Changes Of Plasma Hsp70 Levels And Its Biological Effects In Rat Atherosclerotic Disease Progression

ObjectiveAtherosclerosis is one of the most lethal diseases in cardiovascular diseases. The etiology and pathogenesis of atherosclerosis is so complicated that the development of atherosclerosis hasn't be illuminated completely. Hsp70 has protective effect as intracellular molecular chaperone,but extracellular Hsp70 has been considered to have cytokine function and be associated with atherosclerosis. Our studies want to know the changes of plasma Hsp70 levels in rat atherosclerotic disease progression. Meanwhile, we want to study the effects of extracellular Hsp70 on biological function of vascular endothelial cells through vitro experimental models, and provide scientific evidences for the prevention and therapia of atherosclerosis.Materials and MethodsAdult healthy Wistar rats of male sex, weighing 180²00g,were randomly divided into two groups: control and high-fat diet group. The rats were fed either a standard diet (controls), or given a high fat diet (high-fat diet group) for 12 weeks, the rats of two groups were administered with normal water throughout. The blood samples were collected per two weeks and plasma lipids and Hsp70 levels were measured. In order to observe the formation of atherosclerosis, the aortas were harvested; sections of the aortas were stained with HE. In the vitro experimental models, the cultured vascular endothelial cells were stimulated with different dose of Hsp70 for different times. Cell culture supernatants were collected and the levels of LDH, ICAM-1, and IL-6 were measured by ELISA. Cell survival rate was measured by MTT, The protein expression of TLR4 and NF-κBp65 were detected by western blot. Lymphocytes were isolated from peripheral blood and stimulated by Hsp70; the changes of IL-6 levels in the supernatant were measured by ELISA. To explore the effects of IL-6 on EC, the levels of ICAM-1 and lymphocyte adhesion studies were performed after EC was stimulated by IL-6.Results1 The changes of plasma Hsp70 levels in rat atherosclerotic disease progression: At 2 weeks, plasma total cholesterol and LDL levels in high -fat diet group were significantly higher than control group, and time-dependent response curves were observed. Foam cell accumulation, lipid deposition and proliferation of smooth muscle cells at 10 weeks and necrotic lipid calcic magma in media at 12 weeks were observed in aorta sections of high fat-diet group. No obvious pathological changes were observed in aorta sections of control group in the experiment. Circulating Hsp70 levels in high fat-diet group were significantly higher than control group from 2 weeks, and were characterized positive time-dependent response curves. Immunohistochemical analysis in aorta sections of rats showed SMCs proliferation and higher average HSP70 immunoreactivity at medial tissue at 10 weeks and the higher expression of Hsp70 at the necrotic core at 12 weeks.The evidences have shown that the time of higher circulating Hsp70 levels occurrence were earlier than the initiation of atherosclerosis, and were characterized positive time-dependent response curves in rat atherosclerotic disease progression.2 The injury effect of extracellular Hsp70 on vascular endothelial cell: After vascular endothelial cells were stimulated with different dose of Hsp70 for different times, cell survival rate and the production of LDH, ICAM-1 and IL-6 in the supernatant were not significantly higher than control group. Extracellular Hsp70 couldn't induce higher protein expression of TLR4 and translocation of NF-κBp65 protein from cytoplasm to nucleus.It has been shown that extracellular Hsp70 maybe not induce atherosclerotic injury in blood vessel through activation of vascular endothelial cells.3 The effects of extracellular Hsp70 on endothelial cells through lymphocytes: Lymphocytes were stimulated with Hsp70(1ug/ml) for 24h,the levels of IL^-6 in the supernatant were significantly higher than control group, and were characterized positive dose and time-dependent response curves. When EC were stimulated by IL^-6, the levels of ICAM^-1 in the supernatant and the lymphocyte adhesion rate were higher than control group, and were characterized positive dose and time-dependent response curves.The evidences have shown that the inflammation reaction in vascular endothelial cells induced by extracellular Hsp70 was the cause of atherosclerotic injury in blood vessel.Conclusion1 The time of higher circulating Hsp70 levels occurrence were earlier than the initiation of atherosclerosis, and were characterized positive time-dependent response curves in rats'atherosclerotic disease progression induced by high fat diet.2 The injury of vascular endothelial cells and the release of cytokines induced by extracellular Hsp70 hadn't be observed, Extracellular Hsp70 couldn't induce higher protein expression of TLR4 and translocation of NF-κBp65 protein.It has be shown that extracellular Hsp70 cann't induce dysfunction of vascular endothelial cells.3 The IL^-6 released from activated lymphocytes could induce inflammation of vascular endothelial cells and lymphocytes adhesion。Our studies have shown that extracellular Hsp70 can induce dysfunction of vascular endothelial cells through lymphocytes and mediated the formation of atherosclerosis.