Study Of A Protective Role For OPG In Vascular Endothelial Cell

Background and AbjectiveAtherosclerosis diseases threat humans health most seriously with high morbility and mortality.Experts predict that atherosclerotic cardiovasular diseases would become the most dangerous one leading to death and disability in the 21^th century.Vascular endothelial cells,VEC,spreads out on the vascular inner wall,which plays its critical role in maintaining the vessel permeability barrier,immune defense and inflammatory reaction.The most of experts think of the dysfunction and incompetence of VEC as an initial and prerequisite step of atherogenesis.So the study of function for endothelial cells is a hot point of research for atherosclerosis.The new reseach indicates that serum osteoprotegrin(OPG) is closely associated with the maintenance and protection for endothelial cells function.1997,Simonet for the first time found the sequence of osteoprotegrin from the mouse embro-intestine cDNA library,its protein can dramatically depress the maturation of osteoclast leading to the rising density of bone.Some research manifest that the OPG-/- mice would develop osteoporosis and vessel calcification,then recovery of OPG gene can prevent that changes.Moreever,OPG is an anti-apoptosis factor in the vessel endothelial cell.The immuno-activity of OPG exits the early atherosclerotic lesions.Clinical observations revealed that the level of serum OPG is significantly higher in patients with AS than in the control group,and the higher level of OPG,the greater degree of atherosclerotic lesions.So,the close relationship between OPG and atherosclerosis can be obtained.The roles of OPG in endothelial cells and atherogenesis still remain controversial. Some experts consider that OPG activates the progression of AS,and the other think that the rising level of serum OPG is a compensatory defense mechanism of the development of atherosclerosis with exerting a protective effect on the vessel.In this research,we are intent to investigate the relationship between OPG and atherosclerosis as follows:Ⅰ: Effect of OPG on the AS-related adhension molecules and chemokines.Ⅱ:Effect of OPG on the endothelial cells apoptosis.Ⅲ:Effect of OPG on NF-κB.Ⅳ:Changes of circulating OPG level and their significance in CAD patients. Methods1.Human umbilical vein endothelial cell in cultured media was activated separately by osteoprotegerin(5ng/mL),tumor necrosis factor-α(5ng/mL) and OPG(5ng/mL) combined with TNF-a(5ng/mL).Untreatment of HUVEC acted as the control.The endothelial cell genes and ptoteins,intercellular adhesion molecule-1,vascular cell adhesion molecule-1 and monocyte chemotacticprotein-1,were investigated with ELISA and RT-PCR(3h,6h,12h and 24h).2.Human umbilical vein endothelial cell in cultured media was activated separately by osteoprotegerin(5ng/mL),tumor necrosis factor-α(5ng/mL) and OPG(5ng/mL) combined with TNF-a(5ng/mL).Untreatment of HUVEC acted as the control.The apoptosis cell percentage(apoptosis index,AI) was detected by TUNEL(0.5h,1h,2h).3.HUVEC monolayers were grouped and treated with the same above way.Cytoplasm protein,inhibitory nuclear factor kappa B,and nuclear protein,nuclear factor kappa B p65,were investigated by western blotting.4.88 patients were selected with complete data.Patients were excluded with dysfunction of kidney or liver,abnormality of thyroidgland function,severe heart faliue or myocardial infarction(＜4weeks).64 CHD patients who were identified by coronary angiography constituted the case group and the control group was composed of 26 persons with narmol angiography result.The levels of serum osteoprotegerin were detected with ELISA.At the time of a physical examination,body mass index(BMI), age,historys of cigarette use,hypertention,diabetes were assessed.And,the plasmatic lipid concentration and blood glucose were measured.Results1.An obvious increase in the expression of intercellular adhesion molecule-1,vascular cell adhesion molecule-1 and monocyte chemotacticprotein-1 by endothelial cells in the presence of TNF-α(5ng/mL),was higher than OPG(5ng/mL) treatment,OPG (5ng/mL) combined with TNF-α(5ng/mL) treatment and the control,P＜0.01.The levels of these factors in OPG(5ng/mL)combined TNF-α(5ng/mL)treatment is higher than OPG treatment,P＜0.05.There were no statistical difference of inflammatory factors betweeon OPG treatment and the control.2.TNF-α(5ng/mL)stimulated the rise of apopotosis cell percentage(AI) in HUVEC. The AI of TNF-αtreatment was higher than OPG(5ng/mL)treatment,OPG(5ng/mL) combined with TNF-α(5ng/mL) treatment and the control,P＜0.01.The AI in OPG(5ng/mL)combined with TNF-α(5ng/mL)treatment is higher than OPG treatment, P＜0.05.And,there was no statistical difference betweeon OPG treatment and the control.3.The increase of the expression of cytoplasm protein inhibitory nuclear factor kappa B in the presence of OPG(5ng/mL),was higher than TNF-α(5ng/mL) treatment,OPG (5ng/mL) combined with TNF-α(5ng/mL) treatment and the control,P＜0.01. TNF-α(5ng/mL) downregulated Iκa-B,the level of Iκa-B protein in TNF-αtreatment was lower than in OPG combined with TNF-αtreatment,P＜0.01.No statistical difference existed between OPG combined with TNF-αtreatment and the control.TNF-αstimulated the rise of nuclear factor kappa B p65 protein in HUVEC,and its level of NF-κB was higher than OPG(5ng/mL)treatment,OPG(Sng/mL)combined with TNF-α(5ng/mL)treatment and the control,P＜0.01.There existed the lowest level of NF-κB ptotein in OPG treatment than the other group,P＜0.01.There was no statistical difference between OPG combined with TNF-αtreatment and the control.4.The level of serum OPG was siginificantly higher in patients with CHD (5.16±0.49pmol/L) than in the control group(3.54±0.35pmol/L),P＜0.05.The statistical difference of OPG was found among the patients of various stenosis score group(stenosis score was examined as a categorical variable classified ty tertile values) and significant stenosis vessles group(P＜0.01),and the partial correlation coefficient between serum OPG levels and stenosis score.ConclusionsHUVEC in vitro,OPG can inhibit the TNF-α-induced rise of the level of inflammatory factor and cell apoptosis,perhaps OPG exerted this effect through inhibiting the function of active NF-κB.We presumed that OPG would play its protective role in the development of atherosclerotic lesions.The high level of serum OPG in CHD was a defense,compensatory mechanisms.