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Purpose : To investigate the preventive effects of topical sunitinib, sunitinib-hesperetin and sunitinib-doxycycline combinations on corneal neovascularization (CNV), apoptosis and fibrosis in a corneal alkali burn model. Materials and Methods : The corneas of 32 Wistar albino rats were cauterized with silver nitrate to induce CNV. Four groups were created receiving artificial tears (sham), sunitinib (0.5 mg/ml), sunitinib-hesperetin (0.5 mg/ml-0.2 mg/ml), and sunitinib-doxycycline (0.5 mg/ml-20 mg/ml) treatments. Corneal photographs were taken on days 0, 7 and 15‎. Photographs of the cornea were digitally analyzed to measure the size of the neovascularization area in comparison to the total corneal surface area. On the 15th day, the animals were euthanized, and the eyes were enucleated for immunohistochemical staining to investigate neovascularization, apoptosis, and fibrosis. Results : CNV areas on the 7th day in the sunitinib (4.8% ± 0.07%) and sunitinib-hesperetin (1.1% ± 0.03%) groups were smaller than those in the sham group (33.9% ± 0.12%) (p = 0.001 and, p < 0.001 respectively). On the 15th day, the CNV area in the sunitinib-hesperetin (20.8% ± 0.37%) group was significantly smaller than that of the sham group (74.6% ± 0.32%) (p = 0.039). The combination groups had lower levels of VEGF, TUNEL and α-SMA positivity than the sunitinib monotherapy group. TUNEL positivity was lowest in the sunitinib-hesperetin and sunitinib-doxycycline groups, and α-SMA positivity was lowest in the sunitinib-hesperetin group. Conclusion : Topical sunitinib-hesperetin was more effective than sunitinib alone and the sunitinib-doxycycline combination in the treatment of CNV. The combination of sunitinib and hesperetin seems to be a promising treatment for preventing corneal fibrosis and apoptosis.

Purpose: To determine whether Toll-like receptor 4 knockout protects mice from corneal neovascularization following chemical injury compared to wild-type (WT) mice., Methods: A chemical burn (75% silver nitrate, 25% potassium nitrate) was created under anesthesia in the central right cornea of 32 WT and 31 Toll-like receptor 4 knockout mice. Corneal neovascularization was evaluated at 3, 4, 6, 8, 10, and 35 days after injury using digital photography, fluorescein angiography, gelatin perfusion with fluorescence vascular imaging, immunofluorescence staining, and molecular analysis., Results: There was no significant between-group difference in relative corneal burn area at 10 days after injury (39.0 ± 2.4% vs. 38.8 ± 9.8%, respectively). Neovascularization was detected in all corneas in vivo and perfusion was detected by fluorescence vascular imaging, reaching maximum area on day 10. The relative area of neovascularization was significantly smaller in the knockout than the WT mice on days 6 (33.3 ± 4.2% vs. 46.8 ± 7.4%, respectively, p = 0.005) and 8 (36.6 ± 1.1% vs. 52.2 ± 6.4%, respectively, p = 0.027), although neovascularization was intensive in both groups. In line with the immunostaining findings of angiogenesis and inflammatory infiltration of damaged corneas, molecular analysis (performed on day 3) revealed elevated expression levels of angiogenesis-related genes (vascular endothelial growth factor, VEGFR2, VEGFR1) and inflammation-related genes (CD45 and TGFβ1) in the WT mice. The knockout mice had higher TNF-α expression than the WT mice., Conclusion: In a mouse corneal chemical burn model, lack of Toll-like receptor 4 expression did not completely inhibit angiogenesis, but did have a relative effect to reduce neovascularization as compared to the WT.

Purpose: Corneal ulceration may be treated with human amniotic membrane (HAM) while neovascularization remains a common complication of corneal ulceration. As several factors contained within HAM may contribute to reduced corneal scarring, we investigated if HAM may be additionally loaded with bevacizumab and potentially serve as a carrier for anti-vascular endothelial growth factor (VEGF) drugs to provide constant VEGF blockade., Materials and Methods: Cryo-preserved HAM were incubated with different bevacizumab concentrations in organ culture medium for 2-5 d. Controls were incubated without bevacizumab. Then, all samples were placed into an organ culture medium without bevacizumab for 48 h, 72 h or for 5 d at 37 °C with the medium being changed at all time points. After that, VEGF165 was added to the supernatants for 24 h and free VEGF 165 was measured by ELISA., Results: Free VEGF was significantly blocked at 48 and 72 h (p < 0.01). VEGF blockade was less pronounced after 1 week. However, as compared to control, VEGF was significantly blocked at all times (p < 0.05)., Conclusion: In this in-vitro setting, we could demonstrate effective VEGF-blockade for up to 1 week by incubating HAM with bevacizumab. HAMs might be potentially used as a carrier for drugs delivered to the cornea.

Purpose: To investigate the impact of tacrolimus on vascular endothelial growth factor (VEGF) in experimental corneal neovascularization (NV) immunohistochemically., Material and Methods: Five groups of seven Wistar albino rats were formed. A silver nitrate cauterization technique was used to induce corneal NV in the study groups, excluding Group 1 (Control Group). Rats in group 1 did not receive any treatment. Rats in group 2 (sham 1) were administered 1 ml of saline intraperitoneally once a day and those in group 3 (sham 2) received one drop of saline four times a day. Rats in group 4 were administered 0.3 mg/kg tacrolimus intraperitoneally once a day. For group 5, 0.3 mg/ml tacrolimus was installed four times a day. Digital photography for each cornea was performed and the percentage area of the NV on the total corneal surface was calculated. The intensity of VEGF immunostaining in the epithelial, the stromal, and endothelial layers was performed in a semi quantitative fashion., Results: The mean percentages of the neovascularized areas of intraperitoneally and topically tacrolimus-treated groups were lesser than those of the sham groups (p = 0.002, p = 0.038, respectively). The mean intensity of the epithelial VEGF immunostaining of the intraperitoneally tacrolimus-treated group was less than that of its sham group (p = 0.002), while the mean intensity of the stromal VEGF staining of the topically tacrolimus-treated group was lesser than that of its sham group (p = 0.042). The intensities of the endothelial VEGF immunostaining of the intraperitoneally and topically tacrolimus-treated groups were less than those of the sham groups (p = 0.038, p = 0.032)., Conclusion: Systemic and topical administration of tacrolimus may be beneficial in the prevention of corneal NV because of its effect on VEGF.

Purpose: This study was designed to investigate whether synthetic small interference RNA (siRNA) targeting VEGF-A could inhibit mouse corneal neovascularization or not., Methods: First, the effect of synthetic siRNA targeting VEGF-A on the expression of VEGF in human retinal pigment epithelial line ARPE-19 cells was examined. Corneal neovascularization was induced on the right cornea of C57BL/6 mice by alkali burn. Mice were randomly divided into 4 groups (21 animals in each group): VEGF-A siRNA group, stable negative siRNA group, saline control group, and blank control group. After alkali burn, except for the blank control group, the other 3 groups were subconjunctivally injected with VEGF-A siRNA, stable negative siRNA, and normal saline, respectively. On days 3 and 7 after alkali burn, the concentration of VEGF in the cornea was determined by ELISA kit. On day 10, the neovascularized corneal area was mensurated, the amount of corneal new vessels was quantified by CD31 immunohistochemical assay of corneal sections, and permeability of new vessels was shown by fluorescence angiography., Results: After comparing the histological appearance of the cornea between the VGF-A siRNA group and the 3 control groups on days 10 and 30, it was found that in the VEGF-A siRNA group, not only was VEGF expression in the cornea induced by alkali burn but also neovascularized corneal area and amount of new vessels were reduced, followed by the permeability of new vessels, and corneal histological structure were improved. It was also found that the exposure to VEGF-A siRNA significantly reduced the VEGF-A mRNA expression in AREP-19 cells. These findings suggested synthetic siRNA targeting VEGF-A delivered by subconjunctival injection could inhibit corneal angiogenesis induced by alkali burn., Conclusion: VEGF-A siRNA could potentially serve as an important therapeutic alternative in the management against unwanted angiogenesis including corneal neovascularization.

Purpose: To investigate the effect of bevacizumab in a model of corneal inflammation., Materials and Methods: Epithelium from the cornea and limbus was completely removed using 100% alcohol in rats. Bevacizumab (1.25 mg/0.05 ml) or normal saline was subconjunctivally injected. One week later, corneas were examined and subjected to hematoxylin-eosin and immunofluorescent staining for VEGF. Expression of IL-2, IFN-gamma, IL-6, and TGF-beta 1 were analyzed by ELISA., Results: Bevacizumab showed a borderline reduction in corneal neovascularization (11.0 +/- 4.8% and 18.0 +/- 10.0% in the bevacizumab and control groups, respectively; p = 0.054), while the extent of epithelialization was not affected (5.0 +/- 3.4% and 6.1 +/- 5.2% in the bevacizumab and control groups, respectively; p = 0.715). The infiltration of inflammatory cells was reduced (99.3 +/- 22.3 cells/x 400 in the bevacizumab-injected corneas and 182.3 +/- 58.0 cells/x 400 in the controls; p = 0.013). The levels of IL-2, IFN-gamma, and IL-6 were decreased in the rats with the bevacizumab injections (44 +/- 6 and 79 +/- 9 pg/ml for IL-2 in the bevacizumab-injected group and control, respectively, p = 0.025; 45 +/- 5 and 67 +/- 6 pg/ml for IFN-gamma in the bevacizumab-injected group and controls, respectively, p = 0.043; 45 +/- 6 and 75 +/- 8 pg/ml for IL-6 in the bevacizumab-injected group and controls, respectively, p = 0.030)., Conclusions: Bevacizumab showed a reduction in inflammatory cell infiltration and cytokines in chemically burned rat corneas.

The effects of diced small interfering RNAs (siRNAs) designed for vascular endothelial growth factor (VEGF) on the expression of VEGF in human retinal pigment epithelial cell line ARPE-19 cells in vitro and on corneal angiogenesis in vivo were examined. The exposure to diced siRNAs significantly reduced the VEGF mRNA expression in ARPE-19 cells with minimal toxicity. In suture-induced corneal angiogenesis models, diced siRNAs minimized the severity of angiogenesis. Histological analysis displayed no particular tissue damage in the conjunctiva where siRNA was injected. The approach using diced siRNAs can be a new tool for various neovascular ocular diseases.

Purpose: Eicosanoids that are present in inflamed tissues are thought to play a significant role in angiogenesis. Cyclooxygenase, a key enzyme in eicosanoid synthesis, has recently been shown to exist in two isoforms: the constitutive COX-1 and the inducible COX-2. This study was undertaken to determine the role of COX-2 in the corneal angiogenic response., Methods: Angiogenesis in the rat cornea was provoked by chemical cautery. Either NS-398, a selective COX-2 inhibitor, or indomethacin, a non-selective COX inhibitor, was applied topically 3 times daily for 4 days. Neovascularization was quantitated by digital image analysis in corneal flat preparations. To test their inhibitory effects on eicosanoid synthesis, normal or cauterized corneas were incubated in the culture medium with the inhibitor. Prostaglandin E2 in the medium was assayed using an enzyme-linked immunosorbent assay., Results: Both NS-398 and indomethacin significantly inhibited corneal neovascularization with the % inhibition of 36.4 +/- 9.6%, and 38.5 +/- 9.0%, respectively, when applied topically at a concentration of 0.1% (p <.001). Neither reduced the angiogenic response at a concentration of 0.01% or below. PGE(2) production in the cauterized cornea was 2.0 times higher than that in the controls. In normal corneas, indomethacin inhibited PGE(2) synthesis by 80%, whereas NS-398 inhibited it by no more than 20%. In contrast, in injured corneas, both indomethacin and NS-398 inhibited PGE(2) synthesis in a similar fashion, with a maximal inhibition rate of 75 to 80%., Conclusions: Our results suggest that COX-2 induction in cauterized corneas increases the level of eicosanoids, which result in corneal angiogenesis.

Purpose: Although overnight eye closure is known to result in hypoxia and release of potent angiogenic factors, even prolonged eye closure does not result in corneal neovascularization. This suggests that the closed eye tear film may contain factors that can impede neovascularization. Closed eye tear fluid is known to contain proteases capable of converting plasminogen/plasmin to angiostatin and other angiostatin-like A-chain fragments which are potent inhibitors of angiogenesis. This study was designed to characterize open and closed eye tear fluid for the presence of these entities., Methods: Open and closed eye tears were collected by microcapillaries from normal individuals. Tears were centrifuged and the supernatants analyzed by SDS-PAGE and western blotting. Membranes were probed with antibodies specific for the A-chain of plasmin and plasminogen and with antibodies specific for conformational domains on the smaller N terminal kringles 1-->4 and kringles 1-->3 fragments which are known angiogenesis inhibitors. Supernatants were also analyzed after fractionation by HPLC and binding to lysine sepharose 4B. The isolated fragments were identified based on size, lysine-binding capabilities, antigenic properties and by comparison with standards., Results: Open eye tear fluid from all normal individuals contained low levels of plasminogen, but no detectable antigens consistent with free A-chain or angiostatins. Tears collected after overnight eye closure contained significant amounts of plasminogen, A-chain antigen and various A-chain fragments including kringles 1-->4 and kringles 1-->3 and most likely free kringle 5, all known to have anti-angiogenesis properties. These were often present in concentrations likely to be physiologically significant. In samples collected from an atopic subject, the concentration of angiostatins in CTF increased markedly during active phases of the disease reaching levels of several ng/microl. In these instances and in similar samples obtained from other atopic individuals experiencing active reactions, angiostatin was often detectable in basal-type tear fluid., Conclusion: A-chain fragments, which can inhibit angiogenesis, are often present at physiologically significant levels in human tear fluid collected after overnight eye closure. These fragments may play a role in preventing neovascularization in the hypoxic closed eye environment and may well be up regulated during inflammatory reactions.

Steroids are widely used in the prevention of corneal neovascularization in a wide range of natural and experimental situations. However, no information is available on their effect on the growth of the individual limbal blood vascular cells or of lymphatic cells involved in corneal neovascularization. In addition, tritiated thymidine labelling index is commonly used as an indicator of cell population but doubt exists as to whether it truly represents cell growth. Remote thermal cautery of the rat cornea was used to elicit corneal neovascularization. New cell growth was measured by tritiated thymidine uptake and by the number of cell nuclei per section. Cells investigated were the arteriolar, venular, capillary and lymphatic endothelial cells as well as the arteriolar and venular perivascular cells. A total of 89,320 blood vascular endothelial and perivascular cell nuclei and 12,075 lymphatic nuclei were counted. Thermal cautery elicited a significant increase in labelling index and cell population of all limbal vascular cell types. Steroid application elicited a significant short term inhibition or delay for all six cell types although this was not apparent for venular endothelial cells using labelling index as a growth indicator. At six days only the lymphatic endothelial cell population showed a significant (p < 0.001) increase associated with steroid use.

We assessed the role of platelet-activating factor (PAF) in corneal allograft rejection and evaluated the effects of a PAF antagonist on corneal inflammation, cellular infiltration, vascularization, and edema. Rabbits with vascularized corneas served as recipients of allogeneic cornea grafts. Rabbits with normal corneas underwent autografts as controls. All of the allografts developed the progression of signs characteristic of rejection. Nevertheless, treatment with the PAF antagonist BN52021 significantly inhibited corneal allograft vascularization for up to 10 days after transplantation and reduced the number of eosinophils in the allografts at 28 days after transplantation. In contrast, saline-treated allografts exhibited florid vascularization and intense inflammatory infiltrates. Control autografts survived without developing significant inflammation or vascularization. The retardation of allograft eosinophilia and graft vascularization by the PAF antagonist was most likely the result of suppression of PAF-mediated reactions in the cornea. These results indicate that PAF may play a role in corneal inflammation and vascularization after corneal transplantation, and that PAF antagonists may be clinically useful in delaying some of the pathophysiologic consequences of corneal graft rejection.