Bernardini Chiara, Zannoni Augusta, Turba Maria Elena, Fantinati Paolo, Tamanini Carlo, Bacci Maria Laura, Forni Monica
Department of Veterinary Morphophysiology and Animal Production, University of Bologna, Italy.
Cell Stress Chaperones. 2005 Winter;10(4):340-8. doi: 10.1379/csc-98r1.1.
Lipopolysaccharide (LPS) is a highly proactive molecule that causes in vivo a systemic inflammatory response syndrome and activates in vitro the inflammatory pathway in different cellular types, including endothelial cells (EC). Because the proinflammatory status could lead to EC injury and apoptosis, the expression of proinflammatory genes must be finely regulated through the induction of protective genes. This study aimed at determining whether an LPS exposure is effective in inducing apoptosis in primary cultures of porcine aortic endothelial cells and in stimulating heat shock protein (Hsp)70 and Hsp32 production as well as vascular endothelial growth factor (VEGF) secretion. Cells between third and eighth passage were exposed to 10 microg/mL LPS for 1, 7, 15, and 24 hours (time-course experiments) or to 1, 10, and 100 microg/mL LPS for 7 and 15 hours (dose-response experiments). Apoptosis was not affected by 1 microg/mL LPS but significantly increased in a dose-dependent manner with the highest LPS doses. Furthermore, apoptosis rate increased only till 15 hours of LPS exposure. LPS stimulated VEGF secretion in a dose-dependent manner; its effect became significant after 7 hours and reached a plateau after 15 hours. Both Hsp70 and Hsp32 expressions were induced by LPS in a dose-dependent manner after 7 hours. Subsequent studies were addressed to evaluate the protective role of Hsp32, Hsp70, and VEGF. Hemin, an Hsp32 inducer (5, 20, 50 microM), and recombinant VEGF (100 and 200 ng/mL), were added to the culture 2 hours before LPS (10 microg/mL for 24 hours); to induce Hsp70 expression, cells were heat shocked (42 degrees C for 1 hour) 15 hours before LPS (10 microg/mL for 24 hours). Hemin exposure upregulated Hsp32 expression in a dose-dependent manner and protected cells against LPS-induced apoptosis. Heat shock (HS) stimulated Hsp70 expression but failed to reduce LPS-induced apoptosis; VEGF addition did not protect cells against LPS-induced apoptosis at any dose tested. Nevertheless, when treatments were associated, a reduction of LPS-induced apoptosis was always observed; the reduction was maximal when all the treatments (HS + Hemin + VEGF) were associated. In conclusion, this study demonstrates that LPS is effective in evoking "the heat shock response" with an increase of nonspecific protective molecules (namely Hsp70 and Hsp32) and of VEGF, a specific EC growth factor. The protective role of Hsp32 was also demonstrated. Further investigations are required to clarify the synergic effect of Hsp32, Hsp70, and VEGF, thus elucidating the possible interaction between these molecules.
脂多糖（LPS）是一种具有高度活性的分子，可在体内引发全身炎症反应综合征，并在体外激活包括内皮细胞（EC）在内的不同细胞类型中的炎症信号通路。由于促炎状态可能导致内皮细胞损伤和凋亡，因此必须通过诱导保护性基因来精细调节促炎基因的表达。本研究旨在确定LPS暴露是否能有效诱导猪主动脉内皮细胞原代培养物中的细胞凋亡，并刺激热休克蛋白（Hsp）70和Hsp32的产生以及血管内皮生长因子（VEGF）的分泌。将传代3至8代的细胞分别暴露于10微克/毫升的LPS中1、7、15和24小时（时间进程实验），或暴露于1、10和100微克/毫升的LPS中7和15小时（剂量反应实验）。1微克/毫升的LPS对细胞凋亡无影响，但随着LPS剂量的增加，细胞凋亡呈显著的剂量依赖性增加。此外，细胞凋亡率仅在LPS暴露15小时前增加。LPS以剂量依赖性方式刺激VEGF分泌；其作用在7小时后变得显著，并在15小时后达到平台期。7小时后，Hsp70和Hsp32的表达均被LPS以剂量依赖性方式诱导。后续研究旨在评估Hsp32、Hsp70和VEGF的保护作用。在LPS（10微克/毫升，处理24小时）处理前2小时，向培养物中加入Hsp32诱导剂血红素（5、20、50微摩尔）和重组VEGF（100和200纳克/毫升）；为诱导Hsp70表达，在LPS（10微克/毫升，处理24小时）处理前15小时，将细胞进行热休克处理（42℃，1小时）。血红素暴露以剂量依赖性方式上调Hsp32表达，并保护细胞免受LPS诱导的凋亡。热休克（HS）刺激Hsp70表达，但未能降低LPS诱导的凋亡；在任何测试剂量下，添加VEGF均不能保护细胞免受LPS诱导的凋亡。然而，当联合使用这些处理时，总能观察到LPS诱导的凋亡减少；当所有处理（HS + 血红素 + VEGF）联合使用时，凋亡减少最为显著。总之，本研究表明LPS可有效引发“热休克反应”，使非特异性保护分子（即Hsp70和Hsp32）以及特异性内皮细胞生长因子VEGF增加。同时也证明了Hsp32的保护作用。需要进一步研究以阐明Hsp3hsp70和VEGF的协同作用，从而阐明这些分子之间可能的相互作用。
