PRELP Regulates Cell-Cell Adhesion and EMT and Inhibits Retinoblastoma Progression.

Retinoblastoma (RB) is the most common intraocular pediatric cancer. Nearly all cases of RB are associated with mutations compromising the function of the RB1 tumor suppressor gene. We previously demonstrated that PRELP is widely downregulated in various cancers and our in vivo and in vitro analysis revealed PRELP as a novel tumor suppressor and regulator of EMT. In addition, PRELP is located at chromosome 1q31.1, around a region hypothesized to be associated with the initiation of malignancy in RB. Therefore, in this study, we investigated the role of PRELP in RB through in vitro analysis and next-generation sequencing. Immunostaining revealed that PRELP is expressed in Müller glial cells in the retina. mRNA expression profiling of PRELP-/- mouse retina and PRELP-treated RB cells found that PRELP contributes to RB progression via regulation of the cancer microenvironment, in which loss of PRELP reduces cell-cell adhesion and facilitates EMT. Our observations suggest that PRELP may have potential as a new strategy for RB treatment.

Concentration of acute phase factors in vitreous fluid in diabetic macular edema.

PURPOSE: Diabetic retinal maculopathy is associated with acute and chronic local inflammation. We measured the concentrations of acute phase factors in vitreous fluid of patients with diabetic macular edema (DME) and examined their relations to visual acuity and central retinal thickness (CRT) both before and after vitrectomy. STUDY DESIGN: Retrospective. METHODS: Vitreous fluid was collected during vitreoretinal surgery from 19 patients with DME and 12 control subjects with epiretinal membrane. The concentrations of acute phase factors (α2-macroglobulin, haptoglobin, C-reactive protein, serum amyloid P and A, procalcitonin, ferritin, tissue plasminogen activator, fibrinogen) and vascular endothelial growth factor (VEGF) were measured with multiplex assays. CRT of macular edema was measured by optical coherence tomography (OCT). RESULTS: The levels of serum amyloid P, procalcitonin, ferritin, and fibrinogen in vitreous fluid were increased in DME patients compared with control subjects. The levels of procalcitonin and fibrinogen in DME patients were inversely correlated with visual acuity both before and 3 months after vitrectomy but not 6 months postsurgery. The concentrations of these four factors were not correlated with either CRT or the vitreous levels of VEGF in DME patients. CONCLUSION: Acute phase factors may contribute to local inflammation in DME and may therefore influence disease progression.

Combination management by C-arm fluoroscopy and extraocular muscle severance for penetrating ocular trauma with a retrobulbar foreign body.

We report here the successful removal of a retrobulbar metallic foreign body in a patient with penetrating ocular trauma by a transconjunctival approach and combination management with C-arm fluoroscopy and extraocular muscle severance. A 37-year-old man sustained a penetrating injury to the right eye while using an iron hammer. Initial slitlamp examination revealed a corneoscleral laceration, iridocele, anterior chamber collapse, and a traumatic cataract. Visual acuity in the right eye was limited to the perception of hand motion. Computed tomography revealed an orbital foreign body in the retrobulbar area. The patient underwent corneoscleral suturing, severance of extraocular muscles, removal of the foreign body with guidance by C-arm fluoroscopy, pars plana lensectomy, and pars plana vitrectomy. Combination management with C-arm fluoroscopy and extraocular muscle severance may thus be a suitable approach to the removal of a retrobulbar metallic foreign body.

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

UNLABELLED: 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-ß2 (TGF-ß2)-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-ß2 were also suppressed by the RAR-γ agonist. Furthermore, gelatin zymography and immunoblot analysis revealed that the TGF-ß2-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-ß2-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 MESSAGE: RAR-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 a retinoic acid receptor γ agonist of extracellular matrix remodeling mediated by human Tenon fibroblasts.

PURPOSE: Scar formation is most frequently responsible for the failure of glaucoma filtration surgery. Retinoic acids are vitamin A derivatives that play diverse roles in development, immunity, and tissue repair. The effects of the retinoic acid receptor (RAR) γ agonist R667 on the contractility of human Tenon fibroblasts (HTFs) cultured in a three-dimensional collagen gel as well as on intraocular pressure (IOP) in a rat model of glaucoma filtration surgery were investigated. METHODS: HTFs were cultured in a type I collagen gel, the contraction of which was evaluated by measurement of the gel diameter. The release of matrix metalloproteinases (MMPs) into culture supernatants was assessed with immunoblot analysis and gelatin zymography. Phosphorylation of focal adhesion kinase (FAK) was examined with immunoblot analysis, and production of fibronectin and type I collagen was measured with immunoassays. RESULTS: R667 inhibited transforming growth factor-ß1 (TGF-ß1)-induced collagen gel contraction mediated by HTFs in a concentration- and time-dependent manner, whereas an RARα agonist inhibited this process to a lesser extent and an RARß agonist had no effect. TGF-ß1-induced MMP-1 and MMP-3 release, FAK phosphorylation, and fibronectin and type I collagen production in HTFs were also attenuated by R667. Furthermore, R667 lowered IOP in rats after glaucoma filtration surgery. CONCLUSIONS: R667 inhibited TGF-ß1-induced contraction and extracellular matrix synthesis in HTFs. Such effects might have contributed to the lowering of IOP by R667 in a rat model of glaucoma filtration surgery. RARγ agonists might thus prove effective for inhibition of scar formation after such surgery.

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 17ß-estradiol, progesterone, or dehydro-epiandrosterone. Collagen contraction induced by transforming growth factor-ß2 (TGF-ß2) 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 17ß-estradiol and progesterone inhibited TGF-ß2-induced collagen contraction mediated by RPE cells, whereas the male sex hormone dehydro-epiandrosterone had no such effect. The TGF-ß2-induced release of MMP-2 and MMP-9 from RPE cells was also inhibited by 17ß-estradiol and progesterone, and the MMP inhibitor GM6001 attenuated TGF-ß2-induced collagen contraction. Expression of the mesenchymal markers α-SMA and fibronectin, interleukin-6 release, and Smad2 and MLC phosphorylation induced by TGF-ß2 were all inhibited by 17ß-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-ß2-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.

Role of the JNK signaling pathway in downregulation of connexin43 by TNF-α in human corneal fibroblasts.

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.

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, ß-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.

Role of ß-Pix in corneal epithelial cell migration on fibronectin.

PURPOSE: Corneal epithelial migration during wound healing is important for maintenance of corneal transparency, and fibronectin plays a key role in regulation of the adhesion and migration of corneal epithelial cells. The role of ß-Pix in intracellular signaling that underlies the stimulatory effects of fibronectin on the adhesion and migration of corneal epithelial cells was examined. METHODS: Simian virus 40-transformed human corneal epithelial (HCE) cells were cultured on fibronectin or on bovine serum albumin as a control. The localization and tyrosine phosphorylation of ß-Pix were examined by immunofluorescence and immunoprecipitation analyses, respectively. The actin cytoskeleton and focal adhesions were detected by staining of cells with rhodamine-phalloidin and antibodies to phosphotyrosine, respectively. The effects of depletion of ß-Pix on HCE cell adhesion and migration on fibronectin were investigated by cell transfection with a small interfering RNA specific for ß-Pix mRNA. RESULTS: Fibronectin induced the tyrosine phosphorylation of ß-Pix as well as its apparent accumulation at focal adhesions in HCE cells. Depletion of ß-Pix inhibited the effects of fibronectin on remodeling of the actin cytoskeleton and the formation of focal adhesions. It also inhibited the migration of HCE cells on fibronectin in an in vitro model of wound healing, but it did not affect cell adhesion to fibronectin. CONCLUSIONS: ß-Pix contributes to the regulation of the formation of focal adhesions as well as that of cell migration by fibronectin in HCE cells. This protein therefore likely plays an important role in signal transduction underlying corneal epithelial wound healing.

Inhibition by female sex hormones of collagen degradation by corneal fibroblasts.

PURPOSE: Corneal fibroblasts contribute to collagen remodeling in the corneal stroma in part by mediating collagen degradation. Given that corneal structure is influenced by sex hormone status, we examined the effects of sex hormones on collagen degradation by corneal fibroblasts. METHODS: Rabbit corneal fibroblasts were cultured in three-dimensional collagen gels with or without sex hormones including 17ß-estradiol, progesterone, testosterone, and dehydroepiandrosterone (DHEA). Collagen degradation was determined by measurement of hydroxyproline after acid hydrolysis. The expression and activity of matrix metalloproteinases (MMPs) were evaluated by immunoblot analysis and gelatin zymography. The phosphorylation of mitogen-activated protein kinases (MAPKs) and the nuclear factor-kappa B (NF-κB) inhibitor NF kappa B Inhibitor-alpha (IκB-α) 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: 17ß-Estradiol and progesterone each inhibited interleukin (IL)-1ß-induced collagen degradation by corneal fibroblasts in a concentration-dependent manner, whereas testosterone and DHEA had no such effect. MMP expression and activation in corneal fibroblasts exposed to IL-1ß were also inhibited by 17ß-estradiol and progesterone. These female sex hormones did not affect cell proliferation or viability. Both 17ß-estradiol and progesterone inhibited the IL-1ß-induced phosphorylation of p38 MAPK without affecting that of the MAPKs extracellular Signal-regulated Kinase (ERK) or c-jun N-terminal kinase (JNK). 17ß-Estradiol also inhibited the IL-1ß-induced phosphorylation of IκB-α. CONCLUSIONS: 17ß-Estradiol and progesterone inhibited MMP expression and activity in IL-1ß-stimulated corneal fibroblasts and thereby suppressed collagen degradation by these cells.