Kyu Yeon Han

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.

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.

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.

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.

In Vivo Serial Imaging of Regenerating Corneal Nerves after Surgical Transection in Transgenic Thy1-YFP mice

PURPOSE To determine the effect of lamellar transection surgery on the nerve fiber density (NFD) and pattern of nerve regeneration in the cornea of thy1-YFP transgenic mice. METHODS Wide-field stereo fluorescence microscopy was used to obtain serial images of nerves in live thy1-YFP mice, which express a fluorescent protein in their axons. NFD (mm/mm(2)) was calculated from maximum intensity projection images as the total length of fibers within the area of the contour in which nerves were traced. Whole-mount confocal microscopy was performed to analyze the arrangement of nerves and the types of regenerating fibers. RESULTS NFD in normal corneas was 35.3 ± 1.8 mm/mm(2). Stereo fluorescence microscopy revealed the presence of a subbasal hairpin nerve layer and an intrastromal nerve trunk layer. After surgery, regenerative sprouting was observed from transected distal ends of intrastromal nerve trunks. NFD also increased, with this increase being maximal between 4 and 6 weeks after surgery. NFD approximated baseline values at 6 weeks and did not change any further at 8 weeks. Regenerated nerves did not readopt the normal corneal nerve arrangement. A dense interlacing network of regenerated nerves was present in the corneal bed. Branches from this network traversed the flap to innervate the epithelium. Immunofluorescence staining revealed that regenerating fronds contained peptidergic nociceptive fibers (positive for calcitonin gene-related peptide and substance P) and myelinated non-nociceptive fibers (positive for neurofilament 200). CONCLUSIONS Although corneal NFD recovers to normal levels by 8 weeks after nerve transection, the arrangement of regenerated nerves is abnormal.

Evidence for the involvement of MMP14 in MMP2 processing and recruitment in exosomes of corneal fibroblasts

PURPOSE Matrix metalloproteinase (MMP) 14 has been shown to promote angiogenesis, but the underlying mechanisms are poorly understood. In this study, we investigated exosomal transport of MMP14 and its target, MMP2, from corneal fibroblasts to vascular endothelial cells as a possible mechanism governing MMP14 activity in corneal angiogenesis. METHODS We isolated MMP14-containing exosomes from corneal fibroblasts by sucrose density gradient and evaluated exosome content and purity by Western blot analysis. We then investigated exosome transport in vitro from corneal fibroblasts to two populations of vascular endothelial cells, human umbilical vein endothelial cells (HUVECs) and calf pulmonary artery endothelial cells (CPAECs). Western blot analysis and gelatin zymography were used to determine levels of MMP14 and MMP2, respectively, in exosomal fractions derived from cultured wild-type, MMP14 enzymatic domain-deficient (MMP14Δexon4), and MMP14-null corneal fibroblasts. RESULTS Matrix metalloproteinase 14-containing exosomes isolated from corneal fibroblasts were readily taken up in vitro by HUVECs and CPAECs. We found that MMP14 was enriched in exosomal fractions of cultured corneal fibroblasts. Moreover, loss of the MMP14 enzymatic domain resulted in accumulation of pro-MMP2 protein in exosomes, whereas MMP2 was nearly undetectable in exosomes of MMP14-null fibroblasts. CONCLUSIONS Our results indicate that exosomes secreted by corneal fibroblasts can transport proteins, including MMP14, to vascular endothelial cells. In addition, recruitment of MMP2 into corneal fibroblast exosomes is an active process that depends, at least in part, on the presence of MMP14. The role of exosomal MMP14 transport in corneal angiogenesis has important implications for therapeutic applications targeting angiogenic processes in the cornea.

Characterization of the Interaction Between Endostatin Short Peptide and VEGF Receptor 3

Corneal angiogenesis and lymphangiogenesis are induced by vascular endothelial growth factors (VEGFs) signaling through its receptors VEGFR-1, -2, and -3. Endostatin is a peptide antagonist of these receptors that causes inhibition of bFGF-induced corneal angiogenesis and lymphangiogenesis. Here we show that binding of VEGF-C and endostatin to recombinant VEGFR-3 is competitive. Alignments of the primary amino acid sequences of VEGF-C and the C-terminal endostatin peptide (mEP: LEQKAASCHNSYIVLCIENSFMTSFSK) identified two conserved cysteine residues separated by seven amino acids. Peptides of VEGF-C and mEP containing these conserved residues bound to VEGFR-3. However, substitution of alanine for either of the cysteines in the mEP peptide perturbed the secondary structure, and this mutated peptide was unable to bind to VEGFR-3. Analysis by surface plasmon resonance demonstrated that the binding of the mEP peptide for recombinant VEGFR-3 had a Ka of 1.41 x 10⁷ M⁻¹ s⁻¹, Kd of 0.6718 s⁻¹, and a KD of 4.78 x 10⁻⁸ M. Characterization of the mechanism of endostatin binding to VEGFR-3 may lead to the development of novel therapies for lymphangiogenesis-related disorders, such as transplant rejection, lymphedema, and cancer metastasis.

Semaphorin 7A promotes angiogenesis in an experimental corneal neovascularization model

Purpose: To characterize the involvement of Semaphorin 7A (Sema7a) in corneal neovascularization (NV). Methods: We generated anti-Sema7A antibodies to detect protein expression in corneal fibroblasts. Corneal fibroblast cells were cultured, stimulated with basic fibroblast growth factor (bFGF or FGF-2), immunostained with anti-Sema7A antibodies, and visualized by confocal microscopy. bFGF pellets were implanted in mouse corneal micropockets for 3–10 days, and corneal sections were immunostained with anti-Sema7A antibodies. Mouse corneas were injected with a Sema7A expression vector or a control vector for 3, 7, and 10 days. Mouse corneas were imaged by slit lamp microscopy, and areas of corneal NV were calculated using the ImageJ program. Mouse corneal sections were also immunostained with anti-macrophage marker (F4/80) and anti-vascular endothelial growth factor (VEGF)-A antibodies. Results: Our data showed enhanced Sema7A expression levels in bFGF-stimulated cultured corneal fibroblasts. bFGF corneal implantation also demonstrated enhanced Sema7A expression. Corneas injected with a Sema7A expression vector showed evidence of significant corneal NV compared to controls on day 10 (1.8 mm2 vs. 0.11 mm2; p < 0.02). Additionally, immunolocalization of Sema7A expression vector-injected corneas (at day 7) revealed macrophage recruitment and enhanced VEGF-A levels. Conclusions: We demonstrated that Sema7A was expressed in vascularized corneas and showed pro-angiogenic properties in our corneal model. Understanding the mechanism of Sema7A in angiogenesis may provide a therapeutic target for the treatment of corneal angiogenesis-related disorders.

Simultaneous in vivo imaging of blood and lymphatic vessel growth in Prox1–GFP/Flk1::myr–mCherry mice

The ability to visually observe angiogenesis and lymphangiogenesis simultaneously and repeatedly in living animals would greatly enhance our understanding of the inter‐dependence of these processes. To generate a mouse model that allows such visualization via in vivo fluorescence imaging, we crossed Prox1–GFP mice with Flk1::myr–mCherry mice to generate Prox1–GFP/Flk1::myr–mCherry mice, in which lymphatic vessels emit green fluorescence and blood vessels emit red fluorescence. Corneal neovascularization was induced in these mice using three injury models: implantation of a vascular endothelial growth factor (VEGF) pellet, implantation of a basic fibroblast growth factor (bFGF) pellet, and alkali burn injury. Vessel growth was observed in vivo by stereomicroscopy on days 0, 3, 7 and 10 after pellet implantation or alkali injury as well as in flat‐mounted corneas via confocal microscopy after the final in vivo imaging time point. We observed blood and lymphatic vessel growth in all three models, with the most significant growth occurring from days 0–7. Upon VEGF stimulation, the growth kinetics of blood and lymphatic vessels were similar. Blood vessels exhibited similar growth patterns in VEGF‐ and bFGF‐stimulated corneas. Alkali burn injury induced robust angiogenesis and lymphangiogenesis. The intrinsic fluorescence of blood and lymphatic endothelial cells in Prox1–GFP/Flk1::myr–mCherry mice permitted simultaneous in vivo imaging of angiogenesis and lymphangiogenesis. This allowed us to differentiate the processes as well as observe their inter‐dependence, and will be valuable in development of therapies targeting angiogenesis and/or lymphangiogenesis.