Aortic Vascular Smooth Muscle Cells Research Articles

Osteogenic differentiation and calcification of human aortic smooth muscle cells is induced by the RCN2/STAT3/miR-155-5p feedback loop

BackgroundVascular calcification (VC) is associated with the high morbidity and mortality of cardiovascular diseases in dialysis patients and is a process in which vascular smooth muscle cells (VSMCs) actively differentiate into osteoblast-like cells. Reticulocalbin-2 (RCN2) is involved in the process of osteogenic differentiation under diabetic conditions, but its regulatory role under hyperphosphatemic conditions and the related mechanisms remain unclear. In this study, the importance of the interactions among RCN2, STAT3 and miR-155-5p during the osteogenic differentiation and calcification of human aortic VSMCs (HASMCs) were investigated. MethodsRCN2 was measured in femoropopliteal artery plaque specimens from 6 peripheral arterial disease (PAD) patients with chronic kidney disease (CKD) and 6 PAD patients without CKD. RCN2 protein and mRNA expression were assessed in the high phosphate-induced aortic rings culture ex vivo model. In vitro calcification assays and molecular mechanism studies were performed in HASMCs. ResultsImmunohistochemical staining results revealed increased RCN2 expression in the calcified plaques of femoral arteries of patients with CKD and in a high phosphate-induced aortic culture ex vivo model. RCN2 promoted HASMCs osteogenic differentiation and calcification by inducing STAT3 phosphorylation. Furthermore, inhibition of STAT3 activation by cryptotanshinone (CT) promoted miR-155-5p expression in HASMCs. In turn, miR-155-5p inhibited RCN2 mRNA expression, while RCN2 overexpression partially offset the miR-155-5p-mediated inhibition of HASMC calcification, acting as a positive feedback loop. ConclusionThese results demonstrate that RCN2 is a crucial regulator of VC under hyperphosphatemic conditions. RCN2/STAT3/miR-155-5p feedback loop is important in VC and targeting each member of this feedback loop could potentially reverse high phosphate-induced VC.

Knockdown of H19 Attenuates Ox-LDL-induced Vascular Smooth Muscle Cell Proliferation, Migration, and Invasion by Regulating miR-599/PAPPA Axis.

Long noncoding RNAs could participate in the development of atherosclerosis (AS). However, the underlying mechanism by which long noncoding RNA H19 is implicated in AS remains largely unknown. In this study, we investigated the function of H19 on cell proliferation, migration, and invasion in oxidized low-density lipoprotein (ox-LDL)-treated human aortic vascular smooth muscle cells (HA-VSMCs), and on hyperlipidemia response in high-fat diet (HFD)-treated ApoE-/- mice. Moreover, we explored the target interaction among H19, microRNA (miR)-599, and pappalysin 1 (PAPPA). Our results showed that H19 expression was elevated in serum samples of patients with AS and ox-LDL-treated HA-VSMC. H19 silence mitigated ox-LDL-induced proliferation, migration, and invasion of HA-VSMCs. H19 acted as a sponge for miR-599, and miR-599 knockdown reversed the suppressive effect of H19 silence on proliferation, migration, and invasion of HA-VSMCs. PAPPA was a target of miR-599 and attenuated the inhibitive role of miR-599 in HA-VSMC processes. H19 knockdown repressed PAPPA expression by increasing miR-599. Moreover, H19 interference alleviated hyperlipidemia response in HFD-treated ApoE-/- mice. Collectively, knockdown of H19 inhibited proliferation, migration, and invasion of ox-LDL-treated HA-VSMCs and hyperlipidemia response in HFD-treated ApoE-/- mice by regulating miR-599/PAPPA axis, indicating H19 might act as a potential target for the treatment of AS.

Abstract 16436: Targeting Store Operated Calcium Entry in Vascular Smooth Muscle Cells by a Novel Small-molecule Inhibitor

Vascular smooth muscle cells (VSMC) are physiologically quiescent cells optimised for vascular contractility and the modulation of vascular tone. Platelet-derived growth factor (PDGF) signalling is a critical driver of pathological remodelling of native VSMC to a proliferative synthetic state, which is a process associated with vascular pathologies. Here, we studied the role of store operated calcium entry (SOCE) through Orai1 channels in VSMC plasticity utilizing novel small molecule inhibitor of Orai1 channels. We developed small-molecule Orai1 inhibitor, 4-(2,5-dimethoxyphenyl)-N-[(pyridin-4-yl)methyl]aniline, called JPIII, evaluated its effects on SOCE, and its functional impact in human primary VSMC (hVSMC) from saphenous vein and rat thoracic aorta VSMC cell line. Our Orai1 inhibitor potently suppressed SOCE in hVSMC (IC 50 of 297 nM, n=3) and rat VSMC (IC 50 of 819.5 nM, n=3), leading to significant impairment of PDGF-induced cell migration in hVSMC by 31% (P=0.0071, n=6) and rat VSMC by 24% (P=0.04, n=3). Orai1 inhibition also reduced PDGF-induced proliferation of hVSMC by 20% (P=0.02, n=5) and of rat VSMC by 26% (P=0.003, n=4) at concentration of 30 μM JPIII relative to controls. RNA-Seq analysis of PDGF-stimulated primary human aortic VSMC (n=4/group) was performed to investigate the effects of Orai1 inhibition on the transcriptional response of cells and showed significant downregulation of genes involved in inflammation (C-X-C motif chemokine ligand (CXCL) 1, CXCL3, CXCL5, CXCL6), downregulation of matrix degrading enzymes (metalloproteinase (MMP) 12 and MMP16), and upregulation of cholesterol biosynthesis pathways by Orai1 inhibition. Consistent with the observed functional phenotype, our results revealed an inhibitory effect on transcriptional networks involved in cell proliferation and migration. The effects of Orai1 inhibition on the transcription of genes involved in inflammation, MMPs expression, cholesterol metabolism, cell proliferation, and migration powerfully associate Orai1 signalling with VSMC remodelling. Our results provide insight into the role of Orai1 in VSMC remodelling and the possible therapeutic potential of a small molecule inhibition approach in targeting pathologic remodelling.

MicroRNA-520c-3p targeting of RelA/p65 suppresses atherosclerotic plaque formation

Atherosclerosis is a chronic inflammatory disease, and it's the leading cause of death worldwide. Dysregulation of microRNAs (miRNAs) has been found to be associated with atherosclerosis. miR-520c-3p has been implicated in several types of cancer. However, little is known about the role of miR-520c-3p in atherosclerosis. In this study, we found that miR-520c-3p agomir decreased atherosclerotic plaque size, collagen content, the quantity of PCNA-positive cell and RelA/p65 expression of vascular smooth muscle cells (VSMCs) in the aortic valve of apoE-/- mice in vivo. The possible mechanisms of the protective effects of miR-520c-3p on atherosclerotic mice were then investigated in VSMCs. in vitro experiments showed that miR-520c-3p expressions were significantly reduced in human aortic vascular smooth muscle cell (HASMCs) treated with platelet-derived growth factor (PDGF-BB). miR-520c-3p mimics repress PDGF-BB-mediated the proliferation, migration and decrease in the percentage of cells in G2/M phase, which was associated with downregulation of RelA/p65. Mechanistically, miRNA pull-down, luciferase reporter and mRNA stability assays confirmed miR-520c-3p mimics was able to directly target 3'-UTR of RelA/p65 mRNA and decreased half-life of RelA/p65 mRNA in HASMCs. Overexpression of RelA/p65 reversed the inhibition of cell proliferation induced by miR-520c-3p mimics in HASMCs. In conclusion, our findings suggest that miR-520c-3p inhibits PDGF-BB-mediated the proliferation and migration of HASMCs by targeting RelA/p65, which may provide potential therapeutic strategies in atherosclerosis treatment.

CDKN2B-AS1 Aggravates the Pathogenesis of Human Thoracic Aortic Dissection by Sponge to miR-320d.

In the present study, the role and molecular mechanism of long noncoding RNA CDKN2B-AS1 in human thoracic aortic dissection (TAD), a highly lethal cardiovascular disease, was investigated. The expression of CDKN2B-AS1 in human TAD and normal aortic tissues of donors were examined by quantitative real-time polymerase chain reaction. RNA pull-down assay and a series of luciferase reporter assays were performed to predict the relationships between CDKN2B-AS1, miR-320d, and STAT3. Cell counting kit 8 (CCK-8), TUNEL, and western blot assays were applied to validate the biological functions of CDKN2B-AS1 in rat aortic vascular smooth muscle cells. Results showed that CDKN2B-AS1 was expressed at a higher level in human TAD than in normal aortic tissues. CDKN2B-AS1 overexpression significantly promoted apoptosis and suppressed the proliferation of vascular smooth muscle cells. CDKN2B-AS1 silence exhibited the opposite effects. Mechanistically, CDKN2B-AS1 was identified as a molecular sponge for miR-320d and positively modulated STAT3 expression via repressing miR-320d. In conclusion, our study revealed that CDKN2B-AS1 was dysregulated and displayed multiple potential functions in human TAD. These findings suggested that CDKN2B-AS1 was a novel potential therapeutic target for human TAD.

MicroRNA-342-5p activates the Akt signaling pathway by downregulating PIK3R1 to modify the proliferation and differentiation of vascular smooth muscle cells.

Abnormal cell proliferation and invasion of vascular smooth muscle cells are among the primary causes of cardiovascular disease. Studies have shown that microRNA(miR)-342-5p participates in the development of cardiovascular diseases. The current study aimed to explore the role of miR-342-5p in the proliferation and differentiation of mouse aortic vascular smooth muscle (MOVAS) cells. MOVAS cells were transfected with miR-342-5p mimics, miR-342-5p inhibitor or their respective negative controls, and co-transfected with small interfering (si)RNA targeting phosphatidylinositol 3-kinase regulatory subunit α (PIK3R1) and miR-342-5p inhibitor. The cell proliferation of MOVAS cells was detected using the Cell Counting Kit-8, while cell migration and cell invasion were investigated using a wound healing and Transwell assays, respectively. Target genes for miR-342-5p were confirmed using reverse transcription-quantitative PCR (RT-qPCR) and dual luciferase reporter assay. The relative mRNA and protein expression levels of miR-342-5p were measured using RT-qPCR and western blot analysis. MOVAS cells were treated with a PI3K inhibitor (LY294002) to explore the role of miR-342-5p on the Akt pathway. The results revealed that miR-342-5p mimics promoted cell viability, migration and invasion, and increased the expression of vimentin and phosphorylated-Akt but reduced a-smooth muscle actin (α-SMA) and PIK3R1 expression. However, miR-342-5p inhibitor produced the opposite effects. PIK3R1 was the target gene for miR-342-5p and the effect of siPIK3R1 on MOVAS cells was similar to that of miR-342-5p mimics, while siPIK3R1 partially reversed the effect of miR-342-5p inhibitor on MOVAS cells. The Akt signaling pathway was activated by miR-342-5p mimics or siPIK3R1. Moreover, miR-342-5p mimics partially activated the Akt signaling pathway inhibited by LY294002. MiR-342-5p could promote the proliferation and differentiation of MOVAS and phenotypic transformation. The mechanism behind these processes may be associated with the activation of the Akt signaling pathway induced by PIK3R1 inhibition.

Interleukin-11 is important for vascular smooth muscle phenotypic switching and aortic inflammation, fibrosis and remodeling in mouse models

Transforming growth factor beta-1 (TGFβ1) is a major driver of vascular smooth muscle cell (VSMC) phenotypic switching, an important pathobiology in arterial disease.We performed RNA-sequencing of TGFβ1-stimulated human aortic or arterial VSMCs which revealed large and consistent upregulation of Interleukin 11 (IL11). IL11 has an unknown function in VSMCs, which highly express the IL11 receptor alpha, suggestive of an autocrine loop. In vitro, IL11 activated ERK signaling, but inhibited STAT3 activity, and caused VSMC phenotypic switching to a similar extent as TGFβ1 or angiotensin II (ANGII) stimulation. Genetic or therapeutic inhibition of IL11 signaling reduced TGFβ1- or ANGII-induced VSMC phenotypic switching, placing IL11 activity downstream of these factors. Aortas of mice with Myh11-driven IL11 expression were remodeled and had reduced contractile but increased matrix and inflammatory genes expression. In two models of arterial pressure loading, IL11 was upregulated in the aorta and neutralizing IL11 antibodies reduced remodeling along with matrix and pro-inflammatory gene expression. These data show that IL11 plays an important role in VSMC phenotype switching, vascular inflammation and aortic pathobiology.

Myocardin suppression increases lipid retention and atherosclerosis via downregulation of ABCA1 in vascular smooth muscle cells

Myocardin (MYOCD) plays an important role in cardiovascular disease. However, its underlying impact on atherosclerosis remains to be elucidated. ATP binding cassette transporter A1 (ABCA1), a key membrane-associated lipid transporter which maintains intracellular lipid homeostasis, has a protective function in atherosclerosis progress. The purpose of this study was to investigate whether and how the effect of MYOCD on atherosclerosis is associated with ABCA1 in vascular smooth muscle cells (VSMCs). We found both MYOCD and ABCA1 expression were dramatically decreased in atherosclerotic patient aortas compared to control. MYOCD knockdown inhibited ABCA1 expression in human aortic vascular smooth muscle cells (HAVSMCs), leading to reduced cholesterol efflux and increased intracellular cholesterol contents. MYOCD overexpression exerted the opposite effect. Mechanistically, MYOCD regulates ABCA1 expression in an SRF-dependent manner. Consistently, apolipoprotein E-deficient mice treated with MYOCD shRNA developed more plaques in the aortic sinus, which is associated with reduced ABCA1 expression, increased cholesterol retention in the aorta, and decreased high-density lipoprotein cholesterol levels in the plasma. Our data suggest that MYOCD deficiency exacerbates atherosclerosis by downregulating ABCA1 dependent cholesterol efflux from VSMCs, thereby providing a novel strategy for the therapeutic treatment of atherosclerotic cardiovascular disease.

Quantitative Analysis of Contractile Ability of Vascular Smooth Muscle Cells Using a Micropillar Substrate and a Laser Nanoscissor System

Vascular cell senescence and vascular aging are known to be associated with serious vascular diseases, such as arteriosclerosis and aneurysm generation. However, it is unclear how cellular dedifferentiation and senescence affects cellular contractile force which is especially important for vascular smooth muscle cells. Here we investigated the changes in the cellular contractile ability during repeatedly passage-cultivation that promotes cell dedifferentiation and senescence. Porcine aortic vascular smooth muscle cells exposed to several passage-cultivation were seeded on the polydimethylsiloxane-based elastic micropillar array substrates, and their whole cell contractile forces were preciously estimated by the deflection of the pillars. Furthermore, we dissected intracellular actin stress fibers in living cells by laser irradiation to release their pretension, observed the dynamic behavior of the dissected stress fibers, and estimated the internal tension of individual stress fibers. We found that the both of the whole cell contractile forces and the internal tension of individual stress fibers were significantly decreased in cell dedifferentiation and senescence induced by repeatedly passage-cultivation, and the decrease was more sensitive in the individual stress fibers than in the whole cell. These results indicate that cellular contractile forces are deeply involved in cellular dedifferentiation and senescence.

Role of the JAK2/STAT3 pathway in angiotensin II-induced enhanced expression of Giα proteins and hyperproliferation of aortic vascular smooth muscle cells.

We earlier showed that angiotensin (Ang) II-induced overexpression of Giα proteins contributes to the hyperproliferation of vascular smooth muscle cells (VSMC). In addition, the implication of the JAK2/STAT3 pathway in AngII-induced hyperproliferation of VSMC has also been reported. However, the role of the JAK2/STAT3 pathway in AngII-induced overexpression of Giα proteins and hyperproliferation of VSMC remains unexplored. In the present study, we show that inhibition or knockdown of the JAK2/STAT3 pathway by a specific inhibitor "cucurbitacin I" (CuI) or siRNAs attenuated AngII-induced overexpression of Giα proteins and hyperproliferation of VSMC. In addition, the enhanced expression of cell cycle proteins induced by AngII was also attenuated by CuI. Furthermore, AngII-induced enhanced production of the superoxide anion (O2 -), H2O2, and NADPH oxidase activity, as well as the enhanced expression of NADPH oxidase subunits implicated in enhanced expression of Giα proteins and hyperproliferation, were also attenuated by inhibition of the JAK2/STAT3 pathway. On the other hand, AngII-induced inhibition and augmentation of the levels of nitric oxide and peroxynitrite, respectively, in VSMC were restored to control levels by CuI. In summary, our results demonstrate that AngII through the JAK2/STAT3 pathway increases nitroxidative stress, which contributes to the overexpression of Giα proteins and cell cycle proteins and the hyperproliferation of VSMC.

H2S attenuates oxidative stress via Nrf2/NF-κB signaling to regulate restenosis after percutaneous transluminal angioplasty.

Restenosis after angioplasty of peripheral arteries is a clinical problem involving oxidative stress. Hydrogen sulfide (H2S) participates in oxidative stress regulation and activates nuclear factor erythroid 2-related factor 2 (Nrf2). This study investigated the effect of H2S and Nrf2 on restenosis-induced arterial injury. Using an in vivo rat model of restenosis, we investigated whether H2S inhibits restenosis after percutaneous transluminal angioplasty (PTA) and the oxidative stress-related mechanisms implicated therein. The involvement of Nrf2 was explored using Nrf2-shRNA. Neointimal formation and the deposition of elastic fibers were assessed histologically. Inflammatory cytokine secretion and the expression of proteins associated with oxidative stress and inflammation were evaluated. The artery of rats subjected to restenosis showed increased arterial intimal thickness, with prominent elastic fiber deposition. Sodium hydrosulfide (NaHS), an H2S donor, counteracted these changes in vivo. Restenosis caused a decrease in anti-oxidative stress signaling. This phenomenon was inhibited by NaHS, but Nrf2-shRNA counteracted the effects of NaHS. In terms of inflammation, inflammatory cytokines were upregulated, whereas NaHS suppressed the induced inflammatory reaction. Similarly, Nrf2 downregulation blocked the effect of NaHS. In vitro studies using aortic endothelial and vascular smooth muscle cells isolated from experimental animals showed consistent results as those of in vivo studies, and the participation of the nuclear factor-kappa B signaling pathway was demonstrated. Collectively, H2S played a role in regulating post-PTA restenosis by alleviating oxidative stress, modulating anti-oxidant defense, and targeting Nrf2-related pathways via nuclear factor-kappa B signaling.

Role of SGK1 in the Osteogenic Transdifferentiation and Calcification of Vascular Smooth Muscle Cells Promoted by Hyperglycemic Conditions.

In diabetes mellitus, hyperglycemia promotes the osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs) to enhance medial vascular calcification, a common complication strongly associated with cardiovascular disease and mortality. The mechanisms involved are, however, still poorly understood. Therefore, the present study explored the potential role of serum- and glucocorticoid-inducible kinase 1 (SGK1) during vascular calcification promoted by hyperglycemic conditions. Exposure to high-glucose conditions up-regulated the SGK1 expression in primary human aortic VSMCs. High glucose increased osteogenic marker expression and activity and, thus, promoted the osteogenic transdifferentiation of VSMCs, effects significantly suppressed by additional treatment with the SGK1 inhibitor EMD638683. Moreover, high glucose augmented the mineralization of VSMCs in the presence of calcification medium, effects again significantly reduced by SGK1 inhibition. Similarly, SGK1 knockdown blunted the high glucose-induced osteogenic transdifferentiation of VSMCs. The osteoinductive signaling promoted by high glucose required SGK1-dependent NF-κB activation. In addition, advanced glycation end products (AGEs) increased the SGK1 expression in VSMCs, and SGK1 inhibition was able to interfere with AGEs-induced osteogenic signaling. In conclusion, SGK1 is up-regulated and mediates, at least partly, the osteogenic transdifferentiation and calcification of VSMCs during hyperglycemic conditions. Thus, SGK1 inhibition may reduce the development of vascular calcification promoted by hyperglycemia in diabetes.

Integrin α9 is involved in the pathopoiesis of acute aortic dissection via mediating phenotype switch of vascular smooth muscle cell

Acute aortic dissection (AAD) is a devastating disease with high mortality; however, the pathogenic mechanisms of AAD remain poorly understood. Our present study aimed to identify genes associated with AAD and explore the molecular function of candidate genes in the pathogenesis of AAD. We used a whole-genome transcriptional microarray to identify putative AAD genes using ascending aortic tissues from four patients with AAD and four healthy organ donors. The differentially expressed genes were further validated in eight patients with AAD and eight healthy organ donors. Functional assessments were conducted to analyze the effects of the identified AAD genes on the phenotype of aortic vascular smooth muscle cells (VSMCs). The whole-genome transcriptional microarray analysis found 129 dysregulated genes in the ascending aortic tissues of AAD (fold change≥2), which were mainly associated with the focal adhesion pathway and actin cytoskeleton regulation pathway. Among these genes, integrin α9 (ITGA9) was identified to be involved in both pathways and downregulated by 50% in AAD patients. The association of ITGA9 with AAD was confirmed by Western blotting analysis (P = 0.003). Functional studies showed that knocking down ITGA9 in VSMCs resulted in a decrease in contractile markers (SM22α and α-SMA) and an increase in synthetic markers (OPN and SMemb), suggesting that the VSMCs switched from a contractile to a synthetic phenotype. After overexpression of ITGA9 by a recombinant adenovirus vector in VSMCs, SM22α and α-SMA were upregulated, while SMemb was downregulated, indicating a phenotypic switch from the synthetic to contractile phenotype of VSMCs. In conclusion, our study identified ITGA9 as a novel AAD gene. This gene is downregulated in patients with AAD and is involved in the regulation of the phenotypic switch of VSMCs from a contractile to a synthetic phenotype.

Polycystin-1 Downregulation Induced Vascular Smooth Muscle Cells Phenotypic Alteration and Extracellular Matrix Remodeling in Thoracic Aortic Dissection

ObjectivePolycystin-1 (PC-1) is a protein encoded by the gene of polycystic kidney disease-1 (PKD-1). This study was designed to investigate the regulatory mechanisms of PC-1 on phenotypes of aortic vascular smooth muscle cells (VSMCs) and functions of extracellular matrix (ECM) in thoracic aortic dissection (TAD).MethodsAortic tissues from patients with TAD and healthy controls were collected, primary aortic VSMCs were also isolated. Immunohistochemistry, immunofluorescence, and immunocytochemistry was used to visualize the target proteins. Western blot and RT-qPCR were used to examine the expression of mRNA and proteins. Lentivirus infection was used to downregulate or overexpress PC-1.ResultsCompared with the control group, expression of PC-1 and the contractile phenotypic markers of VSMCs were decreased in TAD group, whereas expression of the synthetic markers of VSMCs, matrix metalloproteinase (MMP)-2, collagen I and collagen III were increased. The phosphorylation of mTOR, S6K and S6 were also elevated in TAD group. PC-1 downregulation of aortic VSMCs inhibited the expression of the contractile markers, but elevated the expression of the synthetic markers, MMP-2, collagen I and collagen III compared with the control group. The phosphorylation of mTOR, S6K and S6 were also increased in PKD-1-knockdown VSMCs. PC-1 upregulation reversed all these expression characteristics in aortic VSMCs. Furthermore, rapamycin treatment to PKD-1-knockdown VSMCs inhibited the effects caused by PC-1 downregulation.ConclusionOur study revealed PC-1 downregulation induces aortic VSMCs phenotypic alteration and ECM remodeling via activation of mTOR/S6K/S6 signaling pathway. Downregulation of PC-1 might be a potential mechanism for the development and progression of TAD. Rapamycin might be a potential inhibitor to attenuate the development and progression of TAD.

Celastrol attenuates arterial and valvular calcification via inhibiting BMP2/Smad1/5 signalling.

Vascular calcification is an important risk factor for the mortality and morbidity in chronic kidney disease (CKD). Unfortunately, until now there is no certain medication targeting vascular calcification in CKD. In this study, we explored the inhibitory effect of celastrol on high calcium–induced vascular calcification and the underlying molecular mechanisms. Cell proliferation assay showed that celastrol inhibited aortic valve interstitial cell (VIC) and vascular smooth muscle cell (VSMC) proliferation when its concentration was higher than 0.6 μmol/L. 0.8 μmol/L celastrol inhibited the expression of osteogenic genes and calcium deposition induced by high‐calcium medium in both AVICs and VSMCs. In mouse vascular calcification model induced by adenine combined with vitamin D, alizarin red and immunostaining showed that celastrol inhibited pro‐calcification gene expression and calcium deposition in aortic wall and aortic valve tissues. At the molecular level, celastrol inhibited the increase of BMP2, phosphorylated Smad1/5 (p‐Smad1/5) and non‐phosphorylated β‐catenin (n‐p‐β‐catenin) induced by high‐calcium medium both in vitro and in vivo. Also, BMP2 overexpression reversed the anti‐calcification effects of celastrol by recovering the decrease of p‐Smad1/5 and n‐p‐β‐catenin. Furthermore, celastrol prevented the up‐regulation of BMPRII and down‐regulation of Smad6 induced by high calcium, and this protectory effect can be abolished by BMP2 overexpression. In conclusion, our data for the first time demonstrate that celastrol attenuates high calcium–induced arterial and valvular calcification by inhibiting BMP2/Smad1/5 signalling, which may provide a novel therapeutic strategy for arterial and valvular calcification in patients with CKD.

Prostacyclin facilitates vascular smooth muscle cell phenotypic transformation via activating TP receptors when IP receptors are deficient.

By activating prostacyclin receptors (IP receptors), prostacyclin (PGI2 ) exerts cardiovascular protective effects such as vasodilation and inhibition of vascular smooth muscle cell (VSMC) proliferation. However, IP receptors are dysfunctional under pathological conditions, and PGI2 produces detrimental effects that are opposite to its physiological protective effects via thromboxane-prostanoid (TP) receptors. This attempted to investigate whether or not IP receptor dysfunction facilitates the shift of PGI2 action. The effects of PGI2 and its stable analog iloprost on VSMC phenotypic transformation and proliferation were examined in A10 cells silencing IP receptors, in human aortic VSMCs (HAVSMCs) knocked down IP receptor by CRISPR-Cas9, or in HAVSMCs transfected with a dysfunctional mutation of IP receptor IPR212C . PGI2 /iloprost treatment stimulated cell proliferation, upregulated synthetic proteins and downregulated contractile proteins, suggesting that PGI2 /iloprost promotes VSMC phenotypic transformation in IP-deficient cells. The effect of PGI2 /iloprost was prevented by TP antagonist S18886 or TP knockdown, indicating that the VSMC detrimental effect of PGI2 is dependent on TP receptor. RNA sequencing and Western blotting results showed that RhoA/ROCKs, MEK1/2 and JNK signalling cascades were involved. Moreover, IP deficiency increased the distribution of TP receptors at the cell membrane. PGI2 induces VSMC phenotypic transformation when IP receptors are impaired. This is attributed to the activation of TP receptor and its downstream signaling cascades, and to the increased membrane distribution of TP receptors. The VSMC detrimental effect of PGI2 medicated by IP dysfunction and TP activation might probably exacerbate vascular remodelling, accelerating cardiovascular diseases.

Abstract P055: Smooth Muscle Cell-specific Conditional Knockout Of Lysine (K)-specific Demethylase 6a Causes Endothelial Dysfunction, Increased Blood Pressure And Arterial Stiffness

Arterial stiffening is the progressive loss of elasticity of the large vessels resulting from age. Increased arterial stiffening has been correlated with hypertension and other cardiovascular co-morbidities. KDM6A is a histone 3 lysine demethylase whose activity increases expression of target genes via removal of silencing methylation marks on lysine 27 of histone 3 (H3K27). The precise role of H3K27me3, or its absence in aortic vascular smooth muscle cells (VSMCs), remains to be investigated. The objectives of this study were to determine the effects of loss of KDM6A on arterial stiffness, blood pressure, and vascular cell function. We generated VSMC-specific Kdm6a knockout mice of C57BL/6 background and confirmed knockout by western blot. Knockout of Kdm6a resulted in increased resting systolic blood pressure and pulse wave velocity in male mice. Pulse wave velocity (PWV) is the gold-standard method for determination of arterial stiffness. PWVs of the knockout mice were significantly increased compared to controls four weeks after Kdm6a knockout (Controls 2.62 + 0.69 mm/ms; KO 3.13 + 0.30 mm/ms; P=0.0228). Systolic blood pressure was directly obtained for each mouse via carotid cannulation. The systolic blood pressures of knockout mice were significantly increased. Mesenteric arteries were isolated from three control and three Kdm6a knockout mice and mounted onto a wire myograph. Vessels were pre-constricted with norepinephrine and vasodilation was measured by treatment with increasing doses of acetylcholine and sodium nitroprusside to assess endothelial and VSMC function. Interestingly, despite Kdm6a knockout in VSMCs, vasodilation of VSMCs was not different between control and knockout arteries, but relaxation in response to acetylcholine, which stimulates the release of nitric oxide from endothelial cells, was decreased. This suggests endothelial dysfunction. These results indicate that loss of KDM6A in VSMCs leads to elevated systolic blood pressure and arterial stiffness.

Abstract P114: Foxo4 Regulates Vascular Smooth Muscle Soluble Guanylyl Cyclase Expression Expression

Soluble guanylyl cyclase (sGC) modulator drugs may offer a new approach to attenuate hypertension by enhancing vasorelaxation through production of cGMP. Loss of sGC leads to impaired vasorelaxation and hypertension, however the mechanisms that regulate sGC transcription are not fully understood. We recently published that Forkhead box subclass O (FoxO) transcription factor inhibition led to decreased sGC mRNA and protein expression and subsequent loss of downstream enzymatic sGC function and vasoreactivity in the aorta. We conducted luciferase experiments in HEK cells which identified several regions which were sensitive to FoxO inhibition and may be responsible for the regulation of sGCβ protein expression (n=4).The objective of this study was to identify which FoxO transcription factor(s) regulates sGC transcription in vascular aortic smooth muscle cells (SMCs) and at what location on the promoter. FoxO1 shRNA produced a 3-fold increase in sGCα and sGCβ mRNA expression (n=3) and a 27% elevation in sGCβ protein expression (n=6). FoxO3a shRNA produced a similar 2-fold increase in sGCα and sGCβ mRNA (n=3) and a 41% increase in sGCβ protein expression (n=6). FoxO4 shRNA, however, produced approximately 55% decrease in sGCα and sGCβ mRNA (n=3) and a similar 52% decrease in sGCβ protein expression (n=6). FoxO1 and FoxO3a shRNA treatment caused no significant change in pVASP expression at baseline or following stimulation with the nitric oxide donor molecule DEA NONOate (n=3). FoxO4 shRNA treatment resulted in a 73% decrease in pVASP expression at baseline and blunted the DEA NONOate-mediated increase in phosphorylation by 33% (n=3). Preliminary ChIP experiments in human aortic SMCs and have identified 4 sites of enriched Foxo4 binding on the human sGCβ promoter consistent with locations previously identified as sensitive to FoxO inhibitor. These data indicate FoxO4 plays a critical role in the maintenance of sGC transcription, protein expression, and downstream enzymatic function of sGC in vascular smooth muscle. Additionally, our data demonstrate that human sGCβ promoter expression requires functional FoxO activity and FoxO4 promoter binding occurs in promoter regions of human smooth muscle found to be important for promoter-luciferase expression.

Quercetin Downregulates Cyclooxygenase-2 Expression and HIF-1α/VEGF Signaling-Related Angiogenesis in a Mouse Model of Abdominal Aortic Aneurysm.

Objective Abdominal aortic aneurysm (AAA) development has been characterized by increased expression of vascular endothelial growth factor (VEGF), which contributes to angiogenesis via cyclooxygenase-2 (COX-2). Quercetin, one of the most common and well-researched flavonoids and abundant in vegetables and fruits, has beneficial effects in inhibiting angiogenesis. This study investigated the antiangiogenic effects of quercetin on experimental aneurysms. Methods We utilized the in vivo AAA mouse model induced by the periaortic application of CaCl2 to examine the effectiveness of quercetin in blocking angiogenesis. Quercetin was administered at 60 mg/kg once daily on the day of the AAA induction and then continued for 6 weeks. Celecoxib, a selective COX-2 inhibitor, was used as the positive control. Results Our results demonstrated that quercetin significantly attenuated aneurysm growth in AAA mice and medial neovascularization. Accordingly, quercetin decreased the expression of proangiogenic mediators, including VEGF-A, intercellular adhesion molecule-1, vascular cell adhesion molecule 1, and vascular endothelial cadherin. Quercetin treatment also inhibited the expression of COX-2 and hypoxia-inducible factor 1α (HIF-1α). It was also found that quercetin-3-glucuronide, a major quercetin metabolite, downregulated the expression of COX-2, HIF-1α, VEGF-A, and matrix metalloproteinase activities in aortic vascular smooth muscle cells isolated from AAA mice. Conclusion Quercetin attenuates neovascularization during AAA growth, and this effect is mediated via the inhibition of COX-2, which decreases HIF-1α/VEGF signaling-related angiogenesis.

RhoA/ROCK Pathway Activation is Regulated by AT1 Receptor and Participates in Smooth Muscle Migration and Dedifferentiation via Promoting Actin Cytoskeleton Polymerization.

Background: In this study, we investigated the mechanism of Rho GTPases signaling on Ang II-mediated cell migration and dedifferentiation in human aortic vascular smooth muscle cells (HA-VSMCs) and an Ang II-infusion mouse model. Methods: Cells were pretreated with different inhibitors or Ang II. Cell migration was detected by Wound healing and Transwell assay. Mice were treated with Ad-RhoA-shRNA virus or Irbesartan or fasudil and then infused with Ang II. Results: Ang II treatment induced HA-VSMCs migration in a dose- and time-dependent manner and reduced the expression of VSMC contractile proteins. These effects were significantly suppressed by the inhibition of Ang II type 1 receptor (AT1 receptor), RhoA, and Rho-associated kinase (ROCK). Furthermore, Ang II treatment promoted the activation of RhoA and ROCK, which was reduced by AT1 receptor inhibition. Meanwhile, Ang II treatment induced F-actin polymerization, which was inhibited after ROCK inhibition. In mice, Ang II infusion increased VSMC migration into the neointima and reduced VSMC differentiation proteins levels, and these effects were shown to be dependent on AT1 receptor and RhoA/ROCK pathway. Conclusion: This study reveals a novel mechanism by which Ang II regulates RhoA/ROCK signaling and actin polymerization via AT1 receptor and then affects VSMC dedifferentiation.