D017 Sonic hedgehog-mediated angiogenesis in the mouse ischemic limb requires osteopontin

Purpose Sonic Hedgehog (Shh), a morphogen involved in embryonic development, stimulates repair of ischemic myocardium and skeletal muscle (Pola et al 2003 and Kusano et al 2005). Shh was shown to induce both the formation of new vessels and their muscularization. However the mechanisms involved in these processes are still poorly understood. We investigated the hypothesis that the angiogenic and chemotactic protein osteopontin (OPN) was involved in the angiogenic effect of Shh. Methods and Results We used a plasmid transfection strategy to increase Shh expression in the ischemic region of the limb and we compared the effect of Shh overexpression in wild type (OPN+/+) and OPN deficient (OPN-/-) 12-weeks-old mice transfected either with Shh plasmid (pShh) or control plasmid (β-gal). Shh overexpression induced a significant increase in the number of new vessels (+ 22 %± 4.2) in OPN+/+ mice in comparaison with β-gal transfected wild type mice, moreover the Shh effect was lost in OPN-/- mice. Analysis of vessel muscularization showed that Shh mainly increased the number of small non-muscularized vessels. In parallel, we observed an increased number of OPN expressing cells in the ischemic lesion mainly localized close to or inside debasing/regenerating muscle fibres. These cells expressed Mac3 and F4/80 markers suggesting a macrophage phenotype. We suggested that shh could increase the migration of macrophages in an OPN-dependent manner in order to accelerate the repair of the ischemic tissue. The experiments related to the function of OPN in ischemic limb stimulated or not by Shh is currently under investigation. Conclusion Our results demonstrated that OPN is required for the Shh angiogenic effect in the ischemic tissue and suggest that osteopontin expression may be associated with a better migration of macrophages in the ischemic lesion under Shh stimulation. Kusano KF et al. Nat Med. 2005 ; 11:1197-1204. Pola R et al. Circulation. 2003 ; 108:479-485.