Differentiation Of Vascular Smooth Muscle Cells Research Articles

Circ-SATB2 upregulates STIM1 expression and regulates vascular smooth muscle cell proliferation and differentiation through miR-939

The prevention and treatment of coronary heart disease (CHD) is a difficult problem to be solved. More and more studies have found that circular RNAs (circRNAs) may play important roles in the development of CHD. Here detection of vascular smooth muscle cells (VSMCs) showed that circ-SATB2 and STIM1 were up-regulated in proliferative VSMCs, while miR-939 were down-regulated. Circ-SATB2 and miR-939 did not affect the expression of each other, but circ-SATB2 could promote while miR-939 inhibited the expression of STIM1 (a target gene of miR-939). Circ-SATB2 overexpression could inhibit the expression of SM22-alpha (SM22α, a marker of contractile VSMCs), while the expression of SM22α was promoted by miR-939. STIM1 could promote cell proliferation and migration, and circ-SATB2 had similar effects, but its linear sequence had no such functions. MiR-939 had the opposite effects, could promote cell apoptosis and inhibit cell proliferation and migration, and siRNAs targeting circ-SATB2 had similar effects. When co-transfected with circ-SATB2 over-expression vector and miR-939 mimics or STIM1 siRNAs, the changes of cell proliferation, apoptosis and migration were not significant. Therefore, circ-SATB2 can regulate VSMC phenotypic differentiation, proliferation, apoptosis and migration by promoting the expression of STIM1. This discovery will provide a theoretical reference for exploring the role of circRNA in VSMCs and the pathogenesis of CHD.

High-serum phosphate and parathyroid hormone distinctly regulate bone loss and vascular calcification in experimental chronic kidney disease.

In chronic kidney disease (CKD), increases in serum phosphate and parathyroid hormone (PTH) aggravate vascular calcification (VC) and bone loss. This study was designed to discriminate high phosphorus (HP) and PTH contribution to VC and bone loss. Nephrectomized rats fed a HP diet underwent either sham operation or parathyroidectomy and PTH 1-34 supplementation to normalize serum PTH. In uraemic rats fed a HP diet, parathyroidectomy with serum PTH 1-34 supplementation resulted in (i) reduced aortic calcium (80%) by attenuating osteogenic differentiation (higher α-actin; reduced Runx2 and BMP2) and increasing the Wnt inhibitor Sclerostin, despite a similar degree of hyperphosphataemia, renal damage and serum Klotho; (ii) prevention of bone loss mostly by attenuating bone resorption and increases in Wnt inhibitors; and (iii) a 70% decrease in serum calcitriol levels despite significantly reduced serum Fgf23, calcium and renal 24-hydroxylase, which questions that Fgf23 is the main regulator of renal calcitriol production. Significantly, when vascular smooth muscle cells (VSMCs) were exposed exclusively to high phosphate and calcium, high PTH enhanced while low PTH attenuated calcium deposition through parathyroid hormone 1 receptor (PTH1R) signalling. In hyperphosphataemic CKD, a defective suppression of high PTH exacerbates HP-mediated osteogenic VSMC differentiation and reduces vascular levels of anti-calcifying sclerostin.

C-Myb regulates transcriptional activation of miR-143/145 in vascular smooth muscle cells.

BackgroundMicroRNAs (miR) are small non-coding RNAs that regulate diverse biological functions. The bicistronic gene miR-143/145 determines cell fate and phenotype of vascular smooth muscle cells (VSMC), in part, by destabilizing Elk-1 mRNA. The transcription factor c-Myb also regulates differentiation and proliferation of VSMC, and here we test whether these effects may be mediated by miR-143/145.Methods & resultsFlow cytometry of cardiovascular-directed d3.75 embryoid bodies (EBs) isolated smooth muscle progenitors with specific cell surface markers. In c-myb knockout (c-myb -/-) EB, these progenitors manifest low levels of miR-143 (19%; p<0.05) and miR-145 (6%; p<0.01) expression as compared to wild-type (wt) EB. Primary VSMC isolated from transgenic mice with diminished expression (c-myblx/lx) or reduced activity (c-mybh/h) of c-Myb also manifest low levels of miR-143 (c-myblx/lx: 50%; c-mybh/h: 41%), and miR-145 (c-myblx/lx: 49%; c-mybh/h: 56%), as compared to wt (P<0.05). Sequence alignment identified four putative c-Myb binding sites (MBS1-4) in the proximal promoter (PP) of the miR-143/145 gene. PP-reporter constructs revealed that point mutations in MBS1 and MBS4 abrogated c-Myb-dependent transcription from the miR-143/145 PP (P<0.01). Chromatin immunoprecipitation (ChIP) revealed preferential c-Myb binding at MBS4 (p<0.001). By conjugating Elk-1 3’-untranslated region (UTR) to a reporter and co-transducing wt VSMC with this plus a miR-143-antagomir, and co-transducing c-myblx/lx VSMC with this plus a miR-143-mimic, we demonstrate that c-Myb’s ability to repress Elk-1 is mediated by miR-143.Conclusionc-Myb regulates VSMC gene expression by transcriptional activation of miR-143/145.

MiR-214 is an important regulator of the musculoskeletal metabolism and disease.

MiR-214 belongs to a family of microRNA (small, highly conserved noncoding RNA molecules) precursors that play a pivotal role in biological functions, such as cellular function, tissue development, tissue homeostasis, and pathogenesis of diseases. Recently, miR-214 emerged as a critical regulator of musculoskeletal metabolism. Specifically, miR-214 can mediate skeletal muscle myogenesis and vascular smooth muscle cell proliferation, migration, and differentiation. MiR-214 also modulates osteoblast function by targeting specific molecular pathways and the expression of various osteoblast-related genes; promotes osteoclast activity by targeting phosphatase and tensin homolog (Pten); and mediates osteoclast-osteoblast intercellular crosstalk via an exosomal miRNA paracrine mechanism. Importantly, dysregulation in miR-214 expression is associated with pathological bone conditions such as osteoporosis, osteosarcoma, multiple myeloma, and osteolytic bone metastasis of breast cancer. This review discusses the cellular targets of miR-214 in bone, the molecular mechanisms governing the activities of miR-214 in the musculoskeletal system, and the putative role of miR-214 in skeletal diseases. Understanding the biology of miR-214 could potentially lead to the development of miR-214 as a possible biomarker and a therapeutic target for musculoskeletal diseases.

Transforming growth factor β1 suppresses proinflammatory gene program independent of its regulation on vascular smooth muscle differentiation and autophagy

Transforming growth factor β (TGFβ) signaling plays crucial roles in maintaining vascular integrity and homeostasis, and is established as a strong activator of vascular smooth muscle cell (VSMC) differentiation. Chronic inflammation is a hallmark of various vascular diseases. Although TGFβ signaling has been suggested to be protective against inflammatory aortic aneurysm progression, its exact effects on VSMC inflammatory process and the underlying mechanisms are not fully unraveled. Here we revealed that TGFβ1 suppressed the expression of a broad array of proinflammatory genes while potently induced the expression of contractile genes in cultured primary human coronary artery SMCs (HCASMCs). The regulation of TGFβ1 on VSMC contractile and proinflammatory gene programs appeared to occur in parallel and both processes were through a SMAD4-dependent canonical pathway. We also showed evidence that the suppression of TGFβ1 on VSMC proinflammatory genes was mediated, at least partially through the blockade of signal transducer and activator of transcription 3 (STAT3) and NF-κB pathways. Interestingly, our RNA-seq data also revealed that TGFβ1 suppressed gene expression of a battery of autophagy mediators, which was validated by western blot for the conversion of microtubule-associated protein light chain 3 (LC3) and by immunofluo-rescence staining for LC3 puncta. However, impairment of VSMC autophagy by ATG5 deletion failed to rescue TGFβ1 influence on both VSMC contractile and proinflammatory gene programs, suggesting that TGFβ1-regulated VSMC differentiation and inflammation are not attributed to TGFβ1 suppression on autophagy. In summary, our results demonstrated an important role of TGFβ signaling in suppressing proinflammatory gene program in cultured primary human VSMCs via the blockade on STAT3 and NF-κB pathway, therefore providing novel insights into the mechanisms underlying the protective role of TGFβ signaling in vascular diseases.

Cartilage oligomeric matrix protein is a novel notch ligand driving embryonic stem cell differentiation towards the smooth muscle lineage

Cartilage oligomeric matrix protein (COMP), a protective component of vascular extracellular matrix (ECM), maintains the homeostasis of mature vascular smooth muscle cells (VSMCs). However, whether COMP modulates the differentiation of stem cells towards the smooth muscle lineage is still elusive. Firstly, purified mouse COMP directly induced mouse embryonic stem cell (ESC) differentiation into VSMCs both in vitro and in vivo, while the silencing of endogenous COMP markedly inhibited ESC-VSMC differentiation. RNA-Sequencing revealed that Notch signaling was significantly activated by COMP during ESC-VSMC differentiation, whereas the inhibition of Notch signaling attenuated COMP-directed ESC-VSMC differentiation. Furthermore, COMP deficiency inhibited Notch activation and VSMC differentiation in mice. Through silencing distinct Notch receptors, we identified that Notch1 mainly mediated COMP-initiated ESC-VSMC differentiation. Mechanistically, COMP N-terminus directly interacted with the EGF11-12 domain of Notch1 and activated Notch1 signaling, as evidenced by co-immunoprecipitation and mammalian two-hybrid assay. In conclusion, COMP served as a potential ligand of Notch1, thereby driving ESC-VSMC differentiation.

Conditioned medium from bone marrow-derived mesenchymal stem cells inhibits vascular calcification through blockade of the BMP2\u2013Smad1/5/8 signaling pathway

BackgroundArterial calcification is associated with cardiovascular disease as a complication of advanced atherosclerosis and is a significant contributor to cardiovascular morbidity and mortality. Osteoblastic differentiation of vascular smooth muscle cells (VSMCs) plays an important role in arterial calcification and is characterized by cellular necrosis, inflammation, and lipoprotein and phospholipid complexes, especially in atherosclerotic calcification. The conditioned medium from bone marrow-derived mesenchymal stem cells (MSC-CM) is well known as a rich source of autologous cytokines and is universally used for tissue regeneration in current clinical medicine. Here, we demonstrate that MSC-CM inhibits beta-glycerophosphate (β-GP)-induced vascular calcification through blockade of the bone morphogenetic protein-2 (BMP2)–Smad1/5/8 signaling pathway.MethodsVSMC calcification was induced by β-GP followed by treatment with MSC-CM. Mineral deposition was assessed by Alizarin Red S staining. Intracellular calcium content was determined colorimetrically by the o-cresolphthalein complexone method and alkaline phosphatase (ALP) activity was measured by the para-nitrophenyl phosphate method. Expression of BMP2, BMPR1A, BMPR1B, BMPR2, msh homeobox 2 (Msx2), Runt-related transcription factor 2 (Runx2), and osteocalcin (OC), representative osteoblastic markers, was assessed using real-time polymerase chain reaction analysis while the protein expression of BMP2, Runx2, and phosphorylated Smad1/5/8 was detected by western blot analysis.ResultsOur data demonstrated that MSC-CM inhibits osteoblastic differentiation and mineralization of VSMCs as evidenced by decreased calcium content, ALP activity, and decreased expression of BMP-2, Runx2, Msx2, and OC. MSC-CM suppressed the expression of phosphorylated Smad1/5/8 and the β-GP-induced translocation from the cytoplasm to the nucleus. Further study demonstrated that human recombinant BMP-2 overcame the suppression of VSMC calcification by MSC-CM.ConclusionMSC-CM may act as a novel therapy for VSMC calcification by mediating the BMP2–Smad1/5/8 signaling pathway

Arterial Calcification Is Regulated Via an miR-204/DNMT3a Regulatory Circuit Both In Vitro and in Female Mice.

Arterial calcification is a common cardiovascular disease that initiates from a process of osteoblastic differentiation of vascular smooth muscle cells (VSMCs). Accumulating evidence has demonstrated that microRNAs play an important role in regulating arterial calcification. miR-204 was significantly downregulated in calcified human renal arteries from patients with uremia; calcified arteries of mice, due to 5/6 nephrectomy with a high-phosphate diet (5/6 NTP); and in VSMCs induced by high phosphate concentration. The overexpression of miR-204 alleviated the osteoblastic differentiation of VSMCs. Bisulphite sequencing PCR revealed that CpG sites upstream of miR-204 DNA were hypermethylated in calcified VSMCs; in calcified arteries of mice, due to 5/6 NTP; and in calcified renal artery tissues from patients with uremia. Moreover, increased DNMT3a resulted in the hypermethylation of miR-204 in high phosphate concentration-induced VSMCs, whereas 5-aza-2'-deoxycytidine could restore the expression of miR-204 in high phosphate concentration-induced VSMCs. Moreover, we found that DNMT3a was the target of miR-204, and the methylation ratio of miR-204 was decreased significantly, meaning that the expression of miR-204 was restored when DNMT3a was knocked down by using DNMT3a small interfering RNA, resulting in abrogation of the effect of high phosphate concentration on VSMC calcification. The progress of arterial calcification is regulated by the miR-204/DNMT3a regulatory circuit.

Selenoprotein S inhibits inflammation-induced vascular smooth muscle cell calcification.

Vascular calcification is a prominent feature of many diseases including atherosclerotic cardiovascular disease (CVD), leading to high morbidity and mortality rates. A significant association of selenoprotein S (SelS) gene polymorphism with atherosclerotic CVD has been reported in epidemiologic studies, but the underlying mechanism is far from clear. To investigate the role of SelS in inflammation-induced vascular calcification, osteoblastic differentiation and calcification of vascular smooth muscle cells (VSMCs) induced by lipopolysaccharide (LPS) or tumor necrosis factor (TNF)-α were compared between the cells with and without SelS knockdown. LPS or TNF-α induced osteoblastic differentiation and calcification of VSMCs, as showed by the increases of runt-related transcription factor2 (Runx2) protein levels, Runx2 and type I collagen mRNA levels, alkaline phosphatase activity, and calcium deposition content. These changes were aggravated when SelS was knocked down by small interfering RNA. Moreover, LPS activated both classical and alternative pathways of nuclear factor-κB (NF-κB) signaling in calcifying VSMCs, which were further enhanced under SelS knockdown condition. SelS knockdown also exacerbated LPS-induced increases of proinflammatory cytokines TNF-α and interleukin-6 expression, as well as increases of endoplasmic reticulum (ER) stress markers glucose-regulated protein 78 and inositol-requiring enzyme 1α expression in calcifying VSMCs. In conclusion, the present study suggested that SelS might inhibit inflammation-induced VSMC calcification probably by suppressing activation of NF-κB signaling pathways and ER stress. Our findings provide new understanding of the role of SelS in vascular calcification, which will be potentially beneficial to the prevention of atherosclerotic CVD.

Abstract 290: The Transcription Factor Tead1 is Critical for Vascular Development in Mouse by Promoting Differentiation of Vascular Smooth Muscle Cells

Background: Our previous study has shown that the Hippo signaling effector YAP plays a crucial role in the phenotypic modulation of vascular smooth muscle cells (VSMCs) in response to arterial injury and cardiovascular development in mice. However, the role of TEAD1, a co-factor of YAP in VSMC development is unknown. Objective: The goal of this study to investigate the functional role of TEAD1 in cardiovascular development in mice. Methods and Results: TEAD1 was specifically ablated in cardiomyocytes and VSMCs by crossing TEAD1 flox mice with SM22alpha-Cre transgenic mice. Cardiac/VSMC-specific deletion of TEAD1 led to embryonic lethality by E14.5 in mice due to cardiovascular defects including severe hypoplastic cardiovascular wall, resulting from impaired cardiac/VSMC proliferation. Whole transcriptomic analysis revealed deletion of TEAD1 in mouse cardiomyocytes and VSMCs significantly down-regulated expression of cardiac/VSMC differentiation genes and key transcription factors including myocardin and pitx2c that are critical for cardiovascular development. In vitro studies demonstrated that myocardin and pitx2c rescued TEAD1-defiency induced defects of VSMC differentiation. Furthermore, we identified pitx2c was a direct transcriptional target of TEAD1 and pitx2c synergistically promoted VSMC differentiation with myocardin. Conclusions: This study revealed a critical role of TEAD1 in cardiovascular development by promoting VSMC proliferation and differentiation and provided novel insights into the regulatory mechanisms for smooth muscle development.

Abstract 698: Functional Analysis of Smad3 Deficiency in VSMCs Derived From Crispr/cas9-modified Human Induced Pluripotent Stem Cells

Introduction: Transcription factor Smad3 plays key roles in both TGF-β signaling transduction and vascular smooth muscle cells(VSMCs) differentiation. SMAD3 gene mutations cause aneurysms-osteoarthritis syndrome(AOS), an autosomal dominant genetic disease with high rate occurrence of thoracic aortic aneurysm(TAA) and early-onset osteoarthritis. The production of patient-derived hiPSCs and genomic-modified ihPSCs by using Crispr/Cas9 technique provide a promise for in vitro disease modeling of SMAD3 mutation in VSMCs as well as drug screening for potential therapeutic targets. Hypothesis: We hypothesis that introducing SMAD3 mutations into hIPSCs that mimic the identified genotypic alteration in AOS patients could recapture the phenotypic defects of differentiation, contractibility and extracellular matrix(ECM) of VSMCs derived from hIPSCs-differentiated neural crest stem cells(NCSCs), the embryonic origin of VSMCs for both aorta root and ascending aorta. Method: hIPSCs were generated from non-TAA patients’ peripheral blood mononuclear cells by overexpression of Yamanaka factors. The indel mutation targeting SMAD3 exon5 were induced by using Crispr/Cas9 technique, which form frameshift mutation close to one in identified AOS patients. Selected cell clones of genomic modified hIPSCs were differentiated into NCSCs-derived VSMCs. Comparing analysis of VSMCs function were proceed in both hIPSCs lines with or without SMAD3 mutation. Result: We generated a homozygote with SMAD3 frameshift mutation. It contains a 7-base pair deletion(c.633_639delGATGTCC), which is similar with a pathogenic Smad3 mutation (c.652 delA). This mutation leads to a premature stop codon and remove the Smad3 protein. The qRCR and WB result showed that the expression of ACTA2 decreased in the differentiated SMC with SMAD3 mutation. Also, the expression of elastin, collagen type III were significantly down-regulated in the SMAD3 mutation cell line while the MMP9 was up-regulated. Conclusion: SMAD3 mutation may cause defects of differentiation, contractibility and ECM synthesis of VSMCs derived from NCSC.

MicroRNA-132 targeting PTEN contributes to cilostazol-promoted vascular smooth muscle cell differentiation

Background and aimsCilostazol, beyond its antiplatelet effect, is also capable of promoting vascular smooth muscle cell (VSMC) differentiation. The aim of this study was to explore the potential role of PTEN, known to associate with VSMC differentiation, and its related microRNA (miRNA) in cilostazol-dependent effects. Methods and resultsMicroarray analysis in balloon-injured rat carotid arteries comparing with and without balloon injury revealed that miR-132 was differentially expressed. Bioinformatic analysis predicts PTEN as a novel target of miR-132. Western blot and quantitative real-time reverse transcription-polymerase chain reaction along with in situ hybridization documented that cilostazol treatment enhanced PTEN and reduced miR-132 expression in the neointima of balloon-injured arteries. Treatment of cultured rat VSMCs with cilostazol resulted in the up-regulation of PTEN mRNA and the down-regulation of miR-132, supporting an in vitro relevance. Co-transfection experiments showed that transfection of miR-132 mimic into VSMCs suppressed PTEN 3′UTR activities, further reflecting that PTEN is the direct target of miR-132. Over-expression of miR-132 in VSMCs led to an attenuation of cilostazol-induced PTEN and its downstream VSMC differentiation marker (calponin) expression, confirming the critical role of miR-132 in VSMC differentiation. Transient transfection studies demonstrated that cilostazol reduced the activity of miR-132 promoter, which was mediated via cyclic AMP response element-binding protein. Notably, the use of lentivirus to over-express miR-132 in the neointima of balloon-injured arteries could reverse the effect of cilostazol in vivo. ConclusionsThese results suggest that miR-132 by targeting PTEN may be an important regulator in mediating cilostazol actions on VSMC differentiation.

FTI-277 inhibits smooth muscle cell calcification by up-regulating PI3K/Akt signaling and inhibiting apoptosis.

BackgroundVascular calcification is associated with increased cardiovascular morbidity and mortality in patients with atherosclerosis, diabetes and chronic kidney disease. However, no viable treatments for this condition have been identified. This study aimed to determine whether farnesyl transferase inhibitors (FTIs) can reduce vascular calcification and the mechanism by which this reduction occurs.ResultsWe demonstrate that FTI-277 significantly inhibits phosphate-induced mineral deposition by vascular smooth muscle cells (VSMC) in vitro, prevents VSMC osteogenic differentiation, and increases mRNA expression of matrix Gla protein (MGP), an inhibitor of mineralization. FTI-277 increases Akt signaling in VSMC in short-term serum-stimulation assays and in long-term mineralization assays. In contrast, manumycin A has no effect on Akt signaling or mineralization. Co-incubation of VSMC with FTI-277 and SH6 (an Akt inhibitor) significantly reduces the inhibitory effect of FTI-277 on mineralization, demonstrating that FTI-277 inhibits calcification by activating Akt signaling. Over-expression of the constitutively active p110 sub-unit of PI3K in VSMC using adenovirus activates Akt, inhibits mineralization, suppresses VSMC differentiation and significantly enhances MGP mRNA expression. FTI-277 also inhibits phosphate-induced activation of caspase 3 and apoptosis of VSMC, and these effects are negated by co-incubation with SH6. Finally, using an ex vivo model of vascular calcification, we demonstrate that FTI-277 inhibits high phosphate-induced mineralization in aortic rings derived from rats with end-stage renal failure.ConclusionsTogether, these results demonstrate that FTI-277 inhibits VSMC mineral deposition by up-regulating PI3K/Akt signaling and preventing apoptosis, suggesting that targeting farnesylation, or Akt specifically, may have therapeutic potential for the prevention of vascular calcification.

MiR-22 Is a Novel Mediator of Vascular Smooth Muscle Cell Phenotypic Modulation and Neointima Formation.

MicroRNA-22 (miR-22) has recently been reported to play a regulatory role during vascular smooth muscle cell (VSMC) differentiation from stem cells, but little is known about its target genes and related pathways in mature VSMC phenotypic modulation or its clinical implication in neointima formation following vascular injury. We applied a wire-injury mouse model, and local delivery of AgomiR-22 or miR-22 inhibitor, as well, to explore the therapeutic potential of miR-22 in vascular diseases. Furthermore, normal and diseased human femoral arteries were harvested, and various in vivo, ex vivo, and in vitro models of VSMC phenotype switching were conducted to examine miR-22 expression during VSMC phenotype switching. Expression of miR-22 was closely regulated during VSMC phenotypic modulation. miR-22 overexpression significantly increased expression of VSMC marker genes and inhibited VSMC proliferation and migration, whereas the opposite effect was observed when endogenous miR-22 was knocked down. As expected, 2 previously reported miR-22 target genes, MECP2 (methyl-CpG binding protein 2) and histone deacetylase 4, exhibited a regulatory role in VSMC phenotypic modulation. A transcriptional regulator and oncoprotein, EVI1 (ecotropic virus integration site 1 protein homolog), has been identified as a novel miR-22 target gene in VSMC phenotypic modulation. It is noteworthy that overexpression of miR-22 in the injured vessels significantly reduced the expression of its target genes, decreased VSMC proliferation, and inhibited neointima formation in wire-injured femoral arteries, whereas the opposite effect was observed with local application of a miR-22 inhibitor to injured arteries. We next examined the clinical relevance of miR-22 expression and its target genes in human femoral arteries. We found that miR-22 expression was significantly reduced, whereas MECP2 and EVI1 expression levels were dramatically increased, in diseased in comparison with healthy femoral human arteries. This inverse relationship between miR-22 and MECP2 and EVI1 was evident in both healthy and diseased human femoral arteries. Our data demonstrate that miR-22 and EVI1 are novel regulators of VSMC function, specifically during neointima hyperplasia, offering a novel therapeutic opportunity for treating vascular diseases.

Hypoxia Supports Epicardial Cell Differentiation in Vascular Smooth Muscle Cells through the Activation of the TGFβ Pathway.

Epicardium-derived cells (EPDCs) are an important pool of multipotent cardiovascular progenitor cells. Through epithelial-to-mesenchymal-transition (EMT), EPDCs invade the subepicardium and myocardium and further differentiate into several cell types required for coronary vessel formation. We previously showed that epicardial hypoxia inducible factor (HIF) signaling mediates the invasion of vascular precursor cells critical for patterning the coronary vasculature. Here, we examine the regulatory role of hypoxia (1% oxygen) on EPDC differentiation into vascular smooth muscle cells (VSMCs). Results: Hypoxia stimulates EMT and enhances expression of several VSMC markers in mouse epicardial cell cultures. This stimulation is specifically blocked by inhibiting transforming growth factor-beta (TGFβ) receptor I. Further analyses indicated that hypoxia increases the expression level of TGFβ-1 ligand and phosphorylation of TGFβ receptor II, suggesting an indispensable role of the TGFβ pathway in hypoxia-stimulated VSMC differentiation. We further demonstrate that the non-canonical RhoA/Rho kinase (ROCK) pathway acts as the main downstream effector of TGFβ to modulate hypoxia’s effect on VSMC differentiation. Conclusion: Our results reveal a novel role of epicardial HIF in mediating coronary vasculogenesis by promoting their differentiation into VSMCs through noncanonical TGFβ signaling. These data elucidate that patterning of the coronary vasculature is influenced by epicardial hypoxic signals.

Brain cytoplasmic RNA 1 suppresses smooth muscle differentiation and vascular development in mice

The cardiovascular system develops during the early stages of embryogenesis, and differentiation of smooth muscle cells (SMCs) is essential for that process. SMC differentiation is critically regulated by transforming growth factor (TGF)-β/SMAD family member 3 (SMAD3) signaling, but other regulators may also play a role. For example, long noncoding RNAs (lncRNAs) regulate various cellular activities and events, such as proliferation, differentiation, and apoptosis. However, whether long noncoding RNAs also regulate SMC differentiation remains largely unknown. Here, using the murine cell line C3H10T1/2, we found that brain cytoplasmic RNA 1 (BC1) is an important regulator of SMC differentiation. BC1 overexpression suppressed, whereas BC1 knockdown promoted, TGF-β-induced SMC differentiation, as indicated by altered cell morphology and expression of multiple SMC markers, including smooth muscle α-actin (αSMA), calponin, and smooth muscle 22α (SM22α). BC1 appeared to block SMAD3 activity and inhibit SMC marker gene transcription. Mechanistically, BC1 bound to SMAD3 via RNA SMAD-binding elements (rSBEs) and thus impeded TGF-β-induced SMAD3 translocation to the nucleus. This prevented SMAD3 from binding to SBEs in SMC marker gene promoters, an essential event in SMC marker transcription. In vivo, BC1 overexpression in mouse embryos impaired vascular SMC differentiation, leading to structural defects in the artery wall, such as random breaks in the elastic lamina, abnormal collagen deposition on SM fibers, and disorganized extracellular matrix proteins in the media of the neonatal aorta. Our results suggest that BC1 is a suppressor of SMC differentiation during vascular development.

Ucma/GRP inhibits phosphate-induced vascular smooth muscle cell calcification via SMAD-dependent BMP signalling

Vascular calcification (VC) is the process of deposition of calcium phosphate crystals in the blood vessel wall, with a central role for vascular smooth muscle cells (VSMCs). VC is highly prevalent in chronic kidney disease (CKD) patients and thought, in part, to be induced by phosphate imbalance. The molecular mechanisms that regulate VC are not fully known. Here we propose a novel role for the mineralisation regulator Ucma/GRP (Upper zone of growth plate and Cartilage Matrix Associated protein/Gla Rich Protein) in phosphate-induced VSMC calcification. We show that Ucma/GRP is present in calcified atherosclerotic plaques and highly expressed in calcifying VSMCs in vitro. VSMCs from Ucma/GRP−/− mice showed increased mineralisation and expression of osteo/chondrogenic markers (BMP-2, Runx2, β-catenin, p-SMAD1/5/8, ALP, OCN), and decreased expression of mineralisation inhibitor MGP, suggesting that Ucma/GRP is an inhibitor of mineralisation. Using BMP signalling inhibitor noggin and SMAD1/5/8 signalling inhibitor dorsomorphin we showed that Ucma/GRP is involved in inhibiting the BMP-2-SMAD1/5/8 osteo/chondrogenic signalling pathway in VSMCs treated with elevated phosphate concentrations. Additionally, we showed for the first time evidence of a direct interaction between Ucma/GRP and BMP-2. These results demonstrate an important role of Ucma/GRP in regulating osteo/chondrogenic differentiation and phosphate-induced mineralisation of VSMCs.

Role of smooth muscle cells in vascular calcification: implications in atherosclerosis and arterial stiffness.

Vascular calcification is associated with a significant increase in all-cause mortality and atherosclerotic plaque rupture. Calcification has been determined to be an active process driven in part by vascular smooth muscle cell (VSMC) transdifferentiation within the vascular wall. Historically, VSMC phenotype switching has been viewed as binary, with the cells able to adopt a physiological contractile phenotype or an alternate ‘synthetic’ phenotype in response to injury. More recent work, including lineage tracing has however revealed that VSMCs are able to adopt a number of phenotypes, including calcific (osteogenic, chondrocytic, and osteoclastic), adipogenic, and macrophagic phenotypes. Whilst the mechanisms that drive VSMC differentiation are still being elucidated it is becoming clear that medial calcification may differ in several ways from the intimal calcification seen in atherosclerotic lesions, including risk factors and specific drivers for VSMC phenotype changes and calcification. This article aims to compare and contrast the role of VSMCs in driving calcification in both atherosclerosis and in the vessel media focusing on the major drivers of calcification, including aging, uraemia, mechanical stress, oxidative stress, and inflammation. The review also discusses novel findings that have also brought attention to specific pro- and anti-calcifying proteins, extracellular vesicles, mitochondrial dysfunction, and a uraemic milieu as major determinants of vascular calcification.

Laminaria japonica Polysaccharide Inhibits Vascular Calcification via Preventing Osteoblastic Differentiation of Vascular Smooth Muscle Cells.

This study aimed to investigate the effect and underlying mechanism of a purified Laminaria japonica polysaccharide (LJP61A) on preventing vascular calcification (VC). In the adenine-induced chronic renal failure (CRF) mice VC model and the β-glycerophosphate (β-GP)-induced vascular smooth muscle cells (VSMC) calcification model, LJP61A was found to significantly inhibit VC phenotypes as determined by biochemical analysis and von Kossa, alizarin red, and immunohistochemical staining. Meanwhile, LJP61A remarkably up-regulated the mRNA levels of VSMC related markers and down-regulated the mRNA levels of sodium-dependent phosphate cotransporter Pit-1. In addition, LJP61A could significantly decrease the protein levels of core-binding factor-1, osteocalcin, bone morphogenetic protein 2, and receptor activator for nuclear factor-κB ligand, and it can increase the protein levels of osteoprotegerin and matrix gla protein. These results indicated that LJP61A ameliorated VC both in vivo and in vitro via preventing osteoblastic differentiation of VSMC, suggesting LJP61A might be a potential therapeutic agent for VC in CRF patients.

Deletion of DGCR8 in VSMCs of adult mice results in loss of vascular reactivity, reduced blood pressure and neointima formation

DiGeorge syndrome chromosomal region 8 (DGCR8), a double-stranded-RNA-binding protein, participates in the miRNA biogenesis pathway and contributes to miRNA maturation by interacting with the RNAase III enzyme Drosha in cell nuclei. To investigate the role of DGCR8 in vascular smooth muscle cells (VSMCs) at the postnatal stages, we generated tamoxifen-inducible VSMC specific knockout (iKO) mice by crossing DGCR8loxp/loxp with VSMC specific tamoxifen-inducible Cre transgenic mice SMA-Cre-ERT2. DGCR8iKO mice display reduced body weight one month following tamoxifen treatment and died around 3 months. Blood pressure and vascular reactivity were significantly reduced in DGCR8iKO mice compared to control. Furthermore, loss of DGCR8 in VSMCs inhibited cell proliferation, migration and neointima formation. VSMC differentiation marker genes, including SMA and SM22, were downregulated in DGCR8 iKO mice. The majority of miRNAs were downregulated in DGCR8iKO mice. Disruption of the DGCR8-mediated miRNA biogenesis pathway attenuated multiple signaling pathways including ERK1/2 and AKT. Our results demonstrate that the DGCR8-mediated miRNA pathway is required for maintaining blood pressure, vascular reactivity and vascular wall remodeling at the postnatal stages.