Gene Silencing in Human Endothelial Colony Forming Cells (ECFC) using siRNA and SilenceMag (Magnetofection)

Human primary ECFC - Endothelial Colony Forming Cells - were transfected with siRNA by Magnetofection using SilenceMag. Gene Silencing was confirmed 24 and 72~h after transfection.

This article demonstres the capacity of Magnetofection technology to efficiently silence gene expression in vivo with siRNA using SilenceMag from OZ Biosciences.

article reference: Cardiovasc Res. 2014 Apr 17. 

Sphingosine kinase 1 expressed by endothelial colony-forming cells has a critical role in their revascularization activity.

Poitevin S, Cussac D, Leroyer AS, Albinet V, Sarlon-Bartoli G, Guillet B, Hubert L, Andrieu-Abadie N, Couderc B, Parini A, Dignat-George F, Sabatier F.

Abstract

AIMS:Cell therapy based on endothelial colony-forming cells (ECFCs) is a promising option for ischemic cardiovascular diseases. A better understanding of the mechanisms by which these cells promote revascularization remains a critical challenge to improving their therapeutic potential. We aimed to identify the critical mechanisms involved in the revascularization activity of ECFCs by using the paracrine properties of mesenchymal stem cells (MSC).Methods and ResultsConditioned medium from human bone marrow-derived MSCs (MSC-CM) increased the angiogenic activity of cord blood ECFCs in vitro (proliferation, migration, and pseudo-tube formation), the survival of ECFCs in mice (Matrigel Plug assay) and the capacity of ECFCs to promote the recovery of blood perfusion in mice with hindlimb ischemia. Furthermore, the capillary density in ischemic gastrocnemius muscle was significantly increased in mice transplanted with the ECFCs pretreated with the MSC-CM. The enhancement of ECFCs activity involved the upregulation of sphingosine kinase 1 (SphK1) expression and activity. The inhibition of SphK1 in ECFCs by using an inhibitor or a siRNA knockdown of SphK1 prevented the stimulation of the ECFCs induced by the MSC-CM. The improvement of ECFC activity by MSC-CM also involved the upregulation of sphingosine-1-phosphate receptor 1 (S1P₁) and a S1P/S1P_1/3-dependent mechanism. Finally, we showed that the stimulation of ECFCs with exogenous S1P increased angiogenesis and promoted blood perfusion in hindlimb ischemia.
CONCLUSION:The upregulation of SphK1 and S1P-dependent pathways is critical for the angiogenic/vasculogenic activity of ECFCs. The identification of this pathway provides attractive targets to optimize cell-based therapy for revascularization in ischemic diseases.