Ken-ichi Miyazaki

Fibrotic disorders in the eye: targets of gene therapy.

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

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

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

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.

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.

Interleukin-7 Modulates Extracellular Matrix Production and TGF-β Signaling in Cultured Human Subconjunctival Fibroblasts

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-β (TGF-β) in cultured human subconjunctival fibroblasts. IL-7 is capable of inducing Smad7, an inhibitory Smad that interferes with TGF-β/Smad signal. Methods: The effects of IL-7 on ECM production, immunolocalization of Smads, type I collagen, fibronectin, α -smooth muscle actin (α -SMA), and cell migration were examined in human subconjunctival fibroblast culture with or without TGF-β1. 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-β1 and type I collagen α1 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-1β than in the control fibroblasts. TGF-β1 strongly showed immunolocalization of phospho-Smad2, and IL-7 also showed immunolocalization of Smad7 in the nuclei. The immunoreactivities of α -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-β1 and col1A1. Conclusions: These findings indicate that IL-7 is involved in ECM production in the subconjunctival fibroblasts activated by exogenous TGF-β1, suggesting that administration of IL-7 can be a novel therapeutic strategy in preventing undesirable bleb scar formation during healing after filtration surgery.

Suppression of Injury-Induced Conjunctiva Scarring by Peroxisome Proliferator-Activated Receptor γ Gene Transfer in Mice

PURPOSE To examine the effects of introduction of the adenoviral peroxisome proliferator-activated receptor (PPAR)-gamma gene on postinjury conjunctival scarring in mice. Its effects on fibrogenic reaction of cultured human subconjunctival fibroblasts (hSCFs) were also evaluated. METHODS The effects of PPARgamma gene introduction on expression of type I collagen, fibronectin, and connective tissue growth factor (CTGF) in hSCFs were examined. A circumferential incision was made in the equatorial conjunctiva of the right eye of generally anesthetized adult C57BL/6 mice (n = 72). PPARgamma cDNA-expressing adenoviral vector was topically applied; the control eye received nonfunctioning adenoviral vector. At 2, 5, 7, and 14 days (each, n = 18), the eyes were processed for histologic or immunohistochemical examination to evaluate tissue scarring. Expression of type I collagen and growth factors was evaluated by real-time reverse transcription-polymerase chain reaction in 32 eyes from control and treatment groups. RESULTS PPARgamma overexpression suppressed type I collagen, fibronectin, and CTGF in cultured hSCFs at the mRNA or protein level. In vivo experiments showed that PPARgamma gene introduction suppressed monocyte/macrophage invasion, generation of myofibroblasts, and mRNA upregulation of cytokines/growth factors and collagen Ialpha2 chain (Col 1A2) in healing conjunctiva. CONCLUSIONS PPARgamma gene transfer suppresses the fibrogenic reaction in hSCFs as well as the injury-induced scarring of conjunctival tissue in mice, suggesting the effectiveness of this strategy in preventing excess scarring after filtration surgery. The mechanism may include suppression of activation of fibroblasts and reduction of macrophage invasion.

Treatment of eyelid epithelial neoplasm by targeting sonic hedgehog signaling : An experimental study

PurposeTo evaluate the effect of cyclopamine, an inhibitor of the Sonic hedgehog (Shh) signal, on the growth of an epithelial neoplasm.MethodsChemically 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 μg/ml recombinant Shh (rShh) and either 0 or 100 μM 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.ResultsHistology 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.ConclusionsCyclopamine 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. Jpn J Ophthalmol 2006;50:305–311