Migration Of Aortic Smooth Muscle Cells Research Articles

Curcumol reduces lower limb arteriosclerosis in rats by inhibiting human arterial smooth muscle cell activity.

Cardiovascular diseases, particularly those involving arterial stenosis and smooth muscle cell proliferation, pose significant health risks. This study aimed to investigate the therapeutic potential of curcumol in inhibiting platelet-derived growth factor-BB (PDGF-BB)-induced human aortic smooth muscle cell (HASMC) proliferation, migration and autophagy. Using cell viability assays, 5-ethynyl-2'-deoxyuridine (EdU) incorporation assays and Western Blot analyses, we observed that curcumol effectively attenuated PDGF-BB-induced HASMC proliferation and migration in a concentration-dependent manner. Furthermore, curcumol mitigated PDGF-BB-induced autophagy, as evidenced by the downregulation of LC3-II/LC3-I ratio and upregulation of P62. In vivo experiments using an arteriosclerosis obliterans model demonstrated that curcumol treatment significantly ameliorated arterial morphology and reduced stenosis. Additionally, curcumol inhibited the activity of the KLF5/COX2 axis, a key pathway in vascular diseases. These findings suggest that curcumol has the potential to serve as a multi-target therapeutic agent for vascular diseases.

Abstract 13109: Empagliflozin Reverses Oxidized LDL-Induced RECK Suppression, MMP Activation, and Human Aortic Smooth Muscle Cell Proliferation and Migration

Empagliflozin, an FDA-approved anti-hyperglycemic agent, is a potent and highly selective inhibitor of sodium glucose co-transporter-2 (SGLT-2) in renal proximal tubule epithelial cells. It blocks glucose reabsorption and promotes glucose excretion via urine. Intriguingly, several clinical trials have reported the pleiotropic beneficial effects of SGLT-2 inhibitors in cardiovascular diseases, including antioxidant and anti-inflammatory effects. Here we investigated whether empagliflozin reverses oxidatively modified LDL (OxLDL)-induced matrix metalloproteinase activation and smooth muscle cell (SMC) migration and proliferation. Quiesced human aortic SMCs (Lonza) were treated with empagliflozin (1 uM) followed by human medium OxLDL (40 ug/mL; Kalen Biomedical). OxLDL stimulated SMC migration (1.8-fold, P<0.05, n=4) and proliferation (1.5-fold, P<0.05, n=4), and empagliflozin attenuated both by 67% (P<0.05, n=4) and 61% (P<0.05, n=4), respectively. Increased migration and proliferation by OxLDL were accompanied by elevated MMP-2 (3.2-fold, P<0.05, n=5) and 9 (3.1-fold, P<0.05, n=5) activities, which were also suppressed by empagliflozin by 65% and 67%, respectively (both P<0.05, n=5). RECK (reversion inducing cysteine rich protein with Kazal motifs) is a membrane anchored MMP inhibitor, and we found that OxLDL dose-dependently downregulated RECK in SMCs (by 60%, P<0.05, n=3) via NF-kB-dependent miR-30b induction. Intriguingly, empagliflozin restored the OxLDL downregulation of RECK to 88% of the control (n=3), as it inhibited miR-30b induction (by 49%, P<0.05, n=5). Consistent with RECK-mediated effects of empagliflozin, adenoviral overexpression of RECK inhibited OxLDL-induced SMC migration (by 67%, P<0.05, n=5) and proliferation (by 50%, P<0.05, n=5). Together, these results suggest that the vasculoprotective effects of empagliflozin are at least in part mediated by restoring RECK, thereby attenuating SMC migration and proliferation under elevated oxidative stress condition. Our results also suggest therapeutic potential of empagliflozin or RECK overexpression in vascular proliferative diseases.

Empagliflozin Reverses Oxidized LDL-Induced RECK Suppression, Cardiotrophin-1 Expression, MMP Activation, and Human Aortic Smooth Muscle Cell Proliferation and Migration.

Persistent oxidative stress and inflammation contribute causally to smooth muscle cell (SMC) proliferation and migration, the characteristic features of vascular proliferative diseases. Oxidatively modified low-density lipoproteins (OxLDL) elevate oxidative stress levels, inflammatory responses, and matrix metallopeptidase (MMP) activation, resulting ultimately in SMC migration, proliferation, and phenotype change. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is a membrane-anchored MMP inhibitor. Empagliflozin is an SGLT2 inhibitor and exerts pleiotropic cardiovascular protective effects, including antioxidant and anti-inflammatory effects. Here, we investigated (i) whether OxLDL regulates RECK expression, (ii) whether ectopic expression of RECK reverses OxLDL-induced SMC migration and proliferation, and (iii) whether pretreatment with empagliflozin reverses OxLDL-induced RECK suppression, MMP activation, and SMC migration, proliferation, and differentiation. Indeed, results show that OxLDL at pathophysiological concentration promotes SMC migration and proliferation via NF-κB/miR-30b-dependent RECK suppression. Moreover, OxLDL changed the SMC phenotype to a more pro-inflammatory type, and this effect is blunted by RECK overexpression. Further, treatment with empagliflozin reversed OxLDL-induced miR-30b induction, RECK suppression, MMP activation, SMC migration, proliferation, and proinflammatory phenotype changes. OxLDL-induced cardiotrophin (CT)-1 expression and CT-1 stimulated SMC proliferation and migration in part via leukemia inhibitory factor receptor (LIFR) and glycoprotein 130 (gp130). Ectopic expression of RECK inhibited these effects by physically associating with LIFR and gp130, as evidenced by immunoprecipitation/immunoblotting and double immunofluorescence. Importantly, empagliflozin inhibited CT-1-induced mitogenic and migratory effects. Together, these results suggest the therapeutic potential of sustaining RECK expression or empagliflozin in vascular diseases characterized by SMC proliferation and migration.

Hsa_circ_0031891 targets miR-579-3p to enhance HMGB1 expression and regulate PDGF-BB-induced human aortic vascular smooth muscle cell proliferation, migration, and dedifferentiation.

Atherosclerosis (AS) is an underlying cause of the majority of coronary artery disease (CAD), in which proliferation, migration, and dedifferentiation of vascular smooth muscle cells (VSMCs) exert vital roles. It has been reported that circular RNAs (circRNAs) are associated with the VSMCs function. Here, we undertook to explore the biological function and mechanism of hsa_circ_0031891 in a platelet-derived growth factor-BB (PDGF-BB)-induced AS cell model. Hsa_circ_0031891 and microRNA-579-3p (miR-579-3p) levels were detected by real-time quantitative polymerase chain reaction (RT-qPCR). Cell proliferation and migration were detected using Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), wound healing, and transwell assay. Protein levels of alpha-smooth muscle actin (α-SMA), smooth muscle protein 22-α (SM22-α), Osteopontin, and High mobility group box-1 (HMGB1) were determined using western blot assay. After predicting via a variety of bioinformatics software, the binding between miR-579-3p and hsa_circ_0031891 or HMGB1 was validated using dual-luciferase reporter and RNA pull-down assays. Increased hsa_circ_0031891 and HMGB1 and reduced miR-579-3p were found in CAD patients and PDGF-BB-induced human aortic vascular smooth muscle cells (HA-VSMCs). Moreover, hsa_circ_0031891 deficiency relieved PDGF-BB-mediated HA-VSMC proliferation, migration, and dedifferentiation. Mechanically, hsa_circ_0031891 modulated HMGB1 expression via sponging miR-579-3p. Hsa_circ_0031891 boosted PDGF-BB-induced proliferation, migration, and dedifferentiation partly by regulating the miR-579-3p/HMGB1 axis, hinting at a feasible therapeutic strategy for AS.

Study on Thyroxine Preventing Coronary Atherosclerosis through TGFBR1 Signaling Pathway

To explore the mechanism of Thyroxine inhibiting Human Aortic Vascular Smooth Muscle Cell (HASMC) proliferation, migration and vascular intimal hyperplasia through the transforming growth factor b receptor 1(TGFBR1). 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium (MTS), 5-ethynyl-2' -deoxyuridine (EdU) staining, Transwell, cell scratch test and Western bloting were performed sequentially to detect the effects of Thyroxine on HASMC and the TGFBR1 signaling pathway. A cell model of TGFBR1 overexpression was constructed to further explore the mechanism of TGFBR1 in Thyroxine inhibiting the activation of HASMC. Thyroxine can inhibit HASMC proliferation and migration, and the activation of TGFBR1 signaling pathway,both in a concentration-dependent manner. Following the overexpression of TGFBR1, the effect of Thyroxine on inhibiting HASMC proliferation, migration and activation of TGFBR1 signaling pathway was partially inhibited. Thyroxine can inhibit the proliferation, migration and vascular intimal hyperplasia of HASMC by inhibiting the activation of TGFBR1 signaling pathway, thereby playing a role in preventing and treating vascular restenosis and related diseases.

Trimethylamine-N-oxide (TMAO) promotes balloon injury-induced neointimal hyperplasia via upregulating Beclin1 and impairing autophagic flux

Background and aimsTMAO is a microbiota-dependent metabolite associated with increased risk of various cardiovascular diseases. However, the relationship between TMAO and vascular injury-related neointimal hyperplasia is unclear. This study aimed to explore whether TMAO promotes neointimal hyperplasia after balloon injury and elucidate the underlying mechanism. Methods and ResultsThrough hematoxylin and eosin staining and immunohistochemistry staining, we found that supplementary TMAO promoted balloon injury-induced neointimal hyperplasia, while reducing TMAO by antibiotic administration produced the opposite result. TMAO showed limited effect on rat aortic vascular smooth muscle cells (RAOSMCs) proliferation and migration. However, TMAO notably induced dysfunction of rat aortic vascular endothelial cells (RAOECs) in vitro and attenuated reendothelialization of carotid arteries after balloon injury in vivo. Autophagic flux was measured by fluorescent mRFP-GFP-LC3, transmission electron microscopy, and western blot. TMAO impaired autophagic flux, as evidenced by the accumulation of p62 and LC3II and high autophagosome to autolysosome ratios. Furthermore, we confirmed that Beclin1 level increased in TMAO-treated RAOECs and carotid arteries. Knocking down Beclin1 alleviated TMAO-induced autophagic flux impairment and neointimal hyperplasia. ConclusionsTMAO promoted neointimal hyperplasia through Beclin1-induced autophagic flux blockage, suggesting that TMAO is a potential target for improvement of vascular remodeling after injury.

The Lnc-RNA APPAT Suppresses Human Aortic Smooth Muscle Cell Proliferation and Migration by Interacting With MiR-647 and FGF5 in Atherosclerosis

Purpose: LncRNA-Atherosclerotic plaque pathogenesis-associated transcript (APPAT) could be detected in circulating blood and has been demonstrated to correlate with the development of atherosclerosis in our previous work. It could be a potential noninvasive biomarker for earlier diagnoses of clinical cardiovascular disease. Moreover, the expression of miR-647 increased in ox-LDL-treated vascular smooth muscle cells and peripheral blood of patients with coronary heart disease. A negative correlation between APPAT and miR-647 was confirmed, and FGF5 was screened as molecular target of miR-647. However, it is largely unclear how APPAT, miR-647, and FGF5 interact and function in disease development. Here, we aim to explore the underlying molecular mechanism in this progression. Materials and Methods: APPAT, miR-647, and FGF5 expression levels were detected by quantitative reverse transcription polymerase chain reaction; cell proliferation was detected by EdU incorporation assay; cell migration was detected by wound-healing assay; the molecular interaction of APPAT/FGF5 with miR-647 was verified by dual-luciferase reporter assay; the western blot was performed to determine the gene expression at protein levels; subcellular localizations of APPAT and miR-647 were observed by fluorescence in situ hybridization; cytosolic and nucleus fractionation assay was performed to further detect the distribution of miR-647. Results: APPAT and miR-647 have inverse effects on human aortic smooth muscle cells’ (HASMCs) proliferation and migration. APPAT negatively regulated the cell activity, whereas miR-647 did it in a positive way (p<0.05). Three pairs of molecular interplay were found: mutual negative regulation between APPAT and miR-647, APPAT downregulated FGF5, miR-647 regulation on FGF5 (p<0.05). Subcellular location assay confirmed the molecular interaction of APPAT and miR-647. Conclusions: APPAT could suppress the migration and proliferation of ox-LDL-treated HASMCs via interacting with miR-647 and FGF5. We revealed a nontypical competing endogenous RNA mechanism of long noncoding RNA in the progression of atherosclerosis.

Interactions between β3‐adrenergic receptors and bFGF‐induced effects in rat aortic smooth muscle cells

BackgroundBasic fibroblast growth factor (bFGF)‐mediated vascular smooth muscle cell (VSMC) proliferation and migration play an important role in vascular injury‐induced neointima formation and subsequent vascular restenosis, a major event that hinders the long‐term success of angioplasty. The function of β3‐adrenergic receptors (β3‐ARs) in vascular injury‐induced neointima formation has not yet been defined.ObjectivesOur current study explored the possible role of β3‐ARs in vascular injury‐induced neointima formation by testing its effects on bFGF‐induced VSMC migration and proliferation.Methodsβ3‐AR expression in rat carotid arteries was examined at 14 days following a balloon catheter‐induced injury. The effects of β3‐AR activation on bFGF‐induced rat aortic smooth muscle cell proliferation, migration, and signaling transduction (including extracellular‐signal‐regulated kinase/ mitogen activated protein kinase, ERK/MAPK and Protein kinase B, AKT) were tested.ResultsWe found that vascular injury induced upregulation of β3‐ARs in neointima. Pretreatment of VSMCs with a selective β3‐AR agonist, CL316,243 significantly potentiated bFGF‐induced cell migration and proliferation, and ERK and AKT phosphorylation. Our results also revealed that suppressing phosphorylation of ERK and AKT blocked bFGF‐induced cell migration and that inhibiting AKT phosphorylation reduced bFGF‐mediated cell proliferation.ConclusionOur results suggest that activation of β3‐ARs enhances bFGF‐mediated effects on VSMCs by interacting with bFGF‐mediated ERK and AKT phosphorylation and β3‐ARs may play a role in vascular injury‐induced neointima formation.

Ginsenoside Rh1 Inhibits Angiotensin II-Induced Vascular Smooth Muscle Cell Migration and Proliferation through Suppression of the ROS-Mediated ERK1/2/p90RSK/KLF4 Signaling Pathway.

Vascular smooth muscle cell (VSMC) proliferation and migration play key roles in the progression of atherosclerosis and restenosis. A variety of ginsenosides exert various cardiovascular benefits. However, whether and how ginsenoside Rh1 (Rh1) inhibits VSMC dysfunction remain unclear. Here, we investigated the inhibitory effects of Rh1 on rat aortic smooth muscle cell (RASMC) migration and proliferation induced by angiotensin II (Ang II) and the underlying mechanisms. Cell proliferation and migration were evaluated using sulforhodamine B and wound-healing assay. The molecular mechanisms were investigated using Western blotting, quantitative reverse-transcription polymerase chain reaction analysis, immunofluorescence staining, and luciferase assay. Reactive oxygen species (ROS) production was measured using dihydroethidium and MitoSOX staining. We found that Rh1 dose-dependently suppressed Ang II-induced cell proliferation and migration. Concomitantly, Ang II increased protein levels of osteopontin, vimentin, MMP2, MMP9, PCNA, and cyclin D1, while these were reduced by Rh1 pretreatment. Notably, Ang II enhanced both the protein expression and promoter activity of KLF4, a key regulator of phenotypic switching, whereas pretreatment with Rh1 reversed these effects. Mechanistically, the effects of Rh1 on VSMC proliferation and migration were found to be associated with inhibition of ERK1/2/p90RSK signaling. Furthermore, the inhibitory effects of Rh1 were accompanied by inhibition of ROS production. In conclusion, Rh1 inhibited the Ang II-induced migration and proliferation of RASMCs by suppressing the ROS-mediated ERK1/2/p90RSK signaling pathway.

Regulation of bFGF-induced effects on rat aortic smooth muscle cells by β3-adrenergic receptors

BackgroundBasic fibroblast growth factor (bFGF)-mediated vascular smooth muscle cell (VSMC) proliferation and migration play an important role in vascular injury-induced neointima formation and subsequent vascular restenosis, a major event that hinders the long-term success of angioplasty. The function of β3-adrenergic receptors (β3-ARs) in vascular injury-induced neointima formation has not yet been defined. ObjectivesOur current study explored the possible role of β3-ARs in vascular injury-induced neointima formation by testing its effects on bFGF-induced VSMC migration and proliferation. Methodsβ3-AR expression in rat carotid arteries was examined at 14 days following a balloon catheter-induced injury. The effects of β3-AR activation on bFGF-induced rat aortic smooth muscle cell proliferation, migration, and signaling transduction (including extracellular-signal-regulated kinase/mitogen activated protein kinase, ERK/MAPK and Protein kinase B, AKT) were tested. ResultsWe found that vascular injury induced upregulation of β3-ARs in neointima. Pretreatment of VSMCs with a selective β3-AR agonist, CL316,243 significantly potentiated bFGF-induced cell migration and proliferation, and ERK and AKT phosphorylation. Our results also revealed that suppressing phosphorylation of ERK and AKT blocked bFGF-induced cell migration and that inhibiting AKT phosphorylation reduced bFGF-mediated cell proliferation. ConclusionOur results suggest that activation of β3-ARs potentiates bFGF-mediated effects on VSMCs by enhancing bFGF-mediated ERK and AKT phosphorylation and that β3-ARs may play a role in vascular injury-induced neointima formation.

EphrinB2 promotes the human aortic smooth muscle cell growth and migration via mediating F-actin remodeling.

To evaluate the potential effect of EphrinB2 in human thoracic aortic dissection (TAD) and to illustrate the mechanisms governing the role of EphrinB2 in the growth of human aortic smooth muscle cells (HASMC). In the study, EphrinB2 expression was investigated by qRT-PCR and immunohistochemistry in 12 pairs of TAD and adjacent human tissues. HASMCs were used for in vitro experiments. Next, EphrinB2 overexpression and depletion in HASMCs were established by EphrinB2-overexpressing vectors and small interfering RNA, respectively. The transfection efficiency was evaluated by qRT-PCR and Western blot. The effects of overexpression and depletion of EphrinB2 on cell proliferation, migration, and invasion were tested in vitro. Cell Counting Kit-8, flow cytometry and transwell migration/invasion, and wound healing assay were used to explore the function of EphrinB2 on HASMC cell lines. The relationship between EphrinB2 and F-actin was assessed by Western blot, immunofluorescence, and Co-IP. We found that EphrinB2 was a prognostic biomarker of TAD patients. Moreover, EphrinB2 expression negatively correlated to aortic dissection tissues, and disease incidence of males, suggesting that EphrinB2 might act as a TAD suppressor by promoting proliferation or decreasing apoptosis in HASMC. Next, over-expression of EphrinB2 in HASMC lines drove cell proliferation, migration, and invasion, and inhibited apoptosis while knockdown EphrinB2 showed the opposite phenomenon, respectively. Furthermore, the level of F-actin in mRNA, protein, and distribution in HASMC cell lines highly matched with the expression of EphrinB2, which indicated that EphrinB2 could mediate the HASMC cytoskeleton via inducing F-actin. In conclusion, our results first provided the pivotal role of EphrinB2 in HASMC proliferation initiated by mediating F-actin and demonstrated a prognostic biomarker and the potential targets for therapy to prevent thoracic aortic dissection.

MiR-150-5p affects AS plaque with ASMC proliferation and migration by STAT1.

We explore miR‐150‐5p in atherosclerosis (AS). The AS model was constructed using Apo E−/− mice with an injection of the miR-150-5p mimic or an inhibitor. Pathological characteristics were assessed using Oil red O staining and Masson staining. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot were used to analyze the expressions of microRNA-150-5p (miR-150-5p), STAT1, α-SMA (α-smooth muscle actin) and proliferating cell nuclear antigen (PCNA). Targetscan and dual-luciferase reporter assay were used to analyze the interaction between miR-150-5p and STAT1. The viability, migration, cell cycle and α-SMA and PCNA expressions in oxidized low-density lipoprotein (ox-LDL)-stimulated primary human aortic smooth muscle cells (ASMCs) were assessed using molecular experiments. miR-150-5p was reduced in both AS mice and ox-LDL-stimulated human aortic smooth muscle cells but STAT1 had the opposite effect. The miR‐150‐5p inhibitor alleviated the increase of lipid plaque and reduced collagen accumulation in the aortas during AS. Upregulation of α-SMA and PCNA was reversed by miR-150-5p upregulation. STAT1 was targeted by miR‐150‐5p, and overexpressed miR-150-5p weakened the ox-LDL-induced increase of viability and migration abilities and blocked cell cycle in ASMCs, but overexpressed STAT1 blocked the effect of the miR‐150‐5p mimic. This paper demonstrates that miR-150-5p has potential as a therapeutic target in AS, with plaque stabilization by regulating ASMC proliferation and migration via STAT1.

Identification of securinine as vascular protective agent targeting atherosclerosis in vascular endothelial cells, smooth muscle cells, and apolipoprotein E deficient mice

BackgroundAtherosclerosis is a chronic vascular disease and characterized by accumulation within the intima of inflammatory cells, smooth muscle cells, lipid, and connective tissue. PurposeThe purpose of the present study was to identify natural agents that commonly reverse advanced atherosclerotic plaque to early atherosclerotic plaque. MethodsDifferentially expressed genes (DEGs) were analyzed in silico. The differentially expressed genes from 9 intimal thickening and 8 fibrous cap atheroma tissue which were collected from GEO data were assessed by the connectivity map. Natural candidate securinine, a main compound from Securinega suffruticosa, was selected and administrated 1, 5 mg/kg/day in apolipoprotein-E-deficient (ApoE KO) mice for 18 weeks. ResultsSecurinine significantly showed lowered blood pressure and improvement of metabolic parameters with hyperlipidemia. The impairment in vasorelaxation was remarkably decreased by treatment with securinine. H&E staining revealed that treatment with securinine reduced atherosclerotic lesions. Securinine suppressed the expression of adhesion molecules and matrix metalloproteinase-2/-9 in both ApoE KO and vascular endothelial cells (HUVEC). In HUVEC pretreatment with securinine significantly inhibited ROS generation and NF-κB activation. Growth curve assays using the real-time cell analyzer showed that securinine significantly decreased TNF-α-induced aortic smooth muscle cell proliferation and migration in a dose-dependent manner. ConclusionSecurinine may be a potential natural candidate for the treatment of atherosclerosis because it attenuates vascular inflammation and dysfunction as well as vascular lesion.

The SGLT2 inhibitor Empagliflozin attenuates interleukin-17A-induced human aortic smooth muscle cell proliferation and migration by targeting TRAF3IP2/ROS/NLRP3/Caspase-1-dependent IL-1β and IL-18 secretion

Chronic inflammation and persistent oxidative stress contribute to the development and progression of vascular proliferative diseases. We hypothesized that the proinflammatory cytokine interleukin (IL)-17A induces oxidative stress and amplifies inflammatory signaling in human aortic smooth muscle cells (SMC) via TRAF3IP2-mediated NLRP3/caspase-1-dependent mitogenic and migratory proinflammatory cytokines IL-1β and IL-18. Further, we hypothesized that these maladaptive changes are prevented by empagliflozin (EMPA), an SGLT2 (Sodium/Glucose Cotransporter 2) inhibitor. Supporting our hypotheses, exposure of cultured SMC to IL-17A promoted proliferation and migration via TRAF3IP2, TRAF3IP2-dependent superoxide and hydrogen peroxide production, NLRP3 expression, caspase-1 activation, and IL-1β and IL-18 secretion. Furthermore, NLRP3 knockdown, caspase-1 inhibition, and pretreatment with IL-1β and IL-18 neutralizing antibodies and IL-18BP, each attenuated IL-17A-induced SMC migration and proliferation. Importantly, SMC express SGLT2, and pre-treatment with EMPA attenuated IL-17A/TRAF3IP2-dependent oxidative stress, NLRP3 expression, caspase-1 activation, IL-1β and IL-18 secretion, and SMC proliferation and migration. Importantly, silencing SGLT2 attenuated EMPA-mediated inhibition of IL-17A-induced cytokine secretion and SMC proliferation and migration. EMPA exerted these beneficial antioxidant, anti-inflammatory, anti-mitogenic and anti-migratory effects under normal glucose conditions and without inducing cell death. These results suggest the therapeutic potential of EMPA in vascular proliferative diseases.

Inhibitory effect of CP-25 on intimal formation and vascular hyperplasia via suppression of GRK2/ERK1/2/EVI1 signaling

Excessive proliferation, migration and dedifferentiation of vascular smooth muscle cells (VSMCs) are the center of intimal formation during in-stent restenosis and vein graft disease. Paeoniflorin-6′-O-benzene sulfonate (CP-25) is known to suppress inflammation and atherogenesis. However, the potential effect of CP-25 on intimal formation remains elusive. In the present study, we found that CP-25 significantly attenuated wire injury-induced intimal formation in C57BL/6 mice (intimal area: 2.64 ± 0.25 × 104 μm2 vs. 1.53 ± 0.21 × 104 μm2, P < 0.05) and vascular hyperplasia indicated by PCNA staining. In vitro experiments showed that CP-25 significantly alleviated human aortic smooth muscle cell (HASMC) proliferation, migration and dedifferentiation induced by PDGF-BB. Mechanistically, CP-25 inhibited GRK2 phosphorylation through PDGF receptor in the presence of PDGF-BB. In accordance with these results, CP-25 disrupted the interaction of GRK2 with ERK1/2 and suppressed the activation of ERK1/2 signaling in HASMCs. EVI1, which is considered as a downstream of ERK1/2 signaling and a novel transcription factor for VSMC differentiation, was also downregulated by CP-25 treatment. Moreover, overexpression of EVI1 partly restored the decreased proliferation and dedifferentiation of HASMCs treated by CP-25. Collectively, these findings suggested that CP-25 could alleviate intimal formation in response to wire injury via suppression of the interaction of GRK2 and ERK1/2 and EVI1 activation, indicating CP-25 might serve as a potent pharmaceutical for intimal formation.

PRDM16 Upregulation Induced by MicroRNA-448 Inhibition Alleviates Atherosclerosis via the TGF-β Signaling Pathway Inactivation.

The dysregulated expression of microRNAs (miRs) has been associated with pathological and physiological processes of atherosclerosis (AS). In addition, PR domain-containing 16 (PRDM16), a transcriptional mediator of brown fat cell identity and smooth muscle cell activities, may be involved in the hypercholesterolemia during development of AS. The bioinformatic analysis identified a regulatory miR-448 of PRDM16. Hence, the current study aimed to explore whether miR-448 influenced the activities of aortic smooth muscle cell (ASMCs) in AS. We validated that miR-448 was highly expressed in peripheral blood of patients with AS and aortic smooth muscle of AS model mice. Whereas, PRDM16 was downregulated in the aortic smooth muscle of AS model mice. PRDM16 overexpression was observed to inhibit oxidative stress injury and cell proliferation, and promote apoptosis of ASMCs. Mechanistic studies revealed that miR-448 targeted PRDM16 and negatively regulated the PRDM16 expression, while PRDM16 blocked the TGF-β signaling pathway. Furthermore, Downregulated miR-448 alleviated oxidative stress injury, and attenuated ASMC cell proliferation, migration and enhanced cell apoptosis through upregulation of PRDM16. Taken together, silencing of miR-448 upregulates PRDM16 and inactivates the TGF-β signaling pathway, thereby impeding development of AS by repressing the proliferation, migration and invasion of ASMCs.

Suppression of circular RNA circDHCR24 alleviates aortic smooth muscle cell proliferation and migration by targeting miR-149-5p/MMP9 axis

Circular RNAs (circRNAs) are involved in many courses of atherosclerosis and coronary artery disease (CHD). However, the role and effect of circRNAs in vascular restenosis after PCI remains unclear. Human aortic vascular smooth muscle cell (HA-VSMC) was cultured and stimulated with PDGF-BB. The expression profile of circRNAs in HA-VSMCs was screened using microarray analysis. A total 257 aberrantly expressed circRNAs were screened with 2 fold change. Has_circ_0113656 (also called circDHCR24) was validated by qRT-PCR to be significantly up-regulated in PDGF-BB induced HA-VSMCs. CircDHCR24 silencing obviously inhibited the proliferation, migration and phenotypic switch. Moreover, bioinformatics analysis predicted that miR-149-5p had complementary binding sites in 3′-UTR of circDHCR24. Luciferase reporter assay and RIP assay further verified the circDHCR24 acts as a spong for miR-149-5p in HA-VSMCs. Besides, bioinformatics analysis, luciferase reporter assay and RIP assay proved MMP9 was a directly target of miR-149-5p. Finally, cells were transfected with si-circDHCR24 with or without miR-149 inhibitor, and the results showed that co-transfection si-circDHCR24 and miR-149 inhibitor reversed the effect of si-circDHCR24 on cell proliferation, migration and phenotypic switch. Taken together, our study suggested for the first time that the knockdown of circDHCR24 alleviates HA-VSMCs proliferation, migration and phenotypic switching, thereby preventing vascular restenosis.

Downregulation of miR‑142‑5p inhibits human aortic smooth muscle cell proliferation and migration by targeting MKL2.

The increased proliferation and migration of vascular smooth muscle cells (VSMCs) are critical in the progression of atherosclerosis (AS). Platelet-derived growth factor type BB (PDGF-BB) may induce VSMC proliferation and migration. miR-142-5p plays a critical role in various biological processes, including tumorigenesis, angiogenesis and inflammation. However, whether miR-142-5p is involved in regulating the pathological process of arteriosclerosis remains to be elucidated. Therefore, in this study, the role of miR-142-5p in PDGF-BB-induced human aortic smooth muscle cell (HSAMC) proliferation and migration was investigated. The results revealed that the expression level of miR-142-5p was enhanced in the serum of patients with AS, while that of its target gene, myocardin-like protein 2 (MKL2) was decreased, compared with that in healthy volunteers. Moreover, there was a negative correlation between miR-142-5p and MKL2 expression in the serum of patients with AS. Furthermore, the downregulation of miR-142-5p inhibited PDGF-BB-induced HASMC proliferation and migration; however, the inhibition of HASMC proliferation and migration was reversed by co-transfection with small interfering RNA (siRNA) against MKL2 (siRNA-MKL2). In addition, transfection with miR-142-5p inhibitor significantly increased the expression levels of MKL2, and decreased those of matrix metalloproteinase (MMP)2 and 9, and these effects were reversed by transfection with siRNA-MKL2. Finally, MKL2 was proven to be a target of miR-142-5p. On the whole, the findings of the present study demonstrate that the downregulation of miR-142-5p inhibits human aortic smooth muscle cell (HSAMC) proliferation and migration possibly by targeting MKL2. Hence, miR-142-5p may prove to be a novel therapeutic target in the treatment of AS.

Securinine protects vascular dysfunction targeting atherosclerosis in vascular endothelial cells, smooth muscle cells, and apolipoprotein E deficient mice

Atherosclerosis is a chronic vascular disease and characterized by accumulation within the intima of inflammatory cells, smooth muscle cells, lipid, and connective tissue. Here, we suggest securinine as an anti‐atherosclerotic agent to attenuate vascular dysfunction and lesion. To identify natural agents that commonly reverse advanced atherosclerotic plaque to early atherosclerotic plaque, significantly regulated genes were analyzed in silico. The differentially expressed genes from 9 intimal thickening and 8 fibrous cap atheroma tissue which were collected from GEO data were assessed by the connectivity map. Natural candidate securinine, a main compound from Securinega suffruticosa, was selected and administrated 1, 5 mg/kg/day in apolipoprotein‐E‐deficient (ApoE KO) mice for 18 weeks. Securinine significantly showed lowered blood pressure and improvement of metabolic parameters with hyperlipidemia. The impairment in vasorelaxation was remarkably decreased by treatment with securinine. H&amp;E staining revealed that treatment with securinine reduced atherosclerotic lesions. Securinine suppressed the expression of adhesion molecules and matrix metalloproteinase‐2/‐9 in both ApoE KO and vascular endothelial cells (HUVEC). In HUVEC pretreatment with securinine significantly inhibited ROS generation and NF‐κB activation. Growth curve assays using the real‐time cell analyzer showed that securinine significantly decreased TNF‐α‐induced aortic smooth muscle cell proliferation and migration in a dose‐dependent manner. Thus securinine may be a potential natural candidate for the treatment of atherosclerosis because it attenuates vascular inflammation and dysfunction as well as vascular lesion.Support or Funding InformationThis work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (2017R1A5A2015805)(2017R1D1A3B03029089) (2019R1A2C1085650).

Sirtuin 1 reduces hyaluronan synthase 2 expression by inhibiting nuclear translocation of NF-κB and expression of the long-noncoding RNA HAS2–AS1

Hyaluronan (HA) is one of the most prevalent glycosaminoglycans of the vascular extracellular matrix (ECM). Abnormal HA accumulation within blood vessel walls is associated with tissue inflammation and is prominent in most vascular pathological conditions such as atherosclerosis and restenosis. Hyaluronan synthase 2 (HAS2) is the main hyaluronan synthase enzyme involved in HA synthesis and uses cytosolic UDP-glucuronic acid and UDP-GlcNAc as substrates. The synthesis of UDP-glucuronic acid can alter the NAD+/NADH ratio via the enzyme UDP-glucose dehydrogenase, which oxidizes the alcohol group at C6 to the COO− group. Here, we show that HAS2 expression can be modulated by sirtuin 1 (SIRT1), the master metabolic sensor of the cell, belonging to the class of NAD+-dependent deacetylases. Our results revealed the following. 1) Treatments of human aortic smooth muscle cells (AoSMCs) with SIRT1 activators (SRT1720 and resveratrol) inhibit both HAS2 expression and accumulation of pericellular HA coats. 2) Tumor necrosis factor α (TNFα) induced HA-mediated monocyte adhesion and AoSMC migration, whereas SIRT1 activation prevented immune cell recruitment and cell motility by reducing the expression levels of the receptor for HA-mediated motility, RHAMM, and the HA-binding protein TNF-stimulated gene 6 protein (TSG6). 3) SIRT1 activation prevented nuclear translocation of NF-κB (p65), which, in turn, reduced the levels of HAS2–AS1, a long-noncoding RNA that epigenetically controls HAS2 mRNA expression. In conclusion, we demonstrate that both HAS2 expression and HA accumulation by AoSMCs are down-regulated by the metabolic sensor SIRT1.