Stimulation of islet revascularization modulating HIF-1ÃŽÂ± expression using siPHD3

A Langlois; E Maillard-Pedracini; M Prinz; R BeaurepÃƒÂ¨re; W Bietiger; C Sookhareea; C Mura; N Je; idier; M Pinget; S Sigrist

doi:10.4172/2161-0991.C1.003

Stimulation of islet revascularization modulating HIF-1ÃÅ½ÃÂ± expression using siPHD3

4^th Global Surgery and Transplantation Congress

October 03-04, 2016 Atlanta, USA

A Langlois, E Maillard-Pedracini, M Prinz, R Beaurep�?¨re, W Bietiger, C Sookhareea, C Mura, N Jeandidier, M Pinget and S Sigrist

University of Strasbourg, France

Posters & Accepted Abstracts: J Transplant Technol Res

Abstract :

Deleterious events for islet engraftment are related to insufficient islets revascularisation inducing �?² cells death. HIF-1�?± is involved in various survival molecular pathways like cell proliferation and angiogenesis whereas for a chronic stabilization, it induces apoptosis. Prolyl-hydroxylase 3 (PHD3) mediates the degradation of HIF1�?± under normal oxygen conditions. It could be interesting to find the best level of HIF-1�?± expression in islets inhibiting PHD3 to induce angiogenesis while limiting apoptosis. The aim of this work was to realize a proof of concept of a new strategy of angiogenesis stimulation inhibiting PHD3 expression using siRNA on �?²-cell lines. RINm5F �?²-cells were lipofected (Lipofectamine RNAiMax) or not with 50 �?¼M of siPHD3. Apoptosis was determined using Apo-ONE�?® Homogeneous Caspase-3/7 Assay, PHD3 expression by western blotting and VEGF secretion using Elisa kit 48 and 72 hours after lipofection. Caspase 3/7 activity is reduced with siPHD3, 48h (Control: 450�?±18 RFU; siPHD3: 155�?±12 RFU, p<0.001) and 72h post lipofection (Control: 689�?±10 RFU; siPHD3: 213�?±21 RFU, p<0.001). A significant decrease of PHD3 protein expression was observed using siPHD3 validating the efficiency of our approach (Control 48h: 0.48�?±0.15 and siPHD3: 0.14�?±0.03 PHD3 protein expression/�?²-actin; p<0.01). Finally, siPHD3 shown a significant pro-angiogenic effect increasing VEGF secretion 72h post lipofection (Control: 9837�?±470 and siPHD3: 17306�?±2118 pg/mg of protein; p<0.05). Inhibition of PHD3 using siRNA, decreases apoptosis and increases VEGF secretion in RINm5F cells. Thus, modulation of HIF-1�?± expression using lipofection of siRNA could increase islet vascularization and could be a realistic approach to improve graft survival.