Effects In Vascular Smooth Muscle Cells Research Articles

Insulin receptors in vascular smooth muscle cells regulate plaque stability of atherosclerosis.

Increased prevalence of acute myocardial infarction related to diabetes and insulin resistance is associated with elevated risk for unstable atherosclerotic plaques, which are characterized by reduced vascular smooth muscle cells (VSMC) and extracellular matrix (ECM) and increased inflammation. Thus, insulin resistance may reduce plaque stability as deleting insulin receptors (IR) in VSMC decreased their proliferation and enhanced apoptosis. Direct effects of insulin on VSMC to alter plaque composition were studied using mice with double knockout of ApoE and IR genes in VSMC with SMIRKO/ApoE-/- and Myh11-CreERT2EYFP+/ApoE-/- and Myh11-CreERT2EYFP+IRKO/ApoE-/- mice, which were also used for lineage tracing studies. Compared to ApoE-/- mice, SMIRKO/ApoE-/-- mice exhibited more atherosclerotic plaques, which contained less VSMC and collagen, but increased levels of VSMC apoptosis and necrotic areas. Lineage tracing studies showed that Icam1+ Vcam1+ VSMC was inflammatory VSMC, which increased in aortas of Myh11-CreERT2EYFP+IRKO/ApoE-/- mice compared to control mice. Isolated VSMC lacking IR expressed higher inflammatory cytokines than cells with IR. Cell based studies indicated that insulin's anti-apoptotic and pro-proliferative effects in VSMC were mediated via activation of IR/Akt pathway, which were decreased in VSMC from SMIRKO or high fat diet (HFD) mice. Analysis of IR targets that regulated inflammatory cytokines in VSMC showed that thrombospondin 1 (Thbs1) and Mmp2 were consistently increased with loss of IR. Insulin inhibited Thbs1 expression, but not Mmp2, by p-Akt/p-FoxO1 pathways in VSMC from ApoE-/- mice, which was impaired in cells from SMIRKO/ApoE-/-- mice. Thbs1 further induced Icam1 and Mmp2 expressions in VSMC. Insulin via IR has significant actions in VSMC to decrease inflammation, apoptosis and ECM turnover via the activation of Akt and FoxO1 pathways. Inhibition of insulin actions and related pathways related to insulin resistance and diabetes may contribute to the formation of unstable atherosclerotic plaques.

Protective effects of Artemisia herba-alba in diabetic peripheral artery disease mediated via inhibition of advanced glycation end products

The rationale for undertaking this study is lethality of diabetes mellitus as predicted by 6.7 million deaths in 2021 and immense pharmacological potential of Artemisa herba-alba. The current research examined how Artemisia herba-alba extract (AHE) affects the peripheral artery disease in diabetic rats through lowering of advanced glycation end products (AGEs). The in-vitro AGE inhibiting potential of AHE was determined by spectrofluorimetric method. The blood glucose levels and HbA1c (A1C) of the rats from each group were determined by automatic analyser. The levels of AGEs in vascular smooth muscle cells (VSMCs) of different rat groups were observed through western blotting. Expression of COX-1 and COX-2 were determined by qRT-PCR. The AHE inhibition of AGEs formation was reported in vitro and exhibited an IC50 of 45 μg/mL which was significantly lower than that of standard AGEs inhibitor aminoguanidine (IC50: 60 μg/mL). Analysis of metabolic profiles revealed that AHE normalised the blood glucose, cholesterol, and triglycerides with no apparent changes on Hb1Ac levels. Western blot analysis showed that AHE exhibited protective effects in VSMCs of diabetic rats by inhibiting fabrication of AGEs. Moreover, the manifestation of COX-2 was inhibited by AHE in diabetic rats. However, the expression of COX-1 remained unaltered. Collectively, the results revealed AHE inhibits AGEs formation in vitro and in VSMCs of diabetic rats. These findings point towards the prospective of AHE applications towards the management of diabetic peripheral artery disease.

Effects of Coronary Artery Disease-Associated Variants on Vascular Smooth Muscle Cells.

Background:Genome-wide association studies have identified many genetic loci that are robustly associated with coronary artery disease (CAD). However, the underlying biological mechanisms are still unknown for most of these loci, hindering the progress to medical translation. Evidence suggests that the genetic influence on CAD susceptibility may act partly through vascular smooth muscle cells (VSMCs).Methods:We undertook genotyping, RNA sequencing, and cell behavior assays on a large bank of VSMCs (n>1499). Expression quantitative trait locus and splicing quantitative trait locus analyses were performed to identify genes with an expression that was influenced by CAD-associated variants. To identify candidate causal genes for CAD, we ascertained colocalizations of VSMC expression quantitative trait locus signals with CAD association signals by performing causal variants identification in associated regions analysis and the summary data–based mendelian randomization test. Druggability analysis was then performed on the candidate causal genes. CAD risk variants were tested for associations with VSMC proliferation, migration, and apoptosis. Collective effects of multiple CAD-associated variants on VSMC behavior were estimated by polygenic scores.Results:Approximately 60% of the known CAD-associated variants showed statistically significant expression quantitative trait locus or splicing quantitative trait locus effects in VSMCs. Colocalization analyses identified 84 genes with expression quantitative trait locus signals that significantly colocalized with CAD association signals, identifying them as candidate causal genes. Druggability analysis indicated that 38 of the candidate causal genes were druggable, and 13 had evidence of drug-gene interactions. Of the CAD-associated variants tested, 139 showed suggestive associations with VSMC proliferation, migration, or apoptosis. A polygenic score model explained up to 5.94% of variation in several VSMC behavior parameters, consistent with polygenic influences on VSMC behavior.Conclusions:This comprehensive analysis shows that a large percentage of CAD loci can modulate gene expression in VSMCs and influence VSMC behavior. Several candidate causal genes identified are likely to be druggable and thus represent potential therapeutic targets.

YAP1/TEAD1 upregulate platelet-derived growth factor receptor beta to promote vascular smooth muscle cell proliferation and neointima formation

We have previously demonstrated that the transcription co-factor yes-associated protein 1 (YAP1) promotes vascular smooth muscle cell (VSMC) de-differentiation. Yet, the role and underlying mechanisms of YAP1 in neointima formation in vivo remain unclear. The goal of this study was to investigate the role of VSMC-expressed YAP1 in vascular injury-induced VSMC proliferation and delineate the mechanisms underlying its action. Experiments employing gain- or loss-of-function of YAP1 demonstrated that YAP1 promotes human VSMC proliferation. Mechanistically, we identified platelet-derived growth factor receptor beta (PDGFRB) as a novel YAP1 target gene that confers the YAP1-dependent hyper-proliferative effects in VSMCs. Furthermore, we identified TEA domain transcription factor 1 (TEAD1) as a key transcription factor that mediates YAP1-dependent PDGFRβ expression. ChIP assays demonstrated that TEAD1 is enriched at a PDGFRB gene enhancer. Luciferase reporter assays further demonstrated that YAP1 and TEAD1 co-operatively activate the PDGFRB enhancer. Consistent with these observations, we found that YAP1 expression is upregulated after arterial injury and correlates with PDGFRβ expression and VSMC proliferation in vivo. Using a novel inducible SM-specific Yap1 knockout mouse model, we found that the specific deletion of Yap1 in adult VSMCs is sufficient to attenuate arterial injury-induced neointima formation, largely due to inhibited PDGFRβ expression and VSMC proliferation. Our study unravels a novel mechanism by which YAP1/TEAD1 promote VSMC proliferation via transcriptional induction of PDGFRβ, thereby enhancing PDGF-BB downstream signaling and promoting neointima formation.

Platonin, a Cyanine Photosensitizing Dye, Ameliorates Inflammatory Responses in Vascular Smooth Muscle Cells by Modulating Inflammatory Transcription Factors

Inflammation of the arterial wall is critical to atherosclerosis pathogenesis. The switch of vascular smooth muscle cells (VSMCs) to macrophage-like cells is essential in the exacerbation of vascular inflammation. Platonin, a cyanine photosensitizing dye, exhibits protective effects in sepsis, trauma, and acute ischemic stroke through its anti-inflammatory capacity in macrophages. The present study investigated the effects and underlying mechanisms of platonin in inflammatory VSMCs. Pretreatment with platonin suppressed the expression of inducible nitric oxide synthetase and mature interleukin-1β but not that of monocyte chemoattractant protein-1 (MCP-1) in VSMCs stimulated by a combination of lipopolysaccharide and interferon-γ (LPS/IFN-γ). Furthermore, platonin inhibited LPS/IFN-γ-induced Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation though the direct reduction of p65Ser536 phosphorylation but not the restoration of Inhibitor of nuclear factor kappa B (IκBα) degradation in VSMCs. However, platonin inhibited Oxidized low-density lipoprotein (ox-LDL)-induced MCP-1 production, possibly through the attenuation of Activator protein 1 (AP-1) binding activity and C-Jun N-terminal kinases ½ (JNK1/2) phosphorylation. Platonin also lowered lipid drop accumulation in VSMCs in Oil red O staining assay. The results collectively indicated that platonin has a vascular protective property with potent anti-inflammatory effects in VSMCs. In conclusion, platonin should be a potential for treating vascular inflammatory diseases such as atherosclerosis.

Diallyl Trisulfide Suppresses Angiotensin II-Induced Vascular Remodeling Via Inhibition of Mitochondrial Fission.

We have shown previously that diallyl trisulfide (DATS) ameliorates mitochondrial fission and oxidative stress in a hyperglycemia-induced endothelial apoptosis and diabetic mouse model. The aim of this study was to investigate whether DATS mitigates Ang II-induced vascular smooth muscle cell (VSMC) phenotypic switching and vascular remodeling, and if so, to determine the underlying molecular events. Male C57BL/6 mice were used to establish a vascular remodeling model by continuous 2-week Ang II infusion using a subcutaneous osmotic pump. Animals were intraperitoneally injected with DATS or vehicle. Physiological parameters, vascular morphology, and molecular markers were assessed. For in vitro studies, VSMCs were pretreated with or without DATS for 1h, then were stimulated with Ang II, and mitochondrial morphology and phenotypic switching of VSMCs were also measured. In primary mouse VSMCs, we found that Drp1-dependent mitochondrial fission regulated mitochondrial reactive oxygen species (mtROS) generation, which eventually promoted Ang II-induced VSMC proliferation, migration, and phenotypic switching. Moreover, Ang II was found to up-regulate the Rho-associated coiled coil-containing protein kinase 1 (ROCK1), which regulated mitochondrial fission and VSMC phenotypic switching by phosphorylating Drp1. However, the biological effect of Ang II was abrogated by DATS. Consistent with the effects in VSMCs, we found that DATS markedly alleviated mitochondrial fission, VSMC differentiation, and vessel wall thickening in an animal model of Ang II-induced vascular remodeling, which was regulated by the ROCK1/Drp1 signal. Our findings showed that DATS mitigated Ang II-induced vascular remodeling by suppressing Drp1-mediated mitochondrial fission in an ROCK1-dependent manner.

The P2RY12 receptor promotes VSMC-derived foam cell formation by inhibiting autophagy in advanced atherosclerosis

ABSTRACT Vascular smooth muscle cells (VSMCs) are an important source of foam cells in atherosclerosis. The mechanism for VSMC-derived foam cell formation is, however, poorly understood. Here, we demonstrate that the P2RY12/P2Y12 receptor is important in regulating macroautophagy/autophagy and VSMC-derived foam cell formation in advanced atherosclerosis. Inhibition of the P2RY12 receptor ameliorated lipid accumulation and VSMC-derived foam cell formation in high-fat diet-fed apoe-/- mice (atherosclerosis model) independent of LDL-c levels. Activation of the P2RY12 receptor blocked cholesterol efflux via PI3K-AKT, while genetic knockdown or pharmacological inhibition of the P2RY12 receptor inhibited this effect in VSMCs. Phosphoproteomic analysis showed that the P2RY12 receptor regulated the autophagy pathway in VSMCs. Additionally, activation of the P2RY12 receptor inhibited MAP1LC3/LC3 maturation, SQSTM1 degradation, and autophagosome formation in VSMCs. Genetic knockdown of the essential autophagy gene Atg5 significantly attenuated P2RY12 receptor inhibitor-induced cholesterol efflux in VSMCs. Furthermore, activation of the P2RY12 receptor led to the activation of MTOR through PI3K-AKT in VSMCs, whereas blocking MTOR activity (rapamycin) or reducing MTOR expression reversed the inhibition of cholesterol efflux mediated by the P2RY12 receptor in VSMCs. In vivo, inhibition of the P2RY12 receptor promoted autophagy of VSMCs through PI3K-AKT-MTOR in advanced atherosclerosis in apoe-/- mice, which could be impeded by an autophagy inhibitor (chloroquine). Therefore, we conclude that activation of the P2RY12 receptor decreases cholesterol efflux and promotes VSMC-derived foam cell formation by blocking autophagy in advanced atherosclerosis. Our study thus suggests that the P2RY12 receptor is a therapeutic target for treating atherosclerosis. Abbreviations: 2-MeSAMP: 2-methylthioadenosine 5′-monophosphate; 8-CPT-cAMP: 8-(4-chlorophenylthio)-adenosine-3ʹ,5ʹ-cyclic-monophosphate; ABCA1: ATP binding cassette subfamily A member 1; ABCG1: ATP binding cassette subfamily G member 1; ACTB: actin beta; ADPβs: adenosine 5′-(alpha, beta-methylene) diphosphate; ALs: autolysosomes; AMPK: AMP-activated protein kinase; APOA1: apolipoprotein A1; APs: autophagosomes; ATG5: autophagy related 5; ATV: atorvastatin; AVs: autophagic vacuoles; CD: chow diet; CDL: clopidogrel; CQ: chloroquine; DAPI: 4ʹ,6-diamidino-2-phenylindole; dbcAMP: dibutyryl-cAMP; DIL-oxLDL: dioctadecyl-3,3,3,3-tetramethylin docarbocyanine-oxLDL; EIF4EBP1/4E-BP1: eukaryotic translation initiation factor 4E binding protein 1; EVG: elastic van gieson; HE: hematoxylin-eosin; HDL: high-density lipoprotein; HFD: high-fat diet; KEGG: Kyoto Encyclopedia of Genes and Genomes; LDL-c: low-density lipoprotein cholesterol; LDs: lipid droplets; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; Masson: masson trichrome; MCPT: maximal carotid plaque thickness; MK2206: MK-2206 2HCL; NBD-cholesterol: 22-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl] amino)-23,24-bisnor-5-cholen-3β-ol; OLR1/LOX-1: oxidized low density lipoprotein receptor 1; ORO: oil Red O; ox-LDL: oxidized low-density lipoprotein; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TIC: ticagrelor; ULK1: unc-51 like autophagy activating kinase 1; VSMCs: vascular smooth muscle cells

CircCBFB-mediated miR-28-5p facilitates abdominal aortic aneurysm via LYPD3 and GRIA4

Heading aimsAbdominal aortic aneurysm (AAA) is featured by the growth impediment and apoptosis surge of VSMCs (vascular smooth muscle cells). MicroRNAs (miRNAs) are suggested to affect cellular behaviors including cell growth and apoptosis. This study concentrated on unraveling the emerging role of miR-28-5p in abdominal aortic aneurysm. Materials and methodsPreviously, miR-28-5p was reported to be highly expressed in AAA. Functional assays were utilized to determine the role of miR-28-5p in VSMC apoptosis. To narrow down the downstream mRNAs, bioinformatics methods were utilized. The interaction between miR-28-5p and GRIA4 (glutamate ionotropic receptor AMPA type subunit 4) or LYPD3 (LY6/PLAUR domain containing 3) was explored. Candidate circRNAs (circular RNAs) of miR-28-5p were identified. Rescue analyses validated function of circCBFB (core-binding factor subunit beta)/miR-28-5p/GRIA4/LYPD3 axis in VSMC apoptosis and growth. Key findingsMiR-28-5p acted as an apoptosis driver while circCBFB, GRIA4 and LYPD3 exerted anti-apoptosis effects in VSMCs. Mechanically, GRIA4 and LYPD3 were suppressed by miR-28-5p. Moreover, circCBFB served as a sponge of miR-28-5p, releasing GRIA4 and LYPD3 from miR-28-5p suppression. Functionally, GRIA4, LYPD3 and miR-28-5p were required in circCBFB-mediated VSMC apoptosis. SignificanceThis work unveiled an innovative axis of circCBFB/miR-28-5p/GRIA4/LYPD3 in VSMC apoptosis, exerting its potential in providing new thoughts in AAA management.

STAT3-induced up-regulation of lncRNA NEAT1 as a ceRNA facilitates abdominal aortic aneurysm formation by elevating TULP3.

Long noncoding RNAs (lncRNAs) were viewed as crucial participants in the pathogenesis of abdominal aortic aneurysm (AAA). LncRNA NEAT1 was recognized as an oncogenic gene in various diseases. However, its function and mechanism in AAA were not precisely documented. Here, we explored the functional role and molecular mechanism of NEAT1 in AAA. Functionally, the effect of NEAT1 on the proliferation was assessed by CCK-8 and EdU assay, while its impact on the apoptosis was evaluated through caspase-3/9 activity and TUNEL assays. As a result, we found that NEAT1 knockdown enhanced the proliferation and impaired the apoptosis of vascular smooth muscle cells (VSMCs). Reversely, overexpressed NEAT1 exerted anti-proliferation and pro-apoptosis effects in VSMCs. Mechanically, we found that STAT3 acted as a transcription factor and contributed to NEAT1 transcription by ChIP and luciferase reporter assays. In addition, NEAT1 was confirmed as a sponge of miR-4688 and thereby increase the expression of TULP3 in VSMCs via RIP assay and RNA pull-down assay. Rescue experiments indicted that TULP3 overexpressing countervailed the impact of NEAT1 depletion on AAA biological processes. Conclusively, lncRNA NEAT1 induced by STAT3 was identified as a ceRNA and facilitated AAA formation by targeting miR-4688/TULP3 axis.

Chemerin inhibits vascular calcification through ChemR23 and is associated with lower coronary calcium in chronic kidney disease.

BackgroundChemerin is an adipokine that signals through the G protein‐coupled receptor ChemR23 and is associated with inflammation, glucose homeostasis, lipid metabolism and renal function, all of which strongly influence cardiovascular risk. However, elevated chemerin provides a survival advantage in patients with chronic kidney disease (CKD), but how this relates to the cardiovascular phenotype is unknown.ObjectivesThe aim of the present study was to establish the association of chemerin with coronary calcification and to determine the effects of chemerin signalling, through ChemR23, in vascular smooth muscle cell (VSMC) calcification.MethodsPlasma chemerin was measured in 113 patients with CKD and 50 healthy controls. All patients underwent computed tomography to determine coronary artery calcium (CAC) score. VSMCs were isolated from wild‐type and ChemR23 knock‐out mice and treated with chemerin.ResultsMultivariate analyses established creatinine, cholesterol, body mass index and tumour necrosis factor as significant confounders for circulating chemerin levels. Despite these positive associations with renal function, cardiometabolic risk factors and inflammation, chemerin was inversely associated with CAC both in an age‐ and sex‐adjusted analysis and in a multivariate analysis adjusting for the aforementioned confounders. In addition, circulating chemerin levels were associated with the calcification inhibitors matrix gla protein (MGP) and fetuin‐A. Finally, chemerin significantly reduced phosphate‐induced calcification and increased MGP expression in VSMCs, whereas chemerin was devoid of these effects in VSMCs lacking ChemR23.ConclusionIn conclusion, these results suggest that chemerin signalling through ChemR23 in VSMCs protects against vascular calcification in CKD.

Aldehyde dehydrogenase 2 deficiency promotes atherosclerotic plaque instability through accelerating mitochondrial ROS-mediated vascular smooth muscle cell senescence

Previous evidence has indicated a beneficial role for aldehyde dehydrogenase 2 (ALDH2) in suppressing atherosclerotic plaque progression and instability. However, the underlying mechanism remains somewhat elusive. This study was designed to examine the effect of ALDH2 deficiency on high-cholesterol diet-induced atherosclerotic plaque progression and plaque vulnerability in atherosclerosis-prone ApoE knockout (ApoE−/−) mice with a focus on foam cell formation in macrophages and senescence of vascular smooth muscle cells (VSMCs). Serum lipid profile, plaque progression, and plaque vulnerability were examined in ApoE−/− and ALDH2/ApoE double knockout (ALDH2−/−ApoE−/−) mice after high-cholesterol diet intake for 8 weeks. ALDH2 deficiency increased the serum levels of triglycerides while it decreased levels of total cholesterol and high-density lipoprotein cholesterol. Unexpectedly, ALDH2 deficiency reduced the plaque area by 58.9% and 37.5% in aorta and aortic sinus, respectively. Plaque instability was aggravated by ALDH2 deficiency along with the increased necrotic core size, decreased collagen content, thinner fibrous cap area, decreased VSMC content, and increased macrophage content. In atherosclerotic lesions, ALDH2 protein was located in both macrophages and VSMCs. Further results revealed downregulated ALDH2 expression in aorta of aged ApoE−/− mice compared with young mice. However, in vitro study suggested that ALDH2 expression was upregulated in bone marrow-derived macrophages (BMDMs) with an opposite effect in VSMCs following 80 μg/ml oxidized low-density lipoprotein (oxLDL) treatment. Interestingly, ALDH2 deficiency displayed little effect in oxLDL-induced foam cell formation from BMDMs, while ALDH2 knockdown by siRNA and ALDH2 overexpression by lentivirus infection promoted and retarded oxLDL-induced VSMC senescence, respectively. Mechanistically, ALDH2 mitigated oxLDL-induced overproduction of mitochondrial reactive oxygen species (mROS) and activation of downstream p53/p21/p16 pathway. Clearance of mROS by mitoTEMPO significantly reversed the promotive effect of ALDH2 knockdown on VSMC senescence. Taken together, our data revealed that ALDH2 deficiency suppressed atherosclerotic plaque area while facilitating plaque instability possibly through accelerating mROS-mediated VSMC senescence.This article is part of a Special Issue entitled: Genetic and epigenetic regulation of aging and longevity edited by Jun Ren & Megan Yingmei Zhang.

Up-regulation of heme oxygenase-1 expression and inhibition of disease-associated features by cannabidiol in vascular smooth muscle cells.

Aberrant proliferation and migration of vascular smooth muscle cells (VSMC) have been closely linked to the development and progression of cardiovascular and cancer diseases. The cytoprotective enzyme heme oxygenase-1 (HO-1) has been shown to mediate anti-proliferative and anti-migratory effects in VSMC. This study investigates the effect of cannabidiol (CBD), a non-psychoactive cannabinoid, on HO-1 expression and disease-associated functions of human umbilical artery smooth muscle cells (HUASMC). HO-1 protein and mRNA were significantly increased by CBD in a time- and concentration-dependent manner. Although the expression of several cannabinoid-activated receptors (CB1, CB2, G protein-coupled receptor 55, transient receptor potential vanilloid 1) was verified in HUASMC, CBD was shown to induce HO-1 via none of these targets. Instead, the CBD-mediated increase in HO-1 protein was reversed by the glutathione precursor N-acetylcysteine, indicating the participation of reactive oxygen species (ROS) signaling; this was confirmed by flow cytometry-based ROS detection. CBD-induced HO-1 expression was accompanied by inhibition of growth factor-mediated proliferation and migration of HUASMC. However, neither inhibition of HO-1 activity nor knockdown of HO-1 protein attenuated CBD-mediated anti-proliferative and anti-migratory effects. Indeed, inhibition or depletion of HO-1 resulted in induction of apoptosis and intensified CBD-mediated effects on proliferation and migration. Collectively, this work provides the first indication of CBD-mediated enhancement of HO-1 in VSMC and potential protective effects against aberrant VSMC proliferation and migration. On the other hand, our data argue against a role of HO-1 in CBD-mediated inhibition of proliferation and migration while substantiating its anti-apoptotic role in oxidative stress-mediated cell fate.

Effect of Ang III on Nuclear Factor Kappa Beta in Wistar Rat VSMCs

ObjectiveWe investigated whether angiotensin (Ang) III induces nuclear factor kappa beta (NF‐kb) phosphorylation in isolated rat vascular smooth muscle cells (VSMCs).BackgroundThe molecular mechanisms by which Ang III induces various biological effects have not been fully investigated. Most studies have shown that NF‐kb mediates Ang II inflammatory responses effects in VSMCs. Inflammation of vascular smooth muscle cells (VSMCs) is a critical action associated with cardiovascular diseases including hypertension. The role of Ang III to induce NF‐kb in VSMCs is unknown and is a focus of these studies.MethodsPrimary cultures of VSMCs were isolated from the thoracic aorta of adult Wistar rats by the explant technique. VSMCs were treated with Ang III ranging in concentration from 0.1 nM to 1000 nM for 10 minutes or with 100 nM Ang III for 1 minute to 30 minutes. The western blotting technique was used to determine the effects of the peptide on NF‐kb protein phosphorylation.ResultsConcentration studies showed that Ang III caused a dose‐dependent increase in NF‐kb protein phosphorylation. The effects of the peptide on NF‐kb phosphorylation were maximal between 10 nM and 100 nM. The peptide's effects were rapid and significant; occurring within minutes of treatment and the maximal effects on NF‐kb phosphorylation was observed by 10 minutes.ConclusionThese findings provide insight into the molecular nature of the actions of Ang III and offer possible mechanism by which Ang III physiological actions occur in VSMCs.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Anti-atherosclerotic Effect of Paeonol against Vascular Smooth Muscle Cell Proliferation by Up-regulation of Autophagy via the AMPK/mTOR Signaling Pathway.

Introduction: Paeonol (2′-hydroxy-4′-methoxyacetophenone), isolated from moutan cortex, is an active component and has been shown to have anti-atherosclerotic and anti-proliferation effects on vascular smooth muscle cells (VSMCs). However, the possible role of Paeonol in protecting against VSMC proliferation as related to autophagy has yet to be elucidated.Materials and Methods: The athero-protective effects of Paeonol were evaluated in apoE-/- mice. The effects of Paeonol on VSMC proliferation and autophagy were examined by staining α-SMA and LC3II spots in the media layer of apoE-/- mice, respectively. CCK8 and BrdU assays were used to investigate the effects of Paeonol on cell proliferation in vitro. The autophagic levels in VSMCs were evaluated by detecting LC3II accumulation and p62 degradation by immunoblot analysis. To investigate if Paeonol could prevent VSMCs proliferation through autophagy induction, we tested the change in autophagy and cell proliferation by inhibition of autophagy. The levels of the AMPK/mTOR pathway in autophagy regulation were detected by immunoblot analysis. An AMPK inhibitor and si-AMPK transfection in VSMCs was used to confirm whether AMPK activity plays a key role in autophagy regulation of Paeonol.Results: In vivo experiments confirmed that Paeonol restricted atherosclerosis development and decreased the amount of VSMCs in the media layer of apoE-/- mice. Paeonol increased protein levels of LC3II and the presence of autophagosomes in the media layer of arteries, which implies that Paeonol may induce VSMCs autophagy in vivo. Paeonol showed potential in inhibiting ox-LDL-induced proliferation in vitro experiments. Paeonol dose-dependently enhanced the formation of acidic vesicular organelles and autophagosmomes, up-regulated the expression of LC3II and increased p62 degradation. The autophagy inhibitor CQ obviously attenuated Paeonol-induced autophagy and the anti-proliferation effect in VSMCs. In addition, Paeonol induced phosphorylation of AMPK and reduced phosphorylation of mTOR. An AMPK inhibitor reversed the Paeonol-induced p-mTOR/mTOR decrease. Paeonol induced LC3II conversion, increased p62 degradation and inhibited cell proliferation in VSMCs, the effects of which were abolished by si-AMPK.Conclusion: These results imply that Paeonol inhibits proliferation of VSMCs by up-regulating autophagy, and activating the AMPK/mTOR signaling pathway, providing new insights into the anti-atherosclerosis activity of Paeonol.

Loss of SPRR3 in ApoE-/- mice leads to atheroma vulnerability through Akt dependent and independent effects in VSMCs.

Vascular smooth muscle cells (VSMCs) represent important modulators of plaque stability in advanced lesions. We previously reported that loss of small proline-rich repeat protein 3 (Sprr3), leads to VSMC apoptosis in a PI3K/Akt-dependent manner and accelerates lesion progression. Here, we investigated the role of Sprr3 in modulating plaque stability in hyperlipidemic ApoE-/- mice. We show that loss of Sprr3 increased necrotic core size and reduced cap collagen content of atheromas in brachiocephalic arteries with evidence of plaque rupture and development of intraluminal thrombi. Moreover, Sprr3-/-ApoE-/- mice developed advanced coronary artery lesions accompanied by intraplaque hemorrhage and left ventricle microinfarcts. SPRR3 is known to reduce VSMC survival in lesions by promoting their apoptosis. In addition, we demonstrated that Sprr3-/- VSMCs displayed reduced expression of procollagen in a PI3K/Akt dependent manner. SPRR3 loss also increased MMP gelatinase activity in lesions, and increased MMP2 expression, migration and contraction of VSMCs independently of PI3K/Akt. Consequently, Sprr3 represents the first described VSMC modulator of each of the critical features of cap stability, including VSMC numbers, collagen type I synthesis, and protease activity through Akt dependent and independent pathways.

INOS-derived peroxynitrite mediates high glucose-induced inflammatory gene expression in vascular smooth muscle cells through promoting KLF5 expression and nitration

Inducible NO synthase (iNOS) expression and peroxynitrite formation are significantly increased in diabetic vascular tissues. Transcription factor KLF5 activates iNOS gene transcription and is involved in vascular inflammatory injury and remodeling. However, mutual regulation between KLF5, iNOS and peroxynitrite in diabetic vascular inflammation, as well as the underlying mechanisms, remain largely unknown. In this study, we found a marked increase in KLF5 and iNOS expression in vascular smooth muscle cells (VSMC) of diabetic patients. High glucose-induced expression of KLF5 and iNOS was also observed in cultured mouse VSMCs. Further investigation showed that high glucose induced KLF5 nitration by iNOS-mediated peroxynitrite generation, and nitrated KLF5 increased its interaction with NF-κB p50 and thus cooperatively activated the expression of inflammatory cytokines TNF-α and IL-1β. Furthermore, we showed that the VSMC-specific knockout of KLF5 dramatically reduced inflammatory cytokine expression in the vascular tissues of diabetic mice. Moreover, 17β-estradiol (E2) inhibited high glucose-mediated effects in VSMCs, and in the response to E2, estrogen receptor (ER) α competed with KLF5 for binding to NF-κB p50, which in turn leads to the suppression of inflammatory gene expression in VSMCs. Together, the present findings were the first to show that KLF5 expression and nitration by iNOS-mediated peroxynitrite are necessary for the induction of TNF-α and IL-1β expression in VSMCs of diabetic vascular tissues.

Gene expression profiles and signaling mechanisms in \u03b12B-adrenoceptor-evoked proliferation of vascular smooth muscle cells

Backgroundα2-adrenoceptors are important regulators of vascular tone and blood pressure. Regulation of cell proliferation is a less well investigated consequence of α2-adrenoceptor activation. We have previously shown that α2B-adrenoceptor activation stimulates proliferation of vascular smooth muscle cells (VSMCs). This may be important for blood vessel development and plasticity and for the pathology and therapeutics of cardiovascular disorders. The underlying cellular mechanisms have remained mostly unknown. This study explored pathways of regulation of gene expression and intracellular signaling related to α2B-adrenoceptor-evoked VSMC proliferation.ResultsThe cellular mechanisms and signaling pathways of α2B-adrenoceptor-evoked proliferation of VSMCs are complex and include redundancy. Functional enrichment analysis and pathway analysis identified differentially expressed genes associated with α2B-adrenoceptor-regulated VSMC proliferation. They included the upregulated genes Egr1, F3, Ptgs2 and Serpine1 and the downregulated genes Cx3cl1, Cav1, Rhoa, Nppb and Prrx1. The most highly upregulated gene, Lypd8, represents a novel finding in the VSMC context. Inhibitor library screening and kinase activity profiling were applied to identify kinases in the involved signaling pathways. Putative upstream kinases identified by two different screens included PKC, Raf-1, Src, the MAP kinases p38 and JNK and the receptor tyrosine kinases EGFR and HGF/HGFR. As a novel finding, the Src family kinase Lyn was also identified as a putative upstream kinase.Conclusionsα2B-adrenoceptors may mediate their pro-proliferative effects in VSMCs by promoting the activity of bFGF and PDGF and the growth factor receptors EGFR, HGFR and VEGFR-1/2. The Src family kinase Lyn was also identified as a putative upstream kinase. Lyn is known to be expressed in VSMCs and has been identified as an important regulator of GPCR trafficking and GPCR effects on cell proliferation. Identified Ser/Thr kinases included several PKC isoforms and the β-adrenoceptor kinases 1 and 2. Cross-talk between the signaling mechanisms involved in α2B-adrenoceptor-evoked VSMC proliferation thus appears to involve PKC activation, subsequent changes in gene expression, transactivation of EGFR, and modulation of kinase activities and growth factor-mediated signaling. While many of the identified individual signals were relatively small in terms of effect size, many of them were validated by combining pathway analysis and our integrated screening approach.

C1q/TNF-related protein 9 inhibits the cholesterol-induced Vascular smooth muscle cell phenotype switch and cell dysfunction by activating AMP-dependent kinase.

Vascular smooth muscle cells (VSMCs) switch to macrophage‐like cells after cholesterol loading, and this change may play an important role in the progression of atherosclerosis. C1q/TNF‐related protein 9 (CTRP9) is a recently discovered adipokine that has been shown to have beneficial effects on glucose metabolism and vascular function, particularly in regard to cardiovascular disease. The question of whether CTRP9 can protect VSMCs from cholesterol damage has not been addressed. In this study, the impact of CTRP9 on cholesterol‐damaged VSMCs was observed. Our data show that in cholesterol‐treated VSMCs, CTRP9 significantly reversed the cholesterol‐induced increases in pro‐inflammatory factor secretion, monocyte adhesion, cholesterol uptake and expression of the macrophage marker CD68. Meanwhile, CTRP9 prevented the cholesterol‐induced activation of the TLR4–MyD88–p65 pathway and upregulated the expression of proteins important for cholesterol efflux. Mechanistically, as siRNA‐induced selective gene ablation of AMPKα1 abolished these effects of CTRP9, we concluded that CTRP9 achieves these protective effects in VSMCs through the AMP‐dependent kinase (AMPK) pathway.

Different effects of neuropeptide Y on proliferation of vascular smooth muscle cells via regulation of Geminin.

The proliferation-promoting effect of neuropeptide Y (NPY) always functions in low-serum-cultured vascular smooth muscle cells (VSMCs), and the phenotypic switch of VSMCs is regulated by concentrations of serum. Whether the property of the NPY proliferative effect in VSMCs relies on phenotype of VSMCs is unclear. We aimed to explore the role of NPY on proliferation of different VSMC phenotypes in the pathogenesis of atherosclerosis. By stimulating A10 cells with 200nM NPY in 0.5 or 10% serum, 3H-thymidine and 5-ethynyl-2'-deoxyuridine (EdU) and CCK8 measurements were used to detect VSMC proliferation. RT-PCR and Flow cytometry were performed to detect the factors involved in different properties of the NPY proliferative effect in VSMCs. Instead of facilitating proliferation, NPY had no significant effect on the growth of VSMCs when cultured in 10% serum (VSMCs stayed at synthetic states). The underlying mechanism may be involved in down-regulation of Y1 receptor (P<0.05 vs. Vehicle) and up-regulation of Geminin (P<0.05 vs. Vehicle) in 10% serum-cultured VSMCs co-incubated with 200nM NPY. Besides, modulation of Geminin was effectively blocked by the Y1 receptor antagonist. The stimulation of NPY on proliferation of VSMCs could be a double-edged sword in the development of atherosclerosis and thus provides new knowledge for therapy of atherosclerosis.

Free Fatty Acids Induce Autophagy and LOX-1 Upregulation in Cultured Aortic Vascular Smooth Muscle Cells.

Elevation of free fatty acids (FFAs) is known to affect microvascular function and contribute to obesity-associated insulin resistance, hypertension, and microangiopathy. Proliferative and synthetic vascular smooth muscle cells (VSMCs) increase intimal thickness and destabilize atheromatous plaques. This study aimed to investigate whether saturated palmitic acid (PA) and monounsaturated oleic acid (OA) modulate autophagy activity, cell proliferation, and vascular tissue remodeling in an aortic VSMC cell line. Exposure to PA and OA suppressed growth of VSMCs without apoptotic induction, but enhanced autophagy flux with elevation of Beclin-1, Atg5, and LC3I/II. Cotreatment with autophagy inhibitors potentiated the FFA-suppressed VSMC growth and showed differential actions of PA and OA in autophagy flux retardation. Both FFAs upregulated lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) but only OA increased LDL uptake by VSMCs. Mechanistically, FFAs induced hyperphosphorylation of Akt, ERK1/2, JNK1/2, and p38 MAPK. All pathways, except OA-activated PI3K/Akt cascade, were involved in the LOX-1 upregulation, whereas blockade of PI3K/Akt and MEK/ERK cascades ameliorated the FFA-induced growth suppression on VSMCs. Moreover, both FFAs exhibited tissue remodeling effect through increasing MMP-2 and MMP-9 expression and their gelatinolytic activities, whereas high-dose OA significantly suppressed collagen type I expression. Conversely, siRNA-mediated LOX-1 knockdown significantly attenuated the OA-induced tissue remodeling effects in VSMCs. In conclusion, OA and PA enhance autophagy flux, suppress aortic VSMC proliferation, and exhibit vascular remodeling effect, thereby leading to the loss of VSMCs and interstitial ECM in vascular walls and eventually the instability of atheromatous plaques. J. Cell. Biochem. 118: 1249-1261, 2017. © 2016 Wiley Periodicals, Inc.