Abstract 654: Isoleukotoxin Promotes Autophagy and Reduces Vascular Cell Adhesion Molecule-1 Expression in Endothelial Cells

Abstract

Rapamycin is a widely used autophagy inducer and has been shown to suppress development of atherosclerosis in animal models. However, the side effect is severe including hyperlipidemia. We identified isoleukotoxin, a metabolite of linoleic acid, as an autophagy inducer and hypothesized that it can be used to reduce inflammation of endothelial cells. To test this hypothesis we treated human umbilical vein endothelial cells (HUVECs) with tumor necrosis factor α (TNFα) along with isoleukotoxin. Rapamycin was used as a positive control to induce autophagy. Isoleukotoxin promotes autophagic flux in HUVECs as well as rapamycin. Isoleukotoxin does not inhibit activity of mammalian target of rapamycin complex, while it inhibits activity of the downstream kinase, S6K. Isoleukotoxin modulates TNFα-induced cell adhesion molecules expression in HUVECs with a similar pattern as rapamycin. In this pattern, only vascular cell adhesion molecule-1 (VCAM-1) expression is reduced but not intercellular cell adhesion molecule-1 (ICAM-1). More interestingly, time-course analysis shows that VCAM-1 expression is only reduced after 20 hours treatment, which is different from that of inhibitors of VCAM-1 transcription such as SB202190. By qPCR we confirmed that isoleukotoxin does not inhibit VCAM-1 transcription. To determine whether isoleukotoxin reduces VCAM-1 expression by inhibiting S6K-mediated protein translation, we compared the effect of isoleukotoxin with S6K siRNA. S6K siRNA reduces expression of VCAM-1 slightly and also ICAM-1, which is different from that of isoleukotoxin. After excluding the regulation of transcription and translation, we hypothesize that isoleukotoxin reduces VCAM-1 expression by promoting VCAM-1 degradation via autophagy. This hypothesis is supported by blocking of autophagy and endocytosis. Blocking of autophagy by Atg5 siRNA enhances VCAM-1 expression, suggesting that VCAM-1 may be degraded by autophagy. Blocking of endocytosis by dynasore blocks the effect of isoleukotoxin on VCAM-1 expression, suggesting that VCAM-1 is degraded after endocytosis. Here we propose that isoleukotoxin is an anti-inflammation and a potential therapeutic molecule for inflammatory vascular diseases such as atherosclerosis.