VEGF-induced retinal angiogenic signalling is critically dependent on Ca2+ signalling via Ca2+/calmodulin-dependent protein kinase II

Purpose. The authors conducted an in vitro investigation of the role of Ca2+-dependent signaling in vascular endothelial growth factor (VEGF)-induced angiogenesis in the retina.

Methods. Bovine retinal endothelial cells (BRECs) were stimulated with VEGF in the presence or absence of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM; intracellular Ca2+ chelator), U73122 (phospholipase C (PLC) inhibitor), xestospongin C (Xe-C), and 2-aminoethoxydiphenyl borate (2APB) (inhibitors of inositol-1,4,5 triphosphate (IP3) signaling). Intracellular Ca2+ concentration ([Ca2+]i) was estimated using fura-2 Ca2+ microfluorometry, Akt phosphorylation quantified by Western blot analysis, and angiogenic responses assessed using cell migration, proliferation, tubulogenesis, and sprout formation assays. The effects of the Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN93 were also evaluated on VEGF-induced Akt signaling and angiogenic activity.

Results. Stimulation of BRECs with 25 ng/mL VEGF induced a biphasic increase in [Ca2+]i, with an initial transient peak followed by a sustained plateau phase. VEGF-induced [Ca2+]i increases were almost completely abolished by pretreating the cells with BAPTA-AM, U73122, Xe-C, or 2APB. These agents also inhibited VEGF-induced phosphorylation of Akt, cell migration, proliferation, tubulogenesis, and sprouting angiogenesis. KN93 was similarly effective at blocking the VEGF-induced activation of Akt and angiogenic responses.

Conclusions. VEGF increases [Ca2+]i in BRECs through activation of the PLC-IP3 signal transduction pathway. VEGF-induced phosphorylation of the proangiogenic protein Akt is critically dependent on this increase in [Ca2+]i and the subsequent activation of CaMKII. Pharmacologic inhibition of Ca2+-mediated signaling in retinal endothelial cells blocks VEGF-induced angiogenic responses. These results suggest that the PLC/IP3/Ca2+/CaMKII signaling pathway may be a rational target for the treatment of angiogenesis-related disorders of the eye.

Development of a Diagnostic Genetic Test for Simplex and Autosomal Recessive Retinitis Pigmentosa

PURPOSE: Retinitis pigmentosa (RP) causes hereditary blindness in adults (prevalence, approximately 1 in 4000). Each of the more than 30 causative genes identified to date are responsible for only a small percentage of cases. Genetic diagnosis via traditional methods is problematic, and a single test with a higher probability of detecting the causative mutation would be very beneficial for the clinician. The goal of this study therefore was to develop a high-throughput screen capable of detecting both known mutations and novel mutations within all genes implicated in autosomal recessive or simplex RP. DESIGN: Evaluation of diagnostic technology. PARTICIPANTS AND CONTROLS: Participants were 56 simplex and autosomal recessive RP patients, with 360 population controls unscreened for ophthalmic disease. METHODS: A custom genechip capable of resequencing all exons containing known mutations in 19 disease-associated genes was developed (RP genechip). A second, commercially available arrayed primer extension (APEX) system was used to screen 501 individual previously reported variants. The ability of these high-throughput approaches to identify pathogenic variants was assessed in a cohort of simplex and autosomal recessive RP patients. MAIN OUTCOME MEASURES: Number of mutations and potentially pathogenic variants identified. RESULTS: The RP genechip identified 44 sequence variants: 5 previously reported mutations; 22 known single nucleotide polymorphisms (SNPs); 11 novel, potentially pathogenic variants; and 6 novel SNPs. There was strong concordance with the APEX array, but only the RP genechip detected novel variants. For example, identification of a novel mutation in CRB1 revealed a patient, who also had a single previously known CRB1 mutation, to be a compound heterozygote. In some individuals, potentially pathogenic variants were discovered in more than one gene, consistent with the existence of disease modifier effects resulting from mutations at a second locus. CONCLUSIONS: The RP genechip provides the significant advantage of detecting novel variants and could be expected to detect at least one pathogenic variant in more than 50% of patients. The APEX array provides a reliable method to detect known pathogenic variants in autosomal recessive RP and simplex RP patients and is commercially available. High-throughput genotyping for RP is evolving into a clinically useful genetic diagnostic tool.

Association of diabetes with age-related macular degeneration in the EUREYE study

Objective: To examine the association between self-reported diabetes history and early or late age-related macular degeneration (AMD) in the European population.

Retinal VEGF mRNA measured by SYBR Green I fluorescence: A versatile approach to quantitative PCR

PURPOSE: To determine whether continuous monitoring of SYBR Green I fluorescence provides a reliable and flexible method of quantitative RT-PCR. Our aims were (i) to test whether SYBR Green I analysis could quantify a wide range of known VEGF template concentrations, (ii) to apply this method in an experimental model, and (iii) to determine whether 20 existing primer pairs could be used to quantify their cognate mRNAs.

Advanced glycation end products in vitreous: Structural and functional implications for diabetic vitreopathy

PURPOSE. Advanced glycation end products (AGES) form irreversible cross- links with many macromolecules and have been shown to accumulate in tissues at an accelerated rate in diabetes. In the present study, AGE formation in vitreous was examined in patients of various ages and in patients with diabetes. Ex vivo investigations were performed on bovine vitreous incubated in glucose to determine AGE formation and cross-linking of vitreous collagen. METHODS. By means of an AGE-specific enzyme-linked immunosorbent assay (ELISA), AGE formation was investigated in vitreous samples obtained after pars plana vitrectomy in patients with and without diabetes. In addition, vitreous AGES were investigated in bovine vitreous collagen after incubation in high glucose, high glucose with aminoguanidine, or normal saline for as long as 8 weeks. AGEs and AGE cross-linking was subsequently determined by quantitative and qualitative assays. RESULTS. There was a significant correlation between AGEs and increasing age in patients without diabetes (r = 0.74). Furthermore, a comparison between age-matched diabetic and nondiabetic vitreous showed a significantly higher level of AGEs in the patients with diabetes (P < 0.005). Collagen purified from bovine vitreous incubated in 0.5 M glucose showed an increase in AGE formation when observed in dot blot analysis, immunogold labeling, and AGE ELISA. Furthermore, there was increased cross-linking of collagen in the glucose-incubated vitreous, when observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein separation. This cross-linking was effectively inhibited by coincubation with 10 mM aminoguanidine. CONCLUSIONS. This study suggests that AGEs may form in vitreous with increasing age. This process seems to be accelerated in the presence of diabetes and as a consequence of exposure to high glucose. Advanced glycation and AGE cross-linking of the vitreous collagen network may help to explain the vitreous abnormalities characteristic of diabetes.

Reduced Nitro-oxidative Stress and Neural Cell Death Suggests a Protective Role for Microglial Cells in TNFα−/− Mice in Ischemic Retinopathy

Purpose. Neovascularization occurs in response to tissue ischemia and growth factor stimulation. In ischemic retinopathies, however, new vessels fail to restore the hypoxic tissue; instead, they infiltrate the transparent vitreous. In a model of oxygen-induced retinopathy (OIR), TNFa and iNOS, upregulated in response to tissue ischemia, are cytotoxic and inhibit vascular repair. The aim of this study was to investigate the mechanism for this effect.

Methods. Wild-type C57/BL6 (WT) and TNFa-/- mice were subjected to OIR by exposure to 75% oxygen (postnatal days 7–12). The retinas were removed during the hypoxic phase of the model. Retinal cell death was determined by TUNEL staining, and the microglial cells were quantified after Z-series capture with a confocal microscope. In situ peroxynitrite and superoxide were measured by using the fluorescent dyes DCF and DHE. iNOS, nitrotyrosine, and arginase were analyzed by real-time PCR, Western blot analysis, and activity determined by radiolabeled arginine conversion. Astrocyte coverage was examined after GFAP immunostaining.

Results. The TNFa-/- animals displayed a significant reduction in TUNEL-positive apoptotic cells in the inner nuclear layer of the avascular retina compared with that in the WT control mice. The reduction coincided with enhanced astrocytic survival and an increase in microglial cells actively engaged in phagocytosing apoptotic debris that displayed low ROS, RNS, and NO production and high arginase activity.

Conclusions. Collectively, the results suggest that improved vascular recovery in the absence of TNFa is associated with enhanced astrocyte survival and that both phenomena are dependent on preservation of microglial cells that display an anti-inflammatory phenotype during the early ischemic phase of OIR.

Docosahexaenoic Acid Improves the Nitroso-Redox Balance and Reduces VEGF-Mediated Angiogenic Signaling in Microvascular Endothelial Cells

Purpose. Disturbances to the cellular production of nitric oxide (NO) and superoxide (O2-) can have deleterious effects on retinal vascular integrity and angiogenic signaling. Dietary agents that could modulate the production of these signaling molecules from their likely enzymatic sources, endothelial nitric oxide synthase (eNOS) and NADPH oxidase, would therefore have a major beneficial effect on retinal vascular disease. The effect of ?-3 polyunsaturated fatty acids (PUFAs) on angiogenic signaling and NO/superoxide production in retinal microvascular endothelial cells (RMECs) was investigated.

Methods. Primary RMECs were treated with docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA) for 48 hours. RMEC migration was determined by scratch-wound assay, proliferation by the incorporation of BrdU, and angiogenic sprouting using a three-dimensional model of in vitro angiogenesis. NO production was quantified by Griess assay, and phospho-eNOS accumulation and superoxide were measured using the fluorescent probe dihydroethidine. eNOS localization to caveolin-rich microdomains was determined by Western blot analysis after subfractionation on a linear sucrose gradient.

Results. DHA treatment increased nitrite and decreased superoxide production, which correlated with the displacement of eNOS from caveolar subdomains and colocalization with the negative regulator caveolin-1. In addition, both ?-3 PUFAs demonstrated reduced responsiveness to VEGF-stimulated superoxide and nitrite release and significantly impaired endothelial wound healing, proliferation, and angiogenic sprout formation.

Conclusions. DHA improves NO bioavailability, decreases O2- production, and blunts VEGF-mediated angiogenic signaling. These findings suggest a role for ?-3 PUFAs, particularly DHA, in maintaining vascular integrity while reducing pathologic retinal neovascularization.