The Study On Proatherosclerotic Effect Of Asymmetric Dimethylarginine And Xanthone Treatment

Atherosclerosis(AS),a chronic inflammatory disease of the large arteries,is the primary cause of heart disease and stroke.Endothelial dysfunction is an early event in the pathophysiology of atherosclerosis. Endothelium-derived nitric oxide(NO)is synthesized from L-arginine by NO synthase(NOS)and plays an important role in the regulation of endothelial function.Asymmetric dimethylarginine(ADMA),an endogenous NOS inhibitor,plays an important role in the development of endothelial dysfunction.ADMA,besides inhibiting NO synthesis,also induces cell apoptosis,participates in inflammation,which facilitates the development of AS.Oxidative stress has been implicated in the development of endothelial dysfunction through decrease NO bioavailability.Oxidized low-density lipoproteins(ox-LDL)is reported to induce oxidative stress as well as elevate ADMA levels.Some antioxidant drugs improve endothelial function via decreasing ADMA levels.Osteopontin(OPN)is a proinflammatory cytokine.It is reported that OPN and its receptor CD44 play important role in the development of AS. Plasma level of osteopontin(OPN)is elevated in patients with atherosclerosis.Diabetes mellitus,hypertension,hypercholesterolemia, and cytokine are major proatherosclerotic factors and are also observed to show increased plasma level of OPN.OPN participates in inflammation, impairs endothelial cells,and promotes vascular smooth muscle cell proliferation,which promote the development of AS.Xanthones are polyphenol compounds existing in many plants. Previous studies in our lab have shown that some xanthones compounds decrease ADMA concentration,and thus attenuate the impairment of vascular endothelial cell.AS is now widely acknowledged as a disease of chronic inflammation. Researchs have established a fundamental role of inflammation in mediating all stages of AS.Recent studies also showed that ADMA participates in inflammation wherease the mechanism remains unexplored. It has been reported that NOS inhibitor N(omega)-nitro-L-argi-nine (L-NNA)promots the expresion of OPN and that antioxidant drugs inhibit the up-regulation of OPN expression.Therefore,we postulated that ADMA could promote the development of vascular inflammation by up-regulating the expression of OPN.This study was designed to investigate the relationship between proatherosclerotic effect of ADMA and OPN pathway,and on the antiatherosclerotic effect of 3,4,5,6-tetrahydroxyxanthone and its mechanism in vitro and in vivo.ChapterⅠThe study on proatherosclerotic effect of asymmetric dimethylarginine and its mechanismsBACKGROUNDEndothelial dysfunction induced by impaired bioavailability of NO is an early event during pathophysiology of atherosclerosis.Recent investigations have indicated that the increased level of endogenous ADMA is associated with the development of AS.ADMA,besides inhibiting the synthesis of NO,inducing oxidative stress and cell apoptosis,could also induce inflammation.However,the mechanism remains unexplored.OPN is a pro-inflammatory cytokine.OPN and its receptor CD44 are reported to be closely related to the development of AS.It is reported that the plasma level of OPN is elevated in patients with atherosclerosis.OPN deficiency reduces atherogenesis in atherosclerotic mice.There is evidence that NOS inhibitor L-NNA could promote the expresion of OPN. Therefore,we hypothesized that ADMA induce inflammation may be related to the OPN-CD44 pathway.Apolipoprotein E deficient(ApoE-/-)mouse is a commonly used model for experimental atherosclerosis research.It has been reported that plasma levels of endogenous ADMA were significantly increased in ApoE-/- mice.However,no inhibitor of ADMA is available presently to confirm its proatherosclerotic effect.In the present study,therefore,we tested the role of endogenous or exogenous ADMA on atherogenesis in ApoE-/- mice.Ox-LDL and lysophosphatidylcholine(LPC),the major component of ox-LDL,are reported to increase ADMA production concomitantly with impairing endothelial cell.In the present study, therefore,we observed the role of endogenous or exogenous ADMA on the expression of OPN and its receptor CD44 in cultured human umbilical vein endothelial cells(HUVECs).METHODS1 In vivo studyMice were divided randomly into 4 groups(n=10,respectively): CJ7BL/6J mice group(as blank control group),CJ7BL/6J mice plus exogenous ADMA(C57BL/6J + ADMA)group,apolipoprotein E deficient(ApoE-/-)mice group(as experimental control group)and ApoE-/- mice plus exogenous ADMA(ApoE-/- + ADMA)group.In the exogenous ADMA-treated groups,ADMA(5 mg/kg per day)were subcutaneously given for 4 weeks.In the control groups,an equal volume of physiological saline was given subcutaneously.After treatment,mice were sacrificed and blood was collected from carotid artery.Plasma was prepared to detect ADMA concentration(HPLC).The aortas were excised to assay the atherosclerotic lesion size(SudanⅣstaining)and OPN expression(RT-PCR and immunohistochemistry).2 In vitro study(1)Effect of LPC on HUVECsHUVECs were divided into 4 groups:control group;LPC treated group: cells were treated with LPC(1,3,10μg/ml)for 24 h.ADMA(HPLC)and OPN(ELISA)level in the conditioned medium,ROS production (fluorescence),and expression of OPN(RT-PCR and western blot)and CD44 mRNA(RT-PCR)in HUVEC-12 cells were detected.(2)Effect of ADMA on HUVECsDose-dependent experiment was divided into 6 groups:control group; ADMA treated group:cells were treated with ADMA(0.3,1,3,10,30μM)for 24 h;Time-dependent experiment was divided into 6 groups: control group;ADMA treated group:cells were treated with 30μM ADMA for 12h,24 h,or 48 h.OPN(ELISA)level in the conditioned medium,expression of OPN(RT-PCR and western blot)and CD44 mRNA(RT-PCR)in HUVEC-12 cells were detected. RESULTS1 In vivo studyIn ApoE-/- mice,atherosclerotic lesions were more obvious as compared with C57BL/6J mice,concomitantly with the elevation of plasma ADMA.Treatment with exogenous ADMA 4 weeks induced or aggravated atherosclerotic lesions in C57BL/6J mice and ApoE-/- mice; OPN expression in ApoE-/- mice was increased significantly compared with C57BL/6J mice;Exogenous ADMA-treatment significant increased OPN expression in C57BL/6J mice and ApoE-/- mice.2 In vitro study(1)Effect of LPC on HUVECsTreatment with 10μg/mL LPC for 24 h markedly increased intracellular levels of ROS,OPN and ADMA,concomitantly with increased the expressions of OPN and CD44 mRNA.(2)Effect of ADMA on HUVECsIn cultured HUVEC-12 cells,30μM ADMA treatment for 24 h increased OPN level in the conditioned medium and expression of OPN and CD44 mRNA.CONCLUSIONADMA is an important contributor to the development of AS,which may be related to the OPN-CD44 pathway.ChapterⅡThe study on anti-atherosclerotic effect of xanthone and its mechanismsBACKGROUNDThere is substantial evidence to suggest that the increased level of endogenous ADMA is associated with the development of endothelial dysfunction in many kinds of cardiovascular diseases including AS.It has been shown that low-density lipoproteins(LDL)or oxidized low-density lipoproteins(ox-LDL)may be a major factor for the increased level of ADMA in atherosclerosis.Some drugs improved endothelial function concomitantly with a decrease in the level of ADMA.As a proinflammatory cytokine,OPN has recently emerged as factor closely related to the development of AS.While nuclear factor-kappaB (NF-κB)is a key regulator of inflammation.The result of our former chapter showed that endogenous or exogenous ADMA up-regulates the expression of OPN.Previous studies of other labs have shown that both OPN and ADMA increase the activity of NF-κB.Xanthones are polyphenol compounds existing in many plants which have potent anti-oxidation,anti-inflammation and endothelial protective effects.It has been reported that some xanthones compounds significantly attenuated the impairment of vascular endothelial cell induced by ox-LDL or LPC,and the protective effect of xanthones on endothelial cell are related to reduction of ADMA concentration.3,4,5,6-tetrahydroxyxanthone is a new xanthones compound synthesized by the department of chemical pharmacy.Based on these facts that ADMA induce oxidative stress concomitantly with elevation of OPN level and that antioxidant compound could inhibit the expression of OPN.In this study,we studied the anti-atherosclerotic effect of xanthone and its mechanisms in ApoE-/-mice and in HUVECs.METHODS1 In vivo studyMice were divided into 4 groups(n=20):①C57BL/6J mice control group,②ApoE-/- mice group,③low dose xanthone-treated group(10 mg/kg body weight per day)and④high dose xanthone-treated group(20 mg/kg body weight per day).After treatment 4 weeks,endothelium-dependent vasorelaxation,atherosclerotic lesion area(SudanⅣstaining), plasm ADMA level(HPLC),dimethylarginine dimethylaminohydrolase (DDAH)ⅡmRNA expression(RT-PCR),DDAH activity,and OPN expression(RT-PCR and immunohistochemistry)were determined. 2 In vitro study(1)Effect of xanthone on the impairement of HUVECs induced by LPCHUVECs were divided into 5 groups:control group;LPC treated group: cells were treated with LPC(10μg/ml)for 24 h;various concentrations of 3,4,5,6-tetrahydroxyxanthone group:cells were treated with 3,4,5,6-tetrahydroxyxanthone(1,3,10μM) for 30 min,then treated with LPC (10μg/ml)for 24 h.ADMA(HPLC)and OPN(ELISA)level in the conditioned medium,ROS production(fluorescence),and the expression of OPN(RT-PCR and immunohistochemistry)and CD44 mRNA in HUVEC-12 cells(RT-PCR)were detected.(2)Effect of xanthone on the impairement of HUVECs induced by ADMAThe experiment was divided into 5 groups:control group;ADMA treated group:cells were treated with ADMA(30μM)for 24 h;different concentration of 3,4,5,6-tetrahydroxyxanthone group:cells were treated with 3,4,5,6-tetrahydroxyxanthone(1,3,10μM)for 30 min,then treated with ADMA(30μM)for 24 h.OPN(ELISA)level in the conditioned medium,expression of OPN(western blot and RT-PCR)and activity of nuclear transcription factors NF-kappaB(NF-κB)(EMSA)in HUVEC-12 cells were detected.RESULTS1 In vivo studyIn ApoE-/-mice,3,4,5,6-tetrahydroxyxanthone treatment decreased atherosclerotic lesion area,improved endothelial function,inhibited the up-regulation in OPN expression,and decreased plasma level of ADMA concomitantly with an increase in DDAHⅡmRNA expression and DDAH activity.2 In vitro study(1)Effect of xanthone on the impairement of HUVECs induced by LPCIn cultured cells,3,4,5,6-tetrahydroxyxanthone significantly inhibited increased intracellular levels of ROS,OPN and ADMA,upregulated expressions of OPN and CD44 mRNA induced by LPC. (2)Effect of xanthoneon the impairement of HUVECs induced by ADMAIn cultured cells,3,4,5,6-tetrahydroxyxanthone significantly inhibited ADMA induced elevated OPN level,and up-regulated OPN expression and NF-κB activity.CONCLUSION3,4,5,6-tetrahydroxyxanthone has protective effects on vascular endothlium,may be related to reduction of ADMA level,inhibition of OPN expression.