Ai Kitano

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.

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 β 1 (TGFβ1) and other proinflammatory gene expression in cultured ocular fibroblasts, but did not affect TGFβ1 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.

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.

Corneal wound healing in an osteopontin-deficient mouse.

PURPOSE To investigate the effects of loss of osteopontin (OPN) in the healing of the injured cornea in mice. Cell culture study was also conducted to clarify the effects of OPN in fibroblast behaviors. METHODS Ocular fibroblasts from wild-type (WT) and OPN-null (KO) mice were used to study the role of OPN on cell behavior. The effect of the lack of OPN on corneal wound healing was evaluated in mice. RESULTS In cell culture, OPN is involved in cell adhesion and in the migration of ocular fibroblasts. Adhesion of the corneal epithelial cell line was not affected by exogenous OPN. OPN was upregulated in a healing, injured mouse cornea. Loss of OPN did not affect epithelial healing after simple epithelial debridement. Healing of an incision injury in cornea was delayed, with less appearance of myofibroblasts and transforming growth factor beta1 expression in a KO mouse than in a WT mouse. The absence of OPN promoted tissue destruction after an alkali burn, resulting in a higher incidence of corneal perforation in KO mice than in WT mice. CONCLUSIONS OPN modulates wound healing-related fibroblast behavior and is required to restore the physiological structure of the cornea after wound healing.

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 α-smooth muscle action (αSMA), a marker of EMT, collagen type I, transforming growth factor β1 (TGFβ1), TGFβ2, 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 αSMA and collagen I, was altered in the absence of OPN with reduced phospho-Smad2/3 signaling. Upregulation of TGFβ1 and TGFβ2 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.

Genipin suppresses subconjunctival fibroblast migration, proliferation and myofibroblast transdifferentiation.

Purpose: Inchin-ko-to is a herbal medicine which has therapeutic effects in ameliorating liver fibrosis or cholestatic liver diseases. Its main bioactive component is genipin, which is an intestinal bacterial metabolite of this medication. Accordingly, we determined whether or not Inchin-ko-to suppresses in a wound healing model subconjunctival fibroblast (SCF) migration proliferation and myofibroblast transdifferentiation since an inhibitory effect could be of value in improving trabeculotomy outcome. Methods: Effects of genipin on SCF cell migration were examined subsequent to wounding confluent monolayer cultures. Alamar blue staining evaluated the effects of genipin (0–50 µg/ml) on fibroblast cell proliferation. Immunostaining determined α-smooth muscle actin (αSMA) expression. Western blotting evaluated (αSMA) expression and phospho-Smad2 formation. Real-time RT-PCR evaluated TGFβ1 and collagen Iα2 mRNA expression. Enzyme-immunoassay determined culture medium collagen I content. Results: Genipin suppressed wound-induced cell migration and proliferation. It also decreased collagen type I TGFβ1 and αSMA mRNA and protein expression. Smad2 signaling was inhibited by genipin in a dose-dependent manner. Conclusion: Genipin suppresses injury-induced fibrogenic responses in SCFs. This result suggests that the herbal medicine Inchin-ko-to might have therapeutic value following trabeculotomy.

Connective tissue growth factor modulates extracellular matrix production in human subconjunctival fibroblasts and their proliferation and migration in vitro

PurposeWe examined the role of connective tissue growth factor (CTGF) in transforming growth factor β1 (TGFβ1)-related behavior in cultured human subconjunctival fibroblasts (SCFs), protein production, mRNA expression of CTGF and type I collagen α1 chain (colIA1), and cell proliferation and migration. TGFβ1 is the major factor involved in bleb scarring following filtration surgery.MethodsAn antisense deoxynucleotide (antisense) (5 μM) for CTGF mRNA was used to block endogenous CTGF expression. Effects of antisense on extracellular matrix (ECM) production and immunolocalization, mRNA expression, and cell proliferation and migration were examined in human SCF cultures with or without TGFβ1 (5 ng/ml). Cell migration was examined in an in vitro wound model of monolayer fibroblast cultures.ResultsCTGF antisense reduced mRNA expression of CTGF and colIA1 and production of the ECM components type I collagen, and fibronectin much more markedly in cells treated with TGFβ1 compared with control fibroblasts, and it inhibited the proliferation of cultured SCFs to 71.9% of that of controls after 13 days of culture. CTGF antisense also delayed defect closure in monolayer cell sheets. In the culture, the defect was closed by TGFβ1 by 36 h, whereas 7.0% of the defect remained at 48 h in the antisense-treated culture.ConclusionsThese findings indicate that CTGF is involved in ECM production in SCFs activated by exogenous TGFβ1 in vitro. Inhibition of CTGF expression may be effective in preventing undesirable scar formation during healing following filtration surgery.

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.

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.