Stem cell marker expression,proliferation and apoptosis of vascular smooth muscle cells

Vascular endothelial Flt-1 and other stem cell markers are variably expressed in vascular smooth muscle cells (SMCs) during normal and pathological conditions, but their biological role remains uncertain. In normal rat aorta, rare flt-1+ and c-kit+ SMCs were detected. Fifteen days after injury, 61.8+3.8, 45.7+3% of the intimal cells resulted flt-1+ and c-kit+ and expressed low level of alpha-smooth muscle actin; CD133+ cells were 5.6+0.7%. BrDU+/flt-1+ largely predominated in the neointima, whereas BrDU+/CD133+ cells were rare. Forty-five and sixty days after injury, intimal proliferation such as BrDU+ cells was greatly reduced. After sixty days, intimal stem marker expression had almost disappeared whereas alpha-smooth muscle actin was restored. Flk-1 and Oct-4 SMC immunodection was consistently negative. In vitro, intimal cells obtained fifteen days after injury exhibited an epithelioid phenotype and increased flt-1 and c-kit protein and mRNA and low smooth muscle markers compared to spindle-shaped medial and intimal SMCs obtained after sixty days. Epithelioid clones, independently from layer of origin, were similar in stem cell marker expression. The anti-flt-1 blocking antibody added to epithelioid SMC cultures reduced serum-deprived apoptosis and migration but not PDGF-BB-induced proliferation, and increased cell-populated collagen lattice contraction. In conclusion, stem marker expression in vascular SMCs was variable, chronologically regulated and prevailed in epithelioid populations and clones; among stem markers, flt-1 expression critically regulates intimal SMC response to microenviromental changes.     

血管平滑肌细胞表型调节机制的研究进展

血管平滑肌细胞（VSMC）的增殖和迁移是动脉粥样硬化斑块形成、高血压和血管再狭窄的共同病理特征,而VSMC表型转化是VSMC增殖和迁移的基础,研究VSMC表型调节的分子机制,对上述疾病的防治具有重要意义。本文对VSMC表型转化的影响因素、信号转导途径和转录因子的研究进展作一综述。     

Lanthanum chloride bidirectionally influences calcification in bovine vascular smooth muscle cells

Vascular calcification (VC) is frequent prevalence in patients with chronic kidney disease (CKD) and atherosclerosis. Lanthanum carbonate is used as an orally administered phosphate-binding agent to reduce the gastrointestinal absorption of phosphate and ameliorate VC in advanced CKD. In this study, we used bovine vascular smooth muscle cells as a model VC in vitro and studied the effects of lanthanum chloride on calcium deposition. Exposure of cells to LaCl(3) at the concentration of 0.1 μM suppressed the β-glycerophosphate-induced alkaline phosphatase activity and calcium deposition. Furthermore, LaCl(3) upregulated the β-glycerophosphate-suppressed expression of calcium-sensing receptor. In contrast to the inhibitory effect of LaCl(3) on calcium deposition, higher level lanthanum (50 μM) was found to promote immediately precipitation of calcium phosphate in cell culture medium. At this concentration, LaCl(3) was found to induce cell apoptosis which involves caspases-9 and -3. These data indicate that the promotory effect of LaCl(3) on calcium deposition is likely mediated by induction of apoptosis. Our in vitro findings do suggest that, in the context of raised lanthanum, greater attention should be paid to potential toxic effects associated to the use of lanthanide-based drugs.     

Rho expression and activation in vascular smooth muscle cells

Phenotypic modulation of smooth muscle cells (SMC) involves dramatic changes in expression and organization of contractile and cytoskeletal proteins, but little is known of how this process is regulated. The present study used a cell culture model to investigate the possible involvement of RhoA, a known regulator of the actin cytoskeleton. In rabbit aortic SMC seeded into primary culture at moderate density, Rho activation was high at two functionally distinct time-points, first as cells modulated to the "synthetic" phenotype, and again upon confluence and return to the "contractile" phenotype. Rho expression increased with time, such that maximal expression occurred upon return to the contractile state. Transient transfection of synthetic state cells with constitutively active RhoA (Val14RhoA) caused a reduction in cell size and reorganization of cytoskeletal proteins to resemble that of the contractile phenotype. Actin and myosin filaments were tightly packed and highly organised while vimentin localised to the perinuclear region; focal adhesions were enlarged and concentrated at the cell periphery. Conversely, inhibition of endogenous Rho by C3 exoenzyme resulted in complete loss of contractile filaments without affecting vimentin distribution; focal adhesions were reduced in size and number. Treatment of synthetic state SMC with known regulators of SMC phenotype, heparin and thrombin, caused a modest increase in Rho activation. Long-term confluence and serum deprivation induced cells to return to a more contractile phenotype and this was augmented by heparin and thrombin. The results implicate RhoA for a role in regulating SMC phenotype and further show that activation of Rho by heparin and thrombin correlates with the ability of these factors to promote the contractile phenotype.     

Extracellular pyrophosphate metabolism and calcification in vascular smooth muscle

Extracellular inorganic pyrophosphate (ePP(i)) is an important endogenous inhibitor of vascular calcification, but it is not known whether systemic or local vascular PP(i) metabolism controls calcification. To determine the role of ePP(i) in vascular smooth muscle, we identified the pathways responsible for ePP(i) production and hydrolysis in rat and mouse aortas and manipulated them to demonstrate their role in the calcification of isolated aortas in culture. Rat and mouse aortas contained mRNA for ectonucleotide pyrophosphatase/phosphodiesterases (NPP1-3), the putative PP(i) transporter ANK, and tissue-nonspecific alkaline phosphatase (TNAP). Synthesis of PP(i) from ATP in aortas was blocked by β,γ-methylene-ATP, an inhibitor of NPPs. Aortas from mice lacking NPP1 (Enpp1(-/-)) did not synthesize PP(i) from ATP and exhibited increased calcification in culture. Although ANK-mediated transport of PP(i) could not be demonstrated in aortas, aortas from mutant (ank/ank) mice calcified more in culture than did aortas from normal (ANK/ANK) mice. Hydrolysis of PP(i) was reduced 25% by β,γ-methylene-ATP and 50% by inhibition of TNAP. Hydrolysis of PP(i) was increased in cells overexpressing TNAP or NPP3 but not NPP1 and was not reduced in Enpp1(-/-) aortas. Overexpression of TNAP increased calcification of cultured aortas. The results show that smooth muscle NPP1 and TNAP control vascular calcification through effects on synthesis and hydrolysis of ePP(i), indicating an important inhibitory role of locally produced PP(i). Smooth muscle ANK also affects calcification, but this may not be mediated through transport of PP(i). NPP3 is identified as an additional pyrophosphatase that could influence vascular calcification.     

Changes in three-dimensional architecture of microfilaments in cultured vascular smooth muscle cells during phenotypic modulation

To investigate changes in the three-dimensional microfilament architecture of vascular smooth muscle cells (SMC) during the process of phenotypic modulation, rabbit aortic SMCs cultured under different conditions and at different time points were either labelled with fluorescein-conjugated probes to cytoskeletal and contractile proteins for observation by confocal laser scanning microscopy, or extracted with Triton X-100 for scanning electron microscopy. Densely seeded SMCs in primary culture, which maintain a contractile phenotype, display prominent linear myofilament bundles (stress fibres) that are present throughout the cytoplasm with alpha-actin filaments predominant in the central part and beta-actin filaments in the periphery of the cell. Intermediate filaments form a meshed network interconnecting the stress fibres and linking directly to the nucleus. Moderately and sparsely seeded SMCs, which modulate toward the synthetic phenotype during the first 5 days of culture, undergo a gradual redistribution of intermediate filaments from the perinuclear region toward the peripheral cytoplasm and a partial disassembly of stress fibres in the central part of the upper cortex of the cytoplasm, with an obvious decrease in alpha-actin and myosin staining. These changes are reversed in moderately seeded SMCs by day 8 of culture when they have reached confluence. The results reveal two changes in microfilament architecture in SMCs as they undergo a change in phenotype: the redistribution of intermediate filaments probably due to an increase in synthetic organelles in the perinuclear area, and the partial disassembly of stress fibres which may reflect a degradation of contractile components.     

Actin cytoskeleton regulates functional anchorage-migration switch during T-cadherin-induced phenotype modulation of vascular smooth muscle cells

Vascular smooth muscle cell (SMC) switching between differentiated and dedifferentiated phenotypes is reversible and accompanied by morphological and functional alterations that require reconfiguration of cell-cell and cell-matrix adhesion networks. Studies attempting to explore changes in overall composition of the adhesion nexus during SMC phenotype transition are lacking. We have previously demonstrated that T-cadherin knockdown enforces SMC differentiation, whereas T-cadherin upregulation promotes SMC dedifferentiation. This study used human aortic SMCs ectopically modified with respect to T-cadherin expression to characterize phenotype-associated cell-matrix adhesion molecule expression, focal adhesions configuration and migration modes. Compared with dedifferentiated/migratory SMCs (expressing T-cadherin), the differentiated/contractile SMCs (T-cadherin-deficient) exhibited increased adhesion to several extracellular matrix substrata, decreased expression of several integrins, matrix metalloproteinases and collagens, and also distinct focal adhesion, adherens junction and intracellular tension network configurations. Differentiated and dedifferentiated phenotypes displayed distinct migrational velocity and directional persistence. The restricted migration efficiency of the differentiated phenotype was fully overcome by reducing actin polymerization with ROCK inhibitor Y-27632 whereas myosin II inhibitor blebbistatin was less effective. Migration efficiency of the dedifferentiated phenotype was diminished by promoting actin polymerization with lysophosphatidic acid. These findings held true in both 2D-monolayer and 3D-spheroid migration models. Thus, our data suggest that despite global differences in the cell adhesion nexus of the differentiated and dedifferentiated phenotypes, structural actin cytoskeleton characteristics per se play a crucial role in permissive regulation of cell-matrix adhesive interactions and cell migration behavior during T-cadherin-induced SMC phenotype transition.     

The role of lysophosphatidic acid receptors in phenotypic modulation of vascular smooth muscle cells

Lysophosphatidic acid (LPA) is a bioactive lipid with diverse physiological effects via activation of G protein-coupled receptors (GPCRs). It has been implicated as a specific dedifferentiation factors that can promote phenotypic modulation of cultured vascular smooth muscle cells (VSMCs) which is critically involved in various vascular disease. However, the role of LPA receptors and details of their signaling in LPA induced phenotypic modulation are largely unexplored. In this study we detect the expression of LPA1 and LPA3 in rat aortic smooth muscle cells (RASMCs). LPA promoted RASMCs phenotypic modulation in a dose-dependent manner and coordinated induced the phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) and extracellular signal-regulated kinase (ERK). LPA-induced cell phenotypic modulation was significantly inhibited by specific LPA1/LPA3-receptor antagonist dioctyl-glycerol pyrophosphate (DGPP8:0) at concentration, but this inhibitive effect was lost when the antagonist was coadministered with a highly selective LPA3 agonist,1-oleoyl-2-Omethyl-rac-glycero-phosphothionate (OMPT). In addition, pertussis toxin (PTX), a Gi protein inhibitor had little affect on the LPA-induced phenotypic modulation in RASMC. These data suggest that LPA-induced phenotypic modulation is mediated through the PTX-insensitive G-protein(s), possibly Gq-coupled LPA3 receptor.     

Endothelin-1 is a potent regulator in vivo in vascular calcification and in vitro in calcification of vascular smooth muscle cells

We observed changes of endothelin content and endothelin mRNA in vivo in vascular calcification and in vitro in calcification of vascular smooth muscle cells to explore the role of endothelin in vascular calcification. Calcification model in vivo was induced by administration of Vitamin D(3) plus nicotine. Calcification of vascular smooth muscle cells (VSMCs) was induced by beta-glycerophosphate. Endothelin content was measured by using radioimmunoassay. Endothelin mRNA amount was determined by using competitive quantitative RT-PCR. The results showed that calcium content, 45Ca(2+) uptake and alkaline phosphatase (ALP) activity were increased in calcified VSMCs, compared with controls, but were decreased, compared with calcified VSMCs plus BQ123 group. The endothelin content in the medium and endothelin mRNA in VSMCs were elevated by 35 and 120% (P<0.05), respectively, compared with those normal VSMCs. Calcium content, 45Ca(2+) accumulation and ALP activity in calcified arteries increased by 5.0-, 1.4-, and 1.4-fold. The endothelin levels in plasma and aorta as well as the amount of endothelin mRNA in calcified aorta were increased by 102, 103, and 22%, respectively, compared with control group. However, calcium content, 45Ca(2+) uptake and ALP activity in VDN plus bosentan group was 33, 36.7, and 40.4% lower than those in VDN group. These results indicated an upregulated endothelin gene expression as well as an increased production of endothelin in calcified aorta and VSMCs with BQ123 and bosentan significantly reducing vascular calcification. This suggested that endothelin might be involved in pathogenesis of vascular calcification.     

MicroRNAs are critical in regulating smooth muscle cell mineralization and apoptosis during vascular calcification

Vascular calcification refers to the pathological deposition of calcium and phosphate minerals into the vasculature. It is prevalent in atherosclerosis, ageing, type 2 diabetes mellitus and chronic kidney disease, thus, increasing morbidity and mortality from these conditions. Vascular calcification shares similar mechanisms with bone mineralization, with smooth muscle cells playing a critical role in both processes. In the last decade, a variety of microRNAs have been identified as key regulators for the differentiation, phenotypic switch, proliferation, apoptosis, cytokine production and matrix deposition in vascular smooth muscle cells during vascular calcification. Therefore, this review mainly discusses the roles of microRNAs in the pathophysiological mechanisms of vascular calcification in smooth muscle cells and describes several interventions against vascular calcification by regulating microRNAs. As the exact mechanisms of calcification remain not fully elucidated, having a better understanding of microRNA involvement in vascular calcification may give impetus to development of novel therapeutics for the control and treatment of vascular calcification.     

Crystallizing nanoparticles derived from vascular smooth muscle cells contain the calcification inhibitor osteoprotegerin

Osteoprotegerin (OPG), a member of the TNF receptor superfamily, was initially found to modulate bone mass by blocking osteoclast maturation and function. Rodent models have also revealed a role for OPG as an inhibitor of vascular calcification. However, the precise mode of how OPG blocks mineralization is unclear. In this study, OPG was found in an in vitro assay to significantly inhibit calcification of vascular smooth muscle cells (VSMC) induced by high calcium/phosphate (Ca/P) treatment (p = 0.0063), although this effect was blunted at high OPG concentrations. By confocal microscopy, OPG was detected in VSMC in the Golgi, the same localization seen in osteoblasts, which express OPG in bone. Treatment of VSMC by minerals (Ca, P, or both) induced OPG mRNA expression as assessed by real-time quantitative PCR, and VSMC derived from atherosclerotic plaque material also exhibited higher OPG expression as compared to control cells (p < 0.05). Furthermore, OPG was detected by Western blotting in matrix vesicles (MV), nanoparticles that are released by VSMC with the capacity to nucleate mineral. In atherosclerotic arteries, OPG colocalized immunohistochemically with annexin VI, a calcium-dependent membrane and phospholipid binding protein found in MV. Thus, the calcification inhibitor OPG is contained in crystallizing MV and has a biphasic effect on VSMC: physiologic concentrations inhibit calcification, whereas high concentrations commonly seen in patients with vascular disease have no effect. Like other calcification inhibitors, OPG may be specifically loaded into these nanoparticles to be deposited at remote sites, where it acts to inhibit calcification.     

Crystallizing nanoparticles derived from vascular smooth muscle cells contain the calcification inhibitor osteoprotegerin

The aim of this study was to investigate the possible pathological relation between mechanical stress and hyperpigmentation. We did this by investigating the influence of cyclic stretch on the expression of keratinocyte- and fibroblast-derived melanogenetic paracrine cytokines in vitro. Using primary human keratinocytes and fibroblasts, alterations of mRNA expression of melanogenetic paracrine cytokines due to cyclic stretch were investigated using a real-time polymerase chain reaction (PCR). The cytokines included basic fibroblast growth factor (bFGF), stem cell factor (SCF), granulocyte/macrophage colony-stimulating factor, interleukin-1α, and endothelin-1 (ET-1) for keratinocytes and bFGF, SCF, and hepatocyte growth factor for fibroblasts. The dose dependence of keratinocyte-derived ET-1 upregulation was further investigated using real-time PCR and an enzyme-linked immunosorbent assay. We also investigated the effects of cyclic stretch on the proliferation and differentiation of keratinocytes. Among the melanogenetic paracrine cytokines investigated, keratinocyte-derived ET-1 was consistently upregulated in all four cell lines. The degree of upregulation increased with the degree of the length and frequency of the stretch; in contrast, cell number and differentiation markers showed no obvious alterations with cyclic stretch. Keratinocyte-derived ET-1 upregulation possibly plays a significant role in the pathogenesis of pigmented disorders, such as friction melanosis, caused by mechanical stress.     

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.     

Extracellular matrix effect on RhoA signaling modulation in vascular smooth muscle cells

Morphological adaptations of vascular smooth muscle cells (VSMC) to the mechanically active environment in which they reside, are mediated by direct interactions with the extracellular matrix (ECM) which induces physiological changes at the intracellular level. This study aimed to analyze the effects of the ECM on RhoA-induced mechanical signaling that controls actin organization and focal adhesion formation. VSMC were transfected with RhoA constructs (wild type, dominant negative or constitutively active) and plated on different ECM proteins used as substrate (fibronectin, collagen IV, collagen I, and laminin) or poly-l-lysine as control. Morphological changes of the VSMC were detected by fluorescence confocal microscopy and total internal reflection fluorescence (TIRF) microscopy, and were independently verified using adhesion assays and Western blot analysis. Our results showed that the ECM has an important role in cell spreading, adhesion and morphology with a direct effect on modulating RhoA signaling. RhoA activity significantly affected the stress fibers and focal adhesions reorganization, but in a context imposed by the ECM. Thus, RhoA activity modulation in VSMC induced an increased activation of stress fibers and FA formation at 5 h, while a significant inhibition was recorded at 24 h after plating on the different ECM. Our findings provide biophysical evidence that ECM modulates VSMC response to mechanical stimuli inducing intracellular biochemical signaling involved in cellular adaptation to the local microenvironment.     

Inhibition of vascular smooth muscle cell calcification by ATP analogues

Purinergic Signalling - Arterial medial calcification (AMC) has been associated with phenotypic changes in vascular smooth muscle cells (VSMCs) that reportedly makes them more osteoblast-like....     

Role of calcium-phosphate deposition in vascular smooth muscle cell calcification

In this work we are studying whether calcium phosphate deposition (CPD) during vascular calcification is a passive or a cell-mediated mechanism. Passive CPD was studied in fixed vascular smooth muscle cells (VSMC), which calcify faster than live cells in the presence of 1.8 mM Ca2(+) and 2 mM P(i). CPD seems to be a cell-independent process that depends on the concentration of calcium, phosphate, and hydroxyl ions, but not on Ca × P(i) concentration products, given that deposition is obtained with 2 × 2 and 4 × 1 Ca × P(i) mM2 but not with 2 × 1 or 1 × 4 Ca × P(i) mM2. Incubation with 4 mM P(i) without CPD (i.e., plus 1 mM Ca) does not induce osteogene expression. Increased expression of bone markers such as Bmp2 and Cbfa1 is only observed concomitantly with CPD. Hydroxyapatite is the only crystalline phase in both lysed and live cells. Lysed cell deposits are highly crystalline, whereas live cell deposits still contain large amounts of amorphous calcium. High-resolution transmission electron microscopy revealed a nanostructure of rounded crystallites of 5-10 nm oriented at random in lysed cells, which is compatible with spontaneous precipitation. The nanostructure in live cells consisted of long fiber crystals, 10-nm thick, embedded in an amorphous matrix. This structure indicates an active role of cells in the process of hydroxyapatite crystallization. In conclusion, our data suggest that CPD is a passive phenomenon, which triggers the osteogenic changes that are involved in the formation of a well organized, calcified crystalline structure.     

The combination of lanthanum chloride and the calcimimetic calindol delays the progression of vascular smooth muscle cells calcification

Phosphate (Pi)-binders are commonly used in dialysis patients to control high Pi levels, that associated with vascular calcification (VC). The aim of this study was to investigate the effects of lanthanum chloride (LaCl(3)) on the progression of high Pi-induced VC, in rat vascular smooth muscle cells (VSMCs). Pi-induced Ca deposition was inhibited by LaCl(3), with a maximal effect at 100μM (59.0±2.5% inhibition). Furthermore, we studied the effects on VC of calcium sensing receptor (CaSR) agonists. Gadolinium chloride, neomycin, spermine, and the calcimimetic calindol significantly inhibited Pi-induced VC (55.9±2.2%, 37.3±4.7%, 30.2±5.7%, and 63.8±5.7%, respectively). To investigate the hypothesis that LaCl(3) reduces the progression of VC by interacting with the CaSR, we performed a concentration-response curve of LaCl(3) in presence of a sub-effective concentration of calindol (10nM). Interestingly, this curve was shifted to the left (IC(50) 9.6±2.6μM), compared to the curve in the presence of LaCl(3) alone (IC(50) 19.0±4.8μM). In conclusion, we demonstrated that lanthanum chloride effectively reduces the progression of high phosphate-induced vascular calcification. In addition, LaCl(3) cooperates with the calcimimetic calindol in decreasing Ca deposition in this in vitro model. These results suggest the potential role of lanthanum in the treatment of VC induced by high Pi.     

TRAIL expression in vascular smooth muscle

TRAIL is a cell-associated tumor necrosis factor-related apoptosis-inducing ligand originally identified in immune cells. The ligand has the capacity to induce apoptosis after binding to cell surface receptors. To examine TRAIL expression in murine vascular tissue, we employed in situ hybridization and immunohistochemistry. In these studies, we found that TRAIL mRNA and protein were specifically localized throughout the medial smooth muscle cell layer of the pulmonary artery. Notably, a similar pattern of expression was observed in the mouse aorta. Consistent with these findings, we found that cultures of primary human aorta and pulmonary artery smooth muscle cells express abundant TRAIL mRNA and protein. We also found that these cells and endothelial cells undergo cell lysis in response to exogenous addition of TRAIL. Last, we confirmed that TRAIL specifically activated a death program by confirming poly(ADP ribose) polymerase cleavage. Overall, we believe that these findings are relevant to understanding the factors that regulate cell turnover in the vessel wall.     

MiR155 modulates vascular calcification by regulating Akt-FOXO3a signalling and apoptosis in vascular smooth muscle cells

microRNA-155 (miR155) is pro-atherogenic; however, its role in vascular calcification is unknown. In this study, we aim to examine whether miR155 regulates vascular calcification and to understand the underlying mechanism. Quantitative real-time PCR showed that miR155 is highly expressed in human calcific carotid tissue and positively correlated with the expression of osteogenic genes. Wound-healing assay and TUNEL staining showed deletion of miR155 inhibited vascular smooth muscle cell (VSMC) migration and apoptosis. miR155 deficiency attenuated calcification of cultured mouse VSMCs and aortic rings induced by calcification medium, whereas miR155 overexpression promoted VSMC calcification. Compared with wild-type mice, miR155^−/− mice showed significant resistance to vitamin D3 induced vascular calcification. Protein analysis showed that miR155 deficiency alleviated the reduction of Rictor, increased phosphorylation of Akt at S473 and accelerated phosphorylation and degradation of FOXO3a in cultured VSMCs and in the aortas of vitamin D3-treated mice. A PI3K inhibitor that suppresses Akt phosphorylation increased, whereas a pan-caspase inhibitor that suppresses apoptosis reduced VSMC calcification; and both inhibitors diminished the protective effects of miR155 deficiency on VSMC calcification. In conclusion, miR155 deficiency attenuates vascular calcification by increasing Akt phosphorylation and FOXO3a degradation, and thus reducing VSMC apoptosis induced by calcification medium.     

Regulation of Na(+) pump expression by vascular smooth muscle cells

The Na(+) pump and its regulation is important for maintaining membrane potential and transmembrane Na(+) gradient in all mammalian cells and thus is essential for cell survival and function. Vascular smooth muscle cells (VSMC) have a relatively low number of pump sites on their membrane compared with other cells. We wished to determine the mechanisms for regulating the number of pump sites in these cells. We used canine saphenous vein VSMC cultured in 10% serum and passaged one time. These cells were subcultured in 5% serum media with low K(+) (1 mM vs. control of 5 mM), and their pump expression was assessed. These VSMC upregulated their pump sites as early as 4 h after treatment (measured by [(3)H]ouabain binding). At this early time point, there was no detectable increase in protein expression of either alpha(1)- or beta(1)-subunits of the pump shown by Western blots. When the cells were treated with the phosphoinositide 3-kinase (PI-3-K) inhibitor LY-294002 (which is known to inhibit cytoplasmic transport processes) in low-K(+) media, the pump site upregulation was inhibited. These data suggest that the low-K(+)-induced upregulation of Na(+) pump number can occur by translocation of preformed pumps from intracellular stores.