Limits...
Sonic hedgehog: its expression in a healing cornea and its role in neovascularization.

Fujita K, Miyamoto T, Saika S - Mol. Vis. (2009)

Bottom Line: The effect of a topical injection of cyclopamine on cauterization-induced corneal neovascularization was then studied.This effect was counteracted by addition of cyclopamine.Cyclopamine did not affect VEGF-enhanced tube formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Wakayama Medical University, Kimiidera, Wakayama, Japan. kyoko@wakayama-med.ac.jp

ABSTRACT

Purpose: 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.

Show MeSH

Related in: MedlinePlus

Effect of cyclopamine on the development of corneal neovascularization in vivo. A, B: Cauterization in the central cornea induced neovascularization (NV) in the peripheral cornea (arrows) in the control group. Such NV formation is seen to be suppressed in the cyclopamine-treated group (B) when compared with the control (A). C: The length of NV in the peripheral cornea from the limbus is shown. The NV length is significantly reduced in the cyclopamine group as compared with the control (p<0.05). D, E: Nuclear translocation of Gli3 (arrows) is detected in the corneal epithelium and stromal cells of the peripheral cornea in PBS-treated control group at day 7 (D) while it is not observed in a cornea treated with topical cyclopamine (E). F, G: Induction of Sonic hedgehog is also seen to be suppressed in a treatment group (F) when compared with the control group (G). Nuclear counterstaining was done with DAPI. Bar, 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2684749&req=5

f7: Effect of cyclopamine on the development of corneal neovascularization in vivo. A, B: Cauterization in the central cornea induced neovascularization (NV) in the peripheral cornea (arrows) in the control group. Such NV formation is seen to be suppressed in the cyclopamine-treated group (B) when compared with the control (A). C: The length of NV in the peripheral cornea from the limbus is shown. The NV length is significantly reduced in the cyclopamine group as compared with the control (p<0.05). D, E: Nuclear translocation of Gli3 (arrows) is detected in the corneal epithelium and stromal cells of the peripheral cornea in PBS-treated control group at day 7 (D) while it is not observed in a cornea treated with topical cyclopamine (E). F, G: Induction of Sonic hedgehog is also seen to be suppressed in a treatment group (F) when compared with the control group (G). Nuclear counterstaining was done with DAPI. Bar, 50 μm.

Mentions: We then examined the role(s) of endogenous Shh on injury-induced corneal NV formation, although the experiment above by using an implantation of a pellet to the cornea suggested both exogenous and endogenous Shh is involved in NV development. Cauterization in the central cornea induced NV in the peripheral cornea as previously reported [20,21]. Topical cyclopamine decreased the length of neovascularization in the peripheral cornea post-cauterization when compared with the control vehicle-treated cornea seven days after injury (Figure 7A-C). In these specimens, we then examined the expression of Shh and the nuclear translocation of Gli3. Immunohistochemistry showed that not only the nuclear translocation but also further induction of Shh in tissue were suppressed by topical treatment with cyclopamine (Figure 7D-G).


Sonic hedgehog: its expression in a healing cornea and its role in neovascularization.

Fujita K, Miyamoto T, Saika S - Mol. Vis. (2009)

Effect of cyclopamine on the development of corneal neovascularization in vivo. A, B: Cauterization in the central cornea induced neovascularization (NV) in the peripheral cornea (arrows) in the control group. Such NV formation is seen to be suppressed in the cyclopamine-treated group (B) when compared with the control (A). C: The length of NV in the peripheral cornea from the limbus is shown. The NV length is significantly reduced in the cyclopamine group as compared with the control (p<0.05). D, E: Nuclear translocation of Gli3 (arrows) is detected in the corneal epithelium and stromal cells of the peripheral cornea in PBS-treated control group at day 7 (D) while it is not observed in a cornea treated with topical cyclopamine (E). F, G: Induction of Sonic hedgehog is also seen to be suppressed in a treatment group (F) when compared with the control group (G). Nuclear counterstaining was done with DAPI. Bar, 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2684749&req=5

f7: Effect of cyclopamine on the development of corneal neovascularization in vivo. A, B: Cauterization in the central cornea induced neovascularization (NV) in the peripheral cornea (arrows) in the control group. Such NV formation is seen to be suppressed in the cyclopamine-treated group (B) when compared with the control (A). C: The length of NV in the peripheral cornea from the limbus is shown. The NV length is significantly reduced in the cyclopamine group as compared with the control (p<0.05). D, E: Nuclear translocation of Gli3 (arrows) is detected in the corneal epithelium and stromal cells of the peripheral cornea in PBS-treated control group at day 7 (D) while it is not observed in a cornea treated with topical cyclopamine (E). F, G: Induction of Sonic hedgehog is also seen to be suppressed in a treatment group (F) when compared with the control group (G). Nuclear counterstaining was done with DAPI. Bar, 50 μm.
Mentions: We then examined the role(s) of endogenous Shh on injury-induced corneal NV formation, although the experiment above by using an implantation of a pellet to the cornea suggested both exogenous and endogenous Shh is involved in NV development. Cauterization in the central cornea induced NV in the peripheral cornea as previously reported [20,21]. Topical cyclopamine decreased the length of neovascularization in the peripheral cornea post-cauterization when compared with the control vehicle-treated cornea seven days after injury (Figure 7A-C). In these specimens, we then examined the expression of Shh and the nuclear translocation of Gli3. Immunohistochemistry showed that not only the nuclear translocation but also further induction of Shh in tissue were suppressed by topical treatment with cyclopamine (Figure 7D-G).

Bottom Line: The effect of a topical injection of cyclopamine on cauterization-induced corneal neovascularization was then studied.This effect was counteracted by addition of cyclopamine.Cyclopamine did not affect VEGF-enhanced tube formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Wakayama Medical University, Kimiidera, Wakayama, Japan. kyoko@wakayama-med.ac.jp

ABSTRACT

Purpose: 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.

Show MeSH
Related in: MedlinePlus