Abstract 138: Cigarette Smoke and Monocytes Induce Endothelial Dysfunction

Abstract

Cigarette smoke induces endothelial cell (EC) dysfunction. Circulating monocytes (HM) adhere to a damaged endothelium and migrate into the intima, differentiate to macrophages and contribute to atherosclerotic plaque growth. We demonstrated that HM exposed to cigarette smoke condensate (CSC) increase the transmigration of HM through EC. Thus, we hypothesized that CSC may affect HM/EC interaction. We analyzed the effects on EC of CSC (30 μg/ml) alone and/or of medium conditioned by HM incubated without (CM) or with CSC (CM/CSC). CSC, CM or CM/CSC did not affect EC vitality (MTT assay). CM and CM/CSC increased the expression (measured by RT-PCR) of adhesion molecule ICAM1 (36-fold and 90-fold, p<0.01 vs control or CM, respectively). The effect on VCAM1 was even more dramatic (63-fold vs control for CM, p<0.01 and 725-fold vs control, p<0.01 with CM/CSC). In addition, CM and CM/CSC enhanced the expression of matrix metalloproteinase (MMP)-3 (+60% vs control, p<0.01; +200% vs control, p<0.01, respectively) and MMP-9 (+70% vs control, p<0.01; +140% vs control, p<0.01, respectively). MMP-1 and MMP-2 were not affected. Then, we used the iCELLigence System to analyze EC biological status (cell number, adhesion, viability, and morphology). Only the addition of CM/CSC to confluent EC monolayer dramatically modified the pattern of the electric flow, indicative of modifications in EC morphology. Indeed, we observed an altered actin cytoskeleton organization by immunofluorescence studies performed with FITC-phalloidin. In particular, CM/CSC caused the shrinking of the EC cytoplasm by about 50% (p<0.01 vs control or CM). Moreover, CM/CSC altered sub-cellular localization of beta-catenin promoting its trafficking from membrane into the cytosol and the nucleus. In contrast, EC morphology was not affected by exposure to CSC or CM. Finally, CM and CM/CSC reduced (by 40% and 60%, p<0.05, respectively) the expression of the small signaling G protein Rac 1 GTPase involved in cytoskeletal remodeling. In conclusion, these results indicate that monocytes, in the presence of cigarette smoke, secrete some soluble factors that affect endothelium morphology, thus increasing its permeability and facilitating monocytes transmigration through the endothelial monolayer.