Viability Of Vascular Smooth Muscle Cells Research Articles

Wogonin attenuates vascular remodeling by inhibiting smooth muscle cell proliferation and migration in hypertensive rat.

Wogonin, extracted from the roots of Scutellaria baicalensis Georgi, has been shown to suppress collagen deposition in spontaneously hypertensive rats (SHRs). This study was performed to investigate the role and mechanism of wogonin underlying vascular remodeling in SHRs. After injection of SHRs with 40 mg/kg of wogonin, blood pressure in rats was measured once a week. Masson's trichrome staining was conducted to observe the changes in aortas and mesenteric arteries. Vascular smooth muscle cells (VSMCs) isolated from rat thoracic aortas were treated with Angiotensin II (Ang II; 100 nM) in the presence or absence of varying concentrations of wogonin. The viability and proliferation of VSMCs were examined using Cell Counting Kit-8 assay and 5-ethynyl-2'-deoxyuridine assay, respectively. The migration of VSMCs was examined using wound healing assay and transwell assay. We found that wogonin administration alleviated hypertension, increased lumen diameter, and reduced the thickness of the arterial media in SHRs. Ang II treatment enhanced the viability of VSMCs, which was inhibited by wogonin in a concentration-dependent manner. Wogonin reversed Ang II-induced increases in the viability, proliferation, and migration of VSMCs. Moreover, wogonin inhibited Ang II-induced activation of mitogen-activated protein kinase (MAPK) signaling in VSMCs. Overall, wogonin repressed the proliferative and migratory capacity of VSMCs by regulating the MAPK signaling pathway, thereby attenuating vascular remodeling in hypertensive rats, indicating that wogonin might be a therapeutic agent for the treatment of vascular diseases.

Knockdown of LINC01128 Downregulates FUT8 to Inhibit OxLDL-Induced Vascular Smooth Muscle Cell Apoptosis in Atherosclerosis.

The apoptosis of vascular smooth muscle cells (VSMCs) contributes to the progression of atherosclerosis (AS). Long intergenic non-protein coding RNA 1128 (LINC01128) has been implicated in AS, and this study aims to uncover the role and mechanism of LINC01128 in regulating oxidized low-density lipoprotein (oxLDL)-induced VSMCs. The position of LINC01128 in cells and its target genes were predicted using bioinformatics. The localization of LINC01128 in human VSMCs was determined through fluorescence in situ hybridization. VSMCs were transfected, and the interaction between LINC01128 and fucosyltransferase 8 (FUT8) was validated by chromatin immunoprecipitation assay. The apoptotic VSMC model was established using oxLDL. LINC01128 expression in VSMCs was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR), and FUT8 expression was detected by qRT-PCR and western blot. VSMC viability, migration, invasion abilities, and apoptosis were assessed using cell counting kit-8, transwell assay, and flow cytometry, respectively. OxLDL (200 μg/mL) upregulated the expression of LINC01128 and FUT8 mRNA, as well as FUT8 protein, in VSMCs. LINC01128 was expressed in the nucleus of VSMCs and bound to FUT8. Knockdown of LINC01128 alleviated the inhibitory effects of oxLDL (200 μg/mL) on viability, migration, and invasion, and mitigated the promotion of apoptosis and FUT8 expression in VSMCs. On the other hand, FUT8 overexpression enhanced the suppressive effects of oxLDL (200 μg/mL) on viability, migration, and invasion activities, and amplified the facilitating effect of oxLDL on apoptosis in VSMCs. Moreover, FUT8 overexpression reversed the impact of LINC01128 silencing on viability, migration, invasion, and apoptosis in oxLDL-stimulated VSMCs. The knockdown of LINC01128 downregulates FUT8, inhibiting the progression of VSMCs in AS.

Longitudinal change of serum exosomal miR-186-5p estimates major adverse cardiac events in acute myocardial infarction patients receiving percutaneous coronary intervention.

Our recently published study discovers that exosomal microRNA (miR)-186-5p promotes vascular smooth muscle cell viability and invasion to facilitate atherosclerosis. This research aimed to explore the prognostic implication of serum exosomal miR-186-5p in acute myocardial infarction (AMI) patients receiving percutaneous coronary intervention (PCI). One hundred and fifty AMI patients receiving PCI and 50 healthy controls (HCs) were screened. Serum exosomal miR-186-5p was detected by reverse transcriptase-quantitative polymerase chain reaction assay in AMI patients at admission and after PCI, as well as in HCs after enrollment. Major adverse cardiac events (MACE) were recorded during follow-up in AMI patients receiving PCI. Serum exosomal miR-186-5p was raised in AMI patients vs. HCs (P < 0.001). Besides, serum exosomal miR-186-5p was positively linked to body mass index (P = 0.048), serum creatinine (P = 0.021), total cholesterol (P = 0.029), and C-reactive protein (P = 0.018); while it was reversely linked with estimated glomerular filtration rate (P = 0.023) in AMI patients. Interestingly, serum exosomal miR-186-5p was correlated with the diagnosis of ST-segment elevation myocardial infarction (P = 0.034). Notably, serum exosomal miR-186-5p was decreased after PCI vs. at admission (P < 0.001). The 6-, 12-, 18-, and 24-month accumulating MACE rates were 4.5%, 8.9%, 14.8%, and 14.8% in AMI patients. Furthermore, serum exosomal miR-186-5p ≥3.39 (maximum value in HCs) after PCI (P = 0.021) and its decrement percentage <median (35%) decrement (P = 0.044) estimated elevated MACE in AMI patients. Serum exosomal miR-186-5p is reduced after PCI, and its post-PCI high level or minor decrease estimates increased MACE risk in AMI patients.

Examining the Effects of Dasatinib, Sorafenib, and Nilotinib on Vascular Smooth Muscle Cells: Insights into Proliferation, Migration, and Gene Expression Dynamics.

Dasatinib, nilotinib, and sorafenib are clinically proven tyrosine kinase inhibitors (TKIs) used for the treatment of leukemia and hepatocellular carcinoma. However, there is a growing concern regarding cardiotoxicity associated with their use. The impact of these TKIs on vascular smooth muscle cells (VSMCs) remains unexplored. This study aims to investigate the effects of TKIs on VSMC proliferation and migration, as well as to elucidate the underlying mechanisms involving inflammatory and apoptotic pathways. VSMCs were extracted from albino rats and cultured in vitro. The cells were divided into four experimental groups: control, dasatinib, sorafenib, and nilotinib. The MTT assay was employed to assess the cytotoxic effects of TKIs on VSMCs. A scratch assay was conducted to evaluate the inhibitory potential of TKIs on VSMC migration. Flow cytometry analysis was used to detect apoptotic cells. Real-Time PCR expression was utilized to determine the differential gene expression of apoptotic and inflammatory markers. Dasatinib, nilotinib, and sorafenib demonstrated significant inhibitory effects on VSMC viability and migration at low concentrations (<1 µmol/L, p < 0.05). Furthermore, gene expression analysis revealed up-regulation of inflammatory biomarkers (TNF-α, IL-6, and IL-1β) and apoptotic markers (P53, BAX), along with down-regulation of the anti-apoptotic biomarker BCL-2 in response to all TKIs. This study demonstrates that dasatinib, nilotinib, and sorafenib inhibit VSMC proliferation and migration, suggesting their potential to induce vascular injury and remodeling by activating inflammation and apoptosis pathways. These findings highlight the need for further investigation into the cardiotoxic effects of these TKIs and the development of strategies to mitigate their adverse vascular effects.

Maximakinin reduced intracellular Ca2+ level in vascular smooth muscle cells through AMPK/ERK1/2 signaling pathways.

We detect the antihypertensive effects of maximakinin (MK) on renal hypertensive rats (RHRs) and further research the influence of MK on vascular smooth muscle cells (VSMCs) to explore its hypotensive mechanism. The effects of MK on arterial blood pressure were observed in RHRs. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assays were performed to detect the effect of MK on VSMC viability. Western blot and flow cytometry were used to investigate the influence of MK on intracellular Ca2+ levels and protein expression changes in VSMCs. In addition, specific protein inhibitors were applied to confirm the involvement of Ca2+-related signaling pathways induced by MK in VSMCs. MK showed a more significant antihypertensive effect than bradykinin in RHRs. MK significantly decreased intracellular Ca2+ concentrations. Furthermore, MK significantly induced the phosphorylation of signaling molecules, including extracellular signal-regulated kinase 1/2 (ERK1/2), P38, AMP-activated protein kinase (AMPK) and Akt in VSMCs. Moreover, only ERK1/2 inhibitor U0126 and AMPK inhibitor Compound C completely restored the decreased intracellular Ca2+ level induced by MK, and further research demonstrated that AMPK functioned upstream of ERK1/2 following exposure to MK. Finally, HOE-140, an inhibitor of the bradykinin B2 receptors (B2Rs), was applied to investigate the potential targets of MK in VSMCs. HOE-140 significantly blocked the AMPK/ERK1/2 pathway induced by MK, suggesting that the B2Rs might play an important role in MK-induced AMPK and ERK1/2 activation. MK significantly reduces blood pressure in RHRs. MK exerts its antihypertensive effect by activating the B2Rs and downstream AMPK/ERK1/2 pathways, leading to significantly reduced Ca2+ levels in VSMCs.

Sarsasapogenin blocks ox-LDL-stimulated vascular smooth muscle cell proliferation, migration, and invasion through suppressing STIM1 expression.

Atherosclerosis (AS) is a pathological vascular disorder responsible for the majority of cardiovascular deaths. Sarsasapogenin (Sar) is a natural steroidal compound which has been extensively applied to multiple human diseases due to its pharmacological properties. In the present paper, the impacts of Sar on oxidized low-density lipoprotein (ox-LDL)-treated vascular smooth muscle cells (VSMCs) and its possible action mechanism were investigated. Firstly, Cell Counting Kit-8 (CCK-8) estimated the viability of VSMCs following treatment with ascending doses of Sar. Then, VSMCs were treated by ox-LDL to stimulate an in vitro cell model of AS. CCK-8 and 5-Ethynyl-2'-deoxyuridine (EDU) assays were used to assess cell proliferation. Wound healing and transwell assays were applied to measure the migratory and invasive capacities, respectively. The expression of proliferation-, metastasis-, and stromal interaction molecule 1 (STIM1)/Orai signaling-associated proteins was measured by western blot. The experimental data illuminated that Sar treatment noticeably protected against ox-LDL-elicited VSMCs proliferation, migration, and invasion. Besides, Sar lowered the elevated STIM1 and Orai expression in ox-LDL-treated VSMCs. Further, STIM1 elevation partially abrogated the impacts of Sar on the proliferation, migration, and invasion of VSMCs challenged with ox-LDL. In conclusion, Sar might reduce STIM1 expression to impede the aggressive phenotypes of ox-LDL-treated VSMCs.

Sulfasalazine induces autophagy inhibiting neointimal hyperplasia following carotid artery injuries in mice.

Background: Neointimal hyperplasia (NH) is a crucial pathophysiological feature in vascular transplant and in-stent restenosis. Excessive proliferation and migration of vascular smooth muscle cells (VSMCs) play important roles in neointimal hyperplasia. This study aims to explore the potentialities and mechanism of sulfasalazine (SSZ) in the prevention of restenosis. Methods: Sulfasalazine was encapsulated in nanoparticles made of poly (lactic-co-glycolic acid) (PLGA). In vivo, carotid ligation injury was induced in mice to induce Neointimal hyperplasia, with or without sulfasalazine containing nanoparticles (NP-SSZ) treatment. After 4weeks, the arteries were collected for histology, immunofluorescence, Western blotting (WB) and qRT-PCR. In vitro, vascular smooth muscle cells were treated with TNF-α to induce cell proliferation and migration, followed by SSZ or vehicle treatment. WB was performed to further explore its mechanism. Results: The ratio of intima to media thickness (I/M) was increased after ligation injury on day 28, while the ratio was significantly reduced in the NP-SSZ treatment group. The dual positive nuclei of Ki-67 and α-SMA were 47.83% ± 9.15%, whereas only 29.83% ± 5.98% in the NP-SSZ-treated group (p < 0.05). Both MMP-2 and MMP-9 were decreased in the NP-SSZ treatment group (p < 0.05, p < 0.05, respectively) compared to the control group. The levels of the targeted inflammatory genes (TNF-α, VCAM-1, ICAM-1, MCP-1) were lower in the NP-SSZ treatment group compared with the control group. In vitro, the proliferating cell nuclear antigen (PCNA) expression was significantly decreased in the SSZ treatment group. The cell viability of VSMCs was markedly increased in the TNF-α treatment group, whereas sulfasalazine treatment inhibited this effect. LC3 II and P62 protein expression were higher in the SSZ group than in the vehicle group both in vitro and in vivo. The phosphorylation of NF-kB (p-NF-kB) and the phosphorylation of mTOR (p-mTOR) were decreased in the TNF-α+ SSZ group, whereas the P62 and LC3 II expression levels were increased. However, the expression level of p-mTOR, P62, and LC3 II was reversed after co-treatment with the agonist of mTOR MHY1485, whereas the p-NF-kB expression level was unchanged. Conclusion: sulfasalazine inhibited vascular smooth muscle cells proliferation and migration in vitro and Neointimal hyperplasia in vivo through NF-kB/mTOR-mediated autophagy.

MALT1 accelerates proatherogenic vascular smooth muscle cell growth, invasion and synthetic phenotype switching via nuclear factor‑κB signaling‑dependent way.

Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) modulates T helper cell differentiation and nuclear factor-κB (NF-κB) pathway-mediated inflammation and potentially regulates lipid metabolism, which are all critical factors involved in atherosclerosis. The present study aimed to investigate the effect of MALT1 on the cellular functions of proatherogenic vascular smooth muscle cells (VSMCs). Therefore, to establish a human proatherogenic VSMC model, VSMCs were treated with different doses of oxidized low-density lipoprotein (oxLDL). Subsequently, the effect of MALT1 overexpression or knockdown in proatherogenic VSMCs treated with or without NF-κB activator was also explored. The results showed that treatment of proatherogenic VSMCs with oxLDL significantly elevated the mRNA and protein expression levels of MALT1 in a dose-dependent manner. Furthermore, MALT1 overexpression enhanced cell viability, invasion and phenotype switching and reduced apoptosis in proatherogenic VSMCs. However, MALT1 knockdown exerted the opposite effect on the above cellular functions. Additionally, the results revealed that MALT1 could positively regulate the NF-κB pathway in proatherogenic VSMCs. Moreover, treatment of proatherogenic VSMCs with NF-κB activator not only exacerbated the dysregulation of cellular functions, but also hampered the effect of MALT1 knockdown on attenuating cell growth, invasion and synthetic phenotype switching, thus suggesting that NF-κB was essential for the regulation of MALT1-triggered functions in proatherogenic VSMCs. In conclusion, the current study suggested that MALT1 could exacerbate cell viability, mobility and synthetic phenotype switching of proatherogenic VSMCs in a NF-κB signaling-dependent manner. Therefore, MALT1 could be considered as a potential therapeutic target for atherosclerosis.

Poly(octamethylene citrate) Modified with Glutathione as a Promising Material for Vascular Tissue Engineering

One of the major goals of vascular tissue engineering is to develop much-needed materials that are suitable for use in small-diameter vascular grafts. Poly(1,8-octamethylene citrate) can be considered for manufacturing small blood vessel substitutes, as recent studies have demonstrated that this material is cytocompatible with adipose tissue-derived stem cells (ASCs) and favors their adhesion and viability. The work presented here is focused on modifying this polymer with glutathione (GSH) in order to provide it with antioxidant properties, which are believed to reduce oxidative stress in blood vessels. Cross-linked poly(1,8-octamethylene citrate) (cPOC) was therefore prepared by polycondensation of citric acid and 1,8-octanediol at a 2:3 molar ratio of the reagents, followed by in-bulk modification with 0.4, 0.8, 4 or 8 wt.% of GSH and curing at 80 °C for 10 days. The chemical structure of the obtained samples was examined by FTIR-ATR spectroscopy, which confirmed the presence of GSH in the modified cPOC. The addition of GSH increased the water drop contact angle of the material surface and lowered the surface free energy values. The cytocompatibility of the modified cPOC was evaluated in direct contact with vascular smooth-muscle cells (VSMCs) and ASCs. The cell number, the cell spreading area and the cell aspect ratio were measured. The antioxidant potential of GSH-modified cPOC was measured by a free radical scavenging assay. The results of our investigation indicate the potential of cPOC modified with 0.4 and 0.8 wt.% of GSH to produce small-diameter blood vessels, as the material was found to: (i) have antioxidant properties, (ii) support VSMC and ASC viability and growth and (iii) provide an environment suitable for the initiation of cell differentiation.

MgCl2 Attenuates ox-LDL-Induced Vascular Smooth Muscle-Derived Foam Cells Pyroptosis by Downregulating the TLR4/NF-κB Signaling Pathway.

Pyroptosis is a type of programmed cell death that is generally upregulated during atherosclerosis (AS). Magnesium, an important cation in the body, has exhibited an antiatherosclerotic effect. We collected AS model datasets from the Gene Expression Omnibus (GEO) and explored the correlation between pyroptosis and AS through a series of bioinformatics methods. We next investigated the impact of oxidized low-density lipoprotein (ox-LDL) on primary cultured vascular smooth muscle cells (VSMCs) foaminess and pyroptosis. Finally, foam cells were preconditioned with different concentrations of MgCl2 to explore its influence on ox-LDL-induced VSMCs pyroptosis. NLRP3-mediated pyroptosis plays a core role in regulating AS progression as shown by bioinformatic analysis. Ox-LDL (50/75/100mg/L) increased CE/TE ratio (> 50%) in VSMCs and prompted VSMC-derived foam cell formation, and (75/100mg/L) ox-LDL-induced pyroptosis. Compared to 1mmol/L MgCl2, 10mmol/L MgCl2 significantly downregulated the expression of pyroptosis related molecules in VSMCs induced by 75mg/L ox-LDL, including NLRP3, ASC, caspase-1, and GSDMD. The secretion of IL-1β, IL-18, and LDH was also inhibited by MgCl2. According to CCK-8 and Hoechst 33,342/PI staining, the damage to VSMCs viability induced by ox-LDL was ameliorated by MgCl2. In addition, MgCl2 attenuated the upregulation of TLR4, IKKβ, and p65 and the downregulation of IκBα in VSMCs induced by ox-LDL. The present study demonstrated that pyroptosis-related genes were the core genes in AS. We also revealed the effect and underlying mechanism of MgCl2 on ox-LDL-induced VSMCs pyroptosis, suggesting that MgCl2 has promising clinical applications for AS pyroptosis prevention and treatment.

Clinical Value of Long Non-Coding RNA HOTAIR in Carotid Artery Stenosis and Its Role in Vascular Smooth Muscle Cell Proliferation.

This study investigated the clinical role of HOTAIR in patients with carotid artery stenosis (CAS) and its mechanism in vascular smooth muscle cells (VSMCs). Patients with CAS were collected. The expression of HOTAIR was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The clinical importance of HOTAIR was revealed by the receiver operating characteristic curve. Overexpression and knockdown of HOTAIR were achieved by transfecting pCDNA3.1-HOTAIR plasmid and si-HOTAIR respectively. CCK-8 assay or Transwell assay were used to analyze the changes in cell viability or migration after transfection treatments. Double luciferase reporter gene assay confirmed the targeted relationship between HOTAIR and miR-148b-3p. The levels of miR-148b-3p in VSMCs and patients were detected by qRT-PCR. Pearson analysis was used to analyze the relationship between HOTAIR and miR-148b-3p in patients with CAS. The expression of HOTAIR in patients with CAS was significantly higher than that in healthy individuals. HOTAIR appeared to discriminate CAS patients from healthy people. The overexpression of HOTAIR increased the viability and migration of VSMCs. Silenced HOTAIR restricted the abnormal viability and migration of VSMCs. A double luciferase reporter revealed a region of complementary binding between HOTAIR and miR-148b-3p. The expression of miR-148b-3p in VSMCs was regulated by the levels of HOTAIR. Reduction of miR-148b-3p expression was substantiated in CAS patients. Pearson analysis exhibited that the expression of HOTAIR was negatively relative to the levels of miR-148b-3p. The long noncoding RNA HOTAIR might be a diagnostic biomarker for CAS patients, and it was involved in the activity of vascular smooth muscle cells.

MiR-7-5p attenuates vascular smooth muscle cell migration and intimal hyperplasia after vascular injury by NF-kB signaling

BackgroundAtherosclerosis (AS) is the primary cause of coronary artery disease, which is featured by aberrant proliferation, differentiation, and migration of vascular smooth muscle cells (VSMCs). MicroRNAs play crucial roles in AS, but the function of miR-7-5p in AS remains unclear. Here, we aimed to explore the effect of miR-7-5p on AS and VSMCs in vitro and in vivo. MethodsThe in vivo rat AS model and apoE−/− mouse model were established. The carotid artery injury was checked by immunohistochemistry staining. The RNA levels of miR-7-5p and p65 were measured by qPCR assay. Protein levels were checked by western blotting. Cell apoptosis was evaluated by flow cytometry. Cell migration was checked by Transwell assay and wound healing assay. The potential interaction between miR-7-5p with p65 was checked by luciferase reporter gene assay. ResultsMiR-7-5p was downregulated and NF-κB p65 was upregulated in injured carotid arteries in rat model. The carotid artery injury in the AS rats and the treatment of miR-7-5p attenuated the phenotype in the model. Immunohistochemistry staining and Western blot analysis revealed that PCNA levels were increased in injured carotid arteries of the model rats and miR-7-5p could reverse the levels. The cell viability of VSMCs was induced by PDGF-BB but miR-7-5p blocked the phenotype. PDGF-BB decreased apoptosis of VSMCs, while miR-7-5p was able to restore the cell apoptosis in the model. PDGF-BB-induced migration of VSMCs was attenuated by miR-7-5p. miR-7-5p mimic remarkably repressed the luciferase activity of p65 in VSMCs. The levels of p65 were inhibited by miR-7-5p in the cells. The PDGF-BB-promoted cell viability and migration of VSMCs was repressed by miR-7-5p and p65 overexpression reversed the phenotype. ConclusionWe concluded that miR-7-5p attenuates vascular smooth muscle cell migration and intimal hyperplasia after vascular injury by NF-kB signaling.

The Protective Effect of miRNA-146a Liposome Nanoparticles on Vascular Smooth Muscle Cells After Coronary Intervention

The abnormal expression of miRNA-146a is related to the progression of coronary arteries. This study intends to explore the protective effect of miRNA-146a on vascular smooth muscle cells (VSMCs) after coronary intervention and the related mechanism. 10 miniature pigs were randomly assigned into control group, model group, blank group, miRNA-146a group, cilostazol group, and STAT3 signaling agonist group followed by analysis of the morphology and viability of VSMCs, expression of miRNA-146a, STAT3, NF-kB, TNF-a, IL-6, and AT-1R as well as the relationship between miR-146a and STAT3. The BNP (192.39±12.32) pg/ml and cTnI (14.20±2.12)μg/L of model group were significantly higher than those of control group (P&lt; 0.05). miRNA-146a level was highest in miRNA-146a group and cilostazol group, while lower in other two groups with the lowest level in agonist group (P&lt;0.05). The cell viability and AngII level of miRNA-146a group and cilostazol group were lower, and higher in the other two groups with highest level in pathway agonist group (P&lt; 0.05). miRNA-146a group and cilostazol group showed lower expressions of STAT3, NF-kB, TNF-a, IL-6, AT-1R than the other two groups. The pathway agonist group showed significantly higher level than blank group (P&lt;0.05). liposome nanoparticles carrying miRNA-146a inhibited the activity of STAT3 signaling, down-regulated the levels of downstream factors including TNF-a, IL-6, and TNF-a and subsequently decreased AngII and AT-1R levels, therefore playing a protective effect on VSMCs after coronary intervention.

MiR-15a-5p accelerated vascular smooth muscle cells viabilities and migratory abilities via targeting Bcl-2.

Aortic dissection (AD) caused by the tear in the aortic wall threatens aorta, causing severe chest pain, syncope and even death. Fortunately, development of genetic technology provides promising approaches for AD treatment. To analyze impacts of miR-15a-5p on modulating cell viability and migratory ability of vascular smooth muscle cells (VSMCs). Ang II (0, 0.05 and 0.1 microM) treatment were applied for inducing inflammatory reactions of VSMCs. RNA expressions of miR-15a-5p with Bcl-2 was examined using RT-qPCR. CCK-8 and transwell evaluated cell viability and migratory ability, respectively. The binding about miR-15a-5p with Bcl-2 were detected by luciferase reporter assay. Western blot detected protein expressions of Bcl-2, MCP-1 and MMP-9. Ang II treatment not only accelerated VSMCs viability and migratory abilities, but also upregulated MCP-1 and MMP-9 protein expressions. MiR-15a-5p was detected to be promoted by Ang II. However, miR-15a-5p inhibitor decreased VSMC cell viability and migratory ability and suppressed protein expressions of MCP-1 and MMP-9. Bcl-2 was targeted and downregulated by miR-15a-5p. Nevertheless, high VSMC cell viability and migration caused by miR-15a-5p overexpression were hindered with overexpressed Bcl-2. MiR-15a-5p mimics also elevated MCP-1 and MMP-9 protein expressions, which were inhibited by Bcl-2 upregulation.

Hsa_circRNA_0008028 Deficiency Ameliorates High Glucose-Induced Proliferation, Calcification, and Autophagy of Vascular Smooth Muscle Cells via miR-182-5p/TRIB3 Axis.

Background It is well-known that dysfunctions of vascular smooth muscle cells (VSMCs) act an essential part in vascular complications of diabetes. Studies have shown that circular RNAs (circRNAs) and microRNAs (miRNAs) play a crucial role in regulating cell functions. However, their influence on the proliferation, calcification, and autophagy of VSMCs remains to be further explored. Therefore, this study elucidates the role and mechanism of hsa_circRNA_0008028 in high glucose- (HG-, 30 mM) treated VSMCs in vitro. Methods Quantitative real-time polymerase chain reaction (qRT-PCR) was chosen to detect the levels of hsa_circRNA_0008028, miR-182-5p, and tribble 3 (TRIB3). Then, dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were used to predict and verify the binding relationship between miR-182-5p and hsa_circRNA_0008028 or TRIB3. Cell counting kit-8 assay, 5-ethynyl-2′-deoxyuridine (EdU) staining, corresponding commercial kits, and western blotting were used to measure indexes reflecting cell viability, proliferation, calcification, and autophagy of VSMCs, respectively. Results In HG-induced VSMCs, hsa_circRNA_0008028 and TRIB3 were highly expressed, whereas miR-182-5p decreased. Meanwhile, cell proliferation, calcification, and autophagy could be repressed by silencing of hsa_circRNA_0008028. However, these effects can be eliminated by miR-182-5p inhibition. Furthermore, it was demonstrated that hsa_circRNA_0008028 could promote the expression of TRIB3, a target of miR-182-5p, by directly sponging miR-182-5p. The expression of TRIB3 was suppressed by hsa_circRNA_0008028 knockout, which was rescued by miR-182-5p inhibition. Conclusion This study reveals that hsa_circRNA_0008028 can act as a sponge of miR-182-5p and promote HG-induced proliferation, calcification, and autophagy of VSMCs partly by regulating TRIB3.

Long Noncoding RNA SBF2-AS1 Promotes Abdominal Aortic Aneurysm Formation through the miRNA-520f-3p/SMARCD1 Axis.

Abdominal aortic aneurysm (AAA) is a chronic vascular inflammatory disease. The regulatory mechanisms during AAA formation remain unclear. Bone marrow stem cells (BMSCs) are pluripotent cells capable of regulating the progression of various diseases by delivering exosomes and exosomal lncRNAs. In this study, we investigated its function in AAA by isolating BMSC exosome-derived lncRNA SBF2-AS1. The results showed that BF2-AS1 could be transferred to vascular smooth muscle cells (VSMCs) and human aortic VSMCs (HASMCs) via BMSC-derived exosomes. Depletion of SBF2-AS1 enhanced the cell viability and proliferation of VSMCs. Conversely, SBF2-AS1 knockdown inhibited VSMC apoptosis. Caspase-3 activity was inhibited by depletion of SBF2-AS1, whereas overexpression of SBF2-AS1 in VSMC promoted Caspase-3 activity. SBF2-AS1 enhances SMARCD1 expression by forming miR-520f-3p in VSMC and HASMC. Overexpression of SMARCD1 or miR-520f-3p inhibitor reversed cell viability and caspase-3 activity mediated by SBF2-AS1 depletion in VSMC and HASMC. Therefore, BMSC exosome-derived SBF2-AS1 promotes AAA formation through the miRNA-520f-3p/SMARCD1 axis. Targeting SBF2-AS1 could serve as a promising therapeutic strategy for AAA.

MicroRNA-4487 regulates vascular smooth muscle cell proliferation, migration and apoptosis by targeting RAS p21 protein activator 1

BackgroundDysregulation of microRNA (miRNA) is involved in the pathogenesis of a variety of diseases, including atherosclerosis (AS). However, the role of miRNA-4487 (miR-4487) in the development of AS is not fully clarified. This study is intended to investigate the regulatory effects of miR-4487 on the proliferation, migration and apoptosis of vascular smooth muscle cells (VSMCs) and the related mechanisms. MethodsOxidized low-density lipoprotein (ox-LDL) was employed to induce the dysfunction of VSMCs. Subsequently, miR-4487 expression was detected by quantitative real-time PCR. Afterward, the expression levels of RAS p21 protein activator 1 (RASA1) and apoptosis-related proteins (Bcl-2, Bax, Cleaved caspase 3, Cleaved caspase 9) were detected by Western blotting. The proliferation, migration and apoptosis of VSMCs were then detected by CCK-8, BrdU, Transwell and flow cytometry assays, respectively. Moreover, a dual-luciferase reporter gene assay was executed to verify the targeting between miR-4487 to the RASA1 3′-untranslated region (3′-UTR). Resultsox-LDL treatment increased miR-4487 expression and decreased RASA1 expression in VSMCs. Additionally, ox-LDL treatment promoted the proliferation and migration of VSMCs, but inhibited apoptosis. Besides, the effects of ox-LDL treatment on the proliferation, migration and apoptosis of VSMCs were attenuated by the transfection of miR-4487 inhibitors. Furthermore, miR-4487 directly targeted the 3′-UTR of RASA1 mRNA and repressed the expression level of RASA1. Also, RASA1 knockdown reversed the effects of miR-4487 inhibition on VSMCs. ConclusionMiR-4487 promotes VSMCs viability and migration and inhibits apoptosis by targeting RASA1 in VSMCs, by which it promotes the pathogenesis of AS.

OxLDL-stimulated macrophage exosomes promote proatherogenic vascular smooth muscle cell viability and invasion via delivering miR-186–5p then inactivating SHIP2 mediated PI3K/AKT/mTOR pathway

The current study aimed to investigate the implication of microRNA (miRNA) profile in the linkage between oxidized low-density-lipoprotein (oxLDL)-stimulated-macrophages (MФ) exosomes and vascular smooth muscle cells (VSMCs) during atherosclerosis. VSMCs were treated by oxLDL-stimulated-MФ with/without GW4869. MiRNA profile in oxLDL-stimulated-MФ and untreated-MФ was detected by microarray, then candidate miRNAs were proposed to RT-qPCR and functional validation in VSMCs. MiR-186–5p mimic/inhibitor was transfected into oxLDL-stimulated-MФ, then its exosomes were used to VSMCs. Subsequently, miR-186–5p, SHIP2 and PI3K/AKT/mTOR pathway were modified alone or in combination in VSMCs. VSMCs viability, invasion and apoptosis were detected. OxLDL-stimulated-MФ induced VSMCs viability, invasion, but repressed apoptosis (all P < 0.01); while after GW4869 treatment to delete exosomes, its effect was weakened (all P < 0.05). Totally 45 dysregulated miRNAs were identified in oxLDL-stimulated-MФ versus untreated-MФ. The top-three dysregulated miRNAs (miR-186–5p, miR-21–5p, miR-320b) were elevated in VSMCs after oxLDL-stimulated-MФ treatment (all P < 0.001); while only miR-186–5p mimic greatly enhanced VSMCs viability and invasion (both P < 0.01). Furthermore, miR-186–5p-overexpressed oxLDL-stimulated-MФ exosomes promoted VSMCs viability, invasion, repressed apoptosis, while miR-186–5p-knockdown oxLDL-stimulated-MФ exosomes exhibited opposite effect (all P < 0.05). MiR-186–5p negatively regulated SHIP2 in VSMCs and bound SHIP2 via luciferase-reporter-gene assay (all P < 0.05). SHIP2 overexpression decreased VSMCs viability, invasion, PI3K/AKT/mTOR pathway, increased apoptosis, and attenuated miR-186–5p-overexpression’s effect on these functions (all P < 0.05). Besides, PI3K activator (740 Y-P) weakened SHIP2-overexpression’s effect on VSMCs viability, invasion and apoptosis (all P < 0.05). In conclusion, oxLDL-stimulated-MФ exosomes deliver miR-186–5p to inactivate SHIP2 mediated PI3K/AKT/mTOR pathway, then promote cell viability and invasion in VSMCs to accelerate atherosclerosis.

Curcumin alleviates lipopolysaccharides-induced inflammation and apoptosis in vascular smooth muscle cells via inhibition of the NF-κB and JNK signaling pathways.

Curcumin plays an important role in inflammation regulation. This study aimed to investigate the effect of curcumin on vascular smooth muscle cells (VSMCs) inflammation induced by lipopolysaccharide (LPS) and its mechanism. VSMCs were treated with different concentrations of curcumin (0, 50, 100 and 150μg/mL). MTT assay and flow cytometry were used to analyze the effects of curcumin on LPS-induced VSMCs viability and apoptosis. The expression and release of inflammatory cytokines in VSMCs were detected by real-time quantitative polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). Moreover, the proteins expressions of NF-κB and JNK signaling pathways were analyzed by western blot. Interestingly, the results showed that curcumin could reduce LPS induced inflammatory injury by increasing VSMC's viability, reducing apoptosis and inhibiting the release of inflammatory cytokines. In addition, curcumin increased the expression of Toll-like receptor 4 (TLR4) in LPS treated VSMCs. Mechanistically, we found that curcumin attenuated LPS-induced cell damage in VSMCs via inhibition of NF-κB and the JNK signal pathway. Curcumin can protect VSMCs from LPS induced inflammatory damage, which may be related to the blocking of NF-κB and the JNK signaling pathway. Herewith, curcumin could be potential therapeutics for the treatment of atherosclerosis.

Molecular mechanism of astragaloside Ⅳ against atherosclerosis by regulating miR-17-5p and PCSK9/VLDLR signal pathway

This study explores the regulatory effect of astragaloside Ⅳ on miR-17-5 p and its downstream proprotein convertase subtillisin/kexin type 9(PCSK9)/very low density lipoprotein receptor(VLDLR) signal pathway, aiming at elucidating the mechanism of astragaloside Ⅳ against atherosclerosis(AS). In cell experiment, oxidized low-density lipoprotein(ox-LDL) was used for endothelial cell injury modeling with vascular smooth muscle cells(VSMCs). Then cells were classified into the model group, miR-17-5 p inhibitor group, blank serum group, and astragaloside Ⅳ-containing serum group based on the invention. Afterward, cell viability and the expression of miR-17-5 p, VLDLR, and PCSK9 mRNA and protein in cells in each group were detected. In animal experiment, 15 C57 BL/6 mice were used as the control group, and 45 ApoE~(-/-) mice were classified into the model group, miR-17-5 p inhibitor group, and astragaloside Ⅳ group, with 15 mice in each group. After 8 weeks of intervention, the peripheral serum levels of interleukin-6(IL-6), interleukin-10(IL-10), and tumor necrosis factor-α(TNF-α), and the expression of miR-17-5 p, VLDLR, and PCSK9 mRNA in the aorta of mice were detected. The pathological changes of mice in each group were observed. According to the cell experiment, VSMC viability in the miR-17-5 p inhibitor group and the astragaloside Ⅳ-containing serum group was higher than that in the model group(P&lt;0.05). The mRNA and protein expression of miR-17-5 p and VLDLR in VSMCs in the miR-17-5 p inhibitor group and the astragaloside Ⅳ-containing serum group was lower than that in the model group(P&lt;0.05), but the mRNA and protein expression of PCSK9 was higher than that in the model group(P&lt;0.05). As for the animal experiment, the levels of IL-6 and TNF-α in the peripheral serum of the miR-17-5 p inhibitor group and the astragaloside Ⅳ group were lower(P&lt;0.05) and the serum level of IL-10 was higher(P&lt;0.05) than that of the model group. The mRNA expression of miR-17-5 p and VLDLR in the aorta in the miR-17-5 p inhibitor group and the astragaloside Ⅳ group was lower(P&lt;0.05), and PCSK9 mRNA expression was higher(P&lt;0.05) than that in the model group. Pathological observation showed mild AS in the miR-17-5 p inhibitor group and the astragaloside Ⅳ group. In summary, astragaloside Ⅳ can prevent the occurrence and development of AS. The mechanism is that it performs targeted regulation of miR-17-5 p, further affecting the PCSK9/VLDLR signal pathway, inhibiting vascular inflammation, and thus alleviating endothelial cell injury.