Abstract 447: Sonic Hedgehog Carried By Microparticles Corrects Endothelial Injury Through Nitric Oxide Release

Abstract

Microparticles (MPs) are small fragments generated from the plasma membrane of cells upon stimulation. Among the candidate proteins harboured by MPs, we have recently shown that Sonic Hedgehog (Shh) is present in MPs generated from activated/apoptotic human T CEM lymphocytes. MPs were isolated following serial centrifugations. Eahy 926 endothelial cells were grown for 24h in the absence or presence of 10 μg/mL MPs pre-incubated (or not) either with inhibitors of PI3-kinase (LY294002, 20 μM), MEK 1/2 (U0126, 10 μM), cyclopamine (30 μM), a specific antagonist of the Sonic Hedgehog receptor (Patched), or siRNA of Patched. Cell lysates were analyzed by Western blot. Cells were used for direct measurement of nitric oxide (NO), and oxidative stress was determined by flow cytometry. In other experiments, after 24h of intravenous injection of MPs to mice, endothelium-dependent relaxation was determined in aortic rings. In addition, ischemia/reperfusion was induced in mice by ligating the left anterior descending coronary artery proximal to its origin and endothelial function of the distal coronary artery was assessed. We show that Shh carried by MPs: a) induces NO release from endothelial cells under basal (2.5 fold) and after a bradykinin-stimulation (20 μM) conditions (2.6 fold) b) increases both the expression and the phosphorylation of enzymes related to the NO pathway, and c) decreases the production of reactive oxygen species (38.6 ± 1.4 % of positive cells in treated vs. 51.4 ± 0.2 % in control). Inhibition of PI3-kinase and ERK signalling reversed the effects of MPs. Injection of MPs to mice improved endothelial function in aorta (EC 50 ; 8.5 x 10 −8 M of acetylcholine in control vs. 7.05 x 10 −8 M in treated mice) and elevated NO release in blood, heart and lungs (1.4, 1.9 and 2.6-fold, respectively). Mice treated with Shh MPs had reduced coronary endothelial dysfunction after ischaemia/reperfusion. Silencing Shh with either cyclopamine or siRNA of Patched caused a 35 % reduction of NO release elicited by MPs. Based on our findings, we propose that the cardiovascular effects of MPs harbouring Shh may represent a new therapeutic approach against endothelial dysfunction caused by endothelial injury e.g. during cardiac ischemia.