Immunolocalization of Matrix Metalloproteinase-2, -9 and Tissue Inhibitor of Metalloproteinase-1, -2 in Suture-induced Corneal Neovascularization

PURPOSE: Metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) have been linked to the angiogenic process in general. In order to understand the potential roles of MMPs and TIMPs in corneal neovascularization process, we examined the expression and activities of MMP-2, MMP-9, TIMP-1 and TIMP-2 during the course of suture-induced corneal neovascularization in rat model. METHODS: Corneal neovascularization of rat cornea was induced by suturing. The expression of MMP-2, MMP-9, TIMP-1 and TIMP-2 in sutured corneas was examined by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). The activities of MMP-2 and MMP-9 were measured before and after suture by gelatin zymography. RESULTS: MMP-2 proenzyme, and TIMP-1, -2 were expressed in normal corneas, predominantly in corneal epithelium. After injury, expression of MMP-2, MMP-9, TIMP-1 and TIMP-2 increased, notably in healing corneal epithelium, infiltrating inflammatory cells, stromal fibroblasts, and ingrowing vascular endothelial cells. The intensity of immunostaining and enzymatic activities of MMP-2 and MMP-9 paralleled the magnitude of inflammatory cell infiltration, which peaked around day 7 after suture. Immunoreactivity of MMP/TIMP decreased significantly two weeks after suturing. At day 35 after suture, staining of MMP-2, TIMP-1, -2 remained visible only in corneal epithelium and vascular endothelial cells. CONCLUSIONS: MMPs as well as TIMPs were upregulated during suture-induced corneal neo-vascularization, suggesting that both may take part in extracellular matrix remodeling in the corneal wound healing, inflammatory, and neovascularization processes.

The Efficacy of Polyhexamethylene Biguanide on Experimental Staphylococcus Aureus Keratitis

To evaluate the efficacy of oplyhexamethylene biguanide(PHMB) in Staphylococcus aureus (S. aureus) keratitis model, 10microliter of S. sureus bacterial suspension(1x10(5) colony-forming unit(cfu)/ml) were infected intrastromally into rabbit corneas. 18 rabbits were divided into three treatment groups : balanced salt solution (BSS) group(n=18 eyes), PHMB(0.02%, 200microgram/ml) group(n=9), cefazolin(40 microgram/ml) group(n=9). Topical antibiotic drops were given hourly starting at 24 hours after inoculation. A subconjunctival injection was given every 24 hours for the study duration(96 hours). The severity of keratitis was scored in masked fashion every 12 hours. Corneal buttons were excised and homogenized at the end of the study to determine viable bacterial counts. In the S. aureus keratitis model, there was no difference of clinical scores between BSS, cefazolin, PHMB group at 72 hours, and the number of viable bacteria recovered from the corneal button in log(10) cfu was 2.65+/-1.85, 3.30+/-0.85, 2.86+/-1.36, in BSS, cefazolin, PHMB group respectively. No differences in either clinical scores or bacterial counts were found between the PHMB and BSS groups. PHMB appears to be ineffective against S. aureus in this model of experimental keratitis.

Evaluation of Efficacy of Polygexamethylene biguanide in Experimental Pseudomonas aeruginosa Keratitis

To evaluate the efficacy of polygexamethylene biguanide(PHMB) in Pseudomonas aeruginosa(P. aeruginosa) keratites model, 10microliter of P. aeruginosa bacterial suspension(1x103 colony-forming unit(cfu)/ml) was injected intrastromally into rabbit corneas. Eighteen rabbits(36 eyes) were divided into three treatment groups: balanced salt solution(BSS) group(N=18 eyes). PHMB(0.02%, 200microliter/ml) group(n=9 eyes), tobramycin(14microliter/ml) group(n=9 eyes). Topical antibiotic drops were given hourly from 12 hours after inoculation. A subconjunctival infection was every 24 hours during the first 72 hours. The Severity of keratitis was scored in basked fashion every 8 hours. Corneal buttons were excised and homogenized at the end of the study to determine the viable bacterial counts. In P. aeruginosa keratitis model, tobramycin was statistically more efficacious than PHMB, according the clinical scores at 58 hours(9.9 vs. 15.1, P<0.0001) and log10 cfu(0.54+/-0.21 vs. 4.87+/-1.07. P<0.0001). No differences were found between the PHMB and BSS groups in either clinical scores or bacterial counts. PHMB appears to be ineffective against P. aeruginosa in experimental keratitis model of rabbit.

Inhibition of Basic Fibroblast Growth Factor Induced Rat Corneal Angiogenesis by a Urokinase Plasminogen Activator Receptor Antagonist

During angiogenesis, binding of urokinase plasminogen activator(uPA) and its receptor(uPAR) has been implicated as an important component of the angiogenesis pathway. We have produced a high-affinity competitive antagonist for the uPA receptor consisting of a fusion protein linking the endothelial growth factor(EGF)-like domain of uPA(residues 1-48) to the Fc domain of IgG. To determine whether this recombinant murine uPA1-48-IgG fusion protein could interfere with angiogenesis, we studied the effect of this compound on rat corneal angiogenesisinduced by basic fibroblast growth factor(bFGF). A hydrogel disk containing 250ng of bFGF and 4.2ug of uPA1-48-IgG fusiong protein in seven eyes, 250ug of bFGF and 4.2ug of phosphate-buffered saline(PBS) in another sseven eyes were implanted intrastromally 1.5mm from the superior limbus. At five days post-implantation of bFGF disk, the eyes treated with uPA1-48IgG fusion protein had reduced angiogenesis (mean score=3.1) compared with the PBS-treated controls(mean score=6.1)(P<0.05, Wilcoxon rank sum test). In a rat corneal pocket assay, murine uPA1-48-IgG fusion protein appears to inhibit bFGF-induced angiogenesis. Compounds that block uPAR binding of uPA may have therapeutic potential as anti-angiogenic agents.

Inhibition of Corneal Angiogenesis by Antibody to Integrin b3 Subunit

Vascular endothelial cell expression of avb3, and integrin receptor that binds von Willebrand factor, vitronectin, and fibrinogen, increases in response to angiogenic stimuli such as basic fibroblast growth factor(bFGF) and during capillary proliferation in vivo. We investigated the importance of avb3 function during bFGF-induced corneal angiogenesis by examining the effects of 9D491, a monoclnal natibody against b3 that blocks avb3-mediated cell adhesion to vitronectin and fibrinogen in vitro. A hydrogel disk containing 500ng of bFGF was implanted into the superior corneal stroma of each of twelve New Zealand white rabbit eyes. Each eye also received a second hydrogel disk placed adjacent of the firtt, randomized to contain either 3ug of 9D491 mAb(n=6) or 6E10, an irrelevant antibody of the same isotype, (n=6). Both disks were positioned 1.2mm from the superior limbus. Eyes were examined daily under a streomicroscope by two masked observers and counted an angiogenesis score based on number and length of new blood vessels. On days 5 through 7 post-implantation, angiogenesis scores were significantly lower in eyes treated with avb3 mAb(averaged score=3.3) as compared to eyes treated with 6E10(averaged score=8.0)(p<0.002, Wilcoxon rank sum test). In a rabbit corneal pocket model of angiogenesis, neutralizing monoclonal antibody to b3 inhibits corneal angiogenesis induced by bFGF. Substances that target the integrin b3 subunit may have therapeutic potential in disorders characterized by ocular neovascularization.

Inhibition of Corneal Angiogenesis by Antibody to Integrin b3 Subunit

Vascular endothelial cell expression of avb3, and integrin receptor that binds von Willebrand factor, vitronectin, and fibrinogen, increases in response to angiogenic stimuli such as basic fibroblast growth factor(bFGF) and during capillary proliferation in vivo. We investigated the importance of avb3 function during bFGF-induced corneal angiogenesis by examining the effects of 9D491, a monoclnal natibody against b3 that blocks avb3-mediated cell adhesion to vitronectin and fibrinogen in vitro. A hydrogel disk containing 500ng of bFGF was implanted into the superior corneal stroma of each of twelve New Zealand white rabbit eyes. Each eye also received a second hydrogel disk placed adjacent of the firtt, randomized to contain either 3ug of 9D491 mAb(n=6) or 6E10, an irrelevant antibody of the same isotype, (n=6). Both disks were positioned 1.2mm from the superior limbus. Eyes were examined daily under a streomicroscope by two masked observers and counted an angiogenesis score based on number and length of new blood vessels. On days 5 through 7 post-implantation, angiogenesis scores were significantly lower in eyes treated with avb3 mAb(averaged score=3.3) as compared to eyes treated with 6E10(averaged score=8.0)(p<0.002, Wilcoxon rank sum test). In a rabbit corneal pocket model of angiogenesis, neutralizing monoclonal antibody to b3 inhibits corneal angiogenesis induced by bFGF. Substances that target the integrin b3 subunit may have therapeutic potential in disorders characterized by ocular neovascularization.

Inhibition of Vascular Endothelial Growth Factor Induced Corneal Angiogenesis by Antibody to Intergrin beta3 Subunit

Vascular cells respond to multiple cytokines, they also express a variety of integrin adhesion receptor. A number of the vascular cell integrins are functionally and structurally homologous, suggesting some level of biologic redundancy. We investigated the importance of alphavbeta3 function during vascular endothelial growth factor(VEGF) induced corneal angiogenesis by examining the effects of 9D491, a monoclonal antibody against beta3 that blocks alphavbeta3-mediated cell adhesion to vitronectin and fibrinogen. A hydrogel disk containing 500ng of VEGF was implanted into the superior corneal stroma of each of twelve New Zealand white rabbit eyes. Each eye also received a second hydrogel disk placed adjacent of the first, randomized to contain either 2.6microgram of 9D491 mAb(n=6) or 6E10, an irrelevant antibody of the same isotype, (n=6). Both disks were positioned 1.2mm from the superior limbus. Eyes were examined daily under a streomicroscope by two masked observers and assigned an angiogenesis score based on number and length of new blood vessels. On days 5 through 7 postimplantation, angiogenesis scores were not significantly lower in eyes treated with anti-alphavbeta3 mAb (averaged score=21.6) as compared to eyes treated with 6E10 (averaged score=24.0) (p<0.2, Wilcoxon rank sum test). In a rabbit corneal pocket assay, monoclonal antibody to beta3 could not inhibit corneal angiogenesis induced by VEGF.

Inhibition of Basic Fibroblast Growth Factor Induced Corneal Angiogenesis by a Urokinase Plasminogen Activator Receptor Antagonist

During angiogenesis, expression of urokinase plasminogen activator(uPA) and its receptor(uPAR) is upregulated in vascular endothelial cells. Binding of uPA to uPAR has been implicated as an important component of the angiogenesis pathway and represents a potential target for the design of anti-angiogenic compounds. We have produced a high-affinity competitive antagonist for the uPA receptor consisting of a fusion protein linking the endothelial growth factor (EGF)-like domain of uPA (residues 1-48) to the Fc domain of IgG. To determine whether this recombinant murine uPA(1-48)-IgG fusion protein could interfere with angiogenesis we studied the effect of this compound on rabbit corneal angiogenesis induced by basic fibroblast growth factor(bFGF). A hydrogel disk containing 1000ng of bFGF was implanted intrastromally into the superior cornea of each of sixteen New Zealand white rabbit eyes. All eyes received a second intrastromal disk, randomized to contain either 35 microgram of uPA(1-48)-IgG fusion protein(n=8) or phosphate-buffered saline(PBS)(n=8). Both disks were positioned side-by-side, 1.2 mm from the superior limbus. At three days post-implantation of bFGF disks, eyes treated with uPA(1-48)-IgG fusion protein had reduced angiogenesis(mean score=2.2) compared to PBS-treated controls (mean score=5.1)(p<0.05, Wilcoxon rank sum test). In a rabbit corneal pocket assay, murine uPA(1-48)-IgG fusion protein appears to inhibit bFGF-induced angiogenesis. Compounds that block uPAR binding of uPA may have therapeutic potential as anti-angiogenic agents.