Modulation Of Angiogenic Process In Cornea By Cystein Cathepsins

Abstract

Purpose: : The avascularization of cornea is essential in normal vision. Several molecules that participate in angiogenesis were identified in cornea as endostatin and angiostatin. Cathepsin V, which is highly expressed in cornea, is able to hydrolyze human plasminogen releasing angiostatin fragments. So, considering these informations we investigated cathepsins as effective angiogenic modulator in human cornea in vitro and in vivo.

Methods: : Techniques used: real time PCR, western blotting, induction of corneal neovascularition and catalytic activity determination using Z-FR-MCA.

Results: : Enzymatic activity of cysteine peptidases was studied in human cornea extracts. The real time PCR confirmed that cathepsin V is highly expressed in human cornea and western blotting showed that plasminogen is also abundant in this tissue. The processing of cornea endogenous plasminogen by the cystein peptidases of this tissue was analyzed by western blotting after incubation of the extract with a cocktail of inhibitors containing PMSF, orto-phenantroline and pepstatin in absence or presence of the cysteine peptidase inhibitor E-64. The results demonstrated that after 2hs of incubation at 37oC, endogenous plasminogen was processed only in the absence of E-64 generating 2 major fragments (40 and 27 kDa). The effects of cathepsins in vivo in the induction of neovascularization were evaluated placing silk sutures in upper left cornea of 12 rabbits. The assays were performed in the absence or presence of E64 during 0, 7, 14, 21 and 28 days. The animals treated with E-64 showed after 21 and 28 days a significant decrease of neovascularization area. The finding suggests that besides cathepsin V other cysteine cathepsins may regulate angiogenesis in cornea. Thus, we analyzed the expression of cathepsins B, L, K and S in human cornea and confirmed by real time PCR the presence of these enzymes in this tissue.

Conclusions: : Our results suggest that cathepsins V, B, L, K and S are importantly involved in the modulation of angiogenesis in cornea.

Keywords: cornea: basic science • gene/expression • proteolysis