Anales RANF

S7-04 EXTRACELLULAR ATP REGULATES ANGIOGENIC RESPONSES IN VASA VASORUM ENDOTHELIAL CELLS VIA THE ACTIVATION OF PI3K-AKT-mTOR SIGNALING AXIS AND C-JUN, FOXO3A, AND C-MYC TRANSCRIPTION FACTORS D. Strassheim 1 , V. Karoor 1 , H. Nijmeh 1 , P. Weston 1 , M. Lapel 1 , J. Schaack 1 , T. Sullivan 1 , M. Fragoso 1 , K.R. Stenmark 1 , E. Dempsey 1,2 , and E. Gerasimovskaya 1* 1 University of Colorado Denver, Aurora, CO, USA; 2 Rocky Mountain Regional VA Medical Center, Aurora, CO, USA Angiogenic vasa vasorum (VV) expansion plays an essential role in the pathogenesis of hypoxia-induced pulmonary hypertension (PH). Previously we showed that extracellular ATP, released in hypoxia, plays an autocrine/paracrine role in angiogenic activation of VV endothelial cells (VVEC), acting via P2Y purinergic receptors (P2YR) and Phosphoinositide 3-kinase (PI3K)- Akt-Mammalian Target of Rapamycin) mTOR signaling axis. To further elucidate the mechanisms of ATP-induced angiogenesis, using TranSignal protein/DNA array we identified ATP-inducible transcription factors (TFs) in VVEC. C-Jun, Foxo3a, and c-Myc were found to be upregulated by extracellular ATP in most tested cell populations and form central nodes connecting several signaling networks. Stimulation with ATP increased phospho-c-Jun Ser73 , phospho-Foxo3a Ser253 , and phospho-c-Myc Ser62 levels in the nuclear fraction, and this effect was reduced by a dual PI3K/mTOR inhibitor, PI-103 (100 nM, 1 h). In addition, PI-103 robustly increased inhibitory phosphorylation of c-Jun at Thr239, that causes a repression of DNA binding activity c-Jun (Fig.1). SiRNA-mediated knockdown of c- Jun, Foxo3a, and c-Myc revealed critical role for these TFs in VVEC angiogenic responses and the regulation of downstream proteins involved in tissue remodeling (MMP-2,9, TIMP-1, PAI-1), cell cycle control (Cyclin D, p21 Cip1/Waf1 , stathmin), expression of endothelial markers (eNOS, PECAM-1), cell adhesion and junction proteins (VE-cadherin, p120 catenin, Connexin 43, ZO-1) (Fig.2). Our findings suggest that pharmacological targeting the components of P2YR-PI3K-Akt-mTOR signaling axis and specific TFs may attenuate VV angiogenesis and pathologic pulmonary vascular remodeling in PH. (Finding: R01-HL-086783 to E.V. Gerasimovskaya)