| BackgroudAtherosclerosis(AS)is a slowly progressive disease.It is the main cause of various cardiovascular and cerebrovascular diseases,mainly involving the large and middle arteries.Numerous studies have proved that vascular endothelial cells(VEC),vascular smooth muscle cells(VSMC)and adventitial fibroblasts(AF)are closely related to vascular remodeling.Previously,endothelial cells and smooth muscle cells were mainly taken as the focus of research.But now a large number of studies have shown that adventitial fibroblasts was able to respond to various stimuli,which plays an important role in proliferation,cell migration,phenotype transformation,adhesion,apoptosis,secretion of cytokines and synthesis of extracellular matrix.Recently,it was found that there were many progenitor cells in vascular adventitia,which can differentiate into vascular smooth muscle cells either in vivo or in vitro.More and more evidences show that the AF plays an important role in vascular proliferative lesions.These suggest that the role of adventitia in the pathogenesis of AS cannot be ignored.Fibroblast specific protein(FSP1)is a cytokine secreted by fibroblasts.Numerous studies have shown that fibroblast specific protein(FSP1)plays an important role in maintaining the biological function of cells.However,the specific mechanism of FSP1 on AF cells remains unclear.Studies have shown that FSP1 mediates a variety of signaling pathways to participate in biological activities.However,it is still unclear which signal pathways are involved in the biological functions in AF cells(such as cell proliferation,migration,adhesion,apoptosis).In conclusion,we proposed the hypothesis that FSP1 can activate the signal transduction network that affects the proliferation of AF cells,thus regulating the biological activity of AF cells.Based on the above ideas,this study was designed to verify this hypothesis in vitro experiments.Objectives1.To investigate the effects of FSP1 and siRNA-FSP1 on the proliferation,migration,adhesion and apoptosis of AF cells.2.To explore the molecular mechanism of FSP1 regulating the biological function of AF cells.3.To investigate the effect of FSP1 on inflammatory factors secreted by AF cells.Methods1.Cell cultureThe adventia was decorticated from the aortic wall of mouse and the primary cells were cultured by a modified explant method.The passage was carried out when the cells reached 80-90%confluence.The primary cells from the fourth to the eighth generations were used for further study.Monoclonal antibody for a-actin(1:200),vimentin(1:200)and secondary antibody conjugated with TRITC(1:100)were used for immunocytochemistry staining to identify AF cells and determine cell purity.2.Experimental groupingAccording to different stimulation,AF cells in the experiment were grouped into AF(untreated),siControl(negative siRNA duplex for FSP1),siRNA-FSP1,FSP1+siRNA-FSP1,FSP1,FSP1+AG490(JAK/STAT specific blocker),FSP1+DKK(Wnt specific blocker),FSP1+FPS-ZM1(RAGE specific blocker),FSP1+Stattic(JAK/STAT specific blocker).3.MTT assay and EdU test:The two experiments were carried out to observe AF proliferative capacity after FSP1,siRNA-FSP1 and signal pathway blockers(AG490,DKK,FPS-ZM1,Stattic)intervention for 48 hours.4.Wound healing test for cell migration:The test was to observe AF migration activity after FSP1,siRNA-FSP1 and signal pathway blockers(AG490,DKK,FPS-ZM1,Stattic)intervention at different time.Three windows were selected randomly in each cell culture plate and the average healing area was taken as comparison.5.Adhesion assay:To observe AF cells adhesion ability in different groups after FSP1,siRNA-FSP1 and signal pathway blockers(AG490,DKK,FPS-ZM1,Stattic)intervention.6.Flow cytometry analysis:The test was to detect AF cell cycle distribution and apoptosis rate in different groups after FSP1,siRNA-FSP1 and signal pathway blockers(AG490,DKK,FPS-ZM1,Stattic)intervention.7.Westm blot analysis:To detect the expression of signal pathway proteins(FSP1,RAGE,JAK2/STAT3,Wnt3a/?-catenin)and inflammatory cytokines(MCP-1,VCAM-1,ICAM-1)after FSP1,siRNA-FSP1 and signal pathway blockers(AG490,DKK,FPS-ZM1,Stattic)intervention.The expression of cell cycle related factors(CDK4,Cyclin-D1 and P53)in AF(normal group),siRNA-FSP1 and FSP1 were also observed.The expression intensity was demonstrated by the ratio of integral optical density(IOD)value between cytokines and P-actin.8.RT-qPCR examination:To detect the expression of mRNA of FSP1,RAGE,JAK2,STAT3,Wnt3a and ?-catenin after FSP1,siRNA-FSP1 and signal pathway blockers(AG490,DKK,FPS-ZM1,Stattic)intervention.The expression intensity was demonstrated by the ratio of integral optical density(IOD)value between cytokines and ?-actin.9.Cell immunofluorescence analysis:After the intervention of FSP1,siRNA-FSP1 and signaling blockers(AG490,DKK,FPS-ZM1,Stattic),the expression of FSP1,RAGE,JAK2/STAT3,Wnt3a/?-catenin signaling pathway proteins in AF cells were detected by cellular immunofluorescence.IOD was used to compare the strength of their expressions in different groups.Results1.Incubation and identification of AF cells:The artery adventitia fibroblast was cultured by the explant method.About 4 days,a small number of cells could be seen swimming out and sticking to the dish wall around the tissue block.These cells were cultured in 10%FBS-DMEM and came to the first confluence about 8-9 days.The passage was carried out when the cells reached 80-90%confluence.The primary cells from the fourth to the eighth generations were used for study.All the cultured cells were proved negative monoclonal antibody stain for a-actin and positive monoclonal antibody stain for Vimentin,suggesting that the purity of AF cells almost came to 100%.2.Effect of FSP1,siRNA-FSP1 and signal pathway blockers on proliferation(1)FSP1 regulating AF proliferation activity in dose manner:MTT demonstrated that the proliferation ability of AF cells was gradually enhanced with the increase of FSP1 concentration.As the concentration of siRNA-FSP1 increased,the proliferation ability of AF cells gradually decreased.(2)MTT and EdU showed that the proliferation ratio of FSP1 group was significantly higher than that of AF,siRNA-FSP1 and siControl group.After the addition of signaling pathway blockers(AG490,DKK,FPS-ZM1,Stattic),cell proliferation rate was significantly reduced compared with that of FSP1 group.3.Effect of FSP1,siRNA-FSP1 and signal pathway blockers on migration(1)Wound healing test showed that the healing area of FSP1 group was higher than that of siRNA-FSP1,AF,siControl and FSP1+siRNA-FSP1 group.(2)Compared with FS1I group,the healing area was decreased obviously when added signal pathway blockers.4.Effect of FSP1,siRNA-FSP1 and signal pathway blockers on adhesion ability(1)Cell adhesion test showed that the AF cell adhesion capacity of FSP1 group was significantly higher than that of AF,siControl,siRNA-FSP1 and FSP1+siRNA-FSP1 group.(2)After the addition of signaling pathway blockers,cell adhesion ability was significantly reduced compared with that of the FSP1 group.5.Effect of FSP1,siRNA-FSP1 and signal pathway blockers on cell cycle and apoptosis(1)Cell cycle and apoptosis tests showed that the proportion of cells in S phase in FSP1 group was significantly higher than that in AF,siControl,siRNA-FSP1 and FSP1+siRNA-FSP1 groups,but the proportion of cells in apoptosis phase was significantly reduced.(2)After the addition of signaling pathway blockers,compared with the FSP1 group,cells in S phase were significantly reduced,while the proportion of AF cell in apoptosis was significantly increased.6.Western blot analysis(1)The signaling pathway proteins(RAGE,FSP1,p-JAK2,p-STAT3,Wnt3a,p-?-catenin)in the FSP1 group were higher than those in any other group.Regardless of which signaling pathway blocker was added,the corresponding phosphorylated proteins in these three pathways were decreased.(2)After adding FSP1 stimulation,the expression of inflammatory factors(MCP-1,VCAM-1,ICAM-1)were significantly higher than that of any other group.After the addition of signaling pathway blockers,the expression of these three inflammatory factors were significantly reduced compared with the FSP1 group.(3)Among the AF normal group,siRNA-FSP1 and FSP1 groups,compared with the AF group,the expression of cyclin-D1 and CDK4 in the FSP1 group were increased,and the expression of P53 was decreased;when the siRNA-FSP1 group was compared with the AF group,the expression of cyclin-D1 and CDK4 decreased,and the expression of P53 increased.7.RT-qPCR analysis(1)The corresponding mRNA expression of RAGE,FSP1,JAK2,STAT3,Wnt3a and P-catenin in FSP1 group was significantly higher than that of AF,siControl,siRNA-FSP1 and FSP1+ siRNA-FSP1 groups.(2)After the addition of signaling pathway blockers,the mRNA expression of the above proteins were significantly reduced compared with that of the FSP1 group.8.Immunocytochemistry analysis(1)Cell immunofluorescence showed that the fluorescence density of RAGE,FSP1,p-JAK2,p-STAT3,Wnt3a,?-catenin in the FSP1 group was higher than that of the AF,siControl,siRNA-FSP1 and FSP1+siRNA-FSP1 groups.(2)Compared with FSP1 group,the fluorescence density was significantly reduced after the addition of signal pathway blockers.Conclusion(1)FSP1 can promote the proliferation,migration and adhesion of AF cells by regulating the cell cycle.SiRNA-FSP1 can effectively inhibit AF proliferation,migration and adhesion.(2)The regulatory networks among the three signaling pathways of RAGE,JAK2/STAT3 and Wnt3a/?-catenin participate in the role of FSP1 in AF cells.(3)FSP1 promotes the expression of inflammatory factors(MCP-1,VCAM-1 and ICAM-1)and promotes vascular remodeling in AF cells.BackgroudAtherosclerosis is a complex,multifactorial disease,which is a major cause of acute cardiovascular events.More and more studies show that autophagy plays an important role in the occurrence and development of atherosclerosis.Evidences from numerous studies show that autophagy is a double-edged sword in cell biology,acting both as a cell growth suppressor and a survival protector.Autophagy could promote cell survival,but excessive autophagy would contribute to cell injury and apoptosis.Autophagy plays an important role in maintaining the metabolism of cardiovascular system,participating in myocardial ischemia reperfusion injury,heart failure(HF),atherosclerosis and other pathophysiological processes.As a fibroblast specific protein,FSP1 can participate in a variety of diseases by regulating autophagy.Understanding the role of FSP1 in autophagy regulation may provide new insights into the prevention and treatment of atherosclerotic diseases.Previous studies have shown that adventitial fibroblasts are involved in the development of atherosclerosis.In fibroblasts,collagen is closely related to autophagy.However,whether FSP1 is involved in autophagy in AF cells and its specific mechanism are still unclear.The relationship between autophagy and collagen remains unclear.Based on the above ideas,we designed this study in vitro:to study whether FSP1 is involved in autophagy in AF cells,then further explore the possible mechanism of FSP1-mediated autophagy,and finally explore the relationship between autophagy and collagen.Purpose1.To observe the effect of FSP1 and siRNA-FSP1 on autophagosomes in AF cells;2.To study the effects of FSP1,siRNA-FSP1 and signaling pathway blockers on relevant autophagy proteins;3.To study the relevant mechanism of FSP1 mediating autophagy in AF cells;4.Explore the relationship between autophagy and collagen under the stimulation of FSP1.Methods1.Cell cultureThe adventia was decorticated from the aortic wall of mouse and the primary cells were cultured by a modified explant method.The passage was carried out when the cells reached 80-90%confluence.The primary cells from the fourth to the eighth generations were used for further study.2.Transmission electron microscopy(TEM):To observe the changes of autophagosomes in different groups after FSP1 and siRNA-FSP1 stimulation.3.Westrn blot analysis:(1)To detect the expression of autophagy proteins(LC3B,Beclin-1,Apg7,p62)after FSP1,siRNA-FSP1 and signal pathway blockers(AG490,DKK,FPS-ZM1,Stattic)intervention.The expression intensity was demonstrated by the ratio of integral optical density(IOD)value between cytokines and P-actin.(2)The expression of LC3,p-?-catenin,p-JAK2,p-STAT3 and RAGE in AF?siRNA-FSP1?FSP1 groups were detected.The expression intensity was demonstrated by the ratio of integral optical density(IOD)value between cytokines and P-actin.(3)The expressions of LC3 and p-catenin in AF,FSP1 and FSP1+cardamonin(?-catenin blocker)groups were detected.The expression of LC3 and RAGE in AF,FSP1 and FSP1+FPS-ZM1(RAGE blocker)groups were detected.The expression of LC3,p-JAK2 and p-STAT3 were detected in AF,FSP1 and FSP1+AG490(JAK2/STAT3 blocker)groups.The expression intensity was demonstrated by the ratio of integral optical density(IOD)value between cytokines and ?-actin.(4)The expression of LC3,p-JNK,p-ERK,p-p38 in AF,siRNA-FSP1 and FSP1 groups were detected.The expression intensity was demonstrated by the ratio of integral optical density(IOD)value between cytokines and ?-actin.(5)The expressions of LC3 and p-JNK in AF,FSP1 and FSP1+SP600125(JNK blocker)groups were detected.The expression of LC3 and p-ERK in AF,FSP1 and FSP1+PD184352(ERK blocker)groups were detected.The expression of LC3 and p-P38 were detected in AF,FSP1 and FSP1+SB203580(P38 blocker)groups.The expression intensity was demonstrated by the ratio of integral optical density(IOD)value between cytokines and ?-actin.(6)The expressions of collagen and LC3B in AF,siRNA-FSP1 and FSP1 were detected.The expression intensity was demonstrated by the ratio of integral optical density(IOD)value between cytokines and p-actin.4.Cell immunofluorescence analysis:After the intervention of FSP1,siRNA-FSP1 and signaling blockers(AG490,DKK,FPS-ZM1,Stattic),the expression of LC3B in AF cells were detected by cellular immunofluorescence.IOD was used to compare the strength of their expressions in different groups.Results1.Incubation of AF cells:The artery adventitia fibroblast was cultured by the explant method.About 4 days,a small number of cells could be seen swimming out and sticking to the dish wall around the tissue block.These cells were cultured in 10%FBS-DMEM and came to the first confluence about 8-9 days.The passage was carried out when the cells reached 80-90%confluence.The primary cells from the fourth to the eighth generations were used for study.2.Autophagosome assayTransmission electron microscopy showed that the autophagosomes in the FSP1 group were higher than those in the AF,siControl and siRNA-FSP1 groups.3.Western blot analysis(1)Autophagy proteins(LC3B,beclin-1 and Apg7)were significantly higher in FSP1 group than in other groups.While the expression of p62 showed the opposite result.(2)The expressions of LC3,p-?-catenin,p-JAK2,p-STAT3,and RAGE in the FSP1 group were higher than those in the normal AF group and the siRNA-FSP1 group.(3)The expressions of p-?-catenin and LC3 in FSP1 group were higher than those in normal AF group and FSP1+cardamonin group.The expressions of p-JAK2,p-STAT3 and LC3 in FSP1 group were higher than in normal AF group and FSP1+AAG490 group.The expression of RAGE and LC3 were higher than that of AF group and FSP1+FPS-ZM1 group.(4)The expressions of LC3,p-JNK,p-P38,p-ERK and RAGE in FSP1 group were higher than those in normal AF group and siRNA-FSP1 group.(5)The expression of p-JNK and LC3 in FSP1 group were higher than those in normal AF group and FSP1+SP600125 group.The expression of p-P38 and LC3 in FSP1 group were higher than those in normal AF group and FSP1+SB203580 group.The expression of p-ERK and LC3 in FSP1 group were higher than those in AF group and FSP1+PD184352 group.(6)In the three groups of AF,siRNA-FSP1 and FSP1,collagens(type ? and ?)in the FSP1 group were significantly higher than that in the AF and siRNA-FSP1 group,and LC3B showed the same trend.4.Immunocytochemistry analysis(1)Cell immunofluorescence showed that the fluorescence density of LC3B in the FSP1 group was higher than that of the AF,siControl,siRNA-FSP1 and FSP1+siRNA-FSP1 groups.Compared with FSP1 group,the fluorescence density was significantly reduced after the addition of signal pathway blockers.Conclusion1.FSP1 promotes autophagy in AF cells.2.FSP1 promotes autophagy through the crosstalk among RAGE,JAK2/STAT3 and Wnt3a/?-catenin signal transduction pathways in AFs.3.After activation of RAGE by FSP1,the downstream signal JNK/ERK/p38 of RAGE was further activated to induce autophagy in AF cells.4.In AF cells,the synthesis of collagens(type ? and ?)increased after the enhancement of autophagy. |
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