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. 2009 Aug;13(8B):2424-2435.
doi: 10.1111/j.1582-4934.2008.00440.x. Epub 2008 Jul 26.

Sonic hedgehog regulates angiogenesis and myogenesis during post-natal skeletal muscle regeneration

Giuseppe Straface  1 , Tamar Aprahamian  2 , Andrea Flex  1 , Eleonora Gaetani  1 , Federico Biscetti  1 , Roy C Smith  3 , Giovanni Pecorini  1 , Enrico Pola  4 , Flavia Angelini  1 , Egidio Stigliano  5 , John J Castellot Jr  6 , Douglas W Losordo  7 , Roberto Pola  1   3   8
Affiliations

Sonic hedgehog regulates angiogenesis and myogenesis during post-natal skeletal muscle regeneration

Giuseppe Straface et al. J Cell Mol Med. 2009 Aug.

Abstract

Sonic hedgehog (Shh) is a morphogen-regulating crucial epithelial-mesenchymal interactions during embryonic development, but its signalling pathway is considered generally silent in post-natal life. In this study, we demonstrate that Shh is de novo expressed after injury and during regeneration of the adult skeletal muscle. Shh expression is followed by significant up-regulation of its receptor and target gene Ptc1 in injured and regenerating muscles. The reactivation of the Shh signalling pathway has an important regulatory role on injury-induced angiogenesis, as inhibition of Shh function results in impaired up-regulation of prototypical angiogenic agents, such as vascular endothelial growth factor (VEGF) and stromal-derived factor (SDF)-1alpha, decreased muscle blood flow and reduced capillary density after injury. In addition, Shh reactivation plays a regulatory role on myogenesis, as its inhibition impairs the activation of the myogenic regulatory factors Myf-5 and MyoD, decreases the up-regulation of insulin-like growth factor (IGF)-1 and reduces the number of myogenic satellite cells at injured site. Finally, Shh inhibition results in muscle fibrosis, increased inflammatory reaction and compromised motor functional recovery after injury. These data demonstrate that the Shh pathway is functionally important for adult skeletal muscle regeneration and displays pleiotropic angiogenic and myogenic potentials in post-natal life. These findings might constitute the foundation for new therapeutic approaches for muscular diseases in humans.

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Figures

Figure 1
Figure 1
Shh pathway activation in injured skeletal muscle. (A, B) Shh mRNA is increased in TA muscle after mechanical crush and CTX‐injection, with maximum up‐regulation 2 days after injury (*P < 0.001). Expression is significantly increased also at day 4 (*P < 0.001) and 7 (§P < 0.01) and returns to normal at day 10. (C–F) Dhh and Ihh mRNA expression levels are unchanged after mechanical crush and CTX injection. (G, H) Ptc1 mRNA is significantly increased 2, 4 and 7 days after mechanical crush and CTX‐injury (*P < 0.001, §P < 0.01, #P < 0.05).
Figure 2
Figure 2
In situ hybridization for Shh after skeletal muscle injury. (A) Two days after mechanical crush, Shh expression is detectable in skeletal muscle fibres surrounding the injured area. (B) Also after CTX injection, there is strong Shh‐positive signal in muscle fibres within the injured tissue.
Figure 3
Figure 3
Inhibition of Shh reduces the angiogenic response to injury. (A) In CTX‐injured muscles, local production of VEGF is significantly reduced by treatment with the Shh inhibitor cyclopamine (cyc) (#P < 0.05). (B) Cyclopamine treatment reduces the up‐regulation of SDF‐1alpha in CTX‐injured muscles (§P < 0.01, #P < 0.05).
Figure 4
Figure 4
Inhibition of Shh decreases local production of myogenic factors and reduces the number of activated MSCs in vivo. (A) In CTX‐injured muscles, local production of IGF‐1 is significantly reduced by cyc (#P < 0.05). (B) Western blotting analysis showing impaired up‐regulation of both Myf5 and MyoD in CTX‐injured muscles of mice treated with cyc. (C) Quantification of Western blotting data by densitometric analysis showing significant difference of Myf5 and MyoD protein expression between saline‐ and cyc‐treated animals (#P < 0.05). (D) Immunofluorescent staining for Myf5 and MyoD 4 days after CTX‐injury, showing substantial reduction of both Myf5‐ and MyoD‐positive cells in injured muscles of mice treated with cyc. (E) Quantification of Myf5 and MyoD histologic analyses, demonstrating that the number of Myf5‐positive cells is significantly reduced in muscles of mice treated with cyc (|P < 0.01). Also the number of MyoD‐positive cells is significantly reduced upon inhibition of Shh activity (§P < 0.01).
Figure 5
Figure 5
Direct effect of Shh on MSCs in vivo and in vitro. (A, B) In NLS‐Ptc1‐LacZ mice, β‐gal‐ositive cells can be detected around and between injured fibres, 4 days after CTX injection. Many β‐gal‐positive cells are characterized by small size and are located on the surface of injured myofibres (black arrowheads) or between them (red arrowheads). (C) Double immunofluorescent staining for β‐gal (nuclear red staining) and Myf5 (green staining) double positive cells in injured skeletal muscle. (D) Double immunofluorescent staining showing β‐gal (nuclear red staining) and MyoD (green staining) double positive cells in injured skeletal muscle. (E) The proliferation of myoblastic C2C12 cells is significantly increased by Shh and reduced by cyc (§P < 0.01).
Figure 6
Figure 6
Inhibition of Shh increases fibrosis and epimysial thickness and reduces motor functional recovery. (A, B) Representative images of fibrosis (blue staining) in muscles of mice treated with cyc and saline. (C) The mean percentage of fibrosis is significantly higher in the cyc group than controls (§P < 0.01). (D, E) Representative images of ET (blue staining) in muscles of mice treated with cyc and saline. (F) The ratio between ET at the injured and contralateral side is significantly higher in mice treated with cyc than controls (§P < 0.01). (G) Cyc treatment reduces motor functional recovery after CTX injury. In saline‐treated animals, grip strength returns to normal levels by day 10 after injury. In comparison, grip strength is significantly reduced in mice treated with cyc at day 6, 8 and 10 after injury (#P < 0.05, §P < 0.01).
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