Thymosin-β4-mediated therapeutic neovascularization: Role of the PI3k/AKT pathway

Objectives: Thymosin β4 (Tβ4) is known to have pro-angogenic abilities in vitro and in vivo, and its cardioprotective effect is PI3/AKT-dependent. Tβ4-induced vessel formation requires transcriptional activation via the MRTF/SRF pathway. However, the relevance of PI3/AKT signaling for Tβ4-induced angiogenesis remains unclear. Here, we analyzed the PI3K/AKT cascade after Tβ4 transduction in models of chronic hindlimb ischemia. Methods: Tube formation assays of endothelial cells transfected with Tβ4 ± AKT-dn or PI3Ka/Rho inhibition were performed. In mice, rAAV.Tβ4 was injected (intramuscular [i.m.]) 14 days before femoral artery ligation. In addition, either rAAV.AKT-dn was co-applied or Rho/PI3K/AKT pathways were inhibited. Capillary density and hindlimb perfusion were obtained. In rabbits, chronic ischemia was induced by femoral artery excision and subsequent i.m. injection of rAAV.Tβ4 ± rAAV.AKT-dn. Analyses of capillary density, collateral formation and perfusion were performed. Results: Tβ4-induced ring formation was blunted by inhibiting the Rho-kinase (ROCK) or the PI3K/AKT pathway. In vivo, Tβ4 transduction induced angiogenesis and perfusion, an effect abrogated by inhibition of Rho-signaling, or PI3Ka/AKT. In the rabbit model, inhibition of AKT in the lower limb not only abolished angiogenesis but also collateral formation. Conclusion: Tβ4 requires PI3Ka/AKT pathway signaling for induction of therapeutic neovascularization in ischemic limb disease.