Takayoshi Sumioka

Fibrotic disorders in the eye: targets of gene therapy.

Fibrotic diseases, e.g., cutaneous and corneal scarring, keloids, and liver and lung fibrosis, etc., are characterized by appearance of myofibroblasts, the key player of the fibrogenic reaction, and excess accumulation of extracellular matrix with resultant tissue contraction and impaired functions. Inflammatory/fibrogenic growth factors/cytokines produced by injured tissues play a pivotal role in fibrotic tissue formation. Ocular tissues are also susceptible to fibrotic diseases. In this article, the pathogenesis of such fibrotic disorders in the eye, i.e., scarring in the cornea and conjunctiva, post-cataract surgery fibrosis of the lens capsule and proliferative vitreoretinopathy are reviewed. Focus is put on the roles of myofibroblast and signals activated by the fibrogenic cytokine, transforming growth factor beta. Modulation of signal transduction molecules, e.g., Smad and mitogen-activated protein kinases, by gene transfer and other technology is beneficial and can be an important treatment regiment to overcome (prevent or treat) these diseases.

Epithelial-mesenchymal transition as a therapeutic target for prevention of ocular tissue fibrosis.

Fibrotic diseases are characterized by the appearance of myofibroblasts, the key cell type involved in the fibrogenic reaction, and by excess accumulation of extracellular matrix with resultant tissue contraction and impaired function. Myofiborblasts are generated by fibroblast-myofibrobalst conversion, and in certain tissues through epithelial-mesenchymal transition (EMT), a process through which an epithelial cell changes its phenotype to become more like a mesenchymal cell. Although inflammatory/fibrogenic growth factors/cytokines produced by injured tissues orchestrate the process of EMT, transforming growth factor beta (TGFbeta) is believed to play a central role in the process. Unlike fibrotic lesions in kidney or other tissues where myofibroblasts are generated from both fibroblasts and epithelial cells, fibrotic lesions in the eye crystalline lens are derived only from lens epithelial cells without contamination of fibroblast-derived myofibroblasts. Thus, this tissue is suitable to investigate detailed mechanisms of EMT and subsequent tissue fibrosis. EMT in retinal pigment epithelium is involved in the development of another ocular fibrotic disease, proliferative vitreoretinopathy, a fibrosis in the retina. EMT-related signal transduction cascades, i. e., TGFbeta/Smad, are a target to prevent or treat unfavorable ocular tissue fibrosis, e. g., fibrotic diseases in the crystalline lens or retina, as well as possibly in other organs.

Impaired cornea wound healing in a tenascin C-deficient mouse model.

We investigated the effects of loss of tenascin C on the healing of the stroma using incision-injured mice corneas. Tenascin C was upregulated in the stroma following incision injury to the cornea. Wild-type (WT) and tenascin C-null (knockout (KO)) mice on a C57BL/6 background were used. Cell culture experiments were also conducted to determine the effects of the lack of tenascin C on fibrogenic gene expression in ocular fibroblasts. Histology, immunohistochemistry and real-time reverse transcription PCR were employed to evaluate the healing process in the stroma. The difference in the incidence of wound closure was statistically analyzed in hematoxylin and eosin-stained samples between WT and KO mice in addition to qualitative observation. Healing of incision injury in corneal stroma was delayed, with less appearance of myofibroblasts, less invasion of macrophages and reduction in expression of collagen Iα1, fibronectin and transforming growth factor β1 (TGFβ1) in KO mice compared with WT mice. In vitro experiments showed that the loss of tenascin C counteracted TGFβ1 acceleration of mRNA expression of TGFβ1, and of collagen Iα1 and of myofibroblast conversion in ocular fibroblasts. These results indicate that tenascin C modulates wound healing-related fibrogenic gene expression in ocular fibroblasts and is required for primary healing of the corneal stroma.

Transforming Growth Factor β Signal Transduction: A Potential Target for Maintenance/Restoration of Transparency of the Cornea

Maintenance of the transparency and regular shape of the cornea are essential to the normal vision, whereas opacification of the tissue impairs vision. Fibrogenic reaction leading to scarring in an injured cornea is characterized by appearance of myofibroblasts, the key player of the fibrogenic reaction, and excess accumulation of fibrous extracellular matrix. Inflammatory/fibrogenic growth factors/cytokines produced by inflammatory cells play a pivotal role in fibrogenic response. Signaling systems involved in myofibroblast formation and fibrogenesis are activated by various growth factors, i.e., transforming growth factor &bgr; or others. Modulation of transforming growth factor &bgr; signal transduction molecules, e.g., Smad and mitogen-activated protein kinases, by gene transfer and other technology provides a new concept of prevention/treatment of unfavorable fibrogenesis in the cornea.

Impairment of Corneal Epithelial Wound Healing in a TRPV1-Deficient Mouse

PURPOSE To examine whether the absence or blockage of an ion channel receptor, transient receptor potential vanilloid subtype 1 (TRPV1), affects the healing of an epithelial injury using an experimental model of an epithelial defect in animal cornea. METHODS The expression of TRPV1 in the corneal epithelium was examined using immunohistochemistry in mice and rats. The migration of the corneal epithelium was examined in epithelium-debrided rat cornea in organ culture in the presence or absence of a TRPV1 agonist or its antagonist. Epithelial migration and cell proliferation following the debridement were examined in the cornea of a TRPV1-null mouse. Real-time RT-PCR was performed in samples of healing corneas to analyze the expression pattern of epithelial migration-related components (i.e., IL-6, substance P, and TGF-β1). RESULTS TRPV1 was detected mainly in the basal layer of mouse or rat corneal epithelium. Adding a TRPV1 receptor agonist to the culture medium enhanced epithelial healing in the rat cornea, and a TRPV1 antagonist retarded it in organ culture. The loss of TRPV1 did not affect the histology of the mouse cornea. In vivo analysis showed the loss of TRPV1-impaired re-epithelialization of the debrided area of the corneal epithelium by the suppression of both cell migration and proliferation. The lack of TRPV1 suppressed the expression of IL-6 and substance P but not of TGF-β1 in response to epithelial debridement in mice. CONCLUSIONS TRPV1 signal is required for the upregulation of IL-6 and substance P and the healing of debrided corneal epithelium in mice.

Expression of cyclooxygenase-2 in corneal cells after photorefractive keratectomy and laser in situ keratomileusis in rabbits

Purpose: To compare the expression pattern of cyclooxygenase‐2 (COX‐2) in rabbit corneal cells after photorefractive keratectomy (PRK) and laser in situ keratomileusis (LASIK) with the same refractive correction. Setting: Department of Ophthalmology, Wakayama Medical University, Wakayama, Japan. Methods: Thirty adult albino rabbits were used in the study. Photorefractive keratectomy or LASIK was performed in 1 eye of each animal for the same refractive correction. Each animal was killed after healing intervals up to 6 months. Paraffin sections of the cornea were processed for immunohistochemistry for COX‐2 and NFκB (p65). Results: After PRK, the central and peripheral corneal epithelia up‐regulated COX‐2 at 3 days; the central epithelium was positive at 4 weeks. Central and peripheral epithelia returned to negative 3 months later. After LASIK, the central epithelium on the corneal flap up‐regulated COX‐2 at 1 and 2 weeks; it returned to negative at 4 weeks. The peripheral epithelium was labeled with the antibody. Keratocytes around the stromal incision between the flap and the stromal bed up‐regulated COX‐2 and returned to negative at 3 months. COX‐1 was not detected immunohistochemically in corneal tissue during the healing intervals after both procedures. Nuclear factor κB was detected in the cytoplasm and nuclei of migrating corneal epithelial cells 1 day after PRK, was positive in the cytoplasm at 3 days and negative in cytoplasm and nuclei at week and later. Conclusions: Migrating injured epithelium expressed COX‐2 until week 4 during post‐PRK healing. Central uninjured epithelium as well as stromal keratocytes expressed COX‐2 from 3 days to 2 weeks after LASIK. Uninjured peripheral epithelium also expressed COX‐2 at 4 weeks. Activation of stromal keratocytes may induce expression of COX‐2 in overlying uninjured epithelium via the inflammatory cytokine(s)/NFκB pathway.

Impaired angiogenic response in the cornea of mice lacking tenascin C.

PURPOSE This study investigated the effects of loss of tenascin C (TNC) in the development of neovascularization in a corneal stroma in mice. Cell culture study was also conducted to clarify the roles of TNC in the expression of vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)β1 in fibroblasts and macrophages. METHODS Ocular fibroblasts and macrophages from wild-type (WT) and TNC-null (KO) mice were used to study the role of TNC in the expression of VEGF and TGFβ1. The effects of the absence of TNC on angiogenic gene expression, inflammatory cell invasion, and cornea neovascularization in the corneal stroma were then evaluated after cauterization of the center of the cornea in mice. Histologic, immunohistochemical, and mRNA expression analyses were performed. RESULTS Absence of TNC suppressed expression of VEGF and counteracted upregulation of TGFβ1 by exogenous TGFβ1 in ocular fibroblast culture. Such effects of the absence of TNC were not observed in cultured macrophages. Absence of TNC attenuated expression of both VEGF and TGFβ1 mRNA as well as neovascularization into the stroma after cauterization at the center of the cornea in mice. Absence of TNC suppressed macrophages, but not neutrophils, invading the cauterized cornea. CONCLUSIONS TNC is involved in angiogenic gene expression in ocular fibroblasts in vitro and in vivo and is required for macrophage invasion and neovascularization of injured corneal stroma.

Cyclooxygenase 2 expression in rat corneas after ethanol exposure

PURPOSE: To evaluate the effects of ethanol exposure of the cornea on inflammation in corneal epithelium. SETTING: Department of Ophthalmology, Wakayama Medical University, Wakayama, Japan. METHODS: One cornea of Wistar rats (n = 60) was exposed to ethanol 20% for 30 seconds. The animals were killed 0.5, 1.0, 1.5, 2.0, 6.0, 12.0, 24.0, 48.0, or 72.0 hours or 7 days after treatment. The paraffin section or cryosection of the treated eyes was processed for histology; immunohistochemistry for cyclooxygenase 2 (COX2); p65 subunit of nuclear factor kappa B (NF‐κB), which is the major transcription factor involved in COX2 expression; phospho‐IκB; or in situ hybridization for COX2 mRNA. RESULTS: In the uninjured corneas, faint immunoreactivity for COX2 was detected in the basal cells of the corneal epithelium, but not in other cell layers. Cyclooxygenase 2 mRNA was not observed in the injured epithelium; it was expressed 2 hours after ethanol exposure, but not 3 hours or later after treatment. The COX2 protein was detected in the corneal epithelium throughout the epithelial layers from 3 to 72 hours, but not at 7 days. The p65 of NF‐κB translocated to the nuclei of corneal epithelium 3 to 24 hours after treatment but was not seen in the nuclei 48 hours after treatment. Phospho‐I κB was detected in corneal epithelium 6 hours after treatment, but not 12 hours or later. CONCLUSION: Ethanol exposure activated NF‐κB and upregulated COX2 expression, which may cause inflammation in corneal tissue.

The role of extracellular matrix in corneal wound healing.

Abstract: During wound healing, cell migration, proliferation, differentiation, and enhanced biosynthetic activities are observed at the sites of injury. Interactions between extracellular matrix (ECM) components and specific cell surface receptors, such as integrins, initiate cascades of signal transduction leading to various cellular responses. This review will focus on the biological functions of several ECM components, such as osteopontin (OPN), tenascin (TCN), and lumican (Lum). The loss of OPN or TCN reduces macrophage invasion and myofibroblast differentiation in the healing stroma and is associated with the suppression of fibrogenic gene expression in response to injury sustained in mice. Gene expression analysis showed that the lack of OPN or TCN resulted in the inhibition of proinflammatory and fibrogenic gene expression. It has been shown that Lum also modulates cell adhesion, migration, and proliferation, thus contributing to corneal epithelial wound healing. Interestingly, OPN-, TCN-, or Lum-null mice had reduced Smad activity and epithelial–mesenchymal transitions. Smads are nuclear effectors of transforming growth factor-&bgr; and regulate transcription. However, it is still not clear as to how individual ECM components initiate the signaling pathway. We hypothesized that a cell surface receptor for these ECM components may mediate the matrikine functions of Lum. To identify the specific receptor for Lum, we developed purified recombinant glutathione S-transferase–Lum. Using this, we identified a potential receptor for Lum that modulated wound healing.

Endothelial mesenchymal transition: a therapeutic target in retrocorneal membrane.

A single layer of corneal endothelial cells covers the posterior surface of the Descemet membrane. Normally, corneal endothelial cells do not proliferate, suggesting that factors inhibit their undergoing mitosis. Fibroblastic transformation of corneal endothelial cells due to syphilitic interstitial keratitis or alkali burn may result in stromal opacification. Various growth factors such as fibroblast growth factor 2 and transforming growth factor β are involved in “endothelial-mesenchymal transition (EnMT).” Endothelial wound-healing assay experiments revealed that cell migration toward artificial wound defect was mediated by p38 and c-Jun N-terminal kinase. To understand whether Smad signal might have an important role in EnMT regulation, gene transfer of Smad7 was employed to block transforming growth factor β/Smad signal in rat corneal endothelium 3 days before alkali burning. EnMT during healing interval after alkali burn was markedly suppressed by Smad7 overexpression associated with upregulation of cell proliferation. Therefore, blocking Smad signal effectively suppresses injury-induced EnMT and fibrogenic reaction by corneal endothelium without impairing repair of wound defect. Strategies that block Smad may be useful for prevention and treatment of fibrogenic disorders in the corneal endothelium.