Abstract 13572: Inhibition of Four-And-A-Half Lim Domain Protein-2 Increases Survival, Migratory Capacity and Paracrine Function of Early Outgrowth Endothelial Progenitor Cells: Implications for Endothelial Regeneration
Inhibition of Four-and-a-half LIM domain protein-2 (FHL-2) attenuates atherosclerotic lesion formation and increases endothelial cell migration. Endothelial progenitor cells (EPCs) substantially contribute to endothelial repair. We investigated the role of FHL-2 in regulation of early outgrowth EPC number and function. Human early outgrowth EPCs were cultured from peripheral blood. FHL-2 knockdown in EPCs by small-interfering RNA (siRNA) induced a 40% reduction in FHL-2 protein expression and resulted in a significant increase in EPC numbers. Cell proliferation was low in EPCs (<1% Ki67-positive cells). FHL-2 knockdown significantly reduced EPC apoptosis, as indicated by a decrease of cleaved caspase-3 and Bax/Bcl-2 expression ratio. This was associated with an increase in sphingosine kinase-1 expression, translocation to the membrane and Akt phosphorylation. Moreover, inhibition of FHL-2 led to an upregulation of alpha-v/beta-3, alpha-v/beta-5, alpha-5, alpha-6 and beta-2 integrins in EPCs and significantly increased EPC migration. Exposure to the conditioned medium of EPCs pretreated with FHL-2 vs. control siRNA significantly decreased apoptosis endothelial cells. In vivo, FHL-2 knockout mice showed higher numbers of sca1/flk1-positive cells in the spleen and higher numbers of spleen-derived, cultured EPCs, as compared to wild-type (WT) mice. This was associated with a reduction of annexin V - and cleaved caspase-3-positive EPCs. Migration was significantly increased in FHL-2 knockout EPCs. In addition, apoptosis was significantly decreased and migration significantly increased in endothelial cells exposed to the conditioned medium of FHL-2 knockout vs. WT EPCs. Finally, reendothelialization after focal carotid endothelial injury in WT mice was significantly increased after intravenous application of spleen-derived progenitor cells from FHL-2 knockout mice compared to transfusion of cells from WT mice. Our findings suggest that FHL-2 negatively regulates early outgrowth EPC numbers and integrin expression. FHL-2 inhibition reduces apoptosis, enhances survival and increases migratory capacity of early outgrowth EPCs and surrounding endothelial cells, resulting in improvement of endothelial regeneration.
- © 2011 by American Heart Association, Inc.