| Chapter 1 The role of ADMA in the inflammatory reactions ofendothelial cells induced by AngⅡand potential mechanismsObjectives: Asymmetric dimethylarginine (ADMA), an endogenous NOSinhibitor, can mediate inflammatory reaction via induction of"NOS uncoupling"to increase intracellular oxygen free radicals generation contributing to theprocess of atherosclerosis (AS). A small scale of clinical studies have shown thattreatment with ACEI or ARB attenuated the elevated levels of ADMA inhypertensive patients, suggesting that activation of RAS may play an importantrole in the elevation of ADMA level. However, the precise relationship betweenAngⅡand ADMA is still unclear. Thus, the present study aims to explore theeffect of AngⅡon the production of ADMA and the role of ADMA in theinflammatory response of endothelial cells induced by AngⅡ, further toinvestigate potential mechanisms.Methods: The activation of endothelial cells was induced by incubationwith different concentrations of AngⅡ(10-9-10-6 M) for various times (6-48 h)in the absence or presence of losartan (1, 3 or 10μM) for 1 h or L-arginine (0.5mM) for 1.5 h. In order to clarify the role of ADMA in inflammatory reaction ofendothelial cells induced by AngⅡ, exogenous ADMA was used in the presentstudy. Endothelial cells were incubated with ADMA (30μM) for 4 or 24 h. Thelevels of ADMA (HPLC), NO (Griess), TNF-αand IL-8 (Elisa) in the medium,intracellular ROS generation, and the adhesion of endothelial cells formonocytes were measured. The expression of IL-8 receptor CXCR2 mRNA(RT-PCR), the protein expression of PRMT (Western blot), the activity ofDDAH (HPLC), and the activity ofNF-κB (EMSA) in cultured endothelial cellswere also determined.Results: 1. Treatment with AngⅡ(10-9-10-6 M) for 24 hconcentration-dependently increased the protein expression of PRMT inendothelial cells. Incubation with AngⅡ(10-6 M) for 6 h seemed to have not asignificant expression of PRMT. However, incubation for 12, 24 or 48 h causeda significant increase in the expression of PRMT, and the maximal effect wasobtained at 24 h. Simultaneously, AngⅡdecreased the activity of DDAH andelevated the level of ADMA. in the medium. 2. Incubation with AngⅡ(10-6 M) for 24 h markedly decreased the level ofnitrite/nitrate, increased the activity of NF-κB and elevated the levels of IL-8and TNF-α, concomitantly with a significant increase in the adhesion ofmonocytes to endothelial cells. AngⅡ(10-6 M) for 4 h significantly enhancedthe intracellular ROS production and upregulated IL-8 receptor CXCR2 mRNAexpression. Pretreatment with losartan (1, 3, 10μM) concentration-dependentlyattenuated the effects induced by AngⅡ. A similar effect was induced bypretreatment with L-arginine.3. Exposed to ADMA (30μM) for 24 h significantly decreased NO level,increased the activity of NF-κB and elevated the levels of IL-8 and TNF-α.ADMA (30μM) for 4 h significantly enhanced intracellular ROS generation andupregulated the expression of CXCR2 mRNA. ADMA also increased theadhesion of endothelial cells for monocytes, the effect which was attenuated bypretreatment with losartan or L-arginine.Conclusions: 1. It was first reported that in cultured endothelial cells, AngⅡelevates the level of ADMA, which is related to increase PRMT proteinexpression and decrease DDAH activity.2. ADMA increases monocytes binding to injured endothelial cells viainduction of oxidant stress and activation of NF-κB then to switch on theexpression of downstream inflammatory factors such as chemokine receptorCXCR2.3. Treatment with losartan attenuates oxidant stress and inflammatoryreaction induced by endogenous or exogenous ADMA, suggesting that ADMAis involved in inflammatory reaction and endothelial injury induced by AngⅡ. Chapter 2 The effect of ADMA on monocyte activity in culturedmonocytes and potential mechanismsObjectives: It was reported that in cultured endothelial cells, ADMAincreases the release of MCP-1, and facilitates monocytes binding to endothelialcells. But whether ADMA directly activates monocytes to induce their adhesionto endothelial cells is still not clear. The results of previous chapter 1 haveshown that incubation of endothelial cells with AngⅡcould elevate the levels ofADMA. However, whether there exist the enzyme systems metabolizing ADMAin monocytes like endothelial cells and AngⅡcan induce a similar effect incultured monocytes to promote the development of AS is worth of furtherinvestigation. Thus, the present study aims to investigate the effect of AngⅡonADMA production and to further explore the molecular mechanisms responsiblefor monocytic activation induced by ADMA.Methods: THP-1 cells were incubated with AngⅡ(10-6 M) for 4 or 24 h inthe absence or presence of losartan (1, 3 or 10μM) for 1 h or antioxidant PDTC(10μM) for 1.5 h. The supematant in the conditioned medium was collected bycentrifugation for determination of ADMA (HPLC), MCP-1, IL-8 and TNF-α(Elisa). Intracellular ROS levels and the adhesion of monocytes to endothelialcells were measured. The expression of IL-8 receptor CXCR2 and MCP-1receptor CCR2 mRNA was determined by RT-PCR. The protein expression ofPRMT (Western blot) and the activity of DDAH (HPLC), NF-κB and AP-1(EMSA) in monocytes were also determined.Results: 1. There is the system of synthetase and hydrolase of ADMA incultured THP-1 cells. AngⅡ(10-6 M) for 24 h significantly increased the proteinexpression of PRMT and decreased the activity of DDAH in monocytes,resulting in elevation of ADMA level. Losartan or PDTC decreased the elevatedADMA level by AngⅡvia down-regulation of PRMT expression andimprovement of DDAH activity.2. Incubation of monocytes with AngⅡ(10-6 M) for 4 h or 24 h markedlyelevated the levels of MCP-1, IL-8 and TNF-α, and enhanced intracellular ROSlevels. Stimulation with AngⅡ(10-6 M) simultaneously activated NF-κB andAP-1 pathways, upregulated the expression of CXCR2 and CCR2 mRNA, andincreased the adhesion of monocytes to endothelial cells. Pretreatment withlosartan (1, 3, 10μM) concentration-dependently attenuated the above effects by AngⅡand inhibited monocytic adhesion. Antioxidant PDTC also inhibitedoxidant stress and monocytic adhesion induced by AngⅡ.3. Culture of monocytes with ADMA (30μM) for 4 h or 24 h significantlyincreased intracellular ROS generation, activated NF-κB and AP-1, andincreased the levels of MCP-1, IL-8 and TNF-αand the expression of CXCR2and CCR2 mRNA, accompanied with increased adhesion of monocytes toendothelial cells. The above effects by ADMA were partly attenuated bypretreatment with losartan or PDTC.Conclusion: 1. There is the system of PRMT and DDAH in THP-1 cells.2. The elevated level of ADMA by AngⅡis related to upregulation ofPRMT protein expression and reduction of DDAH activity.3. ADMA induces the adhesion of monocytes via upregulation ofchemokine receptors expression, in which ROS/NF-rA3/AP-1 pathway isinvolved.4. Losartan attenuates monocytic adhesion induced by endogenous orexogenous ADMA, suggesting that ADMA is involved in monocytic adhesionand activation induced by AngⅡ.Chapter 3 The effect of ADMA on chemokine receptors expressionof monocytes in peripheral blood in hypertensive individualsObjectives: Vascular endothelial dysfunction and adhesion of monocytes toinjured endothelium in the arterial wall are key steps in vascular lesion incardiovascular diseases. In the previous two chapters, the roles of ADMA inendothelial injury and monocytic activation in vitro were investigated. Therefore,the present study aims to determine endothelial function and the activity ofmonocytes in individuals with primary hypertension, further to explore the roleof ADMA in this process.Methods: 42 patients with essential hypertension and 40 healthy controlsin the out-patient clinic and admission department of Xiangya Hospital were enrolled in the present study from 2005.8 to 2006.5. Flow-mediated dilation(FMD) on brachial artery at rest and during reactive hyperemia was assessed byhigh-resolution ultrasonography. Plasma levels of ADMA (HPLC), NO (Griessmethod), MCP-1, IL-8, TNF-αand vWF (Elisa) were measured. Peripheralblood mononuclear cells (PBMC) were separated by gradient centrifugation.Monocytes were collected after adherence and cultured with exogenous ADMA(30μM) for 24 h. The expression of CXCR2 mRNA was determined byRT-PCR and the protein expression of CCR2 in separated mononuclear cells wasanalyzed by flow cytometry.Results: 1. Compared with controls, plasma levels of ADMA weresignificantly elevated (P＜0.05) and the levels of NO were markedly decreased(P＜0.05) in patients with primary hypertension, accompanied with endothelialdysfunction, manifesting that brachial artery FMD significantly decreased andconcentrations of vWF (a marker of endothelial dysfunction) markedlyincreased (P＜0.01).2. Compared with controls, plasma levels of inflammatory factors such asMCP-1, IL-8, TNF-αwere markedly increased in hypertensive patients (P＜0.05,P＜0.01).3. The expression of CXCR2 mRNA and CCR2 protein in separatedperipheral blood monocytes was higher in individuals with hypertension than incontrols. Incubation with ADMA (30μM) for 24 h significantly upregulated theexpression of CXCR2 and CCR2.Conclusions: 1. Endothelial function is impaired and the activity ofmonocytes is increased in patients with essential hypertension.2. The expression of chemokine receptor CXCR2 and CCR2 is upregulatedin individuals with primary hypertension. Incubation of monocytes with ADMAfurther upregulates the expression of CXCR2 and CCR2.3. Vascular endothelial injury and moncytic activation in patients withessential hypertension may promote the development of AS. |
PDF Full Text Request