Diet-induced elevation of circulating HSP70 may trigger cell adhesion and promote the development of atherosclerosis in rats

Abstract

Although accumulating evidence indicates that heat shock protein 70 (HSP70) could be secreted into plasma and its levels have been found to have an ambiguous association with atherosclerosis, our knowledge for the exact role of circulating HSP70 in the development of atherosclerosis is still limited. In the present study, we report an adhesion-promoting effect of exogenous HSP70 and evaluate the potential involvement of elevated circulating HSP70 in the development of atherosclerosis. Time-dependent elevation of plasma HSP70 was found in diet-induced atherosclerotic rats, whose effect was investigated through further in vitro experiments. In rat aortic endothelial cell (RAEC) cultures, exogenous HSP70 incubation neither produced cell injuries by itself nor had protective effects on cell injuries caused by Ox-LDL or homocysteine. However, exogenous HSP70 administration could lead to a higher adhesion rate between rat peripheral blood monocytes (PBMCs) and RAECs. This adhesion-promoting effect appeared only when PBMCs, rather than RAECs, were pretreated with HSP70 incubation. PBMCs in an HSP70 environment released more IL-6 to supernatant, which subsequently up-regulated the expression of ICAM-1 in RAECs. These results indicate that the diet-induced elevation of circulating HSP70 could trigger cell adhesion with the help of IL-6 as a mediator, which provides a novel possible mechanism for understanding the role of circulating HSP70 in the pathogenesis of atherosclerosis.

Keywords: Atherosclerosis; Cell adhesion; Circulating HSP70; Exogenous HSP70; IL-6.

Fig. 1

The level of circulating HSP70 went through a time-dependent elevation in diet-induced atherosclerotic rats. The rats that had received a high-cholesterol diet (HCD) developed a significant dysregulation in cholesterol metabolism. Higher levels of plasma total cholesterol (a) and plasma LDL (b) occurred from the sixth and the second week in the HCD group rats, n = 10. c Atherosclerotic pathological alterations appeared in the thoracic aorta of rats after 6-weeks of HCD feeding. The time-dependent elevation of circulating HSP70 was identified in the HCD group rats by western blot (d) and ELISA assay (e), respectively, n = 10. Western blot images were digitalized and analyzed by software. The gray density of the band of HSP70 was measured and normalized to the gray density of the tubulin band. ELISA results were shown as boxplots because of the non-normal distribution of HSP70 concentrations. Values in a–d represent mean ± SD. Horizontal lines across each box represent medians of plasma HSP70, while heights of each box represent interquartile ranges of HSP70 distribution in e. *p < 0.05 vs. Ctrl

Fig. 2

Exogenous HSP70 neither induced injury or death in rat aortic endothelial cells (RAECs) nor had a cytoprotective role in cell injury in RAECs. Exogenous HSP70 was added with different doses and treatment times in the medium of primary cultured RAECs, while H₂O₂ and LPS were used as positive controls for cell injury and death, respectively. HSP70 did not induce any statistical change in cell LDH activity and survival rate in RAECs (a–d). The potential protective role of exogenous HSP70 was investigated in cell injury models. Different dose of recombinant HSP70 were added in the medium of RAECs simultaneously with the incubation of Ox-LDL or Hcy. The presence of HSP70 did not reverse the Ox-LDL or Hcy-induced increase in RAECs LDH activity (e–f). High dose of HSP70 was also administrated at times before the damaging compound. However, pretreatment with HSP70 for 8 or 24 h failed to decrease the level of LDH activity (g–h). Values represent mean ± SD, n = 3. *p < 0.05 vs. Ctrl

Fig. 3

Exogenous HSP70 promotes the adhesion between rat peripheral blood monocytes (PBMCs) and rat aortic endothelial cells (RAECs) by cytokine release. a RAECs were treated with LPS and HSP70 for 24 h and then incubated with freshly isolated PBMC for an additional 30 min. Adherent PBMCs density indicated that HSP70 has no effect on the adhesion of RAECs. Consistently, 24 h HSP70 treatment failed to induce a higher expression of ICAM-1 in RAECs (b). However, when the experimental procedure was reversed, HSP70 treatment on PBMCs resulted in a higher adhesion rate between PBMCs and RAECs (c). d 24-h HSP70 incubation also induced higher IL-6 release in PBMCs. e RAECs were treated with LPS and IL-6 for 24 h and then incubated with PBMCs for 30 min. Similar adhesion-promoting effects of IL-6 were indicated by higher adherent PBMCs density. f 24-h IL-6 treatment on RAECs led to a dramatic up-regulation of ICAM-1. Values represent mean ± SD, n = 3. *p < 0.05 vs. Ctrl