Yoko Dong

Effect of geranylgeranylacetone on the protection of retinal ganglion cells in a mouse model of normal tension glaucoma

Glaucoma is characterized by axonal degeneration of retinal ganglion cells (RGCs) and apoptotic death of their cell bodies, and lowering intraocular pressure is associated with an attenuation of progressive optic nerve damage. Nevertheless, intraocular pressure (IOP) reduction alone was not enough to inhibit the progression of disease, which suggests the contribution of other factors to the glaucoma pathogenesis. In this study, we investigated the cytoprotective effect of geranylgeranylacetone (GGA) on RGCs degeneration using a normal tension glaucoma (NTG) mouse model, which lacks glutamate/aspartate transporter (GLAST) and demonstrates spontaneous RGC and optic nerve degeneration without elevated intraocular pressure (IOP). Three-week-old GLAST+/− mice were given oral administration of GGA at 100, 300, or 600 mg/kg/day or vehicle alone, and littermate control mice were given vehicle alone for 14 days, respectively. At 5 weeks after birth, the number of RGCs was counted in paraffin sections of retinal tissues stained with hematoxylin and eosin. In addition, retrograde labeling technique was also used to quantify the number of RGC. Expression and localization of heat shock protein 70 (HSP70) in retinas were evaluated by reverse transcription polymerase chain reaction and immunohistochemistry, respectively. Activities of caspase-9 and -3 in retinas were also assessed. The number of RGCs of GLAST+/− mice significantly decreased, as compared to that of control mice. RGC loss was significantly suppressed by administration of GGA at 600 mg/kg/day, compared with vehicle alone. Following GGA administration, HSP70 was significantly upregulated together with reduction in the activities of caspase-9 and -3. Our studies highlight HSP70 induction in the retina is available to suppress RGC degeneration, and thus GGA may be applicable for NTG as a promising therapy.

Advanced glycation endproducts link inflammatory cues to upregulation of galectin-1 in diabetic retinopathy

Diabetic retinopathy (DR) is an inflammatory and progressive vaso-occlusive disease resulting in angiogenesis. Galectin-1 is a hypoxia-induced angiogenic factor associated with cancer and proliferative DR. Here we reveal a significant upregulation of galectin-1 in eyes of DR patients along with progression of clinical stages beginning from the pre-ischemic, inflammatory stage with diabetic macular edema, but not in eyes with non-diabetic retinal vascular occlusions. As for its regulatory mechanism unrelated to hypoxia but selective to DR, in vitro galectin-1/LGALS1 expression was shown to increase after application to Müller glial cells with interleukin (IL)-1β, which was induced in monocyte-derived macrophages and microglial cells via toll-like receptor (TLR) 4 signaling stimulated by advanced glycation endproducts (AGE). In vivo inhibition of AGE generation with aminoguanidine, macrophage depletion with clodronate liposomes, and antibody-based blockade of Il-1β and Tlr4 attenuated diabetes-induced retinal Lgals1 expression in mice. Fibrovascular tissues from proliferative DR eyes were immunoreactive for AGE, TRL4 and IL-1β in macrophages, and IL-1β receptor-positive glial cells expressed galectin-1. Therefore, diabetes-induced retinal AGE accumulation was suggested to activate IL-1β-related inflammatory cues in macrophages followed by Müller cells, linking to galectin-1 upregulation in human DR with time. Our data highlight AGE-triggered inflammation as the DR-selective inducer of galectin-1.

Expression of Vascular Endothelial Growth Factor in Human Ocular Adnexal Lymphoma

PURPOSE To examine the expression of VEGF in extranodal marginal zone B-cell lymphoma (EMZL) and reactive lymphoid hyperplasia (RLH) of human ocular adnexa, and analyze the correlation with the intratumoral microvessel density (MVD). METHODS Twenty-two EMZL and 16 RLH tissues were examined in this study. Paraformaldehyde-fixed, paraffin-embedded tissue sections were processed for immunohistochemistry with antibodies against VEGF and CD20. Vascular endothelial growth factor expression was analyzed using the ELISA and RT-PCR in the EMZL tissues. Microvessel density was determined based on the immunoreactivity for anti-CD34 antibody. RESULTS Vascular endothelial growth factor immunoreactivity was detected in the cytoplasm of lymphoid cells in EMZL and RLH. ELISA and RT-PCR confirmed VEGF protein and mRNA expressions in the EMZL tissue, respectively. Vascular endothelial growth factor-immunopositive rate in B-cells was significantly higher in 12 conjunctival EMZLs than four RLHs (P < 0.01) and 10 orbital EMZLs than 12 RLHs (P < 0.05). The MVD showed a significant positive correlation with the VEGF-immunopositive rate in conjunctival and orbital EMZLs. CONCLUSIONS This study demonstrated increased VEGF expression in human conjunctival and orbital EMZL compared with that in RLH, suggesting that VEGF plays a significant role in the pathogenesis and tumor angiogenesis of ocular adnexal lymphoma.

Localization of Acrolein-Lysine Adduct in Fibrovascular Tissues of Proliferative Diabetic Retinopathy

ABSTRACT Purpose: To determine the presence of Nε-(3-formyl-3,4-dehydropiperidino) lysine adduct (FDP-Lys), unsaturated aldehyde acrolein-derived lipoxidation end-product, in fibrovascular tissues obtained from patients with proliferative diabetic retinopathy (PDR). Methods: Fibrovascular tissues were collected from 11 eyes of 11 patients with PDR and paraffin-embedded tissue sections were prepared. Tissue localization of FDP-Lys was studied by immunohistochemistry. Signal intensity was quantified by two masked evaluators and graded into three discrete categories. The relationship between FDP-Lys staining and vascular density was analyzed. In addition, subcellular localization of FDP-Lys was studied by immunofluorescent microscopy. The impact of acrolein on cell viability and proliferation was assessed and the expression level of heme oxygenase-1 (HO-1) mRNA was quantified by real-time polymerase chain reaction (PCR) in cultured retinal microvascular endothelial cells. Results: In fibrovascular tissues, FDP-Lys staining was found in vascular components containing CD34-positive cells and alpha smooth muscle actin (α-SMA)-positive cells, and clusters of rabbit anti-glial fibrillary acid protein (GFAP)-positive cells. Immunofluorescent staining depicted subcellular localization of FDP-Lys in the nucleus and cytoplasm of the cells. Morphological analysis revealed that fibrovascular tissues with FDP-Lys staining in vascular components showed high vascular density. Exposure of cultured endothelial cells to high concentration of acrolein resulted in the decrease of cell viability and proliferation, whereas lower concentration of acrolein increased cell viability and proliferation. Sublethal concentration of acrolein upregulated HO-1 mRNA expression in retinal microvascular endothelial cells. Conclusions: The current data demonstrated the presence of FDP-Lys in fibrovascular tissues and indicate its involvement in fibrovascular proliferation in PDR.

Pathologic Roles of Receptor-Associated Prorenin System in Idiopathic Epiretinal Membrane

Receptor-associated prorenin system (RAPS) refers to the pathogenic mechanism whereby prorenin binding to (pro)renin receptor [(P)RR] dually activates tissue renin-angiotensin system (RAS) and RAS-independent signaling via (P)RR. The aim of this study is to determine the association of RAPS with idiopathic epiretinal membrane (iERM). Reverse transcription-PCR indicated the expression of RAPS components, including (P)RR and Ang II type 1 receptor (AT1R), in iERM tissues and human Müller glial cell line. Double-labeling analyses demonstrated that (P)RR and AT1R were detected in cells positive for glial fibrillary acidic protein, a marker for glial cells, and co-localized with prorenin and angiotensinogen, respectively. Administration of prorenin to Müller glial cells enhanced mRNA expression of fibroblast growth factor 2, while Ang II application stimulated the expression of glial cell line-derived neurotrophic factor, nerve growth factor, and transforming growth factor-β1. These expression levels induced by prorenin or Ang II were reversed by (P)RR or AT1R blockade, respectively. Immunofluorescence revealed tissue co-localization of (P)RR and AT1R with the products of the upregulated genes in vitro. The present findings suggest the involvement of RAPS in the pathogenesis of iERM.

Soluble Vascular Adhesion Protein-1 Mediates Spermine Oxidation as Semicarbazide-Sensitive Amine Oxidase: Possible Role in Proliferative Diabetic Retinopathy

ABSTRACT Purpose/Aim of the study: To explore the possible role of vascular adhesion protein-1 (VAP-1) via its enzymatic function as a semicarbazide-sensitive amine oxidase (SSAO) in the pathogenesis of proliferative diabetic retinopathy (PDR). Materials and Methods: The levels of soluble VAP-1/SSAO and the unsaturated aldehyde acrolein (ACR)-conjugated protein, Nε-(3-formyl-3, 4-dehydropiperidino) lysine adduct (FDP-Lys), were measured in vitreous fluid samples of PDR and non-diabetic patients using ELISA. Recombinant human VAP-1/SSAO (rhVAP-1/SSAO) was incubated with spermine, with or without semicarbazide or RTU-1096 (a specific inhibitor for VAP-1/SSAO). Immunofluorescence assays were performed to assess the localization of VAP-1/SSAO and FDP-Lys in fibrovascular tissues from patients with PDR. The impact of ACR on cultured retinal capillary endothelial cells was assessed using a cell viability assay and total glutathione (GSH) measurements. Results: The levels of sVAP-1/SSAO and FDP-Lys were elevated in the vitreous fluid of patients with PDR. Incubation of rhVAP-1 with spermine resulted in the generation of hydrogen peroxide and FDP-Lys and the production was inhibited by semicarbazide and RTU-1096. In fibrovascular tissues, FDP-Lys and VAP-1/SSAO were present in endothelial cells. ACR stimulation reduced GSH levels in the cultured endothelial cells in a dose-dependent manner and caused cellular toxicity. Conclusions: Our results indicate the pathological role of sVAP-1/SSAO to generate hydrogen peroxide and toxic aldehyde ACR, both of which are associated with oxidative stress, as a consequence of spermine oxidation in eyes with PDR.

Phosphorylation of alphaB-crystallin in epiretinal membrane of human proliferative diabetic retinopathy.

AIM To examine phosphorylation of alphaB-crystallin (p-αBC), a vascular endothelial growth factor (VEGF) chaperone, and immunohistochemically investigate relationship between p-αBC, VEGF and phosphorylated p38-mitogen-activated protein kinase (p-p38 MAPK) in the epiretinal membrane of human proliferative diabetic retinopathy (PDR). METHODS Eleven epiretinal membranes of PDR surgically excised were included in this study. Two normal retinas were also collected from enucleation tissues due to choroidal melanoma. Paraformaldehyde-fixed, paraffin-embedded tissue sections were processed for immunohistochemistry with anti-p-αBC, VEGF, CD31, and p-p38 MAPK antibodies. RESULTS Immunoreactivity for p-αBC was observed in all of the epiretinal membranes examined, where phosphorylation on serine (Ser) 59 showed strongest immunoreactivity in over 70% of the membranes. The immunolocalization of p-αBC was detected in the CD31-positive endothelial cells, and co-localized with VEGF and p-p38 MAPK in PDR membranes. Immunoreactivity for p-αBC, however, was undetectable in endothelial cells of the normal retinas, where p-p38 MAPK immunoreactivity was less marked than PDR membranes. CONCLUSION Phosphorylation of αBC, in particular, phosphorylation on Ser59 by p-p38 MAPK may play a potential role as a molecular chaperon for VEGF in the pathogenesis of epiretinal membranes in PDR.

Genistein attenuates choroidal neovascularization.

Genistein is a dietary-derived flavonoid abundantly present in soybeans and known to possess various biological effects including anti-inflammation and anti-angiogenic activity. To investigate the effects of genistein on intraocular neovascularization, we used an animal model of laser-induced choroidal neovascularization (CNV). Male C57BL/6J mice were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. CNV was induced by laser photocoagulation. The animals were fed a mixture diet containing 0.5% genistein or a control diet ad libitum for 7 days before laser photocoagulation and the treatment was continued until the end of the study. Seven days after laser injury, the size of CNV lesions was quantified. Retinal pigment epithelium (RPE)-choroid complex was also harvested 1 or 3 days after laser injury and the level of monocyte chemoattractant protein (MCP)-1, intercellular adhesion molecule (ICAM)-1, and matrix metalloproteinase (MMP)-9 were measured by enzyme-linked immunosorbent assay. Expression levels of Ets-1 and F4/80 were examined by real-time PCR. A significant decrease in CNV size was observed in animals treated with genistein (15441.9±1511.8 μm(2)) compared to control mice (21074.0±1940.7μm(2), P<.05). Genistein significantly reduced the protein level of MCP-1, ICAM-1, and MMP-9 in the RPE-choroid complex (P<.05). In addition, genistein suppressed the expression levels of Ets-1 and F4/80 (P<.05). The current data indicate the anti-angiogenic property of genistein during CNV formation.