose-dependently abolished by the treatment of osteoprotegerin. Furthermore, we identified that Msx2, a downstream target of the Notch1/RBP-Jκ signaling, was markedly down-regulated by the treatment of osteoprotegerin.

Conclusion

Osteoprotegerin inhibits vascular calcification through the down regulation of the Notch1-RBP-Jκ signaling pathway.     

The appearance and modulation of osteocyte marker expression during calcification of vascular smooth muscle cells

Background

Vascular calcification is an indicator of elevated cardiovascular risk. Vascular smooth muscle cells (VSMCs), the predominant cell type involved in medial vascular calcification, can undergo phenotypic transition to both osteoblastic and chondrocytic cells within a calcifying environment.

Methodology/Principal Findings

In the present study, using in vitro VSMC calcification studies in conjunction with ex vivo analyses of a mouse model of medial calcification, we show that vascular calcification is also associated with the expression of osteocyte phenotype markers. As controls, the terminal differentiation of murine calvarial osteoblasts into osteocytes was induced in vitro in the presence of calcifying medium (containing ß-glycerophosphate and ascorbic acid), as determined by increased expression of the osteocyte markers DMP-1, E11 and sclerostin. Culture of murine aortic VSMCs under identical conditions confirmed that the calcification of these cells can also be induced in similar calcifying medium. Calcified VSMCs had increased alkaline phosphatase activity and PiT-1 expression, which are recognized markers of vascular calcification. Expression of DMP-1, E11 and sclerostin was up-regulated during VSMC calcification in vitro. Increased protein expression of E11, an early osteocyte marker, and sclerostin, expressed by more mature osteocytes was also observed in the calcified media of Enpp1^−/− mouse aortic tissue.

Conclusions/Significance

This study has demonstrated the up-regulation of key osteocytic molecules during the vascular calcification process. A fuller understanding of the functional role of osteocyte formation and specifically sclerostin and E11 expression in the vascular calcification process may identify novel potential therapeutic strategies for clinical intervention.     

Extracellular transglutaminase 2 activates beta-catenin signaling in calcifying vascular smooth muscle cells

Accumulation of transglutaminase 2 (TG2) is often associated with mineral deposits in vasculature. Here, we demonstrate that purified TG2 stimulated a 3-fold increase in matrix mineralization and up-regulation of osteoblastic markers in cultured primary vascular smooth muscle cells (VSMCs). Extracellular TG2 interacts with the low density lipoprotein related-protein 5 receptor and activates beta-catenin signaling in VSMCs. These results suggest that TG2 may promote vascular calcification by activating the beta-catenin signaling pathway.     

miR-125b/Ets1 axis regulates transdifferentiation and calcification of vascular smooth muscle cells in a high-phosphate environment

Objectives

Vascular calcification is highly prevalent in patients with chronic kidney disease (CKD) and contributes to increased risk of cardiovascular disease and mortality. Accumulated evidences suggested that vascular smooth muscle cells (VSMCs) to osteoblast-like cells transdifferentiation (VOT) plays a crucial role in promoting vascular calcification. MicroRNAs (miRNAs) are a novel class of small RNAs that negatively regulate gene expression via repression of the target mRNAs. In the present work, we sought to determine the role of miRNAs in VSMCs phenotypic transition and calcification induced by β-glycerophosphoric acid.

Approach and results

Primary cultured rat aortic VSMCs were treated with β-glycerophosphoric acid for different periods of time. In VSMCs, after β-glycerophosphoric acid treatment, the expressions of cbf β1, osteocalcin and osteopontin were significantly increased and SM-22β expression was decreased. ALP activity was induced by β-glycerophosphoric acid in a time or dose dependent manner. Calcium deposition was detected in VSMCs incubated with calcification media; then, miR-125b expression was detected by real-time RT PCR. miR-125b expression was significantly decreased in VSMCs after incubated with β-glycerophosphoric acid. Overexpression of miR-125b could inhibit β-glycerophosphoric acid-induced osteogenic markers expression and calcification of VSMCs whereas knockdown of miR-125b promoted the phenotypic transition of VSMCs and calcification. Moreover, miR-125b targeted Ets1 and regulated its protein expression in VSMCs. Downregulating Ets1 expression by its siRNA inhibited β-glycerophosphoric acid-induced the VSMCs phenotypic transition and calcification.

Conclusion

Our study suggests that down-regulation of miR-125b after β-glycerophosphoric acid treatment facilitates VSMCs transdifferentiation and calcification through targeting Ets1.     

RANKL is a downstream mediator for insulin-induced osteoblastic differentiation of vascular smooth muscle cells

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.     

Phosphate and pyrophosphate mediate PKA-induced vascular cell calcification

Vascular calcification is associated with increased cardiovascular risk and occurs by osteochondrogenic differentiation of vascular cells. Many of the same regulatory factors that control skeletal mineralization, including the complex metabolic pathway controlling levels of the activator, inorganic phosphate, and the potent inhibitor, pyrophosphate, also govern vascular calcification. We previously found that the cAMP/PKA signaling pathway mediates in vitro vascular cell calcification induced by inflammatory factors including tumor necrosis factor-alpha 1 and oxidized phospholipids. In this report, we tested whether this signaling pathway modulates phosphate and pyrophosphate metabolism. Treatment of primary murine aortic cells with the PKA activator, forskolin, significantly induced osteoblastic differentiation markers, including alkaline phosphatase (ALP), osteopontin, and osteocalcin as well as the pyrophosphate generator, ectonucleotide-pyrophosphatase/phosphodiesterase-1 (Enpp1) and the pyrophosphate transporter, ankylosis protein, but not the sodium/phosphate cotransporter, Pit-1. In the presence of a substrate for ALP, beta-glycerophosphate, which generates inorganic phosphate, forskolin also enhanced matrix mineralization. Inhibitors of ALP or Pit-1 abrogated forskolin-induced osteopontin expression and mineralization but not forskolin-induced osteocalcin or ALP. These results suggest that phosphate is necessary for PKA-induced calcification of vascular cells and that the extent of PKA-induced calcification is controlled by feedback induction of the inhibitor, pyrophosphate.     

Yap1 protein regulates vascular smooth muscle cell phenotypic switch by interaction with myocardin

The Hippo-Yap (Yes-associated protein) signaling pathway has emerged as one of the critical pathways regulating cell proliferation, differentiation, and apoptosis in response to environmental and developmental cues. However, Yap1 roles in vascular smooth muscle cell (VSMC) biology have not been investigated. VSMCs undergo phenotypic switch, a process characterized by decreased gene expression of VSMC contractile markers and increased proliferation, migration, and matrix synthesis. The goals of the present studies were to investigate the relationship between Yap1 and VSMC phenotypic switch and to determine the molecular mechanisms by which Yap1 affects this essential process in VSMC biology. Results demonstrated that the expression of Yap1 was rapidly up-regulated by stimulation with PDGF-BB (a known inducer of phenotypic switch in VSMCs) and in the injured vessel wall. Knockdown of Yap1 impaired VSMC proliferation in vitro and enhanced the expression of VSMC contractile genes as well by increasing serum response factor binding to CArG-containing regions of VSMC-specific contractile genes within intact chromatin. Conversely, the interaction between serum response factor and its co-activator myocardin was reduced by overexpression of Yap1 in a dose-dependent manner. Taken together, these results indicate that down-regulation of Yap1 promotes VSMC contractile phenotype by both up-regulating myocardin expression and promoting the association of the serum response factor-myocardin complex with VSMC contractile gene promoters and suggest that the Yap1 signaling pathway is a central regulator of phenotypic switch of VSMCs.     

miRNA‐221 and miRNA‐222 synergistically function to promote vascular calcification

Vascular calcification shares many similarities with skeletal mineralisation and involves the phenotypic trans‐differentiation of vascular smooth muscle cells (VSMCs) to osteoblastic cells within a calcified environment. Various microRNAs (miRs) are known to regulate cell differentiation; however, their role in mediating VSMC calcification is not fully understood. miR‐microarray analysis revealed the significant down‐regulation of a range of miRs following nine days in culture, including miR‐199b, miR‐29a, miR‐221, miR‐222 and miR‐31 (p < 0.05). Subsequent studies investigated the specific role of the miR‐221/222 family in VSMC calcification. Real‐time quantitative polymerase chain reaction data confirmed the down‐regulation of miR‐221 (32.4%; p < 0.01) and miR‐222 (15.7%; p < 0.05). VSMCs were transfected with mimics of miR‐221 and miR‐222, individually and in combination. Increased calcium deposition was observed in the combined treatment (two‐fold; p < 0.05) but not in individual treatments. Runx2 and Msx2 expression was increased during calcification, but no difference in expression was observed following transfection with miR mimics. Interestingly, miR‐221 and miR‐222 mimics induced significant changes in ectonucleotide phosphodiesterase 1 (Enpp1) and Pit‐1 expression, suggesting that these miRs may modulate VSMC calcification through cellular inorganic phosphate and pyrophosphate levels. © 2013 The Authors. Cell Biochemistry and Function published by John Wiley & Sons, Ltd.