Abstract
Introduction: Hedgehog signaling is a critical mediator of development, ischemic tissue repair, and carcinogenesis. Sonic hedgehog (Shh) signaling has been shown to mediate ischemic tissue repair in experimental models. However, there is controversy on whether Shh signaling improves tissue perfusion by the canonical hedgehog pathway in endothelial cells or via indirect effects on stromal cells. We tested the hypothesis that direct canonical endothelial Shh signaling via the Hedgehog signal transducer Smoothened (Smo) is required for Shh mediated angiogenesis and ischemic tissue repair. Methods: We utilized Cre-Lox mouse models to test this hypothesis. Two endothelial Cre mouse lines, the Tie2-Cre and Vascular Endothelial Cadherin-Cre lines were crossed with mice in which Smo was homozygously floxed to generate endothelial specific Cre+ Smo flox/flox (eSmoNull) mice. Baseline cardiac phenotype was assessed with echocardiography. eSmoNull and SmoWT mice were compared using the corneal angiogenesis and hind limb ischemia (HLI) mouse models. Results: eSmoNull mice were viable and appeared normal. Baseline echo demonstrated normal LV function (fractional shortening: 34.39±1.9 vs. 34.07±1.1, P=0.93). Collagenase digestion of eSmoNull and SmoWT hearts followed by CD31+ cell selection and RT-PCR confirmed effective deletion of Smo. Corneal angiogenesis was induced with Shh pellets revealing similar degrees of Shh induced angiogenesis in eSmoNull and WT mice. HLI was induced to determine the role of endothelial, Smo-mediated hedgehog signaling after ischemic injury. In striking contrast to the impairment in recovery following antibody-mediated global blockade of Shh (Pola, Nature Med, 2001), laser Doppler perfusion demonstrated no significant difference in perfusion at days 7, 14, 21, and 28 (Day 28 perfusion ratio ischemic/nonischemic: 0.56 vs. 0.74, P=0.07). Limb preservation and functional scores were not significantly different. The ratio of lectin+ capillaries per muscle fiber in the ischemic limb was similar (1.58±0.15 vs. 1.65±0.11, P=0.36). Conclusion: Endothelial canonical Shh signaling is dispensable for vascular development, resting cardiac function, Shh induced angiogenesis, and recovery after ischemic injury.
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