Abstract-Understanding molecular mechanisms regulating angiogenesis may lead to novel therapies for ischemic disorders. Hypoxia-inducible factor 1 (HIF-1) activates vascular endothelial growth factor (VEGF) gene expression in hypoxic/ischemic tissue. In this study we demonstrate that exposure of primary cultures of cardiac and vascular cells to hypoxia or AdCA5, an adenovirus encoding a constitutively active form of HIF-1␣, modulates the expression of genes encoding the angiogenic factors angiopoietin-1 (ANGPT1), ANGPT2, placental growth factor, and platelet-derived growth factor-B. Loss-of-function effects were also observed in HIF-1␣-null embryonic stem cells. Depending on the cell type, expression of ANGPT1 and ANGPT2 was either activated or repressed in response to hypoxia or AdCA5. In all cases, there was complete concordance between the effects of hypoxia and AdCA5. Injection of AdCA5 into mouse eyes induced neovascularization in multiple capillary beds, including those not responsive to VEGF alone. Analysis of gene expression revealed increased expression of ANGPT1, ANGPT2, platelet-derived growth factor-B, placental growth factor, and VEGF mRNA in AdCA5-injected eyes. These results indicate that HIF-1 functions as a master regulator of angiogenesis by controlling the expression of multiple angiogenic growth factors and that adenovirusmediated expression of a constitutively active form of HIF-1␣ is sufficient to induce angiogenesis in nonischemic tissue of an adult animal.

Inflammation affects the formation and the progression of various vitreoretinal diseases. We performed a comprehensive analysis of inflammatory immune mediators in the vitreous fluids from total of 345 patients with diabetic macular edema (DME, n = 92), proliferative diabetic retinopathy (PDR, n = 147), branch retinal vein occlusion (BRVO, n = 30), central retinal vein occlusion (CRVO, n = 13) and rhegmatogenous retinal detachment (RRD, n = 63). As a control, we selected a total of 83 patients with either idiopathic macular hole (MH) or idiopathic epiretinal membrane (ERM) that were free of major pathogenic intraocular changes, such as ischemic retina and proliferative membranes. The concentrations of 20 soluble factors (nine cytokines, six chemokines, and five growth factors) were measured simultaneously by multiplex bead analysis system. Out of 20 soluble factors, three factors: interleukin-6 (IL-6), interleukin-8 (IL-8), and monocyte chemoattractant protein-1 (MCP-1) were significantly elevated in all groups of vitreoretinal diseases (DME, PDR, BRVO, CRVO, and RRD) compared with control group. According to the correlation analysis in the individual patient's level, these three factors that were simultaneously increased, did not show any independent upregulation in all the examined diseases. Vascular endothelial growth factor (VEGF) was significantly elevated in patients with PDR and CRVO. In PDR patients, the elevation of VEGF was significantly correlated with the three factors: IL-6, IL-8, and MCP-1, while no significant correlation was observed in CRVO patients. In conclusion, multiplex bead system enabled a comprehensive soluble factor analysis in vitreous fluid derived from variety of patients. Major three factors: IL-6, IL-8, and MCP-1 were strongly correlated with each other indicating a common pathway involved in inflammation process in vitreoretinal diseases.

Apoptosis-inducing factor (AIF) is a novel mediator in apoptosis. AIF is a flavoprotein that is normally confined to the mitochondrial intermembrane space, yet translocates to the nucleus in several in vitro models of apoptosis. To investigate the role of AIF in the apoptotic process in vivo, we induced retinal detachment (RD) by subretinal injection of sodium hyaluronate, either in Brown Norway rats or in C3H mice. Apoptotic DNA fragmentation, as determined by terminal nick-end labeling, was most prominent 3 days after RD. The subcellular localization of AIF was examined by immunohistochemistry and immunoelectron microscopy. In normal photoreceptor cells, AIF was present in the mitochondrion-rich inner segment. However, AIF was found in the nucleus after RD. Photoreceptor apoptosis developed similarly in C3H control mice, and in mice bearing the gld or lpr mutations, indicating that cell death occurs independently from the CD95/CD95 ligand system. Both the mitochondrio-nuclear transition of AIF localization and the nuclear DNA fragmentation were inhibited by subretinal application of brain-derived neurotrophic factor. To our knowledge, this is the first description of AIF relocalization occurring in a clinically relevant, in vivo model of apoptosis.

The retinal pigment epithelium (RPE) is crucial for the normal development and function of retinal photoreceptors, and mutations in several genes that are preferentially expressed in the RPE have been shown to cause retinal degeneration. We analyzed the 5-upstream region of human VMD2, a gene that is preferentially expressed in the RPE and, when mutated, causes Best macular dystrophy. Transgenic mouse studies with VMD2 promoter/lacZ constructs demonstrated that a ؊253 to ؉38 bp fragment is sufficient to direct RPEspecific expression in the eye. Transient transfection assays using the D407 human RPE cell line with VMD2 promoter/luciferase reporter constructs identified two positive regulatory regions, ؊585 to ؊541 bp for high level expression and ؊56 to ؊42 bp for low level expression. Mutation of a canonical E-box located in the ؊56 to ؊42 bp region greatly diminished luciferase expression in D407 cells and abolished the bands shifted with bovine RPE nuclear extract in electrophoretic mobility shift assays. Independently a candidate approach was used to select microphthalmia-associated transcription factor (MITF) for testing because it is expressed in the RPE and associated with RPE abnormalities when mutated. MITF-M significantly increased luciferase expression in D407 cells in an E-box-dependent manner. These studies define the VMD2 promoter region sufficient to drive RPE-specific expression in the eye, identify positive regulatory regions in vitro, and suggest that MITF as well as other E-box binding factors may act as positive regulators of VMD2 expression. The retinal pigment epithelium (RPE)1 is a monolayer of cuboidal cells located between the photoreceptors and choroid of the eye. It has many specialized functions that support and nourish photoreceptors, including important roles in retinoid metabolism (visual cycle), phagocytosis of shed photoreceptor outer segments, maintenance of the blood-retina barrier, movement of ions and water, and synthesis and transport of substances that constitute the interphotoreceptor matrix (1). The importance of the RPE in maintaining retinal photoreceptors is highlighted by the Royal College of Surgeons rat that exhibits a markedly reduced capacity for phagocytosis of outer segments by the RPE that results in the degeneration and loss of photoreceptor cells (2, 3). In addition, in humans, RPE dysfunction has been implicated in the pathogenesis of age-related macular degeneration, which is the leading cause of irreversible blindness in elderly people in western countries (4, 5).Mutations in several genes that are specifically or preferentially expressed in the RPE, such as RPE65, RLBP1, RGR, TIMP3, and VMD2, are associated with inherited human retinal dystrophies (6 -15). Mutations in VMD2 result in Best disease (vitelliform macular dystrophy (VMD)), an autosomal dominant, juvenile onset macular dystrophy characterized by a striking accumulation of lipofuscin-like material within and beneath the RPE (16 -18). VMD2 encodes a multispan transmembrane protein, bestrophin, tha...

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Aim-To determine the prevalence of age related maculopathy (ARM) in a representative older Japanese population. Methods-1486 residents of Hisayama town, Fukuoka, Japan, aged 50 years or older were examined and the presence of ARM was determined by grading from fundus examination by indirect ophthalmoscope, slit lamp, and colour fundus photographs. Results-The prevalence rate of drusen, which occurred with comparable frequency in men and women, was 9.6%. The frequency of drusen increased with age (p <0.01). Hyperpigmentation and/or hypopigmentation of the retina was present in 3.2%, geographic atrophy in 0.2%, and neovascular age related macular degeneration in 0.67%. The frequency of neovascular age related macular degeneration was significantly higher in the men (1.2% v 0.34%, p <0.01). Conclusions-Early and late stage ARM is less common among Japanese people than among white people in Western countries, while late stage ARM is more common among Japanese than among black people.

Age-related macular degeneration (AMD), the leading cause of irreversible blindness in the world, is a complex disease caused by multiple environmental and genetic risk factors. To identify genetic factors that modify the risk of exudative AMD in the Japanese population, we conducted a genome-wide association study and a replication study using a total of 1,536 individuals with exudative AMD and 18,894 controls. In addition to CFH (rs800292, P = 4.23 × 10(-15)) and ARMS2 (rs3750847, P = 8.67 × 10(-29)) loci, we identified two new susceptibility loci for exudative AMD: TNFRSF10A-LOC389641 on chromosome 8p21 (rs13278062, combined P = 1.03 × 10(-12), odds ratio = 0.73) and REST-C4orf14-POLR2B-IGFBP7 on chromosome 4q12 (rs1713985, combined P = 2.34 × 10(-8), odds ratio = 1.30). Fine mapping revealed that rs13278062, which is known to alter TNFRSF10A transcriptional activity, had the most significant association in 8p21 region. Our results provide new insights into the pathophysiology of exudative AMD.

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