Effect of lipopolysaccharide on viability and secretion function of human umbilical vein endothelial cells

AIM: To observe the direct effect of lipopolysaccharide (LPS) on secretion of endothelin-1 (ET-1) and nitric oxide by human umbilical vein endothelial cell and cell viability of the secretor. METHODS: The third passage of human umbilical vein endothelial cells were incubated with different concentrations of LPS (1 g/L, 100 mg/L, 10 mg/L, 1 mg/L, 100 μg/L, 10 μg/L, 1 μg/L) for 6 hours, and the culture supernatants were collected. The concentrations of ET-1 were determined by radioimmunoassay, the concentrations of nitric oxide were determined using Greiss's method. The viabilities of cells were measured by MTT method. RESULTS: The concentration of ET-1 (pg/L) of normal control group was 251.64±10.90. The concentrations of ET-1 (pg/L) of LPS treated groups were 220.85±19.14, 278.67±15.45, 306.40±11.60, 312.87±33.50, 324.38±17.02, 291.49±14.30, 282.11±13.38, respectively (each group compared with normal control group, P<0.05 or P<0.01). The concentration of NO_x (μmol/L) of normal control group was 629.46±13.36. The concentrations of NO_x (μmol/L) of LPS treated groups were 732.58±23.21, 669.87±9.32, 661.24±16.80, 650.33±13.24, 606.59±12.94, 626.75±9.83, 627.61±5.61, respectively (each group compared with normal control group, P<0.05 or P<0.01). The viabilities of endothelial cells of LPS treated groups were 74%, 81%, 86%, 88%,91%, 93%, 93%, respectively. CONCLUSION: LPS of lower concentrations had no significantly lethal effect on human umbilical vein endothelial cells, but enhanced secretion of ET-1 and inhibited NO production. LPS in higher concentrations showed significant lethal effect on human umbilical vein endothelial cells, inhibited secretion of ET-1 and enhanced NO production.     

Changes of pulmonary apoptosis and NOS mRNA in the lipopolysaccharide-induced acute lung injury

AIM: To observe the chronological changes of pulmonary apoptosis and the expression of iNOS mRNA,nNOS mRNA and eNOS mRNA in lipopolysaccharide (LPS)-induced acute lung injury (ALI) and to investigate the mechanisms of ALI.METHODS: Rats were randomly divided into 2 groups: control group and LPS treated group.The rats were injected with either saline or LPS and killed at 1,3,6,9 and 12 h after LPS injection.The expressions of iNOS mRNA,nNOS mRNA and eNOS mRNA in the lung tissue were respectively measured with RT-PCR methods.Apoptosis and expressions of Bcl-2 and Bax were respectively determined by flow cytometry (FCM) and immunohistochemistry (IHC).The pathological changes of lung tissue were observed under light and electron microscope.RESULTS: Compared with that in control group,the expression of iNOS mRNA was significantly increased at 3,6,9 and 12 h after administration of LPS (P<0.05).The eNOS mRNA was significantly decreased at 3,6,9 and 12 h after administration of LPS (P<0.05).The nNOS mRNA had no significant change during the 12 h in LPS group.Degree of ALI was gradually worsened after administration of LPS.Apoptosis of pulmonary cells was significantly increased,and reached the top level at 9 h after administration of LPS (P<0.01).The expression of Bcl-2 was markedly decreased and the expression of Bax was significantly enhanced in alveolar and airway epithelial cells in LPS treated group.CONCLUSION: The expressions of iNOS mRNA,eNOS mRNA and nNOS mRNA are not identical in LPS-induced acute lung injury.NOS regulates the apoptosis of pulmonary cells through affecting the balance of Bcl-2 and Bax.     

Effects of hypoxia on levels of nitric oxide,endothelin-1 and inducible nitric oxide synthase mRNA expression in cultured human umbilical vein endothelial cells

AIM: To observe the effects of hypoxia on the levels of nitric oxide (NO), endothelin (ET-1) and the expression of inducible nitric oxide synthase (iNOS) mRNA in human umbilical vein endothelial cells (HUVECs), and further investigate the mechanism of hypoxic pulmonary hypertension. METHODS: On the basis of the HUVECs culture model, the methods of nitrate reductase and radioimmunoassay were used to determine the changes of NO and ET-1 in the medium secreted by HUVECs, and the expression of iNOS mRNA was analyzed by semi quantitative RT-PCR after exposure to hypoxia (3% O2) for 6, 12 or 24 h. RESULTS: The contents of NO2-/NO3- and ET-1 in hypoxia group in the medium was significantly higher than that in control group at different time points (P<0.05). Also, iNOS mRNA expression increased significantly (P<0.05). CONCLUSION: Hypoxia stimulates the release of NO and ET-1 from HUVECs, also induces iNOS-mRNA expression. The change of NO may be the result of iNOS mRNA upregulation induced by hypoxia.     

Effect of Sini decoction on expression of caveolin-1 and eNOS in EAhy926 cells injured by homocysteine

AIM：To investigate the mechanism of Sini decoction in treating human vascular endothelial cell injury and the roles of caveolin-1 and nitric oxide (NO) system in this procedure. METHODS：Model of human umbilical vein endothelial EAhy926 cells injured by homocysteine (Hcy) was established. The protective effect of Sini decoction on the injured EAhy926 cells was observed, and the expression of caveolin-1 and endothelial nitric oxide synthase (eNOS) was detected by real-time fluorescence quantitative PCR and Western blotting. RESULTS：Compared with control group, the Hcy-treated EAhy926 cells showed reduced adherent cell number and NO concentration in culture supernatant, decreased expression of eNOS mRNA and protein, and increased expression of caveolin-1 mRNA and protein (all P<0.05). Compared with Hcy group, better growth of adherent cells, elevated NO concentration in culture supernatant, attenuated expression of caveolin-1 mRNA and protein, and enhanced expression of eNOS mRNA and protein in Sini decoction groups were observed (all P<0.05). CONCLUSION：Homocysteine may injure EAhy926 cells by enhancing the expression of caveolin-1 and suppressing the expression of eNOS, while Sini decoction may protect EAhy926 cells by suppressing the expression of caveolin-1 and enhancing the expression of eNOS.     

Role of SIRT1/eNOS/NO signaling pathway in protective effect of ginsenoside Rb1 against replicative senescence of endothelial cells

AIM: To explore the effect of ginsenoside Rb1 on replicative senescence of endothelial cells and the role of SIRT1/eNOS/NO signaling pathway in this process. METHODS: The replicative senescence model of primary human umbilical vein endothelial cells (HUVECs) was established. The morphological change of the cells, the proportion of senescence-associated β-galactosidase (SA-β-Gal) positive cells and the plasminogen activator inhibitor 1 (PAI-1) expression were detected to assess the senescence model. The expression of eNOS and PAI-1 at mRNA and protein levels in the aging cells was determined by real-time PCR and Western blot before and after silencing of SIRT1 was performed. The NO concentration in the cell culture supernatant was measured by nitrate reductase assay. RESULTS: HUVECs with cumulative population-doubling level (CPDL) at 16 were chosen as the replicative senescence model in this research. Ginsenoside Rb1 at 80 μmol/L significantly reduced the expression of PAI-1 at mRNA and protein levels. Furthermore, ginsenoside Rb1 increased the expression of SIRT1 and eNOS at mRNA and protein levels, and increased the NO content. SIRT1 silencing inhibited the expression of eNOS at mRNA and protein levels and reduced NO generation, leading to an increase in the expression of PAI-1 at mRNA and protein levels. Upon intervention of ginsenoside Rb1, the eNOS and PAI-1 expression and the level of NO were not reversed. CONCLUSION: Ginsenoside Rb1 modulates SIRT1/eNOS/NO signaling pathway to prevent the replicative senescence of HUVECs.     

Role of PI3K/Akt/eNOS signaling pathway in inhibitory effects of puerarin on ox-LDL-induced TF expression in vascular endothelial cells

AIM:To explore the role of phosphatidylinositiol 3-kinase/protein kinase B/endothelial nitric oxide synthase (PI3K/Akt/eNOS) signaling pathways in the inhibitory effects of puerarin on oxidized low-density lipoprotein (ox-LDL)-induced tissue factor (TF) expression in vascular endothelial cells.METHODS:The mRNA expression of TF was detected by real-time fluorescent quantitative PCR.The protein levels of TF and Akt was determined by Western blot.The content of the nitric oxide (NO) was measured by nitrate reduction method.RESULTS:Compared with control group,incubating endothelial cells with ox-LDL significantly induced TF expression at mRNA and protein levels and the dephosphorylation of Akt protein,and decreased NO production.Incubation of the endothelial cells with puerarin for 1 h and then treatment of the cells with ox-LDL decreased the TF expression at mRNA and protein levels,increased Akt protein phosphorylation and intracellular NO content.Co-incubation of the endothelial cells with PI3K inhibitor LY294002 and puerarin for 1 h and then treatment of the cells with ox-LDL augmented the TF expression at mRNA and protein levels and the Akt protein dephosphorylation,and decreased NO production.Co-incubation of the endothelial cells with eNOS inhibitor N^G-nitro-L-arginine methyl ester (L-NAME) and puerarin significantly decreased the inhibitory effect of puerarin on ox-LDL-induced TF expression at mRNA and protein levels in the endothelial cells,and reduced Akt protein phosphorylation and NO production.CONCLUSION:Puerarin inhibits ox-LDL-induced TF expression at mRNA and protein levels in the human umbilical vein endothelial cells via activation of PI3K/Akt/eNOS signaling pathway.     

Effects of levcromakalim on pulmonary arterial endothelial cells and smooth muscle cells exposed to hypoxia

AIM:To explore the effects of levcromakalim(Lev) on pulmonary arterial endothelial cells (PAEC) and smooth muscle cells (PASMC) exposed to hypoxia and the mechanisms involved.METHODS:The effects of Lev on [Ca²⁺]_i, and expression of PKC_α, eNOS, iNOS and PDGF-B mRNA and protein levels were observed. The nitrite (NO₂^-) and entothelin-1(ET-1) concentrations in supernatant in cultured PAEC and PASMC were measured. The proliferation and apoptosis of PASMC was also detected.RESULTS:When PASMC were exposed to hypoxia, Lev reduced concentration of ET-1 in cultured cell supernatant, lowed the expression of PKC_α, iNOS and PDGF-B both at mRNA and protein levels, decreased [Ca²⁺]_i concentration, increased proliferation and promoted the apoptosis in PASMC. However, in the presence of Lev, the [Ca²⁺]_i concentration was not changed in the hypoxic PAEC. The NO₂^- concentration in cultured cell supernant and expression of eNOS at mRNA and protein levels in hypoxic PASMC and PAEC were also unchanged. The downregulated ET-1 activity in PASMC and PAEC and proliferation in PASMC involved in the inhibition of PKC_α signaling pathway.CONCLUSIONS:Lev reduce some disadvatage effect of hypoxia on PASMC and PAEC. The mechanism of Lev action may partly involve in the downregulation of PKC_α signaling functions.     

Inhibiting role of polydatin to expression of intercellular adhesion molecule-1 induced by lipopolysaccharide on vascular endothelial cells

AIM and METHODS:To investigate expression of intercellular adhesion molecule-1(ICAM-1) on human umbilical vein endothelial cells (HUVEC) induced by lipopolysaccharide (LPS) , and inhibiting role of polydatin by cellular immune fluorescent staining and laser confocal microscope scanning technology. RESULTS: Compared with basic expression of ICAM-1 on HUVEC, the ICAM-1 expression was enhanced significantly after stimulated by LPS from 8 h to 36 h, dose-dependent relation appeared between expression of ICAM-1 and LPS. ICAM-1 expression on endothelial cells treated only by polydatin had no abvious change,but inducing role of LPS to expression of ICAM-1 was inhibited significantly by polydatin pretreating endothelial cells. CONCLUSION:The expression of ICAM-1 on endothelial cells can be promoted by LPS , and polydatin can inhibit LPS-induced ICAM-1 expression.     

The protective effect of composite salviae dropping pills on human umbilical vein endothelial cells injured by lipopolysaccharide

AIM: To observe the function and morphological change of human umbilical vein endothelial cells (HUVECs) treated with lipopolysaccharide (LPS) and composite salviae dropping pills (DSP). METHODS: HUVECs were cultured and incubated within 10 mg/L LPS for 12 hours. Different final concentrations of composite salviae dropping pills (1 g/L, 0.5 g/L, 0.25 g/L, 0.1 g/L) were added before and after LPS treatment. Cell viability, NO, NOS, ET-1 and intracellular calcium were measured. The cells were observed under inverted microscope, inverted phase contrast microscope, laser confocal scanning microscopy and transmission electron microscope. RESULTS: When given after LPS treatment, different final concentrations of composite salviae dropping pills played a protective role (P<0.05), and the concentration of 0.5 g/L was the most effective (P<0.01). When given before LPS treatment, 0.5 g/L and 0.25 g/L of composite salviae dropping pills protected the human vascular endothelial cells (P<0.05), but 1 g/L and 0.1 g/L didn't play a protective role (P>0.05). The HUVECs injured by LPS underwent apparent morphological change after treatment with composite salviae dropping pills. CONCLUSION: Composite salviae dropping pills have an evident protective effect on human umbilical vein endothelial cells exposed to LPS.     

AngⅡ/AT1R pathway activates PP2A to down-regulate p-eNOS (Ser1177) in human umbilical vein endothelial cells

AIM: To investigate whether angiotensinⅡ (AngⅡ)/angiotensin Ⅱ type 1 receptor (AT₁R) pathway down-regulates endothelial nitric oxide synthase (eNOS) Ser1177 phosphorylation level in human umbilical vein endothelial cells by activating protein phosphatase 2A (PP2A).METHODS: Human umbilical vein endothelial cells were randomly divided into normal control (control) group, Ang Ⅱ group, candesartan (CAN; specific AT₁R blocker) group and CAN pretreatment+AngⅡ group. The protein levels of total eNOS, p-eNOS (Ser1177), PP2Ac, I₂^PP2A and p-PP2Ac (Tyr307) were determined by Western blot. The content of NO in the cell culture medium was detected by chemical colorimetry.RESULTS: Compared with control group, the level of p-eNOS (Ser1177) and the content of NO decreased (P<0.05). Compared with the same concentration of AngⅡ group, CAN pretreatment increased the level of p-eNOS (Ser1177) and the content of NO (P<0.05), but the protein expression of eNOS showed no significant difference. Compared with control group, the levels of p-PP2Ac (Tyr307) and I₂^PP2A decreased (P<0.05). Compared with the same concentration of AngⅡ group, CAN pretreatment increased the levels of p-PP2Ac (Tyr307) and I₂^PP2A (P<0.05), but the protein expression of PP2Ac showed no significant difference.CONCLUSION: AngⅡ down-regulates the level of p-eNOS (Ser1177), and decreases the production of NO in human umbilical vein endothelial cells via AT₁R pathway. This effect may be related to the reduction of p-PP2Ac (Tyr307) and protein expression of I₂^PP2A, which results in the enhancement of PP2A activity. Pretreatment with AT₁R blocker CAN increases p-PP2Ac (Tyr307) level and I₂^PP2A protein expression, thus reducing the PP2A activity, and ultimately restoring eNOS Ser1177 phosphorylation level and eNOS activity.     

Effects of Ginsenosides on LPS-induced TF and PAI-1 expression in vascular endothelial cells

AIM: To study the effect of ginsenosides on lipopolysaccharide-induced expression of tissue factor (TF) and plasminogen activator inhibitor type-1 (PAI-1) in vascular endothelial cells (EC), and to investigate the mechanism of ginsenosides in the healthy protection and treatment of cardiovascular diseases. METHODS: Human umbilical vein endothelial cells (HUVEC) were cultured by trypsin digestion method. PAI-1 was measured in the conditioned medium of HUVEC by a specific enzyme-linked immunosorbent assay (ELISA), whereas TF activity was measured in the lysates of these cells by a single step clotting assay. Specific mRNA expressions were determined by Northern blotting. RESULTS: Treatment of HUVEC with LPS resulted in a significant increase in PAI-1 antigen and TF activity. Ginsenosides inhibited this LPS-induced upregulation of PAI-1 protein and TF activity in HUVEC. These effects were also confirmed on the level of specific PAI-1 and TF mRNA expression by Northern blotting. CONCLUSION: Ginsenosides counteract endothelial cell activation by inhibition LPS-induced PAI-1 and TF expression in these cells. This ability of ginsenosides might explain its efficacy in the healthy protection and the treatment of cardiovascular diseases.     

Norepinephrine attenuates injury of vascular endothelial cells induced by LPS

AIM: To investigate the effects of norepinephrine (NE) on vascular endothelial cell damage induced by lipopolysaccharides (LPS). METHODS: Human umbilical vein endothelial cells (HUVEC-12) were cultured with LPS at 100 mg/L to establish the cell damage model. Real-time PCR and Western blot were used to determine the expressions of VE-cadherin at mRNA and protein levels. The levels of TNF-α, IL-1β, IL-2 and IL-10 in culture supernatant were measured by ELISA. The reactive oxygen species (ROS) production in the endothelial cells was detected by ROS assay kit. RESULTS: LPS decreased both mRNA and protein levels of VE-cadherin accompanied by increased levels of TNF-α, IL-1β, IL-2 and intracellular ROS, and decreased level of IL-10 in the endothelial cells. NE reversed the expression of VE-cadherin at mRNA and protein levels under the condition of LPS treatment in a dose-dependent manner, and also alleviated the intracellular oxidative stress. CONCLUSION: NE reverses the endothelial damage induced by LPS, which may be related to the up-regulation of VE-cadherin level and the decreases in oxidative stress and inflamatory mediators.     

Role of MEK1/2 in ICAM-1 over expression induced by lipopolysaccharide in human umbilical vein endothelial cells

AIM: To investigate the role of MEK1/2, a subfamily of mitogen activated protein kinase-kinase (MAPKK), in expression of intercellular adhesion molecule-1 (ICAM-1) induced by lipopolysaccharide (LPS) in human umbilical vein endothelial cells (HUVECs). METHODS: Expression levels of ICAM-1 mRNA and its protein were assayed using RT-PCR and Western blotting, respectively, in HUVECs pretreated with different concentrations of LPS for different times with or without PD98059, a specific inhibitor of MEK1/2. RESULTS: LPS up-regulated the expression of ICAM-1 mRNA and its protein in HUVECs in a concentration- and time-dependent manners. The expression levels of ICAM-1 mRNA and its protein began to elevate at 2 h after LPS treatment, and reached nearly a peak value at 6 h after LPS (100 μg·L-1) treatment. PD98059 (10 μg·L-1) significantly inhibited LPS-induced expression of ICAM-1 mRNA and protein, the expression inhibitory rates of which were 54.4% and 44.9%, respectively (P<0.01). CONCLUSION: Modulation of MEK1/2 signaling pathway might be a new and useful strategy for the prevention and treatment of vascular endothelial injury induced by LPS.     

Role of miRNA-24 in regulation of endothelial nitric oxide synthase expression and vascular endothelial cell proliferation

AIM：To investigate whether miRNA-24 is involved in the regulation of endothelial nitric oxide synthase (eNOS) expression and vascular endothelial cell proliferation. METHODS：A plasmid that highly expressed miRNA-24 was constructed, and was transfected into the human umbilical vein endothelial cells (HUVECs) by liposome. The cell proliferation was detected by MTT assay. The expression of eNOS and Sp1 at mRNA and protein levels was exa-mined by real-time PCR, immunohistochemistry and Western blotting.RESULTS：Compared with control group, the proliferation of endothelial cells in miRNA-24 group was significantly decreased by 41.97 % (0.47±0.04 vs 0.81±0.03, P<0.01), and the expression of eNOS at mRNA and protein levels was decreased by 44.8% (0.48±0.01 vs 0.87±0.03, P<0.05) and 71.92% (0.16±0.06 vs 0.57±0.08, P<0.05), respectively. Meanwhile, the mRNA and protein levels of Sp1 were significantly decreased by 53.00% (0.45±0.02 vs 0.93±0.01, P<0.05) and by 62.31% (0.13±0.07 vs 0.31±0.09, P<0.05), respectively. In miRNA-24 inhibitor group, the above indexes were decreased compared with control group, but significantly increased compared with miRNA-24 group. CONCLUSION：miRNA-24 significantly inhibits the proliferation of HUVECs and the eNOS expression. Sp1 possibly acts as one of the important factors in the regulation of eNOS expression by miRNA-24.     

Effects of LPS on the organization and localization of VE-cadherin and F-actin in cultured endothelial cells

AIM:This experiment is to investigate the effects of LPS on the organization and localization of VE-cadherin and F-actin in cultured human umbilical endothelial cells.METHODS:The human umbilical vein endothelial cell lines ECV-304 were incubated with LPS at different concentrations for 30 min. VE-cadherin was detected by immunofluorescence with primary mAb of VE-cadherin and FITC-conjugated secondary antibody. F-actin was detected with fluorescence staining with rodamine-phalloidin.RESULTS:At high concentration, LPS could induce reorganization of VE-cadherin with the formation of serrata cellular border and enlargement of intercellular gaps, which were apparently different from that in normal conditions with the high fluorescence intensity at cell-cell junction. F-actin depolymerization could also be induced by LPS at high concentration with the formation of stress fiber and filopodia.CONCLUSION:LPS(300 μg/L) could induce reorganization of VE-cadherin and F-actin in human umbilical vein endothelial cells.     

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 peroxisome proliferator activated receptor δ on mRNA expression of MCP-1 induced by homocysteine in cultured human umbilical vein endothelial cells

AIM: To investigate the effects of peroxisome proliferator activated receptor δ (PPARδ) on the mRNA expression of monocyte chemoattractant protein 1 (MCP-1) induced by homocysteine (Hcy) in human umbilical vein endothelial cells (HUVECs). METHODS: Collagenase was used to isolate endothelial cells from human umbilical vein, and the cells were cultured in vitro . The HUVECs were divided into blank control group, Hcy group, GW0742 (a specific agonist of PPARδ) group and diphenyleneiodonium (DPI,a specific inhibitor of NADPH oxidase) group. RT-PCR was used to examine the mRNA expression of MCP-1 and PPARδ. The protein level of PPARδ was detected by Western blotting.2',7'-Dichlorofluorescin diacetate(DCFH-DA) was added to monitor intracellular production of reactive oxygen species (ROS). RESULTS: Compared with control group, Hcy promoted the mRNA expression of MCP-1 in a concentration-dependent manner, and decreased the mRNA expression of PPARδ in HUVECs. The mRNA expression of MCP-1 was significantly elevated by Hcy at the concentration of 10^-5 mol/L, and the mRNA expression of PPARδ was decreased remarkably (P<0.01). GW0742 decreased the mRNA expression of MCP-1 compared with Hcy group (P<0.01). Hcy remarkably increased the production of ROS compared with control group. Hcy-induced production of ROS was also significantly attenuated by GW0742. CONCLUSION: The activation of PPARδ decreases the Hcy-induced mRNA expression of MCP-1 by suppressing Hcy-stimulated production of ROS.