The Protective Effects Of Xanthone On Intercellular Communication Dysfunction In Endothelial Cells: Role Of Endogenous Nitric Oxide Synthase Inhibitor

Atherosclerosis (AS) represents the major cause of death anddisability in human adult populations. The loss of functional or structuralintegrity of vascular endothelium is closely related to the initiation of AS.Nitric oxide (NO) is synthesized from L-arginine under catalysis of NOsynthase (NOS) and is believed to be a crucial endogenous anti-atherosclerotic factor.It was reported that an endogenous NOS inhibitor, asymmetricdimethylarginine (ADMA) can competitively inhibit NOS activity andreduce NO production and consequently damage the function of bloodvessels. In addition, ADMA can induce oxidative stress, production ofpro-inflammatory factors and endothelial dysfunction. ADMA is acceptedas a new endogenous atherogenic factor. Dimethylargininedimethylaminohydrolase (DDAH) is responsible for the degradation ofADMA and the reduced DDAH activity is believed to be the main causeof elevated ADMA levels. Some drugs exert protective effects onendothelial dysfunction via improving DDAH activity and decreasingaccumulation of ADMA. The DDAH/ADMA pathway is believed to be anew target for prevention and treatment of AS. The thesis systematicallystudied the role of DDAH/ADMA pathway in the progression andprevention of AS through the mechanism of ADMA induced endothelialdysfunction, the role of DDAH/ADMA pathway in AS, and the effects ofxanthone.ChapterⅠEffects of ADMA on the development of atherosclerosisPart One Effects of ADMA on the development of atherosclerosisBACKGROUNDIt is suggested that gap junction intercellular communication (GJIC)is involved in the progression of AS. GJIC has been demonstrated to beinvolved in the regulation of a variety of endothelial activities. And thealteration of GJIC activity in endothelial cells could lead to a disorder ofhomeostasis. The alternation in expression, distribution, and the structure of Cx43 is of great significance in AS. Recent studies imply thatendothelial gap junctions are involved in atherogenesis and atherogenicfactors are potential regulators of endothelial gap junctions.It is reported that various stimuli oxidize or change the structure ofCxs and then lead to GJIC dysfunction. Based on the facts that oxidativestress results in GJIC dysfunction and ADMA can induce oxidative stress,we suppose that ADMA might cause oxidization of Cx43 via inducingoxidative stress, which contributes to the development of AS.METHODSMice were divided randomly into 4 groups (n=8): (1) WT group:C57BL/6J mice treated with saline, (2) exogenous ADMA-treatedC57BL/6J mice group, (3) ApoE^-/-mice group: apoE^-/-mice treated withsaline, and (4) exogenous ADMA-treated ApoE^-/-mice group. ADMA (5mg/kg, s.c) was given for 4 weeks. After treatment, mice were sacrificedand serum was colleted to detect paraoxonase (PON1) activity andADMA concentration (HPLC). The aortas were excised to assay theatherosclerotic lesion area (SudanⅣstaining) and Cx43 expression(immunohistochemistry).RESULTSIn ApoE^-/- mice, atherosclerotic lesions were more obviouscompared with C57BL/6J mice, concomitantly with the elevation ofADMA. Exogenous ADMA-treatment induced or aggravatedatherosclerotic lesions in WT and ApoE^-/-mice; Plasma PON1 activitywas significantly reduced in ApoE^-/-mice and exogenous ADMAtreatment further decreased PON1 activity; Exogenous ADMA-treatmentsignificantly decreased Cx43 expression in both ApoE^-/- and WT mice.CONCLUSIONADMA plays a key role in facilitating the development of AS, whichmay be related to the down-regulation of Cx43 and inhibition of GJIC. Part Two: Lysophosphatidylcholine-induced elevation of asymmetricdimethylarginine level by the NADPH oxidase pathwayBACKGROUNDEndothelial dysfunction is closely related to oxidative stress. Recentstudies have suggested that endothelium is a major source of reactiveoxygen species (ROS) and the main source was via NADPH oxidasepathway. NADPH oxidase is involved in multiple pathophysiologicalprocesses such as cell proliferation and apoptosis, NO-mediatedvasorelaxation and anoxia/reoxygenation injury in endothelial cells.Much evidence showed that ADMA was closely related toendothelial dysfunction. Various stimuli including ox-LDL can induceADMA accumulation. Lysophosphatidylcholine (LPC), a majorcomponent of ox-LDL, can induce ADMA elevation and enhance theactivity of NADPH oxidase. In present study, we tested whether theincreased level of ADMA induced by LPC is due to reduction of DDAHactivity and up-regulation of PRMT expression by the NADPH oxidasepathway.METHODSIn LPC-treated endothelial cells (HUVEC-12), ROS production(fluorescence), cell viability (MTT), ADMA (HPLC) and NO levels(Griess), activity of DDAH (HPLC) and expression of PRMTⅠ(westernblot) were detected.RESULTSTreatment with LPC (10 mg/L) markedly increased intracellularROS production, expression of PRMTⅠ, levels of ADMA, decreased theconcentration of NO and activity of DDAH. These effects wereattenuated by diphenyliodonium, the NADPH oxidase inhibitor.CONCLUSIONLPC-induced elevation of ADMA was due to reduction of DDAH activity and the up-regulation of PRMT expression by stimulation of ROSproduction via NADPH oxidase pathway.Part Three: Effects of ADMA on intercellular communicationBACKGROUNDThe functional integrity of endothelium requires highly coordinationbetween cells, among which GJIC plays an important role. It has beenreported that dysfunction of GJIC is closely related to initiation anddevelopment of AS. Cx43 is the main isotype expressed in culturedendothelial cells. The alternation in expression, distribution, and thestructure of Cx43 is of great significance in AS.LDL is the main component of serum lipids and can inducefunctional and structural changes of endothelial cells. The effects of LDLare partly mediated by ADMA. Recent studies suggest that inhibition ofGJIC induced by LDL is partly responsible for LDL-induced endothelialdysfunction. In the present study, we further observed the effects of LPCon GJIC activity and Cx43 expression.Over 90% percent of ADMA was metabolized by DDAH. As aresult, the level of ADMA depends on DDAH activity and expression. Bygene transfection, we established DDAH2 overexpressed HUVECs andobserved the role of DDAH/ADMA pathway in LPC-induced GJICinhibition in DDAH2 overexpressed endothelial cells.METHODS(1) Effects of LPC on GJIC and the role of DDAH/ADMA pathwayUntransfected cells, DDAH2 transfected cells and empty vectortransfected cells were treated with LPC (10 mg/L) for 24 h. GJICfunction (Lucifer yellow) and Cx43 expression (RT-PCR, western blot)were determined.(2) The mechanism of ADMA on GJIC function and Cx43expression in HUVEC-12 ①dose and time effects of ADMA on Cx43 expressionCells were incubated with different concentrations of ADMA fordifferent time courses, and then the expression of Cx43 was detected.②the mechanism of ADMA on Cx43 expression1) role of NADPH oxidaseControl group; ADMA treated group: cells were treated with ADMA(10μM) for 4 h; +DPI (100μM) group; +PDTC (10μM) group; + allo(100μM) group; Vehicle group. After treatment with drugs for half anhour, then added ADMA for 4 h.2) role of p38MAPKControl group; ADMA treated group: cells were treated with ADMA(10μM) for 20 min; +DPI (100μM) group; +specific p38MAPKinhibitor SB203580 (20μM) group; +PDTC (10μM group); + allo (100μM) group; Vehicle group. After treatment with drugs for30 min, thenadded ADMA for 20 min.3) role of PKCControl group; ADMA treated group: cells were treated with ADMA(10μM) for 24 h; +DPI (100μM) group; +PDTC (10μM) group;+specific PKC inhibitor H7 (10μM) group. After treatment with drugsfor 30 min, then added ADMA for 20 min. PKC activator12-O-tetradecanoylpforbal 13-acetate (TPA) group: cells were treatedwith TPA (100 nM) for 1 h.RESULTS1. The expression of DDAH2 was increased in DDAH2 transfected cellscompared with empty vector transfected and untransfected cells.2. DDAH2 overexpression can enhance cell viability, decreaseintracellular ROS production and ADMA concentration.3. DDAH2 overexpression can enhance GJIC function, concomitantlywith up-regulation of Cx43.4. LPC inhibited GJIC and down-regulated expression of Cx43, which isrelated to the excessive production of ROS via activation of NADPHoxidase. DDAH2 overexpression can reverse such effects of LPC.5. ADMA dose- and time-dependently down-regulated Cx43 expression.NADPH oxidase inhibitor, p38MAPK inhibitor and PKC inhibitor can inhibit the effects of ADMA.CONCLUSIONLPC induced inhibition of GJIC was mediated by DDAH/ADMApathway, which may involve ROS-p38MAPK signal pathway.ChapterⅡThe suppression of xanthone on atherogenesis and itsmechanismsBACKGROUNDRecent studies suggested that the alternation of GJIC function playsan important role in LDL induced AS. LDL can dose dependently inhibitGJIC function in smooth muscle cells and endothelial cells. LDL andox-LDL are the main causes of elevated ADMA in AS and the elevationof ADMA also mediates the effects of LDL. Some drugs can inhibit theeffects of LDL via decreasing ADMA levels.Xanthones are polyphenol compounds existing in many plants whichhave potent anti-inflammation, anti-oxidation and endothelial protectiveeffects. Some polyphenol compounds have cholesterol-lowing effects.3,4,5,6-tetrahydroxyxanthone is synthesized by the department ofchemical pharmacy. The previous work of our lab has shown that3,4,5,6-tetrahydroxyxanthone has protective effects on endothelium andmyocardium. In this study, we systematically studied the effects ofxanthone on the progression of AS and its relationship to GJIC both invivo and in vitro.There are two parts: 1) to test the effects of xanthone on serum lipidand oxidative state of hypercholesterolemic rats; 2) to observe the effectsof 3,4,5,6-tetrahydroxyxanthone on the progression of AS in ApoEdeficient mice and its relationship to the alternation of GJIC; and furtherexplored the mechanism of 3,4,5,6-tetrahydroxyxanthone on LPCinduced inhibition of GJIC. Part One Effects of xanthone on serum lipid profiles inhypercholesterolemic ratsMETHODSThe experiments were divided into two parts: in the preventiveexperiment, the rats were treated with xanthone (10, 30 or 90 mg/kg, i.g)for 2 days and then treated with cholesterol emulsion for 10 days in thepresence of xanthone; In the therapeutic experiment, after being fed withfoods rich in cholesterol for 4 weeks, the rats were treated with xanthonefor 3 weeks (i.g.). Serum lipids and malondialdohyde were measured.RESULTSXanthone, given preventively or therapeutically, markedly decreasedtotal cholesterol and LDL level. Moreover, in the therapeutic experimentxanthone also decreased malondialdohyde content.CONCLUSIONXanthone decreased total cholesterol and LDL level inhypercholesterolemic rats, which may be related to its anti-oxidativeeffects.Part Two Effects of xanthone on expression of Cx43 and itsmechanismMETHODS1 animal studyThe experiment was divided into 4 groups (n=8):①Wild typeC57BL/6J mice;②ApoE^-/- mice group;③low dose xanthone treatmentgroup, ApoE^-/- mice were given xanthone (10 mg/kg, i.g.)④high dosexanthone treatment group, ApoE^-/- mice were given xanthone (20 mg/kg,i.g.). After treatment, serum was colleted to detect PON1 activity andADMA concentration (HPLC); aortas were excised to assay atherosclerotic lesion area (SudanⅣstaining) and Cx43 expression(immunohistochemistry).2 cell cultureThe experiment was divided into 4 groups: control group; LPCtreated group: cells were treated with LPC (10 mg/L) for different time;different concentration of 3,4,5,6-tetrahydroxyxanthone group: cells weretreated with 3,4,5,6-tetrahydroxyxanthone (1, 3, 10μM) for 30 min, thentreated with LPC (10 mg/L) for different time. ROS production(fluorescence), cell viability (MTT), ADMA (HPLC) level and expressionof Cx43 (western blot and RT-PCR) were detected.RESULTSIn ApoE^-/- mice, 3,4,5,6-tetrahydroxyxanthone significantlydiminished atherosclerotic lesion area, increased serum PON1 activity,decreased ADMA and up-regulated Cx43 expression. In cultured cells,3,4,5,6-tetrahydroxyxanthone significantly inhibited LPC induceddecreased cell viability, increased ROS production and down-regulatedCx43 expression.CONCLUSION3,4,5,6-tetrahydroxyxanthone has anti-atherosclerotic effects whichis related to the reduction of ADMA level and consequently improvementof GJIC function.