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Corneal angiogenic privilege: angiogenic and antiangiogenic factors in corneal avascularity, vasculogenesis, and wound healing (an American Ophthalmological Society thesis).

ABSTRACT: PURPOSE: To determine the molecular basis of corneal avascularity during wound healing and determine the role of angiogenic and antiangiogenic factors in corneal vasculogenesis. METHODS: The expression of proangiogenic factors (vascular endothelial growth factor [VEGF]; basic fibroblast growth factor [bFGF]; matrix metalloproteinase-2 [MMP-2]; and membrane-type 1-MMP [MT1-MMP]) and antiangiogenic factors (pigment epithelium-derived factor [PEDF]; angiostatin; restin; and endostatin) was analyzed in avascular corneas and in models of corneal neovascularization (bFGF pellet implantation, intrastromal injection of MT1-MMP cDNA, and surgically induced partial limbal deficiency). RESULTS: Immunohistochemistry demonstrated the presence of antiangiogenic factors (PEDF, angiostatin, restin, and endostatin) and proangiogenic molecules (VEGF, bFGF, MMP-2, and MT1-MMP) in the cornea after wounding. Proangiogenic MMPs were upregulated in stromal fibroblasts in the vicinity of invading vessels following bFGF pellet implantation. Corneal neovascularization (NV) was also induced by intrastromal injection of MT1-MMP naked cDNA in conjunction with de-epithelialization. Partial limbal deficiency (HLD-) resulted in corneal NV in MMP-7 and MMP-3 knockout mice but not in wild type controls. CONCLUSIONS: Corneal angiogenic privilege is an active process involving the production of antiangiogenic factors to counterbalance the proangiogenic factors (which are upregulated after wound healing even in the absence of new vessels). Our finding that the potent antiangiogenic factors, angiostatin and endostatin, are colocalized with several MMPs during wound healing suggests that MMPs may be involved in the elaboration of these antiangiogenic molecules by proteolytic processing of substrates within the cornea.

SUBMITTER: Azar DT

PROVIDER: S-EPMC1809914 | biostudies-literature | 2006

REPOSITORIES: biostudies-literature

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Corneal angiogenic privilege: angiogenic and antiangiogenic factors in corneal avascularity, vasculogenesis, and wound healing (an American Ophthalmological Society thesis).

Azar Dimitri T DT

Transactions of the American Ophthalmological Society 20060101

<h4>Purpose</h4>To determine the molecular basis of corneal avascularity during wound healing and determine the role of angiogenic and antiangiogenic factors in corneal vasculogenesis.<h4>Methods</h4>The expression of proangiogenic factors (vascular endothelial growth factor [VEGF]; basic fibroblast growth factor [bFGF]; matrix metalloproteinase-2 [MMP-2]; and membrane-type 1-MMP [MT1-MMP]) and antiangiogenic factors (pigment epithelium-derived factor [PEDF]; angiostatin; restin; and endostatin) ...[more]

PMID: 17471348

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Project description:PurposeMutations of keratoepithelin (KE) and myocilin (MYOC) have been linked to certain types of inherited corneal stromal dystrophy and open-angle glaucoma, respectively. In this study, the feasibility of using small interfering RNAs (siRNAs) to suppress the expression of keratoepithelin and myocilin and their capabilities to reduce the related cytotoxic effects caused by mutant myocilins were investigated.MethodscDNAs of human KE gene and myocilin gene were amplified by polymerase chain reaction and subcloned into pEGFP-N1 to construct respective plasmids, KEpEGFP and MYOCpEGFP, to produce fluorescence-generating fusion proteins. Short hairpin RNAs (shRNAs) were generated from an RNA polymerase III promoter-driven vector (pH1-RNA). Transformed HEK293 and trabecular meshwork (TM) cells were cotransfected via liposomes with either KEpEGFP or MYOCpEGFP and respective shRNA-generating plasmids to evaluate the suppression efficacy of shRNAs. Suppression of KE-EGFP by KE-specific shRNAs was evaluated by fluorescence microscopy and Western blotting. Suppression of MYOC-EGFP by myocilin-specific shRNAs was quantified with UN-SCAN-IT software on digitized protein bands of Western blots. A BiP promoter-driven luciferase reporter assay was used to evaluate the stress response of TM cells induced by misfolded mutant myocilins.ResultsTwo KE-specific shRNAs that effectively suppressed the expression of KE-EGFP in HEK293 cells were identified. One shRNA (targeting the coding sequence starting at 1528bp of KE) reduced the expression of KE-EGFP approximately by 50%, whereas the other shRNA (targeting the 3'-UTR region of KE) suppressed greater than 80% of the expression. Cotransfection of MYOCpEGFP and various shRNA-generating plasmids targeting different regions of myocilin (containing amino acid residues R76, E352, K423, or N480 associated with inherited glaucoma) showed effective reduction of MYOC-EGFP, ranging from 78% to 90% on average. The activation of BiP gene (as a stress response induced by mutant myocilins) in transformed TM cells was significantly reduced when mutant myocilin proteins were suppressed by myocilin-specific shRNAs.ConclusionsKE- or myocilin-specific shRNAs could effectively suppress the expression of recombinant KE or myocilin proteins and the related cytotoxicity of mutant myocilins. RNA interference may have future therapeutic implications in suppressing these genes.

Dataset Information

Corneal angiogenic privilege: angiogenic and antiangiogenic factors in corneal avascularity, vasculogenesis, and wound healing (an American Ophthalmological Society thesis).

Publications

Corneal angiogenic privilege: angiogenic and antiangiogenic factors in corneal avascularity, vasculogenesis, and wound healing (an American Ophthalmological Society thesis).

Similar Datasets

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