Apoptosis of human umbilical vein endothelial cells (HUVEC) induced by IgA1 isolated from Henoch-Schonlein purpura children.
Yuan Ping,Bo Yan,Ming Gui,Wen-Jun Fei,Qin Zhang,Bo Hu
Asian Pacific journal of allergy and immunology
OBJECTIVE:It had been shown that apoptosis of vascular endothelial cells played an important role in the pathogenesis of HSP. The present study was designed to investigate the apoptosis of vascular endothelial cells induced by isolated IgA1 from sera of HSP patients. METHODS:HUVEC were cultured in 3 different types of media with IgA1 from HSP patients, normal healthy children and simply medium (blank control). Serum IgA1 was purified by jacalin affinity chromatography. The rates of apoptosis in HUVEC incubated with IgA1 were determined by the TUNEL method and flow cytometry, respectively. The expression of bax/bcl-2 and p53 was detected with the methods of Real-time PCR and Westernblot, respectively. RESULT:The results showed that the apoptosis rate of HUVEC by IgA1 isolated from HSP patients was higher than that the normal controls (14.77±2.23% vs 9.97±1.48%) and blank controls (14.77±2.23% vs 2.25±0.77%) (P <0.01). Moreover the rate of HUVEC by IgA1 from normal healthy children was higher than the blank controls (9.97±1.48% vs 2.25±0.77%) (P <0.01). In addition, the bax and P53 expression were up-regulated and the Bcl-2 expression was down-regulated in HUVEC co-cultured with IgA1 isolated from HSP patients for 24 hours. CONCLUSIONS:These findings suggested that IgA1 from HSP patients could induce the apoptosis of HUVEC, which might be related to the vascular endothelial injury of HSP.
Down-Regulation of miR-218-5p Promotes Apoptosis of Human Umbilical Vein Endothelial Cells Through Regulating High-Mobility Group Box-1 in Henoch-Schonlein Purpura.
Yu Shao-Fei,Feng Wan-Yu,Chai Shao-Qing,Meng Xiao-Bo,Dou Zhong-Xia,Zhu Hua
The American journal of the medical sciences
BACKGROUND:Apoptosis of human umbilical vein endothelial cells (HUVECs) plays an important role in the progression of Henoch-Schonlein purpura (HSP). In the present study, we explored the function of miR-218-5p in HUVEC apoptosis and HSP development. MATERIALS AND METHODS:HSP rat model was established and peripheral blood mononuclear cells (PBMC) were isolated. The expression of miR-218-5p and high-mobility group box-1 (HMGB1) protein in HUVECs was determined by quantitative real-time polymerase chain reaction and western blot, respectively. Cell apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. The association between miR-218-5p and HMGB1 was determined by luciferase assay. The endogenous expression of related genes was modulated with recombinant plasmids and cell transfection. RESULTS:MiR-218-5p was down-regulated and HMGB1 was up-regulated in vessels of the lower limb of HSP rats and in HUVECs co-cultured in HSP PBMC supernatant. MiR-218-5p negatively regulated HMGB1 by targeting its 3'-untranslated regions. Over expression of miR-218-5p reversed the increased apoptosis and HMGB1 expression observed in HUVECs co-cultured in PBMC supernatant, whereas miR-218-5p knockdown showed the opposite outcomes. Furthermore, the miR-218-5p mimic demonstrated an inhibitory effect on the apoptosis of HUVECs co-cultured in PBMC supernatant, which was reversed by over expression of HMGB1. In HSP rats, over expression of miR-218-5p attenuated HSP and decreased the level of HMGB1. CONCLUSIONS:MiR-218-5p attenuated HSP at least partly through regulating HMGB1 expression and affecting the function of HUVECs.
Antiendothelial cells autoantibodies in vasculitis-associated systemic diseases.
Guilpain Philippe,Mouthon Luc
Clinical reviews in allergy & immunology
Antiendothelial cell antibodies (AECA) have been detected in healthy individuals, as well as in autoimmune and systemic inflammatory diseases, including systemic vasculitides. AECA have been reported in large vessel vasculitides such as giant cell arteritis and Takayasu arteritis; medium-sized vessel vasculitides, such as polyarteritis nodosa related to hepatitis B virus infection and Kawasaki disease; and small-sized vessel vasculitides, such as Wegener's granulomatosis, microscopic polyangiitis, and Henoch-Schonlein purpura. In Takayasu arteritis and antineutrophil cytoplasm antibody-positive vasculitides, AECA have been reported to correlate with disease activity. A cell-based enzyme-linked immunosorbent assay (ELISA) using cultured human umbilical vein endothelial cells (HUVEC) represent one of the reference techniques for AECA detection, although flow cytometry and immunobloting have also been proposed. AECA might contribute to the pathogenesis of systemic vasculitides and vasculitis-associated diseases through (1) activation of endothelial cells (EC), (2) direct cytotoxic effect due to complement-dependent cytotoxicity or indirect cytotoxic effect secondary to antibody-dependent cytotoxicity, (3) induction of coagulation, (4) induction of apoptosis through the binding of phospholipids or heat-shock protein 60, and (5) induction of EC activation. None of the identified target antigens of AECA is specific for EC, and EC-specific target antigens of AECA remain to be identified in systemic vasculitides.
HMGB1/RAGE pro-inflammatory axis promotes vascular endothelial cell apoptosis in limb ischemia/reperfusion injury.
Mi Lei,Zhang Ying,Xu Yugang,Zheng Xiao,Zhang Xia,Wang Zhu,Xue Ming,Jin Xing
Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie
OBJECTIVE:High-Mobility Group Box 1 (HMGB1) promotes vascular injuries induced by limb Ischemia and Reperfusion (IR), but the molecular mechanisms are not well understood. This study aimed to investigate the role of Receptor for Advanced-Glycation End products (RAGE) in HMGB1-regulated inflammatory response and vascular injury in limb IR using the rat IR and cellular Hypoxia and Reoxygenation (HR) models. METHODS:We analyzed the vascular structure and elastic fiber deposition in rat femoral arteries by histological staining. We determined gene expression in vascular tissues and cells by quantitative RT-PCR, Western blotting and immunofluorescence; analyzed the protein levels in rat serum or cell supernatant by ELISA; and assessed protein interaction by co-immunoprecipitation. We used CCK-8 for analyzing cell viability, and assessed apoptosis by Hoechst staining and flow cytometry. RESULTS:RAGE inhibition by FPS-ZM1 significantly repressed rat vascular injury that was induced by limb IR treatment. HMGB1 and RAGE expression, P38, ERK1/2, P65 and IKBa phosphorylation, as well as HIF-1a, NLRP3, Caspase-1, TNF-a and IL-6 expression and P65 in nucleus, increased in femoral arteries of a rat IR model and HUVEC undergoing HR treatment, whereas all the factors except HMGB1 and RAGE were inhibited by FPS-ZM1 treatment. In addition, we found that HMGB1 binds with RAGE in HUVEC undergoing HR treatment, which was suppressed by FPS-ZM1. Finally, the apoptosis of HUVEC also increased by HR treatment, but repressed under FPS-ZM1 treatment. CONCLUSION:HMGB1 binds with RAGE to promote vascular inflammation and endothelial cell apoptosis, which mediates vascular injury during acute limb IR.
Diallyl trisulfide attenuates hyperglycemia-induced endothelial apoptosis by inhibition of Drp1-mediated mitochondrial fission.
Hao Ying,Liu Hui-Min,Wei Xin,Gong Xue,Lu Zhao-Yang,Huang Zhen-Hao
AIMS:Hyperglycemia induces endothelial cell apoptosis and blood vessel damage, while diallyl trisulfide (DATS) has shown cardiovascular protection in animal models and humans. The aim of this study was to investigate the effects of DATS on inhibition of high glucose-induced endothelial cell apoptosis and the underlying molecular events. METHODS:Human umbilical vein endothelial cells (HUVECs) were incubated with DATS (100 μM) for 30 min and then cultured in high-glucose medium (HG, 33 mM) for 24 h for assessment of apoptosis, glutathione (GSH), reactive oxygen species (ROS), superoxide dismutase (SOD), and gene expression using the terminal deoxyuridine triphosphate nick end labeling (TUNEL), flow cytometry, caspase-3 activity, ROS, SOD, and western blot assays as well as JC-1 and MitoTracker Red staining, respectively. RESULTS:DATS treatment significantly inhibited high glucose-induced HUVEC apoptosis by blockage of intracellular and mitochondrial ROS generation, maintenance of the mitochondrial membrane potential, and suppression of high glucose-induced dynamin-related protein 1 (Drp1) expression. Furthermore, DATS blockage of high glucose-induced mitochondrial fission and apoptosis was through adenosine monophosphate-activated protein kinase (AMPK) activation-inhibited Drp1 expression in HUVECs. CONCLUSIONS:DATS demonstrated the ability to inhibit high glucose-induced HUVEC apoptosis via suppression of Drp1-mediated mitochondrial fission in an AMPK-dependent fashion.
Overexpression of miR‑17‑5p protects against high glucose‑induced endothelial cell injury by targeting E2F1‑mediated suppression of autophagy and promotion of apoptosis.
Yuan Yifeng,Li Xue,Li Maoquan
International journal of molecular medicine
E2 promoter binding factor 1 (E2F1) has been reported to have an important regulatory role in cell survival during hyperglycemic conditions; however, the mechanisms remain to be fully elucidated. Bioinformatics analyses have suggested that microRNA (miR)‑17‑5p targets the 3'untranslated region (3'UTR) of E2F1. The aim of the present study was to characterize the protective effect of miR‑17‑5p/E2F1 on human umbilical vein endothelial cells (HUVECs) under high glucose (HG) conditions, to confirm the regulatory effect of miR‑17‑5p on E2F1/AMP‑activated protein kinase α2 (AMPKα2)‑mediated apoptosis and E2F1/mammalian target of rapamycin complex 1 (mTORC1)‑mediated autophagy. Bifluorescein experiments were performed to characterize the interaction between miR‑17‑5p and E2F1. The Cell Counting Kit‑8 assay, flow cytometry, immunofluorescence, and reverse transcription‑quantitative polymerase chain reaction and western blot analyses were used to detect cell viability, apoptosis, autophagy, and relative mRNA and protein expression, respectively. The results showed that HG induced the downregulation of miR‑17‑5p and upregulation of E2F1 during HUVEC injury. The downregulation of E2F1 inhibited HG‑induced HUVEC dysfunction by suppressing mTORC1‑mediated inhibition of autophagy and AMPKα2‑mediated promotion of apoptosis. The results suggested that inhibiting the expression of E2F1 protected against HG‑induced HUVEC injury via the activation of autophagy. The overexpression of miR‑17‑5p inhibited E2F1‑mediated HUVEC injury under HG conditions, which was reversed following transfection with an E2F1‑overexpression vector. The bifluorescein experiments showed that miR‑17‑5p targeted the 3'UTR of E2F1. Taken together, the results suggested that the expression of miR‑17‑5p inhibited HG‑induced endothelial cell injury by targeting E2F1.
[Gly14]-Humanin Prevents Lipid Deposition and Endothelial Cell Apoptosis in a Lectin-like Oxidized Low-density Lipoprotein Receptor-1-Dependent Manner.
Ding Yu,Feng Yue,Zhu Wawa,Zou Yutian,Xie Ying,Wang Fen,Liu Chun-Feng,Zhang Yanlin,Liu Huihui
Oxidized low-density lipoprotein (Ox-LDL) may induce apoptosis and dysfunction of vascular endothelial cells, contributing to the initiation and development of atherosclerosis and lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) plays a central role in Ox-LDL uptake in the course of atherogenesis. Humanin (HN), a mitochondrial-derived peptide, was recently demonstrated to exert a protective role against endothelial dysfunction and Ox-LDL-induced progression of atherosclerosis. The HN analog HNGF6A (HNG) modulates cholesterol metabolism in macrophage RAW 264.7 cells. However, whether HNG affects Ox-LDL metabolism in endothelial cells is unknown. In this study, we investigated the effect of HNG on Ox-LDL accumulation in human umbilical vein endothelial cell (HUVEC) and its underlying mechanisms. HUVEC were preincubated with HNG for 1 h before addition of Ox-LDL. Total cholesterol content was measured by using a tissue total cholesterol assay kit and flow cytometry. Cell viability was measured by CCK8 assay. Protein content was examined by Western blot assays. Flow cytometry was used to identify apoptotic cells. Flow cytometry and tissue total cholesterol assays showed that HNG reduced Ox-LDL accumulation in HUVEC. In addition, HNG inhibited Ox-LDL-induced apoptosis of HUVEC. Western blot results showed that HNG reduced LOX-1 protein content. However, when LOX-1 was knocked down or inhibited, the effect of HNG in reducing Ox-LDL aggregation and apoptosis in HUVEC disappeared. Our study demonstrated that HNG reduces lipid aggregation and apoptosis in HUVEC in a LOX-1-dependent manner.
Propranolol suppresses HUVEC viability, migration, VEGF expression, and promotes apoptosis by downregulation of miR-4295.
Zhao Feng,Yang Xiaoliang,Xu Guangqi,Bi Jianhai,Lv Renrong,Huo Ran
Journal of cellular biochemistry
Infantile hemangioma (IH) is a common benign tumor. Human umbilical vein endothelial cells (HUVECs) have the potential of stem cells, which has been widely used in vascular endothelial cell experiments. Oral propranolol was first reported to treat hemangioma in 2008. However, the role of propranolol in IH remains unclear. Therefore, in this study, we investigated the effects of propranolol on HUVECs in vitro, to explore the underlying mechanism of propranolol in IH. HUVECs were treated with 0.15, 1.5, and 15 μM of propranolol, and transfected with microRNA-4295 (miR-4295) mimic. Cell viability, migration, and apoptosis were examined using Cell Counting Kit-8, transwell assay, and flow cytometry analysis, respectively. In addition, the expressions and concentrations of miR-4295, vascular endothelial growth factor (VEGF), VEGF-A, FLT1, FLT2, and FOXF1 were assessed using real-time polymerase chain reaction, Western blot assay, and enzyme-linked immunosorbent assay. We found that 15 μM of propranolol decreased HUVEC viability the most. Then, cell migration and the concentrations of VEGF and VEGF-A were reduced, and apoptosis was increased when treated with propranolol. Meanwhile, the expressions of VEGF, VEGF-A, FLT1, FLT2, and FOXF1 were downregulated by propranolol exposure. Further study showed that miR-4295 expression was upregulated in IH tissues, and propranolol treatment downregulated miR-4295 expression in HUVECs. MiR-4295 overexpression alleviated the reductions of viability, migration, and factors expression, as well as the increase of apoptosis. Propranolol suppressed HUVEC viability, migration, the expression of VEGF, VEGF-A, FLT1/2, FOXF1, and promoted apoptosis via downregulation of miR-4295. This study lays a foundation for further study of the effect of propranolol on IH.
The suppression of ox-LDL-induced inflammatory response and apoptosis of HUVEC by lncRNA XIAT knockdown via regulating miR-30c-5p/PTEN axis.
Hu W-N,Duan Z-Y,Wang Q,Zhou D-H
European review for medical and pharmacological sciences
OBJECTIVE:Exposure of oxidized low-density lipoprotein (ox-LDL) could cause dysfunction of HUVEC, thus leading to atherosclerosis development, which is a common inflammatory vascular disease. Long noncoding RNA X-inactive specific transcript (XIST) has been reported to be implicated in atherosclerosis. However, the mechanism by which this lncRNA participates in the progression of atherosclerosis is poorly defined. MATERIALS AND METHODS:HUVEC challenged by ox-LDL were used as a cellular model of atherosclerosis. Cell viability, apoptosis, LDH release, and inflammatory cytokines secretion were detected by MTT, flow cytometry, and ELISA assays. The expression levels of XIST, microRNA (miR)-30c-5p, and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) were measured by quantitative Real Time-Polymerase Chain Reaction and Western blot. The target interaction between XIST and miR-30c-5p or miR-30c-5p and PTEN was validated by the Luciferase reporter assay and RNA immunoprecipitation. RESULTS:Treatment of ox-LDL induced cell apoptosis and inflammatory cytokines release in HUVEC. XIST expression was enhanced in HUVEC treated by ox-LDL, and its knockdown decreased cell apoptosis and inflammatory response in ox-LDL-treated cells. MiR-30c-5p was a target of XIST and its overexpression suppressed cell apoptosis and inflammatory response induced by ox-LDL, which was weakened by the introduction of XIST. PTEN was a target of miR-30c-5p, and its interference led to great inhibition of cell apoptosis and inflammatory response induced by ox-LDL in HUVEC, while this effect was attenuated by miR-30c-5p deficiency or XIST overexpression. CONCLUSIONS:XIST knockdown suppresses inflammatory response and apoptosis of HUVEC stimulated by ox-LDL by increasing miR-30c-5p and decreasing PTEN.