Ryoji Yanai

Cell–matrix and cell–cell interactions during corneal epithelial wound healing

The corneal epithelium serves as a barrier and contributes to the maintenance of corneal transparency and rigidity. In most instances, corneal epithelial defects caused by simple injury are resurfaced promptly. However, in individuals with certain clinical conditions, such as herpes simplex virus infection, neurotrophic keratopathy or diabetic keratopathy, corneal epithelial defects persist and do not respond to conventional treatment regimens because of delayed epithelial wound healing. After the corneal epithelium is removed by injury, the remaining epithelial cells migrate over the denuded surface of the cornea in a manner that is dependent both on the interaction of the cells with the underlying substrate and on cell-cell adhesion. In this review, we describe the specific roles of cell-matrix and cell-cell interactions during the course of corneal epithelial wound healing. The clinical implications of the basic research findings are also discussed.

Open clinical study of eye-drops containing tetrapeptides derived from substance P and insulin-like growth factor-1 for treatment of persistent corneal epithelial defects associated with neurotrophic keratopathy

Background/aims: Loss of corneal sensation results in the development of persistent corneal epithelial defects. The combination of a substance P-derived peptide (FGLM-amide) and an insulin-like growth factor-1 (IGF-1)-derived peptide (SSSR) stimulates rabbit corneal epithelial migration in vitro and rabbit corneal epithelial wound closure in vivo. The clinical efficacy of eye-drops containing FGLM-amide and SSSR for the treatment of persistent corneal epithelial defects in individuals with neurotrophic keratopathy was examined in a prospective open study. Methods: Twenty-five consecutive patients (26 eyes) with persistent corneal epithelial defects associated with neurotrophic keratopathy were treated by administration of eye-drops containing FGLM-amide and SSSR. The course of epithelial healing was monitored by slit-lamp examination. Results: Epithelial defects resurfaced completely in 19 of the 26 eyes (73%) within 4 weeks after treatment initiation. Complete resurfacing of epithelial defects was apparent in 18 of 22 (82%) or in one of four (25%) eyes without or with limbal stem cell deficiency, respectively. No adverse effects of treatment were observed in any subject. Conclusion: Eye-drops containing FGLM-amide and SSSR induced the rapid resurfacing of persistent epithelial defects in stem cell-positive individuals with neurotrophic keratopathy.

Cytokine, Chemokine, and Adhesion Molecule Expression Mediated by MAPKs in Human Corneal Fibroblasts Exposed to Poly(I:C)

PURPOSE Polyinosinic-polycytidylic acid [poly(I:C)], an analog of viral double-stranded RNA, interacts with Toll-like receptor (TLR)-3 and thereby elicits immunoinflammatory responses characteristic of viral infection. The effects of poly(I:C) on the expression of proinflammatory cytokines, chemokines, and adhesion molecules, as well as the signaling pathways that underlie such effects, were investigated in cultured human corneal fibroblasts. METHODS Expression of the adhesion molecules intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 was evaluated by immunoblot and immunofluorescence analyses. Release of the proinflammatory cytokine IL-6 and of the chemokines interleukin (IL)-8, granulocyte colony-stimulating factor (G-CSF), macrophage inflammatory protein (MIP)-1beta, eotaxin, and RANTES was measured with assay kits. Subcellular localization of the p65 subunit of the transcription factor nuclear factor (NF-kappaB) was examined by immunofluorescence analysis. Expression of TLR3, phosphorylation (activation) of mitogen-activated protein kinases (MAPKs), and phosphorylation and degradation of the NF-kappaB-inhibitory protein IkappaB-alpha was assessed by immunoblot analysis. RESULTS Poly(I:C) induced the up-regulation of TLR3, the release of IL-6, IL-8, G-CSF, MIP-1beta, eotaxin, and RANTES, and the expression of ICAM-1 and VCAM-1 in corneal fibroblasts. It also activated the MAPKs ERK, p38, and JNK and induced the phosphorylation and degradation of IkappaB-alpha and the nuclear translocation of p65 in these cells. Poly(I:C)-induced expression of IL-6, IL-8, G-CSF, MIP-1beta, exotaxin, RANTES, and ICAM-1 was inhibited differentially by the MAPK inhibitors PD98059 and SB203580 and by JNK inhibitor II. CONCLUSIONS Poly(I:C) induces the up-regulation of TLR3, the MAPK-dependent expression of proinflammatory cytokines, chemokines, and adhesion molecules and the activation of NF-kappaB in human corneal fibroblasts. Corneal fibroblasts may thus play an important role in the modulation of local immune and inflammatory responses to viral infection in the corneal stroma.

Persistent epithelial defects due to neurotrophic keratopathy treated with a substance p-derived peptide and insulin-like growth factor 1.

PurposeThe loss of corneal sensation results in the development of persistent corneal epithelial defects. The combination of a substance P-derived peptide, phenylalanine -glycine-leucine-methionine (FGLM)-amide, and insulin-like growth factor 1 (IGF-1) stimulates corneal epithelial migration in vitro and corneal epithelial wound closure in vivo. The clinical efficacy of eye drops containing FGLM-amide and IGF-1 for the treatment of persistent epithelial defects in individuals with neurotrophic keratopathy was examined in a prospective open study.MethodsEleven patients with persistent corneal epithelial defects associated with neurotrophic keratopathy were treated for up to 28 days by the administration of eye drops containing FGLM-amide and IGF-1. The course of epithelial healing was monitored by slit-lamp examination, and visual acuity was measured before and after treatment.ResultsComplete epithelial resurfacing was achieved in eight of the nine (89%) cases of persistent epithelial defects in the nine patients who received the full course of treatment. Epithelial defects had completely resurfaced in two of nine (22%) and five of nine (56%) cases within 1 and 2 weeks, respectively, after treatment initiation. No adverse effects of treatment were observed in any of the 11 patients.ConclusionsEye drops containing FGLM-amide and IGF-1 induced the rapid resurfacing of persistent epithelial defects in individuals with neurotrophic keratopathy. Jpn J Ophthalmol 2007;51:442–447

Cytochrome P450-generated metabolites derived from ω-3 fatty acids attenuate neovascularization

Significance The ω-3 long-chain polyunsaturated fatty acids are a class of dietary lipids that are highly enriched in the central nervous system and the retina. We demonstrate that dietary enrichment with ω-3s suppresses choroidal neovascularization in a mouse model of age-related macular degeneration (AMD), a leading cause of blindness. The ω-3s have anti-inflammatory properties and compete with ω-6s for downstream lipid metabolite synthesis at the cytochrome P450 (CYP) level. Specifically, 17,18- epoxyeicosatetraenoic acid and 19,20- epoxydocosapentaenoic acid derived from the CYP pathway were identified by liquid chromatography-tandem MS and found to confer protection. Systemic immune-cell recruitment and adhesion-molecule regulation were dampened significantly in mice receiving ω-3s. These findings provide a unique mechanism whereby specific CYP-derived lipid metabolites regulate angiogenesis in a mouse model of AMD. Ocular neovascularization, including age-related macular degeneration (AMD), is a primary cause of blindness in individuals of industrialized countries. With a projected increase in the prevalence of these blinding neovascular diseases, there is an urgent need for new pharmacological interventions for their treatment or prevention. Increasing evidence has implicated eicosanoid-like metabolites of long-chain polyunsaturated fatty acids (LCPUFAs) in the regulation of neovascular disease. In particular, metabolites generated by the cytochrome P450 (CYP)–epoxygenase pathway have been shown to be potent modulators of angiogenesis, making this pathway a reasonable previously unidentified target for intervention in neovascular ocular disease. Here we show that dietary supplementation with ω-3 LCPUFAs promotes regression of choroidal neovessels in a well-characterized mouse model of neovascular AMD. Leukocyte recruitment and adhesion molecule expression in choroidal neovascular lesions were down-regulated in mice fed ω-3 LCPUFAs. The serum of these mice showed increased levels of anti-inflammatory eicosanoids derived from eicosapentaenoic acid and docosahexaenoic acid. 17,18-epoxyeicosatetraenoic acid and 19,20-epoxydocosapentaenoic acid, the major CYP-generated metabolites of these primary ω-3 LCPUFAs, were identified as key lipid mediators of disease resolution. We conclude that CYP-derived bioactive lipid metabolites from ω-3 LCPUFAs are potent inhibitors of intraocular neovascular disease and show promising therapeutic potential for resolution of neovascular AMD.

Upregulation of Tight-Junctional Proteins in Corneal Epithelial Cells by Corneal Fibroblasts in Collagen Vitrigel Cultures

PURPOSE To investigate the effects of corneal fibroblasts on the differentiation of corneal epithelial cells in a coculture system based on a collagen vitrigel membrane. METHODS Simian virus 40-transformed human corneal epithelial (HCE) cells and human corneal fibroblasts were cultured on opposite sides of a collagen vitrigel membrane. The distribution of HCE cells and corneal fibroblasts on the collagen membrane was determined by immunofluorescence staining and immunoblot analysis of marker proteins. Expression of the tight-junctional proteins ZO-1, occludin, and claudin and of the adherens-junctional proteins E- and N-cadherin in HCE cells was determined at the mRNA and protein levels by reverse transcription-polymerase chain reaction analysis and immunoblot analysis, respectively. RESULTS The abundance of ZO-1, occludin, and claudin mRNA and proteins in HCE cells was markedly increased by coculture with corneal fibroblasts. The expression of E- or N-cadherin did not differ between HCE cells cultured with corneal fibroblasts and those cultured without them. PD98059, a specific inhibitor of signaling by extracellular signal regulated kinase (ERK), prevented the upregulation of tight-junctional proteins in HCE cells by corneal fibroblasts. CONCLUSIONS Human corneal fibroblasts regulated the expression of tight-junctional proteins in HCE cells, suggesting that corneal fibroblasts may play an important role in the differentiation of corneal epithelial cells.

Complement Involvement in Neovascular Ocular Diseases

Pathological neovascularization (NV) is a hallmark of late stage neovascular age-related macular degeneration (AMD), diabetic retinopathy (DR), and retinopathy of prematurity (ROP). There is accumulating evidence that alterations in inflammatory and immune system pathways that arise from genetic differences, injury, and disease can predispose individuals to retinal neovascular eye diseases. Yet the mechanism of disease progression with respect to the complement system in these maladies is not fully understood. Recent studies have implicated the complement system as an emerging player in the etiology of several retinal diseases. We will summarize herein several of the complement system pathways known to be involved in ocular neovascular pathologies. Current treatment for many neovascular eye diseases focuses on suppression of NV with laser ablation, photodynamic therapy, or anti-VEGF angiogenic inhibitors. However, these treatments do not address the underlying cause of many of these diseases. A clear understanding of the cellular and molecular mechanisms could bring a major shift in our approach to disease treatment and prevention.

Effects of antiglaucoma drugs on collagen gel contraction mediated by human corneal fibroblasts.

PurposeMeasurement of intraocular pressure is affected by the shape and thickness of the cornea, and corneal shape is thought to be maintained by contraction of corneal fibroblasts. The effects of the antiglaucoma drugs latanoprost, timolol maleate, and pilocarpine on the contraction of corneal fibroblasts cultured in a 3-dimensional collagen gel were investigated. The effects of these drugs on collagen degradation by corneal fibroblasts and their possible cytotoxicity were also examined. Materials and MethodsHuman corneal fibroblasts were cultured in a 3-dimensional gel of type I collagen and in the presence of various concentrations of latanoprost, timolol maleate, or pilocarpine for various times. Collagen gel contraction was evaluated by daily measurement of gel diameter. The extent of collagen degradation was determined by measurement of the amount of hydroxyproline generated by acid-heat hydrolysis of culture supernatants. The release of lactate dehydrogenase from corneal fibroblasts was determined as an index of drug cytotoxicity. ResultsLatanoprost stimulated collagen gel contraction mediated by corneal fibroblasts in a concentration- and time-dependent manner, whereas timolol maleate and pilocarpine had no such effect. None of the 3 drugs affected collagen degradation by corneal fibroblasts or exhibited cytotoxicity at concentrations as high as 100 μM. ConclusionsAmong the antiglaucoma drugs examined, only latanoprost stimulated collagen gel contraction mediated by human corneal fibroblasts. This action of latanoprost might affect corneal shape and thereby influence measurement of intraocular pressure.

Up-regulation of ZO-1 expression and barrier function in cultured human corneal epithelial cells by substance P

The effects of the sensory neurotransmitter substance P on the expression of tight junction proteins and on barrier function in human corneal epithelial cells were investigated. The expression of ZO‐1, but not that of occludin or claudin‐1, was increased by substance P in a concentration‐ and time‐dependent manner. This effect was inhibited by the NK‐1 receptor antagonist GR82334 and by KN62, an inhibitor of Ca2+‐ and calmodulin‐dependent protein kinase II. Substance P also increased the transepithelial electrical resistance of a cell monolayer in a manner sensitive to GR82334. Substance P may therefore play a role in maintenance of tight junctions in the corneal epithelium.

Up-regulation of semaphorin 3A in human corneal fibroblasts by epidermal growth factor released from cocultured human corneal epithelial cells

Semaphorins are a family of glycoproteins that play an important role in repulsive axon guidance during embryogenesis. We have now investigated the effect of corneal epithelial cells on the expression of Sema3A in corneal fibroblasts with the use of a coculture system in which the two cell types are separated by a collagen vitrigel membrane. Reverse transcription-polymerase chain reaction and immunoblot analyses revealed that the presence of immortalized human corneal epithelial (HCE) cells increased the expression of Sema3A in human corneal fibroblasts at both the mRNA and protein levels. This effect of HCE cells was mimicked by recombinant human epidermal growth factor (EGF) in a concentration- and time-dependent manner. An inhibitor of the tyrosine kinase activity of the EGF receptor, PD153035, blocked the EGF-induced up-regulation of both Sema3A mRNA and protein in corneal fibroblasts. Depletion of EGF in HCE cells by RNA interference largely abolished the effect of these cells on Sema3A expression in corneal fibroblasts. These findings indicate that EGF released from corneal epithelial cells up-regulates the expression of Sema3A in corneal fibroblasts. This effect of EGF may play an important role in maintenance of corneal structure and repair of corneal damage.