The Role Of ALK7 In The Activation Of Adventitial Fibroblasts Induced By AGEs And Its Mechanism

Background:Diabetic arteriosclerosis is a common pathological basis for a variety of cardiovascular and cerebrovascular diseases-coronary heart disease,stroke and peripheral vascular disease.Adventitial fibroblasts(AFs)are the most abundant cell types in the adventitia.In diabetic state,phenotypic changes,proliferation,migration,and increased collagen secretion are one of the main pathological mechanisms leading to arteriosclerosis.AGEs are stable covalent addition products of macromolecules such as proteins,lipoproteins or nucleic acids that are non-enzymatically catalyzed and spontaneously reacted with glucose or other reducing monosaccharides.They are important intermediates in the development of diabetic complications.The study found that in the long-term chronic high glucose state,the production of AGEs increased,and the cardiovascular complications of diabetes was promoted by AGEs through various ways.Activin receptor-like kinase-7(ALK7)is a new member of the Activin receptor family cloned from human placenta and one of the TGF-? superfamily type I receptors.A variety of ways to participate in the development of diabetes.The study found that the degree of arterial fibrosis was significantly improved after the ALK7 was down-regulated in diabetic rats,and the development of ALK7 diabetes and diabetic ventricular remodeling were closely related to diabetic myocardial fibrosis.Therefore,this study proposes the hypothesis that in the diabetic state,AGEs regulate the proliferation,activation,and collagen secretion of AFs through the ALK7 signaling pathway,causing collagen deposition in the adventitia,and changes in the wall structure,eventually leading to arterial stiffness.Objective:1.To observe the effect of AGEs on the activation of adventitial fibroblasts;2.To investigate the role and possible mechanism of ALK7 in the activation of adventitial fibroblasts induced by AGEs;Methods:1.In vitro culture of SD rat adventitial fibroblasts,AGEs stimulation was given,and the cell proliferation activity,phenotypic transformation and collagen expression levels of type I and III were detected.2.AGEs stimulated the adventitial fibroblasts,and the control group and BSA group was set,and observed the expression level of ALK7 protein;3.The ALK7 siRNA plasmid was constructed to infect the adventitial fibroblasts to inhibit the expression of ALK7.After stimulation with AGEs,the cell proliferation,phenotypic transformation and expression of type I and type III collagen were observed.Results:1.Morphological observation and immunological identification of primary adventitial fibroblasts The cells were flat spindle-shaped,with large nuclei,1-2 /cell,and cytoplasmic transparency;The immunofluorescence of vimentin was positive for green fluorescence,and the red fluorescence of alpha-SMA was negative,confirmed as adventitial fibroblasts.2.AGEs promote AFs cell proliferation,phenotypic transformation and Collagen I/III secretion AGEs promoted the proliferation of vascular fibroblasts for 24 h,and compared with the control group,the proliferation activity was increased(P<0.05).There was no significant difference between the BSA group and the control group.AGEs stimulated the adventitial fibroblasts for 48 h.Compared with the control group,the expression of ?-SMA was increased(P<0.05).There was no significant difference between the BSA group and the control group.Collagen I/III secretion increased compared with the control group(P <0.05),there was no significant difference between the BSA group and the control group.3.AGEs can promote the expression of ALK7 Western blotting results confirmed that ALK7 was expressed in primary rat vascular adventitial fibroblasts.The adventitial fibroblasts were incubated with low glucose DMEM medium and low glucose DMEM medium + BSA and low glucose DMEM medium + AGEs for 0h,4h,8h,12 h,24h,48 h.The results showed that AGEs stimulated for 8h ~ 48 h,the expression level of AKL7 was significantly higher than that of the CON group(P<0.05),and it was time-dependent,but decreased slightly at 48 h,but the difference was still statistically significant.There was no significant difference between the BSA group and the CON group.4.ALK7 to participate in the regulation of the AFs cells proliferation,migration,phenotypic transformation and increased Collagen I/III secretion induced by the AGE After ALK-siRNA was used to inhibit the expression of ALK7 in the adventitial fibroblasts,the proliferation of fibroblasts was decreased after AGEs stimulated fibroblasts for 48 h(P<0.05).The scratch test showed that after down-regulating ALK7,Compared with AGE group,the migration ability of adventitial fibroblasts was significantly decreased(P<0.05);the expression level of ?-SMA was significantly decreased(P<0.01),and the expression level of type I/III collagen was significantly decreased(P<0.05).Conclusions:1.AGEs can promote the proliferation migration,phenotypic transformation and Collagen I/III expression of adventitial fibroblasts;2.AGEs can promote the expression of ALK7 protein in adventitial fibroblasts;3.The ALK7 is involved in the regulation of AGEs-induced proliferation,migration,phenotypic transformation,and increased expression of Collagen I/III of adventitial fibroblasts;...