Mizuki Kitamura

αB Crystallin Is Apically Secreted within Exosomes by Polarized Human Retinal Pigment Epithelium and Provides Neuroprotection to Adjacent Cells

αB Crystallin is a chaperone protein with anti-apoptotic and anti-inflammatory functions and has been identified as a biomarker in age-related macular degeneration. The purpose of this study was to determine whether αB crystallin is secreted from retinal pigment epithelial (RPE) cells, the mechanism of this secretory pathway and to determine whether extracellular αB crystallin can be taken up by adjacent retinal cells and provide protection from oxidant stress. We used human RPE cells to establish that αB crystallin is secreted by a non-classical pathway that involves exosomes. Evidence for the release of exosomes by RPE and localization of αB crystallin within the exosomes was achieved by immunoblot, immunofluorescence, and electron microscopic analyses. Inhibition of lipid rafts or exosomes significantly reduced αB crystallin secretion, while inhibitors of classic secretory pathways had no effect. In highly polarized RPE monolayers, αB crystallin was selectively secreted towards the apical, photoreceptor-facing side. In support, confocal microscopy established that αB crystallin was localized predominantly in the apical compartment of RPE monolayers, where it co-localized in part with exosomal marker CD63. Severe oxidative stress resulted in barrier breakdown and release of αB crystallin to the basolateral side. In normal mouse retinal sections, αB crystallin was identified in the interphotoreceptor matrix. An increased uptake of exogenous αB crystallin and protection from apoptosis by inhibition of caspase 3 and PARP activation were observed in stressed RPE cultures. αB Crystallin was taken up by photoreceptors in mouse retinal explants exposed to oxidative stress. These results demonstrate an important role for αB crystallin in maintaining and facilitating a neuroprotective outer retinal environment and may also explain the accumulation of αB crystallin in extracellular sub-RPE deposits in the stressed microenvironment in age-related macular degeneration. Thus evidence from our studies supports a neuroprotective role for αB crystallin in ocular diseases.

AlphaB crystallin regulation of angiogenesis by modulation of VEGF

alphaB-crystallin is a chaperone belonging to the small heat shock protein family. Herein we show attenuation of intraocular angiogenesis in alphaB-crystallin knockout (alphaB-crystallin(-/-)) mice in 2 models of intraocular disease: oxygen-induced retinopathy and laser-induced choroidal neovascularization. Vascular endothelial growth factor A (VEGF-A) mRNA and hypoxia inducible factor-1alpha protein expression were induced during retinal angiogenesis, but VEGF-A protein expression remained low in alphaB-crystallin(-/-) retina versus wild-type mice, whereas VEGF-R2 expression was not affected. Both alphaB-crystallin and its phosphorylated serine59 formwere expressed, and immunoprecipitation revealed alphaB-crystallin binding to VEGF-A but not transforming growth factor-beta in cultured retinal pigment epithelial (RPE) cells. alphaB-crystallin and VEGF-A are colocalized in the endoplasmic reticulum in RPE cells under chemical hypoxia. alphaB-crystallin(-/-) RPE showed low VEGF-A secretion under serum-starved conditions compared with wild-type cells. VEGF-A is polyubiquitinated in control and alphaB-crystallin siRNA treated RPE; however, mono-tetra ubiquitinated VEGF-A increases with alphaB-crystallin knockdown. Endothelial cell apoptosis in newly formed vessels was greater in alphaB-crystallin(-/-) than wild-type mice. Proteasomal inhibition in alphaB-crystallin(-/-) mice partially restores VEGF-A secretion and angiogenic phenotype in choroidal neovascularization. Our studies indicate an important role for alphaB-crystallin as a chaperone for VEGF-A in angiogenesis and its potential as a therapeutic target.

Clinical features of intraocular inflammation in Hokkaido, Japan

Purpose:  We aimed to investigate the clinical features of intraocular inflammation/uveitis in Hokkaido, Japan.

Osteopontin Aggravates Experimental Autoimmune Uveoretinitis in Mice

Human endogenous uveitis is a common sight-threatening intraocular inflammatory disease and has been studied extensively using a murine model of experimental autoimmune uveoretinitis (EAU). It is possibly mediated by Th1 immune responses. In the present study, we investigated the role of osteopontin (OPN), a protein with pleiotropic functions that contributes to the development of Th1 cell-mediated immunity. Accompanying EAU progression, OPN was elevated in wild-type (WT) mice that had been immunized with human interphotoreceptor retinoid-binding protein (hIRBP) peptide 1–20. OPN-deficient (OPN−/−) mice showed milder EAU progression in clinical and histopathological scores compared with those of WT mice. The T cells from hIRBP-immunized OPN−/− mice exhibited reduced Ag-specific proliferation and proinflammatory cytokine (TNF-α and IFN-γ) production compared with those of WT T cells. When hIRBP-immunized WT mice were administered M5 Ab reacting to SLAYGLR sequence, a cryptic binding site to integrins within OPN, EAU development was significantly ameliorated. T cells from hIRBP-immunized WT mice showed significantly reduced proliferative responses and proinflammatory cytokine production upon stimulation with hIRBP peptide in the presence of M5 Ab in the culture. Our present results demonstrate that OPN may represent a novel therapeutic target to control uveoretinitis.

Amelioration of experimental autoimmune uveoretinitis (EAU) with an inhibitor of nuclear factor-κB (NF-κB), pyrrolidine dithiocarbamate

Experimental autoimmune uveoretinitis (EAU) is a T helper type 1 cell‐mediated autoimmune disease, which serves as a model of human chronic uveitis. In this model, cells of a monocyte/macrophage lineage and retinal antigen (Ag)‐specific T cells infiltrate into the retina and cause inflammatory lesion, where proinflammatory cytokines and various stimuli activate a transcriptional factor, nuclear factor‐κB (NF‐κB), which modulates inflammation and enhances immune responses. In the present study, the therapeutic effect of administration of a NF‐κB inhibitor, pyrrolidine dithiocarbamate (PDTC), was examined in a murine EAU model. It was shown that PDTC ameliorated the clinical symptoms of EAU mice and significantly reduced the histopathological score compared with those in untreated mice. mRNA expressions of tumor necrosis factor α and interleukin‐1β were suppressed in eyes of PDTC‐treated EAU mice. However, when T cells from PDTC‐treated EAU mice, Ag‐presenting cells (APC), and the retinal Ag peptides were cocultured, these T cells showed the same level of proliferation as those from control mice. Furthermore, addition of PDTC in the culture of T cells from EAU mice, Ag, and APC completely abrogated the T cell‐proliferative response and cytokine production. Pretreatment of Ag‐primed T cells or APC with PDTC in vitro also reduced these responses. These results indicate that the inhibitory effect of PDTC is attributed mainly to the suppression of effector‐phase responses including inflammation but not to the inhibition of T cell priming. Regulation of NF‐κB pathway in the lesion could be a novel target for the successful control of uveoretinitis.

Increased Osteopontin Levels in the Vitreous of Patients with Diabetic Retinopathy

Purpose: Osteopontin (OPN) has diverse functions such as cell adhesion, chemoattraction, immunomodulation, and angiogenesis. The aim of this study is to analyze the OPN levels in vitreous fluid obtained from diabetic retinopathy (DR) and non-DR patients. Methods: Nineteen patients out of 11 with DR and 8 without DR underwent pars plana vitrectomy and vitreous fluid was obtained simultaneously. Two distinct sandwich enzyme-linked immunosorbent assay systems (systems 1 and 2) were applied, which have been developed in our laboratories to quantify the OPN concentrations in vitreous fluid. Results: The non-thrombin-cleaved full-length OPN levels in the vitreous fluid were 921.63 ± 45.38 ng/ml in DR and 632.80 ± 83.43 ng/ml in non-DR using system 1. Also, vitreous thrombin-cleaved and noncleaved OPN levels were increased to 2,109.22 ± 151.651 and 1,651.13 ± 229.82 ng/ml in patients with DR and non-DR using system 2. The vitreous OPN levels were significantly higher in DR than those in non-DR (p < 0.01 by system 1 and p < 0.05 by system 2). Conclusion: Thrombin-cleaved and noncleaved vitreous OPN levels in patients with DR were increased compared with control subjects, suggesting that OPN plays a potential role in the pathogenesis of diabetic retinal ischemia.

Prevention of experimental autoimmune uveoretinitis by blockade of osteopontin with small interfering RNA.

Osteopontin (OPN) is elevated during the progression of experimental autoimmune uveoretinitis (EAU) in C57BL/6 (B6) mice. Furthermore, EAU symptoms are ameliorated in OPN knockout mice or in B6 mice treated with anti-OPN antibody (M5). Recently, OPN has been shown to promote the Th1 response not only in the extracellular space as a secretory protein but also in cytosol as a signaling component. Thus, we attempted to reduce OPN in both compartments by using a small interfering RNA (siRNA) targeting the OPN coding sequence (OPN-siRNA). EAU was induced in B6 mice by immunization with human interphotoreceptor retinoid-binding protein (hIRBP) peptide sequence 1-20. The OPN- or control-siRNA was administered with hydrodynamic methods 24 h before and simultaneously with immunization (prevention regimen). When plasma OPN levels were quantified following siRNA administration with the prevention regimen, the level in the OPN-siRNA-treated group was significantly lower than that in the control-siRNA-treated group. Accordingly, the clinical and histopathological scores of EAU were significantly reduced in B6 mice when siRNA caused OPN blockade. Furthermore, TNF-alpha, IFN-gamma, IL-2, GM-CSF and IL-17 levels in the culture supernatants were markedly suppressed in the OPN-siRNA-treated group, whereas the proliferative responses of T lymphocytes from regional lymph nodes against immunogenic peptides was not significantly reduced. On the other hand, the protection was not significant if the mice received the OPN-siRNA treatment on day 7 and day 8 after immunization when the clinical symptoms appeared overt (reversal regimen). Our results suggest that OPN blockade with OPN-siRNA can be an alternative choice for the usage of anti-OPN antibody and controlling uveoretinitis in the preventive regimen.

Efficacy and complications of intravitreal injection of triamcinolone acetonide for refractory cystoid macular edema associated with intraocular inflammation

PurposeTo assess the effects and complications of intravitreal injection of triamcinolone acetonide (IVTA) for posterior sub-Tenon injection of triamcinolone acetonide (PSTA)-resistant cystoid macular edema (CME) with intraocular inflammation.MethodsMedical records of eight eyes of six patients with PSTA-resistant CME were retrospectively examined. Each eye received a 4-mg IVTA, and an additional injection was performed when CME recurred. Visual acuity as logarithm of the minimum angle of resolution (logMAR), intraocular pressure (IOP), and central macular thickness (CMT) were assessed before and after each treatment.ResultsCME improved in six eyes (75%) with mean visual acuity recovering from 0.56 ± 0.29 to 0.41 ± 0.195 (logMAR, P = 0.13) and mean CMT decreasing from 470 μm (range, 275–660 μm) to 297 μm (range, 150–697 μm) (P = 0.04) 2 months after the initial IVTA. CME recurred an average of 9 months (range, 5–11 months) after IVTA. A higher dose (16-mg) IVTA was effective for two eyes refractory to repeated 4-mg IVTA. IOP was elevated in two eyes (25%), of which one required filtration surgery (12.5%). In phakic eyes, cataracts progressed and necessitated surgery.ConclusionsIVTA is effective for PSTA-resistant CME with intraocular inflammation, and its efficacy might be dose dependent.

Decrease in the glyceraldehyde derived advanced glycation end products in the sera of patients with Vogt-Koyanagi-Harada disease.

Background/aims: Advanced glycation end products (AGEs) are considered to act as mediators of both age related pathologies and diabetic complications. It was recently reported that glyceraldehyde derived AGE (AGE-2) has a strong biological effect on various diseases. The aim of this study was to investigate the serum AGE-2 levels in Vogt-Koyanagi-Harada (VKH) disease. Methods: Sera were obtained from 31 patients with active VKH. 20 of these 31 patients were treated with systemic corticosteroids. As controls, 33 healthy volunteers were also examined. The serum AGE-2 levels were determined with a competitive enzyme linked immunosorbent assay using AGE-2 polyclonal antibody. Results: The mean AGE-2 level in the sera of patients with VKH disease was 4.91 (SD 2.23) U/ml, which was significantly lower than that of the healthy control subjects (8.32 (2.94), p<0.001). The average serum AGE-2 level significantly increased to 13.49 (2.17) U/ml after the patients were treated with systemic corticosteroids (p<0.001). Conclusions: These results suggest that AGE-2 may be involved in the onset of VKH disease.

Amelioration of experimental autoimmune uveoretinitis by inhibition of glyceraldehyde‐derived advanced glycation end‐product formation

AGEs are permanently modified macromolecule derivatives that form through nonenzymatic glycation of amino groups of proteins. Glycer‐AGEs are highly toxic and play an important role in the pathogenesis of chronic inflammatory diseases. However, the contribution of glycer‐AGEs to the pathogenesis of uveitis is unclear. In this study, we measured serum levels of glycer‐AGEs in 100 patients with endogenous uveitis (22 with HLA‐B27‐associated uveitis, 20 with VKH disease, 14 with Behçets disease, and 44 with sarcoidosis) and 33 healthy volunteers. We then examined the effect of the AGE inhibitor in a mouse model of human endogenous uveitis (EAU) by continuous oral administration of pyridoxamine at 200 or 400 mg/kg/day. Regardless of the etiology, serum glycer‐AGE levels were significantly higher in patients with uveitis than in healthy subjects. Treatment with 400 mg/kg pyridoxamine significantly reduced the clinical and histological severity of EAU and was accompanied by a significant decrease in serum and retinal glycer‐AGE levels and suppression of translocation of NF‐κB p65 into the nucleus of retinal cells. Serum glycer‐AGE levels may therefore serve as a biomarker of human uveitis, as well as systemic inflammation, and may contribute to the progression of uveitis, including diabetic iritis, via the activation of NF‐κB.