Sonic hedgehog: its expression in a healing cornea and its role in neovascularization.
OBJECTIVE
To examine if sonic hedgehog (Shh) is involved in tissue neovascularization by using cell culture and an animal cornea.


METHODS
The effects of exogenous Shh (5.0 nM), vascular endothelial growth factor (VEGF), and/or a Shh signal inhibitor (2.5 or 10.0 muM cyclopamine) on vessel-like tube formation of vascular endothelial cells were examined in vitro. The effects of Shh on the expression of angiogenic cytokines in cultured cell types were examined in cultured cells. The expression of Shh and its receptor, Patched 1 (Ptc), was examined in a vascularized mouse cornea during post-alkali burn healing. The effect of exogenous Shh on corneal neovascularization in vivo was assayed using a rat cornea system. The effect of a topical injection of cyclopamine on cauterization-induced corneal neovascularization was then studied.


RESULTS
Adding Shh promoted vessel-like tube formation of vascular endothelial cells. This effect was counteracted by addition of cyclopamine. Cyclopamine did not affect VEGF-enhanced tube formation. Shh did not affect the expression levels of angiogenic cytokines in cultured cell types. mRNA and protein expression levels of Shh and Ptc were under the detection limit in an uninjured cornea, but Shh but not Ptc was upregulated in a healing, alkali-burned, vascularized cornea. Exogenous Shh promoted neovascularization (NV) formation in vivo in a rat cornea. Topical cyclopmine blocked Gli signaling (blocked translocation of Gli3) and the length of neovascularization in the peripheral cornea post-cauterization as compared with the control vehicle-treated cornea.


CONCLUSIONS
Shh enhances endothelial tube formation independently through VEGF signaling in vitro. Shh signaling is involved in the development of unfavorable corneal neovascularization in animal corneas.
Fujita
Kyoko
K
Department of Ophthalmology, Wakayama Medical University, Kimiidera, Wakayama, Japan. kyoko@wakayama-med.ac.jp
Miyamoto
Takeshi
T
Saika
Shizuya
S
D000468:Alkalies / Q000009:adverse effects; D000818:Animals; D002460:Cell Line; D018932:Chemokine CCL2 / Q000378:metabolism; D003315:Cornea / Q000098:blood supply / Q000648:ultrastructure; D003316:Corneal Diseases / Q000139:chemically induced; D065306:Corneal Injuries; D015870:Gene Expression / Q000187:drug effects; D053823:Hedgehog Proteins / Q000037:antagonists & inhibitors / Q000494:pharmacology* / Q000502:physiology*; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D018919:Neovascularization, Physiologic / Q000187:drug effects; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D053773:Transforming Growth Factor beta1 / Q000378:metabolism; D014471:Umbilical Veins / Q000166:cytology; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D014704:Veratrum Alkaloids / Q000378:metabolism; D014945:Wound Healing / Q000187:drug effects / Q000502:physiology
D000468:Alkalies; C491768:Ccl2 protein, mouse; D018932:Chemokine CCL2; D053823:Hedgehog Proteins; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D053773:Transforming Growth Factor beta1; D042461:Vascular Endothelial Growth Factor A; D014704:Veratrum Alkaloids; C467480:vascular endothelial growth factor A, mouse; C000541:cyclopamine