Human Aortic Smooth Muscle Cells Proliferation Research Articles

HMGB2 promotes smooth muscle cell proliferation through PPAR-γ/PGC-1α pathway-mediated glucose changes in aortic dissection

Background and aimsAortic dissection (AD) is a fatal condition with a complicated pathogenesis. High mobility group protein B2 (HMGB2) is a member of the high mobility group protein family; HMGB2 is involved in innate immunity and inflammatory diseases, but its role in AD remains unclear. MethodsHMGB2-/- mice were generated and treated with β-aminopropionitrile and angiotensin II (Ang II) to an establish AD model. An F12 gel containing AAV9-HMGB2 was applied to overexpress HMGB2 in mice. Pathological changes in the aorta were assessed by visualizing vascular collagen deposition and elastic fiber fracture via H&E, Masson and EVG staining. HMGB2 expression was measured by Western blotting and immunohistochemistry. The MTS, CCK-8 and EdU assays were used to test cell proliferation. ResultsHMGB2 expression was increased in samples from AD patients, samples from AD model mice and human aortic smooth muscle cells (HASMCs). HMGB2 promoted HASMC proliferation. Immunofluorescence staining and plasma membrane protein isolation revealed that HMGB2 decreased GLUT1 expression and promoted GLUT4 translocation. HMGB2 was also found to inhibit the expression of SIRT1/PGC-1α, but blocking the PPAR-γ pathway attenuated this effect. HMGB2-/- significantly reduced the incidence and mortality rates of AD, whereas treatment with AAV9-HMGB2 exacerbated AD. ConclusionsThis study suggested that HMGB2 promotes HASMC proliferation and vascular remodeling by regulating glucose metabolism through the PPAR-γ/SIRT1/PGC-1α pathway. HMGB2 knockdown reduced, whereas HMGB2 overexpression promoted, the occurrence of AD in mice. This study may help elucidate the underlying mechanisms and provide a new preventive target for AD.

Local vascular Klotho mediates diabetes-induced atherosclerosis via ERK1/2 and PI3-kinase-dependent signaling pathways

Background and aimsDiabetes is one of the major causes of cardiovascular disease (CVD). As high as 29 % of patients with diabetes develop atherosclerosis. Vascular Smooth Muscle Cells (VSMCs) are a key mediator in the pathogenesis of atherosclerosis, generating pro-inflammatory and proliferative characteristics in atherosclerotic lesions. MethodsWe used human atherosclerotic samples, developed diabetes-induced atherosclerotic mice, and generated loss of function and gain of function in Klotho human aortic smooth muscle cells to investigate the function of Klotho in atherosclerosis. ResultsWe found that Klotho expression is decreased in smooth muscle actin-positive cells in patients with diabetes and atherosclerosis. Consistent with human data, we found that Apoe knockout mice with streptozotocin-induced diabetes fed on a high-fat diet showed decreased expression of Klotho in SMCs. Additionally, these mice showed increased expression of TGF-β, MMP9, phosphorylation of ERK and Akt. Further, we utilized primary Human Aortic Smooth Muscle Cells (HASMCs) with d-glucose under dose-response and in time-dependent conditions to study the role of Klotho in these cells. Klotho gain of function and loss of function studies showed that Klotho inversely regulated the expression of atherosclerotic markers TGF-β, MMP2, MMP9, and Fractalkine.Further, High Glucose (HG) induced Akt, and ERK1/2 phosphorylation were enhanced or mitigated by endogenous Klotho deficiency or its overexpression respectively. PI3K/Akt and MAPK/ERK inhibition partially abolished the HG-induced upregulation of TGF-β, MMP2, MMP9, and Fractalkine. Additionally, Klotho knockdown increased the proliferation of HASMCs and enhanced α-SMA and TGF-β expression. ConclusionsTaken together, these results indicate that local vascular Klotho is involved in diabetes-induced atherosclerosis, which is via PI3K/Akt and ERK1/2-dependent signaling pathways.

Mechanism of salidroside in inhibiting proliferation, migration and promoting phenotypic switching of arterial smooth muscle cells

This study aims to examine the effect of salidroside(SAL) on the phenotypic switching of human aortic smooth muscle cells(HASMC) induced by the platelet-derived growth factor-BB(PDGF-BB) and investigate the pharmacological mechanism. Firstly, the safe concentration of SAL was screened by the lactate dehydrogenase release assay. HASMC were divided into control, model, and SAL groups, and the cells in other groups except the control group were treated with PDGF-BB for the modeling of phenotypic switching. Cell proliferation and migration were detected by the cell-counting kit(CCK-8) assay and Transwell assay, respectively. The cytoskeletal structure was observed by F-actin staining with fluorescently labeled phalloidine. The protein levels of proliferating cell nuclear antigen(PCNA), migration-related protein matrix metalloprotein 9(MMP-9), fibronectin, α-smooth muscle actin(α-SMA), and osteopontin(OPN) were determined by Western blot. To further investigate the pharmacological mechanism of SAL, this study determined the expression of protein kinase B(Akt) and mammalian target of rapamycin(mTOR), as well as the upstream proteins phosphatase and tensin homologue(PTEN) and platelet-derived growth factor receptor β(PDGFR-β) and the downstream protein hypoxia-inducible factor-1α(HIF-1α) of the Akt/mTOR signaling pathway. The results showed that the HASMCs in the model group presented significantly increased proliferation and migration, the switching from a contractile phenotype to a secretory phenotype, and cytoskeletal disarrangement. Compared with the model group, SAL weakened the proliferation and migration of HASMC, promoted the expression of α-SMA(a contractile phenotype marker), inhibited the expression of OPN(a secretory phenotype marker), and repaired the cytoskeletal disarrangement. Furthermore, compared with the control group, the modeling up-regulated the levels of phosphorylated Akt and mTOR and the relative expression of PTEN, HIF-1α, and PDGFR-β. Compared with the model group, SAL down-regulated the protein levels of phosphorylated Akt and mTOR, PTEN, PDGFR-β, and HIF-1α. In conclusion, SAL exerts a protective effect on the HASMCs exposed to PDGF-BB by regulating the PDGFR-β/Akt/mTOR/HIF-1α signaling pathway.

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.

High Phosphate-Induced JAK-STAT Signalling Sustains Vascular Smooth Muscle Cell Inflammation and Limits Calcification.

Vascular calcification (VC) is an age-related complication characterised by calcium-phosphate deposition in the arterial wall driven by the osteogenic transformation of vascular smooth muscle cells (VSMCs). The JAK-STAT pathway is an emerging target in inflammation. Considering the relationship between VC and inflammation, we investigated the role of JAK-STAT signalling during VSMC calcification. Human aortic smooth muscle cells (HASMCs) were cultured in high-inorganic phosphate (Pi) medium for up to 7 days; calcium deposition was determined via Alizarin staining and colorimetric assay. Inflammatory factor secretion was evaluated via ELISA and JAK-STAT members' activation using Western blot or immunohistochemistry on HASMCs or calcified aortas of Vitamin D-treated C57BL6/J mice, respectively. The JAK-STAT pathway was blocked by JAK Inhibitor I and Von Kossa staining was used for calcium deposits in murine aortic rings. During Pi-induced calcification, HASMCs released IL-6, IL-8, and MCP-1 and activated JAK1-JAK3 proteins and STAT1. Phospho-STAT1 was detected in murine calcified aortas. Blocking of the JAK-STAT cascade reduced HASMC proliferation and pro-inflammatory factor expression and release while increasing calcium deposition and osteogenic transcription factor RUNX2 expression. Consistently, JAK-STAT pathway inhibition exacerbates mouse aortic ring calcification ex vivo. Intriguingly, our results suggest an alternative link between VSMC inflammation and VC.

The SGLT2 inhibitor empagliflozin attenuates atherosclerosis progression by inducing autophagy.

Cardiovascular disease due to atherosclerosis is one of the leading causes of death worldwide; however, the underlying mechanism has yet to be defined. The sodium-dependent glucose transporter 2 inhibitor (SGLT2i) empagliflozin is a new type of hypoglycemic drug. Recent studies have shown that empagliflozin not only reduces high glucose levels but also exerts cardiovascular-protective effects and slows the process of atherosclerosis. The purpose of this study was to elucidate the mechanism by which empagliflozin ameliorates atherosclerosis. Male apolipoprotein E-deficient (ApoE-/-) mice were fed a high-fat Western diet to establish an atherosclerosis model. The area and size of atherosclerotic lesions in ApoE-/- mice were then assessed by performing hematoxylin-eosin (HE) staining after empagliflozin treatment. Concurrently, oxidized low-density lipoprotein (oxLDL) was used to mimic atherosclerosis in three different types of cells. Then, following empagliflozin treatment of macrophage cells (RAW264.7), human aortic smooth muscle cells (HASMCs), and human umbilical vein endothelial cells (HUVECs), western blotting was applied to measure the levels of autophagy-related proteins and proinflammatory cytokines, and green fluorescent protein (GFP)-light chain 3 (LC3) puncta were detected using confocal microscopy to confirm autophagosome formation. Oil Red O staining was performed to detect the foaming of macrophages and HASMCs, and flow cytometry was used for the cell cycle analysis. 5-ethynyl-2'-deoxyuridine (EdU), cell counting kit-8 (CCK-8), and scratch assays were also performed to examine the proliferation and migration of HASMCs. Empagliflozin suppressed the progression of atherosclerotic lesions in ApoE-/- mice. Empagliflozin also induced autophagy in RAW246.7 cells, HASMCs, and HUVECs via the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway, and it significantly increased the levels of the Beclin1 protein, the LC3B-II/I ratio, and p-AMPK protein. In addition, empagliflozin decreased the expression of P62 and the protein levels of inflammatory cytokines, and it inhibited the foaming of RAW246.7 cells and HASMCs, as well as the expression of inflammatory factors by inducing autophagy. Empagliflozin activated autophagy through the AMPK signaling pathway to delay the progression of atherosclerosis. Furthermore, the results of flow cytometry, EdU assays, CCK-8 cell viability assays, and scratch assays indicated that empagliflozin blocked HASMCs proliferation and migration. Empagliflozin activates autophagy through the AMPK signaling pathway to delay the evolution of atherosclerosis, indicating that it may represent a new and effective drug for the clinical treatment of atherosclerosis.

HIF-1α Contributes to Hypoxia-induced VSMC Proliferation and Migration by Regulating Autophagy in Type A Aortic Dissection.

Type A aortic dissection (AD) is a catastrophic cardiovascular disease. Hypoxia-inducible factor-1α (HIF-1α) and autophagy are reported to be upregulated in the AD specimens. However, the interaction between HIF-1α and autophagy in the pathogenesis of AD remains to be explored. HIF-1α and LC3 levels are evaluated in 10 AD and 10 normal aortic specimens. MDC staining, autophagic vacuoles, and autophagic flux are detected in human aortic smooth muscle cells (HASMCs) under hypoxia treatment. CCK-8, transwell, and wound healing assay are used to identify proliferation and migration under hypoxia treatment. Furthermore, 3-MA is used to inhibit autophagy in hypoxia-treated HASMCs. This study reveals that AD tissues highly express HIF-1α and the LC3. Autophagy is induced under hypoxia in a time-dependent manner, and autophagy is positively related to HIF-1α in HASMCs. Moreover, the proliferation and migration of HASMCs are enhanced by hypoxia, whereas the knockdown of HIF-1α attenuates this effect. Additionally, inhibiting autophagy with 3-MA ameliorates hypoxia-induced proliferation and migration of HASMCs. In summary, the above results indicate that HIF-1α facilitates HASMC proliferation and migration by upregulating autophagy in a hypoxic microenvironment. Thus, inhibition of autophagy may be a novel therapeutic target for the prevention and treatment of AD.

Human aortic smooth muscle cell regulation by METTL3 via upregulation of m6A NOTCH1 modification and inhibition of NOTCH1.

Thoracic aortic dissection (TAD) is a very serious vascular condition that requires immediate treatment. Phenotypic conversion of human aortic smooth muscle cells (HASMCs) has been reported to be a causal factor for TAD development. Genetic variations affecting RNA modification may play a functional role in TAD. In this study, we aimed to explore the potential role of the methyltransferase like 3 (METTL3) and notch homolog 1 (NOTCH1) N6-methyladenosine (m6A) modification mechanisms in HASMCs. HASMCs were cultured. METTL3 was knocked down and overexpressed. Then, both METTL3 and NOTCH1 were simultaneously knocked down in HASMCs. HASMC proliferation was determined using Cell Counting Kit-8(CCK-8). METTL3, NOTCH1, α-smooth muscle actin (α-SMA), smooth muscle protein 22-alpha (SM22α), and calponin expressions were monitored with quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. An m6A dot blot assay was used to examine the m6A modification levels. The NOTCH1 3' untranslated region (3'UTR) m6A modification was analyzed using SRAMP and RMBase v. 2.0. A methylated RNA immunoprecipitation (MeRIP) assay was used to evaluate the METTL3 overexpression effect on m6A modification of NOTCH1 messenger RNA (mRNA). A dual-luciferase assay was used to investigate the effect of METTL3 binding of the NOTCH1 mRNA m6A modification site. YTH domain family 2 (YTHDF2)-RNA immunoprecipitation (RIP) was used to detect the change in YTHDF2's ability to bind to NOTCH1 mRNA after METTL3 overexpression. Overexpression of METTL3 inhibited α-SMA, SM22α, calponin, and NOTCH1 expressions and promoted HASMC proliferation. Knocking down METTL3 had the opposite effect. The cointerference of the METTL3 and NOTCH1 results suggested that METTL3 regulated NOTCH1, contributing to HASMC phenotypic changes. The MeRIP assay showed that the m6A modification of NOTCH1 mRNA increased after METTL3 overexpression. The dual-luciferase assay indicated that the NOTCH1 mRNA m6A modification site and METTL3 overexpression promoted NOTCH1 mRNA degradation. YTHDF2-RIP further demonstrated that the binding ability of YTHDF2 and NOTCH1 mRNA was enhanced after METTL3 overexpression. METTL3 regulated the phenotypic changes of HASMC by upregulating m6A modification of NOTCH1 and inhibiting NOTCH1.

LncRNA MCM3AP-AS1 is downregulated in atherosclerosis and sponges miR-448 to suppress vascular smooth muscle cell proliferation.

Long noncoding RNA MCM3AP-AS1 plays critical roles in cancers, but its role in atherosclerosis is yet to be elucidated. The expression of MCM3AP-AS1 in atherosclerosis and control plasma samples were measured by RT-qPCR. IntaRNA was used to predict potential base pairings between MCM3AP-AS1 and miR-448, and the results were confirmed by a dual luciferase activity assay. Cell proliferation assay was performed to explore the role of overexpression of MCM3AP-AS1, miR-448, and myocyte enhancer factor 2 (MEF2)-C in the proliferation of human aortic smooth muscle cells (HAOSMCs). MCM3AP-AS1 was downregulated in atherosclerosis and directly interacted with miR-448, which is a critical player in the proliferation of HAOSMCs, indicating its involvement in atherosclerosis. However, MCM3AP-AS1 and miR-448 showed no role in regulating the expression of each other. In contrast, overexpression of MCM3AP-AS1 increased the expression levels of MEF2-C, which can be targeted by miR-448. Moreover, MCM3AP-AS1 was found to inhibit the effects of miR-448 overexpression on both HAOSMC proliferation and MEF2-C expression. MCM3AP-AS1 is downregulated in atherosclerosis and sponges miR-448 to suppress the proliferation of HAOSMCs.

FAM3A mediates the phenotypic switch of human aortic smooth muscle cells stimulated with oxidised low-density lipoprotein by influencing the PI3K-AKT pathway.

Family with sequence similarity 3 member A (FAM3A) is a multifunctional protein that is related to the pathological process of various disorders. FAM3A is reportedly able to affect the phenotypic change of vascular smooth muscle cells under a hypertensive state. Whether FAM3A mediates the phenotypic switch of vascular smooth muscle cells under an atherosclerotic state remains unaddressed. This work investigated the roles and mechanisms of FAM3A in mediating the phenotypic switch of human aortic smooth muscle cells (HASMCs) stimulated with oxidised low-density lipoprotein (ox-LDL) in vitro. FAM3A expression was elevated in HASMCs following ox-LDL treatment. FAM3A silencing led to a suppressive effect on ox-LDL-provoked proliferation, migration and inflammation of HASMCs, whereas FAM3A overexpression had an opposite effect. Ox-LDL elicited a change in HASMCs from a contractile phenotype to a synthetic phenotype, which was inhibited by FAM3A silencing or enhanced by FAM3A overexpression. Further investigation elucidated that FAM3A silencing repressed and FAM3A overexpression promoted ox-LDL-induced activation of the PI3K-AKT pathway in HASMCs. Reactivation of AKT reversed the suppressive effect of FAM3A silencing on the ox-LDL-induced phenotypic switch of HASMCs. Restraining AKT blocked the promoting effect of FAM3A overexpression on the ox-LDL-induced phenotypic switch of HASMCs. In summary, this work elucidates that FAM3A mediates the ox-LDL-induced phenotypic switch of HASMCs by influencing the PI3K-AKT pathway, indicating a potential role for FAM3A in atherosclerosis.

Senomorphic properties of miR-181b in aged human aortic smooth muscle cells

Introduction: Age associated large artery stiffness (LAS) is an independent predictor of adverse cardiovascular health outcomes in humans and has also been shown to be associated with the development of heart failure, kidney disease, and even dementia. Furthermore, the development of cellular senescence in the aorta, the permanent arrest of the cell cycle and development of pro-inflammatory, pro-fibrotic senescence associated secretory phenotype (SASP), has also been shown to contribute to age associated LAS. MicroRNAs (miR) are small, non-coding RNAs that down-regulate protein expression in a semi-specific manner. miR-181b has been shown to be downregulated with aging, targets many of the secreted factors associated with the aortic SASP and has previously been shown to have a beneficial effect on vascular health. With smooth muscle cells being the predominant cell type in the large arteries, we wanted to address the hypothesis that miR-181b has the potential to act as a senomorphic in human aortic smooth muscle cells (HASMCs) and promote beneficial changes in individual cells that influence LAS. Methods: To test our hypothesis, we obtained young and old HASMCs and transfected them with miR-181b or a scrambled miR control utilizing a lipofectamine transfection protocol. Total RNA was isolated from the HASMCs and quantitative PCR was performed to assess changes of the SASP profile. Cell proliferation was measured using electric cell-substrate impedance sensing (ECIS) technology. Lastly, individual HASMC stiffness measurements were made using twisting magnetic microscopy. Results: Transfection of HASMCs with miR-181b resulted in reduced expression of markers of cell cycle arrest (p16 and p21) and the SASP profile (IL-6, TNF-alpha, MCP1, TGF-b1, IL-1alpha, and MMP2) when compared to scrambled control (p<0.05). HASMC proliferation was decreased in old HASMCs compared to young (p<0.05) and was increased with miR-181b transfection when compared to scramble control (p<0.05). Lastly, individual HASMC stiffness was lower in young cells compared to old (0.4382 ± 0.29 vs. 0.9959 ± 0.72 Pa/nm, n=120, p<0.001). Cell stiffness was decreased in old HASMCs when treated with miR-181b when compared with control (0.7856 ± 0.144 vs. 1.18 ± 0.288 Pa/nm, n=76, p<0.001), but there was no difference in young HASMCs. Conclusions: miR-181b appears to act as a senomorphic agent when transfected in old HASMCs. This is evidenced by noticeable changes in the expression of markers of cellular senescence which corresponded with positive changes in cellular characteristics that are associated with LAS, namely the improvement in cellular proliferation and reduction of individual cell stiffness in the old HASMC groups. Together, these results suggest the modulation of miR-181b may be a potential therapeutic target to improve age associated LAS through an effect on cellular senescence. K08 AG070281 (ET), Western Institute for Veterans Research (ET), U-CARES (ET), R01 AG048366 (LAL), R01 AG050238 (AJD), R01 AG060395 (AJD) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The PPAR-γ Agonist Pioglitazone Modulates Proliferation and Migration in HUVEC, HAOSMC and Human Arteriovenous Fistula-Derived Cells.

The failure of arteriovenous fistulas (AVFs) following intimal hyperplasia (IH) increases morbidity and mortality rates in patients undergoing hemodialysis for chronic kidney disease. The peroxisome-proliferator associated receptor (PPAR-γ) may be a therapeutic target in IH regulation. In the present study, we investigated PPAR-γ expression and tested the effect of pioglitazone, a PPAR-γ agonist, in different cell types involved in IH. As cell models, we used Human Endothelial Umbilical Vein Cells (HUVEC), Human Aortic Smooth Muscle Cells (HAOSMC), and AVF cells (AVFCs) isolated from (i) normal veins collected at the first AVF establishment (T0), and (ii) failed AVF with IH (T1). PPAR-γ was downregulated in AVF T1 tissues and cells, in comparison to T0 group. HUVEC, HAOSMC, and AVFC (T0 and T1) proliferation and migration were analyzed after pioglitazone administration, alone or in combination with the PPAR-γ inhibitor, GW9662. Pioglitazone negatively regulated HUVEC and HAOSMC proliferation and migration. The effect was antagonized by GW9662. These data were confirmed in AVFCs T1, where pioglitazone induced PPAR-γ expression and downregulated the invasive genes SLUG, MMP-9, and VIMENTIN. In summary, PPAR-γ modulation may represent a promising strategy to reduce the AVF failure risk by modulating cell proliferation and migration.

Inhibition of tiRNA-Gly-GCC ameliorates neointimal formation via CBX3-mediated VSMCs phenotypic switching.

tRNA-derived fragments (tRFs) are a new class of non-coding RNAs involved in a variety of pathological processes, but their biological functions and mechanisms in human aortic smooth muscle cells (HASMCs) phenotype transition and vascular intimal hyperplasia are unclear. tiRNA-Gly-GCC is upregulated in synthetic HASMCs, atherosclerotic arteries, plasma, and the balloon injured carotid artery of rats. Functionally, the inhibition of tiRNA-Gly-GCC represses HASMCs proliferation, migration, and reversed dedifferentiation, whereas the overexpression of tiRNA- Gly-GCC have contrary effects. Mechanistically, tiRNA-Gly-GCC performs these functions on HASMCs via downregulating chromobox protein homolog 3 (CBX3). Finally, the inhibition of tiRNA-Gly-GCC could ameliorate neointimal formation after vascular injury in vivo. tiRNA-Gly-GCC is a mediator of HASMCs phenotypic switching by targeting CBX3 and inhibition of tiRNA-Gly-GCC suppresses neointimal formation.

Porphyromonas gingivalis Lipopolysaccharides Promote Proliferation and Migration of Human Vascular Smooth Muscle Cells through the MAPK/TLR4 Pathway

Atherosclerosis is a major cause of mortality worldwide. The initial change in atherosclerosis is intimal thickening due to muscle cell proliferation and migration. A correlation has been observed between periodontal disease and atherosclerosis. Here, we investigated the proliferation and migration of human aortic smooth muscle cells (HASMCs) using Porphyromonas gingivalis-derived LPS (Pg-LPS). To elucidate intracellular signaling, toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88) of HASMCs were knocked down, and the role of these molecules in Pg-LPS-stimulated proliferation and migration was examined. The role of mitogen-activated protein kinase (MAPK) in HASMC proliferation and migration was further elucidated by MAPK inhibition. Pg-LPS stimulation increased the proliferation and migration of HASMCs and activated the TLR4/MyD88 pathway. TLR4 knockdown inhibited Pg-LPS stimulated HASMCs proliferation and migration. Pg-LPS stimulation led to the phosphorylation of P38 MAPK, JNK, and ERK, and MyD88 knockdown inhibited the phosphorylation of P38 MAPK and JNK but not ERK. P38 MAPK and SAPK/JNK inhibition did not suppress the proliferation of HASMCs upon Pg-LPS stimulation, but ERK inhibition significantly inhibited proliferation. SAPK/JNK and ERK inhibition suppressed Pg-LPS-stimulated migration of HASMCs. In conclusion, our findings suggest that Pg-LPS may promote atherosclerosis via the activation of MAPK through TLR4.

LncRNA GAS5 promotes abdominal aortic aneurysm formation through regulating the miR-185-5p/ADCY7 axis

One of the causes of abdominal aortic aneurysm (AAA) is the apoptosis of vascular smooth muscle cells. Many long noncoding RNA (lncRNAs) have been implicated in AAA formation. However, the mechanism of growth arrest-specific 5 (GAS5) in AAA formation is not yet clear. The expression levels of GAS5, microRNA-185-5p (miR-185-5p) and adenylate cyclase 7 (ADCY7) were determined by quantitative real-time PCR. Angiotensin II (ANGII) was used to induce AAA cell models. Cell viability was detected by MTT assay, and cell apoptosis was assessed by flow cytometry. Western blot analysis was used to test the protein expression levels. Besides, a dual-luciferase reporter assay was used to identify the mechanism of GAS5. GAS5 was upregulated in AAA tissues and ANGII-induced human aortic smooth muscle cells (HASMCs). GAS5 overexpression inhibited proliferation and promoted apoptosis and inflammatory response in ANGII-induced HASMCs, while its knockdown had the opposite effects. MiR-185-5p could be absorbed by GAS5, and its inhibitor could invert the effects of GAS5 silencing on proliferation, apoptosis and inflammatory response in ANGII-induced HASMCs. ADCY7 was a target of miR-185-5p. ADCY7 knockdown increased proliferation, while decreased apoptosis and inflammatory response in ANGII-induced HASMCs. Also, overexpressed ADCY7 reversed the effect of miR-185-5p overexpression on proliferation, apoptosis and inflammatory response in ANGII-induced HASMCs. GAS5 positively regulated the ADCY7 expression to inhibit the activity of the AKT signaling pathway by sponging miR-185-5p. LncRNA GAS5 contributed to AAA formation through regulating HASMCs proliferation, apoptosis and inflammatory response, which might provide new ideas for the treatment of AAA.

Cyclic tensile strain facilitates proliferation and migration of human aortic smooth muscle cells and reduces their apoptosis via miRNA-187-3p

ABSTRACT The cardiovascular is a system that contains extremely complex mechanical factors, in which the circulatory flow of blood has rich mechanical laws. Many studies have revealed that mechanical factors play a very important role in the process of revascularization. Hence, it is essential to investigate the mechanical factors in the process of revascularization in depth. A cyclic tensile strain (CTS) was applied to human aortic smooth muscle cells (HASMCs) at a frequency of 1 Hz and amplitudes of 5%, 10% and 15%, respectively. SmallRNA-seq was used to identify differentially expressed miRNAs (DE-miRNAs) responding to CTS in HASMCs. Starbase database predicted the target genes of DE-miRNAs. Metascape was applied for GO and KEGG pathway enrichment analysis and protein–protein interaction network construction. The proliferation and migration of CTS-treated HASMCs were significantly enhanced, and apoptosis were significantly reduced compared to the control group. SmallRNA-seq results demonstrated that 55, 16 and 16 DE-miRNAs were present in 5%, 10% and 15% CTS-treated HASMCs, respectively. Compared to controls, with miR-26a-2-3p and miR-187-3p being the intersection of these DE-miRNAs. Starbase database identified 189 common target genes for miR-26a-2-3p and miR-187-3p. Common target genes are mainly enriched in the basolateral plasma membrane and endocytosis. Further, in vitro experiments exhibited that CTS upregulated miR-187-3p expression, and miR-187-3p enhanced the proliferation and migration of HASMCs and reduced their apoptosis. It is suggested that miR-187-3p may be an important target for CTS participate in the process of cardiovascular disease.

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.

LncRNA SCIRT is downregulated in atherosclerosis and suppresses the proliferation of human aortic smooth muscle cells (HAOSMCs) by sponging miR-146a in cytoplasm

BackgroundSCIRT has been characterized as a key player in cancer biology, while its role in other human diseases is unclear. This study explored its role in atherosclerosis, with a specific focus on its interaction with SCIRT and miR-146a.MethodsThe expression of SCIRT and miR-146a in atherosclerosis-affected tissues and healthy tissues from 56 atherosclerosis patients were analyzed by RT-qPCR. The expression of SCIRT in nuclear and cytoplasm samples was detected by RNA fractionation assay. The direct interaction between SCIRT and miR-146a was detected by RNA pull-down assay. SCIRT and miR-146a were overexpressed in human aortic smooth muscle cells (HAOSMCs) to study the crosstalk between them. The role of SCIRT and miR-146a in the proliferation of HAOSMCs was analyzed with BrdU assay.ResultsSCIRT was downregulated by atherosclerosis, while miR-146a was upregulated by atherosclerosis. SCIRT was detected in both cytoplasm and nuclear samples, and it directly interacted with miR-146a. In HAOSMCs, overexpression of SCIRT and miR-146a did not affect the expression of each other. Interestingly, SCIRT suppressed the proliferation of HAOSMCs and reduced the enhancing effects of miR-146a on cell proliferation.ConclusionTherefore, SCIRT is downregulated in atherosclerosis and it suppresses the proliferation of HAOSMCs by sponging miR-146a in cytoplasm.

Expression and Functional Analysis of lncRNAs Involved in Platelet-Derived Growth Factor-BB-Induced Proliferation of Human Aortic Smooth Muscle Cells.

Abnormal proliferation of vascular smooth muscle cells (VSMCs) is a common feature of many vascular remodeling diseases. Because long non-coding RNAs (lncRNAs) play a critical role in cardiovascular diseases, we analyzed the key lncRNAs that regulate VSMC proliferation. Microarray analysis identified 2,643 differentially expressed lncRNAs (DELs) and 3,720 differentially expressed coding genes (DEGs) between fetal bovine serum (FBS) starvation-induced quiescent human aortic smooth muscle cells (HASMCs) and platelet-derived growth factor-BB (PDGF-BB)-stimulated proliferative HASMCs. Gene Ontology and pathway analyses of the identified DEGs and DELs demonstrated that many lncRNAs were enriched in pathways related to cell proliferation. One of the upregulated lncRNAs in proliferative HASMC was HIF1A anti-sense RNA 2 (HIF1A-AS2). HIF1A-AS2 suppression decreased HASMC proliferation via the miR-30e-5p/CCND2 mRNA axis. We have thus identified key DELs and DEGs involved in the regulation of PDGF-BB induced HASMC proliferation. Moreover, HIF1A-AS2 promotes HASMC proliferation, suggesting its potential involvement in VSMC proliferative vascular diseases.

The Expression Patterns and Roles of Lysyl Oxidases in Aortic Dissection.

Background: Lysyl oxidases (LOXs), including LOX, LOXL1, LOXL2, LOXL3, and LOXL4, catalyze the formation of a cross-link between elastin (ELN) and collagen. Multiple LOX mutations have been shown to be associated with the occurrence of aortic dissection (AD) in humans, and LOX-knockout mice died during the perinatal period due to aortic aneurysm and rupture. However, the expression levels and roles of other LOX members in AD remain unknown.Methods: A total of 33 aorta samples of AD and 15 normal aorta were collected for LOXs mRNA and protein levels detection. We also analyzed the datasets of AD in GEO database through bioinformatics methods. LOXL2 and LOXL3 were knocked down in primary cultured human aortic smooth muscle cells (HASMCs) via lentivirus.Results: Here, we show that the protein levels of LOXL2 and LOXL3 are upregulated, while LOXL4 is downregulated in AD subjects compared with non-AD subjects, but comparable protein levels of LOX and LOXL1 are detected. Knockdown of LOXL2 suppressed MMP2 expression, the phosphorylation of AKT (p-AKT) and S6 (p-S6), but increased the mono-, di-, tri-methylation of H3K4 (H3K4me1/2/3), H3K9me3, and p-P38 levels in HASMCs. These results indicate that LOXL2 is involved in regulation of the extracellular matrix (ECM) in HASMCs. In contrast, LOXL3 knockdown inhibited PCNA and cyclin D1, suppressing HASMC proliferation. Our results suggest that in addition to LOX, LOXL2 and LOXL3 are involved in the pathological process of AD by regulating ECM and the proliferation of HASMCs, respectively. Furthermore, we found that LOXL2 and LOXL4 was inhibited by metformin and losartan in HASMCs, which indicated that LOXL2 and LOXL4 are the potential targets that involved in the therapeutic effects of metformin and losartan on aortic or aneurysm expansion.Conclusions: Thus, differential regulation of LOXs might be a novel strategy to prevent or treat AD.