Immunohistochemical Findings of Lens Capsules Obtained from Dead Bag Syndrome Patients.

PURPOSE: To investigate the extracellular matrix and cellular components in lens capsules extracted from patients with dead bag syndrome (DBS) through immunohistochemistry. SETTING: Department of Ophthalmology, Wakayama Medical University School of Medicine and Department of Ophthalmology and Visual Sciences, John A. Moran Eye Center, University of Utah. DESIGN: Immunohistochemical experimental study. METHODS: Nine capsular bag specimens from DBS cases, as well as 2 control specimens from late-postoperative in-the-bag intraocular lens dislocation cases related to previous vitrectomy, pseudoexfoliation, and blunt trauma were included. They were processed for histopathology; unstained sections were obtained from each one, and analyzed by immunohistochemistry targeting collagen type IV, laminin, vimentin, collagen type I and fibronectin. RESULTS: Immunohistochemistry in DBS showed lens capsule stained for basement membrane components. The outer part of the anterior capsule that was split from the inner part was more markedly stained for type IV collagen as compared with the posterior part. Faint staining for fibrous posterior capsular opacification (PCO) components, e. g., collagen type I and fibronectin, was detected in limited areas, but the major portion of the capsule was free from these components. Small spotty vimentin-positive materials, suggesting the presence of cell debris, was also detected in limited samples. CONCLUSION: Small amounts of fibrotic PCO components were detected in capsules extracted from patients with DBS, but their major parts were free from PCO components. Current findings suggest small amounts of lens epithelial cells were present after surgery and secreted fibrous components before undergoing cell death process.

A Glaucoma Patient with an Intraocular Pressure Decrease following Total Gastrectomy and Postoperative Anticancer Treatment.

We herein report a case of glaucoma with an intraocular pressure (IOP) decrease following total gastrectomy (TG) and anticancer treatment for gastric cancer. A 62-year-old male underwent trabeculectomy of the left and right eyes in August 2011 and July 2012, respectively. During the follow-up, IOP of the right eye was 9-12 mmHg (with bimatoprost, dorzolamide, and timolol maleate), and that of the left eye ranged between 14 and 26 mmHg (with bimatoprost, dorzolamide, timolol maleate, and brimonidine tartrate). In December 2014, TG was performed due to gastric cancer. After surgery, the patient received S-1+CDDP, weekly PAC, and CPT-11 therapies. The patient died on March X, 2017. Before TG, the body mass index (BMI) was 29.5 but decreased to 24.8 before the start of the two courses of weekly PAC therapy. IOP of the right eye was 6 mmHg (with bimatoprost), and that of the left eye was 10 mmHg (with bimatoprost, dorzolamide, and brimonidine tartrate), showing decreases. After the initiation of weekly PAC therapy, BMI was approximately 19. IOP of the right eye ranged between 6 and 10 mmHg until the final ophthalmological examination (January 11, 2017), while that of the left eye ranged between 8 and 15 mmHg. In this patient with glaucoma, IOP was not controlled by eye drop treatment, and TG for gastric cancer and postoperative treatment with anticancer drugs resulted in weight loss and a decrease in IOP.

Loss of TRPV4 Cation Channel Inhibition of Macrophage Infiltration and Neovascularization in a Mouse Cornea.

Corneal injury-associated inflammation could induce inward-growing neovascularization from the periphery of the tissue. Such neovascularization could cause stromal opacification and curvature disturbance, and both potentially impair visual function. In this study, we determined the effects of the loss of transient receptor potential vanilloid 4 (TRPV4) expression on the development of neovascularization in the corneal stroma in mice by producing a cauterization injury in the central area of the cornea. New vessels were immunohistochemically labeled with anti-TRPV4 antibodies. TRPV4 gene knockout suppressed the growth of such CD31-labeled neovascularization in association with the suppression of infiltration of macrophages and tissue messenger RNA expression of the vascular endothelial cell growth factor A level. Treatment of cultured vascular endothelial cells with supplementation of HC-067047 (0.1 µM, 1 µM, or 10 µM), a TRPV4 antagonist, attenuated the formation of a tube-like structure with sulforaphane (15 µM, for positive control) that modeled the new vessel formation. Therefore, the TRPV4 signal is involved in injury-induced macrophagic inflammation and neovascularization activity by vascular endothelial cells in a mouse corneal stroma. TRPV4 could be a therapeutic target to prevent unfavorable postinjury neovascularization in the cornea.

Delayed regression of laser-induced choroidal neovascularization in TNFα-null mice.

We investigated the effects of lacking TNFα on the development and regression of Argon-laser-induced choroidal neovascularization (CNV) in mice. We lasered ocular fundus for induction of CNV in both wild-type (WT) and TNFα-null (KO) mice. Fluorescence angiography was performed to examine the size of CNV lesions. Gene expression pattern of wound healing-related components was examined. The effects of exogenous TNFα on apoptosis of human retinal microvascular endothelial cells (HRMECs) and on the tube-like structure of the cells were investigated in vitro. The results showed that Argon-laser irradiation-induced CNV was significantly larger in KO mice than WT mice on Day 21, but not at other timepoints. Lacking TNFα increased neutrophil population in the lesion. The distribution of cleaved caspase3-labelled apoptotic cells was more frequently observed in the laser-irradiated tissue in a WT mouse as compared with a KO mouse. Exogenous TNFα induced apoptosis of HRMECs and accelerated regression of tube-like structure of HRMECs in cell culture. Taken together, TNFα gene knockout delays the regression of laser-induced CNV in mice. The mechanism underlying the phenotype might include the augmentation of neutrophil population in the treated tissue and attenuation of vascular endothelial cell apoptosis.

Long-term intraocular pressure after switching a combination ophthalmic medication of ß-blocker/prostaglandin.

PURPOSE: We examined intraocular pressure (IOP)-reducing effects 12 months after switching timolol maleate/travoprost combination ophthalmic solution in one bottle (TM/TR-COMBI-SOL) to carteolol hydrochloride/latanoprost combination ophthalmic solution in one bottle (CR/LT-COMBI-SOL). CASES: The participants included 25 patients (25 eyes) who could be followed up for 12 months after a switch from TM/TR-COMBI-SOL to CR/LT-COMBI-SOL in Saiseikai Arida Hospital between March 1, 2017, and August 31, 2018. They consisted of patients in whom antiglaucoma eye drop other than TM/TR-COMBI-SOL had not been used (monotherapy group, 12 patients [12 eyes], 12.8 ± 3.0 mmHg) and those in whom antiglaucoma eye drop other than TM/TR-COMBI-SOL had been concomitantly used (multitherapy group, 13 patients [13 eyes], 13.8 ± 2.4 mmHg). We excluded patients in whom drugs for glaucoma were changed or added during the follow-up and those who underwent intraocular surgery. MATERIALS AND METHODS: We retrospectively and statistically examined the IOP before eye drop switching and after 1, 6, and 12 months, using the paired t-test. RESULTS: The IOPs 1 month after eye drop switching in the monotherapy group and multitherapy group were 12.5 ± 3.3 and 13.8 ± 2.5 mmHg, respectively. The values after 6 months were 13.5 ± 3.0 and 11.5 ± 2.7 mmHg, respectively. Those after 12 months were 12.8 ± 2.7 and 11.7 ± 2.5 mmHg, respectively. In the monotherapy group, there was no significant difference during the follow-up period. In the multitherapy group, there were significant decreases in comparison with the preswitching value after 6 and 12 months (P < 0.05, respectively). CONCLUSION: The IOP-reducing effects of CR/LT-COMBI-SOL were similar to those of TM/TR-COMBI-SOL. However, the effects may be enhanced after switching from TM/TR-COMBI-SOL in patients receiving multitherapy.

Effects of a prostaglandin F2alpha derivative glaucoma drug on EGF expression and E-cadherin expression in a corneal epithelial cell line.

Purpose: We examined the effects of travoprost on cell proliferation-related signals and E-cadherin expression in vitro and in situ in order to obtain evidence to support the hypothesis that topical travoprost impairs the integrity of the corneal epithelium.Methods: A human corneal epithelial cell culture was treated with travoprost (0.4 mg/ml) and/or PD168393 (an EGF receptor inhibitor, 10 µM). The culture was then processed for cell proliferation, an mRNA expression analysis of epidermal growth factor (EGF) and E-cadherin, and protein expression analysis of E-cadherin by immunocytochemistry and Western blotting. The eyes of C57/BL6 mice were incubated in serum-free medium plus travoprost (0.4 mg/ml) and/or PD168393 (10 µM). After being cultured for 24 h, the expression patterns of phospho-EGFR, phospho-ERK, E-cadherin, and Ki67 were immunohistochemically examined in paraffin sections.Results: The addition of travoprost up-regulated EGF mRNA expression and cell proliferation in the corneal epithelial cell culture, and this was cancelled by the addition of PD168393. This FP agonist also decreased E-cadherin expression levels in the cell-cell contact zone, and this was cancelled by the addition of PD168393. In the organ culture, the addition of travoprost to the medium up-regulated the expression of phospho-EGFR and phospho-ERK as well as cell proliferation, and down-regulated the expression of E-cadherin in the corneal epithelium, particularly in basal cells, whereas PD168393 reversed these effects.Conclusions: Travoprost activates epithelial cell proliferation by up-regulating an EGF-related signal in association with the suppression of E-cadherin localization in the cell-cell contact zone. Modulation of the EGF signal may be a strategy to minimize the negative impact of this mitogen on reformation of corneal barrier function during epithelial renewal.

Suppression of neovascularization in corneal stroma in a TRPA1-null mouse.

Corneal neovascularization and inflammatory fibrosis induced by severe injury or infection leads to tissue opacification and even blindness. Transient receptor potential (TRP) channel subtypes contribute to mediating these maladaptive responses through their interactions with other receptors. TRPV1 is one of the contributing channel isoforms inducing neovascularization in an alkali burn mouse wound healing model. VEGF-A upregulation contributes to neovascularization through interaction with its cognate receptors (VEGFR). Since the TRP isoform in this tissue, TRPA1, is also involved, we determined here if one of the pathways mediating neovascularization and immune cell infiltration involve an interaction between VEGFR and TRPA1 in a cauterization corneal mouse wound healing model. Localization of TRPA1 and endothelial cell (EC) CD31 immunostaining pattern intensity determined if TRPA1 expression was EC delimited during cauterization induced angiogenesis. Quantitative RT-PCR evaluated the effects of the absence of TRPA1 function on VEGF-A and TGF-ß1 mRNA expression during this process. Macrophage infiltration increased based on rises in F4/80 antigen immunoreactivity. TRPA1 immunostaining was absent on CD31-immunostained EC cells undergoing neovascularization, but it was present on other cell type(s) adhering to EC in vivo. Absence of TRPA1 expression suppressed both stromal neovascularization and inhibited macrophage infiltration. Similarly, the increases occurring in both VEGF-A and TGF-ß1 mRNA expression levels in WT tissue were blunted in the TRPA1-/- counterpart. On the other hand, in the macrophages their levels were invariant and their infiltration was inhibited. To determine if promotion by TRPA1 of angiogenesis was dependent on its expression on other unidentified cell types, the effects were compared of pharmacological manipulation of TRPA1 activity on EC proliferation tube formation and migration. In the presence and absence of a fibroblast containing feeder layer. Neither VEGF-induced increases in human vascular endothelial cell (HUVEC) proliferation nor migration were changed by a TRPA1 antagonist HC-030031 in the absence of a feeder layer. However, on a fibroblast feeder layer this antagonist suppressed HUVEC tube formation. In conclusion, during corneal wound healing transactivation by VEGFR of TRPA1 contributes to mediating neovascularization and macrophage infiltration. Such crosstalk is possible because of close proximity between VEGFR delimited expression on EC and TRPA1 expression restricted to cell types adhering to EC.

Loss of tenascin X gene function impairs injury-induced stromal angiogenesis in mouse corneas.

To determine the contribution by tenascin X (Tnx) gene expression to corneal stromal angiogenesis, the effects were determined of its loss on this response in TNX knockout (KO) mice. In parallel, the effects of such a loss were evaluated on vascular endothelial growth factor (VEGF) and transforming growth factor ß1 (TGFß1) gene and protein expression in fibroblasts and macrophages in cell culture. Histological, immunohistochemical and quantitative RT-PCR changes determined if Tnx gene ablation on angiogenic gene expression, inflammatory cell infiltration and neovascularization induced by central corneal stromal cauterization. The role was determined of Tnx function in controlling VEGF-A or TGFß1 gene expression by comparing their expression levels in ocular fibroblasts and macrophages obtained from wild-type (WT) and body-wide Tnx KO mice. Tnx was up-regulated in cauterized cornea. In Tnx KO, macrophage invasion was attenuated, VEGF-A and its cognate receptor mRNA expression along with neovascularization were lessened in Tnx KOs relative to the changes occurring in their WT counterpart. Loss of Tnx instead up-regulated in vivo mRNA expression of anti-angiogenic VEGF-B but not VEGF-A. On the other hand, TGFß1 mRNA expression declined in Tnx KO cultured ocular fibroblasts. Loss of Tnx gene expression caused VEGF-A expression to decline in macrophages. Tnx gene expression contributes to promoting TGFß1 mRNA expression in ocular fibroblasts and VEGF-A in macrophages, macrophage invasion, up-regulation of VEGF-A expression and neovascularization in an injured corneal stroma. On the other hand, it suppresses anti-angiogenic VEGF-B mRNA expression in vivo.

Bacteriology of the conjunctiva in pre-cataract surgery patients with occluded nasolacrimal ducts and the operation outcomes in Japanese patients.

BACKGROUND: Contamination of the conjunctiva in association with nasolacrimal duct obstruction is by all accounts a risk factor for infectious endophthalmitis post-cataract surgery. METHODS: All patients who underwent cataract day surgery routinely received nasolacrimal duct syringing with normal saline at the Wakayama Medical University Hospital, Japan, from 2011 to 2013. The microorganisms isolated from conjunctival swab samples of patients with occluded nasolacrimal ducts and their susceptibility to antibiotics, as well as the operation outcomes in all the patients were retrospectively investigated. RESULTS: Nasolacrimal duct obstruction was observed in 125 eyes of 90 patients (3.3%; 42 eyes of 30 male individuals, and 83 eyes of 60 female individuals) from a total of 3754 eyes of 2384 patients by using irrigation samples of nasolacrimal ducts. The mean age of the subjects with duct obstruction was 79 ± 8.5 years.. In bacterial cultures of swabs from these 125 individuals, microbial growth was detected in 56 samples (i.e. 44.8%). Coagulase-negative Staphylococcus was detected in 28 eyes, and Corynebacterium species was detected in 17 eyes. Staphylococcus aureus, excluding methicillin-resistant S. aureus was detected in seven eyes with nasolacrimal duct obstruction. Methicillin-resistant S. aureus was isolated in two eyes with nasolacrimal duct obstruction. Each case was treated with topical antibiotics based on the results of antibiotic sensitivity tests. After culturing of cotton swab samples from the conjunctiva, and using direct micrography of bacteria every 2 or 3 days after starting treatment, and once the results were negative (consecutively tested three times), the patients received cataract surgery. In the current case series, bacteria were not detected in conjunctival swabs obtained consecutively three times for 3 weeks after starting topical antibiotics in 118 eyes from 125 eyes (94.4%), and later in the remaining patients. No patient required dacryocystorhinostomy to eliminate bacterial contamination in the conjunctiva following topical antibiotic therapy. No patient developed infectious endophthalmitis at least 1-year post-cataract surgery. CONCLUSIONS: All the patients receiving cataract day surgery underwent the operation after the elimination of conjunctival microorganism contamination in association with nasolacrimal duct obstruction by using appropriate topical antibiotics.

Loss of TRPV4 Function Suppresses Inflammatory Fibrosis Induced by Alkali-Burning Mouse Corneas.

In humans suffering from pulmonary disease and a mouse model, transient receptor potential vanilloid 4 (TRPV4) channel activation contributes to fibrosis. As a corneal alkali burn induces the same response, we determined if such an effect is also attributable to TRPV4 activation in mice. Accordingly, we determined if the alkali burn wound healing responses in wild-type (WT) mice are different than those in their TRPV4-null (KO) counterpart. Stromal opacification due to fibrosis in KO (n = 128) mice was markedly reduced after 20 days relative to that in WT (n = 157) mice. Immunohistochemistry revealed that increases in polymorphonuclear leukocytes and macrophage infiltration declined in KO mice. Semi-quantitative real time RT-PCR of ocular KO fibroblast cultures identified increases in proinflammatory and monocyte chemoattractant protein-1 chemoattractant gene expression after injury. Biomarker gene expression of fibrosis, collagen1a1 and α-smooth muscle actin were attenuated along with macrophage release of interleukin-6 whereas transforming growth factor ß, release was unchanged. Tail vein reciprocal bone marrow transplantation between WT and KO chimera mouse models mice showed that reduced scarring and inflammation in KO mice are due to loss of TRPV4 expression on both corneal resident immune cells, fibroblasts and infiltrating polymorphonuclear leukocytes and macrophages. Intraperitoneal TRPV4 receptor antagonist injection of HC-067047 (10 mg/kg, daily) into WT mice reproduced the KO-phenotype. Taken together, alkali-induced TRPV4 activation contributes to inducing fibrosis and inflammation since corneal transparency recovery was markedly improved in KO mice.

A Case of Solitary Nonvascularized Corneal Epithelial Dysplasia.

Background. Epithelial dysplasia is categorized as conjunctival/corneal intraepithelial neoplasia which is a precancerous lesion. The lesion is usually developed at the limbal region and grows towards central cornea in association with neovascularization into the lesion. Here, we report a case of isolated nonvascularized corneal epithelial dysplasia surrounded by normal corneal epithelium with immune histochemical finding of ocular surface tissues cytokeratins, for example, keratin 13 and keratin 12. Case Presentation. A 76-year-old man consulted us for visual disturbance with localized opacification of the corneal epithelium in his left eye. His visual acuity was 20/20 and 20/200 in his right and left eye, respectively. Slit lamp examination showed a whitish plaque-like lesion at the center of his left corneal epithelium. No vascular invasion to the lesion was found. The lesion was surgically removed and subjected to histopathological examination and diagnosed as epithelial dysplasia. Amyloidosis was excluded by direct fast scarlet 4BS (DFS) staining. Immunohistochemistry showed that the dysplastic epithelial cells express keratin 13 and vimentin, but not keratin 12, indicating that the neoplastic epithelial cells lacked corneal-type epithelium differentiation. Conclusions. The lesion was diagnosed as nonvascularized epithelial dysplasia of ocular surface. Etiology of the lesion is not known.

Modulation of Smad signaling by non-TGFß components in myofibroblast generation during wound healing in corneal stroma.

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 ß (TGFß). Although it is established that TGFß/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-TGFß 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.

Histological analysis of a cornea following experimental femtosecond laser ablation.

Corneal photorefractive surgery is currently performed by ablation of corneal stroma under the stromal flap. A stromal flap is created using a femtosecond (FS) laser or mechanical microkeratome, although the FS laser procedure is considered safer and more accurate. This review assesses and compares the use of FS laser versus mechanical microkeratome ablation for corneal stromal characteristics mainly examined by histology and cellular biological responses. Supporting data from our studies, using corneas of enucleated porcine eye globes, are included in this review. Histological analysis and experimental studies of cellular/tissue responses to FS laser irradiation should be further investigated, and the equipment used to perform these techniques should be improved.

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)ß1 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 TGFß1. 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 TGFß1 by exogenous TGFß1 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 TGFß1 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.

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 ß 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 ß/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.

TGF beta in fibroproliferative diseases in the eye.

Transforming growth factor b (TGF beta) is believed to be the most important ligand in the pathogenesis of fibrotic diseases in the eye. Such ocular fibrotic diseases include scarring in the cornea and conjunctiva, fibrosis in the corneal endothelium, post-cataract surgery fibrosis of the lens capsule, excess scarring the tissue around the extraocular muscles in the strabismus surgery and proliferative vitreoretinopathy. In the proliferative stage of diabetic retinopathy, fibrogenic reaction causes tractional retinal detachment in association with contraction of the tissue. A myofibroblast, the major cellular component in the fibrotic lesions, is derived from both mesenchymal cells (in cornea and conjunctiva) and epithelial cell types (lens or retinal pigment epithelium or corneal endothelium) through epithelial-mesenchymal transition (EMT). The myofibroblasts cause excess accumulation of fibrogenic extracellular matrix with resultant tissue contraction and impaired functions. Although various cytokine signaling pathways are involved in the fibrogenic reaction in tissues, TGF beta/Smad signal is the critical one. Blocking Smad signal by chemical or natural inhibitors or anti-Smad gene introduction effectively suppress fibrogenic reaction; inhibition of both fibroblast-myofibroblast conversion or EMT. Such strategies can be clinically tested.

Inhibitory effect of blocking TGF-beta/Smad signal on injury-induced fibrosis of corneal endothelium.

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.

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.

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.

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-kappaB), which is the major transcription factor involved in COX2 expression; phospho-IkappaB; 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-kappaB 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 kappaB was detected in corneal epithelium 6 hours after treatment, but not 12 hours or later. CONCLUSION: Ethanol exposure activated NF-kappaB and upregulated COX2 expression, which may cause inflammation in corneal tissue.