Up-regulation of ZO-1 expression and barrier function in cultured human corneal epithelial cells by substance P

AbstractThe effects of the sensory neurotransmitter substance P on the expression of tight junction proteins and on barrier function in human corneal epithelial cells were investigated. The expression of ZO-1, but not that of occludin or claudin-1, was increased by substance P in a concentration- and time-dependent manner. This effect was inhibited by the NK-1 receptor antagonist GR82334 and by KN62, an inhibitor of Ca2+- and calmodulin-dependent protein kinase II. Substance P also increased the transepithelial electrical resistance of a cell monolayer in a manner sensitive to GR82334. Substance P may therefore play a role in maintenance of tight junctions in the corneal epithelium

Severe panuveitis in neuro-Behçet’s disease in Malaysia: a case series

Behçet’s disease (BD) is a multisystemic disease that is very rare in Malaysia. About 5% of patients develop central nervous system involvement, termed neuro-Behçet’s. Neuro- Behçet’s is one of the most serious causes of long-term morbidity and mortality. We report two cases of neuro-Behçet’s associated with uveitis (ocular BD) highlighting the clinical presentation, diagnostic measurement, and therapeutic management of these cases

EGF-Like-Domain-7 Is Required for VEGF-Induced Akt/ERK Activation and Vascular Tube Formation in an Ex Vivo Angiogenesis Assay

EGFL7 is a secreted angiogenic factor, which in contrast to the well-known secreted angiogenic molecules VEGF and FGF-2, is almost exclusively expressed by endothelial cells and may act in an autocrine fashion. Prior studies have shown EGFL7 to mediate its angiogenic effects by interfering with the Notch pathway and/or via the intronic miR126. Less is known about its effects on VEGF signaling. We wanted to investigate the role of epidermal growth factor-like domain 7 (EGFL7) in VEGF-driven angiogenesis using an ex vivo Matrigel-embedded mouse eye cup assay and siRNA mediated knockdown of EGFL7 by siRNA. Our results suggested that VEGF-induced vascular tube formation was significantly impaired after siRNA downregulation of EGFL7. In addition, knockdown of EGFL7 suppressed VEGF upregulation of phospho-Akt and phospho-Erk(1/2) in endothelial cells, but did not alter VEGFR phosphorylation and neuropilin-1 protein expression or miR126 expression. Thus, in conclusion, EGFL7 is required for VEGF upregulation of the Akt/Erk (1/2) pathway during angiogenesis, and may represent a new therapeutic target in diseases of pathological neovascularization

EGF-Like-Domain-7 Is Required for VEGF-Induced Akt/ERK Activation and Vascular Tube Formation in an Ex Vivo Angiogenesis Assay

EGFL7 is a secreted angiogenic factor, which in contrast to the well-known secreted angiogenic molecules VEGF and FGF-2, is almost exclusively expressed by endothelial cells and may act in an autocrine fashion. Prior studies have shown EGFL7 to mediate its angiogenic effects by interfering with the Notch pathway and/or via the intronic miR126. Less is known about its effects on VEGF signaling. We wanted to investigate the role of epidermal growth factor-like domain 7 (EGFL7) in VEGF-driven angiogenesis using an ex vivo Matrigel-embedded mouse eye cup assay and siRNA mediated knockdown of EGFL7 by siRNA. Our results suggested that VEGF-induced vascular tube formation was significantly impaired after siRNA downregulation of EGFL7. In addition, knockdown of EGFL7 suppressed VEGF upregulation of phospho-Akt and phospho-Erk(1/2) in endothelial cells, but did not alter VEGFR phosphorylation and neuropilin-1 protein expression or miR126 expression. Thus, in conclusion, EGFL7 is required for VEGF upregulation of the Akt/Erk (1/2) pathway during angiogenesis, and may represent a new therapeutic target in diseases of pathological neovascularization

Efficacy and Safety of Intravitreal Aflibercept Treat-and-Extend Regimens in Exudative Age-Related Macular Degeneration: 52- and 96-Week Findings from ALTAIR : A Randomized Controlled Trial.

PURPOSE:To evaluate efficacy and safety of intravitreal injections of aflibercept (IVT-AFL) treat-and-extend (T&E) dosing regimens in treatment-naïve patients with exudative age-related macular degeneration (AMD).METHODS:Adults aged at least 50 years old with exudative AMD and best-corrected visual acuity (BCVA) of 73-25 Early Treatment Diabetic Retinopathy Study (ETDRS) letters were included. Patients received three monthly doses of IVT-AFL 2 mg. At week 16, patients were randomized 1:1 to IVT-AFL T&E with either 2- or 4-week adjustments. The primary endpoint was mean change in BCVA from baseline to week 52. Outcomes were assessed at weeks 52 and 96.RESULTS:Baseline characteristics were comparable between the groups (n = 123 each). Over 52 weeks, mean number of injections was 7.2 and 6.9 and mean last injection interval was 10.7 and 11.8 weeks, for the 2- and 4-week groups, respectively. From baseline, mean change in BCVA was + 9.0 and + 8.4 letters (week 52) and + 7.6 and + 6.1 letters (week 96); mean change in central retinal thickness was - 134.4 µm and - 126.1 µm (week 52) and - 130.5 µm and - 125.3 µm (week 96). Last injection interval before week 52 was at least 12 weeks in 42.3% and 49.6% of patients and 56.9% and 60.2% before week 96. Over 96 weeks, mean number of injections was 10.4 (both groups). The safety profile of IVT-AFL was consistent with previous reports.CONCLUSIONS:IVT-AFL administered using two different T&E regimens for treatment-naïve exudative AMD improved functional and anatomic outcomes at week 52 and outcomes were maintained to week 96. Outcomes were similar between the 2- and 4-week groups.TRIAL REGISTRATION:ClinicalTrials.gov identifier, NCT02305238

Inhibition of the alternative complement pathway preserves photoreceptors after retinal injury

* Degeneration of photoreceptors is a primary cause of vision loss worldwide, making the underlying mechanisms surrounding photoreceptor cell death critical to developing new treatment strategies. Retinal detachment, characterized by the separation of photoreceptors from the underlying retinal pigment epithelium, is a sight-threatening event that can happen in a number of retinal diseases. The detached photoreceptors undergo apoptosis and programmed necrosis. Given that photoreceptors are nondividing cells, their loss leads to irreversible visual impairment even after successful retinal reattachment surgery. To better understand the underlying disease mechanisms, we analyzed innate immune system regulators in the vitreous of human patients with retinal detachment and correlated the results with findings in a mouse model of retinal detachment. We identified the alternative complement pathway as promoting early photoreceptor cell death during retinal detachment. Photoreceptors down-regulate membrane-bound inhibitors of complement, allowing for selective targeting by the alternative complement pathway. When photoreceptors in the detached retina were removed from the primary source of oxygen and nutrients (choroidal vascular bed), the retina became hypoxic, leading to an up-regulation of complement factor B, a key mediator of the alternative pathway. Inhibition of the alternative complement pathway in knockout mice or through pharmacological means ameliorated photoreceptor cell death during retinal detachment. Our current study begins to outline the mechanism by which the alternative complement pathway facilitates photoreceptor cell death in the damaged retina

Disruption of Zonula Occludens-1 Localization in the Rabbit Corneal Epithelium by Contact Lens-Induced Hypoxia

PURPOSE. Hypoxia impairs the barrier function of the corneal epithelium. This function depends on tight junctions, of which zonula occludens (ZO)-1 is a major component. The authors have investigated the effects of hypoxia on ZO-1 localization and expression in the rabbit corneal epithelium in vivo. METHODS. A polymethylmethacrylate (PMMA) or rigid gas-permeable (RGP) lens was applied to one eye each of albino rabbits for 24 hours. The structure of the corneal epithelium was examined by in vivo confocal microscopy, and epithelial barrier function was evaluated by measurement of central corneal thickness. The distribution and expression of ZO-1 in the corneal epithelium were examined by immunofluorescence analysis and by immunoblot and reverse transcriptionpolymerase chain reaction analyses, respectively. RESULTS. Application of a PMMA lens, but not that of an RGP lens, resulted in a reduction in cell size at the surface of the corneal epithelium, compared with that in control eyes, and an increase in central corneal thickness. Immunofluorescence analysis revealed a continuous pattern of ZO-1 immunoreactivity around the perimeter of superficial corneal epithelial cells in control eyes or in eyes treated with an RGP lens. In contrast, the pattern of ZO-1 staining was discontinuous and patchy in eyes treated with a PMMA lens. Amounts of ZO-1 mRNA and protein in the corneal epithelium were reduced by application of a PMMA lens but not by that of an RGP lens. T he corneal epithelium plays important roles in the refraction of light at the ocular surface, stabilization of the tear film, and provision of a renewable protective barrier for the eye. 1,2 The rapid turnover of corneal epithelial cells at the ocular surface is associated with a high metabolic activity that requires a consistent supply of oxygen. When the eye is open, a substantial portion of the oxygen consumed by the corneal epithelium is derived by diffusion from the atmosphere through the tear film. 3 During eye closure, oxygen is derived by diffusion from the aqueous humor and the blood vessels of the overlying palpebral conjunctiva. Hypoxia affects corneal glucose metabolism by inducing a switch from aerobic to anaerobic respiration, the latter of which yields less energy and generates lactic acid as a waste product. 4 -6 Tight junctions play an important role in the establishment and maintenance of barrier function. 7-10 The tight junction complex includes transmembrane proteins such as claudin and occludin; membrane-associated proteins such as zonula occludens (ZO)-1, ZO-2, and ZO-3; and actin filaments. 11,12 Tight junctions in the corneal epithelium contribute to the barrier function at the ocular surface that protects the inner tissues of the eye. 13,14 Hypoxia has been shown to disturb the distribution of the tight junction protein ZO-1 in vascular endothelial cells. 17 ZO-1 is expressed in superficial and sub-superficial cell layers of the corneal epithelium and contributes to its barrier function. 20 The wearing of contact lenses results in a substantial reduction in the oxygen supply to the cornea. The extent of hypoxia depends on the gas permeability of the lens material; greater oxygen permeability results in higher oxygen tension under the lens. METHODS Antibodies and Reagents Rabbit polyclonal antibodies to ZO-1 were obtained from Zymed (Carlsbad, CA), mouse monoclonal antibodies to ␣-tubulin and a protease inhibitor cocktail were from Sigma (St. Louis, MO), and horseradish peroxidase-conjugated secondary antibodies were obtained from Promega (Madison, WI). Dulbecco modified Eagle medium (DMEM)-F12 (50:50, vol/vol) and Alexa Fluor 488 -labeled goat antibodies to rabbit immunoglobulin G were obtained from InvitrogenGibco (Rockville, MD), red fluorescent nucleic acid for probing DNA From th

Attenuation of choroidal neovascularization by dietary intake of ω-3 long-chain polyunsaturated fatty acids and lutein in mice

Dietary ω-3 long-chain polyunsaturated fatty acids (LCPUFAs) and lutein each protect against age-related macular degeneration (AMD). We here examined the effects of ω-3 LCPUFAs and lutein supplementation in a mouse model of AMD. Mice were assigned to four groups: (1) a control group fed an ω-3 LCPUFA–free diet, (2) a lutein group fed an ω-3 LCPUFA–free diet with oral administration of lutein, (3) an ω-3 group fed an ω-3 LCPUFA–supplemented diet, and (4) an ω-3 + lutein group fed an ω-3 LCPUFA–supplemented diet with oral administration of lutein. Mice were fed the defined diets beginning 2 weeks before, and received lutein with an oral gavage needle beginning 1 week before, induction of choroidal neovascularization (CNV) by laser photocoagulation. The area of CNV measured in choroidal flat-mount preparations was significantly reduced in mice fed ω-3 LCPUFAs or lutein compared with those in the control group, and it was reduced in an additive manner in those receiving both ω-3 LCPUFAs and lutein. The concentrations of various inflammatory mediators in the retina or choroid were reduced in mice fed ω-3 LCPUFAs or lutein, but no additive effect was apparent. The generation of reactive oxygen species (ROS) in chorioretinal lesions revealed by dihydroethidium staining as well as the expression of NADPH oxidase 4 (Nox4) in the retina revealed by immunohistofluorescence and immunoblot analyses were attenuated by ω-3 LCPUFAs and lutein in a synergistic manner. Our results thus show that dietary intake of ω-3 LCPUFAs and lutein attenuated CNV in an additive manner and in association with suppression of inflammatory mediator production, ROS generation, and Nox4 expression. Dietary supplementation with both ω-3 LCPUFAs and lutein warrants further study as a means to protect against AMD

Promotion of Corneal Epithelial Wound Healing by a Tetrapeptide (SSSR) Derived from IGF-1

PURPOSE. A prior study showed that a tetrapeptide (FGLMamide) derived from the carboxyl terminus of substance P (SP) and a 12-residue peptide corresponding to the C domain of insulin-like growth factor (IGF)-1 mimic the synergistic effect of the full-length molecules on corneal epithelial wound healing. To develop an effective treatment for persistent corneal epithelial defects, the current study was conducted to investigate the minimal sequence within the C domain of IGF-1 that is required for such synergism with SP or FGLM-amide. METHODS. The effects of IGF-1-derived peptides on corneal epithelial migration were evaluated with a rabbit corneal organ-culture system. RESULTS. A tetrapeptide (SSSR; Ser 33 -Ser-Ser-Arg) derived from the C domain of IGF-1 was sufficient for the synergistic promotion with FGLM-amide both of corneal epithelial migration in vitro and of wound closure in vivo. The activity of the SSSR peptide was sequence specific and its potency was similar to that of IGF-1. The SSSR peptide by itself also promoted corneal epithelial migration in vitro at higher concentrations. It was devoid, however, of both the mitogenic action of IGF-1 and the ability of the full-length molecule to induce neovascularization. CONCLUSIONS. The SSSR sequence mediates the synergistic effect of IGF-1 with SP on corneal epithelial wound healing. Clinical application of the SSSR peptide would be expected to be free of potentially deleterious side effects associated with treatment with full-length IGF-1. Local administration of the SSSR tetrapeptide, alone or in combination with FGLM-amide, is thus a potential new strategy for the treatment of nonhealing epithelial wounds. (Invest Ophthalmol Vis Sci. 2006;47: 3286 -3292) DOI:10.1167/iovs.05-1205 E pithelia provide an essential barrier that protects organs and tissues from the external environment and contributes to maintenance of the internal environment of the body. Although epithelial defects in general heal relatively quickly, the persistence of such defects under certain conditions, such as those that exist in individuals with diabetes mellitus or peripheral neuropathy, can have serious pathologic consequences. The persistence of epithelial defects of the cornea often results in the development of corneal ulcer, which can lead to corneal perforation and loss of vision. There is currently no consistently effective mode of treatment for persistent corneal epithelial defects that is free of potentially adverse consequences. Damage to the corneal epithelium is followed by three phases of epithelial wound healing. Immediately after injury, the remaining epithelial cells begin to migrate to cover the area of the defect. After the defect is covered, contact inhibition results in the termination of epithelial cell migration and the epithelial cells enter a proliferative phase. Finally, the newly generated cells differentiate to form the stratified structure of the corneal epithelium. 1 Epithelial migration is thus an important first step in corneal epithelial wound healing. To develop an effective treatment for persistent corneal epithelial defects, we have established an organ-culture system for the rabbit cornea that allows quantitative evaluation of the effects of test agents on epithelial migration. 2 With this system, we have previously shown that several biological factors, including epidermal growth factor, 3 fibronectin, 2 interleukin-6, 4 and the combination of insulin-like growth factor (IGF)-1 and substance P (SP) 5 promote epithelial migration in vitro. Verification of the relevance of this in vitro system was provided by the observation that the combination of IGF-1 and SP also facilitates closure of rabbit corneal epithelial wounds in vivo. 11 The carboxyl-terminal four amino acids of SP (Phe-Gly-Leu-Metamide, or FGLM-amide) are sufficient for this effect. 12 The synergistic effect of IGF-1 with SP on epithelial migration is mimicked by IGF-2 but not by insulin. 13 IGFs and insulin share many structural similarities in their A and B domains. 14 However, the C domain of IGFs does not exhibit sequence homology to the C peptide of proinsulin, which is not retained in the mature insulin molecule. We have recently shown that the C domain of IGF-1 or -2 is responsible for the synergistic facilitation with SP of corneal epithelial migration