Jin Hong Chang

Corneal Neovascularization: An Anti-VEGF Therapy Review

Corneal neovascularization is a serious condition that can lead to a profound decline in vision. The abnormal vessels block light, cause corneal scarring, compromise visual acuity, and may lead to inflammation and edema. Corneal neovascularization occurs when the balance between angiogenic and antiangiogenic factors is tipped toward angiogenic molecules. Vascular endothelial growth factor (VEGF), one of the most important mediators of angiogenesis, is upregulated during neovascularization. In fact, anti-VEGF agents have efficacy in the treatment of neovascular age-related macular degeneration, diabetic retinopathy, macular edema, neovascular glaucoma, and other neovascular diseases. These same agents have great potential for the treatment of corneal neovascularization. We review some of the most promising anti-VEGF therapies, including bevacizumab, VEGF trap, siRNA, and tyrosine kinase inhibitors.

Diminished corneal angiogenesis in gelatinase A‐deficient mice

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 mm2) was significantly less than that of wild‐type littermates (0.53±0.35 mm2; P<0.01). Similarly, aortic ring assays showed significant reduction of endothelial outgrowth in gelatinase A‐deficient mice (0.26±0.14 mm2) as compared to wild‐type littermates (0.44±0.06 mm2; P<0.05). These results suggest that gelatinase A may play an important role in the regulation of corneal angiogenesis.

Novel aspects of corneal angiogenic and lymphangiogenic privilege

In this article, we provide the results of experimental studies demonstrating that corneal avascularity is an active process involving the production of anti-angiogenic factors, which counterbalance the pro-angiogenic/lymphangiogenic factors that are upregulated during wound healing. We also summarize pertinent published reports regarding corneal neovascularization (NV), corneal lymphangiogenesis and corneal angiogenic/lymphangiogenic privilege. We outline the clinical causes of corneal NV, and discuss the angiogenic proteins (VEGF and bFGF) and angiogenesis regulatory proteins. We also describe the role of matrix metalloproteinases MMP-2, -7, and MT1-MMP, anti-angiogenic factors, and lymphangiogenic regulatory proteins during corneal wound healing. Established and potential new therapies for the treatment of corneal neovascularization are also discussed.

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 28 kDa 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.

Corneal Neurotoxicity Due to Topical Benzalkonium Chloride

PURPOSE The aim of this study was to determine and characterize the effect of topical application of benzalkonium chloride (BAK) on corneal nerves in vivo and in vitro. METHODS Thy1-YFP+ neurofluorescent mouse eyes were treated topically with vehicle or BAK (0.01% or 0.1%). Wide-field stereofluorescence microscopy was performed to sequentially image the treated corneas in vivo every week for 4 weeks, and changes in stromal nerve fiber density (NFD) and aqueous tear production were determined. Whole-mount immunofluorescence staining of corneas was performed with antibodies to axonopathy marker SMI-32. Western immunoblot analyses were performed on trigeminal ganglion and corneal lysates to determine abundance of proteins associated with neurotoxicity and regeneration. Compartmental culture of trigeminal ganglion neurons was performed in Campenot devices to determine whether BAK affects neurite outgrowth. RESULTS BAK-treated corneas exhibited significantly reduced NFD and aqueous tear production, and increased inflammatory cell infiltration and fluorescein staining at 1 week (P < 0.05). These changes were most significant after 0.1% BAK treatment. The extent of inflammatory cell infiltration in the cornea showed a significant negative correlation with NFD. Sequential in vivo imaging of corneas showed two forms of BAK-induced neurotoxicity: reversible neurotoxicity characterized by axonopathy and recovery, and irreversible neurotoxicity characterized by nerve degeneration and regeneration. Increased abundance of beta III tubulin in corneal lysates confirmed regeneration. A dose-related significant reduction in neurites occurred after BAK addition to compartmental cultures of dissociated trigeminal ganglion cells. Although both BAK doses (0.0001% and 0.001%) reduced nerve fiber length, the reduction was significantly more with the higher dose (P < 0.001). CONCLUSION Topical application of BAK to the eye causes corneal neurotoxicity, inflammation, and reduced aqueous tear production.

MT1-MMP-Mediated Cleavage of Decorin in Corneal Angiogenesis

Background/Aims: Decorin has been shown to have antiangiogenic properties. In this study, we evaluate the involvement of membrane type 1-matrix metalloproteinase (MT1-MMP), a proangiogenic enzyme, in decorin cleavage in the cornea. Methods: MT1-MMP expression was confirmed immunohistochemically in keratocytes and immortalized corneal fibroblast cell lines. Corneal micropockets of bFGF were used to assess the expression of decorin and MT1-MMP. Western blotting was used to evaluate decorin degradation by MT1-MMP. Aortic ring tube formation assays were used to assay the inhibitory effect of decorin and stimulatory effect of MT1-MMP on vascular endothelial cells in vitro. Results: We show that MT1-MMP expression is upregulated following bFGF pellet implantation in the cornea in vivo, and that MT1-MMP cleaves decorin in a time- and concentration-dependent manner in vitro. Furthermore, the addition of MT1-MMP reduces the inhibitory effects of decorin on aortic ring tube formation in vitro. Cleavage of decorin by MT1-MMP-deficient corneal cell lysates is diminished relative to that by wild-type corneal cell lysates, and an MT1-MMP knockin restores decorin processing in vitro. Conclusion: The proangiogenic role of MT1-MMP in the cornea may be mediated, in part, by facilitated cleavage of corneal decorin.

Neostatin‐7 regulates bFGF‐induced corneal lymphangiogenesis

Neostatin‐7, with an anti‐angiogenic potential, is generated from the proteolytic action of matrix metalloproteinase‐7 on collagen XVIII. We previously reported that neostatin‐7 inhibited angiogenesis in vitro and in vivo. Here we demonstrate that neostatin‐7/collagen XVIII may possess anti‐lymphangiogenic activities by: (1) corneal micropellet implantation of neostatin‐7 reduced bFGF‐induced corneal lymphangiogenesis; (2) neostatin‐7 bound to VEGF receptor‐3 in vitro; and (3) enhanced corneal lymphangiogenesis and VEGF‐C expression in collagen XVIII knockout mice in a corneal wounding model. Understanding the mechanism of neostatin‐7/collagen XVIII on corneal lymphangiogenesis may provide therapeutic interventions to treat lymphangiogenesis‐related disorders, such as lymphedema, transplantation rejection and cancers.

Membrane Type-1 Matrix Metalloproteinase Potentiates Basic Fibroblast Growth Factor-Induced Corneal Neovascularization

Corneal neovascularization is one of the leading causes of blindness. The aim of this study was to evaluate the pro-angiogenic role of corneal fibroblast-derived membrane type-1 matrix metalloproteinase (MT1-MMP) on basic fibroblast growth factor (bFGF)-induced corneal neovascularization in vivo and in vitro. Immunohistochemical studies demonstrated that MT1-MMP was expressed in keratocytes and immortalized corneal fibroblast cell lines. Vascular endothelial growth factor protein levels were increased after bFGF-stimulation of wild-type fibroblast cells compared with MT1-MMP knockout fibroblast cells. Corneal vascularization was significantly increased after a combination of bFGF pellet implantation and naked MT1-MMP DNA injection in wild-type mouse corneas compared with either bFGF pellet implantation or naked MT1-MMP DNA-injected corneas. Western blotting analysis of the phosphorylation levels of the key signaling molecules (p38, JNK, and ERK) demonstrated that phosphorylation levels of both p38 and JNK were diminished after bFGF stimulation of MT1-MMP knockout cells compared with wild-type and MT1-MMP knockin cells. These results suggest that MT1-MMP potentiates bFGF-induced corneal neovascularization, likely by modulating the bFGF signal transduction pathway.

Wound healing after keratorefractive surgery: review of biological and optical considerations.

Abstract: The introduction of the excimer laser for keratorefractive surgery in the 1990s permanently reshaped the treatment landscape for correcting refractive errors, such as myopia, hyperopia, and astigmatism. Until that point, these treatments had relied on less predictable techniques, such as radial keratotomy and automated lamellar keratectomy. In recent years, other new technologies, along with increased understanding of the basic science of refractive errors, higher-order aberrations, biomechanics, and the biology of corneal wound healing, have allowed for a reduction in the surgical complications of keratorefractive surgery. Novel technologies, such as eye tracking, anterior segment imaging, the femtosecond laser, and asphericity-optimized and wavefront-guided custom laser in situ keratomileusis, have assisted refractive surgeons in achieving greater predictability of their laser vision correction procedures. Understanding the cascade of events involved in the corneal wound healing process and examination of how corneal wound healing influences corneal biomechanics and optics are crucial to improve the efficacy and safety of laser vision correction.

VEGF TrapR1R2 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.