Change Of Plasma Anti-hsp70 Autoantibody Level And Its Biological Effects During The Formation Of Atherosclerosis

Atherosclerosis (atherosclerosis, AS) is a serious impact on human health of cardiovascular disease. The pathogenesis of atherosclerosis is very complicated. Recently, the study suggests that inflammatory and immune mechanisms are the major pathological mechanism during the development of atherosclerosis. However, the existing studies have been difficult to fully explain the molecular mechanism of AS. During explore the etiology mechanism of atherosclerosis, more and more studies indicate that there was a correlation between the heat shock protein 70 (heat shock protein 70, HSP70) and HSP70 antibodies with atherosclerosis. Studies found that there were not only significantly increased levels of plasma HSP70, but the presence of antibodies in coronary heart disease, atherosclerosis and hypertension patients. Further epidemiological investigation showed there was a correlation between significantly elevated plasma HSP70 antibodies and the severity of disease, this relationship regardless of age, gender and other risk factors. These results show that the increased plasma HSP70 and appeared HSP70 antibodies were involved in the pathological mechanism of the progression of cardiovascular disease. However, it is not clear that plasma levels of HSP70 antibody changes the formation of atherosclerosis. And it is also not clear that the pathological mechanism of plasma HSP70 antibody involved in the development of atherosclerosis. To elucidating the role of plasma HSP70 autoantibody in the procession of atherosclerosis, in this study, we developed animal models of atherosclerosis. We systematically observed the dynamic level changes of plasma HSP70 antibody, and to explore the resource of plasma HSP70 antibody in the procession of atherosclerosis. And we further study the role of plasma HSP70 antibody in the formation of atherosclerosis and its pathological mechanism of atherosclerosis. These results will reveal the pathogenesis of atherosclerosis, and provide us new scientific evidence of atherosclerosis prevention and treatment. 1. Change of plasma HSP70 antibody levels during the formation of AtherosclerosisIn this study, atherosclerotic animal model was developed by fed on high fat diet for 10 weeks. Atherosclerotic animal model was assessed by serum lipid levels and pathological confirmation of aortic atherosclerosis. At 2w of high fat diet, rat plasma total cholesterol (TC), low density lipoprotein (LDL) levels were significantly higher than the control group, and with the development of disease showing the trend of gradual increase. While the high-density lipoprotein and triglycerides in plasma were not significantly different with control. Pathological examination showed that rats vessel wall intima thickening, formation of flakes or punctuates calcification at 10w in HCD, Which were the arteries pathological changes in atherosclerotic plaque. These results suggest that AS animal model was developed successfully. The plasma HSP70 antibody levels determined by ELISA showed: rat plasma total HSP70 IgG and IgM antibodies were significantly higher in HCD than control at 4w, which were earlier than pathological changes occur in rat aortic time point (6w). And with the occurrence of atherosclerosis development occurs gradually increased. At 10w, the total IgG, IgG1, IgM subtype were significantly higher in AS group than control. While the IgA, IgG2a, IgG2b subclass levels were not significantly changed. HSP70 antibody subclass levels between 0w and 10w in AS group were showed the same trend. These result showed that the level of plasma HSP70 antibodies were significantly increased during the formation of atherosclerosis. And the plasma HSP70 antibody subclass levels were changed during the progression of atherosclerosis.To explore the mechanism of plasma HSP70 antibody production, the levels of plasma HSP70 were further tested. Plasma HSP70 ELISA results showed that HSP70 plasma levels in AS group were significantly increased at 2w (0.18±0.021 g/ml vs 0.087±0.014 g/ml), which was gradually increased with time trend. Plasma HSP70 complex was detected by western Blotting. The results showed that plasma HSP70 complex species in AS group were significantly higher than control. Prompted increased plasma levels of HSP70 and plasma HSP70 complex may be the source of plasma HSP70 antibodies.2. The role of HSP70 antibody and its pathological mechanism during the formation of atherosclerosisTo observe the role of plasma HSP70 antibody during the formation of atherosclerosis, BD091 monoclonal antibody was developed by hybridoma technology. BD091 had the same binding site with plasma HSP70 antibody by competition ELISA. Rats were treated with HSP70 antibody BD091(IgG1,aa 384-642) or SPA-810 (aa 437-504) by intravenous injection. BD091 could induce vascular injury, and increase the areas of atherosclerotic plaque and the numbers of SMCs,MΦ,T in plaque. BD091 also could improve the expression of ICAM-1/MCP-1 in plaque. The SPA-810 antibody had no effect on the formation of atherosclerosis. The serum lipid levels were not changed between antibody treated group and untreated group. These results suggest that specific site of HSP70 antibody can promote the formation of atherosclerosis; this role will not change the blood lipid levels in rats.OxLDL is atherosclerosis risk factors, which could induce elevated level of HSP70 expression of vascular endothelial cells. Plasma OxLDL levels were significantly higher than control, and were significantly correlated with levels of plasma HSP70 antibody. High level of HSP70 was expressed in vascular endothelial cells which were treated with 20 g/ml OxLDL for 12h, and induced increased HSP70 localized in the cell membrane. In order to study the damaged effect of HSP70 antibodies on vascular endothelial cell, vascular endothelial cell culture LDH were not significantly increased after treated with 20 g/ml OxLDL for 12h. Cell culture LDH were significantly increased after added HSP70 antibody and complement or peripheral blood lymphocytes. This suggested that HSP70 antibody could induce vascular endothelial cell injury. The level of plasma HSP70-IC in AS rat was significantly higher than control at 8w, increased gradually with time. This suggested that HSP70-IC were involved in the formation of atherosclerosis. Application of PEG precipitation method HSP70-IC (BD091-IC), the numbers of peripheral blood lymphocytes adhere to endothelial cells determined by ELISA. HSP70-IC can adhere to endothelial cells and induced elevated levels of ICAM-1 in endothelial cells, and increase the cell number of peripheral blood lymphocyte adhere to endothelial cells. There were no significant effect of peripheral blood lymphocytes adhered to endothelial cells treated with HSP70 or BD091alone.This suggest that HSP70-IC may mediate lymphocyte adhesion to vascular endothelial cells.To observe the HSP70-IC on the smooth muscle cells, rat vascular smooth muscle cells were cultured using tissue culture. Smooth muscle cells proliferations were determined by MTT and flow cytometry. 50 g/ml concentration of HSP70-IC above can induce smooth muscle cell proliferation, increased the levels of smooth muscle cell culture medium PDGF-BB, MCP-1. HSP70-IC could induce smooth muscle cell migration by using wound healing assay and Boyden Chamber experiment. Fcγreceptors (FcγRs) are located on cell surface which specifically interacted with IC. By using fluorescence real-time quantitative PCR, the level of SMCs FcγRI, FcγRIII expression were increased after incubated with 50 g/ml BD091-IC for 6h. The levels of FcγRs expression were depended on BD091-IC concentration. After incubated with BD091 or HSP70 alone, the level of SMCs FcγRI, FcγRIII expression was not increased. These results suggested that HSP70-IC could induce FcγRI and FcγRIII expression on smooth muscle cells. 50 g/ml HSP70-IC could induce smooth muscle NF-κB/ERK pathway activated by western blotting. These results suggested that, HSP70-IC could induce increased FcγRs expression of smooth muscle cell, HSP70-IC interaction with FcγRs, activated NF-κB/ERK pathway to promote smooth muscle cells to produce cytokines, leading to smooth muscle cell proliferation and migration.In summary, during the formation of AS induced by high-fat diet, it may be the cause of HSP70 antibody production owing to high plasma HSP70 level or changed plasma HSP70 complex, And then induced aortic endothelial damage, increased atherosclerotic plaque size and the number of infiltrating cells in plaque, raising the level of the inflammatory factors in plaque, playing a harmful role in promoting the formation of atherosclerosis. The atherosclerotic rat plasma HSP70 antibodies were involved in the pathogenesis of AS by accelerating the process. HSP70 antibody play a damaged role through endothelial cells injury induced by HSP70 antibody, or formatting HSP70-IC, mediated PBL adhesion to endothelial cells, promoted smooth muscle cell proliferation and migration.