Silencing of circARHGAP12 inhibits the progression of atherosclerosis via miR-630/EZH2/TIMP2 signal axis.

Atherosclerosis (AS) is a common disease that seriously threatens human health. So far, the pathogenesis of AS has not been fully understood. This project investigates the effects of circARHGAP12 on AS and its regulatory mechanism. ApoE-/- knockout mice (ApoE) were adopted and reared with a high-fat diet to construct an AS model. Lentivirus was established to knock down the expression of circARHGAP12 in mice. After 12 weeks, the aorta was removed and the expression of circARHGAP12 was detected. Vascular oil red O staining was used to detect the degree of AS. The expression of inflammatory factors was detected by ELISA. Aortic smooth muscle cells (MASMCs) were cultured to evaluate the effects of circARHGAP12 on the phenotype of MASMCs. RNA pull-down and luciferase assay were used to verify the downstream target genes of circARHGAP12. In addition, the effects of circARHGAP12 on MASMCs proliferation and migration were detected by MTT and transwell assay. Compared with the normal group, the expression of circARHGAP12 in the MASMCs under ox-LDL treatment was elevated, and circARHGAP12 silencing could inhibit AS in vitro and in vivo. The results of the mechanism study showed that circARHGAP12 can directly bind with miR-630. In addition, miR-630 can also target EZH2 to modulate the transcription of TIMP2 and to influence the migration of MASMCs. circARHGAP12 is upregulated in AS. CircARHGAP12 knockdown can inhibit the progression of AS. This study expands on the role of circRNA in AS and provides potential targets for the treatment of AS.

MiR-18a-5p contributes to enhanced proliferation and migration of PASMCs via targeting Notch2 in pulmonary arterial hypertension.

AIM: This study is undertaken to investigate the role and molecular mechanisms of miR-18a-5p in regulating pulmonary arterial hypertension (PAH) pathogenesis. METHODS: Gene expression and protein levels were determined by qRT-PCR and western blot, respectively; Cell counting kti-8 and Transwell migration assays were used to determine the biological functions of miR-18a-5p in pulmonary arterial smooth muscle cells (PASMCs); bioinformatics analysis, luciferase reporter assays were used to elucidate the mechanisms of miR-18a-5p. RESULTS: MiR-18a-5p was up-regulated in the clinical samples from PAH patients. PASMCs treated with hypoxia exhibited enhanced proliferative ability and upregulated miR-18a-5p expression. Knockdown of miR-18a-5p attenuated hypoxia-induced hyper-proliferation and enhanced migratory potential of PASMCs; while miR-18a-5p overexpression promoted PASMC proliferation and migration. Further mechanistic studies showed that Notch2 was a direct target of miR-18a-5p and was repressed by miR-18a-5p overexpression. The rescue studies indicated that Notch2 overexpression counteracted the enhanced proliferation and migration induced by miR-18a-5p mimics in PASMCs. Similarly, Notch2 overexpression also block the effects caused by hypoxia in PASMCs. Moreover, Notch2 expression was down-regulated in the PAH patients and was negatively correlated with miR-18a-5p expression. In vivo animal studies further revealed the up-regulation of miR-18a-5p and the down-regulation of Notch2 in the PAH rats. CONCLUSIONS: Collectively, this study identified the up-regulated miR-18a-5p in the PAH patients; our data suggest that miR-18a-5p contributes to the enhanced proliferation and migration of PASMCs via repressing Notch2 expression.

Exosomes derived from oxLDL-stimulated macrophages induce neutrophil extracellular traps to drive atherosclerosis.

This study aimed to investigate the role and underlying mechanism of exosomes secreted by oxidized low-density lipoprotein (oxLDL)-stimulated macrophages in the progression of atherosclerosis (AS). Exosomes from peripheral blood of AS patients or oxLDL-treated macrophages were co-cultured with human neutrophils. Neutrophil extracellular traps (NETs) were detected by immunofluorescence staining. The levels of inflammatory cytokines were quantified by enzyme-linked immunosorbent assay (ELISA). The expression levels of miR-146a and superoxide dismutase 2 (SOD2) were determined by quantitative real-time PCR (qRT-PCR) and western blot. The generation of intracellular reactive oxygen species (ROS) was observed by using dichlorofluorescin diacetate (DCFH-DA). ApoE-deficient mice were fed with high-fat diet (HFD) to induce AS. Atherosclerotic plaques were evaluated by Oil red O (ORO) and hematoxylin-eosin (HE) staining. Our results showed that miRNA-146a was enriched in serum-derived exosomes of AS patients and oxLDL-treated macrophage THP-1-derived exosomes. Importantly, exosomal miR-146a secreted by oxLDL-treated macrophages promoted ROS and NETs release via targeting SOD2. In addition, intravenous administration of oxLDL-treated THP-1 cells-derived exosomes into AS mice significantly deteriorated AS in vivo. Our findings indicate that exosomal miR-146a derived from oxLDL-treated macrophages promotes NETs formation via inducing oxidative stress, which might provide a novel scientific basis for the understanding of AS progression.

Effects of integrin α5ß1 on the proliferation and migration of human aortic vascular smooth muscle cells.

Integrin (ITG) α5ß1 is a dominant fibronectin receptor that is abundantly expressed on the surface of vascular smooth muscle cells (VSMCs). However, the association between integrin α5ß1 and the proliferation and migration of VSMCs has yet to be elucidated. The aim of the present study was to characterize the roles of ITGα5 and ITGß1 in the proliferation and migration of VSMCs, and to determine the effects of ITGα5ß1 on integrin-linked kinase (ILK) and focal adhesion kinase (FAK) mRNA expression. Lentiviral expression vectors as well as RNA interference vectors of ITGα5 and ITGß1 were successfully constructed and transfected into VSMCs to obtain ITGα5­ and ITGß1­overexpressing or -silenced cells, respectively. Cell cycle distribution, proliferation and migration were analyzed in the transfected VSMCs in order to clarify the roles of ITGß1 and ITGα5 in the proliferation and migration of VSMCs. ITGß1 was markedly associated with the proliferation and migration of VSMCs, and FAK was shown to be involved in the signaling pathways of ITGß1. ITGα5 did not exert any effects on VSMCs. The results of the present study may provide a possible therapeutic target for the prevention and treatment of early vascular disease associated with VSMCs.

Effects of Citral on Lipopolysaccharide-Induced Inflammation in Human Umbilical Vein Endothelial Cells.

Citral is an active compound of lemongrass oil which has been reported to have anti-inflammatory effects. In this study, we investigated the effects of citral on lipopolysaccharide (LPS)-induced inflammatory response in a rat model of peritonitis and human umbilical vein endothelial cells (HUVECs). LPS was intraperitoneally injected into rats to establish a peritonitis model. The HUVECs were treated with citral for 12 h before exposure to LPS. The levels of TNF-α and IL-8 were measured using ELISA. Western blotting was used to detect the expression of VCAM-1, ICAM-1, NF-κB, and PPAR-γ. The results showed that citral had a protective effect against LPS-induced peritonitis. Citral decreased the levels of WBCs and inflammatory cytokines TNF-α and IL-6. Citral also inhibited LPS-induced myeloperoxidase (MPO) activity in the peritoneal tissue. Treatment of HUVECs with citral significantly inhibited TNF-α and IL-8 expression induced by LPS. LPS-induced VCAM-1 and ICAM-1 expression were also suppressed by citral. Meanwhile, we found that citral inhibited LPS-induced NF-κB activation in HUVECs. Furthermore, we found that citral activated PPAR-γ and the anti-inflammatory effects of citral can be reversed by PPAR-γ antagonist GW9662. In conclusion, citral inhibits LPS-induced inflammatory response via activating PPAR-γ which attenuates NF-κB activation and inflammatory mediator production.

Meta-analysis of the association between angiotensin-converting enzyme I/D polymorphism and aortic aneurysm risk.

BACKGROUND: Angiotensin-converting enzyme (ACE) I/D polymorphism has been indicated to be correlated with aortic aneurysm (AA) susceptibility, but study results are still debatable. Thus, a meta-analysis was conducted. METHODS: Databases including PubMed, Embase, Web of Science, and Chinese National Knowledge Infrastructure (CNKI) were searched. Data were extracted and pooled odds ratios (OR) with 95% confidence intervals (CI) were calculated. RESULTS: Ten studies with 3557 cases and 5231 controls were included in this meta-analysis. The association between ACE I/D genotype and AA risk was significant (OR=1.30; 95%CI, 1.07-1.57; p<0.01; I(2)=68%). When stratified by ethnicity, a significantly elevated risk was observed in Caucasians (OR=1.31; 95%CI, 1.07-1.61; p<0.01; I(2)=71%). In the abdominal AA subgroup, a significantly increased risk was observed (OR=1.29; 95%CI, 1.03-1.62; p=0.02; I(2)=73%). However, ACE I/D polymorphism was not associated with thoracic AA risk (OR=1.33; 95%CI, 0.85-2.07; p=0.21; I(2)=52%). Subgroup analysis on blood pressure status showed that an increased risk was found in hypertensive patients (OR=1.52; 95%CI, 1.02-2.26; p=0.04; I(2)=0%) but not in normotensive subjects (OR=1.46; 95%CI, 0.72-2.96; p=0.30; I(2)=25%). CONCLUSIONS: In conclusion, this meta-analysis suggested that ACE I/D polymorphism is a risk factor for AA.