Effects Of Hunan Dark Tea On Vascular Endothelium And Endothelial Progenitur Cells Functian

Chapter 1 Effects of the extract of ten-liang tea on endothelial dysfunction and atherogenesis in hypercholesterolemic animalsBACKGROUND:Atherosclerosis(AS)is the leading cause of death in the cardiovascular diseases.Endothelial dysfunction is recognized as an early event in the pathogenesis of atherosclerosis.Hypercholesterolemia is an independent risk factor of AS.It has been shown that endothelium-dependent vasodilation is attenuated in hypercholesterolemic animals and patients. The elevated concentration of low-density lipoprotein(LDL)has been suggested to play a key role in endothelial dysfunction in hypercholesterolemia.LDL has been shown to accumulate and be oxidized in the arterial wall and induce vascular inflammatory reaction, which further decreases endothelial function and increases adhesion of monocytes to endothelial cells.Recently,much interest focused on the L-arginine analogs asymmetric dimethylarginine(ADMA),a potent endogenous inhibitor of nitric oxide synthase(NOS),which competitively inhibits NOS activities. Besides inhibiting NO production,ADMA could injury endothelial structure,induce oxidative stress,promote vascular inflammatory responses and increase lipids intake.Moreover,exogenous ADMA exerted direct pro-atherogenetic activity in several animal models.Thus, ADMA is thought as a novel endogenous pro-atherogenetic factor.There is substantial evidence that endothelium dysfunction in hypercholesterolemic animal and patient is associated with elevation of ADMA.Tea is very traditional drink in China,which contains some polyphenols and catechin.Tea have extentive pharmacological activities including antioxidation,lowering blood lipids and anti-atherosclerosis. Dark tea is a main kind of tea in Hunan province such as Hua-jian tea and Fu tea,which is produced in the region of Anhua County.However,the studies about the cardiovascular effects of Hunan dark tea are very litter. In the present study,we observed that the effects of Shi-liang tea,a kind of Hua-juan tea,on blood lipids,endothelial function and atherogenesis in two kinds of hypercholesterolemic animal models and investigated whether these effects of Shi-liang tea is related to regulating endothelial ADMA/NO pathway.The Chapter was divided into two parts:Partâ… :The effects of the extract of Shi-liang tea on blood lipids and endothelial function in hypercholesterolemic rats;Partâ…¡:The effects of the extract of Shi-liang tea on atherosclerosis in hypercholesterolemic rabbits. Partâ… Effects of the extract of Shi-liang tea on blood lipids and endothelial function in hypercholesterolemic ratsMETHODSMale SD rats were fed with food containing cholesterol for 4 weeks to induce hypercholesterolemia.For drug treatment,the animals were orally treated with different doses of the extract of shi-liang tea(50,100 or 200 mg/kg per day)during the last two-week period of the experiment. At the end of the experiment,blood samples were collected to measure the plasma levels of NO,ADMA and malondialdohyde(MDA),and the thoracic aortas were isolated to determine endothelium-dependent vasodilator response.RESULTS1.The extract of shi-liang tea could dose-dependently decrease plasma levels of total cholesterol,LDL and triglyceride in hypercholesterolemic rats.2.The extract of shi-liang tea could improve the endothelium-dependent vasodilator response in hypercholesterolemic rats.3.The extract of shi-liang tea could decrease plasma levels of ADMA and MDA as well as increase NO content in hypercholesterolemic rats..CONCLUSIONSThe extract of shi-liang tea lowers blood lipids and protects endothelial function,and its effects may be related to inhibiting peroxidation and modulating ADMA/NO system. Partâ…¡Antiatherosclerotic properties of the extract of Shi-liang tea in hypercholesterolemic diet-fed rabbitsMETHODSMale rabbits were fed with food containing cholesterol for 8 weeks to induce atherosclerosis.For drug treatment,the animals were orally treated with different doses of the extract of shi-liang tea(25,50 or 100 mg/kg per day)during the last four-week period of the experiment.At the end of the experiment,blood samples were collected to measure the plasma levels of NO,ADMA and MDA,and the aortas were isolated to determine endothelium-dependent vasodilator response and lesion area.RESULTSThe extract of shi-liang tea could dose-dependently decrease plasma levels of total cholesterol,low-density lipoprotein and lesion area in atherosclerotic rabbits.Moreover,such treatment could improve endothelium-dependent vasodilator response and erythrocyte deformability,decrease plasma levels of ADMA and MDA and increase NO content.CONCLUSIONSThe extract of shi-liang tea lowers blood lipids and inhibits atherogenesis,which may be related to improving endothelial function and erythrocyte deformability via inhibiting lipid peroxidation and modulating ADMA/NO system. Chapterâ…¡Effects of the extract of shi-liang tea on ox-LDL-induced dysfunction of endothelial cells and endothelial progenitor cellsBACKGROUND:The senescence of endothelial cells contributes to endothelial dysfunction.Oxidative stress plays a key role in cells senescence.The elevated level of LDL plays a key role in endothelial dysfunction in hypercholesterolemia.It has been reported that the aggression and oxidation of LDL in endothelium can promote the senescence of endothelial cells,which results in endothelium-dependent vasorelaxating responses.The proliferation,differentiation and repair ability of matured endothelial cells are very limited after endothelium is damaged.Thus, there is significance how to protect endothelial function and promote repair of damaged endothelium for therapy of AS.Recent studies found that bone marrow-derived endothelial progenitor cells(EPCs)could differentiate into matured endothelial cells,which promoted vascular reendothelialization and were involved in the repair of damaged endothelium.Numbers of studies showed that the development of AS in subjects with AS risk factors is closely related to the decreased number and dysfunction of circulating EPCs.As mentioned previously,endogenous NOS inhibitor ADMA can induce senescence of endothelial cells,damage endothelial function and promote atherogenesis.In vivo most of ADMA is degraded by dimethylarginine dimethylaminohydrolase(DDAH),which hydrolyzes ADMA to L-citrulline and dimethylamine.In cultured endothelial cells, oxidized LDL(ox-LDL)markedly decreased DDAH activity and increase ADMA accumulation,and ox-LDL-induced cells damage could significantly blocked by improving DDAH activity.Several recent studies reported that the DDAH/ADMA could modulate EPCs function via regulating NO and vascular endothelial growth factor(VEGF)production. A clinical study showed that the plasma level of ADMA in CAD patients was negatively correlated to the number of circulating EPCs,and exogenous ADMA could concentration-dependently inhibit the proliferation of EPCs.In Chapter I,we showed that the extract of shi-liang tea could markedly decrease plasma level of ADMA and improve endothelial function in hypercholesterolemic rats and rabbits.To further investigate the precise mechanisms of protective effects of the extract of shi-liang tea on endothelial function,in the present study,we observed the effects of he extract of shi-liang tea on ox-LDL-induced damages in endothelial cells and EPCs,and their relationship with the DDAH/ADMA pathway.The Chapter was divided into two parts:Partâ… :The effects of the extract of shi-liang tea on ox-LDL-induced senescence in cultured human umbilical vein endothelial cells(HUVECs);Partâ…¡:The effects of the extract of shi-liang tea on dysfunction of EPCs induced by ox-LDL.Partâ… Effects of the extract of shi-liang tea on ox-LDL-induced senescence of endothelial cellsMETHODSHUVECs were cultured and used for all these studies.Human plasma LDL was isolated and oxidized by Cu²⁺.The senescence of endothelial cells was reflected by both senescence-associated beta-galactosidase activity and telomerase activity.Intracellular oxidant productions were measured using H2DCF,a fluoresent indicator.The level of ADMA in cultured medium and the activity of DDAH in endothelial cells were determined by HPLC.The mRNA level of DDAH2 was determined using real time PCR.DDAH2 siRNA was transfected into endothelial cells to silent DDAH2 expression.RESULTS1.Treatment with ox-LDL(100μg/ml)for 48 h significantly increased beta-galactosidase-positive cells ratio and reduced telomerase activity,which could be markedly attenuated by pretreatment with the extract of shi-liang tea(1,2.5 and 5mg/ml).2.Treatment with ox-LDL(100μg/ml)for 48h significantly increased intracellular ROS production,which could be markedly attenuated by pretreatment with the extract of shi-liang tea(1,2.5 and 5 mg/ml).3.Treatment with ox-LDL(100μg/ml)for 48 h significantly increased the level of ADMA in medium and decreased the activity of DDAH in endothelial cells,which could be markedly attenuated by pretreatment with the extract of shi-liang tea(1,2.5 and 5 mg/ml).These treatments fail to affect the mRNA expression of DDAH2 in endothelial cells.4.DDAH2 siRNA markedly blocked the inhibitory effects of the extract of shi-liang tea(5 mg/ml)on ox-LDL-induced increase in beta-galactosidase-positive cells ratio and reduction of telomerase activity.CONCLUSIONThe extract of shi-liang tea inhibits ox-LDL-induced senescence of endothelial cells,and such effect may be related to inhibiting oxidative stress,improving DDAH activity and decreasing ADMA level. Partâ…¡Effects of the extract of shi-liang tea on ox-LDL-induced EPCs dysfunctionMETHODSEPCs were isolated from human umbilical vein blood.Human plasma LDL was isolated and oxidized by Cu2+.The function of EPCs was reflected by both migrated and tube-formatting capabilities.The levels of ADMA and VEGF in cultured medium were determined by HPLC and ELISA,respectively.RESULTS1.Treatment with ox-LDL(100μg/ml)for 48 h significantly decreased both migrated and tube-formatting capabilities,which could be markedly attenuated by pretreatment with the extract of shi-liang tea(1, 2.5 and 5 mg/ml).2.Treatment with ox-LDL(100μg/ml)for 48 h significantly increased the level of ADMA and decreased the level of VEGF in medium,which could be markedly attenuated by pretreatment with the extract of shi-liang tea(1,2.5 and 5 mg/ml).CONCLUSIONThe extract of shi-liang tea inhibits ox-LDL-induced EPCs dysfunction,and such effect may be related to decreasing ADMA level and increasing VEGF production.