Tomoko Orita

Protection of Human Corneal Epithelial Cells from TNF-α-Induced Disruption of Barrier Function by Rebamipide.

PURPOSE TNF-α disrupts the barrier function of cultured human corneal epithelial (HCE) cells. We investigated the effects of the cytoprotective drug rebamipide on this barrier disruption by TNF-α as well as on corneal epithelial damage in a rat model of dry eye. METHODS The barrier function of HCE cells was evaluated by measurement of transepithelial electrical resistance. The distribution of tight-junction (ZO-1, occludin) and adherens-junction (E-cadherin, β-catenin) proteins, and the p65 subunit of nuclear factor-κB (NF-κB) was determined by immunofluorescence microscopy. Expression of junctional proteins as well as phosphorylation of the NF-κB inhibitor IκB-α and myosin light chain (MLC) were examined by immunoblot analysis. A rat model of dry eye was developed by surgical removal of exorbital lacrimal glands. RESULTS Rebamipide inhibited the disruption of barrier function as well as the downregulation of ZO-1 expression, and the disappearance of ZO-1 from the interfaces of neighboring HCE cells induced by TNF-α. It also inhibited the phosphorylation and downregulation of IκB-α, the translocation of p65 to the nucleus, the formation of actin stress fibers, and the phosphorylation of MLC induced by TNF-α in HCE cells. Treatment with rebamipide eyedrops promoted the healing of corneal epithelial defects as well as attenuated the loss of ZO-1 from the surface of corneal epithelial cells in rats. CONCLUSIONS Rebamipide protects corneal epithelial cells from the TNF-α-induced disruption of barrier function by maintaining the distribution and expression of ZO-1 as well as the organization of the actin cytoskeleton. Rebamipide is, thus, a potential drug for preventing or ameliorating the loss of corneal epithelial barrier function associated with ocular inflammation.

Inhibition by medroxyprogesterone acetate of interleukin-1β-induced collagen degradation by corneal fibroblasts.

PURPOSE To examine the effect of medroxyprogesterone 17-acetate (MPA) on interleukin-1β (IL-1β)-induced collagen degradation by corneal fibroblasts. METHODS Rabbit corneal fibroblasts were cultured in three-dimensional collagen gels with or without MPA. Collagen degradation was determined by measurement of hydroxyproline after acid hydrolysis. The expression or activity of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) was evaluated by immunoblot analysis or gelatin zymography. The phosphorylation of mitogen-activated protein kinases (MAPKs) in corneal fibroblasts was examined by immunoblot analysis. Cell proliferation and viability were evaluated by measurement of bromodeoxyuridine incorporation and the release of lactate dehydrogenase, respectively. RESULTS MPA inhibited IL-1β-induced collagen degradation by corneal fibroblasts in a concentration- and time-dependent manner. MMP expression and activation as well as TIMP expression in corneal fibroblasts exposed to IL-1β were also inhibited by MPA. MPA had no effect on cell proliferation or viability. MPA inhibited the IL-1β-induced phosphorylation of p38 MAPK without affecting that of the MAPKs ERK or JNK. IL-1β-induced MMP expression and activation as well as collagen degradation were also blocked by the p38 MAPK inhibitor SB203580. CONCLUSIONS MPA inhibited MMP expression and thereby suppressed collagen degradation by corneal fibroblasts induced by IL-1β. Furthermore, inhibition of p38 MAPK phosphorylation by MPA may contribute to its inhibition of collagen degradation.

Inhibition by Female Sex Hormones of Collagen Gel Contraction Mediated by Retinal Pigment Epithelial Cells

PURPOSE Collagen contraction mediated by retinal pigment epithelial (RPE) cells contributes to the pathogenesis of proliferative vitreoretinopathy (PVR). We examined the effects of sex hormones on this process. METHODS Mouse RPE cells were cultured in a type I collagen gel and exposed to 17β-estradiol, progesterone, or dehydro-epiandrosterone. Collagen contraction induced by transforming growth factor-β2 (TGF-β2) was determined by measurement of gel diameter. Expression of α-smooth muscle actin (α-SMA), as well as phosphorylation of Smad2 and myosin light chain (MLC), was examined by immunoblot analysis. Matrix metalloproteinase (MMP) release was evaluated by gelatin zymography. Fibronectin and interleukin-6 secretion was measured with immunoassays. RESULTS The female sex hormones 17β-estradiol and progesterone inhibited TGF-β2-induced collagen contraction mediated by RPE cells, whereas the male sex hormone dehydro-epiandrosterone had no such effect. The TGF-β2-induced release of MMP-2 and MMP-9 from RPE cells was also inhibited by 17β-estradiol and progesterone, and the MMP inhibitor GM6001 attenuated TGF-β2-induced collagen contraction. Expression of the mesenchymal markers α-SMA and fibronectin, interleukin-6 release, and Smad2 and MLC phosphorylation induced by TGF-β2 were all inhibited by 17β-estradiol and progesterone. Immunohistochemical analysis also detected nuclear immunoreactivity for estrogen and progesterone receptors in proliferative fibrocellular membranes of PVR patients. CONCLUSIONS Female sex hormones inhibited TGF-β2-induced collagen contraction mediated by RPE cells. This action appeared to be mediated through inhibition both of MMP, α-SMA, and fibronectin expression as well as of Smad2 and MLC phosphorylation. Female sex hormones might thus prove effective for the treatment of PVR.

Upregulation of Matrix Metalloproteinase Expression by Poly(I:C) in Corneal Fibroblasts: Role of NF-κB and Interleukin-1β

PURPOSE To characterize the mechanism of corneal ulceration associated with viral keratitis, the authors investigated the effects of polyinosinic-polycytidylic acid [poly(I:C)], a synthetic analog of viral double-stranded RNA, on the expression of matrix metalloproteinases (MMPs) in human corneal fibroblasts. METHODS The expression of MMPs in human corneal fibroblasts cultured in the absence or presence of poly(I:C) was examined by immunoblot analysis, gelatin zymography, and quantitative reverse transcription-polymerase chain reaction analysis. The phosphorylation of the NF-κB inhibitor protein IκB-α was assessed by immunoblot analysis, and the concentration of interleukin (IL)-1β in culture supernatants was measured by enzyme-linked immunosorbent assay. RESULTS Poly(I:C) increased the expression of MMP-1 and MMP-3 in corneal fibroblasts at the mRNA and protein levels. It also induced the phosphorylation of IκB-α and the secretion of IL-1β in these cells. The poly(I:C)-induced expression of MMP-1 and MMP-3 was attenuated by a synthetic inhibitor of NF-κB signaling and by IL-1 receptor antagonist; the latter also inhibited poly(I:C)-induced phosphorylation of IκB-α. CONCLUSIONS Poly(I:C) induces the expression of MMP-1 and MMP-3 in human corneal fibroblasts in a manner dependent on activation of the transcription factor NF-κB and on IL-1β secretion. These effects may play an important role in corneal ulceration associated with viral keratitis.

Attenuation of EMT in RPE cells and subretinal fibrosis by an RAR-γ agonist

Subretinal fibrosis contributes to the loss of vision associated with age-related macular degeneration (AMD). Retinal pigment epithelial (RPE) cells play a key role in the pathogenesis of AMD including the fibrotic reaction. We examined the role of retinoic acid receptor-γ (RAR-γ) in the epithelial-mesenchymal transition (EMT) and other fibrosis-related processes in mouse RPE cells cultured in a type I collagen gel. Transforming growth factor-β2 (TGF-β2)–induced collagen gel contraction mediated by the RPE cells was inhibited by the RAR-γ agonist R667 in a concentration- and time-dependent manner. Expression of the mesenchymal markers α-smooth muscle actin and fibronectin, the release of interleukin-6, and the phosphorylation of paxillin, mitogen-activated protein kinases (ERK, p38, and JNK), Smad2, and AKT induced by TGF-β2 were also suppressed by the RAR-γ agonist. Furthermore, gelatin zymography and immunoblot analysis revealed that the TGF-β2-induced release of matrix metalloproteinase (MMP)-2, MMP-3, MMP-8, and MMP-9 from RPE cells was inhibited by R667, and the MMP inhibitor GM6001 attenuated TGF-β2-induced RPE cell contraction. Finally, immunohistofluorescence analysis with antibodies to glial fibrillary acidic protein showed that R667 inhibited the development of subretinal fibrosis in a mouse model in vivo. Our results thus suggest that RAR-γ agonists may prove effective for the treatment of subretinal fibrosis associated with AMD.Key messageRAR-γ agonist R667 suppressed collagen gel contraction mediated by RPE cells.Epithelial-mesenchymal transition (EMT) in RPE cells was inhibited by RAR-γ agonist R667.RAR-γ agonist R667 inhibited fibrosis-related processes in RPE cells.RAR-γ agonists may attenuate AMD-associated fibrosis.

Inhibition by All-Trans-Retinoic Acid of Transforming Growth Factor-β–Induced Collagen Gel Contraction Mediated by Human Tenon Fibroblasts

PURPOSE Excessive wound contraction can lead to scar formation in the conjunctiva. The effects of all-trans-retinoic acid (ATRA) on the contractility of human Tenon fibroblasts (HTFs) cultured in three-dimensional (3D) collagen gels were investigated. METHODS Human Tenon fibroblasts were cultured in 3D gels of type I collagen and in the absence or presence of TGF-β, ATRA, or various inhibitors. Collagen gel contraction was evaluated by measurement of gel diameter. Phosphorylation of various signaling molecules was examined by immunoblot analysis. The formation of actin stress fibers and focal adhesions was detected by laser confocal microscopy. RESULTS All-trans-retinoic acid inhibited TGF-β-induced collagen gel contraction mediated by HTFs in a concentration- and time-dependent manner. The TGF-β-induced phosphorylation of focal adhesion kinase (FAK) and formation of stress fibers and focal adhesions in HTFs were attenuated by ATRA. All-trans-retinoic acid also inhibited the TGF-β-induced phosphorylation of the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase (ERK), p38, and c-Jun NH2-terminal kinase (JNK) as well as that of c-Jun and Smad2/3. Furthermore, TGF-β-induced collagen gel contraction was blocked by inhibitors of ERK, p38, or JNK signaling. CONCLUSIONS All-trans-retinoic acid inhibited TGF-β-induced collagen gel contraction mediated by HTFs, most likely by attenuating the formation of actin stress fibers and focal adhesions as well as signaling by MAPKs, c-Jun, and Smads. All-trans-retinoic acid may therefore prove effective for inhibition of conjunctival scarring through attenuation of the contractility of Tenon fibroblasts.

Poly(I:C)-Induced Adhesion Molecule Expression Mediated by NF-κB and Phosphoinositide 3-Kinase–Akt Signaling Pathways in Human Corneal Fibroblasts

PURPOSE Viral infection at the ocular surface can lead to the chronic condition of viral stromal keratitis. Polyinosinic-polycytidylic acid [poly(I:C)], an analog of viral double-stranded RNA, induces the expression of adhesion molecules in cultured corneal fibroblasts. The authors investigated the roles of nuclear factor (NF)-κB and phosphoinositide 3-kinase (PI3K)-Akt signaling pathways in the poly(I:C)-induced expression of adhesion molecules in corneal fibroblasts. METHODS Human corneal fibroblasts were cultured with poly(I:C) in the absence or presence of IκB kinase 2 (IKK2) inhibitor (an inhibitor of NF-κB activation) or the PI3K inhibitor LY294002. The expression of vascular cell adhesion molecule (VCAM)-1, intercellular adhesion molecule (ICAM)-1, ICAM-2, and E-selectin, as well as the phosphorylation of the NF-κB inhibitory protein IκB-α and Akt, were examined by immunoblot analysis. The subcellular localization of adhesion molecules was determined by immunofluorescence analysis. RESULTS Poly(I:C) increased the expression of VCAM-1 and ICAM-1 but not that of ICAM-2 or E-selectin in corneal fibroblasts. Poly(I:C) also induced the phosphorylation of IκB-α and Akt. The poly(I:C)-induced expression of VCAM-1 and ICAM-1 was attenuated by both IKK2 inhibitor and LY294002. CONCLUSIONS The NF-κB and PI3K-Akt signaling pathways mediate the poly(I:C)-induced upregulation of VCAM-1 and ICAM-1 in corneal fibroblasts, with PI3K acting upstream of NF-κB activation. These pathways thus likely modulate local immune and inflammatory responses to viral infection in the corneal stroma.

Cytokine and chemokine secretion induced by poly(I:C) through NF-κB and phosphoinositide 3-kinase signaling pathways in human corneal fibroblasts.

Purpose/Aim: Viral infection of the cornea can result in inflammation and scarring and eventually lead to blindness. Polyinosinic-polycytidylic acid [poly(I:C)], an analog of viral double-stranded RNA, induces the secretion of cytokines and chemokines from cultured corneal fibroblasts. We have now investigated the role of nuclear factor (NF)-κB and phosphoinositide 3-kinase (PI3K) signaling pathways in poly(I:C)-induced cytokine and chemokine secretion from corneal fibroblasts. Materials and Methods: Human corneal fibroblasts were cultured with poly(I:C) in the absence or presence of IKK-2 inhibitor or LY294002, which are inhibitors of NF-κB and PI3K signaling, respectively. The release of the pro-inflammatory cytokine interleukin (IL)-6 and the chemokines IL-8, IP-10, and RANTES from the cells was measured with an enzyme-linked immunosorbent assay. Results: Poly(I:C) induced the secretion of IL-6, IL-8, IP-10, and RANTES from corneal fibroblasts. Whereas the poly(I:C)-induced secretion of IL-6, IP-10, and RANTES was inhibited by both IKK-2 inhibitor and LY294002, that of IL-8 was blocked only by IKK-2 inhibitor. Conclusions: The poly(I:C)-induced secretion of IL-6, IP-10, and RANTES from human corneal fibroblasts is mediated by both NF-κB and PI3K signaling pathways, whereas that of IL-8 is mediated by the NF-κB pathway. These signaling pathways thus likely contribute to local inflammation in the corneal stroma induced by viral infection.

Role of the JNK Signaling PathWay in Downregulation of Connexin43 by TNF-α in Human Corneal Fibroblasts

Abstract Purpose/Aim: Proinflammatory cytokines such as tumor necrosis factor (TNF)-α contribute to corneal inflammation. Corneal stromal fibroblasts are connected to each other via gap junctions. We have now examined the role of mitogen-activated protein kinase (MAPK) signaling pathways in TNF-α-induced downregulation of the gap junction protein connexin43 (Cx43) in human corneal fibroblasts. Materials and Methods: Cultured human corneal fibroblasts were exposed to TNF-α in the absence or presence of inhibitors of MAPK signaling pathways. Expression of Cx43 was evaluated by immunofluorescence and immunoblot analyses. Gap-junctional intercellular communication (GJIC) was measured with a dye-coupling assay. Results: TNF-α reduced the abundance of Cx43 in human corneal fibroblasts (as revealed by immunoblot analysis) as well as induced the loss of specific staining for this protein (as revealed by immunofluorescence analysis). These effects of TNF-α were attenuated by an inhibitor of c-Jun NH2-terminal kinase (JNK inhibitor II) but not by inhibitors of signaling by extracellular signal-regulated kinase (PD98059) or by p38 MAPK (SB203580). JNK inhibitor II also attenuated the inhibitory effect of TNF-α on GJIC. Conclusions: The inhibitory effects of TNF-α on Cx43 expression and GJIC in human corneal fibroblasts are mediated, at least in part, by the JNK signaling pathway, which therefore likely plays a role in corneal inflammation.

Identification of common secreted factors in human corneal fibroblasts exposed to LPS, poly(I:C), or zymosan.

Infection of the cornea with bacteria, viruses, or fungi can result in corneal ulceration. Corneal stromal cells participate in the immune and inflammatory responses to such infection in part by producing various cytokines and chemokines. The effects of lipopolysaccharide (LPS), polyinosinic-polycytidylic acid [poly(I:C)], and zymosan as surrogates for bacteria, viruses, and fungi, respectively, on the release of cytokines and chemokines from cultured human corneal fibroblasts were examined in order to identify common factors in infectious corneal keratitis. The secretion of various cytokines and chemokines by human corneal fibroblasts exposed to LPS, poly(I:C), or zymosan was measured with a multiplex assay system. LPS induced the release of interleukin (IL)-6, IL-8, MCP-1, RANTES, IP-10, eotaxin, and IL-12 from corneal fibroblasts. Poly(I:C) stimulated the secretion of IL-6, IL-8, MCP-1, RANTES, IP-10, eotaxin, MIP-1β, and interferon-γ, whereas zymosan triggered the production of IL-6, IL-8, and MCP-1. LPS, poly(I:C), and zymosan thus each induced a distinct pattern of cytokine and chemokine release from human corneal fibroblasts, with the release of IL-6, IL-8, and MCP-1 being commonly elicited by all three agents. Our results suggest that IL-6, IL-8, and MCP-1 may therefore play a key role in the inflammatory response to corneal infection.