Smooth Muscle Cell Differentiation Research Articles

Moderate substrate stiffness induces vascular smooth muscle cell differentiation through cellular morphological and tensional changes.

Vascular smooth muscle cells (VSMCs) are one of the main components of arterial walls and actively remodel the arterial walls in which they reside through biomechanical signals applied to themselves. Contractile or differentiated VSMCs have been observed in normal blood vessels. In pathological vascular conditions, they become dedifferentiated from contractile to non-contractile or synthetic cells, and a similar change is observed when VSMCs are placed in culture conditions. The mechanisms regulating VSMC differentiation remain unclear at this stage. In this paper we investigated the effects of substrate stiffness on the morphology, intercellular tension, and differentiation of VSMCs. Rat VSMCs were cultured on polyacrylamide (PA) gels, with elastic moduli of 15 kPa, 40 kPa, and 85 kPa, and PDMS substrate with elastic modulus of 1 MPa, and their morphology, intercellular tension, and contractile differentiation were assessed. Using fluorescence microscope image-based analysis and nano-indentation imaging with atomic force microscopy, we found that cell spreading and stiffening were induced by substrate stiffening in VSMCs. Interestingly, VSMCs on PA gel substrates with medium stiffness (40 kPa) showed significant elongation and shape polarization, and their 𝛼-SMA with F-actin cytoskeleton expression ratio was significantly higher than those of cells on other substrates. The results indicate an existing optimal substrate stiffness for promoting VSMC differentiation, and also indicate that cell shape polarization might be a key factor for VSMC differentiation.

The interaction between 30b-5p miRNA and MBNL1 mRNA is involved in vascular smooth muscle cell differentiation in patients with coronary atherosclerosis.

Vascular smooth muscle cells (VSMCs) in the arterial wall have diverse functions. In pathological states, the interplay between transcripts and microRNAs (miRNAs) leads to phenotypic changes. Understanding the regulatory role of miRNAs and their target genes may reveal how VSMCs modulate the pathogenesis of coronary artery disease. Laser capture microdissection was performed on aortic wall tissues obtained from coronary artery bypass graft patients with and without recent acute myocardial infarction (MI). The mSMRT-qPCR miRNA assay platform (MiRXES, Singapore) was used to profile miRNA. The miRNA data were co-analyzed with significant mRNA transcripts. TargetScan 7.1 was applied to evaluate miRNA–mRNA interactions. The miRNA profiles of 29 patients (16 MI and 13 non-MI) were evaluated. Thirteen VSMC-related miRNAs were differentially expressed between the MI and non-MI groups. Analysis revealed seven miRNA-targeted mRNAs related to muscular tissue differentiation and proliferation. TargetScan revealed that among the VSMC-related transcripts, MBNL1 had a recognition site that matched the hsa-miR-30b-5p target seed sequence. In addition to predicted analysis, our experiment in vitro with human VSMC culture confirmed that hsa-miR-30b-5p negatively correlated with MBNL1. Our data showed that overexpression of hsa-miR-30b-5p led to downregulation of MBNL1 in VSMCs. This process influences VSMC proliferation and might be involved in VSMC differentiation.

Adipocyte enhancer binding protein 1 (AEBP1) knockdown suppresses human glioma cell proliferation, invasion and induces early apoptosis

BackgroundGlioma is the most common primary malignant tumor with poor prognosis due to the lack of understanding the mechanism underlying the disease and the early diagnosis indexs. It is necessary to identify molecular signatures for predicting the overall prognosis of glioma. Adipocyte enhancer binding protein1 (AEBP1) acts as a transcriptional repressor and plays a role in adipogenesis and smooth muscle cell differentiation. However, its role in glioma remains unclear. Materials and methodsAEBP1 expression was analyzed by bioinformatics using the public database and by qPCR and western blotting in human glioma tissues. AEBP1 downregulation was performed by lipofectamine3000-mediated siRNA transfection. Cell proliferation and invasion were determined by cell counting kit-8 and transwell assays, while early cell apoptosis was determined by flow cytometry. The proteins of downstream NF-κB signaling pathway were determined by western blotting. ResultsAEBP1 is highly expressed in human gliomas. Lipofectamine 3000-mediated siRNA transfection stably and efficiently suppressed AEBP1 mRNA and protein expression in human glioma cells. AEBP1 downregulation inhibited cell proliferation and invasion, but promoted early cell apoptosis. Also, AEBP1 knockdown in glioma cells decreased the expression of NF-κB1. Furthermore, the downstream of NF-κB signaling pathway, Bax, caspase-3 are increased, while MMP2 and Bcl-2 are decreased in glioma cells. ConclusionElevated AEBP1 is positively associated with poor prognosis of glioma. AEBP1 downregulation suppressed cell proliferation and invasion, but promoted early cell apoptosis. AEBP1 downregulation suppressed the cell proliferation and invasion may by inhibiting the NF-κB signaling pathway.

Rho-associated protein kinase and cyclophilin a are involved in inorganic phosphate-induced calcification signaling in vascular smooth muscle cells

Arterial calcification, a risk factor of cardiovascular events, develops with differentiation of vascular smooth muscle cells (VSMCs) into osteoblast-like cells. Cyclophilin A (CypA) is a peptidyl-prolyl isomerase involved in cardiovascular diseases such as atherosclerosis and aortic aneurysms, and rho-associated protein kinase (ROCK) is involved in the pathogenesis of vascular calcification. CypA is secreted in a ROCK activity-dependent manner and works as a mitogen via autocrine or paracrine mechanisms in VSMCs. We examined the involvement of the ROCK-CypA axis in VSMC calcification induced by inorganic phosphate (Pi), a potent cell mineralization initiator. We found that Pi stimulated ROCK activity, CypA secretion, extracellular signal-regulated protein kinase (ERK) 1/2 phosphorylation, and runt-related transcription factor 2 expression, resulting in calcium accumulation in rat aortic smooth muscle cells (RASMCs). The ROCK inhibitor Y-27632 significantly suppressed Pi-induced CypA secretion, ERK1/2 phosphorylation, and calcium accumulation. Recombinant CypA was found to be associated with increased calcium accumulation in RASMCs. Based on these results, we suggest that autocrine CypA is mediated by ROCK activity and is involved in Pi-induced ERK1/2 phosphorylation following calcification signaling in RASMCs.

Coronary Disease-Associated Gene TCF21 Inhibits Smooth Muscle Cell Differentiation by Blocking the Myocardin-Serum Response Factor Pathway.

The gene encoding TCF21 (transcription factor 21) has been linked to coronary artery disease risk by human genome-wide association studies in multiple racial ethnic groups. In murine models, Tcf21 is required for phenotypic modulation of smooth muscle cells (SMCs) in atherosclerotic tissues and promotes a fibroblast phenotype in these cells. In humans, TCF21 expression inhibits risk for coronary artery disease. The molecular mechanism by which TCF21 regulates SMC phenotype is not known. To better understand how TCF21 affects the SMC phenotype, we sought to investigate the possible mechanisms by which it regulates the lineage determining MYOCD (myocardin)-SRF (serum response factor) pathway. Modulation of TCF21 expression in human coronary artery SMC revealed that TCF21 suppresses a broad range of SMC markers, as well as key SMC transcription factors MYOCD and SRF, at the RNA and protein level. We conducted chromatin immunoprecipitation-sequencing to map SRF-binding sites in human coronary artery SMC, showing that binding is colocalized in the genome with TCF21, including at a novel enhancer in the SRF gene, and at the MYOCD gene promoter. In vitro genome editing indicated that the SRF enhancer CArG box regulates transcription of the SRF gene, and mutation of this conserved motif in the orthologous mouse SRF enhancer revealed decreased SRF expression in aorta and heart tissues. Direct TCF21 binding and transcriptional inhibition at colocalized sites were established by reporter gene transfection assays. Chromatin immunoprecipitation and protein coimmunoprecipitation studies provided evidence that TCF21 blocks MYOCD and SRF association by direct TCF21-MYOCD interaction. These data indicate that TCF21 antagonizes the MYOCD-SRF pathway through multiple mechanisms, further establishing a role for this coronary artery disease-associated gene in fundamental SMC processes and indicating the importance of smooth muscle response to vascular stress and phenotypic modulation of this cell type in coronary artery disease risk.

Vanadium Derivative Exposure Promotes Functional Alterations of VSMCs and Consequent Atherosclerosis via ROS/p38/NF-κB-Mediated IL-6 Production.

Vanadium is a transition metal widely distributed in the Earth’s crust, and is a major contaminant in fossil fuels. Its pathological effect and regulation in atherosclerosis remain unclear. We found that intranasal administration of the vanadium derivative NaVO3 significantly increased plasma and urinary vanadium levels and induced arterial lipid accumulation and atherosclerotic lesions in apolipoprotein E-deficient knockout mice (ApoE−/−) murine aorta compared to those in vehicle-exposed mice. This was accompanied by an increase in plasma reactive oxygen species (ROS) and interleukin 6 (IL-6) levels and a decrease in the vascular smooth muscle cell (VSMC) differentiation marker protein SM22α in the atherosclerotic lesions. Furthermore, exposure to NaVO3 or VOSO4 induced cytosolic ROS generation and IL-6 production in VSMCs and promoted VSMC synthetic differentiation, migration, and proliferation. The anti-oxidant N-acetylcysteine (NAC) not only suppresses IL-6 production and VSMC pathological responses including migration and proliferation but also prevents atherosclerosis in ApoE−/− mice. Inhibition experiments with NAC and pharmacological inhibitors demonstrated that NaVO3-induced IL-6 production is signaled by ROS-triggered p38-mediated NF-κB-dependent pathways. Neutralizing anti-IL-6 antibodies impaired NaVO3-mediated VSMC migration and proliferation. We concluded that NaVO3 exposure activates the ROS-triggering p38 signaling to selectively induce NF-κB-mediated IL-6 production. These signaling pathways induce VSMC synthetic differentiation, migration, and proliferation, leading to lipid accumulation and atherosclerosis.

TBX2-positive cells represent a multi-potent mesenchymal progenitor pool in the developing lung

BackgroundIn the embryonic mammalian lung, mesenchymal cells act both as a signaling center for epithelial proliferation, differentiation and morphogenesis as well as a source for a multitude of differentiated cell types that support the structure of the developing and mature organ. Whether the embryonic pulmonary mesenchyme is a homogenous precursor pool and how it diversifies into different cell lineages is poorly understood. We have previously shown that the T-box transcription factor gene Tbx2 is expressed in the pulmonary mesenchyme of the developing murine lung and is required therein to maintain branching morphogenesis.MethodsWe determined Tbx2/TBX2 expression in the developing murine lung by in situ hybridization and immunofluorescence analyses. We used a genetic lineage tracing approach with a Cre line under the control of endogenous Tbx2 control elements (Tbx2cre), and the R26mTmG reporter line to trace TBX2-positive cells in the murine lung. We determined the fate of the TBX2 lineage by co-immunofluorescence analysis of the GFP reporter and differentiation markers in normal murine lungs and in lungs lacking or overexpressing TBX2 in the pulmonary mesenchyme.ResultsWe show that TBX2 is strongly expressed in mesenchymal progenitors in the developing murine lung. In differentiated smooth muscle cells and in fibroblasts, expression of TBX2 is still widespread but strongly reduced. In mesothelial and endothelial cells expression is more variable and scattered. All fetal smooth muscle cells, endothelial cells and fibroblasts derive from TBX2+ progenitors, whereas half of the mesothelial cells have a different descent. The fate of TBX2-expressing cells is not changed in Tbx2-deficient and in TBX2-constitutively overexpressing mice but the distribution and abundance of endothelial and smooth muscle cells is changed in the overexpression condition.ConclusionThe fate of pulmonary mesenchymal progenitors is largely independent of TBX2. Nevertheless, a successive and precisely timed downregulation of TBX2 is necessary to allow proper differentiation and functionality of bronchial smooth muscle cells and to limit endothelial differentiation. Our work suggests expression of TBX2 in an early pulmonary mesenchymal progenitor and supports a role of TBX2 in maintaining the precursor state of these cells.

P.775 The impact of mental illness on compliance with therapy in patients with Parkinson's disease

Calcification is a hallmark of advanced atherosclerosis and an active process akin to bone remodeling. Heparanase (HPSE) is an endo-β-glucuronidase, which cleaves glycosaminoglycan chains of heparan sulfate proteoglycans. The role of HPSE is controversial in osteogenesis and bone remodeling while it is unexplored in vascular calcification. Previously, we reported upregulation of HPSE in human carotid endarterectomies from symptomatic patients and showed correlation of HPSE expression with markers of inflammation and increased thrombogenicity. The present aim is to investigate HPSE expression in relation to genes associated with osteogenesis and osteolysis and the effect of elevated HPSE expression on calcification and osteolysis in vitro.Transcriptomic and immunohistochemical analyses were performed using the Biobank of Karolinska Endarterectomies (BiKE). In vitro calcification and osteolysis were analysed in human carotid smooth muscle cells overexpressing HPSE and bone marrow-derived osteoclasts from HPSE-transgenic mice respectively.HPSE expression correlated primarily with genes coupled to osteoclast differentiation and function in human carotid atheromas. HPSE was expressed in osteoclast-like cells in atherosclerotic lesions, and HPSE-transgenic bone marrow-derived osteoclasts displayed a higher osteolytic activity compared to wild-type cells. Contrarily, human carotid SMCs with an elevated HPSE expression demonstrated markedly increased mineralization upon osteogenic differentiation.We suggest that HPSE may have dual functions in vascular calcification, depending on the stage of the disease and presence of inflammatory cells. While HPSE plausibly enhances mineralization and osteogenic differentiation of vascular smooth muscle cells, it is associated with inflammation-induced osteoclast differentiation and activity in advanced atherosclerotic plaques.

Identification of the intermediate filament protein synemin/SYNM as a target of myocardin family coactivators.

Myocardin (MYOCD) is a critical regulator of smooth muscle cell (SMC) differentiation, but its transcriptional targets remain to be exhaustively characterized, especially at the protein level. Here we leveraged human RNA and protein expression data to identify novel potential MYOCD targets. Using correlation analyses we found several targets that we could confirm at the protein level, including SORBS1, SLMAP, SYNM, and MCAM. We focused on SYNM, which encodes the intermediate filament protein synemin. SYNM rivalled smooth muscle myosin (MYH11) for SMC specificity and was controlled at the mRNA and protein levels by all myocardin-related transcription factors (MRTFs: MYOCD, MRTF-A/MKL1, and MRTF-B/MKL2). MRTF activity is regulated by the ratio of filamentous to globular actin, and SYNM was accordingly reduced by interventions that depolymerize actin, such as latrunculin treatment and overexpression of constitutively active cofilin. Many MRTF target genes depend on serum response factor (SRF), but SYNM lacked SRF-binding motifs in its proximal promoter, which was not directly regulated by MYOCD. Furthermore, SYNM resisted SRF silencing, yet the time course of induction closely paralleled that of the SRF-dependent target gene ACTA2. SYNM was repressed by the ternary complex factor (TCF) FLI1 and was increased in mouse embryonic fibroblasts lacking three classical TCFs (ELK1, ELK3, and ELK4). Imaging showed colocalization of SYNM with the intermediate filament proteins desmin and vimentin, and MRTF-A/MKL1 increased SYNM-containing intermediate filaments in SMCs. These studies identify SYNM as a novel SRF-independent target of myocardin that is abundantly expressed in all SMCs.

Aloe-emodin inhibits osteogenic differentiation and calcification of mouse vascular smooth muscle cells

Vascular calcification increases the risk of morbidity and mortality in patients with cardiovascular diseases, chronic kidney diseases, and diabetes. However, viable therapeutic methods to target vascular calcification are limited. Aloe-emodin (AE), an anthraquinone is a natural compound found in the leaves of Aloe-vera. In this study, we investigated the underlying mechanism of AE in the calcification of vascular smooth muscle cells (VSMCs) and murine thoracic aorta. We demonstrate that AE repressed not only the phenotypes of Ca2+ induced calcification but also level of calcium in VSMCs. AE has no effect on cell viability in VSMC cells. Alizarin red, von Kossa stainings and calcium quantification showed that Ca2+ induced vascular calcification is significantly decreased by AE in a concentration-dependent manner. In contrast, AE attenuated Ca2+ induced calcification through inhibiting osteoblast differentiation genes such as SMAD4, collagen 1α, osteopontin (OPN), Runt-related transcription factor (RUNX-2) and Osterix. AE also suppressed Ca2+ induced osteoblast-related protein expression including collagen 1α, bone morphogenic protein 2 (BMP-2), RUNX-2 and smooth muscle actin (SMA). Furthermore, Alizarin red, von Kossa stainings and calcium quantification showed that AE significantly inhibited the calcification of ex vivo ring formation in murine thoracic aorta, and markedly inhibited vitamin D3 induced medial aorta calcification in vivo. Taken together, our findings suggest that AE may have therapeutic potential for the prevention of vascular calcification program.

Folate deficiency promotes differentiation of vascular smooth muscle cells without affecting the methylation status of regulated genes.

Elevated serum homocysteine, an intermediate of cellular one-carbon metabolism, is an independent risk factor for cardiovascular disease (CVD). Folate deficiency increases serum homocysteine and may contribute to CVD progression. Vascular smooth muscle cells (VSMCs) regulate vascular contractility, but also contribute to repair processes in response to vascular injury. Nutritional deficiencies, like folate deficiency, are thought to impact on this phenotypic plasticity, possibly by epigenetic mechanisms. We have investigated the effect of folate deficiency on VSMCs in two cell culture systems representing early and late stages of smooth muscle cells differentiation. We find that folate deficiency promotes differentiation towards a more contractile phenotype as indicated by increased expression of respective marker genes. However, microarray analysis identified markers of striated muscle as the predominant gene expression change elicited by folate deficiency. These changes are not merely a reflection of cell cycle arrest, as foetal calf serum restriction or iron deficiency do not replicate the gene expression changes observed in response to folate deficiency. Folate deficiency only has a marginal effect on global DNA methylation. DNA methylation of CpG islands associated with genes regulated by folate deficiency remains unaffected. This supports our earlier findings in a mouse model system which also did not show any changes in global DNA methylation in response to folate and vitamin B6/B12 deficiency. These data suggest that folate deficiency enhances the expression of smooth muscle marker gene expression, promotes a shift towards a skeletal muscle phenotype, and does not regulate gene expression via DNA methylation.

CDC42 promotes vascular calcification in chronic kidney disease.

Vascular calcification is prevalent in patients with chronic kidney disease (CKD) and a major risk factor of cardiovascular disease. Vascular calcification is now recognised as a biological process similar to bone formation involving osteogenic differentiation of vascular smooth muscle cells (VSMCs). Cell division cycle 42 (CDC42), a Rac1 family member GTPase, is essential for cartilage development during endochondral bone formation. However, whether CDC42 affects osteogenic differentiation of VSMCs and vascular calcification remains unknown. In the present study, we observed a significant increase in the expression of CDC42 both in rat VSMCs and in calcified arteries during vascular calcification. Alizarin red staining and calcium content assay revealed that adenovirus-mediated CDC42 overexpression led to an apparent VSMC calcification in the presence of calcifying medium, accompanied with up-regulation of bone-related molecules including RUNX2 and BMP2. By contrast, inhibition of CDC42 by ML141 significantly blocked calcification of VSMCs in vitro and aortic rings ex vivo. Moreover, ML141 markedly attenuated vascular calcification in rats with CKD. Furthermore, pharmacological inhibition of AKT signal was shown to block CDC42-induced VSMC calcification. These findings demonstrate for the first time that CDC42 contributes to vascular calcification through a mechanism involving AKT signalling; this uncovered a new function of CDC42 in regulating vascular calcification. This may provide a potential therapeutic target for the treatment of vascular calcification in the context of CKD. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Contribution of transcription factor EB to adipoRon-induced inhibition of arterial smooth muscle cell proliferation and migration.

Abnormal vascular smooth muscle cell (SMC) dedifferentiation with increased proliferation and migration during pathological vascular remodeling is associated with vascular disorders, such as atherosclerosis and in-stent restenosis. AdipoRon, a selective agonist of adiponectin receptor, has been shown to protect against vascular remodeling by preventing SMC dedifferentiation. However, the molecular mechanisms that mediate adipoRon-induced SMC differentiation are not well understood. The present study aimed to elucidate the role of transcription factor EB (TFEB), a master regulator of autophagy, in mediating adipoRon's effect on SMCs. In cultured arterial SMCs, adipoRon dose-dependently increased TFEB activation, which is accompanied by upregulated transcription of genes involved in autophagy pathway and enhanced autophagic flux. In parallel, adipoRon suppressed serum-induced cell proliferation and caused cell cycle arrest. Moreover, adipoRon inhibited SMC migration as characterized by wound-healing retardation, F-actin reorganization, and matrix metalloproteinase-9 downregulation. These inhibitory effects of adipoRon on proliferation and migration were attenuated by TFEB gene silencing. Mechanistically, activation of TFEB by adipoRon is dependent on intracellular calcium, but it is not associated with changes in AMPK, ERK1/2, Akt, or molecular target of rapamycin complex 1 activation. Using ex vivo aortic explants, we demonstrated that adipoRon inhibited sprouts that had outgrown from aortic rings, whereas lentiviral TFEB shRNA transduction significantly reversed this effect of adipoRon on aortic rings. Taken together, our results indicate that adipoRon activates TFEB signaling that helps maintain the quiescent and differentiated status of arterial SMCs, preventing abnormal SMC dedifferentiation. This study provides novel mechanistic insights into understanding the therapeutic effects of adipoRon on TFEB signaling and pathological vascular remodeling.

Wnt Signaling Modulates Routes of Retinoic Acid-Induced Differentiation of Embryonic Stem Cells.

Smooth muscle cells (SMCs) are important cell type for regenerative medicine. Previous studies showed that retinoic acid (RA) induces differentiation of SMCs from monolayer-cultured embryonic stem cells (ESCs) with high efficiency. However, the underlying mechanisms are still poorly defined. Here, we identified Wnt signaling as a primary regulator for RA-induced ESC differentiation. The activation of Wnt signaling inhibited the epithelial-mesenchymal transition during ESC differentiation, leading to inhibition of RA-induced SMC differentiation and promoting differentiation of ESCs toward primitive endoderm (PrE) lineage instead, while the inhibition of Wnt signaling promoted RA-induced SMC differentiation. Loss-of-function studies revealed that 7-like 2 (Tcf7l2) was the key transcription factor that Wnt operate through during RA-induced differentiation. Thus, this study revealed that the Tcf7l2-mediated Wnt signaling is a switch in determining the mesoderm/PrE fates in RA-induced ESC differentiation.

ALDH1A3 Regulations of Matricellular Proteins Promote Vascular Smooth Muscle Cell Proliferation.

SummaryVascular smooth muscle cell (VSMC) proliferation promotes intimal hyperplasia (IH) in occluding vascular diseases. Here we identified a positive role of ALDH1A3 (an aldehyde dehydrogenase) in this pro-IH process. The expression of ALDH1A3, but not that of 18 other isoforms of the ALDH family, was substantially increased in cytokine-stimulated VSMCs. PDGF(BB) stimulated VSMC total ALDH activity and proliferation, whereas ALDH1A3 silencing abolished this effect. ALDH1A3 silencing also diminished the expression of two matricellular proteins (TNC1 and ESM1), revealing a previously unrecognized ALDH1A3 function. Loss-of-function experiments demonstrated that TNC1 and ESM1 mediated ALDH1A3's pro-proliferative function via activation of AKT/mTOR and/or MEK/ERK pathways. Furthermore, ALDH inhibition with disulfiram blocked VSMC proliferation/migration in vitro and decreased TNC1 and ESM1 and IH in angioplasty-injured rat carotid arteries. Thus, ALDH1A3 promotes VSMC proliferation at least partially through TNC1/ESM1 upregulation; dampening excessive ALDH1A3 activity represents a potential approach to IH mitigation.

The effects of hyperuricemia on the differentiation and proliferation of osteoblasts and vascular smooth muscle cells are implicated in the elevated risk of osteopenia and vascular calcification in gout: An in vivo and in vitro analysis.

In this study, we established both an animal model and a cellular model of hyperuricemia (HUC). Subsequently, we treated these models with allopurinol (ALLO) to study the effect of uric acid (UA) and ALLO on the differentiation and proliferation of osteoblasts and vascular smooth muscle cells (VSMC). Western Blot, immunohistochemistry assay, and real-time polymerase chain reaction were conducted to measure the changes in the expression of differentiation-related factors in osteoblasts and VSMCs in HUC and HUC+ALLO groups. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and flow cytometry were utilized to observe the changes in the proliferation of osteoblasts in HUC and HUC+ALLO groups. Von Kossa staining was performed along with calcium content measurement to investigate the effect of HUC/ALLLO on vascular calcification. In this study, the levels of Wnt3a and differentiation-related factors, including Runx2, Sp7, Ibsp, Bglap, Dmp1, and Col1a1, were all evidently decreased in HUC rats, while the presence of ALLO increased the levels of above factors. In addition, the viability of osteoblasts was reduced while their apoptosis was elevated in the HUC group, and ALLO treatment reduced the apoptosis and increased the viability of osteoblasts to a certain extent. Moreover, HUC elevated the levels of Wnt3a, Runx2, Sp7, Bglap, Col1a1, SM22a, and Acta2 in VSMCs of HUC rats, leading to greatly increased calcium content and obvious vascular calcification. In contrary, ALLO treatment reduced the effect of HUC. Furthermore, the effect of UA and ALLO on osteoblasts and VSMCs was also validated in cellular models treated with monosodium urate (MSU) crystals or MSU+ALLO. HUC can suppress the differentiation and proliferation of osteoblasts while promoting the differentiation of VSMCs both in vivo and in vitro. The treatment by ALLO exhibited a therapeutic effect on HUC by promoting the differentiation and proliferation of osteoblasts while reducing vascular calcification.

Reticulocalbin 2 enhances osteogenic differentiation of human vascular smooth muscle cells in diabetic conditions

AimDiabetes accelerates pro-atherogenic and pro-osteogenic phenotypes of vascular smooth muscle cells (VSMCs), an important process for vascular calcification. Reticulocalbin 2 (RCN2) is a candidate gene for atherosclerosis and involved in vascular remodeling in hypertension. However, the role of RCN2 in VSMCs calcification under diabetic conditions is unclear. Materials and methodsExpression of RCN2 and Runt-related transcription factor 2 (Runx2) in femoropopliteal arterial plaques was compared between type 2 diabetes mellitus (DM) and non-DM patients using immunohistochemical staining (IHCS). Human aortic VSMCs (HAVSMCs) were analyzed under RCN2 gene knockdown and overexpression conditions. Alizarin red staining and intracellular calcium deposition quantification were used to observe calcification induced in vitro under normal glucose or high glucose combined with β-glycerol phosphoric acid conditions. The cells were investigated for gene modulation of osteogenic differentiation markers using Western blotting. Key findingsThe expression of RCN2 and Runx2 in femoropopliteal artery plaques was significantly higher in DM than in non-DM patients. In addition, a significant positive correlation was observed between RCN2 and Runx2 levels. RCN2 was highly expressed when HAVSMCs were treated with high glucose and the expression levels correlated with the calcification characteristics. RCN2 upregulated osteogenic transformation markers Runx2 and Osterix in HAVSMCs and downregulated contractile phenotype markers α-SMA and SM22α. SignificanceThe results from this study indicate RCN2 is a major factor in mediating the calcification process of HAVSMCs in diabetic conditions. Thus, RCN2 may serve as a future therapeutic target for vascular calcification in diabetes.

Adiponectin inhibits vascular smooth muscle cell calcification induced by beta-glycerophosphate through JAK2/STAT3 signaling pathway

Vascular calcification is a common problem in the elderly with diabetes, heart failure and end-stage renal disease. The differentiation of vascular smooth muscle cells (VSMCs) into osteoblasts is the main feature, but the exact mechanism remains unclear. It is not clear whether adiponectin (APN) affects osteogenic differentiation of VSMCs. This study aims to explore the effect of APN on vascular calcification by using a cell model induced by beta-glycerophosphate (beta-GP). VSMCs were isolated and treated with beta-GP and APN in this study. The alkaline phosphatase (ALP) activity and expression levels of Runx2, BMP-2, collagen type I and osteocalcin were determined. The expression levels of STAT3 and p-STAT3 in nucleus and cytoplasm of VSMCs were analyzed. The results showed that APN significantly inhibited the expression of ALP, Runx2, BMP-2, collagen I, osteocalcin and the formation of the mineralized matrix in VSMCs induced by beta-GP. APN reduces the osteogenic differentiation of VSMCs induced by beta-GP and down-regulates the expression of the osteogenic transcription factor osterix by inhibiting STATS3 phosphorylation and nuclear transport. APN may be one of the potential candidates for clinical treatment of vascular calcification.

Lipoproteins And Their Modified Forms Regulate Early Stage Vascular Smooth Muscle Cell Calcification

Background and Aims: Vascular calcification (VC), a hallmark of atherosclerosis, appears as inflammatory associated microcalcifications and plaque stabilising macrocalcifications, resulting from the differentiation of vascular smooth muscle cells (VSMC) into bone-like cells. Although atherogenic lipoproteins are well characterised in atherosclerosis and implicated in VC, their effects on VC remain unknown to this prevalent, untreatable disease. This study therefore examines the roles of lipoproteins on VC.

MicroRNA-125b in vascular diseases: An updated systematic review of pathogenetic implications and clinical applications.

Epigenetic changes, particularly non‐coding RNAs, have been implicated extensively in the pathogenesis of vascular diseases. Specific miRNAs are involved in the differentiation, phenotypic switch, proliferation, apoptosis, cytokine production and matrix deposition of endothelial cells and/or vascular smooth muscle cells. MicroRNA‐125b has been studied in depth for its role in carcinogenesis with a double‐edged role; that is, it can act as an oncogene in some cancer types and as a tumour suppressor gene in others. However, cumulative evidence from the use of advanced miRNA profiling techniques and bioinformatics analysis suggests that miR‐125b can be a potential mediator and useful marker of vascular diseases. Currently, the exact role of miR‐125b in vascular diseases is not known. In this systematic review, we intend to provide an updated compilation of all the recent findings of miR‐125b in vascular diseases, using a systematic approach of retrieving data from all available reports followed by data summarization. MiR‐125b serves as a pathogenic player in multiple vascular pathologies involving endothelia and vascular smooth muscle cells and also serves as a diagnostic marker for vascular diseases. We further provide a computational biologic presentation of the complex network of miR‐125b and its target genes within the scope of vascular diseases.