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The synthetic peptide of lumican C-terminal 13 amino acids with the cysteine replaced by an alanine, hereafter referred to as lumikine (LumC13 C-A : YEALRVANEVTLN), binds to TGFβ type I receptor/activin-like kinase5 (TBR1/ALK5) in the activated TGFβ receptor complex to promote corneal epithelial wound healing. The present study aimed to identify the minimum essential amino acid epitope necessary to exert the effects of lumikine via ALK5 and to determine the role of the Y (tyrosine) residue for promoting corneal epithelium wound healing. This study also aimed to determine the signaling pathway(s) triggered by lumican-ALK5 binding. For such, adult Lum knockout ( Lum -/- ) mice (~8-12 weeks old) were subjected to corneal epithelium debridement using an Agerbrush ® . The injured eyes were treated with 10 µL eye drops containing 0.3 µM synthetic peptides designed based on the C-terminal region of lumican for 5-6 h. To unveil the downstream signaling pathways involved, inhibitors of the Alk5 and EGFR signaling pathways were co-administered or not. Corneas isolated from the experimental mice were subjected to whole-mount staining and imaged under a ZEISS Observer to determine the distance of epithelium migration. The expression of EGFR ligands was determined following a scratch assay with HTCE (human telomerase-immortalized cornea epithelial cells) in the presence or not of lumikine. Results indicated that shorter LumC-terminal peptides containing EVTLN and substitution of Y with F in lumikine abolishes its capability to promote epithelium migration indicating that Y and EVTLN are essential but insufficient for Lum activity. Lumikine activity is blocked by inhibitors of Alk5, EGFR, and MAPK signaling pathways, while EGF activity is only suppressed by EGFR and MAPK inhibitors. qRT-PCR of scratched HTCE cells cultures treated with lumikine showed upregulated expression of several EGFR ligands including epiregulin (EREG). Treatment with anti-EREG antibodies abolished the effects of lumikine in corneal epithelium debridement healing. The observations suggest that Lum/lumikine binds Alk5 and promotes the noncanonical Smad-independent TGFβ/TBRs signaling pathways during the healing of corneal epithelium debridement.

Corneal scarring/fibrosis disturbs normal transparency and curvature of the tissue and thus impairs vision. The lesion is characterized by appearance of myofibroblasts, the key player of the fibrogenic reaction, and excess accumulation of extracellular matrix. Inflammatory/fibrogenic growth factors or cytokines expressed in inflammatory cells that infiltrate into injured tissues play a pivotal role in fibrotic tissue formation. In this article the pathogenesis of fibrosis/scarring in the corneal stroma is reviewed focusing on the roles of myofibroblast, the key player in corneal stromal wound healing and fibrosis, and cytoplasmic signals activated by the fibrogenic cytokine, transforming growth factor β (TGFβ). Although it is established that TGFβ/Smad signal is essential to the process of keratocyte-myofibroblast transformation in a healing corneal stroma post-injury. This article emphasizes the involvement of non-TGFβ molecular mechanisms in modulating Smad signal. We focus on the roles of matricellular proteins, i.e., osteopontin and tenascin C, and as cellular components, the roles of transient receptor potential (TRP) cation channel receptors are discussed. Our intent is to draw attention to the possibility of signal transduction cascade modulation (e.g., Smad signal and mitogen-activated protein kinases, by gene transfer and other related technology) as being beneficial in a clinical setting to reduce or even prevent corneal stromal tissue fibrosis/scarring and inflammation., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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 beta or others. Modulation of transforming growth factor beta 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.

Purpose: Injury-mediated corneal epithelial wound healing in vivo is mediated through different cell signaling pathways depending on whether or not the basement membrane is removed. Given this dependence, we ascertained if c-jun N-terminal kinase (JNK/SAPK) mitogen-activated protein kinase (MAPK) cell signaling mediates this response in vivo and in vitro, irrespective of the presence or absence of the basement membrane. Furthermore, in vitro the relative contribution was determined by the JNK/SAPK pathway to that of its p38 and ERK MAPK counterparts in mediating injury-induced increases in cell migration., Methods: Corneal epithelial debridement was performed in C57BL/6 mice and their organ-cultured eyes without removal of the basement membrane. In rabbits, following basement membrane removal by keratectomy, fluorescein-staining monitored reepithelialization was performed as in the mice. Immunohistochemistry evaluated changes in JNK phosphorylation status and localization. JNK inhibitor I and its inactive analogue determined if JNK signaling activation contributes to wound healing. BrdU staining assessed cell proliferation. A scratch wound assay of healing rates in SV40-immortalized human corneal epithelial cell line (HCEC) evaluated the relative contributions by p38 and ERK and JNK MAPK signaling activation to wound healing. A TUNEL assay probed for apoptosis after wound closure of HCEC. MTT assay evaluated corneal epithelial viability., Results: Two hours following mice corneal epithelial debridement, phospho-JNK was transiently upregulated in the nucleus, whereas total JNK was constitutively expressed. JNK inhibitor I suppressed epithelial spreading in organ-cultured mouse eyes and rabbit corneal blocks, irrespective of the presence or absence of basement membrane. No proliferation was detected at the wound edges. In HCEC, a p38 (SB203580) and a JNK pathway inhibitor (JNK inhibitor I) inhibited migration rates more than U0126-induced ERK, whereas the JNK inhibitor I inactive analogue had no effect. JNK pathway inhibition wound closure in this region was not associated with either any TUNEL or BrdU-positive cells. Cell viability was unaffected by any of these MAPK inhibitors., Conclusion: JNK/SAPK pathway activation stimulates wound healing in vitro and in vivo, irrespective of the presence or absence of the basement membrane. Therefore, studies on how wound closure is elicited in HCEC are relevant for identifying how MAPK signaling mediates this response in vivo and in organ-cultured eyes. This realization suggests that the JNK signaling system has a role in vivo that is intermediate to those of ERK and p38 in mediating increases in cell migration., (2009 S. Karger AG, Basel.)

Purpose: To understand the role of TGF-beta related signals in the repair of a corneal endothelium defect and also to evaluate the therapeutic effect of Smad7 gene transfer on injury induced fibrosis of the corneal endothelium in rats., Methods: (1) Japanese albino rabbits (n=108) were used. Blocks of central cornea (4 x 4 mm) were prepared. After partially scraping the endothelium to produce a defect, the blocks were organ cultured for 24 h in the presence of either exogenous growth factors, transforming growth factor beta (TGF-beta)-neutralizing antibody, or inhibitors of each TGF-beta related signal. Endothelium repair was assayed under light microscopy. (2) Adult Wistar rats (n=62) were then used. Smad7 expressing adenoviral vector (Smad7-Ad) or non-functioning control vector (Cre-Ad) was administered to the anterior chamber of an eye. The cornea was burned with topical 1 N NaOH (10 microl) three days later. After specific intervals, the eye was histologically observed., Results: (1) The endothelial layer that elongated toward the defect lacked proliferation after 24 h in organ culture. Endogenous TGF-beta was required for endothelium defect repair. Inhibition of p38 and Erk but not c-Jun NH(2)-terminal kinase (JNK) and ALK5 signal (Smad) retarded such cell spreading. (2) Adenoviral Smad7 overexpression suppressed fibrogenic reaction of the endothelium of an alkali-burned cornea as evaluated by immunohistochemistry for phospho-Smad2, collagen I, and alpha-smooth muscle actin, a marker for endothelial-mesenchymal transition (EnMT), and by electron microscopy., Conclusions: Inhibition of Smad and JNK signals do not affect corneal endothelium defect repair. Inhibition of Smad suppresses fibrogenic reaction via EnMT of corneal endothelium in vivo.

Wound healing involves both local cells and inflammatory cells. Alkali burn of ocular surface tissue is a serious clinical problem often leading to permanent visual impairment resulting from ulceration, scarring and neovascularization during healing. Behaviors of corneal cells and inflammatory cells are orchestrated by growth factor signaling networks that have not been fully uncovered. Here we showed that adenoviral gene introduction of peroxisome proliferator-activated receptor-gamma (PPARgamma) inhibits activation of ocular fibroblasts and macrophages in vitro and also induced anti-inflammatory and anti-fibrogenic responses in an alkali-burned mouse cornea. PPARgamma overexpression suppressed upregulation of inflammation/scarring-related growth factors and matrix metalloproteinases (MMPs) in macrophages. It also suppressed expression of such growth factors and collagen Ialpha2 and myofibroblast generation upon exposure to TGFbeta1. Exogenous PPARgamma did not alter phosphorylation of Smad2, but inhibited its nuclear translocation. PPARgamma overexpression enhanced proliferation of corneal epithelial cells, but not of fibroblasts in vitro. Epithelial cell expression of MMP-2/-9 and TGFbeta1 and its migration were suppressed by PPARgamma overexpression. In vivo experiments showed that PPARgamma gene introduction suppressed monocytes/macrophages invasion and suppressed the generation of myofibroblasts, as well as upregulation of cytokines/growth factors and MMPs in a healing cornea. In vivo re-epitheliazation with basement membrane reconstruction in the healing, burned, cornea was accelerated by PPARgamma-Ad expression, although PPARgamma overexpression was considered to be unfavorable for cell migration. Together, these data suggest that overexpression of PPARgamma may represent an effective new strategy for treatment of ocular surface burns.

Purpose: To evaluate the effect of cyclopamine, an inhibitor of the Sonic hedgehog (Shh) signal, on the growth of an epithelial neoplasm., Methods: Chemically induced eyelid tumors in XPC-null mice (n=40) were treated daily with a subcutaneous injection of cyclopamine (1 mg/animal) for 7 days. The animals were killed after bromodeoxyuridine (BrdU) labeling, and the tumors were histologically examined. An in vitro study was conducted by using a squamous cell carcinoma (SCC) cell line. The SCC cells were treated with 0, 12.5, or 25.0 microg/ml recombinant Shh (rShh) and either 0 or 100 microM cyclopamine, and cell proliferation was evaluated by using an MTT assay. Cells from this cell line were also implanted subcutaneously in nude mice (n=8) to develop tumors, and the effect of cyclopamine administration was examined in the developed tumors., Results: Histology showed that cyclopamine treatment suppressed BrdU incorporation and induced apoptosis in the majority of cells in tumors chemically induced in the eyelid of the XPC-null mice. Cell proliferation of the SCC cell line was enhanced by adding rShh, and this effect was abolished by adding cyclopamine. Proliferation of the SCC cell line was not affected by adding cyclopamine in the absence of rShh. On the other hand, the SCC cells expressed Shh in vivo in tumors developed in nude mice, but cyclopamine suppressed cell proliferation in the tumors, and the Shh-signaling pathway was inhibited by cyclopamine-induced apoptosis., Conclusions: Cyclopamine inhibits proliferation and induces apoptosis in epithelial tumor cells in vivo. The Shh-signaling pathway may be a potential therapeutic target for patients with eyelid tumors., (Copyright (c) Japanese Ophthalmological Society 2006.)

Purpose: We examined the role of interleukin-7 (IL-7) in modulation of production of extracellular matrix (ECM), immunolocalization of Smads, and cell migration and expressions of transforming growth factor-beta (TGF-beta) in cultured human subconjunctival fibroblasts. IL-7 is capable of inducing Smad7, an inhibitory Smad that interferes with TGF-beta/Smad signal., Methods: The effects of IL-7 on ECM production, immunolocalization of Smads, type I collagen, fibronectin, alpha -smooth muscle actin (alpha -SMA), and cell migration were examined in human subconjunctival fibroblast culture with or without TGF-beta1. ECM production, such as type I collagen and fibronectin, was measured by immunoassay or real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Cell migration was examined using an in vitro wound model in monolayer cultures. We also examined the effects of IL-7, PKC inhibitor, and STAT inhibitor on the expressions of TGF-beta1 and type I collagen alpha1 chain (col1A1) m-RNA by using real-time RT-PCR., Results: IL-7 reduced the ECM production much more markedly in the cells treated with TGF-1beta than in the control fibroblasts. TGF-beta1 strongly showed immunolocalization of phospho-Smad2, and IL-7 also showed immunolocalization of Smad7 in the nuclei. The immunoreactivities of alpha -SMA and fibronectin were weaker in the presence of IL-7 than in the control cells. IL-7 also delayed defect closure in the monolayer cell sheets, and the delay was recovered by exogenous type I collagen or fibronectin. Each of IL-7, BIS I, or AGS 490 reduced the mRNA expressions of TGF-beta1 and col1A1., Conclusions: These findings indicate that IL-7 is involved in ECM production in the subconjunctival fibroblasts activated by exogenous TGF-beta1, suggesting that administration of IL-7 can be a novel therapeutic strategy in preventing undesirable bleb scar formation during healing after filtration surgery.

Cell proliferation and related cellular behavior in ocular neoplastic disease and in the healing process in ocular surgery or post-injury management, as well as new treatment strategy were investigated. The roles of growth factors and their signal transduction pathways were studied. Cell proliferation-related signals were found to be activated to a greater extent in malignant ocular tumors than in benign tumor cells regardless of the similarity of simple histological findings. Suppression of cell proliferation-related signals can be a new treatment for ocular neoplastic diseases. The causes of complications associated with tissue repair response include acceleration of cell proliferation and extracellular matrix expression and cellular phenotypic alteration, i. e., epithelial-mesenchymal transition. These cellular activities can be controlled by modulation of growth factor signaling by employing such strategy including gene introduction.

Abstract: 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 β. 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. [Copyright &y& Elsevier]