Extract of Ginkgo Biloba decreased expression of lectin-like oxidized low density lipoprotein receptor-1 induced by TNF-α in bovine aortic endothelial cells

AIM: To investigate the effects of extract of Ginkgo biloba (EGB) on the expression of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) induced by tumor necrosis factor a(TNF-α) in bovine aortic endothelial cells(BAECs). METHODS: The BAECs were incubated with TNF-α, followed by EGB treatment. The effect of EGB on LOX-1 expression was investigated by RT-PCR and Western blotting. The content of endothelial nitric oxide synthase (eNOS) was determined by Western blotting. Potential involvement of signaling pathways of the effects was explored by using related inhibitors of the signal molecules. The concentration of NO^-₂/NO^-₃ was also tested. RESULTS: Increased LOX-1 expression was induced by TNF-α. EGB markedly prevented the increase of LOX-1 expression induced by TNF-α (P<0.05), and this effect was inhibited by inhibitor of NOS (P<0.05). EGB significantly prevented the decrease of eNOS expression induced by TNF-α (P<0.05). EGB also significantly prevented the decrease of NO^-₂/NO^-₃ production induced by TNF-α (P<0.05). CONCLUSION: EGB markedly prevents the increase of LOX-1 expression induced by TNF-α and the effect is mediated by eNOS.     

Recent advances in lectin-like oxidized low-density lipoprotein receptor-1 and atherosclerosis

Lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) is a type-Ⅱ membrane protein belonging to the C-type lectin family molecules, which acts as a cell surface endocytosis receptor for atherogenic oxidized LDL (Ox-LDL). LOX-1 supports the binding internalization and proteolytic degradation of oxidized LDL, but not of significant amounts of acetylated LDL. LOX-1 is initially synthesized as a 40 kD precursor protein with N-linked high mannose-type carbohydrate, which is further glycosylated and processed into a 48-kD mature form. In vivo, endothelial cells that cover early therosclerotic lesions, intimal macrophages and smooth muscle cells in advanced atherosclerotic plaques express LOX-1. LOX-1 is cleaved at membrane proximal extracellular domain and released from the cell surface. Measurement of soluble LOX-1 in vivo may provide novel diagnostic strategy for the evaluation and prediction of atherosclerosis and vascular diseases.     

Effect of oxidized LDL on expression of connexin43 in cultured human umbilical vein endothelial cells

AIM: To investigate the effect of oxidized LDL (ox-LDL) on the expression of gap junction protein connexin43 in cultured human umbilical vein endothelial cells (HUVECs) in vitro. METHODS: Human umbilical vein endothelial cells cultured in normal condition were divided into blank control group, 50 mg/L,100 mg/L and 200 mg/L ox-LDL intervention groups. The mRNA expression of connexin43 in cultured HUVECs was detected with RT-PCR method; while the protein level of connexin43 was determined by the method of immunocytochemistry in the control and 100 mg/L ox-LDL intervention groups 24 h after ox-LDL was given. RESULTS: Different concentrations (50 mg/L, 100 mg/L, 200 mg/L) of ox-LDL up-regulated mRNA expression of connexin43 in cultured HUVECs after 24 h intervention (P<0.01). The protein level of connexin43 in cultured HUVECs intervened with 100 mg/L Ox-LDL for 24 h was up-regulated as compared to the control cells (P<0.01).CONCLUSION: Ox-LDL may up-regulate the expression of connexin43 at mRNA and protein levels in cultured human umbilical vein endothelial cells within short time, indicating that connexin43 plays an important role in the pro-atherosclerotic effect of Ox-LDL.     

Dual roles of oxidized LDL in modulating expression of inflammatory molecules in HUVECs

AIM:To determine the role of LOX-1/PPAR pathway in regulating expression of adhesion molecules elicited by oxidizing low density lipoprotein(Ox-LDL) through Lectin-like oxidized low-density lipoprotein receptor-1(LOX-1) in human umbilical vein endothelial cells(HUVECs). METHODS:HUVECs were incubated with Ox-LDL,poly(I),carrageenan or 15-deoxy-△12,14-prostaglanding J2 (15d-PGJ₂ ). PPAR mRNA and protein were examined by real time RT-PCR and Western blotting. ICAM-1 and E-selectin were detected by RT-PCR and Western blotting respectively. RESULTS:Ox-LDL increased PPAR expression in HUVECs,which was inhibited by pretreatment of HUVECs with LOX-1 blockers. Preincubation of HUVECs with 15d-PGJ₂ attenuated the expression of intercellular adhesion molecule-1(ICAM-1) and E-selectin in response to Ox-LDL. Upregulation of ICAM-1 and E-selectin mediated by Ox-LDL were suppressed more significantly by the combination of 15d-PGJ₂ and polyinosonic acid as compared to either 15d-PGJ₂ or polyinosonic acid alone. CONCLUSION:The results indicate that Ox-LDL exerts a biphasic effects on inflammatory response. It evokes harmful effects by inflammatory injury on one side and protective effects by triggering the LOX-1/ PPAR signaling pathway on the other hand.     

Insulin protects endothelial progenitor cells against functional damage caused by high glucose

AIM: To investigate the impact of various levels of glucose on endothelial progenitor cells (EPCs) proliferation, senescence, and nitric oxide (NO) secretion,and the effect of insulin under high glucose conditions.METHODS: Mononuclear cells were collected from rat bone marrow by density gradient centrifugation, cultured with medium 199, and identified to be EPCs at 7th day by flk-1 and AC133 double staining. EPCss were harvested and incubated with glucose (5, 10, 20, 40 mmol/L) or insulin (0.1, 1, 10, 100 nmol/L) under high glucose conditions for 24 h or 7 days. Proliferative capacity, senescence level and NO secretion (after 24 h of incubation) were subsequently determined.RESULTS: High glucose (40 mmol/L) markedly inhibited EPCs proliferation, accelerated EPCs senescence, and decreased NO production (all P<0.05). Compared with high glucose (40 mmol/L) group, insulin intervention promoted EPCs proliferation, inhibited EPCs senescence (prominent at 1 nmol/L, P<0.05), and enhanced NO secretion (prominent at 10 nmol/L, P<0.05).CONCLUSION: High glucose harms EPCs proliferation and function, while insulin alleviates this jeopardy, indicating the protective role of insulin for the cardiovascular system.     

Effects of insulin and glucose on secretion of tissue-type plasminogen activator and its inhibitor-1 in hypoxic vascular endothelial cells

AIM: To study the effects of insulin and glucose on tissue-type plamingen activator (tPA) and its inhibitor-1 (PAI-1) secretion in cultured human endothelial cells. METHODS: Human endothelial cell line ECV-304 was cultured with glucose and/or insulin at different concentrations with or without hypoxic exposure. RESULTS: The tPA, PAI-1 secretion and ratio of tPA/PAI-1 increased in endothelial cells during hypoxia. Insulin and glucose increased the tPA and PAI-1 secretion in endothelial cells exposed to hypoxia, and increase in tPA/PAI-1 ratio was also observed at 4 h and 8 h. CONCLUSION: Hypoxia stimulates the release of tPA and PAI-1. Insulin and glucose also stimulate the tPA and PAI-1 secretion during hypoxia.     

Proliferation and apoptosis of human umbilical vein endothelial cells induced by oxidized low density lipoprotein

AIM:To investigate proliferation and apoptosis of cultured endothelial cell (ECV-304 cell line) induced by varied concentrations of oxidized low density lipoprotein (ox-LDL). METHODS:Cell morphology, Typan blue test, MTT test, LDH release test, flow cytometry and micro-molecular weight DNA fragment gel electrophoresis of apoptosis were used for the detection of the cytotoxic effects of ox-LDL on ECV-304 cell line.RESULTS:0.1, 1, 10 mg/L ox-LDL could promote proliferation of ECV-304 cells. When the concentration of ox-LDL reached up to 100 mg/L and above, the distinct cytotoxic effect appeared. Further study showed that the apoptosis rate of endothelial cells, induced by ox-LDL of 150 mg/L and 200 mg/L for 12 hours, are 15.86% and 21.89%, respectively. 18 h and above hours after incubation, the apoptosis rate began to decrease and rate of necrosis increased. CONCLUSION:ox-LDL has strong cytotoxic effects on endothelial cells and could give rise to different pathologic process, such as proliferation, apoptosis prophase, apoptosis and necrosis.     

Role of endothelial cell glucose metabolism in vascular development

As an important organ in the body, blood vessels transport oxygen and nutrients to the tissues of the body. Endothelial cells are layers of cells lined with blood vessels whose metabolism is involved in regulating multiple links of vascular development and vascular diseases. Glucose metabolism provides the main energy for endothelial cells, and changes in glucose metabolism regulation affect vascular development and even lead to various diseases. Recently, strategies have been proposed to target endothelial cell metabolism for the treatment of vascular diseases. This article reviews the role of endothelial cell glucose metabolism in vascular development and vascular diseases in recent years.     

Unfractionated heparin ameliorates lipopolysaccharide-induced expression of interleukin-8 in human endothelial cells through Toll-like receptor 4 signaling

AIM:To determine the effect of unfractionated heparin(UFH) on lipopolysaccharide(LPS)-induced expression of interleukin-8(IL-8) and the role of Toll-like receptor 4(TLR4) signaling. METHODS:Human pulmonary microvascular endothelial cells(HPMECs) were either exposed to LPS alone(10 mg/L) or in combination with 100 U/L or 10³ U/L UFH. UFH was added to the cells 15 min prior to stimulation with LPS. Those samples not receiving LPS or UFH received an equal volume of phosphate-buffered saline. The concentrations of IL-8 in the cell culture supernatants were detected by ELISA at 2, 6 and 12 h. The mRNA expression of IL-8, CD14 and TLR4 in HPMECs was detected by real-time fluorescence quantitative polymerase chain reaction at 2, 6 and 12 h. RESULTS:Compared with normal control group, the mRNA expression of IL-8 in LPS group was increased, and reached the peak at 6 h. The protein level of IL-8 reached the peak at 12 h. The mRNA expression of TLR4 in LPS group reached the peak at 6 h. They were down-regulated in UFH group. The mRNA expression of CD14 was not detected. CONCLUSION:The expression of IL-8 is obviously increased in LPS-treated HPMECs. UFH might exert its therapeutic effect through TLR4 signaling.     

High glucose up-regulates TRAIL-R4 expression in THP-1 cells and smooth muscle cells

AIM: To evaluate the expression of TNF-related apoptosis inducing ligand receptor 4 (TRAIL-R4) of THP-1 cells and human aorta smooth muscle cells under high glucose intervention. METHODS: Monocytic cell line THP-1 was incubated with PMA to induce to mature macrophage, Adhesion molecules CD11b and CD11c were assessed by FACS. TRAIL-R4 levels in THP-1 cells treated with different glucose concentrations were determined by Western blotting. The changes of TRAIL-R4 protein expressions were observed at different time points in human aorta smooth muscle cells. Western blotting was employed to evaluate TRAIL-R4 levels after the intervention of PKC activator. RESULTS: Incubation with 160 nmol/L PMA induced mature macrophages. TRAIL-R4 expression was up-regulated after incubation with 20 mmol/L glucose in macrophages. TRAIL-R4 was elevated in a time course manner under high glucose level in human aorta smooth muscle cells. Moreover, activation of PKC induced TRAIL-R4 expressions. CONCLUSION: Up-regulated TRAIL-R4 protein levels induced by high glucose levels might inhibit apoptosis of monocytes and smooth muscle cells and contribute to the progression of atherosclerosis.     

Lectin-like oxidized low density lipoprotein receptor-1 mediates expression of MCP-1 induced by ox-LDL in cultured human vascular endothelial cells

AIM: To investigate the effects of lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) on the expression of MCP-1 in the cultured human unbilical vein endothelial cells (HUVECs). METHODS: Cultured HUVECs was incubated with ox-LDL, or preincubated with carrageenan and polyinosinic acid. LOX-1 and MCP-1 mRNA and protein were determined by RT-PCR and Western blot. RESULTS: Incubation of HUVECs with ox-LDL (from 0-100 mg/L) for 24 h markedly increased the expression of LOX-1 and MCP-1 (mRNA and protien) in a concentration-dependent fashion. Preincubation of HUVECs with carrageenan and polyinosinic acid, the chemical inhibitors of LOX-1, for 2 h, ox-LDL-mediated upregulation of LOX-1 and MCP-1 was suppressed (P<0.05). CONCLUSION: These findings indicate that ox-LDL upregulates MCP-1 and its own endothelial receptor, and ox-LDL-induced MCP-1 is mediated by the action of LOX-1. LOX-1 plays a critical role in the pathogenesis of atherosclerosis.     

Caveolin-1 expression is downregulated by shear stress and TNF-α in human aortic endothelial cells

AIM: To observe effects of shear stress and TNF-α on caveolin-1 expression. METHODS: Cultured human aortic endothelial cells (HAECs) of passage 3-5 were used in the experiment. Cells were exposed to a laminar flow (shear stress 1.0 Pa) by using a parallel rectangular flow chamber for different time. Caveolin-1 mRNA and protein expression were measured by RT-PCR and Western blot, respectively. Caveolin-1 expression of the cells stimulated by TNF-α were also studied to elucidate the influence of this inflammatory factor. RESULTS: After 24 h of exposure to 1.0 Pa shear stress, both of caveolin-1 protein and mRNA expression decreased in HAECs, especially caveolin-1 mRNA expression (P<0.05). No significant decrease in caveolin-1 protein expression was found after 4 h exposure to the shear flow, although there was a decrease in caveolin-1 mRNA expression (P<0.05). TNF-α induced decreases in caveolin-1 protein and mRNA expression in the cells stimulated for 24 h (P<0.05). CONCLUSION: Laminar flow of 1.0 Pa and TNF-α induce decreases in caveolin-1 protein and mRNA expression, which could play an important role in atherogenesis.     

Sinomenine inhibits TNF-α-induced VCAM-1 expression on human umbilical vein endothelial cells

AIM: To investigate whether sinomenine(SN) can decrease TNF-α-induced VCAM-1 expression in human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were isolated from freshly collected umbilical cords.Positive control samples were stimulated with TNF-α, omitting SN. Negative control samples were treated in the same way, omitting TNF-α and SN. Experiment samples were co-cultured with TNF-α and SN at different concentration (0.25, 0.5, and 1.0 mol/L),or TNF-α and dexamethasone(Dex) at concentration of 1.0×10^-6mol/L.Cells were harvested after cultivation with the drugs for 12 hours. VCAM-1 mRNA expression was detected by real-time quantitative PCR, and VCAM-1 expression was detected by flow cytometry (FCM).RESULTS: VCAM-1 mRNA and VCAM-1 were induced by TNF-α. Compared with the positives, the relative VCAM-1 mRNA expression decreased to varying degrees in the experiment groups (P<0.05), and SN at concentration of 0.5 mol/L and 1.0 mol/L inhibited expression of VCAM-1 (P<0.05). SN at concentration of 1.0 mol/L decreased VCAM-1 expression by 28.8%(P<0.05), and SN at concentration of 0.5 mol/L reduced VCAM-1 expression by 21.68%(P<0.05). But SN at concentration of 0.25 mol/L and Dex at concentration of 1.0×10^-6mol/L didnt depress expression of VCAM-1. CONCLUSION: SN may inhibit TNF-α-induced VCAM-1 expression in HUVECs in vitro.     

Overexpression of Jagged1 promotes aged rat-derived endothelial proge-nitor cells differentiating into mature endothelial cells

AIM: To investigate the effect of Jagged1 overexpression on endothelial cell-directional differentiation of aged rat-derived endothelial progenitor cells (EPC).METHODS: Mononuclear cells were obtained from bone marrow of young (1 to 2 months old) or aged (19 to 26 months old) Sprague-Dawley rats and cultured in DMEM/F12 medium supplemented with 10% FBS. EPC were characterized as double positive for DiI-ac-LDL uptake and lectin binding. The experiments were divided into control group, PIRES2-EGFP transfection group, PIRES2-EGFP-Jagged1 transfection group and young rat-derived EPC group in which transfection was not performed. The GFP expression positive cell number was acquired by fluorescence microscopy and the transfection efficiency was calculated. Immunofluorescence, RT-PCR and Western blotting were used to detect the mRNA and protein expression. In vitro vasculogenesis kit was used to test the tube formation ability of EPC.RESULTS: EGFP-Jagged1 transfection induced a significant increase in the expression of Jagged1 in aged rat-derived EPC (P<0.01). Compared with the control, Jagged1 overexpression markedly enhanced the mRNA expression of von Willebrand factor (vWF) and kinase insert domain receptor (KDR) of vascular endothelial grouth factor vWF in aged rat-derived EPC (P<0.01) and improved the EPC-related tube formation (P<0.01). No significant difference between Jagged1 transfection and young rat-derived EPC groups in vWF and KDR mRNA expression and the ability of tube formation was found. CONCLUSION: In endothelial cell-conditioning medium, Jagged1 overexpression significantly promotes aged rat-derived EPC differentiation into mature endothelial cells.     

Effects of high-glucose on proliferation and apoptosis in endothelial progenitor cells of type 2 diabetes mellitus

AIM: This study aimed to observe the effects of high-glucose on proliferation and apoptosis of endothelial progenitor cells (EPCs) in type 2 diabetes mellitus patients,and tried to elucidate their possible role.METHODS: Various concentrations of glucose were added to the culture system of EPCs from 25 cases of type 2 diabetes mellitus patients (DM group) and 25 cases of healthy volunteers (control group).MTT assays were used to detect the proliferative rates.Annexin-V/PI stains were used to detect the apoptotic rates,and RT-PCR to detect the expression level of bcl-2 and bax.RESULTS: Proliferative activity of EPCs in both control group and DM group were attenuated when concentration of glucose was 33 mmol/L,while apoptotic rates increased.No significant change of proliferative rate and apoptotic rate of EPCs in DM group and control group in the presence of 5 mmol/L glucose was observed.The expression level of bax of EPCs in both DM group and control group increased while expression level of bcl-2 did not change much in the presence of 33 mmol/L glucose.CONCLUSION: High-glucose attenuates proliferative activity of EPCs and increases the apoptotic rate.Upregulation of bax may be its possible role.     

Chlamydia pneumoniae enhanced the expression of ICAM-1 in cultured human umbilical vein endothelial cells

AIM: To investigated the effects of Chlamydia pneumoniae (C.pneumoniae) infection on the expression of intercellular adhesion molecule-1 (ICAM-1) in cultured human umbilical vein endothelial cells (HUVECs). METHODS: After propagated in HEP-2 cells, C. pneumoniae organisms were infected to HUVECs. The infection was assessed by ectromicroscope and PCR. The expression of ICAM-1 on HUVECs was detected by flow cytometry before infection and 12 h, 24 h, 48 h, 72 h after infection. RT-PCR was used to detect the ICAM-1 mRNA expression. RESULTS: C.pneumoniae was able to infect cultured HUVECs. After infection, the expression of ICAM-1 on the surface of cultured HUVECs was increased,, the peak was at the time of about 24-48 h; The light quantative RT-PCR analysis demonstrated that the infection also enhanced the ICAM-1 mRNA expression. CONCLUSION: The ability of C.pneumoniae to grow in HUVECs and to stimulate the expression of ICAM-1 protein and mRNA suggests that C.pneumoniae may playa role in atheriosclerosis.