Abstract 461: Alcohol Modulates Chemokine- And Hypoxia-induced Endothelial-to-mesenchymal Transition In Vitro And Following Ligation Injury In Vivo

Abstract

Endothelial-to-mesenchymal transition (EndMT) plays a role in arteriosclerotic cardiovascular disease (CVD). Low-to-moderate alcohol consumption (i.e., 1-3 drinks/d range) protects against endothelial dysfunction and arteriosclerosis, in part by stimulating Notch, but whether it does so by influencing EndMT is unknown. Our aim was to address this question. Human coronary artery endothelial cells (HCAEC) (p5-p14) were treated (7 d) with transforming growth factor β (TGFβ, 10 ng/ml) or exposed to hypoxia (5% O 2 ), in the absence or presence of ‘moderate’ level EtOH (25 mM), or the Notch ligand DLL4 +/- the Notch inhibitor DAPT. EndMT was assessed by determining endothelial marker CD31 and smooth muscle cell marker αSMA expression by immunohistochemistry and qRT-PCR, as well as by cell morphology and extracellular matrix protein Fibronectin production. Both TGFβ and hypoxia induced EndMT of HCAEC as determined by simultaneously decreased CD31 and increased αSMA expression, together with a change in morphology from cobblestone (typical of endothelial cells) to spindle-shaped (typical of mesenchymal cells), and increased fibronectin expression. EtOH inhibited EndMT caused by both TGFβ and hypoxia as indicated by maintenance of CD31 expression and cobblestone morphology, and attenuated αSMA and fibronectin expression in the EtOH treated group vs controls. Similar to EtOH, treatment with DLL4 inhibited TGFβ-induced myogenic αSMA and fibronectin mRNA expression in HCAEC, effects blocked by DAPT. Following carotid ligation in C57Bl/6 mice, ~25% of neointimal and medial cells in the remodeled vessel co-expressed αSMA and CD31 (indicative of myo-endothelial cells), suggesting the involvement of EndMT. In the ‘daily moderate’ alcohol group (2 drinks per day equivalent; peak BAC=15 mM) carotid neointimal hyperplasia was reduced compared to controls concomitant with fewer (<10%) αSMA + /CD31 + myo-endothelial cells suggesting repression of EndMT in that cohort. In conclusion, these data illuminate an inhibitory effect of alcohol on EndMT, potentially mediated via Notch. This effect on endothelial transdifferentiation may underlie, in part, the protective effect of moderate alcohol consumption on CVD.