Change Of Plasma Hsp70 Level And Its Mechanism During The Formation Of Atherosclerosis

Objective: In this study, we would investigate the change of plasma Hsp70 during the formation of atherosclerosis, elucidate the source and mechanism underlying plasma Hsp70 change, and explore the effect of plasma Hsp70 on cardiovascular disease. Our study would provide new scientific evidences for the pathogenesis and prevention method of atherosclerosis. Methods: To study the regularity of plasma Hsp70 level in the development of atherosclerosis, and to investigate whether plasma Hsp70 could be an early marker for the cardiovascular disease process, we determined the alteration of plasma Hsp70 level during the atherogenesis of rats from the experiment beginning to the end. Atherosclerosis model in rats fed with high fat diet was established. Plasma was collected for detection of cholesterol and plasma Hsp70 level. For conventional histology, the aortas were sectioned for hematoxylin and eosin (H&E) staining. Plasma lipid levels were measured using biochemistry method. The Hcy concentration in rat plasma was determined by high performance liquid chromatography (HPLC)-fluorometry. The index for atherosclerosis formation was evaluated by histological study and plasma lipid. Sandwich ELISA was used to detect plasma Hsp70 level. Immunohistochemistry was used to detect Hsp70 expression of atherosclerotic lesions. To explore the source and release mechanism of plasma Hsp70, Rat vascular endothelia cells (RVECs) were treated with Ox-LDL or Hcy, the Western blotting method and Sandwich ELISA were used to investigate the expression of Hsp70 in RVECs and the extracellular Hsp70 level in medium. Western blotting method and EMSA were selected to determine the activity of ERK1/2 in RVECs treated with Ox-LDL or Hcy. Discontinuous density gradient ultracentrifugation and four times centrifugation method was used to extract lipid rafts and exosomes, and Western blotting was selected to identify the Hsp70 release pathway of RVECs treated with Ox-LDL or Hcy. To investigate the effect of plasma Hsp70 to the development of atherosclerosis, ELISA method was used to observe the effect of extracellular Hsp70 on cytokine and inflammatory factors production from RVECs or blood cells. Lymphocyte adhesion studies were performed to indentify the effect of extracellular Hsp70 on lymphocyte adhesion to RVECs. Results: Blood samples were collected for the measurement of serum TC, TG, Ox-LDL, HDL. A significantly rise of plasma TC and Ox-LDL was observed at 2 weeks, and time-dependent response curves were observed. A widening of the first interlamellar spaces, more accumulation of foam cells in interlamellar spaces, rectitude and fragmentation of medial elastic lamellae aortic lesions, proliferated and disoriented smooth muscle cells, lipid-calcic plaque was observed in the aorta of hypercholesterolemic rats. Hsp70 level was markedly elevated in the high fat diet rats compared with chow diet rats at 4 weeks, and there was an increase trend in plasma Hsp70 level during the atherogenisis. There was significantly positive correlation between plasma Hsp70 level and the progress of atherosclerosis and Ox-LDL or Hcy level. The elevation of plasma Ox-LDL or Hcy was earlier than the elevation of plasma Hsp70 level. Lesion sites of the aortic arch displayed strong Hsp70 expression in necrotic intimal plaques and media underlying necrotic plaques. The necrotic core and fibrous cap regions of advanced lesions exhibited increased expression of Hsp70. We found that treatment of endothelial cells with low dose Ox-LDL or Hcy increased the release of Hsp70 into the supernatant, without significant increase in cell death, but augmentation of Hsp70 release from cells induced by high dose Ox-LDL or Hcy was partly due to cell death, because the LDH concentration of medium was significantly elevated. ERK1/2 signal transduction pathway was activated in RVECs treated with Ox-LDL or Hcy. Activated ERK1/2 induced HSF1 phosphorylation and translocation from cytoplasm to nucleus, and increased the Hsp70 expression.The overexpression of Hsp70 in RVECs might be the reason of Hsp70 release. We further found that the Ox-LDL and Hcy induced robust releases of HSP70 independent of the classical route of endoplasmic reticulum/Golgi protein trafficking or the formation of lipid rafts. In contrast, Ox-LDL and Hcy significantly enhanced exosomal secretory rate with increases in content of HSP70 in exosomes., exosomes may be the pathway of Hsp70 active release from RVECs. We also found that extracellular Hsp70 had no effect on cell disruption and ICAM-1 expression of RVECs, but it could induce lymphocytes secrete IL-6, and then promote lymphocytes adhesion to RVECs. Conclusions: During the formation of rat atherosclerosis induced by high fat diet, the change of plasma Hsp70 level was elevated siginificantly compared with control group and was characterized positive time-dependent response curves in disease progression, and was positively correlated with the plasma Ox-LDL and Hcy level. The important reason of plasma Hsp70 rise was Hsp70 overexpression in RVECs induced by Ox-LDL or Hcy by activating ERK1/2 signal transduction pathway, and then Hsp70 was released into extracellular space from RVECs by active or passive pathway, exosomes may be the pathway of Hsp70 release actively. Plasma Hsp70 could be involved in the initiation of atherosclerosis by stimulating lymphocytes to secrete cytokines and evoking inflammatory reaction.