Inhibition of Four-and-a-Half LIM Domain Protein 2 Increases Survival, Migratory Capacity and Paracrine Function of Human Early Outgrowth Cells Through Activation of the Sphingosine Kinase-1 Pathway: Implications for Endothelial Regeneration
Rationale: Inhibition of Four-and-a-half LIM domain protein 2 (FHL2) attenuates atherosclerotic lesion formation and increases endothelial cell migration. Early outgrowth cells (EOCs) contribute substantially to endothelial repair.
Objective: We investigated the role of FHL2 in the regulation of EOCs.
Methods and Results: Human EOCs were cultured from peripheral blood. FHL2 knockdown in EOCs by small-interfering RNA (siRNA) resulted in increased EOC numbers and reduced apoptosis, as indicated by decreased cleaved caspase-III and reduced Bax/Bcl-2 expression ratio. This was mediated through increased phosphorylation and membrane translocation of sphingosine kinase-1 (SK-1), increased sphingosine-1-phosphate (S1P) levels, and Akt phosphorylation. FHL2 knockdown increased SDF-1-induced EOC migration through upregulation of αv/β3, αv/β5 and β2 integrins, associated with increased cortactin expression. Reduced apoptosis, increased EOC migration and cortactin upregulation by FHL2 siRNA were prevented by CAY10621, the SK-1 inhibitor and the S1P receptor-1/-3 antagonist VPC23019. These findings were confirmed using spleen-derived EOCs from FHL2-/- mice. Apoptosis was decreased and migration increased in endothelial cells (ECs) exposed to the conditioned medium of FHL2-/- vs. wild-type (WT) EOCs. These paracrine effects were abolished by VPC23019. Importantly, reendothelialization after focal carotid endothelial injury in WT mice was significantly increased after intravenous injection of FHL2-/- vs. WT EOCs.
Conclusions: Our findings suggest that FHL2 negatively regulates EOC survival, migration and paracrine function. FHL2 inhibition in EOCs reduces apoptosis and enhances survival and migratory capacity of both EOCs and surrounding ECs by activation of the SK-1/S1P pathway, resulting in improvement of endothelial regeneration.
- early outgrowth cells
- signal transduction
- progenitor cell
- vascular biology
- Received August 1, 2013.
- Revision received September 27, 2013.
- Accepted October 1, 2013.