The Expression Of CRP In HUVECs And The Effect Of CRP On Function Of Endothelial Cells

Objective:The CRP elevation in the blood has been emerged as one of the most powerful predictor in cardiovascular disease.CRP,produced mainly in hepatocytes, will rise up to 100-200μg/mL during the acute phase of inflammatory.However as a predictive risk factor,its concentration level only reach to 1-3μg/mL.Thus identification another source of CRP production could give a better explaination to the problem of lower CRP level as a useful factor for cardiovascular risk prediction. Accumulated evidences have suggested that atherosclerotic arteries also can produce CRP.However the cell types producing CRP locally in the atherosclerotic arterial wall have not been clearly identified.There are a lot of evidence suggest that CRP, uric acid(UA)and hyperinsulinemia in the blood are related to endothelial dysfunction.In this study,based on the culturing human umbilical vein endothelial cells(HUVEC),we explored that:(1)whether the HUVEC can synthesize CRP;(2) observing the influence of CRP on the secrete function of endothelial.(3)the impact of other interventions such as UA,Angâ…¡,glucose and insulin on the secreting function of endothelial was observed simutanenously.Methods:(1)HUVEC were incubated with UA,Angâ… ,glucose,insulin in different concentrations for 48 hours.The supernatants were concentrated and analyzed by a high-sensitivity enzyme-linked immunosorbent assay(ELISA)specific for human CRP.RNA was extracted from the HUVECs by reverse transcriptase-polymerase chain reaction(RT-PCR)using specific primers for the CRP.(2)HUVEC were incubated with CRP in different concentrations for 24 hours and the effects of CRP on HUVEC secreting Angâ…¡,NO,eNOS,O₂^-,SOD were investigated.(3)HUVEC were treated with Angâ… separately,or together with ACEI,antichymotrypin,aprotinin,ARB, or combinationing ACEI,antichymotrypin and aprotinin together to observe the feature of Angâ…¡generation pathways in HUVEC and the influences of HUVEC Angâ…¡formation on generating NO and O₂^- in situ.Result:(1)after treated with UA,Angâ… ,Glucose and insulin,CRP production were not detected in HUVEC by ELISA and rt-PCR.(2)incubated with different concentrations of CRP(5mg/l,25mg/l,50mg/l,100mg/l),the level of NO and the activaty of eNOS were decreased significantly,the level of O₂^- increased,all in a dose-dependent manner.there was no changes on the activity of SOD(p＞0.05).(3) Incubated with Angâ… (10^-2μM),the HUVEC Angâ…¡production was significantly increased(p＜0.05).Compared to the Angâ… treated group,captopril(1000μM)can inhibt Angâ…¡generation by 21.18%,antichymotrypin by 50.93%,and Aprotinin by 20.04%.Captopril only can block Angâ…¡generation in small part.Combinating captopril with other two blockers,the generation of Angâ…¡were almost blocked completely.The increased Angâ…¡in the HUVEC decrease the releasing of NO, inhibting the activity of eNOS and SOD,elevating O₂^- production.All the changes can be reversed by blocking AT1 receptors in irbesartan.(4)The different concentrations of UA(6,12mg/dl)can reduce the NO releasing and eNOS activity,but had no obvious effect on SOD.(5)Both glucose(5,25mM)and insulin(100, 1000mU/L)can prompt the Angâ…¡generation in dose-dependent manner,so does glucose(25mM)combined with insulin(1000mU/L).High dosage of glucose(25mM) can elvate NO releasing,eNOS activity and O₂^- production,but SOD decreased. Insulin(100,1000mU/L)can increase eNOS and NO in dose-dependent manner but not SOD and O₂^-.Combination of glucose(25mM)and Insulin(1000mU/L)can reduce NO production,eNOS and SOD activity,but increase O₂^-(p＜0.05).Conclusion:(1)CRP production could not be detected in HUVEC after stimulated by UA,Angâ… ,glucose and insulin.(2)Human recombinate CRP can reduce NO releasing,decrease the NOS activity and elevate the O₂^- level.(3)ACE and chymase are both Angâ…¡-forming enzymes in cultured HUVEC,chymase was dominant.(4) The increased Angâ…¡formation in situ may decrease NOS and SOD activity,NO generation while increase O₂^- level.(5)UA in normal concentration can reduce NO releasing,decrease the NOS activity and elevate O₂^- level.(6)NOS activity,the NO concentration are decreased while Angâ…¡and O₂^- elevated in the HUVEC culture supernatant in the intervention of High concentration of glucose and insulin.