Abstract 5445: Gli2 and Gli3 Are Over-expressed in the Ischemic Tissue and Participate in Ischemia-induced Angiogenesis and Myogenesis

Abstract

Gli transcription factors are mediators of hedgehog signaling and have been shown to be critical in several steps during development. In addition, Gli transcription factors have also been implicated as potent oncogenes. We have shown that the Hedgehog pathway is reactivated in the adult cardiovascular system under ischemic conditions and that the hedgehog pathway can be exploited therapeutically in ischemic disease; however the role and expression of the Gli transcription factors in the response to injury following ischemia have not been characterized. We induced hind-limb ischemia by resection of the left femoral artery of FVB mice and found that Gli2 and Gli3 mRNA were overexpressed in the ischemic tissue up to 4-fold and 9-fold respectively while Gli1 expression was not modulated. Immunostaining revealed that several cell types overexpressed Gli2 and Gli3 including myocytes and endothelial cells (ECs). To investigate the role and mechanism of action of Gli2 and Gli3, in vitro experiments were first performed. Overexpression of Gli2 and Gli3 promotes myoblast survival and proliferation. In ECs, overexpression of Gli2 and Gli3 promote survival and migration as well as induce expression of proteins involved in angiogenesis such as MMP-9 and osteopontin. To confirm the hypothesis of a favorable role of Gli2 and Gli3 in neovascularization and tissue repair following ischemic injury, adenovirus encoding LacZ, Gli2 and Gli3 were administrated locally in the tibialis anterior muscle, 2 days before hind limb ischemia surgery was performed. Both Ad-Gli2 and Ad-Gli3 treated mice exhibited higher proliferation in the regenerating muscle 7 days after ischemia. Moreover, Ad-Gli3 treated mice display higher capillary density (38.9±10.2 vessels/HPF vs. 26.1±6.1 in the control mice, p=0.012) and limb perfusion (the ratio of the perfusion of the ischemic leg vs. the control leg was 1.07±0.20 vs. 0.62±0,16 for the control mice, p=0.0003) 7 and 14 day after ischemia respectively. These data indicate that Gli2 and Gli3, transcription factors of the hedgehog pathway, are overexpressed in ischemic tissue and participate in tissue repair by promoting both neovascularization and proliferation and survival of myoblasts.