title: Corneal angiogenesis modulation by cysteine cathepsins: In vitro and in vivo studies.
abstract: Corneal avascularization is essential for normal vision. Several antiangiogenic factors were identified in cornea such as endostatin and angiostatin. Cathepsin V, which is highly expressed in the cornea, can hydrolyze human plasminogen to release angiostatin fragments. Herein, we describe a detailed investigation of the expression profile of cathepsins B, L, S and V in the human cornea and the role of cysteine peptidases in modulating angiogenesis both in vitro and in vivo. We used various methodological tools for this purpose, including real-time PCR, SDS-PAGE, western blotting, catalytic activity assays, cellular assays and induction of corneal neovascularity in rabbit eyes. Human corneal enzymatic activity assays revealed the presence of cysteine proteases that were capable of processing endogenous corneal plasminogen to produce angiostatin-like fragments. Comparative real-time analysis of cathepsin B, L, S and V expression revealed that cathepsin V was the most highly expressed, followed by cathepsins L, B and S. However, cathepsin V depletion revealed that this enzyme is not the major cysteine protease responsible for plasminogen degradation under non-pathological conditions. Furthermore, western blotting analysis indicated that only cathepsins B and S were present in their enzymatically active forms. In vivo analysis of angiogenesis demonstrated that treatment with the cysteine peptidase inhibitor E64 caused a reduction in neovascularization. Taken together, our results show that human corneal cysteine proteases are critically involved in angiogenesis.
authors: Departamento de Biofísica, Universidade Federal de São Paulo, São Paulo, SP, Brazil.
authors: Larissa P
authors: LP
authors: Coppini
authors: Departamento de Psicobiologia, Universidade Federal de São Paulo, São Paulo, SP, Brazil.
authors: Bruna
authors: B
authors: Visniauskas
authors: Departamento de Medicina I, Universidade Federal do Maranhão, São Luís, MA, Brazil.
authors: Elaine F
authors: EF
authors: Costa
authors: Departamento de Oftalmologia e Ciências Visuais, Instituto da Visão (IPEPO), Universidade Federal de São Paulo, São Paulo, SP, Brazil.
authors: Milton N
authors: MN
authors: Filho
authors: Departamento de Oftalmologia e Ciências Visuais, Instituto da Visão (IPEPO), Universidade Federal de São Paulo, São Paulo, SP, Brazil.
authors: Eduardo B
authors: EB
authors: Rodrigues
authors: Departamento de Ciências da Saúde, Universidade Federal de São Paulo, Santos, SP, Brazil.
authors: Jair R
authors: JR
authors: Chagas
authors: Departamento de Oftalmologia e Ciências Visuais, Instituto da Visão (IPEPO), Universidade Federal de São Paulo, São Paulo, SP, Brazil.
authors: Michel E
authors: ME
authors: Farah
authors: Departamento de Biofísica, Universidade Federal de São Paulo, São Paulo, SP, Brazil; Departamento de Ciências Exatas e da Terra, Universidade Federal de São Paulo, Diadema, SP, Brazil. Electronic address: nilana.barros@unifesp.br.
authors: Nilana M T
authors: NM
authors: Barros
authors: Departamento de Biofísica, Universidade Federal de São Paulo, São Paulo, SP, Brazil. Electronic address: ak.carmona@unifesp.br.
authors: Adriana K
authors: AK
authors: Carmona
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002403:Cathepsins / Q000235:genetics / Q000378:metabolism*; D016510:Corneal Neovascularization / Q000201:enzymology* / Q000473:pathology; D004195:Disease Models, Animal; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation / Q000502:physiology; D006801:Humans; D010958:Plasminogen / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D011817:Rabbits; D060888:Real-Time Polymerase Chain Reaction; D014019:Tissue Donors
chemical_list: D012333:RNA, Messenger; D010958:Plasminogen; D002403:Cathepsins
keywords: Cathepsins; Cysteine proteases; Ocular angiogenesis; Plasminogen