Inhibition of transient receptor potential vanilloid 1 reduces shedding and transmission during Streptococcus pneumoniae co-infection with influenza.

Transmission is the first step for a microorganism to establish colonization in the respiratory tract and subsequent development of infectious disease. Streptococcus pneumoniae is a leading pathogen that colonizes the mucosal surfaces of the human upper respiratory tract and causes subsequent transmission and invasive infections especially in co-infection with influenza A virus. Host factors contributing to respiratory contagion are poorly understood. Transient receptor potential vanilloid (TRPV) channels have various roles in response to microoorganism. Inhibition of TRPV exacerbates invasive infection by Streptococcus pneumoniae, but it is unclear how TRPV channels influence pneumococcal transmission. Here, we describe the effect of inhibition of TRPV1 on pneumococcal transmission. We adopted a TRPV1-deficient infant mouse model of pneumococcal transmission during co-infection with influenza A virus. We also analyzed the expression of nasal mucin or pro-inflammatory cytokines. TRPV1 deficiency attenuated pneumococcal transmission and shedding during co-infection with influenza A virus. TRPV1 deficiency suppressed the expression of nasal mucin. In addition, there were increases in the expression of tumor necrosis factor-α and type I interferon, followed by the suppressed replication of influenza A virus in TRPV1-deficient mice. Inhibition of TRPV1 was shown to attenuate pneumococcal transmission by reducing shedding through the suppression of nasal mucin during co-infection with influenza A virus. Inhibition of TRPV1 suppressed nasal mucin by modulation of pro-inflammatory responses and regulation of replication of influenza A virus. TRPV1 could be a new target in preventive strategy against pneumococcal transmission.

Effects of Ripasudil Hydrochloride on Epithelial Repair in a Mouse Cornea.

PURPOSE: Effect of topical administration of a Rho kinase inhibitor, ripasudil, on epithelial wound healing in a mouse cornea was investigated. Effects of treatment of cultured human corneal epithelial cell (HCEC) line and organ-cultured corneal epithelium with ripasudil on expression of p-ERK was also examined. METHODS: Epithelial defects with a diameter of 2.0 mm were prepared in the central corneas of C57BL/6 mice with or without 1-week travoprost pre-treatment, to which ripasudil or PBS as a control was instilled every 6 h immediately after preparation. The mice eyes were cultured with or without travoprost for 24-hrs. The expression levels of p-ERK in epithelium of mice eyes were compared by immunostaining after further 24-hrs culture with or without ripasudil for 24-hrs. HCEC were cultured with or without ripasudil and processed for examination for proliferation activity and protein expression of p-ERK by either immunostaining or Western blotting. The cells were also treated with or without travoprost for 24-hrs, and were further cultured with or without ripasudil. Expression levels of p-ERK were examined by Western blotting. RESULTS: Ripasudil treatment suppressed post-debridement epithelial healing in association with reduced proliferation activity in peripheral (limbal) epithelium in cornea with or without pre-treatment with travoprost. Ripasudil treatment accelerated p-ERK expression. Ripasudil supplementation upregulated proliferation with increased p-ERK in HCEC. CONCLUSION: Ripasudil treatment promotes wound healing of the mouse corneal epithelium by enhancing cell proliferation on peripheral (limbal) epithelium.

Immunohistochemical findings of lens capsules obtained from patients with dead bag syndrome.

PURPOSE: To investigate the extracellular matrix and cellular components in lens capsules extracted from patients with dead bag syndrome (DBS) through immunohistochemistry. SETTING: Department of Ophthalmology, Wakayama Medical University School of Medicine, Wakayama, Japan, and Department of Ophthalmology and Visual Sciences, John A. Moran Eye Center, University of Utah, Salt Lake City, Utah. DESIGN: Immunohistochemical experimental study. METHODS: 9 capsular bag specimens from DBS cases, as well as 2 control specimens from late-postoperative in-the-bag intraocular lens dislocation cases related to previous vitrectomy, pseudoexfoliation, and blunt trauma were included. They were processed for histopathology; unstained sections were obtained from each one and analyzed by immunohistochemistry targeting collagen type IV, laminin, vimentin, collagen type I, and fibronectin. RESULTS: Immunohistochemistry in DBS showed lens capsule stained for basement membrane components. The outer part of the anterior capsule that was split from the inner part was more markedly stained for type IV collagen as compared with the posterior part. Faint staining for fibrous posterior capsular opacification (PCO) components, for example, collagen type I and fibronectin, was detected in limited areas, but the major portion of the capsule was free from these components. Small spotty vimentin-positive materials, suggesting the presence of cell debris, were also detected in limited samples. CONCLUSIONS: Small amounts of fibrotic PCO components were detected in capsules extracted from patients with DBS, but their major parts were free from PCO components. Current findings suggest small amounts of lens epithelial cells were present after surgery and secreted fibrous components before undergoing cell death process.

Impaired healing in an incision wound in corneal stroma in a lumican-null mouse.

PURPOSE: We investigated healing pattern of an incisional wound in corneal stroma of lumican-null (KO) mice. METHODS: C57BL/6 mice (wild-type, WT) and lumican-null (knockout, KO) mice were used. A linear full-thickness incision was produced in one cornea of each mouse. After intervals of healing, the corneas were processed for the following analyses. Histology was employed to measure the distance between each edge of the disrupted Descemet's membrane at the center of the cornea. Immunohistochemistry and real-time RT-PCR were employed to evaluate the expression of wound healing-related components in the tissue. Cultured ocular fibroblasts were obtained from cornea and sclera of WT and KO postnatal day 1 pups. The cells were subjected to examination for cell proliferation and expression of wound healing-related gene products. In vitro gel contraction assay was used to asses cell contractile activity of WT and KO cells. RESULTS: At day 5 of incision, the distance between the disrupted Descemet's membrane was larger in a KO mouse as compared with a WT mouse. Myofibroblast appearance in the wound was suppressed by the loss of lumican. The loss of lumican downregulated TGFß1's effects on mRNA expression of α-smooth muscle actin and collagen Ia1 in cultured ocular fibroblasts. Cell proliferation rate increased in injured stroma, which was further supported by in vitro datum of cell proliferation augmentation by the loss of lumican. Loss of lumican suppressed cell-mediated gel contraction. CONCLUSION: Loss of lumican perturbs the healing of penetrating incision in mouse corneal stroma in association with suppression of myofibroblast generation.

Engineered Knockout of TRPA1 Inhibits Laser-Induced Choroidal Neovascularization Along With Associated TGFß1 Expression and Neutrophil Infiltration.

We examined the effects of gene ablation and chemical inhibition of transient receptor potential ankyrin 1 (TRPA1) on the growth of experimental argon laser-induced choroidal neovascularization (CNV) in mice. CNV was induced in the eyes of 6- to 8-week-old TRPA1-null (knockout [KO]) and wild-type (WT) mice by argon laser irradiation. Gene expression analysis was performed in laser-injured tissues at days 1 and 3. CNV growth was evaluated at day 14. Reciprocal bone marrow transplantation was performed between each genotype to identify the components responsible for either recipient tissue or bone marrow-derived inflammatory cells. Our results show that laser irradiation successfully induced CNV growth at the site of laser injury. The size of induced CNV was significantly smaller in KO mice than in WT mice at day 14, as determined by angiography with fluorescein isothiocyanate-dextran. Invasion of neutrophils, but not macrophages, was suppressed in association with suppression of the expression of transforming growth factor ß1 and interleukin 6 in laser-irradiated KO tissue. Bone marrow transplantation indicated that the genotype of the recipient mouse, but not of inflammatory cells, is attributable to the KO phenotype. Systemic administration of a TRPA1 antagonist also reduced the CNV in a WT mouse. In conclusion, TRPA1 signaling in local cells is involved in growth of laser-induced CNV. The phenotype was not attributable to vascular endothelial cells and inflammatory cells. Blocking TRPA1 signal may therefore be a potential treatment strategy for CNV-related ocular diseases.

Tenascins and osteopontin in biological response in cornea.

The structural composition, integrity and regular curvature of the cornea contribute to the maintenance of its transparency and vision. Disruption of its integrity caused by injury results in scarring, inflammation and neovascularization followed by losses in transparency. These sight compromising effects is caused by dysfunctional corneal resident cell responses induced by the wound healing process. Upregulation of growth factors/cytokines and neuropeptides affect development of aberrant behavior. These factors trigger keratocytes to first transform into activated fibroblasts and then to myofibroblasts. Myofibroblasts express extracellular matrix components for tissue repair and contract the tissue to facilitate wound closure. Proper remodeling following primary repair is critical for restoration of transparency and visual function. Extracellular matrix components contributing to the healing process are divided into two groups; a group of classical tissue structural components and matrix macromolecules that modulate cell behaviors/activities besides being integrated into the matrix structure. The latter components are designated as matricellular proteins. Their functionality is elicited through mechanisms which modulate the scaffold integrity, cell behaviors, activation/inactivation of either growth factors or cytoplasmic signaling regulation. We discuss here the functional roles of matricellular proteins in mediating injury-induced corneal tissue repair. The roles are described of major matricellular proteins, which include tenascin C, tenascin X and osteopontin. Focus is directed towards dealing with their roles in modulating individual activities of wound healing-related growth factors, e. g., transforming growth factor ß (TGF ß). Modulation of matricellular protein functions could encompass a potential novel strategy to improve the outcome of injury-induced corneal wound healing.

A Glaucoma Patient with an Intraocular Pressure Decrease following Total Gastrectomy and Postoperative Anticancer Treatment.

We herein report a case of glaucoma with an intraocular pressure (IOP) decrease following total gastrectomy (TG) and anticancer treatment for gastric cancer. A 62-year-old male underwent trabeculectomy of the left and right eyes in August 2011 and July 2012, respectively. During the follow-up, IOP of the right eye was 9-12 mmHg (with bimatoprost, dorzolamide, and timolol maleate), and that of the left eye ranged between 14 and 26 mmHg (with bimatoprost, dorzolamide, timolol maleate, and brimonidine tartrate). In December 2014, TG was performed due to gastric cancer. After surgery, the patient received S-1+CDDP, weekly PAC, and CPT-11 therapies. The patient died on March X, 2017. Before TG, the body mass index (BMI) was 29.5 but decreased to 24.8 before the start of the two courses of weekly PAC therapy. IOP of the right eye was 6 mmHg (with bimatoprost), and that of the left eye was 10 mmHg (with bimatoprost, dorzolamide, and brimonidine tartrate), showing decreases. After the initiation of weekly PAC therapy, BMI was approximately 19. IOP of the right eye ranged between 6 and 10 mmHg until the final ophthalmological examination (January 11, 2017), while that of the left eye ranged between 8 and 15 mmHg. In this patient with glaucoma, IOP was not controlled by eye drop treatment, and TG for gastric cancer and postoperative treatment with anticancer drugs resulted in weight loss and a decrease in IOP.

Loss of TRPV4 Cation Channel Inhibition of Macrophage Infiltration and Neovascularization in a Mouse Cornea.

Corneal injury-associated inflammation could induce inward-growing neovascularization from the periphery of the tissue. Such neovascularization could cause stromal opacification and curvature disturbance, and both potentially impair visual function. In this study, we determined the effects of the loss of transient receptor potential vanilloid 4 (TRPV4) expression on the development of neovascularization in the corneal stroma in mice by producing a cauterization injury in the central area of the cornea. New vessels were immunohistochemically labeled with anti-TRPV4 antibodies. TRPV4 gene knockout suppressed the growth of such CD31-labeled neovascularization in association with the suppression of infiltration of macrophages and tissue messenger RNA expression of the vascular endothelial cell growth factor A level. Treatment of cultured vascular endothelial cells with supplementation of HC-067047 (0.1 µM, 1 µM, or 10 µM), a TRPV4 antagonist, attenuated the formation of a tube-like structure with sulforaphane (15 µM, for positive control) that modeled the new vessel formation. Therefore, the TRPV4 signal is involved in injury-induced macrophagic inflammation and neovascularization activity by vascular endothelial cells in a mouse corneal stroma. TRPV4 could be a therapeutic target to prevent unfavorable postinjury neovascularization in the cornea.

Delayed regression of laser-induced choroidal neovascularization in TNFα-null mice.

We investigated the effects of lacking TNFα on the development and regression of Argon-laser-induced choroidal neovascularization (CNV) in mice. We lasered ocular fundus for induction of CNV in both wild-type (WT) and TNFα-null (KO) mice. Fluorescence angiography was performed to examine the size of CNV lesions. Gene expression pattern of wound healing-related components was examined. The effects of exogenous TNFα on apoptosis of human retinal microvascular endothelial cells (HRMECs) and on the tube-like structure of the cells were investigated in vitro. The results showed that Argon-laser irradiation-induced CNV was significantly larger in KO mice than WT mice on Day 21, but not at other timepoints. Lacking TNFα increased neutrophil population in the lesion. The distribution of cleaved caspase3-labelled apoptotic cells was more frequently observed in the laser-irradiated tissue in a WT mouse as compared with a KO mouse. Exogenous TNFα induced apoptosis of HRMECs and accelerated regression of tube-like structure of HRMECs in cell culture. Taken together, TNFα gene knockout delays the regression of laser-induced CNV in mice. The mechanism underlying the phenotype might include the augmentation of neutrophil population in the treated tissue and attenuation of vascular endothelial cell apoptosis.

Anomaly of cornea and ocular adnexa in spinster homolog 2 (Spns2) knockout mice.

Spinster 2 (Spns2) is a transporter that pumps sphingosine-1-phosphate (S1P), a bioactive lipid mediator synthesized in the cytoplasm, out of cells into the inter cellular space. S1P is a signal that modulates cellular behavior during embryonic development, inflammation and tissue repair, etc. A Spns2-null (KO) mouse is born with failure of eyelid closure (eyelid-open-at birth; EOB) and develop corneal fibrosis in adulthood. It remains elusive whether corneal lesion is caused by exposure to keratitis (lagophthalmos) of EOB phenotype or the loss of Spns2 directly perturbs the corneal tissue morphogenesis and intra-eyelid structures. Therefore, we investigated differences between the cornea and ocular adnexa morphogenesis in KO and wild-type (WT) embryos and adults as well. The loss of Spns2 perturbs cornea morphogenesis during embryonic development as early as E16.5 besides EOB phenotype. Histology showed that the corneal stroma was thinner with less extracellular matrix accumulation, e.g., collagen and keratocan in the KO mouse. Epithelial stratification, expression of keratin 12 and formation of desmosomes and hemidesmosomes were also perturbed in these KO corneas. Lacking Spns2 impaired morphogenesis of the Meibomian glands and of orbicularis oculi muscles. KO glands were labeled for ELOVL4 and PPARγ and were Oil-Red O-positive, suggesting KO acinar cells possessed functionality as the glands. This is the first report on the roles of Spns2 in corneal and Meibomian gland morphogenesis. Corneal tissue destruction in an adult KO mouse might be due to not only lagophthalmos but also to an impaired morphogenesis of cornea, Meibomian glands, and orbicularis oculi muscle.

Therapeutic Penetrating Keratoplasty in a Case of Corneal Perforation Caused by Colletotrichum gloeosporioides Infection.

Background: Corneal infection of Colletotrichum gloeosporioides is uncommon and usually limited to the anterior stroma. However, we observed a case of corneal stromal perforation caused by this fungus under a compromised condition. Case: A 73-year-old woman consulted us with a severe corneal ulceration. She was a tangerine orange farmer who suffered from rheumatoid arthritis for more than ten years. Before consultation with us, she received pterygium excision in her right eye. She then developed a corneal ulceration and received topical glucocorticoid therapy upon diagnosis of rheumatoid arthritis-related stromal ulcer in the eye. At the first consultation with us, a corneal ulceration was observed in the inferotemporal area of her right cornea. Biological examination detected a filamentous fungus, Colletotrichum gloeosporioides. Topical and systemic antifungal treatments were not significantly effective. Fourteen days after consultation, the lesion grew worse, leading to stromal perforation, which was treated by therapeutic penetrating keratoplasty using a preserved corneal button. Conclusions: Topical glucocorticoid could accelerate the growth of Colletotrichum gloeosporioides before diagnosis, even though the primary cause of corneal ulceration development might be rheumatoid arthritis.

Impairment of corneal epithelial wound healing is association with increased neutrophil infiltration and reactive oxygen species activation in tenascin X-deficient mice.

The purpose of the study was to uncover the role of tenascin X in modulation of healing in mouse corneas subjected to epithelium debridement. Healing in corneas with an epithelial defect was evaluated at the levels of gene and protein expression. Wound healing-related mediators and inflammatory cell infiltration were detected by histology, immunohistochemistry and real-time RT-PCR. Tenascin X protein was upregulated in the wounded wild-type (WT) corneal epithelium. The lack of tenascin X impaired closure of an epithelial defect and accelerated infiltration of neutrophils into the wound periphery as compared to the response in WT tissue. Expression of wound healing-related proinflammatory and reparative components, i.e., interleukin-6, transforming growth factor ß, matrix metalloproteinases, were unaffected by the loss of tenascin X expression. Marked accumulation of malondialdehyde (a lipid peroxidation-derived product) was observed in KO healing epithelia as compared with its WT counterpart. Neutropenia induced by systemic administration of a specific antibody rescued the impairment of epithelial healing in KO corneas, with reduction of malondialdehyde levels in the epithelial cells. Finally, we showed that a chemical scavenging reactive oxygen species reversed the impairment of attenuation of epithelial repair with a reduction of tissue levels of malondialdehyde. In conclusion, loss of tenascin X prolonged corneal epithelial wound healing and increased neutrophilic inflammatory response to debridement in mice. Tenascin X contributes to the control of neutrophil infiltration needed to support the regenerative response to injury and prevent the oxidative stress mediators from rising to cytotoxic levels.

Roles of Epithelial and Mesenchymal TRP Channels in Mediating Inflammatory Fibrosis.

The maintenance of normal vision is dependent on preserving corneal transparency. For this to occur, this tissue must remain avascular and its stromal architecture needs to be retained. Epithelial transparency is maintained provided the uppermost stratified layers of this tissue are composed of terminally differentiated non-keratinizing cells. In addition, it is essential that the underlying stromal connective tissue remains avascular and scar-free. Keratocytes are the source of fibroblasts that are interspersed within the collagenous framework and the extracellular matrix. In addition, there are sensory nerve fibers whose lineage is possibly either neural crest or mesenchymal. Corneal wound healing studies have been undertaken to delineate the underlying pathogenic responses that result in the development of opacification following chemical injury. An alkali burn is one type of injury that can result in severe and long- lasting losses in ocular transparency. During the subsequent wound healing process, numerous different proinflammatory cytokines and proteolytic enzymes undergo upregulation. Such increases in their expression levels induce maladaptive expression of sustained stromal inflammatory fibrosis, neovascularization, and losses in the smooth optical properties of the corneal outer surface. It is becoming apparent that different transient receptor potential channel (TRP) isoforms are important players in mediating these different events underlying the wound healing process since injury upregulates both their expression levels and functional involvement. In this review, we focus on the involvement of TRPV1, TRPA1 and TRPV4 in mediating some of the responses that underlie the control of anterior ocular tissue homeostasis under normal and pathological conditions. They are expressed on both different cell types throughout this tissue and also on corneal sensory nerve endings. Their roles have been extensively studied as sensors and transducers of environmental stimuli resulting from exposure to intrinsic modulators and extrinsic ligands. These triggers include alteration of the ambient temperature and mechanical stress, etc., that can induce pathophysiological responses underlying losses in tissue transparency activated by wound healing in mice losses in tissue transparency. In this article, experimental findings are reviewed about the role of injury-induced TRP channel activation in mediating inflammatory fibrotic responses during wound healing in mice.

Loss of sphingosine 1-phosphate receptor 3 gene function impairs injury-induced stromal angiogenesis in mouse cornea.

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid generated through sphingosine kinase1 (SPK1)-mediated phosphorylation of sphingosine. We show here that injury-induced S1P upregulation increases corneal neovascularization through stimulating S1PR3, a cognate receptor. since this response was suppressed in S1PR3-knockout mice. Furthermore, Cayman10444, a selective S1PR3 inhibitor, reduced this response in WT mice. Such reductions in neovascularization were associated with reduced vascular endothelial growth factor A (VEGF-A) mRNA expression levels in WT TKE2 corneal epithelial cells and macrophages treated with CAY10444 as well as macrophages isolated from S1PR3 KO mice. S1P increased tube-like vessel formation in human vascular endothelial cells (HUVEC) and human retinal microvascular endothelial cells (HRMECs) cells expressing S1PR3. In S1PR3 KO mice, TGFß1-induced increases in αSMA gene expression levels were suppressed relative to those in the WT counterparts. In S1PR3 deficient macrophages, VEGF-A expression levels were lower than in WT macrophages. Transforming growth factor ß1(TGFß1) upregulated SPK1 expression levels in ocular fibroblasts and TKE2 corneal epithelial cells. CAY10444 blocked S1P-induced increases in VEGF-A mRNA expression levels in TKE2 corneal epithelial cells. Endogenous S1P signaling upregulated VEGF-A and VE-cadherin mRNA expression levels in HUVEC. Unlike in TKE2 cells, SIS3 failed to block TGFß1-induced VEGF-A upregulation in ocular fibroblasts. Taken together, these results indicate that injury-induced TGFß1 upregulation increases S1P generation through increases in SPK1 activity. The rise in S1P formation stimulates the S1PR3-linked signaling pathway, which in turn increases VEGF-A expression levels and angiogenesis in mouse corneas.

Long-term intraocular pressure after switching a combination ophthalmic medication of ß-blocker/prostaglandin.

PURPOSE: We examined intraocular pressure (IOP)-reducing effects 12 months after switching timolol maleate/travoprost combination ophthalmic solution in one bottle (TM/TR-COMBI-SOL) to carteolol hydrochloride/latanoprost combination ophthalmic solution in one bottle (CR/LT-COMBI-SOL). CASES: The participants included 25 patients (25 eyes) who could be followed up for 12 months after a switch from TM/TR-COMBI-SOL to CR/LT-COMBI-SOL in Saiseikai Arida Hospital between March 1, 2017, and August 31, 2018. They consisted of patients in whom antiglaucoma eye drop other than TM/TR-COMBI-SOL had not been used (monotherapy group, 12 patients [12 eyes], 12.8 ± 3.0 mmHg) and those in whom antiglaucoma eye drop other than TM/TR-COMBI-SOL had been concomitantly used (multitherapy group, 13 patients [13 eyes], 13.8 ± 2.4 mmHg). We excluded patients in whom drugs for glaucoma were changed or added during the follow-up and those who underwent intraocular surgery. MATERIALS AND METHODS: We retrospectively and statistically examined the IOP before eye drop switching and after 1, 6, and 12 months, using the paired t-test. RESULTS: The IOPs 1 month after eye drop switching in the monotherapy group and multitherapy group were 12.5 ± 3.3 and 13.8 ± 2.5 mmHg, respectively. The values after 6 months were 13.5 ± 3.0 and 11.5 ± 2.7 mmHg, respectively. Those after 12 months were 12.8 ± 2.7 and 11.7 ± 2.5 mmHg, respectively. In the monotherapy group, there was no significant difference during the follow-up period. In the multitherapy group, there were significant decreases in comparison with the preswitching value after 6 and 12 months (P < 0.05, respectively). CONCLUSION: The IOP-reducing effects of CR/LT-COMBI-SOL were similar to those of TM/TR-COMBI-SOL. However, the effects may be enhanced after switching from TM/TR-COMBI-SOL in patients receiving multitherapy.

A Case of Repeating Transient Increase in Intraocular Pressure by Instability of an Intraocular Lens Implanted in the Capsular Bag.

We observed repeated episodes of rapid increases in intraocular pressure (IOP) considered to be caused by an in-the-bag intraocular lens (IOL) instability in a patient with an implanted IOL. As acute glaucoma attack-like increase in IOP was noted in the left eye on November 8, she was admitted to Wakayama Medical University Hospital. The findings at the first examination included an IOP of 62 mm Hg, instability of a PMMA one-piece IOL, shallow anterior chamber, narrow angle, moderate mydriasis, and loss of pupillary light reaction in the left avitreous eye. On November 15, a 6-mm Hg increase in IOP was observed during 60-min dark room prone provocative testing. After the first examination, the patient perceived pain and reduced visual acuity of the left eye and emergently consulted our hospital twice. Despite miosis, normalization of the anterior chamber depth and IOP with widening of the angle were achieved by resting in the supine position. These episodes were thought to be caused by instability and anterior shift of the IOL. On January 17, 2018, suture fixation of the in-the-bag IOL was performed. The IOL was fixed by transscleral suturing of the bilateral supporting parts to the sclera. Recurrence of sudden ophthalmalgia, instability of the in-the-bag IOL, and an increase in IOP have not been observed for 1 year after surgical treatment. Instability of an in-the-bag IOL caused repeated acute angle-closure glaucoma-like attacks. The situation was well treated by suturing and fixing the haptics of IOL to the sclera.

Effects of a prostaglandin F2alpha derivative glaucoma drug on EGF expression and E-cadherin expression in a corneal epithelial cell line.

Purpose: We examined the effects of travoprost on cell proliferation-related signals and E-cadherin expression in vitro and in situ in order to obtain evidence to support the hypothesis that topical travoprost impairs the integrity of the corneal epithelium.Methods: A human corneal epithelial cell culture was treated with travoprost (0.4 mg/ml) and/or PD168393 (an EGF receptor inhibitor, 10 µM). The culture was then processed for cell proliferation, an mRNA expression analysis of epidermal growth factor (EGF) and E-cadherin, and protein expression analysis of E-cadherin by immunocytochemistry and Western blotting. The eyes of C57/BL6 mice were incubated in serum-free medium plus travoprost (0.4 mg/ml) and/or PD168393 (10 µM). After being cultured for 24 h, the expression patterns of phospho-EGFR, phospho-ERK, E-cadherin, and Ki67 were immunohistochemically examined in paraffin sections.Results: The addition of travoprost up-regulated EGF mRNA expression and cell proliferation in the corneal epithelial cell culture, and this was cancelled by the addition of PD168393. This FP agonist also decreased E-cadherin expression levels in the cell-cell contact zone, and this was cancelled by the addition of PD168393. In the organ culture, the addition of travoprost to the medium up-regulated the expression of phospho-EGFR and phospho-ERK as well as cell proliferation, and down-regulated the expression of E-cadherin in the corneal epithelium, particularly in basal cells, whereas PD168393 reversed these effects.Conclusions: Travoprost activates epithelial cell proliferation by up-regulating an EGF-related signal in association with the suppression of E-cadherin localization in the cell-cell contact zone. Modulation of the EGF signal may be a strategy to minimize the negative impact of this mitogen on reformation of corneal barrier function during epithelial renewal.

Suppression of neovascularization in corneal stroma in a TRPA1-null mouse.

Corneal neovascularization and inflammatory fibrosis induced by severe injury or infection leads to tissue opacification and even blindness. Transient receptor potential (TRP) channel subtypes contribute to mediating these maladaptive responses through their interactions with other receptors. TRPV1 is one of the contributing channel isoforms inducing neovascularization in an alkali burn mouse wound healing model. VEGF-A upregulation contributes to neovascularization through interaction with its cognate receptors (VEGFR). Since the TRP isoform in this tissue, TRPA1, is also involved, we determined here if one of the pathways mediating neovascularization and immune cell infiltration involve an interaction between VEGFR and TRPA1 in a cauterization corneal mouse wound healing model. Localization of TRPA1 and endothelial cell (EC) CD31 immunostaining pattern intensity determined if TRPA1 expression was EC delimited during cauterization induced angiogenesis. Quantitative RT-PCR evaluated the effects of the absence of TRPA1 function on VEGF-A and TGF-ß1 mRNA expression during this process. Macrophage infiltration increased based on rises in F4/80 antigen immunoreactivity. TRPA1 immunostaining was absent on CD31-immunostained EC cells undergoing neovascularization, but it was present on other cell type(s) adhering to EC in vivo. Absence of TRPA1 expression suppressed both stromal neovascularization and inhibited macrophage infiltration. Similarly, the increases occurring in both VEGF-A and TGF-ß1 mRNA expression levels in WT tissue were blunted in the TRPA1-/- counterpart. On the other hand, in the macrophages their levels were invariant and their infiltration was inhibited. To determine if promotion by TRPA1 of angiogenesis was dependent on its expression on other unidentified cell types, the effects were compared of pharmacological manipulation of TRPA1 activity on EC proliferation tube formation and migration. In the presence and absence of a fibroblast containing feeder layer. Neither VEGF-induced increases in human vascular endothelial cell (HUVEC) proliferation nor migration were changed by a TRPA1 antagonist HC-030031 in the absence of a feeder layer. However, on a fibroblast feeder layer this antagonist suppressed HUVEC tube formation. In conclusion, during corneal wound healing transactivation by VEGFR of TRPA1 contributes to mediating neovascularization and macrophage infiltration. Such crosstalk is possible because of close proximity between VEGFR delimited expression on EC and TRPA1 expression restricted to cell types adhering to EC.

Sensory nerve supports epithelial stem cell function in healing of corneal epithelium in mice: the role of trigeminal nerve transient receptor potential vanilloid 4.

In order to understand the pathobiology of neurotrophic keratopathy, we established a mouse model by coagulating the first branch of the trigeminal nerve (V1 nerve). In our model, the sensory nerve in the central cornea disappeared and remaining fibers were sparse in the peripheral limbal region. Impaired corneal epithelial healing in the mouse model was associated with suppression of both cell proliferation and expression of stem cell markers in peripheral/limbal epithelium as well as a reduction of transient receptor potential vanilloid 4 (TRPV4) expression in tissue. TRPV4 gene knockout also suppressed epithelial repair in mouse cornea, although it did not seem to directly modulate migration of epithelium. In a co-culture experiment, TRPV4-introduced KO trigeminal ganglion upregulated nerve growth factor (NGF) in cultured corneal epithelial cells, but ganglion with a control vector did not. TRPV4 gene introduction into a damaged V1 nerve rescues the impairment of epithelial healing in association with partial recovery of the stem/progenitor cell markers and upregulation of cell proliferation and of NGF expression in the peripheral/limbal epithelium. Gene transfer of TRPV4 did not accelerate the regeneration of nerve fibers. Sensory nerve TRPV4 is critical to maintain stemness of peripheral/limbal basal cells, and is one of the major mechanisms of homeostasis maintenance of corneal epithelium.