scholarly journals Silencing of OIP5-AS1 Protects Endothelial Cells From ox-LDL-Triggered Injury by Regulating KLF5 Expression via Sponging miR-135a-5p

2021 ◽  
Vol 8 ◽  
Author(s):  
Minghu Zhao ◽  
Yuanyuan Yang ◽  
Jingchao Li ◽  
Min Lu ◽  
Yu Wu
Keyword(s):  
Endothelial Cells ◽  
Animal Studies ◽  
Umbilical Vein ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Enzyme Linked Immunosorbent Assay ◽  
Klf5 Expression

Background: Long non-coding RNAs (lncRNAs) have been implicated in the pathogenesis of atherosclerosis. LncRNA OIP5 antisense RNA 1 (OIP5-AS1) has been found to be associated with the development of atherosclerosis. In this study, we further investigated the molecular basis of OIP5-AS1 in atherosclerosis pathogenesis.Methods: Oxidative low-density lipoprotein (ox-LDL) was used to treat human umbilical vein endothelial cells (HUVECs). The levels of OIP5-AS1, miR-135a-5p, and Krüppel-like factor 5 (KLF5) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot. Cell viability, migration, and apoptosis were evaluated using the Cell Counting Kit-8 (CCK-8), Transwell, and flow cytometry, respectively. The levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and malondialdehyde (MDA) were determined with enzyme-linked immunosorbent assay (ELISA). Targeted interactions among OIP5-AS1, miR-135a-5p, and KLF5 were confirmed by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. Animal studies were performed to assess the role of OIP5-AS1 in atherosclerosis progression in vivo.Results: Our data showed the significant upregulation of OIP5-AS1 in atherosclerosis serum and ox-LDL-stimulated HUVECs. The silencing of OIP5-AS1 protected against ox-LDL-triggered cytotoxicity in HUVECs and diminished lipids secretion in ApoE−/− mice. Moreover, OIP5-AS1 functioned as a molecular sponge of miR-135a-5p, and miR-135a-5p was a functional mediator of OIP5-AS1 in regulating ox-LDL-induced HUVEC injury. KLF5 was a direct target of miR-135a-5p, and the increased expression of miR-135a-5p alleviated ox-LDL-induced cytotoxicity by downregulating KLF5. Furthermore, OIP5-AS1 influenced KLF5 expression through sponging miR-135a-5p.Conclusion: The current work identified that the silencing of OIP5-AS1 protected against ox-LDL-triggered cytotoxicity in HUVECs at least in part by influencing KLF5 expression via acting as a miR-135a-5p sponge.

Exosomal MALAT1 derived from ox-LDL-treated endothelial cells induce neutrophil extracellular traps to aggravate atherosclerosis

2020 ◽  
Vol 401 (3) ◽  
pp. 367-376 ◽  
Author(s):  
Hailai Gao ◽  
XiaoLi Wang ◽  
Chaolan Lin ◽  
Zhujun An ◽  
Jiangbo Yu ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Low Density Lipoprotein ◽  
Atherosclerotic Lesion ◽  
Density Lipoprotein ◽  
Neutrophil Extracellular Traps ◽  
Human Neutrophils ◽  
Enzyme Linked Immunosorbent Assay ◽  
Extracellular Traps ◽  
Umbilical Vein Endothelial Cells

AbstractThe objective of this study was to reveal a novel mechanism underlying the progression of atherosclerosis (AS) associated with endothelial cells (ECs) and neutrophils. Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) were used to observe the morphology and particle size of isolated exosomes. Western blotting was applied to examine exosomal markers, while the expression of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) was measured by quantitative real-time polymerase chain reaction (qRT-PCR). The production of inflammatory cytokines and reactive oxygen species (ROS) was determined by an enzyme-linked immunosorbent assay (ELISA) and a dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay. Circulating neutrophil extracellular traps (NETs) were represented by myeloperoxidase (MPO)-DNA complexes. NETs formation was assessed using immunofluorescence microscopy. Atherosclerotic lesion development was measured by Oil Red O (ORO) staining. In the results, MALAT1 expression was increased in exosomes extracted from oxidized low-density lipoprotein (ox-LDL)-treated human umbilical vein endothelial cells (HUVECs). When co-cultured with human neutrophils, exosomes derived from ox-LDL-treated HUVECs were revealed to promote NETs formation, which was mediated by exosomal MALAT1. Furthermore, ox-LDL-treated HUVECs-derived exosomes were demonstrated to trigger hyperlipidemia, inflammatory response and NETs release in a mouse model of AS. In conclusion, exosomal MALAT1 derived from ox-LDL-treated ECs initiated NETs formation, which in turn deteriorated AS.

Curcumin improves atherosclerosis by inhibiting the epigenetic repression of lncRNA MIAT to miR-124

Vascular ◽  
2022 ◽  
pp. 170853812110409
Author(s):  
Shang Ouyang ◽  
Ou Zhang ◽  
Hua Xiang ◽  
Yuan-Hui Yao ◽  
Zhi-Yong Fang
Keyword(s):  
Myocardial Infarction ◽  
Cell Apoptosis ◽  
Cell Model ◽  
Umbilical Vein ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Umbilical Vein Endothelial Cells

Objectives: Atherosclerosis is a dominant cardiovascular disease. Curcumin has protective effect on atherosclerosis. However, the mechanisms remain to be explored. Methods: Atherosclerosis was induced by feeding mice with high-fat diet (HFD) and ox-low-density lipoprotein (LDL)-induced human umbilical vein endothelial cells (HUVECs) were structured. Oil Red O staining was used to evaluate the plaques in the artery. Quantitative real-time PCR (qRT-PCR) was conducted to detect the level of myocardial infarction associated transcript (MIAT), miR-124, and enhancer of zeste homolog 2 (EZH2). We performed western blotting and enzyme linked immunosorbent assay to examine the expression of EZH2 and cytokines including IL-1β, TNFα, IL-6, and IL-8, respectively. RNA immunoprecipitation and chromatin immunoprecipitation (ChIP) were used to validate the interaction between myocardial infarction associated transcript and EZH2. Flow cytometry and CCK-8 assay were used to examine cell apoptosis and proliferation, respectively. Results: Curcumin suppressed inflammation in atherosclerosis mouse model and ox-LDL-induced cell model. MIAT overexpression and miR-124 inhibition relieved the anti-inflammation effect of curcumin in ox-LDL-induced cell. MIAT regulated miR-124 by interacting with EZH2. Curcumin relieved ox-LDL-induced cell inflammation via regulating MIAT/miR-124 pathway. Conclusion: MIAT/miR-124 axis mediated the effect of curcumin on atherosclerosis and altered cell apoptosis and proliferation, both in vivo and in vitro. These data further support the application of curcumin in control of atherosclerosis advancement.

scholarly journals Overexpressed lncRNA ROR Promotes the Biological Characteristics of ox-LDL-Induced HUVECs via the let-7b-5p/HOXA1 Axis in Atherosclerosis

2021 ◽  
Vol 8 ◽  
Author(s):  
Cong Yu ◽  
Bin Wu ◽  
Jinsong Jiang ◽  
Guangwei Yang ◽  
Chao Weng ◽  
...  
Keyword(s):  
Cell Viability ◽  
Umbilical Vein ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Biological Characteristics ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Associated Proteins ◽  
And Migration ◽  
Umbilical Vein Endothelial Cells

The long non-coding RNA regulator of reprogramming (lncRNA ROR) is involved in atherosclerosis (AS), but the specific mechanism remains unclear. The expressions of lncRNA ROR, let-7b-5p, Homeobox A1 (HOXA1), and apoptosis-associated proteins in the serum of AS patients and human umbilical vein endothelial cells (HUVECs) were determined by quantitative real-time PCR (qRT-PCR) and Western blot. The relationships of lncRNA ROR, let-7b-5p, and HOXA1 were analyzed by Pearson's correlation test. The viability and the migration of HUVECs were measured by Cell Counting Kit-8, wound healing, and Transwell assays. The predicted target gene and the potential binding sites were confirmed by dual-luciferase reporter assay. lncRNA ROR was highly expressed in AS, which promoted the cell viability and migration of HUVECs, while lncRNA ROR silencing produced the opposite results. The expression of let-7b-5p, which bound to lncRNA ROR, was downregulated in AS, indicating that the two genes were negatively correlated. Besides this, let-7b-5p reversed the effects of upregulated lncRNA ROR expression on let-7b-5p expression, cell viability, and migration as well as the expressions of apoptosis-related proteins of ox-LDL-treated HUVECs. HOXA1 was targeted by let-7b-5p and upregulated in AS, with its expression being negatively correlated with let-7b-5p but positively correlated with lncRNA ROR. In ox-LDL-treated HUVECs, overexpressed HOXA1 reversed the effects of let-7b-5p, and HOXA1 silencing reversed the effects of lncRNA ROR. In AS, lncRNA ROR promoted the biological characteristics of oxidation of low-density lipoprotein-induced HUVECs via the let-7b-5p/HOXA1 axis.

scholarly journals Exosome-encapsulated miR-505 from ox-LDL-treated vascular endothelial cells aggravates atherosclerosis by inducing NET formation

2019 ◽  
Vol 51 (12) ◽  
pp. 1233-1241 ◽  
Author(s):  
Li Chen ◽  
Liqun Hu ◽  
Qing Li ◽  
Jian Ma ◽  
Hongqi Li
Keyword(s):  
Endothelial Cells ◽  
Peripheral Blood ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Density Lipoprotein ◽  
Luciferase Reporter ◽  
Vascular Endothelial ◽  
Healthy Donors ◽  
Net Release

Abstract Neutrophil extracellular traps (NETs) play an important role in the pathological process of atherosclerosis (AS). This study aims to evaluate whether exosomes from oxidized low-density lipoprotein (ox-LDL)-treated vascular endothelial cells (VECs) aggravate AS by inducing NET formation. Exosomes from the peripheral blood of healthy donors and AS patients (namely NC-EXO and AS-EXO, respectively) and exosomes from human umbilical vein endothelial cells (HUVECs) treated without or with ox-LDL (namely normal EXO and ox-LDL-EXO, respectively) were isolated, identified, and co-cultured with neutrophils from peripheral blood of healthy donors. NET formation was evaluated by immunofluorescence staining and determining the content of cell-free DNA and myeloperoxidase-DNA complex. Dual-luciferase reporter assay, chromatin immunoprecipitation assay, quantitative reverse transcription polymerase chain reaction, and western blot analysis were performed to explore the underlying mechanisms. We found that AS-EXO and ox-LDL-EXO induced NET release from neutrophils. Mechanistically, ox-LDL treatment in HUVECs might activate the NF-κB pathway, which transcriptionally activates miR-505, and then the exosome-encapsulated high miR-505 expression targeted and inhibited SIRT3 in neutrophils, thereby inducing reactive oxygen species (ROS) level increase and NET release by neutrophils. Further in vivo experiments showed that ox-LDL-EXO accelerated AS progression in AS mice. In summary, exosome-encapsulated miR-505 from ox-LDL-treated VECs aggravates AS by inducing NET formation.

scholarly journals Knockdown of circ_0003204 alleviates oxidative low-density lipoprotein-induced human umbilical vein endothelial cells injury: Circulating RNAs could explain atherosclerosis disease progression

Open Medicine ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. 558-569
Author(s):  
Qiuxia Su ◽  
Xianhua Dong ◽  
Chonghui Tang ◽  
Xiaojie Wei ◽  
Youguo Hao ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Endothelial Cells ◽  
Signaling Pathway ◽  
Umbilical Vein ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Luciferase Reporter ◽  
Low Density ◽  
Human Umbilical Vein ◽  
Umbilical Vein Endothelial Cells

Abstract Atherosclerosis (AS) is a serious cardiovascular disease. Circular RNAs (circRNAs) play an important role in the progression of many diseases, including AS. However, the role of circ_0003204 in AS is not clear. Oxidized low-density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cells (HUVECs) were used to construct an AS cell model in vitro. Cell viability was assessed using cell counting kit 8 (CCK8) assay. Flow cytometry and caspase-3 activity were used to measure cell apoptosis. The contents of inflammatory cytokines were measured using enzyme-linked immunosorbent assay (ELISA). Oxidative stress marker expression and cell injury marker activity were detected by their corresponding Assay Kits. Besides, the expression levels of circ_0003204, miR-330-5p, and toll-like receptor 4 (TLR4) were tested by real-time polymerase chain reaction (qPCR). The interaction between miR-330-5p and circ_0003204 or TLR4 was examined by dual-luciferase reporter assay and RNA pull-down assay. Western blot (WB) analysis was used to determine the levels of TLR4 protein and nuclear factor-kappa B (NF-κB) signaling pathway-related protein. Our data suggested that ox-LDL could suppress viability and promote apoptosis, inflammatory response, and oxidative stress in HUVECs. circ_0003204 was highly expressed in ox-LDL-induced HUVECs, and its silencing could inhibit ox-LDL-induced HUVECs injury. miR-330-5p could be sponged by circ_0003204, and its inhibitor could reverse the inhibition effect of silenced circ_0003204 on ox-LDL-induced HUVECs injury. Further, TLR4 could be targeted by miR-330-5p, and its overexpression could invert the suppression effect of miR-330-5p on ox-LDL-induced HUVECs injury. The activity of the NF-κB signaling pathway was regulated by the circ_0003204/miR-330-5p/TLR4 axis. Our results indicated that circ_0003204 silencing could alleviate ox-LDL-induced HUVECs injury, suggesting that circ_0003204 might be a novel target for AS treatment.

LncRNA OIP5-AS1 accelerates ox-LDL-treated HUVECs injury by NF-κB pathway via miR-30c-5p

2021 ◽  
pp. 1-12
Author(s):  
Lei Zhang ◽  
Qiulai Li ◽  
Yanxia Chen ◽  
Qiao Zhu
Keyword(s):  
Oxidative Stress ◽  
Vascular Endothelial Cells ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Vital Role ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Sod Activity ◽  
Interacting Protein ◽  
Rna Immunoprecipitation

BACKGROUND: Oxidized low-density lipoprotein (ox-LDL) could induce endothelial injury and played a vital role in the progression and development of atherosclerosis. This study aimed to investigate the role of Opa-interacting protein 5 antisense RNA 1 (OIP5-AS1) in ox-LDL-induced human umbilical vascular endothelial cells (HUVECs) injury and the potential mechanisms. METHODS: Cell proliferation and apoptosis were evaluated by Cell Counting Kit-8 (CCK-8) assay and flow cytometry assay, respectively. The levels of lactate dehydrogenase (LDH), reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD) and nitric oxide (NO) were detected by corresponding detection kits, respectively. Quantitative real-time PCR (qRT-PCR) was conducted to measure the expression of OIP5-AS1 or microRNA-30c-5p (miR-30c-5p) in HUVECs. Binding between OIP5-AS1 and miR-30c-5p was predicted through bioinformatics analysis and confirmed by dual-luciferase reporter assay and RNA immunoprecipitation (RIP). Western blot was used to analyze p-IκB, IκB, p-p65 and p65 levels. RESULTS: In HUVECs, exposure to ox-LDL led to a decrease in cell viability and an increase in LDH release and apoptosis with concomitant enhancement of oxidative stress, as evidenced by increased ROS and MDA generation, as well as decreased SOD activity and NO levels, while OIP5-AS1 knockdown or miR-30c-5p upregulation could rescue these effects above. Mechanically, OIP5-AS1 functioned as a sponge of miR-30c-5p. OIP5-AS1-induced injury and apoptosis, oxidative stress and activation of NF-κB pathway were reversed by miR-30c-5p in ox-LDL-treated HUVECs. CONCLUSION: OIP5-AS1 contributed to ox-LDL-treated HUVECs injury by activation of NF-κB pathway via miR-30c-5p.

scholarly journals Abnormally expressed miR-23b in Chinese Mongolian at high cardiovascular risk may contribute to monocyte/macrophage inflammatory reaction in atherosclerosis

2018 ◽  
Vol 38 (6) ◽  
Author(s):  
Li-ping He ◽  
Xing-sheng Zhao ◽  
Le-ping He
Keyword(s):  
Cardiovascular Risk ◽  
Inflammatory Response ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Nuclear Factor Κb ◽  
Luciferase Reporter ◽  
Enzyme Linked Immunosorbent Assay ◽  
High Cardiovascular Risk ◽  
Peripheral Blood Mononuclear

Background: The prevalence of coronary heart disease (CHD) appears to be high among Chinese Mongolians. MiR-23b has been proven to play a key role in atherosclerosis. The expression and role of miR-23b in the Mongolians at high cardiovascular risk were explored in the present study. Methods: Forty cases of blood samples from the Mongolians at high cardiovascular risk were enrolled in the present study. The expression of miR-23b was quantified by quantitative real-time PCR. To induce monocytes differentiation into macrophages, HP-1 cells were cultured with phorbol 12-myristate 13-acetate. The level of inflammatory markers was determined by the enzyme-linked immunosorbent assay. The interaction between miR-23b and A20 was explored by the dual luciferase reporter assay. Results: The expression of miR-23b in the Mongolian at high cardiovascular risk was higher than that in healthy Mongolian volunteers. Decrease in ATP-binding cassette transporter A1 caused by miR-23b is responsible for TC accumulation in the Mongolian at high cardiovascular risk. MiR-23b enhanced the oxidized low-density lipoprotein (oxLDL)-induced inflammatory response of THP-1 derived macrophage. MiR-23b regulated nuclear factor-κB (NF-κB) pathway through targeting A20. MiR-23b mediated oxLDL-induced inflammatory response of peripheral blood mononuclear cell in the Mongolian at high cardiovascular risk. Conclusion MiR-23b enhanced oxLDL-induced inflammatory response of macrophages in the Mongolian at high cardiovascular risk through the A20/NF-κB signaling pathway, and thus contributing to atherosclerosis.

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