Stimulation of islet revascularization modulating HIF-1α expression using siPHD3

Transplantation Technologies & Research

ISSN: 2161-0991

Open Access

Stimulation of islet revascularization modulating HIF-1α expression using siPHD3

4th 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.

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