Effects of asymmetric dimethylarginine on apoptosis and expression of p38 mitogen-activated protein kinase in endothelial outgrowth cells

AIM: To investigate the effects of asymmetric dimethylarginine (ADMA) on apoptosis, adhesion ability and phosphorylation of p38 mitogen-activated protein kinase in endothelial outgrowth cells (EOCs). METHODS: The mononuclear cells were harvested from umbilical cord blood by Ficoll density gradient centrifugation, and induced into EOCs and then expanded in vitro. The endothelial characteristics of EOCs were identified by immunostaining and fluorescent staining. The EOCs were treated with different concentrations of ADMA (0, 1, 5, 10, 30 μmol/L) for 48 h. Apoptotic incidences of EOCs were quantitatively determined by flow cytometry. The morphologic changes of the apoptotic cells were visualized by DAPI staining and Annexin-V/PI staining. Adhesion ability of EOCs was measured by replacing the cells on dishes and adherent cells were counted under the inverted microscopy. The p38MAPK activity was evaluated by immunoblotting technique with phospho-p38MAPK antibody. RESULTS: EOCs possessed many endothelial characteristics. Immunostaining showed that the surface antigen factor VIII, CD34 and Flk-1 were positive. The fluorescent staining revealed that EOCs were double positive for Dil-ac-LDL-uptaking and FITC-UEA-I-lectin binding. ADMA (1-30 μmol/L) induced apoptosis of EOCs in a dose-dependent manner (P<0.01). Obvious change of apoptotic morphology in EOCs incubated with ADMA was observed with DAPI staining and Annexin-V/PI staining. ADMA (5-30 μmol/L) inhibited the adhesion ability of EOCs, whereas ADMA at concentration of 1 μmol/L had no effect. ADMA (5-30 μmol/L) induced phosphorylation of p38MAPK in a dose-dependent manner (P<0.01). CONCLUSION: ADMA induces apoptosis and inhibits adhesion ability in EOCs. Activated p38MAPK might be involved in the course of apoptotic effects of ADMA.     

Effects of high hydrostatic pressure on ADMA metabolism in human vascular endothelial cells and the role of RAS

AIM:To observe the effects of high hydrostatic pressure on asymmetric NG, NG-dimethyl-L-arginine (ADMA) metabolism of human vascular endothelial cells (HUVECs), and the role of renin-angiotensin system (RAS). METHODS:Cultured HUVECs of 3-6th passage were exposed to atmosphere (0 mmHg, APC), 120 mmHg (MPC), 180 mmHg (HPC). There were three groups in each pressure condition, one as control, the other two were interfered with captopril （Cap, 10 μmol/L or 100 μmol/L） or irbesartan （Irb, 10 μmol/L or 100 μmol/L） respectively. Cell proliferation was quantified by determining hexosaminidase activity at 12 h. Concentration of ADMA in conditioned medium was measured by high performance liquid chromatography (HPLC) at 12 h. RESULTS:Compared with APC group, ADMA concentration increased prominently in MPC and HPC (4.69±0.37 and 4.48±0.39 vs 0.75±0.05，P<0.01), but no difference was found between MPC and HPC group. ADMA concentration was not influenced by Cap and Irb in APC, but obviously reduced in MPC and HPC in a dose-dependent manner. CONCLUSION:ADMA is upregulated by high hydrostatic pressure and RAS is involved.     

Fenofibrate increases endothelial nitric oxide synthase expression in bovine aortic endothelial cells

AIM: We hypothesize that peroxisome proliferator-activated receptor α(PPARα) agonists act directly on nitric oxide (NO) production in vascular endothelium. Thus, the purpose of this study is to investigate the effects of fenofibrate on endothelial NO synthase(eNOS) activity and its expression in cultured vascular endothelial cells. METHODS: Bovine aortic endothelial cells (BAECs) were treated with the PPARα activator fenofibrate. The eNOS activity and the expression of eNOS protein and its mRNA were determined. RESULTS: Our data show that fenofibrate increased eNOS activity in a dose-and time-dependent manner. At the concentration of 10 μmol/L or more, fenofibrate treatment caused a significant increase in eNOS activity. The maximal increase in eNOS activity(2.32±0.47 fold of the control) was observed with 50 μmol/L fenofibrate treatment for 48 h. Fenofibrate failed to increase eNOS activity at 1 and 12 h. RT-PCR analysis demonstrated that eNOS mRNA relative to β-actin mRNA significantly increased at concentrations of 5 μmol/L or more. It reached 2.08±0.33 fold of the control with 50 μmol/L fenofibrate. Significant increase in eNOS mRNA levels was observed after 6 h, and lasted for 48 h. The peak increase in eNOS mRNA levels(2.13±0.30 fold of the control,P<0.01) was observed with 50 μmol/L fenofibrate treatment for 12 h. Longer incubation of cells with 50 μmol/L fenofibrate caused no further increase. The treatment of BAECs with fenofibrate for 48 h demonstrated a concentration-dependent increase in eNOS protein levels as measured by Western blot analysis. Densitometric analysis indicated that there was a significant increase in eNOS to β-actin ratios after fenofibrate treatment at concentrations of 10,50 and 100 μmol/L(1.80±0.45, 2.70±0.42 and 2.20±0.32 fold of the control, respectively, P<0.01). The significant increase in eNOS protein levels was observed 12 h after treatment and lasted for 48 h. CONCLUSION: PPARα activator fenofibrate, enhances endothelial NO production by directly upregulating eNOS expression and activity.     

Effects of carvedilol on metabolic mechanism of endogenous NOS inhibitor in human umbilical vein endothelial cells treated with oxidized free radical

AIM: To observe the effects of oxidized free radical (OFR) on dimethylarginine dimethylaminohydrolase (DDAH) activity and concentration in human umbilical vein endothelial cells (HUVECs), and to investigate the metabolic mechanism of endogenous NOS inhibitor and the role of carvedilol. METHODS: HUVECs of 3-6th passage, cultured with modified Jaffes method, were divided into three groups: (1) cells cultured with equivalent DMEM medium as control; (2) OFR intervention groups, 0.01 mmol/L, or 0.1 mmol/L OFR was added respectively; (3) drug intervention groups: 0.1 mmol/L OFR plus 10 μmol/L carvedilol. ADMA, nitric oxide (NO), endothim (ET), L-citrulline concentrations and the activity of NOS in conditioned medium were measured after 24 h exposure. ADMA concentration in the conditioned medium was determined by high-performance liquid chromatography. Western blotting was performed to evaluate DDAH expression. RESULTS: Compared with control group, ADMA and ET concentrations were increased, while the level of NO and the activity of NOS decreased and relevant to the concentrations of OFR. We assayed DDAH activity by determining L-citrulline formation from ADMA. The concentration of L-citrulline was decreased, while the DDAH expression had no obvious change. With the role of carvedilol, ADMA, ET concentrations were decreased, while the level of NO, L-citrulline and the activity of NOS increased. CONCLUSION: Endothelial dysfunction induced by OFR is associated with the increase in ADMA concentration and reduction of DDAH activity, but not DDAH expression. Carvedilol promotes the degradation of ADMA through increasing activity of DDAH and improving endothelium function.     

Effect of 15(S)-hydroxyeicosate traenoic acid (15-HETE) on eNOS Thr495 phosphorylation in pulmonary artery endothelial cells

AIM: To examine the effect of 15(S)-hydroxyeicosate traenoic acid (15-HETE) on eNOS activity in pulmonary artery endothelial cells (PAECs).METHODS: 1.Rat pulmonary artery (PA) rings of intrapulmonary arteries were suspended under isometric tension in oxygenated Krebs buffer in order to observe the differences on constriction induced by 15-HETE using nitro-L-arginine methyl ester (L-NAME, 10-4 mol/L, an inhibitor of eNOS) and removal of endothelium.2.The bovine PAECs was cultured for NO assay by Greiss reaction.3.Immunoprecipitation (IP) and Western blotting (WB) were used to analyze the cell extracts from bovine PAECs treated with 2×10-6 mol/L 15-HETE for 30 and 60 min, using phosphatase-eNOS (Thr495) antibody for IP and eNOS antibody for WB.RESULTS: 1.Inhibition of eNOS with L-NAME and removal of endothelium significantly enhanced 15-HETE induced contractions in rat PA rings in a concentration-dependent manner (P＜0.05, P＜0.01).2.15-HETE increased phospho-eNOS (Thr495) levels (P＜0.01) in cultured bovine PAECs.3.10-6 mol/L 15-HETE (P＜0.05) did significantly reduce nitrite production, whereas 10-5 mol/L cinnamy l 3, 4-dihydroxy-［alpha］-cyanocinnamate (CDC, P＜0.05), 10-4 mol/L nordihydroguiairetic acid (NDGA, P＜0.01) significantly increased nitrite production, as measured by a spectrophotometric assay.CONCLUSION: 15-HETE inhibits eNOS activity and decreases production of NO (NO-2/NO-3).The pathway of eNOS/NO is involved in 15-HETE-induced contraction in the rat PA.     

Effects of hypoxia on endothelial nitric oxide synthase expression in cerebral artery endothelial cells

AIM: To investigate the molecular mechanism by which hypoxia affect the endothelial nitric oxide synthase (eNOS) expression in cerebral artery endothelial cells (CAECs). METHODS: Primary cultured porcine CAECs were exposed to hypoxia for 2 h, 6 h, 12 h, 24 h and 48 h. The eNOS mRNA level was determined by RT-PCR. The level of eNOS protein was detected by Western blotting. After specific PKC inhibitors BIM Ⅰ(1 μmol/L) and G6983 (1 μmol/L) were added, CAECs were exposed to hypoxia for 24 h. The effect of hypoxia on eNOS mRNA stability was analyzed after actinomycin D was added. RESULTS: After exposure to hypoxia for 2 h, the levels of eNOS mRNA and protein in CAECs were increased. The levels of eNOS mRNA and protein reached peak after 12 h of hypoxia (about 2.5 fold and 2.0 fold, respectively, compared to control), and remained at higher level even after 48 h of hypoxia. Moreover, hypoxia did not change the stability of eNOS mRNA. The specific PKC inhibitors BIM Ⅰ and G6983 attenuated significantly the effects of hypoxia on eNOS gene expression. CONCLUSION: These results suggest that hypoxia may enhance the expression of eNOS gene in CAECs through PKC signaling pathway, which might be one of the mechanisms of cerebral artery dilation and neuroprotection during cerebral hypoxia.     

Roles of protein phosphatase 4 in down-regulation of eNOS Ser633 phosphorylation induced by palmitic acid in human umbilical vein endotheli?al cells

AIMTo investigate the roles of protein phosphatase 4 (PP4) in down-regulation of endothelial nitric oxide synthase (eNOS) Ser633 phosphorylation induced by palmitic acid (PA). METHODSHuman umbilical vein endothelial cells (HUVECs) were treated with PA at 25 μmol/L, 50 μmol/L, 100 μmol/L and 200μmol/L for 36 h, or treated with PA at 100 μmol/L for 12 h, 24 h, 36 h and 48 h. Protein phosphatase 2A (PP2A) family inhibitor fostriecin (FST, 20 nmol/L) or okadaic acid (OA, 5 nmol/L) was selected to pretreat the HUVECs for 30 min. Protein phosphatase 4 catalytic subunit (PP4c) siRNA or protein phosphatase 2A catalytic subunit (PP2Ac) siRNA was transfected into the HUVECs. The protein expression levels of of eNOS, PP4c and PP2Ac, as well as the level of eNOS Ser633 phosphorylation, were detected by Western blot. The intracellular nitric oxide (NO) content was measured by DAF-FM DA. RESULTS(1) Compared with control group, the levels of eNOS Ser633 phosphorylation were decreased in PA groups in which the HUVECs were treated with 25 μmol/L, 50 μmol/L, 100 μmol/L and 200 μmol/L PA for 36 h (P<0.05) and 100 μmol/L PA for 24 h, 36 h and 48 h (P<0.05). No significant difference in the level of total eNOS protein expression among all the groups was observed. (2) Compared with control group, both FST and OA pretreatment reversed the reduction of eNOS Ser633 phosphorylation (P<0.05) and the decrease in intracellular NO content (P<0.05) induced by PA. No significant difference in the level of total eNOS protein expression among all the groups was observed. (3) Compared with si-Control group, the PP4c protein expression was significantly reduced (P<0.05), while the level of eNOS Ser633 phosphorylation was significantly increased in si-PP4c group (P<0.05). Although the levels of PP2Ac protein expression declined significantly (P<0.05), the level of eNOS Ser633 phosphorylation remained unchanged in si-PP2Ac group. No significant differencein the level of total eNOS protein expression among all the groups was found. CONCLUSION PA significantly reduces the level of eNOS Ser633 phosphorylation and the content of NO in the HUVECs, which may be due to PA inducing the activation of the PP2A family member PP4 rather than PP2A.     

Rosiglitazone inhibits osteoclastogenesis in rheumatoid arthritis by down-regulating RANKL expression and suppressing ERK phosphorylation

AIM: To investigate the effects of rosiglitazone on fibroblast-like synoviocyte (FLS)-induced osteoclastogenesis in rheumatoid arthritis (RA) and the related mechanism. METHODS: RA-FLS were cocultured with peripheral blood monocytes from healthy volunteers in the presence of macrophage colony-stimulating factor (M-CSF) and rosiglitazone. Osteoclasts were assayed by tartrate-resistant acid phosphatase (TRAP) staining. Resorption lacunae area was identified by toluidine blue staining and quantified by image analysis software. The mRNA expression of RANKL and OPG was evaluated by real-time PCR, and the protein levels of RANKL, OPG, p-ERK, p-p38 and p-JNK were measured by Western blot. RESULTS: Compared with control group (without rosiglitazone treatment), rosiglitazone at concentration of 15 μmol/L significantly decreased the number of osteoclasts (P<0.01) and resorption lacunae area (P<0.05). The expression of RANKL at mRNA and protein levels was significantly down-regulated by rosiglitazone at concentration of 15 μmol/L, while the mRNA and protein expression of OPG was up-regulated (P<0.01). Rosiglitazone (15 μmol/L) significantly decreased the protein level of p-ERK (P<0.05), but not the protein level of p-p38 or p-JNK (P>0.05). CONCLUSION: Rosiglitazone inhibits RA-FLS-induced osteoclast formation and its resorption activity by down-regulating RANKL expression and ERK phosphorylation, suggesting that rosiglitazone may inhibit RA osteoclastogenesis and bone resorption.     

Puerarin pretreatment protects human umbilical vein endothelial cells against hypoxia/reoxygenation injury via increasing protein expression of endothelial nitric oxide synthase

AIM: To investigate the protective effects of puerarin (PUE) pretreatment on hypoxia/reoxygenation (H/R) injury in human umbilical vein endothelial cells (HUVECs), as well as its possible mechanism and the signal transduction pathways involved. METHODS: HUVECs were randomly divided into normal control group, H/R group, PUE pretreatment group and PUE+H/R group (1.0×10^-3 mol/L, PUE pretreated the cells for 24 h before H/R). The protein expression of endothelial nitric oxide synthase (eNOS) was measured by Western blot. The activity of constitutive NOS (cNOS) was determined via chemical colorimetric methods. Apoptosis of HUVECs was detected by TUNEL assay. In addition, the cells were treated with ERK inhibitor U0126 (1.0×10^-5 mol/L) or PKB/Akt inhibitor LY294002 (5.0×10^-5 mol/L) for 1 h before PUE pretreatment, and then H/R was performed.RESULTS: Compared with control group, H/R decreased the protein expression of eNOS (P<0.05), and PUE pretreatment up-regulated it (P<0.05). This effect of PUE was inhibited by U0126 or LY294002 (P<0.05). Compared with control group, the activity of cNOS decreased in H/R group (P<0.05), while it increased after PUE pretreatment (P<0.05). Compared with control group, the apoptotic index significantly increased in H/R group (P<0.01). PUE pretreatment reduced the apoptotic index (P<0.01). CONCLUSION: H/R decreases the protein expression and enzyme activity of eNOS in HUVECs, and induces apoptosis of HUVECs. PUE pretreatment up-regulates the protein expression and enzyme activity of eNOS, and reduces the apoptosis of HUVECs with H/R injury. The protective effect of PUE might be through increasing eNOS protein expression via ERK1/2 and PKB/Akt signaling pathways.     

Rosuvastatin inhibits up-regulation of inflammatory factors in endothelial outgrowth cells induced by C-reactive protein

AIM：To study the effects of rosuvastatin on C-reactive protein (CRP)-induced expression of inflammatory factors in endothelial outgrowth cells (EOCs). METHODS：Mononuclear cells (MNCs) were isolated from human umbilical cord blood by density gradient centrifugation. EOCs were treated with CRP at concentration of 5, 10, 25 and 50 mg/L and were exposed to 50 mg/L CRP for different time (0, 3, 6 and 12 h). In addition, EOCs were pre-incubated with rosuvastatin at different concentrations (10-8, 10-7 and 10-6mol/L) for 12 h and then stimulated with 50 mg/L CRP. The mRNA levels of IL-8, MCP-1, VCAM-1 and ICAM-1 in EOCs were measured by quantitative PCR. The protein levels of MCP-1 and VCAM-1 were detected by ELISA. RESULTS：CRP dose-dependently increased the mRNA levels of inflammatory factors (IL-8, MCP-1, VCAM-1 and ICAM-1) and protein levels of MCP-1 and VCAM-1. The mRNA levels of IL-8, VCAM-1, and ICAM-1 in EOCs reached to the peak at 6 h, while MCP-1 peaked at 3 h when treated with 50 mg/L CRP. The protein levels of MCP-1 and VCAM-1 increased in a time-dependent manner. The mRNA and protein levels of the inflammatory factors were significantly decreased by treatment with rosuvastatin at different concentrations. CONCLUSION：CRP is not just an inflammatory marker as CRP induces inflammation in EOCs. Rosuvastatin attenuates CRP-induced inflammatory response in EOCs, indicating a new target for the prevention of atherosclerosis.     

2,3,5,4’-Tetrahydroxystilbene-2-O-β-D-glucoside protects against LPC-induced endothelial cell injury

AIM: To observe the protective effect of 2,3,5,4’-tetrahydroxystilbene-2-O-β-D-glucoside (TSG) on lysophosphatidylcholine (LPC)-induced vascular endothelial cell injury. METHODS: The 3rd and 4th generations of human umbilical vein endothelial cells (HUVECs) were cultured in vitro and propagated. The cells were randomly divided into 3 groups: control group, model group (LPC) and experimental group (TSG+LPC). The cells in control group were not treated with any reagent. To establish endothelial cell injury model, LPC was administered to HUVECs at concentration of 10 mg/L and incubated with the cells for 24 h. In TSG+LPC group, TSG was administered to HUVECs at concentrations of 10.0, 1.0 and 0.1 μmol/L 1 h before administration of LPC, and then the cells were incubated for 24 h. The cell viability, the content of asymmetric dimethyl arginine (ADMA) and NO, and apoptotic rate were detected. RESULTS: Compared with control group, ADMA content in the cell culture supernatants and apoptotic rate of the HUVECs in LPC group were significantly increased, while the NO content and cell viability were notably decreased. Compared with LPC group, ADMA content and apoptotic rate in TSG+LPC group was significantly decreased, while the NO content and cell viability were notably increased. CONCLUSION: TSG may protect LPC-injured vascular endothelial cells by attenuating the expression of ADMA and enhancing the production of NO, thus inhibiting apoptosis and increasing cell survival rate.     

PPARs ligands antagonizes Ang-induced decrease in endothelial NO production by upregulating eNOS expression

AIM: We hypothesized that PPARγ ligands stimulate endothelial-derived nitric oxide (NO) release to protect the vascular wall. Thus, the purpose of this study is to investigate the effects of ciglitazone (Cig) and fenofibrate (Fen) on angiotensin Ⅱ (AngⅡ)-induced decrease in endothelial NO synthase (eNOS) expression and NO production in human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were preincubated for 24 h with Cig (10-7, 10-6, 10-5, 10-4 mol/L) or Fen (10-5 and 10-4 mol/L), then incubated for 12 h with 10-7 mol/L AngⅡ. Total RNA was extracted, and the expression of mRNA and protein of eNOS was assessed by RT-PCR and Western blotting. NO production was measured by Griees method. RESULTS: In the presence of 10-7 mol/L AngⅡ for 12 h, NO production in cultured HUVECs was decreased (P<0.01). Cig and Fen pretreatments enhanced NO production (P<0.01) and antagonized Ang-induced decrease in eNOS mRNA and protein levels in HUVECs. CONCLUSION: PPARγ activator, ciglitazone, and PPARα activator, fenofibrate, antagonize Ang-induced decrease in endothelial NO production by directly upregulating eNOS expression.     

Effects of histamine and hypoxia on eNOS gene expression in cultured porcine pulmonary and aorta endothelial cells

AIM: To investigate the effect of histamine and hypoxia on the expression of eNOS mRNA and protein in cultured porcine pulmonary artery and aorta endothelial cells. METHODS: Semi-quantitative RT-PCR and immuno-cytochemistry were used. RESULTS: (1) Histamine increased eNOS mRNA expression in a dose-and time dependent manner. For pulmonary endothelial cells, the effect reached peak when exposed to 10-5 mol/L histamine in 24 h. eNOS mRNA level was increased to 178.2%±7.7% (P<0.01) compared with control. eNOS protein was also enhanced to 173%±47% (P<0.01) compared with control. For aorta endothelial cells, the effect reach peak when exposed to 10-6 mol/L histamine in 24 h. The eNOS mRNA level was increased to 177.4%±14.3% (P<0.01) compared with control. The eNOS protein was also enhanced to 165%±54% (P<0.01). (2) The eNOS mRNA was enhanced in pulmonary endothelial cells after exposed to hypoxia for 12 h and reached peak in 24 h, increasing to 151.0%±9.1% (P<0.01). The protein expression was also enhanced to 216%±44% (P<0.01) compared with control. But there was no significant change in eNOS mRNA and protein expression in aorta endothelial cells during hypoxia. CONCLUSION: The experiments show that histamine increases the endothelial eNOS expression in both pulmonary and aorta endothelial cells, whereas hypoxia only increases eNOS expression in pulmonary endothelial cells. This may account partly for the different responses of pulmonary circulation and systemic circulation to hypoxia.     

Effect of protein S-nitrosylation on cAMP response element-binding protein activity in RSC-364 cells

AIM: To investigate the effect of protein S-nitrosylation on cAMP response element-binding protein(CREB) activity in RSC-364 cells. METHODS: ① RSC-364 cells cytoplasmic extracts were incubated with 100 μmol/L GSH (control chemical), 100 μmol/L GSNO (a donor of NO) in the presence or absence of 10 mmol/L DTT (inhibitor of S-nitrosylation) for 15 min at room temperature. The S-nitrosylated proteins were determined by biotin switch assay. The expression of S-nitrosylated proteins was assayed by Western blotting. ② RSC-364 cell nuclear extracts were subjected to S-nitrosylation and electrophoretic mobility shift assay (EMSA) to analyze the CREB DNA binding activity after 1 h stimulation of rIL-1β (10 μg/L). RESULTS: GSNO obviously increased the expression of nitrosylated proteins and inhibited the CREB activity, which was reversed by DTT. CONCLUSION: S-nitrosylation may inhibit the CREB activity in RSC-364 cells.     

Naringin protects human umbilical vein endothelial cells against injury induced by high glucose through PI3K/AKT/eNOS pathway

AIM: To investigate the protective effect of naringin (Nar) on the injury of human umbilical vein endothelial cells (HUVECs) induced by 33 mmol/L high glucose (HG) and to explore its possible mechanisms.METHODS: The injury model was established by treating HUVECs with HG medium for the indicated time (6, 12, 24, 48 and 72 h), and then the levels of NO, eNOS and p-eNOS were detected, respectively. The effects of Nar on high glucose-induced endothelial cell injury were observed. HUVECs were treated with Nar at concentrations of 5, 10, 25, 50 and 100 mg/L for 6 h, 12 h, 24 h, 36 h and 48 h. The levels of NO in the supernatants were measured. The effects of Nar on HG-injured HUVECs were explored by treating the cells with 10 μmol/L of LY294002, a PI3K inhibitor, or 0.5 μmol/L of AKT inhibitor Ⅳ, an AKT inhibitor, and then the levels of NO, PI3K, AKT, eNOS and their phosphorylated proteins were determined by Western blot.RESULTS: Nar at concentration of 50 mg/L significantly attenuated the injury of endothelial cells induced by high glucose (P<0.01), and the protective effects of Nar were abolished by pretreating with the inhibitor of PI3K or AKT (P<0.01).CONCLUSION: Nar protects endothelial cells against the injury induced by high glucose through PI3K/AKT/eNOS pathway.     

Effect of rosiglitazone on expression of AQP1, VEGF-A and COX-2 in human peritoneal microvascular endothelial cells

AIM: To investigate the effect of rosiglitazone on the expression of aquaporin-1 (AQP1), vascular endothelial growth factor-A (VEGF-A) and cyclooxygenase-2 (COX-2) in human peritoneal microvascular endothelial cells.METHODS: Cultured peritoneal microvascular endothelial cells were divided into 4 groups. The morphological changes of the cells were observed under inverted microscope. The protein expression of AQP1, VEGF-A and COX-2 in human peritoneal microvascular endothelial cells was determined by Western blotting. The mRNA expression of AQP1, VEGF-A and COX-2 in the cells was measured by real-time PCR. RESULTS: Rosiglitazone stimulated the proliferation of the cells. Rosiglitazone up-regulated the expression of AQP1, and down-regulated the expression of VEGF-2 and COX-2 at mRNA and protein levels in the cells. The PPAR-γ antagonist GW9662 partly inhibited the up-regulation of AQP1 expression by rosiglitazone (P<0.05), but had no obvious effect on the expression of VEGF-A and COX-2 (P>0.05). CONCLUSION: Rosiglitazone up-regulates the expression of AQP1 and down-regulates the expression of VEGF-A and COX-2 in human peritoneal microvascular endothelial cells, thus promoting water transportation and attenuating peritoneal fibrosis during peritoneal dialysis.     

Effects of oxygen free radicals and captopril on endogenous NOS inhibitor in human vascular endothelial cells

AIM: To observe the effects of oxygen free radical (OFR) and captopril on the level of asymmetric NG, NG-dimethyl-L-arginine (ADMA) in human vascular endothelial cells (HUVECs).METHODS: HUVECs of 3-6 th passage, cultured with modified Jaffes’ method, were used in the experiment and divided into three groups: (1)Cells cultured with equivalence of DMEM medium as control; (2)OFR intervention groups, OFR at concentrations of 0.01 mmol/L, or 0.1 mmol/L, respectively, were added to the cell culture; (3)Drug intervention groups: the cell culture was treated with 0.1 mmol/L of OFR combined with 50 mg/L or 100 mg/L of captopril, respectively. Concentrations of ADMA, L-arginine, nitric oxide(NO), endothelin(ET) and the activity of angiotensin-converting enzyme(ACE) in conditioned medium were measured after 24 h exposure. RESULTS: Concentrations of ADMA, ET and the activity of ACE were increased, while the amount of NO decreased in OFR intervention groups compared with control group. After treatment with captopril, ADMA, ET concentrations and the activity of ACE were decreased, while the amount of NO increased, but the level of L-arginine had no obvious change. CONCLUSIONS: OFR induces endothelial dysfunction through increasing ADMA concentration, while captopril relieves endothelial dysfunction induced by ox-LDL through decreasing ADMA concentration.     

Protective effect of onychin on the human umbilical vein endothelial cells injured by menadione

AIM: To investigate the protective effect of onychin on the endothelial cells injured by oxidative stress. METHODS: The injured model was established by endothelial cells treated with menadione. The growth inhibitory rate of endothelial cell was determined by MTT assay; NO₂^-／NO₃^- concentration in the medium was determined by nitrate reductase assay; eNOS and caveolin-1 protein levels were determined by Western blot. RESULTS: Onychin significantly decreased the growth inhibitory rate of endothelial cells injured by menadione, increased NO₂^-／NO₃^- concentration in the medium and eNOS activity and up-regulated caveolin-1 expression. CONCLUSION: Onychin possesses a protective effect against endothelial cell injury induced by menadione via caveolin-1/eNOS pathway.