Clinical Significance of HSCARG for Atherosclerotic Coronary Heart Disease and Reduced ROS-Oxidative Stress in in Vivo and in Vitro Models via p47phox by NF-κB Activity
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Abstract:
Coronary heart disease (CHD) is a dynamic process in which there are interactions between endothelial dysfunction, oxidative stress, and inflammatory responses. The aim of the present study was to investigate the function and mechanism of HSCARG in the treatment of CHD.Male apolipoprotein E/low-density lipoprotein receptor-deficient mice were given a high-fat diet with 21% fat and 0.15% cholesterol for the in vivo model. Human umbilical vein endothelial cells were incubated with angiotensin II for the in vitro model. HSCARG expression was inhibited in patients or mice with CHD.HSCARG reduced oxidative stress in mice with CHD. HSCARG also reduced reactive oxygen species (ROS)-oxidative stress in the in vitro model. HSCARG induced p47phox expression in the in vitro model by NF-κB activity. The regulation of nuclear factor kappa B (NF-κB) activity or p47phox expression participates in the effects of HSCARG in CHD.Altogether, our data indicate that HSCARG reduced ROS-oxidative stress in in vivo and in vitro models of CHD via p47phox by NF-κB activity and may be a clinical target for CHD.Keywords:
Endothelial Dysfunction
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.[
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Objective To investigate the effect of testosterone on expression of transforming growth factor-β1(TGF-β1) and hemeoxygenase-1(HO-1)in endothelial cells of human umbilical vein.Methods Effect of testosterone on expression of TGF-β1 and HO-1 in endothelial cells of human umbilical vein was detected by ELISA.Results Testosterone at a physiological concentration of 3.0×10-8mol/L or at an approximately physiological concentration promoted the expression of TGF-β1 and HO-1 in endothelial cells of human umbilical vein in a time-and concentration-dependent manner.Conclusion Testosterone at a physiological concentration or at an approximately physiological concentration promotes the expression of TGF-β1 and HO-1 in endothelial cells of human umbilical vein.
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AIM:To observe the effects of porphyromonas gingivalis(Pg) to the proliferation and apoptosis of human umbilical vein endothelial cells(HUVECs) in vitro,so as to evaluate the virulence of Pg to HUVECs.METHODS:An aeropack was used to culture Pg,and HUVECs were derived from primary culture of human umbilical vein.The MTT was employed to assay the proliferation of HUVECs.The apoptosis of HUVECs was detected by in situ cell apoptosis detection kit I.RESULTS:Pg obviously inhibited the proliferation of HUVECs compared with lipopolysaccharide,and apoptosis can also be observed in HUVECs after treated by Pg.CONCLUSION:The Pg might injure the human umbilical vein endothelial cells(HUVECs) and to aggravate the inflammatory reaction in periodontitis,which could be one of the pathologic mechanisms in cardiovascular disease.
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Objective To explore the time-effect of damage in human umbilical vein endothelial cell lines induced by Trichlorfon and to analyze its mechanism.Methods HUVECs-12 were cultured in DMEM containing Trichlorfon for 24 h,the growth inhibition was measured by MTT assay.Cells were incubated in IC50 concentration Trichlorfon(400 μmol/L) for different times(0,4,6,8,12,24,48 h)) to test the time-effect of damage in human umbilical vein endothelial cells;the level of malondialde-hyde(MDA) in supernatant-conditioned media was monitored by thiobituric acid reaction(TBA).Results Trichlorfon inhibited HUVECs-12 growth in a dose-dependent manner and increased the production of MDA.Conclusion Trichlorfon can induce damage in human umbilical vein endothelial cells.
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Objective: To observe the effect of simvastatin on the secretion of IL-6 andIL-8 in human umbilical vein endothelial cells. Method: Human umbilical vein endothelial cells were stimulated by LPS (100 ng/ml)and co-incubated with different concentrations of simvastatin (5 μg/mL,10 μg/mL) for 1-24 hours (1 h,12 h and 24 h). The levels of IL -6 and IL -8 in the cells were measured by ELISA. Result: LPS could increase the production of both IL-6 and IL-8 in umbilical vein endothelial cells significantly . The levels of these cytokines were reduced significantly by simvastatin when compared with blank control group (P0.01). Conclusion: Simvastatin can reduce the levels of IL-6 and IL-8 in the human umbilical vein endothelial cells, which is stimulated by LPS.
Interleukin 8
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Objective To investigate the effect of TNF-α on production of nitric oxide (NO) and activity of nitric oxide synthase(eNOS) in human umbilical vein endothelial cells(HUVECs). Methods HUVECs were incubated with TNF-α at different concentrations (2-20ng/ml) and different times(2-48h) or pretreatment with L-NMMA or DXM. Nitric oxide production and activity of nitric oxide synthase in HUVECs were measured by NO and NOS assay kits. Results (1) Activity of eNOS was down-regulated by TNF-α in a concentration-dependent manner, while production of NO was upregulated. (2) The activity of eNOS attenuated after long-time treatment with TNF-α, but marked increased production of NO was detected after 24h. (3) The induction of NO by TNF-α can be inhibited by L-NMMA and DXM. Conclusion TNF-α can reduce activity of eNOS and induce iNOS, so that production of NO is increased.
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Objective: To explore the molecular mechanism of the proliferation of human umbilical vein endothelial cells induced by AngⅡ.Methods: The lines of human umbilical vein endothelial cell cultured in vitro were divided into 3 groups which were treated by AngⅡ, AngⅡ+NAC, and normal culture medium respectively. First we observed the proliferous effect of human umbilical vein endothelial cells induced by AngⅡ at different concentration at different time with biochemical methods. Then the contents of ROS(·OH) in 3 groups were detected by spectrophotometer. Finally we detected the related gene expression in the process of human umbilical vein endothelial cells proliferation via the technique of genechips.Results: Human umbilical vein endothelial cells incubated with AngⅡ(0.03125～1μmol/L ) for 12 hours increased the proliferation rate (P0.05);the negative correlation between proliferation rate and the contents of ROS(·OH)(r=-0.8, P0.01) was significant;NAC can reduce the content of ROS(·OH) and inhibit the proliferation of human umbilical vein endothelial cells;The technique of genechips analysis suggests that the genes expression related to proliferation such as ERK, Akt, CCN and PCNA increased, while the gene expression related to apoptosis such as DR6, Caspases6 BAK1 and PDCD8 decreased when human umbilical vein endothelial cell were induced by AngⅡ(0.0625μmol/L) for 12 hours; In contrast, when human umbilical vein endothelial cells were induced by AngⅡ(1μmol/L) for 12 hours, we can obtain reverse results. Conclusion: Human umbilical vein endothelial cells induced by AngⅡcan produce ROS(·OH).The target gene expression related to proliferation of human umbilical vein endothelial cells may be mediated by ROS(·OH), ROS(·OH) may be the molecules which play an important role in the signal transduction and gene expression of proliferation.
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Objective: To study the effects and mechanisms of chitosan on cultured human umbilical vein endothelial cells in vitro. Methods: After human umbilical vein endothelial cells (HUVEC) had been cultured with chitosan and rapamycin in different concentration for 48 hours,the cell proliferation were evaluated by MTT assay, and c-Myc, Bcl-2, Bax protein expression were detected by immunocytochemistry respectively. Results: With the concentration of chitosan increasing (≥0.01 mg/ml), the number of human umbilical vein endothelial cells increased (P 0.05) and the expression of Bax was decreased. Conclusions: Through lowering the expression of Bax protein, chitosan can inhibit the apoptosis of HUVEC and develop its proliferation effects.
MTT assay
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Objective:To investigate the effect of leptin on the expression of VEGF in cultured human umbilical vein endothelial cells (HUVECs). Method:The leptins in different concentrations were used to stimulate the primary cultured HUVECs and then detected the level that the HUVECs express the VEGF. Result:With the same time, the expression of VEGF protein and VEGF mRNA increased when the level of leptin increased; meanwhile, with the same concentration of leptin, the expression of VEGF protein and VEGF mRNA increased when the time extended. Both are significant when they are analyzed by the statistics. Conclusion: The expression of VEGF is concerned with the level of leptin and the time that the leptin acts on the HUVECs.
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Objective To study the culture method in vitro and identification of human umbilical vein endothelial cells(HUVECs).Methods HUVECs obtained from human umbilical vein by the technique of irrigative digestion,and were cultured with DMEM/F12 mixed culture medium in the carbon dioxide incubator,and were passaged when confluensing.The cultured cells were identified by CD34 immunohisto-chemitry.Results HUVECs could adhere to the plate completely after 24 hours,and confluens a monolayer 4～5 days later.The cultured cells were HUVECs confirmed by CD34 immmunohistochemitry.Conclusion HUVECs can be cultivated and passaged successfully in vitro.
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