416 Inhibition of Four- and- a- Half LIM Domain Protein 2 Increases Survival and Migratory Capacity of Human Early Outgrowth Endothelial Progenitor Cells Through Upregulation of Sphingosin Kinase- 1: Implications for Endothelial Regeneration

Abstract

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 the regulation of early outgrowth EPC number and function. Early outgrowth EPCs were obtained from human peripheral blood. FHL-2 knockdown in EPCs by small-interfering RNA (siRNA) resulted in a significant increase in EPC number and a reduction of apoptosis (by 40%), as indicated by a decrease of cleaved caspase-3, through activation and translocation to the membrane, of sphingosine kinase-1 (SK-1), enzyme that metabolizes sphingosine- 1 phosphate (s1p). Furthermore, FHL-2 siRNA increased significantly (2 fold) stromal derived factor (SDF) -1- induced EPC migration; through upregulation of α-v/β-3 and α-v/β-5 integrins; this was associated with an increase of the F-actin binding protein cortactin, known to promote migration. Interestingly, increased SDF-1- induced EPC migration and upregulation of cortactin by FHL-2 siRNA were totally prevented by CAY10621, a specific inhibitor of SK-1. In addition stimulation of EPCs with exogenous s1p peptide significantly decreased apoptosis and increased SDF-1- induced migration. These results were confirmed In vivo using FHL-2 knockout (FHL-2 −/−) mice. Moreover, apoptosis was significantly decreased and migration increased in endothelial cells exposed to the conditioned medium of FHL-2 −/− vs. WT EPCs. These effects were abolished by VPC23019, an antagonist of sphingosine- 1- phosphate receptor- 1 and 3. Finally, reendothelialization after focal carotid endothelial electric injury in WT mice was significantly increased after application of spleen-derived progenitor cells from FHL-2 −/− mice vs. WT mice. Our findings suggest that FHL-2 negatively regulates early outgrowth EPC function and secretion of paracrine factors. FHL-2 inhibition reduces apoptosis, enhances survival and migratory capacity of EPCs and ECs by upregulating SK-1/s1p pathway, integrin subunits and cortactin; which results in the improvement of endothelial regeneration.