The murine lens: A model to investigate in vivo epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) produces myofibroblasts that contribute to the formation of fibrotic tissue with an impairment of tissue homeostasis and functionality. The crystalline lens of the eye is a unique transparent and isolated tissue. The lens vesicle becomes isolated from the surface ectoderm, its cells are all contained as they line the inner surface of the lens capsule. Clinically the formation of fibrotic tissue by the lens epithelial cells causes a type of cataract or opacification and contraction of the lens capsule postcataract surgery. Production of EMT in the intact animal lens by using specific gene transfer to the lens or experimental lens injury has been shown to be a powerful tool to investigate EMT processes. It is not easy to uncover whether the origin of the myofibroblast is epithelial cell-derived or from other cell lineages in fibrotic tissues. However, myofibroblasts that appear in the crystalline lens pathology are totally derived from the lens epithelial cells for the reasons mentioned above. Here, we report on different animal models of lens EMT, using either transgenic approaches or injury to study the biological aspects of EMT. Developmental Dynamics 247:340-345, 2018. © 2017 The Authors Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

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.

Ocular surface mucins and local inflammation--studies in genetically modified mouse lines.

Mucins locate to the apical surfaces of all wet-surfaced epithelia including ocular surface. The functions of the mucins include anti-adhesive, lubrication, water retention, allergens and pathogen barrier function. Ocular surface pathologies, i.e. dry eye syndrome or allergic conjunctivitis, are reportedly associated with alteration of expression pattern of mucin components. Recent investigations indicated anti-bacterial adhesion or anti-inflammatory effects of members of mucins in non-ocular tissues, i.e., gastrointestinal tracts or airway tissues, by using genetically modified mouse lines that lacks an expression of a mucin member. However, examination of ocular phenotypes of each of mucin gene-ablated mouse lines has not yet fully performed. Muc16-deficient mouse is associated with spontaneous subclinical inflammation in conjunctiva. The article reviews the roles of mucin members in modulation of local inflammation in mucous membrane tissues and phenotype of mouse lines with the loss of a mucin gene. Analysis of ocular surface of mucin-gene related mutant mouse lines are to be further performed.

Effects of the loss of conjunctival Muc16 on corneal epithelium and stroma in mice.

PURPOSE: To examine the role of conjunctival Muc16 in the homeostasis of the ocular surface epithelium and stroma using Muc16-null knockout (KO) mice. METHODS: We used KO mice (n = 58) and C57/BL6 (WT) mice (n = 58). Histology and immunohistochemistry were employed to analyze the phenotypes in the ocular surface epithelium. The expression of phospho-Stat3, AP-1 components, interleukin 6 (IL-6), and tumor necrosis factor-α (TNFα) in the cornea and conjunctiva was examined. The shape of the nuclei of corneal epithelial cells was examined to evaluate intraepithelial cell differentiation. Epithelial cell proliferation was studied using bromo-deoxyuridine labeling. Finally, the wound healing of a round defect (2-mm diameter) in the corneal epithelium was measured. The keratocyte phenotype and macrophage invasion in the stroma were evaluated after epithelial repair. RESULTS: The loss of Muc16 activated Stat3 signal, affected JunB signal, and upregulated the expression of IL-6 in the conjunctiva. Basal-like cells were observed in the suprabasal layer of the corneal epithelium with an increase in proliferation. The loss of Muc16 accelerated the wound healing of the corneal epithelium. The incidence of myofibroblast appearance and macrophage invasion were more marked in KO stroma than in WT stroma after epithelial repair. CONCLUSIONS: The loss of Muc16 in the conjunctiva affected the homeostasis of the corneal epithelium and stroma. The mechanism might include the upregulation of the inflammatory signaling cascade (i.e., Stat3 signal, and IL-6 expression in the KO conjunctiva). Current data provides insight into the research of the pathophysiology of dry eye syndrome.

TRPV1 involvement in inflammatory tissue fibrosis in mice.

We examined whether absence or blocking of transient receptor potential vanilloid subtype 1 (TRPV1) affects the level of inflammation and fibrosis/scarring during healing of injured tissue using an alkali burn model of cornea in mice. A cornea burn was produced with 1 N NaOH instilled into one eye of TRPV1-/- (KO) (n = 88) or TRPV1+/+ (n = 94) mice. Examinations of the corneal surface and eye globe size suggested that the loss of TRPV1 suppressed inflammation and fibrosis/scarring after alkali burn, and this was confirmed by histology, IHC, and gene expression analysis. The loss of TRPV1 inhibited inflammatory cell invasion and myofibroblast generation in association with reduction of expression of proinflammatory and profibrogenic components. Experiments of bone marrow transplantation between either genotype of mice showed that KO corneal tissue resident cells, but not KO bone marrow-derived cells, are responsible for KO-type wound healing with reduced inflammation and fibrosis. The absence of TRPV1 attenuated expression of transforming growth factor ß 1 (TGFß1) and other proinflammatory gene expression in cultured ocular fibroblasts, but did not affect TGFß1 expression in macrophages. Loss of TRPV1 inhibited myofibroblast transdifferentiation in cultured fibroblasts. Systemic TRPV1 antagonists reproduced the KO type of healing. In conclusion, absence or blocking of TRPV1 suppressed inflammation and fibrosis/scarring during healing of alkali-burned mouse cornea. TRPV1 is a potential drug target for improving the outcome of inflammatory/fibrogenic wound healing.

Two cases of congenital retrobulbar cyst or peripapillary staphyloma with an alcoholic mother.

The authors report two cases of congenital retrobulbar cyst or large peripapillary staphyloma with alcoholic mothers. In the first case, a 53-day-old newborn was referred for evaluation of microphthalmia in her left eye. Ophthalmic examination showed microcornea, microphthalmia, and an abnormally enlarged optic disc with an excavation in her left eye. Imaging revealed the presence of a cystic lesion in her left orbit that seemed to be communicated with the vitreous cavity. She did not have chromosomal abnormality. In the second case, a large peripapillary staphyloma was detected in the right eye of a 7-month-old newborn. The mother of each patient was an alcoholic. Retrobulbar cystic lesion or peripapillary staphyloma could be considered to be a series of abnormalities caused by the degree of the failure of the optic cup. Effects of each mother's alcohol intake on the development of the eye, especially the closure of the optic cup, is to be further investigated.

Immunohistochemical observation of anterior subcapsular cataract in eye with spontaneously regressed retinoblastoma.

We report the histological findings of secondary cataract in an eye with a spontaneously regressed retinoblastoma to obtain keys to clarify the mechanism of this phenomenon. During phacoemulsification, opacified anterior capsule was obtained, fixed in formalin, and embedded in paraffin. Paraffin sections of the specimen were histologically observed. Hematoxylin-eosin staining showed extracellular matrix accumulation in the extracted fibrous anterior subcapsular opacification. Immunohistochemistry revealed the presence of fibrous collagen types and cellular fibronectin. Presumed lens cells amid matrix were positively labeled for vimentin, alpha-smooth muscle actin, and phospho-Smad2. Histology of the fibrous anterior subcapsular opacification tissue showed the possibility of epithelial-mesenchymal transition of the lens epithelial cells in the secondary cataract following a spontaneously regressed retinoblastoma.

Therapeutic potential of trichostatin A to control inflammatory and fibrogenic disorders of the ocular surface.

PURPOSE: To examine the effects of a histone deacetylase inhibitor, Trichostatin A (TSA), on the behavior of macrophages and subconjunctival fibroblasts in vitro and on ocular surface inflammation and scarring in vivo using an alkali burn wound healing model. METHODS: Effects of TSA on expression of inflammation-related growth factors or collagen I were examined by real-time RT-PCR or immunoassay in mouse macrophages or human subconjunctival fibroblasts. Effects of TSA on trans forming growth factor ß (TGFß)/Smad signaling were evaluated with western blotting and/or immunocytochemistry. Alkali-burn injuries on the eyes of mice were performed with three µl of 0.5 N NaOH under general and topical anesthesia. TSA (600 µg/Kg daily) or vehicle was administered to animals via intraperitoneal (i.p.) injection. Histology and real-time RT-PCR investigations evaluated the effects of TSA on the healing process of the cornea. RESULTS: TSA inhibited TGFß 1 and vascular endothelial growth factor (VEGF) expression in macrophages, and TGFß1 and collagen I in ocular fibroblasts. It elevated the expression of 5'-TG-3'-interacting factor (TGIF) and Smad7 in fibroblasts and blocked nuclear translocation of phospho-Smad2. Real-time PCR and immunocytochemistry studies showed that systemic administration of TSA suppressed the inflammation and fibrotic response in the stroma and accelerated epithelial healing in the alkali-burned mouse cornea. CONCLUSIONS: Systemic administration of TSA reduces inflammatory and fibrotic responses in the alkali-burned mouse ocular surface in vivo. The mechanisms of action involve attenuation of Smad signal in mesenchymal cells and reduction in the activation and recruitment of macrophages. TSA has the potential to treat corneal scarring in vivo.

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.

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.

Perturbed intraepithelial differentiation of corneal epithelium in c-Fos-null mice.

PURPOSE: AP-1 is a transcription factor that plays a pivotal role in regulating cellular homeostasis and which may modulate the differentiation of corneal epithelial cells. We examined the role of c-Fos in the differentiation of corneal epithelial cells by using c-Fos-deficient (c-fos (-/-)) mice. METHODS: Ten adult c-fos (-/-) mice and ten control (c-fos (+/-) or c-fos (+/+)) mice were used. The expression patterns of the mRNA and protein of keratin 12 (K12) were determined to examine the differentiation of cornea-type epithelium. To evaluate the intraepithelial differentiation from basal cells to superficial cells, the ultrastructure of the corneal epithelium was studied. We focused on the formation of desmosomes in the superficial, suprabasal, and basal cell layers, and also on the hemidesmosomes. The number of desmosomes in each epithelial layer was statistically analyzed by using an unpaired t test. The expressions of keratin 14 (K14), desmoglein, E-cadherin, occludin, connexin 43, filaggrin, loricrin, and involucrin were examined to analyze epithelial differentiation. RESULTS: The mRNA and protein of K12 were expressed in the corneal epithelium of c-fos (-/-) and control mice. Ultrastructural observations showed that the number of desmosomes between the basal cells of the corneal epithelia was similar in c-fos (-/-) and control mice. However, there were fewer desmosomes between suprabasal cells and between superficial cells in c-fos (-/-) mice than in control mice. The number of hemidesmosomes in the corneal epithelial cells in c-Fos-null mice was similar to that in control mice. The expressions of the other epithelial cell differentiation markers were not affected by the absence of c-Fos. Ultrastructural observations showed a disarrangement of the corneal epithelium in the c-Fos-null mice. CONCLUSIONS: The absence of c-Fos disturbs the formation of desmosomes in the superficial layers of the corneal epithelium, suggesting a perturbation of intraepithelial differentiation from the basal epithelial cells to the suprabasal and superficial epithelial cells.

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.

Emodin suppression of ocular surface inflammatory reaction.

PURPOSE: To determine whether a Chinese herbal medicine component, emodin, suppresses inflammatory/fibrogenic reaction in cultured subconjunctival fibroblasts and reduces injury-induced increases in ocular surface inflammation in mice. METHODS: Effects of emodin were measured in human subconjunctival fibroblasts on proliferation and migration with colorimetry and scratch wound assay, respectively. Neovascularization was evaluated using an endothelial cell-fibroblast coculture model. Proinflammatory mediator and extracellular matrix component gene and protein expression was characterized with real-time reverse transcription-polymerase chain reaction, enzyme immunoassay, and immunocytochemistry, respectively. Western blotting and immunohistochemistry evaluated the activation of nuclear factor-kappaB (NF-kappaB) and c-Jun N-terminal kinase (JNK). In a mouse corneal alkali-burn model, the effects of emodin on ocular surface inflammation and fibrosis were evaluated. RESULTS: Emodin suppressed tumor necrosis factor alpha (TNF-alpha)-induced fibroblast migration and fibronectin deposition in vitro. VEGF induced neovascularization but did not affect cell proliferation and collagen type 1 production. Monocyte/macrophage-chemoattractant protein-1 gene and protein expression declined. Emodin inhibited TNF-alpha-induced NF-kappaB p65 and JNK activation but did not affect transforming growth factor beta1-induced Smad2/3 signaling. In vivo, emodin inhibited proinflammatory and fibrogenic reactions. CONCLUSIONS: Emodin suppressed in vitro TNF-alpha-induced stimulation of proinflammatory reaction. In a mouse ocular alkali burn model, this herbal component lessened inflammation and scarring. Additional studies are warranted to evaluate the therapeutic potential of emodin in lessening ocular tissue inflammation and resultant fibrosis after injury.

AP-1 expression in ethanol-treated corneal epithelium in vivo.

PURPOSE: To examine the expression pattern of stress-related genes, c-fos and c-jun, both the major components of activator protein-1 (AP-1), in rat corneal epithelium treated with a short-term ethanol exposure. The purpose of the current study was to examine if the ethanol exposure during laser epithelial keratomileusis (LASEK) may stimulate or damage the corneal epithelial cells. METHOD: Sixty male Wistar rats were used. Fifty microliters of 20% ethanol was placed onto a surface 2.4 mm in diameter of the central corneal epithelium for 30 s. The affected eyes, washed with saline, were then enucleated after various intervals of healing. To know the expression pattern of c-fos and c-jun mRNAs and c-Fos, c-Jun and Jun D proteins, in situ hybridization and immunohistochemistry were carried out. The expression level of c-fos and c-jun mRNAs was determined by real-time reverse-transcriptase polymerase chain reaction (RT-PCR). Apoptotic nuclei in the tissue sections were identified by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay. RESULTS: Thirty to 60 min after the treatment, c-fos and c-jun mRNAs were detected in the corneal epithelium. These signals were no longer evident at 90 min. c-Fos protein was detected in the corneal epithelium around the area of ethanol exposure from 60 to 120 min after the treatment, while c-Jun protein was not detected. Jun D protein was detected in control whole corneal epithelium and not affected by ethanol exposure in the periphery. The levels of c-fos and c-jun mRNAs were increased approximately 8 times at 30 min compared with the control level. TUNEL-positive apoptotic nuclei in the tissue sections were identified. CONCLUSION: Corneal epithelial cells, especially those surrounding the ethanol-exposed area, are transiently transcriptionally activated at a very early phase after the ethanol exposure. mRNA expression for c-fos is followed by protein synthesis, but that of c-jun is not followed by protein synthesis. Resistance of Jun D protein expression to ethanol suggests that it might be a candidate for an AP-1 complex with c-Fos.

Loss of osteopontin perturbs the epithelial-mesenchymal transition in an injured mouse lens epithelium.

We previously reported that osteopontin (OPN), a matrix structural glycophosphoprotein, is upregulated in the injured mouse lens prior to the epithelial-mesenchymal transition (EMT). Here, we investigated the role of this protein in EMT of the lens epithelium during wound healing. The crystalline lens was injured by needle puncture in OPN-null (KO, n=40) and wild-type (WT, n=40) mice. The animals were killed at day 1, 2, 5, and 10 postinjury. Immunohistochemistry was employed to detect alpha-smooth muscle action (alphaSMA), a marker of EMT, collagen type I, transforming growth factor beta1 (TGFbeta1), TGFbeta2, and phospho-Smad2/3. Cell proliferation was assayed by examining uptake of bromodeoxyuridine (BrdU). The results showed that injury-induced EMT of mouse lens epithelium, as evaluated by histology, expression pattern of alphaSMA and collagen I, was altered in the absence of OPN with reduced phospho-Smad2/3 signaling. Upregulation of TGFbeta1 and TGFbeta2 in the epithelium was also inhibited. Cell proliferation was more active in KO mice as compared with WT mice at day 1 and 2, but not at day 5 and 10. An in vitro experiment shows OPN facilitates cell adhesion of lens epithelial cell line. OPN is required for activation of Smad2/3 signal in an injured lens epithelium and lens cell EMT.

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.

Genipin suppression of fibrogenic behaviors of the alpha-TN4 lens epithelial cell line.

PURPOSE: To determine in a lens epithelial cell line, alpha-TN4, whether genipin, an intestinal metabolite component of the herbal medicine inchin-ko-to, suppresses profibrogenic myofibroblast generation and upregulation of fibrogenic cytokines and to evaluate the potential benefit of the medicine in preventing posterior capsule opacification (PCO). SETTING: Department of Ophthalmology, Wakayama Medical University, Wakayama, Japan. METHODS: In this study, alpha-TN4 cell proliferation, migration, and expression of alpha-smooth muscle actin (alpha-SMA), the hallmark of myofibroblast generation, were assayed with a colorimetric assay, scratch wound assay, immunohistochemistry, and Western blot analysis. Gene expression of transforming growth factor-beta1 (TGF-beta1) and connective tissue growth factor (CTGF) was characterized with real-time reverse transcription-polymerase chain reaction. In addition, p38 mitogen-activated protein kinase (p 38 MAPK), extracellular signal-regulated kinase (ERK) limb, and Smad signalings were evaluated by Western blotting and immunohistochemistry. Cytotoxicity of genipin was evaluated using a commercial colorimetric assay kit for nuclear matrix protein 41/7 (NMP41/7) in culture medium. RESULTS: Genipin suppressed cell proliferation and migration in association with inhibition of Smad and p38 MAPK phosphorylation, although ERK signaling was enhanced. Genipin suppressed mRNA expression of TGF-beta1 and CTGF. Cytoplasmic fiber formation declined based on less intense alpha-SMA immunocytochemical staining. However, alpha-SMA protein expression was actually not altered. This negative result suggests that genipin attenuated formation of alpha-SMA-containing cytoskeleton. Treatment of the cells with genipin for 48 hours did not increase the release of NMP41/7 to the medium, indicating this compound is not cytotoxic. CONCLUSION: Because genipin suppressed alpha-TN4 lens cell fibrogenic behaviors, it may be of therapeutic value in preventing PCO.

A new model of anterior subcapsular cataract: involvement of TGFbeta/Smad signaling.

PURPOSE: To develop a new animal model of anterior subcapsular cataract formation by topical application of alkali to the eye and to examine the role of Transforming growth factorbeta/Smad3 (TGFbeta/Smad3) signaling in the formation of this cataract model. METHODS: Under anesthesia, one eye of adult Wistar rats (n=142) was subjected to alkali burn by topical application of 1 N NaOH. The eye was then histologically examined at specific time intervals. Immunohistochemistry with a battery of antibodies was carried out to examine the epithelial-mesenchymal transition (EMT) in lens epithelium. Enzyme immunoassay was employed to determine the level of growth factors in aqueous humor and lens tissue. Smad3-null mice were also used to examine the role of Smad3 signaling in cataractogenesis in this model. RESULTS: Two days post-burn of the ocular surface, lens epithelium underwent EMT as evidenced by the upregulation of Snail and alpha-smooth muscle actin and formed a multilayer of cells beneath the capsule. Smad signaling was found to be activated in EMT-type lens cells. The majority of myofibroblast-type lens cells expressed proliferative cell nuclear antigen (PCNA). The total amount of active TGFbeta2, total TGFbeta2, and Fibroblast growth factor 2 (FGF2) increased in the aqueous humor and lens. Loss of Smad3 attenuated, but did not completely abolish, EMT in the lens epithelium. CONCLUSIONS: Topical alkali treatment of the ocular surface readily induces an EMT-type anterior subcapsular cataract. Smad3 signaling is involved, but not required, for achievement of EMT in the lens epithelium in this cataract model.

Transcriptional activation in lens epithelial cells following an ocular blunt trauma.

PURPOSE: To determine whether an ocular blunt trauma activates anterior ocular segment (cornea and lens) by examining the expression patterns of c-fos and c-jun mRNAs in these tissues of an eye of adult rat following a blunt trauma. SETTING: Department of Ophthalmology, Wakayama Medical University School of Medicine, Kimiidera, Wakayama, Japan. METHODS: Adult Wistar rats (n=36) were generally anesthetized by ether inhalation. One eye was hit with an iron sphere (30 gram) that fell to the eye from 1 m. After the procedure, the animals were killed and the affected eye was enucleated at 15, 30, 60, 120, and 180 minutes. In situ hybridization using radiolabeled oligoprobes was used to detect mRNAs of c-fos and c-jun in tissue. RESULTS: The c-fos and c-jun mRNAs were not detected in the epithelium of uninjured cornea and lens by in situ hybridization. The mRNAs for c-fos and c-jun were then detected in corneal epithelium from 15 to 60 minutes posttreatment, and were no longer observed thereafter. In lens epithelium, mRNA for c-fos or c-jun were transiently detected from 15 to 60 minutes or 30 minutes posttreatment, respectively. CONCLUSION: The c-fos and c-jun mRNAs were transiently expressed in corneal and lens epithelial cells after blunt trauma. Ocular blunt trauma activates corneal and lens epithelial cells without apparent corneal ablation or direct injury in the lens epithelium. Such activation in lens epithelium might be involved in cataractogenesis.

[Investigation of mechanism of cell proliferation regulation and its clinical application].

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.