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.     

PDGF induces osteoprotegerin expression in vascular smooth muscle cells by multiple signal pathways

Acetohydroxyacid synthase (AHAS; EC 4.1.3.18) contains catalytic and regulatory subunits, the latter being required for sensitivity to feedback regulation by leucine, valine and isoleucine. The regulatory subunit of Arabidopsis thaliana AHAS possesses a sequence repeat and we have suggested previously that one repeat binds leucine while the second binds valine or isoleucine, with synergy between the two sites. We have mutated four residues in each repeat, based on a model of the regulatory subunit. The data confirm that there are separate leucine and valine/isoleucine sites, and suggest a complex pathway for regulatory signal transmission to the catalytic subunit.     

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.     

Regulation of vascular smooth muscle cells and mesenchymal stem cells by mechanical strain

Vascular smooth muscle cells (SMCs) populate in the media of the blood vessel, and play an important role in the control of vasoactivity and the remodeling of the vessel wall. Blood vessels are constantly subjected to hemodynamic stresses, and the pulsatile nature of the blood flow results in a cyclic mechanical strain in the vessel walls. Accumulating evidence in the past two decades indicates that mechanical strain regulates vascular SMC phenotype, function and matrix remodeling. Bone marrow mesenchymal stem cell (MSC) is a potential cell source for vascular regeneration therapy, and may be used to generate SMCs to construct tissue-engineered vascular grafts for blood vessel replacements. In this review, we will focus on the effects of mechanical strain on SMCs and MSCs, e.g., cell phenotype, cell morphology, cytoskeleton organization, gene expression, signal transduction and receptor activation. We will compare the responses of SMCs and MSCs to equiaxial strain, uniaxial strain and mechanical strain in three-dimensional culture. Understanding the hemodynamic regulation of SMC and MSC functions will provide a basis for the development of new vascular therapies and for the construction of tissue-engineered vascular grafts.     

Adult vascular smooth muscle cells in culture express neural stem cell markers typical of resident multipotent vascular stem cells

Differentiation of resident multipotent vascular stem cells (MVSCs) or de-differentiation of vascular smooth muscle cells (vSMCs) might be responsible for the SMC phenotype that plays a major role in vascular diseases such as arteriosclerosis and restenosis. We examined vSMCs from three different species (rat, murine and bovine) to establish whether they exhibit neural stem cell characteristics typical of MVSCs. We determined their SMC differentiation, neural stem cell marker expression and multipotency following induction in vitro by using immunocytochemistry, confocal microscopy, fluorescence-activated cell sorting analysis and quantitative real-time polymerase chain reaction. MVSCs isolated from rat aortic explants, enzymatically dispersed rat SMCs and rat bone-marrow-derived mesenchymal stem cells served as controls. Murine carotid artery lysates and primary rat aortic vSMCs were both myosin-heavy-chain-positive but weakly expressed the neural crest stem cell marker, Sox10. Each vSMC line examined expressed SMC differentiation markers (smooth muscle α–actin, myosin heavy chain and calponin), neural crest stem cell markers (Sox10⁺, Sox17⁺) and a glia marker (S100β⁺). Serum deprivation significantly increased calponin and myosin heavy chain expression and decreased stem cell marker expression, when compared with serum-rich conditions. vSMCs did not differentiate to adipocytes or osteoblasts following adipogenic or osteogenic inductive stimulation, respectively, or respond to transforming growth factor-β1 or Notch following γ-secretase inhibition. Thus, vascular SMCs in culture express neural stem cell markers typical of MVSCs, concomitant with SMC differentiation markers, but do not retain their multipotency. The ultimate origin of these cells might have important implications for their use in investigations of vascular proliferative disease in vitro.     

Mesenchymal stem/stromal cells-derived exosomes for osteoporosis treatment

Osteoporosis is a systemic bone disease, which leads to decreased bone mass and an increased risk of fragility fractures. Currently, there are many anti-resorption drugs and osteosynthesis drugs, which are effective in the treatment of osteoporosis, but their usage is limited due to their contraindications and side effects. In regenerative medicine, the unique repair ability of mesenchymal stem cells(MSCs) has been favored by researchers. The exosomes secreted by MSCs have signal transduction an...     

Calmodulin levels are dynamically regulated in living vascular smooth muscle cells

The total unbound calmodulin (i.e., not bound to target proteins) level in living smooth muscle cells from the ferret portal vein was monitored with a low-affinity, calmodulin-binding peptide tagged with an environmentally sensitive fluorophore. GS17C, a previously characterized peptide, from the calmodulin-binding domain of caldesmon was tagged with iodoacetyl nitrobenz-2-oxa-1,3-diazole (NBD) or, as a negative control, with iodoacetylfluorescein isothiocyanate. Increases in NBD-GS17C fluorescence were detected by using confocal microscopy when chemically loaded cells were stimulated with solutions of elevated [K(+)] or the calcium ionophore 4-bromoA-23187 to elicit increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) quantified by fura 2. Increases in peptide fluorescence were detected in response to a phorbol ester in the absence of changes in [Ca(2+)](i). These changes were blocked by the addition of the calmodulin antagonist calmidazolium. These results suggest that the total unbound intracellular calmodulin levels may be sufficient to regulate the activity of caldesmon and, furthermore, that phosphorylation of protein kinase C substrates may increase the level of available calmodulin in living smooth muscle cells.     

Mesenchymal stem cells-derived exosomal microRNAs contribute to wound inflammation

Clinical and experimental studies have highlighted the significance of inflammation in coordinating wound repair and regeneration.However,it remains challenging to control the inflammatory response and tolerance at systemic levels without causing toxicity to injured tissues.Mesenchymal stem cells(MSCs) possess potent immunomodulatory properties and facilitate tissue repair by releasing exosomes,which generate a suitable microenvironment for inflammatory resolution.Exosomes contain several effective bioactive molecules and act as a cell-cell communication vehicle to influence cellular activities in recipient cells.During this process,the horizontal transfer of exosomal microRNAs(miRNAs) to acceptor cells,where they regulate target gene expression,is of particular interest for understanding the basic biology of inflammation ablation,tissue homeostasis,and development of therapeutic approaches.In this review,we describe a signature of three specific miRNAs(miR-21,miR-146 a,and miR-181) present in human umbilical cord MSC-derived exosomes(MSC-EXO) identified microarray chip analysis and focus on the inflammatory regulatory functions of these immune-related miRNAs.We also discuss the potential mechanisms contributing to the resolution of wound inflammation and tissue healing.     

Calcification of human vascular smooth muscle cells: associations with osteoprotegerin expression and acceleration by high-dose insulin

Arterial medial calcifications occur often in diabetic individuals as part of the diabetic macroangiopathy. The pathogenesis is unknown, but the presence of calcifications predicts risk of cardiovascular events. We examined the effects of insulin on calcifying smooth muscle cells in vitro and measured the expression of the bone-related molecule osteoprotegerin (OPG). Human vascular smooth muscle cells (VSMCs) were grown from aorta from kidney donors. Induction of calcification was performed with beta-glycerophosphate. The influence of insulin (200 microU/ml or 1,000 microU/ml) on calcification was judged by measuring calcium content in the cell layer and by von Kossa staining. OPG was measured in the medium by ELISA. Histochemistry was used for determination of alkaline phosphatase (ALP). Bone sialoprotein (BSP) and OPG mRNA expressions were done by RT-PCR. beta-Glycerophosphate was able to induce calcification in human smooth muscle cells from a series of donors after variable time in culture. Decreased OPG amounts were observed from the cells during the accelerated calcification phase. High dose of insulin (1,000 microU/ml) accelerated the calcification, whereas lower concentrations (200 microU/ml) did not. Calcified cells expressed ALP and BSP activity in high levels. In conclusion, high concentration of insulin enhances in vitro-induced calcification in VSMCs. Altered OPG levels during the calcification raise the possibility that OPG may have a potent function in regulating the calcification process or it may represent a consequence of mineralization. Effects of insulin and modulations by OPG on the calcification process in arterial cells may play a role in the development of calcifications as part of the diabetic macroangiopathy.     

Leptin increases endothelin type A receptor levels in vascular smooth muscle cells

Leptin, one of the adipocyte-secreted peptides, is involved in the control of appetite and body weight. Several studies have demonstrated that plasma leptin levels are elevated in obese subjects and are positively correlated with body weight. The arterial endothelin (ET) system plays an important role in the regulation of vascular tone, and ET-1 overexpression may be involved in the pathogenesis of the hypertension associated with insulin resistance. This study was performed to explore the regulatory effects of leptin on ET receptor expression and ET binding in A10 vascular smooth muscle cells (VSMCs) by use of Northern blotting, immunoblotting, and a (125)I-labeled ET-1 binding assay. The effect of leptin on ET receptor-mediated cell proliferation was also tested. The results showed that leptin caused a significant increase in [(125)I]-ET-1 binding, which was time- and dose-dependent. Immunoblotting showed that expression of the ET type A receptor (ET(A)R) in leptin (10(-7) M)-treated cells was increased by up to 2.3-fold compared with controls. Levels of ET(A)R mRNA measured by Northern blotting were also increased by up to 2.2-fold in leptin (10(-7) M)-treated cells. Pretreatment with an ERK inhibitor, PD-98059 (2.5 x 10(-5) M), blocked the leptin-induced increase in (125)I-ET-1 binding. Finally, ET-1 (10(-7) M)-stimulated cell proliferation was enhanced by leptin (10(-7) M) pretreatment, with a maximal increase of twofold compared with controls. In conclusion, leptin increases ET(A)R expression in VSMCs in a time- and dose-dependent manner. This effect is ERK dependent and is associated with increased ET-1-stimulated cell proliferation. These findings provide support for roles for leptin and the ET system in the pathogenesis of obesity-associated hypertension.     

Resident multipotent vascular stem cells exhibit amplitude dependent strain avoidance similar to that of vascular smooth muscle cells

Atherosclerosis is one of the leading causes of mortality worldwide, and presents as a narrowing or occlusion of the arterial lumen. Interventions to re-open the arterial lumen can result in re-occlusion through intimal hyperplasia. Historically only de-differentiated vascular smooth muscle cells were thought to contribute to intimal hyperplasia. However recent significant evidence suggests that resident medial multipotent vascular stem cells (MVSC) may also play a role. We therefore investigated the strain response of MVSC since these resident cells are also subjected to strain within their native environment. Accordingly, we applied uniaxial 1 Hz cyclic uniaxial tensile strain at three amplitudes around a mean strain of 5%, (4–6%, 2–8% and 0–10%) to either rat MVSC or rat VSMC before their strain response was evaluated. While both cell types strain avoid, the strain avoidant response was greater for MVSC after 24 h, while VSMC strain avoid to a greater degree after 72 h. Additionally, both cell types increase strain avoidance as strain amplitude is increased. Moreover, MVSC and VSMC both demonstrate a strain-induced decrease in cell number, an effect more pronounced for MVSC. These experiments demonstrate for the first time the mechano-sensitivity of MVSC that may influence intimal thickening, and emphasizes the importance of strain amplitude in controlling the response of vascular cells in tissue engineering applications.     

Nicotine-induced vascular endothelial growth factor release via the EGFR-ERK pathway in rat vascular smooth muscle cells

Cigarette smoke has been firmly established as an independent risk factor for atherosclerosis and other vascular diseases. The proliferation and migration of vascular smooth muscle cells (VSMC) induced by growth factors have been proposed to play an important role in the progression of atherosclerosis. In the present study, we investigated the effects of nicotine, which is one of the important constituents of cigarette smoke, on vascular endothelial growth factor (VEGF) release, in rat VSMC. The stimulation of cells with nicotine resulted in a time- and concentration-dependent release of VEGF. Hexamethonium, an antagonist of nicotinic acetylcholine receptor (nAChR), inhibited nicotine-induced VEGF release. We next investigated the mechanisms by which nicotine induces VEGF release in the cells. The nicotine-induced VEGF release was inhibited by treatment with U0126, a selective inhibitor of MEK, which attenuated the nicotine-induced ERK phosphorylation. Nicotine induced a transient phosphorylation of ERK. Furthermore, AG1478, a selective inhibitor of epidermal growth factor receptor (EGFR) kinase, inhibited nicotine-induced ERK phosphorylation and VEGF release. These data suggest that nicotine releases VEGF through nAChR in VSMC. Moreover, VEGF release induced by nicotine is mediated by an EGFR-ERK pathway in VSMC. VEGF may contribute to the risk of cardiovascular diseases in cigarette smokers.     

27nt-miRNA对间充质干细胞向血管平滑肌细胞分化的影响

为探讨27nt-miRNA对间充质干细胞向血管平滑肌细胞分化影响,构建27nt-miRNA过表达、反义序列Anti-27nt-miRNA以及阴性对照的表达质粒,慢病毒包装后分别转染人脐带间充质干细胞(hUCMSC),加入Ⅳ型胶原诱导hUCMSC定向分化为血管平滑肌细胞。四唑盐(MTT)比色法检测分化后细胞活力,免疫细胞化学染色法检测分化后细胞SM22α(兔抗平滑肌22α,smooth muscle 22α)的表达,Western印迹法和RT-PCR检测分化后细胞内的SMA (兔抗平滑肌肌动蛋白,smooth muscle actin) mRNA、SM 22α mRNA及其蛋白质表达情况。经检测,27nt-miRNA过表达分化组与阴性对照组相比,细胞活力下降20.48%(P0.05),SMA mRNA、SM22α mRNA及其蛋白质表达量明显升高(P0.05);而Anti-27nt-miRNA分化组细胞活力上升了18.07%(P0.05),SMA mRNA、SM22α mRNA及其蛋白质表达量下降(P0.05)。综上所述,27nt-miRNA能够促进间充质干细胞向血管平滑肌细胞分化,并且抑制分化后的细胞活力。     

Hemoglobin causes release of inositol trisphosphate from vascular smooth muscle

To test the hypothesis that oxyhemoglobin causes contraction of vascular smooth muscle by production of inositol 1,4,5-trisphosphate which results in a release of intracellular calcium, smooth muscle cells were exposed to oxyhemoglobin and inositol trisphosphate was measured. Oxyhemoglobin, but not methemoglobin which has much less contractile action, stimulated inositol trisphosphate production. The time course was consistent with an early role for this compound in the contraction produced by hemoglobin. The increase in production of inositol trisphosphate was inhibited by pertussis toxin and also by neomycin, an inhibitor of phospholipase C, although the actions of the latter compound cannot be attributed only to an inhibition of the enzyme responsible for the production of inositol trisphosphate.     

血管平滑肌细胞的分化与血管内壁的构建

血管平滑肌细胞(vascular smooth muscle cells,VSMCs)的发育与血管壁的构建是目前相关领域中的重要学科前沿．国内外同行的工作多集中在血管发育初始阶段内皮细胞及其前体细胞在血管新生中的作用、调节因素及生物学机制．VSMCs参与血管壁早期构建,特别是VSMCs的募集与分化机制已经成为血管新生研究中的一个新领域． 本期发表的《 抑制Rac1蛋白活化阻碍胚胎发育早期血管新生 》(见696～701页)报道了韩雅玲教授及其合作者在这一领域取得的最新研究结果．Rac1是真核细胞内重要的一类信号传递分子,在细胞信号传递过程中发挥分子开关作用．他们采用胚胎干细胞(ESCs)为模型,建立稳定表达持续型Rac1和显性失活型Rac1编码序列的小鼠ESCs并制备胚胎小体,诱导分化后观察其对内皮细胞分化和迁移的影响,发现抑制Rac1可以干扰血管内皮细胞连接成血管网状结构,细胞骨架F-actin排列紊乱,细胞的迁移受到明显抑制,表明Rac1在胚胎早期血管发育过程中与内皮细胞的迁移有关[1]． 近年来,韩雅玲教授及其研究集体在VSMCs发育与血管构建、胚胎干细胞来源的拟胚体血管平滑肌发育与血管新生机制以及胚胎主动脉VSMCs起源等方面开展了研究,取得了一系列有价值的成果[2～11],可能为闭塞性和增生性血管病的发生及防治提供理论依据和候选基因．详见“相关链接”．     

Regulation of vascular smooth muscle cells by micropatterning

Vascular smooth muscle cells (SMCs) undergo morphological and phenotypic changes when cultured in vitro. To investigate whether SMC morphology regulates SMC functions, bovine aortic SMCs were grown on micropatterned collagen strips (50-, 30-, and 20-microm wide). The cell shape index and proliferation rate of SMCs on 30- and 20-microm strips were significantly lower than those on non-patterned collagen (control), and the spreading area was decreased only for cells patterned on the 20-microm strips, suggesting that SMC proliferation is dependent on cell shape index. The formation of actin stress fibers and the expression of alpha-actin were decreased in SMCs on the 20- and 30-microm collagen strips. SMCs cultured on micropatterned biomaterial poly-(D,L-lactide-co-glycolide) (PLGA) with 30-microm wide grooves also showed lower proliferation rate and less stress fibers than SMCs on non-patterned PLGA. Our findings suggest that micropatterned matrix proteins and topography can be used to control SMC morphology and that elongated cell morphology decreases SMC proliferation but is not sufficient to promote contractile phenotype.     

Impedance analysis of renal vascular smooth muscle cells

Impedance of renal vascular smooth muscle cells (VSMCs) cultured on microelectrodes was measured by electric cell-substrate impedance sensing. Changes in measured impedance as a function of frequency were compared with the calculated values obtained from an extended cell-electrode model to estimate the junctional resistance, distance between the ventral cell surface and the substratum, and apical and basolateral membrane capacitances of renal VSMCs. This cell-electrode model was derived to accommodate the slender and rectangular shape of VSMCs. The calculated changes in impedance (Z_cal) based on the model agreed well with the experimental measurement (Z_exp), and the percentage error defined as |(Z_cal – Z_exp)/Z_exp| was 1.0%. To test the sensitivity of the new model for capturing changes in cell-cell and cell-substrate interactions induced by changes in cellular environment, we then applied this model to analyze timpedance changes induced by an integrin binding peptide in renal VSMCs. Our result demonstrates that integrin binding peptide decreases junctional resistance between cells, increases the distance between the basolateral cell surface and substratum, and increases the apical membrane capacitance, whereas the basolateral membrane capacitance stays relatively stable. This model provides a generic approach for impedance analysis of cell layers composed of slender, rectangular cells. electric cell-substrate impedance sensing; cell attachment; cell adhesion; extracellular matrix; integrin     

Stem cells, vascular smooth muscle cells and atherosclerosis

Stem cells have the ability to differentiate into a variety of cells to replace dead cells or to repair tissue. Recently, accumulating evidence indicates that mechanical forces, cytokines and other factors can influence stem cell differentiation into vascular smooth muscle cells (SMCs). In developmental process, SMCs originate from several sources, which show a great heterogenicity in different vessel walls. In adult vessels, SMCs display a less proliferative nature, but are altered in response to risk factors for atherosclerosis. Traditional view on SMC origins in atherosclerotic lesions is challenged by the recent findings that stem cells and smooth muscle progenitors contribute to the development of atherosclerotic lesions. Vascular progenitor cells circulating in human blood and the presence of adventitia in animals are recent discoveries, but the source of these cells is still unknown. The present review gives an update on the progress of stem cell and SMC research in atherosclerosis, and discusses possible mechanisms of stem/progenitor cell differentiation that contribute to the disease process.     

Effects of adenine nucleotides on inositol 1,4,5-trisphosphate-induced calcium release in vascular smooth muscle cells

Effects of adenine nucleotides on the inositol 1,4,5-trisphosphate (IP3)-induced Ca release (IICR) mechanism were studied in smooth muscle cells of the guinea pig portal vein. A microfluorometry method of fura-2 was used to measure Ca release from saponin-skinned thin muscle strips (width approximately 200 microns, thickness 50-70 microns, length 2-3 mm). About 80% of ionomycin-releasable Ca store was sensitive to IP3, of which approximately 20% was also sensitive to caffeine. The rate of Ca release by 0.1 microM IP3 depended biphasically on ATP concentration in the absence of Mg2+; it was dose-dependently enhanced by ATP up to approximately 0.5 mM, and above this concentration the enhancement became smaller. However, the decline of enhancement of the IICR at the higher ATP concentrations was absent at IP3 concentrations greater than 1 microM. This suggests competitive antagonism between IP3 and ATP. Clear effects of ATP were observed not only at pCa 7 or 8, where the Ca-induced Ca release was not activated, but after a ryanodine treatment to excise the functional compartment that possessed the Ca-induced Ca release mechanism. ATP had no effect on the rate of Ca leakage in the absence of IP3 even at pCa 5.5 after the ryanodine treatment. Therefore, ATP has direct biphasic effects on the IP3-induced Ca release mechanism. The Ca release induced by 0.1 microM IP3 at pCa 7 was potentiated not only by ATP, but by 0.5 mM ADP, AMP, or beta, gamma-methyleneadenosine 5'-triphosphate. 0.5 mM GTP had only a little effect on the IP3-induced Ca release. These results extend the functional similarities between Ca- and IP3-induced Ca release mechanisms in that adenine nucleotides enhance Ca release. Millimolar concentration of ATP, which is present physiologically, will shift the dose-response relation of IP3 toward the higher IP3 concentration and enhance the maximal effect of IP3. Thus, ATP is expected to assist the Ca release by higher concentrations of IP3 while having less effect on the Ca release by low levels of IP3. These effects of ATP may be important in the switching of Ca release from the intracellular Ca store by IP3.