Exosomes,the message transporters in vascular calcification

Vascular calcification (VC) is caused by hydroxyapatite deposition in the intimal and medial layers of the vascular wall, leading to severe cardiovascular events in patients with hypertension, chronic kidney disease and diabetes mellitus. VC occurrences involve complicated mechanism networks, such as matrix vesicles or exosomes production, osteogenic differentiation, reduced cell viability, aging and so on. However, with present therapeutic methods targeting at VC ineffectively, novel targets for VC treatment are demanded. Exosomes are proven to participate in VC and function as initializers for mineral deposition. Secreted exosomes loaded with microRNAs are also demonstrated to modulate VC procession in recipient vascular smooth muscle cells. In this review, we targeted at the roles of exosomes during VC, especially at their effects on transporting biological information among cells. Moreover, we will discuss the potential mechanisms of exosomes in VC.     

Endothelial cell and macrophage regulation of vascular smooth muscle cell calcification modulated by cholestane-3beta, 5alpha, 6beta-triol

The cellular and molecular mechanisms that mediate vascular calcification remain poorly understood. In our previous study, oxysterol cholestane-3beta, 5alpha, 6beta-triol (Triol) was shown to promote vascular smooth muscle cells (VSMCs) calcification. In this study, by using direct coculture, non-contact transwell coculture, and culture with conditioned media, we investigated the roles of endothelial cells (ECs) and macrophages in the regulation of VSMCs calcification in the absence or presence of Triol. In vitro calcification was induced by incubation of VSMCs with beta-glycerophosphate. The results showed that ECs inhibited VSMCs calcification, as manifested by the reduction of calcium deposition in extracellular matrix. This effect of ECs on calcification was via the secreted soluble factors. Furthermore, the stimulation of ECs by Triol had no influence on ECs inhibition of calcification. On the other hand, macrophages promoted VSMCs calcification via the secreted soluble factors such as reactive oxygen species, which was further enhanced by Triol. Our results supported the roles for ECs and macrophages in vascular calcification, modulated by oxysterols in atherosclerotic plaque.     

New insight of complement system in the process of vascular calcification

The complement system defences against pathogenic microbes and modulates immune homeostasis by interacting with the innate and adaptive immune systems. Dysregulation, impairment or inadvertent activation of complement system contributes to the pathogenesis of some autoimmune diseases and cardiovascular diseases (CVD). Vascular calcification is the pivotal pathological basis of CVD, and contributes to the high morbidity and mortality of CVD. Increasing evidences indicate that the complement system plays a key role in chronic kidney diseases, atherosclerosis, diabetes mellitus and aging-related diseases, which are closely related with vascular calcification. However, the effect of complement system on vascular calcification is still unclear. In this review, we summarize current evidences about the activation of complement system in vascular calcification. We also describe the complex network of complement system and vascular smooth muscle cells osteogenic transdifferentiation, systemic inflammation, endoplasmic reticulum stress, extracellular matrix remodelling, oxidative stress, apoptosis in vascular calcification. Hence, providing a better understanding of the potential relationship between complement system and vascular calcification, so as to provide a direction for slowing the progression of this burgeoning health concern.     

Microtubule stabilization attenuates vascular calcification through the inhibition of osteogenic signaling and matrix vesicle release

Vascular calcification is a strong predictor of cardiovascular morbidity and mortality, especially in individuals with chronic kidney disease or diabetes. The mechanism of vascular calcification has remained unclear, however, and no effective therapy is currently available. Our study was aimed at identifying the role of dynamic remodeling of microtubule cytoskeletons in hyperphosphatemia-induced vascular calcification. Exposure of primary cultures of mouse vascular smooth muscle cells (VSMCs) to inorganic phosphate (Pi) elicited ectopic calcification that was associated with changes in tubulin dynamics, induction of osteogenic signaling, and increased release of matrix vesicles. A microtubule depolymerizing agent enhanced Pi-dependent calcification, whereas microtubule stabilization by paclitaxel suppressed calcification both in VSMC cultures and in an ex vivo culture system for the mouse aorta. The inhibition of Pi-stimulated calcification by paclitaxel was associated with down-regulation of osteogenic signal and attenuation of matrix vesicle release. Our results indicate that microtubule plays a central role in vascular calcification, and that microtubule stabilization represents a potential new approach to the treatment of this condition.     

Apposite insulin-like growth factor (IGF) receptor glycosylation is critical to the maintenance of vascular smooth muscle phenotype in the presence of factors promoting osteogenic differentiation and mineralization

Vascular calcification is strongly linked with increased morbidity and mortality from cardiovascular disease. Vascular calcification is an active cell-mediated process that involves the differentiation of vascular smooth muscle cells (VSMCs) to an osteoblast-like phenotype. Several inhibitors of this process have been identified, including insulin-like growth factor-I (IGF-I). In this study, we examined the role of the IGF receptor (IGFR) and the importance of IGFR glycosylation in the maintenance of the VSMC phenotype in the face of factors known to promote osteogenic conversion. IGF-I (25 ng/ml) significantly protected VSMCs from β-glycerophosphate-induced osteogenic differentiation (p < 0.005) and mineral deposition (p < 0.01). Mevalonic acid depletion (induced by 100 nm cerivastatin) significantly inhibited these IGF protective effects (p < 0.01). Mevalonic acid depletion impaired IGFR processing, decreased the expression of mature IGFRs at the cell surface, and inhibited the downstream activation of Akt and MAPK. Inhibitors of N-linked glycosylation (tunicamycin, deoxymannojirimycin, and deoxynojirimycin) also markedly attenuated the inhibitory effect of IGF-I on β-glycerophosphate-induced mineralization (p < 0.05) and activation of Akt and MAPK. These results demonstrate that alterations in the glycosylation of the IGFR disrupt the ability of IGF-I to protect against the osteogenic differentiation and mineralization of VSMCs by several interrelated mechanisms: decreased IGFR processing, reduced IGFR cell-surface expression, and reduced downstream signaling via the Akt and MAPK pathways. IGF-I thus occupies a critical position in the maintenance of normal VSMC phenotype and protection from factors known to stimulate vascular calcification.     

非编码RNA调控血管平滑肌细胞成骨样表型转化的研究进展

分化成熟的血管平滑肌主要功能是收缩血管、调节血管周径及血压等.在高磷、高糖、维生素D₃、炎症等因素的作用下,平滑肌细胞可转分化为成骨样细胞参与血管钙化的形成,诱发心脑血管不良事件.非编码RNA是经基因转录但不翻译为蛋白质的一类RNA总称,其通过调控多种细胞活动来参与机体的生理和病理过程.已有研究表明,非编码RNA可通过调控血管平滑肌细胞成骨样表型转化影响血管钙化的发生、发展.本文从微小RNA、长链非编码RNA、环状RNA几方面综述非编码RNA在血管平滑肌成骨样表型转化中的调节作用,有助于进一步了解血管钙化的分子机制以及发现防治血管钙化的新靶点.     

Release of the cytokines colony-stimulating factor-1, granulocyte-macrophage colony-stimulating factor, and IL-6 by cloned murine vascular smooth muscle cells

Vascular smooth muscle cells were cultured from the mesenteric arteries of MRL lpr/lpr, MRL +/+, CBA/J, or C3H/HeJ mice and evaluated for their ability to synthesize a range of cytokines. Vascular smooth muscle cells of MRL +/+, MRL lpr/lpr, and CBA/J origin released biologically significant amounts of CSF-1 and IL-6 and relatively low but detectable amounts of granulocyte macrophage-CSF (GM-CSF) but not IL-2, IL-3, or IL-4. Vascular smooth muscle cells of C3H/HeJ origin produced lower amounts of CSF-1 and IL-6, and GM-CSF was barely detectable. Production of these cytokines did not require the exogenous growth factors present in FCS and occurred, although at lower levels, in serum-free medium supplemented with insulin, transferrin, and albumin. Cloned lines of MRL +/+ vascular smooth muscle cells, with electron microscopic and immunochemical properties of vascular smooth muscle cells, produced CSF-1, IL-6, and GM-CSF, establishing that vascular smooth muscle cells were a direct source of CSF-1, IL-6, and GM-CSF. These observations highlight the need for experiments to directly address the question of whether vascular smooth muscle cells constitutively produce these cytokines under physiologic conditions in vivo and suggest that vascular smooth muscle cells may participate actively in inflammation by releasing cytokines that are active on lympho-hemopoietic and other cells.     

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.     

Neuromedin B modulates phosphate-induced vascular calcification

Vascular calcification is the heterotopic accumulation of calcium phosphate salts in the vascular tissue and is highly correlated with increased cardiovascular morbidity and mortality. In this study, we found that the expression of neuromedin B (NMB) and NMB receptor is upregulated in phosphate-induced calcification of vascular smooth muscle cells (VSMCs). Silencing of NMB or treatment with NMB receptor antagonist, PD168368, inhibited the phosphate-induced osteogenic differentiation of VSMCs by inhibiting Wnt/β-catenin signaling and VSMC apoptosis. PD168368 also attenuated the arterial calcification in cultured aortic rings and in a rat model of chronic kidney disease. The results of this study suggest that NMB–NMB receptor axis may have potential therapeutic value in the diagnosis and treatment of vascular calcification.     

De la physiopathologie des calcifications vasculaires aux nouveaux marqueurs biologiques chez l’insuffisant rénal chronique

Vascular calcifications constitute an important risk factor for mortality in chronic kidney disease patients. A better knowledge of physiopathologic phenomena responsible for vascular mineralization leads to emerging biological markers of vascular calcifications. In calcified arteries, presence of bone matrix as well as osteoblast cells suggest that vascular calcification is an active and highly regulated process. In uremic environment, vascular smooth muscle cells can transdifferentiate into osteoblast-like cells. The OPG–RANK–RANKL system is clearly of central significance in controlling vascular calcifications as in bone metabolism. Converging results suggest that circulating OPG determination should be a relevant marker of calcifications. Impairment in inhibitory system such as Matrix Gla Protein and fetuin-A promotes bone matrix calcification. Finally, FGF-23, an early and sensitive marker of bone and mineral disorders in chronic kidney disease patients, appears as a promising marker.     

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.     

细胞外基质、基质水解酶与血管钙化

血管钙化常见于动脉粥样硬化、糖尿病、慢性肾功能衰竭及衰老的血管.近年来的研究证实血管钙化的发生是一种类似于生理性矿化的主动调节过程,而非单纯的钙磷的被动沉积.血管细胞外基质是血管的主要组成成分,对血管起支持、保护作用,且与血管壁细胞相互作用影响其粘附、增殖、迁移、分化等功能,同时又是各种生长因子和细胞因子的储存库.目前的研究显示,在血管钙化过程中细胞外基质的组成和表达可能发生了变化,并参与了对钙化进程的主动调节.基质水解酶可能通过基质降解依赖或非依赖的机制,在钙化的发生发展中起到重要作用.本文主要综述了在血管钙化过程中细胞外基质的变化及其对血管钙化的作用,以及基质水解酶对血管钙化过程可能的影响.     

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.     

Cartilage formation in growth plate and arteries: from physiology to pathology

Vascular calcifications are the consequence of several pathological conditions such as atherosclerosis, diabetes, hypercholesterolemia and chronic renal insufficiency. They are associated with risks of amputation, ischemic heart disease, stroke and increased mortality. A growing body of evidence indicates that vascular smooth muscle cells (VSMCs) undergo chondrogenic commitment eventually leading to vascular calcification, by mechanisms similar to those governing ossification in the cartilage growth plate. Our knowledge of the formation of cartilage growth plate can therefore help us to understand why and how arteries calcify and, consequently, develop new therapeutic strategies. Reciprocally, thorough consideration of the events leading to ectopic chondrocyte differentiation appears crucial to further increase our understanding of growth plate formation. In this context, we will review the effects of known or suspected factors that promote chondrogenic differentiation in growth plate and arteries.     

C-型钠尿肽与血管损伤性疾病

C-型钠尿肽(C-type natriuretic peptide, CNP)作为钠尿肽家系的一员, 主要是由血管内皮分泌,CNP与血管平滑肌细胞钠尿肽受体-B(NPR-B)结合,激活颗粒型鸟苷酸环化酶,促进细胞内cGMP 水平升高,以旁分泌和/或自分泌方式调节循环系统功能稳态.CNP广泛分布于血管系统,尤其在内皮细胞中高表达.CNP具有利钠、利尿、调节血管张力、抑制血管平滑肌细胞迁移、增殖等作用,与高血压、动脉粥样硬化、血栓形成、冠脉成形术后再狭窄和血管钙化等多种血管损伤性疾病密切相关.