The Study Of Adiponectin Interferring Nitric Oxide Stress Induced By LPS Via Vascular Adventitia

BackgroundTraditionally,adipose has been regarded as a simple energy storage organ,but mounting evidence suggests it produces and secretes many bioactive substances, collectively referred to adipocytokines such as adiponectin(APN),resistin,TNFα,etc. Now,Adiponectin is found to be the most important protective adipocytokine in the body.Low adiponectin has been found to play an important role in the occurance and development of atherosclerosis,diabetes and metabolic syndrome.Deeply studying the machanism and effects of adiponectin has important theoretical and clinical significance to provent and cure the occurance and development of atherosclerosis and metabolic syndrome.In traditional theory,the function of adventitia is protecting,supporting and nourishing the vessel.But the recent study showed thatadventitia can secret a lot of vaso-active substancs which regulate the vasomotion and change of vessel stucture by paracrine mode.The main cell component in adventitia is adventitial fibroblas. Adventitial fibroblasts can paracrine and antocrine and can secret many biological active factors such as TGF-β1,AB,MCP-1,IL-1,IL-6,TNF-αand MMPs,etc which have important functions to the stucture and function of vessels.When injury in the vessels such as vasodilatation,endothelium strippe,hyperlipoidemia or injury out of the vessels such as adventtia stripped and nourishing bleeding obstructed,adventitial fibroblasts will proliferate,product extracellular matrix,and transform to myofibroblasts,then lead to tissue recovery and vessel remodeling.In the whole course of injury,adventitial cell proliferate more than those of medium.Adventitial fibroblats expressedα-SM-actin which implyed phenotype of adventitial fibroblasts is transformated to myofibroblasts.Though the studies were more and more in recent years,but the relationship between adiponectin and adventitia is also unclear.Many studies showed that anti-inflammatory and anti-atherosclerotic function of adiponectin not only depend on the availability of adiponectin but also on the expression levels of AdipoRs.Whether AdipoRs espressed in adventitia and adventitial fibroblast are not found in documents. To sum up,we hypothesize that there were expression of AdipoRs in adventitia and adventitial fibroblasts.And in inflammatory conditions,the expression of AdipoRs is lowwer.These will lay the foundation of the study of adiponectin's anti-inflammation and anti-atherosclerosis via adventitia.Methods1 Cell cultureAFs were prepared by a modified explant method.The adventitia was carefully decorticated from the aortic wall of C57BL/6J mice and used for AF culture.When cell confluence reached 80%-90%,the cells were removed again and those at passage 4-8 were selected for further study.Monoclonal antibody for a-actin(1:200), vimentin(1:300) and secondary antibody conjugated with TRITC(1:100) were used for immunocytochemistry(ICC) staining to identify AF cells and determine cell purity.2 RT-PCRTo examine some cytokines mRNA expression in AFs after APN and LPS intervention.The expression intensity was demonstrated by the ratio of integral optical density(IOD) value between cytokines andβ-actin.3 Western blot analysisTo examine some cytokines express after APN and LPS intervention.The expression intensity was demonstrated by the ratio of integral optical density(IOD) value between cytokines andβ-actin.4 Immunoeyte fluorescent staining: To observe the express of some cytokines labled with TRITC second antibody in AFs niter APN and LPS intervention 24h.The cytokine expression intensity was demonstrated by IOD value.Results1 AF incubation and identification:The fragments from artery adventitia were cultivated by the explant method for 4-7d days and small amounts of cells started to imigrate out of the specimens and adhere to flask bottom.These cells were cultured for 7-10 days with 10% FBS-DMEM at 37℃and appeared the first confluence.When these cells reached 80%-90%confluence,their passage was carried out,The forth to tenth passages were used for experiment.By means of ICC method,all cultured cells demonstrated negative monoclonal antibody stain for a-SM-actin and positive monoclonal antiboby stain for Vimentin,which suggested that the purity of cultured AFs was almost 100%.2 The expression of Adiponectin and adipoRsAdipoR1 mRNA expression was approximately 85%of that in skeletal muscle. AdipoR1 protein expression in rat adventitial tissues showed a pattern similar to that in skeletal muscle(1.96±1.12 vs 1.69±0.82,P＞0.05).mRNA and protein of AdipoR2 were low-expressed in adventitia than in liver tissues(5.72±3.90 vs 36.67±15.28;0.0059±0.0002 vs 0.6653±0.0270,P＜0.01).Furthermore,no local expression of adiponectin has been detected in adventitial tissues by RT-PCR.3 Effects of Lipopolysaeeharide on expression of AdipoR1Real-time PCR showed that mRNA expression of AdipoR1 is gradually down-regulated as the dose increased,and reached the bottom in 50μg/ml(Fig.3A). The protein expression of AdipoR1 by westernblot analysis also decreased as the dose increased.Real-time PCR revealed that LPS 10μg/ml induced down-regulation of AdipoR1 mRNA in cultured adventitial fibroblasts,and reached its bottom at～48 hours,adipoR1 protein expression levels decreased during the time of 10μg/ml LPS treatment compared to vehicle-treated adventitial fibroblasts. Conclusion1 Adiponectin receptors can effectively express in adventitia and adventitial fibroblasts.AdipoR1 expressed highly,otherwise AdipoR2 expressed lowly. Adiponectin almost no expressed in AFs.2 The expression of AdipoR1 was decreasing with LPS dose increasing and infection time prolonging. BackgroundAdiponectin has been found to play an important role in preventing atherosclerosis in which it reduces the size of atherosclerotic lesions,inhibits neointimal thickening and proliferation of vascular smooth muscle cells in injured arteries,and suppresses expression of vascular adhesion molecules.Vessel endothelial injury initiated the development of atherosclcrosis.Adventitia is the outer most layer of vessel,which made it considered as a bystander.However, lots of studies performed recently indicated that inflammation in the adventitia was related to the development of atherosclerosis and activated during early stage.The degree of adventitial inflammation was positive correlated with the severity of atherosclcrosis,and adventitial inflammation could result in neointimal formation. Both collar and endotoxin infiltrated silk arround adventitia could induce intimal hyperplasia.These researches suggested that adventitia might be involved in neointimal formation,but the mechanism is unclear.It has been well known that oxidative stress is one of the major mechanisms involved in the development of atherosclerosis,which is represented as increased activity of oxidant enzymes and/or reduced activity of antioxidant enzymes.ROS (reactive oxygen species) produced by oxidant enzymes participates in the development and progression of atherosclerosis in two ways:one is the direct injury to vascular cells;the other is as an intracellular second messenger to induce expression of cytokines and participate in the regulation of proliferation and migration of VSMC.Plenty of NO producted by iNOS combined with oxygen or superoxide anion and product ONOO-quickly.ONOO-canlead to cell apoptosis and tissue injury.TheⅡtype NOS locating in the No.17 euchromosomeis is called induced NOS(iNOS).Only after cells are stimulated by inflammatary factors,iNOS will be activated and expressed highly.Plenty of NO producted by iNOS combined with oxygen or superoxide anion and product ONOO-quickly.ONOO-can lead to cell apoptosis and tissue injury.Amounts of studies have shown that lipoprotein overoxidation and endothelium membrane injury are the main causes of atherosclerosis.And ONOO-producted by endothelialcells is the main substance to overoxidate lipoprotein and injure endothelial cells membrane.Adventitial fibroblasts have individual NOS/NO system and can product NO.In normal conditions,iNOS in adventitial fibroblasts is low expressed,and product little NO.But when adventitial fibroblasts are stimulated by LPS,TNF-αand IL-1β,cultured adventitial fibroblasts highly expressed iNOS mRNA.The protein of iNOS and production of NO increases significantly.All above showwed that adventitia is an important resource of NO in vessel walls.To sum up,we hypothesize that adiponectin maybe secreted by adipose tissue arround adventitia mediates anti-atherosclerosis via adventitia-AMPK-iNOS pathway.The results will lay the theoretical foundation to deeply study the mechanism of adiponectin anti-atherosclerosis.According to the ideas mentioned above,this study was therefore designed the experiment in vivo and in vitro to test our hypothesis.Methods1 in vivo test1.1 Animal Protocol:Male apoE-/-mice(n=90),10 to 12 weeks of age,were randomly divided into our groups,including Ad-APN transfer via adventitia, Ad-βgal transfer via adventitia;and control.(30 for each group).Carotid atherosclerotic lesions were induced by perivascular constrictive collars placement on the left common carotid arteries.Four weeks after surgery,mice in Ad-APN transfer via adventitia group,Ad-βgal transfer via adventitia group and control group were taken off the collars and transfected by Ad-APN,Ad-βgal and saline,respectively.1.2 ELISA assay:Aiponectin levels in rabbit serum and in the supernatant of cultured Pichia pastoris were quantitatively determined via immunoassays.A kit consisting of rat anti-human adiponectin enzyme was applied and the measurements were performed following the manufacturer's instructions.1.3 Micro-ultrasound Measurement:Plaque areas were compared with micro-ultrasound imaging techonology.1.4 Biological Measurements:Blood samples were taken from retro-orbital bleeding. Serum lipid was measured.1.5 NO production assay:Serum NO concentrations were measured.1.6 Histological and Morphology Analyses:Sections were stained with hematoxylin and eosin(H&E).iNOS and ONOO-were detected by inmmunostaining.2 in vitro test2.1 Cell culture:The same as Article I.2.2 The inhibitive effect of siRNA on the adipoR1 protein level:To detect protein expression ofβ-aetin and adipoR1 by western blot in transfection cells.2.3 MTT assay:To observe AF proliferative activity after APN and LPS intervention at different time.2.4 Scratch method and transwell method for cells migration:To detect AF migration activity after APN and LPS intervention at different time.2.5 NO production assay:To observe NO production of AFs after APN and LPS intervention.2.6 RT-PCR:To examine mRNA expression of iNOS andβ-actin in AFs after APN and LPS intervention.The expression intensity was demonstrated by the ratio of integral optical density(IOD) value between cytokines andβ-actin. 2.7 Western blot analysis:To examine iNOS,nitrotyrosine,p-AMPK,p-ACC andβ-actin express after APN and LPS intervention.The expression intensity was demonstrated by the ratio of integral optical density(IOD) value between cytokines andβ-actin.2.8 Immunocyte fluorescent staining:To observe the express ofα-SM-actin labled with TRITC second antibody in AFs after APN and LPS intervention 24h.The cytokine expression intensity was demonstrated by IOD value.2.9 Experimental grouping2.9.1 According to APN stimulating doses and time:In order to inspect the change of adventitial fibroblasts stimulated by different doses and time of APN,AFs was grouped into 4 groups according to APN stimulating doses:3μg/ml APN + LPS 10μg/ml group,10μg/ml APN + LPS 10μg/ml group,30μg/ml APN + LPS 10μg/ml group,LPS 10μg/ml(control) group and AFs was grouped into 6 groups according to APN stimulating time:10μg/ml APN + LPS 10μg/ml for 1/2 hr group,10μg/ml APN + LPS 10μg/ml for 2 hr group,10μg/ml APN + LPS 10μg/ml for 6 hr group,10μg/ml APN + LPS 10μg/ml for 12 hr group,10μg/ml APN + LPS 10μg/ml for 24 hr group,and LPS 10μg/ml(control) group.2.9.2 According to siRNA transfection or AMPK inhibitor:In order to observe APN and LPS affecting respectively some cytokines expression in AF related signal transduction pathway,AFs were grouped into 6 groups:10μg/ml APN + LPS 10μg/ml group,siRNA-AdipoR1+10μg/ml APN + LPS 10μg/ml group,nonsense siRNA+ 10μg/ml APN + LPS 10μg/ml group,Compound C+10μg/ml APN + LPS 10μg/ml group,10μg/ml APN group and LPS 10μg/ml(control) group.Results1 in vivo test:1.1 Body Weigh:In total,one mouse in Ad-APN group and Ad-βgal group respectively died after surgury.There were no significant differences in the body weights between groups.After 8 weeks,the body weughts were decreased significantly in Ad-APN group vs control group or Ad-βgal groups(27.35±1.26 vs 31.44±1.24.27.35±1.26 vs 30.75±1.21,P all＜0.01) 1.2 Serum adiponectin concentrations:Serum adiponectin concentrations in mice after Ad-APN group increased about 9 times as much as those before transfer(P＜0.01).After transfer,the serum adiponectin concentrations elevated to a level about 10 times higher than the level of endogenous adiponectin in Ad-GFP treated mice(P＜0.01).1.3 Micro-ultrasound Measurement:Micro-ultrasound imaging revealed that in mice infected via adventitia,Ad-APN treatment reduced plaque area by 26.72%as compared with the area before treatment(P＜0.01) and 24.53%compared with that in Ad-βgal group(P＜0.01).1.4 The effect of APN on serum lipid:There was no significant differences in the lipid profile between groups before surgury(P＞0.05).Compared to control group, serum TC,TG and LDL decreased significantly in Ad-APN group(11.56±1.07 vs 19.28±1.69;0.13±1.47 vs 0.86±1.57;3.46±1.54 vs 7.32±1.63,P all＜0.01,and serum HDL increased(2.07±1.64 vs 1.03±1.25,P＜0.01).Compared to Ad-βgal group, serum TC,TG and LDL decreased significantly in Ad-APN group(11.56±1.07 vs 19.32±1.45;0.13±1.47 vs 0.97±1.69;3.46±1.54 vs 7.83±±1.35,P all＜0.01),and serum HDL increased(2.07±1.64 vs 0.99±±1.46,P＜0.01).1.5 The effect of APN on NO production and expression of iNOS and ONOO-: Compared with Ad-βgal group and control group,the serum NO concentration and expression of iNOS and ONOO-decreased significantly(3.57±1.53 vs 14.62±1.47; 3.57±1.53 vs 15.25±1.38,P all＜0.01).1.6 The effect of APN on expression of iNOS and ONOO-:Compared with Ad-βgal group and control group,the expression of iNOS and ONOO- decreased significantly (iNOS:4.36±1.58 vs 6.75±1.76;4.36±1.58 vs 7.31±1.65,ONOO-:6.29±1.74 vs 11.34±2.20;6.29±1.74 vs13.01±1.78,P all＜0.01).2 in vitro test2.1 cell culture:the same as Article I.2.2 The effect of siRNA on the adipoR1 protein level:Using different dose of siRNA-AdipoR1(50,100,200,250pmol/L) transfect AFs,the results of western blot showwed that according to the increasing doses of siRNA-AdipoR1,expression of adipoR1 decreased(P＜0.01) and expression ofβ-actin has no change.2.3 The effect of APN and LPS on AF proliferation(1) LPS increasing AF proliferation activity:Through MTT assay confirmation, compared with control group,each group of LPS could effectively increase AF's growth(0.564±1.46 vs 0.265±1.34,P＜0.01).(2) APN affecting AF proliferation induced by LPS:Through MTT assay confirmation,no change was accured in normal AF treated by APN(0.273±1.59 vs 0.265±1.34,P＞0.05).There were lowwer OD value in APN + LPS group than LPS group(0.241±1.52 vs 0.564±1.46,P＜0.01).There were higher OD value in siRNA-adipoR1+APN+LPS(0.607±1.65 vs 0.241±1.52) and Compound C+APN+LPS group(0.584±1.36 vs 0.241±1.52) than APN + LPS group(P all＜0.01).2.4 The effect of APN and LPS on AF migration(1) LPS regulating AF migration in dose and time dependent manner:Scratch test showwed that with LPS transfected time prolonging,LPS's effect on AF migration was increasing.Just like the result of scratch test,transwell test showwed that with LPS transfected time prolonging,cell numbers migarted cross the membrane increaed.(2) APN affecting AF migration induced by LPS:There were shorter migration lenth and migration cells in APN +LPS group than LPS group(16.12±4.58 vs 48.60±5.12,P＜0.01).There were longer migration lenth and cells in siRNA-adipoR1+APN+LPS(45.66±5.23 vs 16.12±4.58) and Compound C+APN+LPS group(44.76±4.97 vs 16.12±4.58)than APN + LPS group(P all＜0.01).2.5 The effect of APN and LPS on AF transition to MF(1) LPS affecting AF transition to MF:Through immunocyte fluorescent staining confirmation,AF showwed positive monoclonal antibody stain for a-SM-actin in LPS group.(2) APN affecting AF transition to MF induced by LPS:Compared to normal AFs, LPS treatment lead to increased a-SM-actin positive cell numbers(27.65±3.27 vs 3.58±2.16,P＜0.01).There were little numbers of a-SM-actin positive AFs in APN+LPS group and LPS group(5.79±3.06 vs 27.65±3.27,P＜0.01).There were more numbers of a-SM-actin positive AFs in siRNA-adipoR1+APN+LPS(28.46±4.51 vs 5.79±3.06) and Compound C+APN+LPS group(27.99±4.53 vs 5.79±3.06) than APN + LPS group(P all＜0.01).2.6 The effect of APN and LPS on AF's NO production(1) LPS affecting AF's NO production:In normal conditions,AF can product low level of NO.Under LPS treatment,NO production increased significantly(9.12±1.52 vs 1.96±1.14μM,P＜0.01).(2) APN affecting AF's NO production:There were lowwer NO production in APN+LPS group than in LPS group(1.99±1.05 vs 9.12±1.52μM,P＜0.01).In different doses of APN interfer(3,10,30μg/ml),NO production reached the lowwest level in 10μg/ml.When APN(10μg/ml) treated in 1/2,1,2,6,12,24,48hr,NO production reached the lowwest level in 2 hr,then according to the time of APN treatment,NO production increaed lowwly.There were more NO production in siRNA-adipoR1+APN+LPS(8.23±2.04 vs 1.99±1.05μM) and Compound C+APN+LPS group(8.75±1.69 vs 1.99±1.05μM) than APN + LPS group(P all＜0.01).2.7 The effect of APN and LPS on AF's expression of iNOS and ONOO-:There were lowwer expression of iNOS and ONOO- in APN+LPS group than LPS group(P all＜0.01).There were higher expression of iNOS and ONOO- in siRNA-adipoR1+APN+LPS(23.47±4.37 vs 4.59±1.53) and Compound C+APN+LPS group(21.75±3.96 vs 4.59±1.53) than APN + LPS group(P all＜0.01).2.8 The effect of APN and LPS on AF signal transduction pathway:There were more protein expression of p-AMPK and p-ACC in APN+LPS group than LPS group (P all＜0.01).There were lowwer expression of p-AMPK and p-ACC in siRNA-adipoR1+APN+LPS(p-AMPK:11.07±1.33 vs 15.36±2.57;p-ACC:9.75±1.24 vs 23.41±3.55) and Compound C+APN+LPS group(p-AMPK:3.54±1.33 vs 22.48±3.69;p-ACC:3.72±1.03 vs 18.62±3.41)than APN + LPS group(P all＜0.01).Conclusion 1 Local transfer of Ad-APN via adventitia can effect serum lipid concentration and body weight which imply APN can promote metabolic function.2 Local transfer of Ad-APN via adventitia reduced serum NO concentration and the expression of iNOS and ONOO- in vascular walls.It implied that Local transfer of Ad-APN via adventitia may be have the effect on atherosclerosis by reducing expression of iNOS and ONOO-.3 LPS promotes AF migration,proliferation and transition to myofibroblast,otherwise APN can reverse these effects of AF induced by LPS.4 LPS can activate iNOS of AFs and increases NO and ONOO- production.APN can inhibit iNOS expression induced by LPS.5 APN may be activate AMPK signal pathway via adipoR1,inhibit iNOS expression and NO and ONOO- production and then inhibit AF migration,proliferation and transition to myofibroblast induced by LPS.