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Abstract
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 PI3Kα/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 PI3Kα/AKT. In the rabbit model, inhibition of AKT in the lower limb not only abolished angiogenesis but also collateral formation.
Conclusion: Tβ4 requires PI3Kα/AKT pathway signaling for induction of therapeutic neovascularization in ischemic limb disease.
Acknowledgments
The authors specially thank Tien Cuong Kieu and Elisabeth Raatz for their excellent technical assistance. All animal experiments were conducted at the Walter Brendel Centre of Experimental Medicine.
Declaration of interest
This paper is part of a supplemental issue, sponsored by SciClone. This work was supported by the Deutsche Forschungsgemeinschaft (DFG, KU 1019/10-1), the DZHK, the Fritz Bender Stiftung (E Deindl) and the German Ministry of Education and Research (BMBF) (C Kupatt and R Hinkel), as well as the Else-Kröner-Fresenius Foundation (2009_A61 to C Kupatt and R Hinkel) and FöFoLe grants of the Ludwig-Maximilians-University (T Trenkwalder, C Kupatt, R Hinkel). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.