The Study On The Promoting Effect Of Adventitial Activation On The Formation And Development Of Atherosclerotic Lesion

The "Response-to-Injury Hypothesis" and the "Inflammation Hypothesis" put forward and revised by R.Ross,are the dominant hypotheses in the mechanism study of atherosclerosis(AS),which indicate that injury to the endothelium and the proliferation of vascular smooth muscle cells play an important role in the development of AS and the injury to the endothelium is recognized as the initiating event.However the role of the adventitia in the development of AS has not been mentioned.The adventitia is traditionally defined as nothing more than "loose connective tissue" surrounding blood vessels.It has been considered to be simply a support for the blood vessel and a scaffold for sympathetic nerve endings and the vasa vasorum.Thus,given its apparently marginal importance,it has not been the focus of much attention.The contribution of the adventitia and adventitial fibroblasts to atherogenesis have not been clear.Much attention was gradually paid to the role of the adventitia as an innocent bystander or an active participant in cardiovascular disease in recent years.Some reports have proposed that the vascular adventitia acts as a critical regulator to modulate,directly or indirectly,the structure and function of the vascular wall.As the main cell type in the adventitia,the fibroblast exhibits histological,biochemical and functional characteristics that relate to regulate vascular response to injury or stress. In 1962 Schwartz and Mitchell investigated over 440 tissue blocks from 111 randomly selected men and women over 35 years of age and found a positive relationship between the degree of adventitial inflammation and the severity of atherosclerotic disease in the overlying arterial wall.Kim Rayner studied the arterial serial sections of apoE(-/-)mice after 0-12 weeks' feeding of hyperlipidic diet at the mRNA level and found that the earliest expression of JE/MCP-1 and CCR2 mRNA was seen in mesenchymal cells in the adventitia,macrophages were subsequently found to accumulate in these affected regions.The result showed that inflammatory cells infiltrated in the adventitia before the intimal lesion formation.Mori transfected the mutant of MCP-1 gene into skeletal muscles around the carotid artery in rabbits after balloon injury and he found the suppression of the inflammation mediated by MCP-1 and the attenuation of monocyte infiltration and the adventitial fibrosis and thus attenuated the pathologic artery remodeling.The composition of the adventitial matrix was altered during the process of diseases such as atherosclerosis and restenosis. Marked increases in the production and accumulation of collagen and elastin were observed in the adventitia during the development of pulmonary hypertension. Zalewski has postulated that fibroblasts proliferation precedes modulation to myofibroblasts,which then migrate to the neointima of the coronary artery after balloon angioplasty.Furthermore,in carotid artery-vein grafts,neointimal proliferation is preceded by activation and proliferation of adventitial fibroblasts, modulation to myofibroblasts,and migration to the neointima.However many reports related to the advantitia,recognized the adventitial inflammation and other reactions as the late pathologic changes of AS.There are few data about the adventitial participation in the early stage of atherosclerosis.Our previous findings indicated that accumulation of inflammatory cells in the adventitia of coronary artery trees and branches in the myocardial tissue of apoE(-/-) mice induced the formation and extending of atherosclerotic lesions.Objectives:In the present study we aimed to systemically study the relationship between the activation of adventitia and the atherosclerotic lesion formation in vivo by observing the adventitia and specifically the fibroblasts phenotype differentiation,proliferation, migration,collagen composition and the changes of various cytokines at the different stage of atherosclerosis in the apoE(-/-)mice.In addition we further cultured the adventitial fibroblasts derived from apoE(-/-)mice and C57BL/6 mice to observe the differentiation of phenotype,functional characteristics and gene expression.Then we choose the different genes to analyze the mechanism of the participation of the adventitial fibroblasts in the atherosclerotic lesion formation.Thus given the special position of the adventitia we devise strategy for gene therapy from the adventitia.It may be a new way to the therapy of AS.At the same time the data will provide proof for the hypothesis-the activation of the adventitial fibroblasts promote the formation and development of the early atherosclerotic lesion.The experiment contains three parts as following:Partâ… :The study on the role of the adventitial activation in the atherosclerotic lesion formation and development in vivoMethods:ApoE(-/-)mice of 6 weeks were used in this experiment.Wild-type C57BL/6 black mice were used as controls.All animals were fed hyperlipidic diet(84.75% basic feed,15%w/w saturated fat,0.25%cholesterol)and water ad libitum. 5-bromo-2-deoxyuridine(BrdU)was administered 24 h(30 mg/kg ip)before the animals were killed at 0,2,4 or 8 weeks after onset of hyperlipidic diet.The ascending aorta was removed with the heart attached for serial sectioning.Some sections were stained by the HE and Movat methods in order to observe the morphological changes of the tissues and measure the thickness of the adventitia. Some sections were stained with Sirius red to identify the typeâ… and typeâ…¢collagen under the polarizing microscope.Some sections were stained with immunohistochemistry to observe the changes in the expression ofα-SM-actin,vimentin, desmin,BrdU,MCP-1,ICAM-1,VCAM-1,TGF-β₁ and p47phox in the adventitia and intima of different stages.FISH was performed to examine the expression of the mRNA for JE/MCP-1.Results:1.The adventitial thickness of apoE(-/-)mice increased gradually with increasing duration of the hyperlipidic diet.After hyperlipidic diet for 4 weeks the foam cells were showed in the intima and the atherosclerotic lesion developed after hyperlipidic diet for 8 weeks in apoE(-/-)mice.However C57BL /6 mice did not show any changes of adventitial thickness nor lesion development at any time point.2.Minority adventitial cells expressedα-SM-actin in apoE(-/-)mice after hyperlipidic diet for 2 and 4 weeks but norα-SM-actin expression after hyperlipidic diet for 8 weeks.Desmin expression was not found at any time point.Most adventitial cells expressed vimentin.It showed the cells mentioned in adventitia were fibroblasts and some of them transdifferentiated into myofibroblasts.However there was noα-SM actin expression in the adventitia of C57BL/6 mice all the time.3.The expression of collagen increased gradually with increasing duration of the hyperlipidic diet in apoE(-/-)mice and was significantly increased than the control group.The proliferation of collagenâ…¢was predominated till the hyperlipidic diet of 2 weeks.After hyperlipidic diet for 4 weeks,the increase of collagen was attributable mainly to collagenâ… .There were no significant changes of collagen in C57BL/6 mice.4.After hyperlipidic diet for 2 weeks in apoE(-/-)mice,the BrdU-labeled cells were found in the adventitia when there was no visible intimal lesion formation and then subsequently emerged in the intimal lesion.But no expression of BrdU was found in C57BL/6 mice at any time point.5.Before the lesion formation in apoE(-/-)mice,the expression of MCP-1, TGF-β₁,p47phox,VCAM-1 were firstly found in the adventitia and the expression of ICAM-1 increased gradually in the adventitial vasa vasorum and the intima with increasing duration of the hyperlipidic diet.However the expression of ICAM-1 was infrequent and stable in C57BL/6 mice.In addition only minority fibroblasts in C57BL/6 mice expressed p47phox but there was not prominent changes after hyperlipidic diet.Conclusions:1.The activation of the adventitia and specifically adventitial fibroblasts before the atherosclerotic lesion formation promotes the formation and development of atherosclerotic lesion in apoE(-/-)mice.2.The adventitial fibroblasts exhibit the characteristic of differentiation into myofibroblasts,the increase of collagen composition,proliferation and the production of active cytokines before the atherosclerotic lesion formation,which are induced by hyperlipoproteinemia and other risk factors.3.The expression of adhesion molecules in the adventitial vasa vasorums increases before the atherosclerotic lesion formation which is induced by the hyperlipoproteinemia and other risk factors.It is one of the exhibition of the activation of the adventitia.4.Oxidative stress is one of the cause of the activation of the adventitial fibroblasts and is also the manner of autocrine and paracrine by which the adventitial fibroblasts promote the formation and development of the atherosclerotic lesion.5.Adventitial inflammation is an early event in the formation and development of atherosclerotic lesion.Partâ…¡:The study on the role of the adventitial fibroblasts in the atheroselerotie lesion formation and development in vitroMethods:In present study the adventitial fibroblasts derived from apoE(-/-)mice and C57BL/6 mice of 6 weeks after hyperlipidic diet for 2 weeks were cultured by the explanting method.Fibroblasts in culture between third and seventh passages were used.The cell phenotype was observed by immunofluorescence staining of vimentin, desmin andα-SM- actin and by transmission electromicroscope.The synthesis of collagens was examined using the hydroxyproline kit.The proliferation of cells were examined by CCK-8 kit and the method of Brdu incorporation.Cell cycle was examined by PI staining by flow cytometry.Cell migration was measured in a transwell chamber apparatus.The NADPH oxidase activity and superoxide anion production were measured by NADPH oxidase kit and superoxide anion kit.We examined cell proliferation and migration after preincubation of cells with NAD(P)H oxidase inhibitor(DPI).The difference of gene expression was observed from the gene chip.Some different genes were verified by RT-PCR and western-blot.We transfected p47phox stealth siRNA into apoE(-/-)mice fibroblasts to observe the effect on NADPH oxidase activity,the cell proliferation and migration. Results:1.Cells of two groups showed vimentin positive and desmin negative.Most cells of apoE(-/-)mice exhibited the characteristic of fibroblasts and minority cells showed the ultrastructural changes,such as the obviously increase of filaments.Minority cells of apoE(-/-)mice showedα-SM-actin positive but noα-SM-actin positive was found in C57BL/6 mice.2.The adventitial fibroblasts of apoE(-/-)mice exhibited a higher capacity to synthesize collagens compared with C57BL/6 mice.3.The adventitial fibroblasts of apoE(-/-)mice possessed an increased capacity to proliferate when compared with C57BL/6 mice in culture with 5,10 and 20%serum. The same result was gained by the method of Brdu incorporation.The adventitial fibroblsts of apoE(-/-)mice exhibited higher percent of S and G₂/M phase than C57BL/6 mice.4.The adventitial fibroblasts of apoE(-/-)mice possessed increased capacity of migration than that of C57BL/6 mice.5.The adventitial fibroblasts of apoE(-/-)mice possessed increased NADPH oxidase activity and superoxide anion production than that of C57BL/6 mice.6.The preincubation of cells with NAD(P)H oxidase inhibitor(DPI)reduced NADPH oxidase activity and O₂^- production.The cell proliferation and migration of apoE(-/-)mice were significantly inhibitted.7.The gene chip assay of the adventitial fibroblasts of apoE(-/-)mice showed the up-regulation ofoncogenes such as Rab3a,Rabl,Fyn,c-Met,grow factor genes such as PDGF,FGF,TGF,inflammatory cytokine genes such as IL-4,Tnfsf,chemokines such as MCP-5,CCR-2,CCR-5,adhesion molecule genes such as ICAM-2, Itga3,CD36,extracellular matrix genes such as collagen,Fbn2,TN-C,Fibulin4,and some enzyme genes such as Noxo 1,Mapk,Prkcbp 1,but the down-regulation of cancer suppressor genes and apoptosis genes.The different genes were mostly associated with cell growth,oxidation stress,signal transduction,inflammation and cell adhesion.8.The adventitial fibroblasts of apoE(-/-)mice possessed increased mRNA level of collagenâ… ,collagenâ…¢,TGF-β₁,MCP-1,CCR-2,PDGF-b and p47phox.The protein levels of TGF-β₁ MCP-1,PDGF-b,p47phox were coincident with the results of mRNA expression mentioned above.9.After 48 hours of the transfection of p47phox siRNA(MSS206956),the NADPH oxidase activity was reduced and the fibroblasts proliferation and migration was attenuated in apoE(-/-)mice.Conclusions:1.Part of the adventitial fibroblasts of apoE(-/-)mice exhibit the characteristic of transdifferentiation into myofibroblasts.2.The adventitial fibroblasts of apoE(-/-)mice exhibit stronger activity of proliferation,migration,NADPH oxidase,synthesis of collagens and active cytokines compared with C57BL/6 mice.3.Oxidative stress activates the adventitial fibroblasts.The increase of NADPH oxidase activity promotes the activity of proliferation and migration of the adventitial fibroblasts.4.The subunit of NADPH oxidase,p47phox,may be one target of the gene therapy of AS.Partâ…¢.The suggestion of a new hypothesisOn the basis of the experimental data in vivo and in vitro,we put forward a new hypothesis—The activation of the adventitial fibroblasts promotes the formation and development of early atherosclerotic lesion.If verified,this hypothesis would a complement of the "Response-to-Injury Hypothesis" and the "Inflammation Hypothesis"...