The Alternative Complement System Mediates Cell Death in Retinal Ischemia Reperfusion Injury.

Ischemia reperfusion (IR) injury induces retinal cell death and contributes to visual impairment. Previous studies suggest that the complement cascade plays a key role in IR injury in several systemic diseases. However, the role of the complement pathway in the ischemic retina has not been investigated. The aim of this study is to determine if the alternative complement cascade plays a role in retinal IR injury, and identify which components of the pathway mediate retinal degeneration in response to IR injury. To accomplish this, we utilized the mouse model of retinal IR injury, wherein the intraocular pressure (IOP) is elevated for 45 min, collapsing the retinal blood vessels and inducing retinal ischemia, followed by IOP normalization and subsequent reperfusion. We found that mRNA expression of complement inhibitors complement receptor 1-related gene/protein-y (Crry), Cd55 and Cd59a was down-regulated after IR. Moreover, genetic deletion of complement component 3 (C3-/-) and complement factor b (Fb-/-) decreased IR-induced retinal apoptosis. Because vascular dysfunction is central to IR injury, we also assessed the role of complement in a model of shear stress. In human retinal endothelial cells (HRECs), shear stress up-regulated complement inhibitors Cd46, Cd55, and Cd59, and suppressed complement-mediated cell death, indicating that a lack of vascular flow, commonly observed in IR injury, allows for complement mediated attack of the retinal vasculature. These results suggested that in retinal IR injury, the alternative complement system is activated by suppression of complement inhibitors, leading to vascular dysfunction and neuronal cell death.

Cytochrome P450 monooxygenase lipid metabolites are significant second messengers in the resolution of choroidal neovascularization.

Age-related macular degeneration (AMD) is the most common cause of blindness for individuals age 50 and above in the developed world. Abnormal growth of choroidal blood vessels, or choroidal neovascularization (CNV), is a hallmark of the neovascular (wet) form of advanced AMD and leads to significant vision loss. A growing body of evidence supports a strong link between neovascular disease and inflammation. Metabolites of long-chain polyunsaturated fatty acids derived from the cytochrome P450 (CYP) monooxygenase pathway serve as vital second messengers that regulate a number of hormones and growth factors involved in inflammation and vascular function. Using transgenic mice with altered CYP lipid biosynthetic pathways in a mouse model of laser-induced CNV, we characterized the role of these lipid metabolites in regulating neovascular disease. We discovered that the CYP-derived lipid metabolites epoxydocosapentaenoic acids (EDPs) and epoxyeicosatetraenoic acids (EEQs) are vital in dampening CNV severity. Specifically, overexpression of the monooxygenase CYP2C8 or genetic ablation or inhibition of the soluble epoxide hydrolase (sEH) enzyme led to increased levels of EDP and EEQ with attenuated CNV development. In contrast, when we promoted the degradation of these CYP-derived metabolites by transgenic overexpression of sEH, the protective effect against CNV was lost. We found that these molecules work in part through their ability to regulate the expression of key leukocyte adhesion molecules, on both leukocytes and endothelial cells, thereby mediating leukocyte recruitment. These results suggest that CYP lipid signaling molecules and their regulators are potential therapeutic targets in neovascular diseases.

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.

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.

Short Communication: Increase of Sialylated N-Glycansin Eyes with Neovascular Glaucoma Secondary to Proliferative Diabetic Retinopathy.

PURPOSE: To investigate the alteration of N-glycans in the vitreous fluid of patients with neovascular glaucoma (NVG) secondary to proliferative diabetic retinopathy (PDR). METHODS: Vitreous samples were collected from 18 patients with PDR (including 7 with NVG and 11 without NVG), and 17 patients without diabetes. Profiles of N-glycans were analyzed by glycoblotting-based high throughput protocol, which we recently developed. Protein levels of vascular endothelial growth factor (VEGF)-A were measured by ELISA. RESULTS: The concentration of total N-glycans and the concentration of N-glycans with sialic acids were significantly higher in NVG group compared with those in non-NVG group or control group, whereas there was no significant difference in concentrations of high-mannose N-glycans among three groups. There was a moderate correlation between the concentrations of sialylated N-glycans and VEGF-A. CONCLUSIONS: Our data demonstrate the distinct changes of N-glycan profile and the increase of sialylated N-glycans in eyes with NVG secondary to PDR.

Alteration of N-Glycan Profiles in Diabetic Retinopathy.

PURPOSE: To investigate the alteration of vitreal N-glycans in patients with proliferative diabetic retinopathy (PDR). METHODS: Plasma and vitreous samples were collected from 17 patients (10 females and 7 males) with PDR (PDR group) and 17 nondiabetic patients (8 females and 9 males) with epiretinal membrane (ERM) and idiopathic macular hole (MH) (non-diabetes mellitus [DM] group). Profiles of N-glycans were analyzed by a glycoblotting-based high-throughput protocol that we recently developed. Human retinal microvascular endothelial cells (HRMECs) were cultivated with culture media containing either low glucose (5 mM) or high glucose (25 mM), and expression levels of sialyltransferases were analyzed by real-time PCR and ELISA. RESULTS: Amount of N-glycans in the vitreous fluid of the PDR group was significantly higher than that of the non-DM group (495.5 ± 37.4 vs. 142.7 ± 30.8 pmol/100 µg protein, P < 0.005), whereas there was no significant difference in the plasma samples between the PDR and the non-DM group. In addition, profile analysis showed that N-glycans with sialic acids increased in the vitreous of the PDR group (328.4 ± 25.8 pmol/100 µg protein) compared to the non-DM group (92.1 ± 21.2 pmol/100 µg protein, P < 0.0005). Expression levels of sialyltransferases ST3GAL1 and ST3GAL4 were upregulated in the HRMECs after high-glucose stimulation. Consistent with the real-time PCR data, high-glucose stimulation elevated the protein levels of ST3GAL1 (117.4 ± 14.9 pg/mg, P < 0.01) and ST3GAL4 (6.1 ± 0.9 pg/mg, P < 0.05) in the HRMECs compared with the cells cultured with low-glucose culture media (ST3GAL1, 64.4 ± 5.8 pg/mg; ST3GAL4, 3.8 ± 0.3 pg/mg). CONCLUSIONS: Our data demonstrate distinct changes in the N-glycan profile and an increase in sialylated N-glycans in eyes with 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.

A comparison of N-glycan profiles in human plasma and vitreous fluid.

PURPOSE: To investigate the concentration and composition of N-glycans in plasma and vitreous samples obtained from patients with non-proliferative vitreoretinal diseases. METHODS: Plasma and vitreous samples were collected from 11 patients with idiopathic macular hole (MH) and 9 patients with epiretinal membrane (ERM). The samples were pretreated for enzymatic cleaving, and subsequently glycans released from proteins were captured on BlotGlyco H beads. Sialic acids were methyl-esterified. Processed glycans were tagged with aminooxy-functionalized peptide reagent (aoWR) and released from the beads, followed by detection by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The concentration and composition of N-glycans in the samples were assessed. RESULTS: Concentration of N-glycans in vitreous samples (132 ± 29 pmol/100 µg protein) was significantly lower compared with those in plasma samples (714 ± 29 pmol/100 µg protein, p < 0.001). Predominant N-glycan in both plasma (39.7 ± 1.1 %) and vitreous fluid (37.2 ± 3.1 %) was identical, and the composition was presumed as [(Hex)2(HexNAc)2(NeuAc)2+ (Man)3(GlcNAc)2]. By contrast, the second-ranked N-glycan in vitreous samples (15.6 ± 1.5 %) was the seventh in plasma (2.3 ± 0.2 %). CONCLUSIONS: The current data provide useful information on N-glycan profile in the vitreous fluid, which is distinct from that in the plasma.

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.