ABSTRACT
Metalloproteinases (MMPs) are essential regulators during various phases of the angiogenic process. These include the degradation of the basement membrane and the extracellular matrix, the mobilisation and activation of growth factors and the production of fragments with pro- or anti-angiogenic activity. In addition to their role in migration and invasion, MMPs can influence endothelial cell proliferation and survival by modifying the balance between angiogenic and anti-angiogenic molecules.
Subject(s)
Metalloendopeptidases/metabolism , Neovascularization, Physiologic , Animals , Basement Membrane , Cell Division , Cell Survival , Endothelium, Vascular/cytology , Extracellular Matrix , HumansABSTRACT
The goal of the present study was to define the role of gelatinase A in angiogenesis. We performed corneal micropocket assays in gelatinase A-deficient mice and their age-matched wild-type littermates. The corneal neovascular area in gelatinase A-deficient mice (0.15+/-0.14 mm(2)) was significantly less than that of wild-type littermates (0.53+/-0.35 mm(2); P<0.01). Similarly, aortic ring assays showed significant reduction of endothelial outgrowth in gelatinase A-deficient mice (0.26+/-0.14 mm(2)) as compared to wild-type littermates (0.44+/-0.06 mm(2); P<0.05). These results suggest that gelatinase A may play an important role in the regulation of corneal angiogenesis.
Subject(s)
Cornea/blood supply , Corneal Neovascularization/enzymology , Matrix Metalloproteinase 2/deficiency , Matrix Metalloproteinase 2/metabolism , Animals , Aorta/cytology , Aorta/drug effects , Cell Movement/drug effects , Cornea/enzymology , Cornea/metabolism , Cornea/pathology , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Fibroblast Growth Factor 2/pharmacology , Gene Deletion , Genotype , Immunohistochemistry , Matrix Metalloproteinase 2/genetics , Mice , Mice, Knockout , Microscopy, Confocal , Neovascularization, Physiologic/drug effectsABSTRACT
PURPOSE: To localize endostatin and collagen type XVIII in human corneas and to characterize the enzymatic action of matrix metalloproteinases (MMPs) in the cleavage of collagen type XVIII and generation of endostatin in the cornea. METHODS: Anti-endostatin and anti-hinge antibodies were generated using peptide fragments corresponding to the endostatin region and the adjacent nonendostatin hinge region of collagen XVIII noncollagenous (NC)1 domain, respectively. Confocal immunostaining was performed to localize collagen XVIII in human corneas. SV40-immortalized corneal epithelial cells were immunoprecipitated and incubated with active MMP-1, -2, -3, -7, or -9, and Western blot analysis was performed to study collagen XVIII cleavage. Incubation with MMP-7 was performed at various concentrations (0, 2, 4, and 6 microg/ml) and time intervals (0, 1, 5, and 12 hours). Purified recombinant NC1 fragment of collagen XVIII was also digested with MMP-7, and the cleavage product was sequenced. RESULTS: Collagen XVIII was immunolocalized to the human corneal epithelium, epithelial basement membrane, and Descemet membrane. Western blot analysis demonstrated a 180- to 200-kDa band corresponding to collagen XVIII. MMP-7 (but not MMP-1, -2, -3, and -9) cleaved corneal epithelium-derived collagen XVIII to generate a 28-kDa endostatin-spanning fragment in a time- and concentration-dependent fashion. MMP-7 cleaved purified recombinant 34-kDa NC1 fragment of collagen XVIII in the hinge region to generate a 28-kDa fragment. CONCLUSIONS: Collagen XVIII is present in human cornea. MMP-7 cleaves the collagen XVIII NC1 domain to generate a 28-kDa fragment in the cornea.
Subject(s)
Angiogenesis Inhibitors/metabolism , Collagen/metabolism , Cornea/drug effects , Matrix Metalloproteinase 7/pharmacology , Peptide Fragments/metabolism , Amino Acid Sequence , Angiogenesis Inhibitors/chemistry , Animals , Antibody Formation , Basement Membrane/metabolism , Blotting, Western , Collagen/chemistry , Collagen Type XVIII , Cornea/metabolism , Descemet Membrane/metabolism , Dose-Response Relationship, Drug , Endostatins , Epithelium, Corneal/metabolism , Fluorescent Antibody Technique, Indirect , Humans , Matrix Metalloproteinase 7/immunology , Microscopy, Confocal , Molecular Sequence Data , Molecular Weight , Peptide Fragments/chemistry , RabbitsABSTRACT
Corneal neovascularization (NV) is a sight-threatening condition usually associated with inflammatory or infectious disorders of the ocular surface. It has been shown in the field of cancer angiogenesis research that a balance exists between angiogenic factors (such as fibroblast growth factor and vascular endothelial growth factor) and anti-angiogenic molecules (such as angiostatin, endostatin, or pigment epithelium derived factor) in the cornea. Several inflammatory, infectious, degenerative, and traumatic disorders are associated with corneal NV, in which the balance is tilted towards angiogenesis. The pathogenesis of corneal NV may be influenced by matrix metalloproteinases and other proteolytic enzymes. New medical and surgical treatments, including angiostatic steroids, nonsteroidal inflammatory agents, argon laser photocoagulation, and photodynamic therapy have been effective in animal models to inhibit corneal NV and transiently restore corneal "angiogenic privilege."
