VEGF Trap(R1R2) suppresses experimental corneal angiogenesis.

PURPOSE: To determine the effect of vascular endothelial growth factor (VEGF) TrapR1R2 on bFGF-induced experimental corneal neovascularization (NV). METHODS: Control pellets or pellets containing 80 ng bFGF were surgically implanted into wild-type C57BL/6 and VEGF-LacZ mouse corneas. The corneas were photographed, harvested, and the percentage of corneal NV was calculated. The harvested corneas were evaluated for VEGF expression. VEGF-LacZ mice received tail vein injections of an endothelial-specific lectin after pellet implantation to determine the temporal and spatial relationship between VEGF expression and corneal NV. Intraperitoneal injections of VEGF TrapR1R2 or a human IgG Fc domain control protein were administered, and bFGF pellet-induced corneal NV was evaluated. RESULTS: NV of the corneal stroma began on day 4 and was sustained through day 21 following bFGF pellet implantation. Progression of vascular endothelial cells correlated with increased VEGF-LacZ expression. Western blot analysis showed increased VEGF expression in the corneal NV zone. Following bFGF pellet implantation, the area of corneal NV in untreated controls was 1.05+/-0.12 mm2 and 1.53+/-0.27 mm2 at days 4 and 7, respectively. This was significantly greater than that of mice treated with VEGF Trap (0.24+/-0.11 mm2 and 0.35+/-0.16 mm2 at days 4 and 7, respectively; p<0.05). CONCLUSIONS: Corneal keratocytes express VEGF after bFGF stimulation and bFGF-induced corneal NV is blocked by intraperitoneal VEGF TrapR1R2 administration. Systemic administration of VEGF TrapR1R2 may have potential therapeutic applications in the management of corneal NV.

Basement membrane and collagen deposition after laser subepithelial keratomileusis and photorefractive keratectomy in the leghorn chick eye.

OBJECTIVE: To evaluate corneal scar formation and new collagen deposition after laser subepithelial keratomileusis (LASEK) compared with photorefractive keratectomy (PRK) in the leghorn chick corneal model. METHODS: Leghorn chick corneas treated with LASEK surgery (using 20% ethanol for 30 seconds) or PRK were evaluated by indirect confocal immunofluorescence and transmission electron microscopy at 1, 2, 7, 14, and 28 days after surgery. New collagen deposition was determined by dichlorotriazinylaminofluorescein staining 2 and 4 weeks after surgery. RESULTS: Laminin was detected around the basal layers during the immediate postoperative period and 4 weeks after LASEK surgery, and from day 2 onwards after PRK. Collagen III deposition in the cornea was about 3 times greater with PRK than with LASEK. The thickness of new collagen deposition at 4 weeks was 34 microm +/- 2.5 microm in the PRK group, which was significantly greater than that of the LASEK group (11 microm +/- 1 microm; P<.001). CONCLUSIONS: Reduced subepithelial stromal tissue deposition was observed in LASEK-treated eyes compared with PRK-treated eyes. Postoperative preservation of the epithelial basement membrane and survival of epithelial cells in LASEK and possibly in epithelial laser in situ keratomileusis may contribute to this phenomenon. CLINICAL RELEVANCE: An advantage of LASEK over PRK is the reduction of postoperative haze.

Proteomic identification of activin receptor-like kinase-1 as a differentially expressed protein during hyaloid vascular system regression.

The hyaloid vascular system (HVS) is a transient network of capillaries that nourishes the embryonic lens and the primary vitreous of the developing eye. We used proteomic analysis and immunohistochemical staining to identify activin receptor-like kinase-1 (ALK1), a type I receptor for transforming growth factor-beta1, during the HVS regression phase. In addition, we overexpressed ALK1 in corneas implanted with bFGF (basic fibroblast growth factor) pellets and observed that ALK1 overexpression resulted in inhibition of bFGF-induced corneal neovascularization in vivo. Our data suggest that ALK1 may play a role in HVS regression during ocular development.

Functional characterization of neostatins, the MMP-derived, enzymatic cleavage products of type XVIII collagen.

Several anti-angiogenic factors are derived from proteolytic processing of large molecules including endostatin from type XVIII collagen and angiostatin from plasminogen. In previous studies we showed that neostatin-7, the C-terminal 28kDa endostatin-spanning proteolytic fragment, is generated from the proteolytic action of matrix metalloproteinase matrilysin (MMP)-7 on type XVIII collagen. Now, we report a second member of the neostatin family of proteins, neostatin-14. Given the small quantities of neostatin-7 and -14 generated by the breakdown of naturally occurring collagen XVIII (using MMP-7 and -14, respectively), we used two other approaches to characterize the anti-angiogenic properties of these molecules: murine recombinant neostatin in vitro, and gene therapy. We demonstrate that murine recombinant neostatin-7 inhibits calf pulmonary artery endothelial cell proliferation and that microinjection of neostatin-7 and neostatin-14 naked DNA into the corneal stroma of mice results in significant reduction of basic fibroblast growth factor-induced corneal neovascularization. These results provide supportive evidence of the possible anti-angiogenic effect of neostatins.

Anti-angiogenic role of angiostatin during corneal wound healing.

The purpose of this study is to determine whether angiostatin is involved in maintaining corneal avascularity after wounding. We generated polyclonal rabbit anti-mouse angiostatin antibodies directed against each of the five kringle domains, (K1-5) and anti-mouse plasmin B chain antibodies. Mouse corneas were immunostained with anti-K1 angiostatin antibody after excimer laser keratectomy. Corneal epithelial cell lysate was harvested and angiostatin was isolated using lysine sepharose. Purified plasminogen was incubated with lysate of mouse corneal epithelial cells from wild type mice in the presence or absence of MMP inhibitors. Angiostatin activity was determined using calf pulmonary artery endothelial (CPAE) cell proliferation assay with and without angiostatin immunoprecipitation; and corneal neovascularization was assayed by intrastromal injection of anti-plasminogen, anti-K1-3 or anti-B chain antibodies after corneal wounding. Using the anti-mouse angiostatin antibodies that we generated, we confirmed that angiostatin-like molecules were expressed in the corneal epithelium and in cultured corneal epithelial cells. Western blotting after incubation of scraped corneal epithelial cell lysate with purified plasminogen showed reduction of the plasminogen bands at 6, 12, and 24 hr, respectively. Complete cleavage of plasminogen occurred by 48 hr. Functional assays in which corneal epithelial cell extracts were incubated with CPAE cells resulted in inhibition of vascular endothelial cell proliferation. Depletion experiments using anti-angiostatin (K1) antibodies resulted in a 25 +/- 1.2% increase in vascular endothelial cell proliferation as compared to 12 +/- 1.8% using the protein A control (p < 0.05). Corneal neovascularization was observed after excimer laser keratectomy when anti-angiostatin antibodies were injected into the cornea (65 +/- 13%) which was significantly higher than when plasmin B chain antibodies were injected (10 +/- 2.6%; p < 0.05). Plasminogen and angiostatin are produced in the cornea. They may play a role in preventing vascularization and may contribute to the maintenance of corneal avascularity after excimer laser keratectomy.

Confocal and electron microscopic studies of laser subepithelial keratomileusis (LASEK) in the white leghorn chick eye.

OBJECTIVES: To evaluate the effect of 20% alcohol on the white leghorn chick cornea and to determine the confocal and electron microscopic findings of laser subepithelial keratomileusis surgery in the white leghorn chick corneal model. METHOD: Laser subepithelial keratomileusis surgery was performed on chick corneas and the morphologic changes were examined by transmission electron microscopy. Chick corneas were exposed to 20% alcohol for 30 and 45 seconds or 1 and 2 minutes (5 chicks per group) to evaluate the effect on the corneal epithelium. Photorefractive keratectomy using either mechanical or 20% alcohol-assisted debridement (5 chicks per group) was also performed. Keratocyte and epithelial cell deaths were analyzed 4 hours after surgery using terminal deoxynucleotidyl transfer-mediated biotin-dexoyuridine 5-triphosphate nick-end labeling (TUNEL) staining and transmission electron microscopy. RESULTS: Exposure of the corneal epithelium to 20% alcohol for 30 seconds or longer allowed reproducible separation of epithelial flaps in white leghorn chick eyes. Transmission electron microscopy immediately after alcohol treatment showed that exposure to 20% alcohol for 30 seconds or less had minimal adverse effects on the corneal epithelium. The TUNEL staining of corneas obtained 4 hours after surgery revealed TUNEL-positive cells in the central superficial stroma and more abundantly in the peripheral superficial stroma around the epithelial flap margin and in the epithelial flap itself, particularly in the basal epithelial layer. Transmission electron microscopy showed similar evidence of apoptosis in the epithelium and anterior stroma. CONCLUSIONS: The white leghorn chick eye seems to be a reasonable model for laser subepithelial keratomileusis surgery. Treatment with 20% alcohol for 30 seconds results in reproducible epithelial flap creation in the chick cornea and in relatively low levels of stromal and epithelial cell death after surgery.

Human corneal epithelial cell viability and morphology after dilute alcohol exposure.

PURPOSE: To determine the effect of dilute alcohol on human corneal epithelial cellular morphology and viability. Dilute alcohol is used for epithelial removal during photorefractive keratectomy (PRK) and laser subepithelial keratomileusis (LASEK). METHODS: Corneal epithelial sheets harvested from human eyes after alcohol application during PRK were examined by light and electron microscopy (specimens I-IV). In addition, tissue cultures of human epithelial sheets were monitored for epithelial migration and attachment (specimens V-VII). To determine the effect of dilute alcohol on epithelial cell viability, simian virus (SV)40-immortalized human corneal epithelial cells were exposed to dilute alcohol in distilled water (EtOH-H2O) or to keratinocyte serum-free medium (EtOH-KSFM) for incubation periods of 20 to 45 seconds and concentrations of 10% to 70%. Cell membrane permeability and intracellular esterase activity were analyzed by calcein-acetoxymethyl ester (AM)/ethidium homodimer assay. TdT-mediated dUTP nick-end labeling (TUNEL) assay was used to detect apoptotic cells at 0, 8,12, 24, and 72 hours. RESULTS: Electron microscopy showed varying degrees of basement membrane alterations after alcohol application, including disruptions, discontinuities, irregularities, and duplication (specimens I-IV). Cellular destruction and vacuolization of basal epithelial cells associated with absent basement membrane were also observed (specimen III). One of three cultured epithelial sheets showed attachment and outgrowth in the tissue culture until day 15 (specimen V). Twenty-second exposure of cultured immortalized human cells to various concentrations of EtOH-H2O showed significant reduction of viable cells when EtOH-H2O concentration exceeded 25% (P = 0.005). Increasing the duration of application of 20% EtOH-H2O beyond 30 seconds resulted in a significant reduction in viable cells (69.69% +/- 16.34% at 30 seconds compared with 2.14% +/- 2.29%, 10.45% +/- 7.11%, and 11.09% +/- 15.73% at 35, 40, and 45 seconds, respectively; P = 0.01). TUNEL assay of cultured human corneal epithelial cells exposed to 20% EtOH-H(2)O for 20 and 40 seconds showed maximal labeling at 24 hours (58.05% +/- 33.10%) and 8 hours (94.12% +/- 1.21%), respectively. Exposure to 20% EtOH-KSFM for 20 and 40 seconds resulted in substantially lower TUNEL positivity (3.51% +/- 0.20% at 24 hours and 7.11% +/- 0.49% at 8 hours). CONCLUSIONS: The viability and electron microscopic findings in the basement membrane zone showed significant variation after treatment of the epithelium in vivo with dilute alcohol. The application of dilute alcohol on the monolayer of cultured corneal epithelial cells resulted in increasing cell death in a dose- and time-dependent manner.