Investigating the role of the AKT1/eNOS/TSP2 signaling axis in angiogenesis and tissue repair

Vascular remodeling is essential for tissue repair and is regulated by multiple factors including thrombospondin-2 (TSP2) and hypoxia/VEGF-induced activation of Akt. In contrast to TSP2 knockout (KO) mice, Akt1 KO mice have elevated TSP2 expression and delayed tissue repair. To investigate the contribution of increased TSP2 to Akt1 KO mice phenotypes, we generated Akt1/TSP2 double KO (DKO) mice. Full thickness excisional wounds in DKO mice healed at an accelerated rate, and blood flow recovery to the lower leg following induction of hindlimb ischemia was improved when compared to Akt1 KO mice. Both phenotypes were associated with increased angiogenesis in injured tissues in DKO mice. In addition, DKO mice exhibited normal arterial branching following hindlimb ischemia, which was not evident in Aktl KO mice. Isolated dermal Akt1 KO fibroblasts and immortalized Akt1 KO mouse lung endothelial cells (MLEC's) expressed increased TSP2 and displayed altered morphology and defects in migration and adhesion. In primary dermal fibroblasts, these defects were rescued in DKO fibroblasts or after TSP2 knockdown. Conversely, addition of exogenous TSP2 to WT fibroblasts induced cell morphology and migration rates that were similar to Akt1 KO cells. Akt1 KO fibroblasts displayed reduced adhesion to fibronectin with manganese stimulation when compared to WT and DKO cells, revealing an Akt1-dependent role for TSP2 in regulating integrin-mediated adhesions, however, this effect was not due to changes in beta1 integrin surface expression or activation. Consistent with these results, Akt1 KO fibroblasts displayed reduced Rac l activation that was dependent upon expression of TSP2 and could be rescued by expression of a constitutively active Rac mutant. Our observations show that repression of TSP2 expression is a critical aspect of Akt1 function in tissue repair.