The Effects Of Swiprosin-1 Expressed In Macrophage On Atherosclerosis And Its Mechanisms

Atherosclerosis?AS?is a chronic disorder,tending to lead to myocardial infarction,stroke and other ischemic cardiovascular diseases.So far,although there are numerous researches on AS,yet its mechanisms of pathogenesis still remain unclear.Macrophage is not only one of the most important component of atherosclerotic plaque,but also plays an important role in the initiation and development of AS.It was demonstrated that macrophages involed in apoptosis,autophagy,inflammation in atherosclerotic plaque.Swiprosin-1 is a kind of new protein,which was first found and reported by a Swiss scientist,Vuadens F.The subsequent reports were majorly focused on its roles in lymphocyte and its other immune-related functions.Our previous study showed that swiprosin-1 existed in the atherosclerotic plaque and was mainly expressed in macrophage in the atherosclerotic plaque.However,the role of swiprosin-1 during the progression of atherosclerosis remains unknown.We found that genetic ablation of swiprosin-1 in macrophage largely reduced the extent of the formation of foam cell.The transformation from macrophage to foam cell is considered as the foundation of the pathogenesis of AS,which produces an important effect on the regulation of the severity of AS.Taken together,our previous studies suggest that swiprosin-1 might be a critical regulator during the progression of atherosclerosis by regulating macrophage activation.Thus,we aimed to investigate the contribution of swiprosin-1 and its mechanisms during the progression of atherosclerosis both in vitro and in vivo.Methods and resultsFirstly,the expression and location of swiprosin-1 were investigated in atherosclerotic plaque.1.Swiprosin-1 was expressed in atherosclerotic plaque and localized in macrophage by immunofluorescence.2.Swiprosin-1 in macrophage was marked by immunofluorescence and the expression of swiprosin-1 in macrophage was detected by immunoblotting.We found that swiprosin-1 was majorly expressed in cellular membrane but not in nuclei.Swiprosin-1 was highly expressed in macrophage.3.The expression of swiprosin-1 in endothelial layer,smooth muscle layer,and the outer layer of the aorta was detected by immunoblotting.We found that there was relatively higher expressed only in macrophage but not in endothelial layer,smooth muscle layer and the outer layer of the aorta.Secondly,the expression of swiprosin-1 in the atherosclerotic plaque in aorta was detected.1.ApoE^-/-mice was fed with western diet to construct the AS mice model.Aorta was separated to analyzed plague by immunohistochemical staining.The results showed that there is atherosclerotic plague in aorta,indicating the mice model was successful.2.The expression of swiprosin-1 in aorta from both control group and mice model group was detected by immunoblotting.We found that the expression of swiprosin-1 in aorta was increase from model group compared with from the control group.Thirdly,the expression of swiprosin-1 in macrophages was investigated with the stimulation of lipids.1.RAW264.7 macrophage was stimuluted with Ox-LDL for 24 h to construct the cellular model of the foam cell transformed from macrophage.With the staining of red oil O,we found that macrophage transformed into foam cell in a dose-dependent manner with Ox-LDL stimulation from 10 to 80 ?g/mL.2.The effects of Ox-LDL in different doses?10,20 40 80 ?g/m L?on the expression of swiprosin-1 in RAW264.7 macrophage was detected by immunoblotting.We found that with the expression of swiprosin-1 was increased with the increasing of the concentration of Ox-LDL.The expression of swiprosin-1 reached to the peak value at the dose of 40 ?g/mL.3.The expression of swiprosin-1 in RAW264.7 macrophage was detected by immunoblotting after Ox-LDL stimulation in different time?0,6,12,24,48 h?.We found that the expression of swiprosin-1 was increased with the increasing of the stimulation time of Ox-LDL.The expression of swiprosin-1 reached to the peak value after Ox-LDL stimulation in 24 h.4.The expression of swiprosin-1 was detected in peritoneal macrophage.We got the similar results in peritoneal macrophage and demonstrated that the expression of swiprosin-1 increased significantly during macrophage transforming into foam cell.Fourthly,the effects of swiprosin-1 on the development of AS in mice AS model was investigated.The bone marrow of swiprosin-1^-/-and swiprosin-1^+/+ mice was transplanted into ApoE^-/-mice and then the mice was fed with western diet for the induction of AS,the former was the model group and the latter was the control group.1.The aorta was separated from mice and opened longitudinally,and the plaque in aorta was stained with red oil O.We found that the total area of plaque was significantly decreased in aorta from swiprosin-1 knockout mice compared with that from wild type mice.2.The aorta in mice was prepared and stained by red oil O.We found that the area of plaque in aorta root was significantly decreased in aorta from swiprosin-1 knockout mice compared with that from wild type mice.3.The macrophages in the atherosclerotic plaque was detected by immunohistochemistry.We found that the number of macrophages was relatively smaller in swiprosin-1 knockout mice compared with in wild type mice.4.The level of cholestrol in serum was detected from the AS mice model.We found that the serum cholestrol level in swiprosin-1 knockout mice was relatively smaller compared with that in wild type mice.Fifthly,the effects of knocking out swiprosin-1 on the transformation from macrophage to foam cell was investigated.Primary peritoneal macrophages were extracted from swiprosin-1^-/-and swiprosin-1^+/+ mice,the former were the model group and the latter were the control group.1.Macrophage was stimulated with Ox-LDL,then stained with red oil O.We found that the extent of the transformation from macrophage to foam cell was relatively reduced compared with that of the wild type mice.2.The level of cholesterol in macrophages was detected after the stimulation with Ox-LDL.We found that the level of cholesterol was relatively lower compared with that of the wild type mice.3.The level of cholesterol efflux from foam cell was detected with the fluorescent labeling.We found that the level of cholesterol efflux of swiprosin-1 overexpression cell was relatively decreased compared with that of the wild type mice.4.The extent of swallowing by macrophage was detected by fluorescent labeling Ox-LDL.We found that the level of swallowing was relatively increased compared with that of the wild type mice.Sixthly,the potential mechanism of swiprosin-1 expressed in macrophages in the development of AS was explored.Primary peritoneal macrophages were extracted from swiprosin-1^-/-and swiprosin-1^+/+ mice,the former were the model group and the latter were the control group.1.The apoptotic proteins in macrophage was detected by immunoblotting.We found that the expressions of apoptosis-positive proteins like Bax,caspase3 and caspase9 were down-regulated and those of apoptosis-negative protein Bcl-2 in macrophages deficiency of swiprosin-1 were up-regulated compared with those from the wild type mice.The level of apoptosis was detected by Annexin V-FITC and TUNEL apoptosis detection assay.We found that the level of apoptosis in macrophages deficiency of swiprosin-1 was relatively lower compared with that from the wild type mice?7.93±1.52% vs 0.41±0.12%?.2.The concentrations of inflammatory cytokines in serum and the cultural medium of macrophage was detected by ELISA kit.We found that the concentrations of IL-1? and in serum of swiprosin-1^-/-mice was relatively decreased compared with those of swiprosin-1^+/+ mice.Inflammatory cytokines IL-1? and TNF-? in the cultural medium of swiprosin-1^-/-macrophages were relatively decreased compared with those of swiprosin-1^+/+ macrophages.3.The expression of autophagy related proteins including LC3 and P62 in macrophage was detected by immunoblotting.We found that the level of LC3 in primary peritoneal macrophages from swiprosin-1^-/-mice was relatively increased and the level of P62 was decreased compared with that from swiprosin-1^+/+ mice.The mRNA levels of P62 was detected by realtime PCR.The result suggested that the autophagy function of macrophage may be enhanced.4.Extracted primary peritoneal macrophages from swiprosin-1^-/- and swiprosin-1^+/+ mice,the ability of migration of macrophage was detected by Transwell.We found that the ability of migration of macrophage deficiency of swiprosin-1 was relatively down-regulated compared with that from the wild type mice.Conclusion1.Swiprosin-1 is highly expressed in macrophages in atherosclerotic plaque and the expression increased with the increasing degree of foamy;2.Swiprosin-1 inhibits phagocytosis of macrophages with oxidized low density lipoprotein;3.Swiprosin-1 inhibits macrophage autophagy process;4.Swiprosin-1 promotes macrophage apoptosis,migration,and cytokine secretion;5.Deficiency of swiprosin-1 in macrophage may inhibit the occurrence of atherosclerosis development.