Desert Hedgehog Promotes Ischemia-Induced Angiogenesis by Ensuring Peripheral Nerve Survival

Abstract

Rationale: Blood vessel growth and patterning have been shown to be regulated by nerve-derived signals. Desert hedgehog (Dhh), one of the Hedgehog family members, is expressed by Schwann cells of peripheral nerves. Objective: The purpose of this study was to investigate the contribution of Dhh to angiogenesis in the setting of ischemia. Methods and Results: We induced hindlimb ischemia in wild-type and Dhh –/– mice. First, we found that limb perfusion is significantly impaired in the absence of Dhh. This effect is associated with a significant decrease in capillary and artery density in Dhh –/– . By using mice in which the Hedgehog signaling pathway effector Smoothened was specifically invalidated in endothelial cells, we demonstrated that Dhh does not promote angiogenesis by a direct activation of endothelial cells. On the contrary, we found that Dhh promotes peripheral nerve survival in the ischemic muscle and, by doing so, maintains the pool of nerve-derived proangiogenic factors. Consistently, we found that denervation of the leg, immediately after the onset of ischemia, severely impairs ischemia-induced angiogenesis and decreases expression of vascular endothelial growth factor A, angiopoietin 1, and neurotrophin 3 in the ischemic muscle. Conclusions: This study demonstrates the crucial roles of nerves and factors regulating nerve physiology in the setting of ischemia-induced angiogenesis.

Hedgehog-Dependent Regulation of Angiogenesis and Myogenesis Is Impaired in Aged Mice

“…Those results were confirmed by immunostaining of ischemic muscle sections; Shh-expressing cells were found in the skeletal muscle of both young and old mice 2 days after HLI surgery was performed and became undetectable at day 10 in both mice ( Figure 2C). As previously described, 10 Dhh is mainly expressed by nerves and consistently with mRNA expression analysis, Dhh staining was weaker on muscle cross-section from old mice ( Figure 2D). This first set of data suggests that impaired activation of Hh signaling in old mice is, at least in part, because of the decrease in Dhh expression.…”

“…9,10 A recent study has suggested that activation of this pathway, in the setting of HLI, is impaired in middle-aged mice (ie, 1 year old). 15 The present study confirms and extends those observations in 2-year-old mice and further investigates the reason why Hh signaling is not fully functional in the setting of aging.…”

“…9,10 The third member of Hh family, Indian hedgehog, is barely expressed in the muscle. 10 As shown in Figure 2A, Shh is strongly overexpressed (by >200-fold) 2 days after HLI surgery (ie, in the not repaired ischemic skeletal muscle) in both old and young mice. Shh expression level was not significantly different between young and old animals.…”

“…6,11,13 We found that endogenous desert Hh (Dhh) is an essential actor of Hh-dependent ischemia-induced angiogenesis by promoting peripheral nerve survival and subsequently nerve-driven angiogenesis. 10 Besides, Hh signaling has been shown to be involved in postnatal myogenesis after mechanical crush or cardiotoxin injection in mice, by regulating satellite cell proliferation. 14 Although Shh gene therapy has been shown to promote ischemia-induced angiogenesis in aged mice, 6 a recent study has reported that activation of Hh signaling observed in the setting of ischemic muscle repair is impaired in middle-aged mice because Gli1 is not upregulated.…”

Sonic hedgehog mediates a novel pathway of PDGF-BB–dependent vessel maturation

“…This finding reveals a new effect of Shh on SMCs, in addition to its previously demonstrated role in proliferation of these cells. [16][17][18] Even if Hh proteins are suggested to act on angiogenesis only indirectly, 12,31,32 ECs were shown to respond to Shh by increasing their migration and the impermeability of the brain endothelium. 33 In the context of growing vessels, SMC-derived Shh may not only act on SMCs themselves, but also on the neighborhood ECs by stimulating their migration and consequently by promoting vessel growth.…”