Characteristics of the study population.

<p>AMD: age-related macular degeneration; SD: standard deviation.</p

Association of <i>C3</i> haplotype blocks with wet AMD.

<p>OR: odds ratio; CI: confidence intervals.</p><p>The association of haplotype CC in block 4 remained statistically significant after correction for multiple testing (permutation <i>P</i> = 0.011).</p><p>*The <i>P</i>-values were calculated by the chi-square test on haplotype counts (1 degree of freedom).</p>†<p>The omnibus <i>P</i>-values were calculated by the PLINK software (4 degrees of freedom for block 1; 2 degrees of freedom for block 2, 4, and 5; 3 degrees of freedom for block 3).</p

Four-marker sliding window haplotype analysis over the entire <i>C3</i> locus.

<p>SNP: single nucleotide polymorphism.</p><p>*Omnibus <i>P</i> value corresponding to the haplotype with the listed SNP as the first SNP in the haplotype.</p

Multilocus logistic regression analysis of <i>C3</i> rs2241394, <i>ARMS2</i> A69S, <i>CFH</i> I62V, and <i>CFH</i> Y402H.

<p>AIC: Akaike information criterion.</p

Linkage disequilibrium (LD) structure of the <i>C3</i> locus.

<p>LD was measured using data from all subjects in the present study. The haplotype blocks were determined by the solid spine of LD method implemented in the Haploview software. Each box provides estimated statistics of the coefficient of determination (r²), with darker shades representing stronger LD.</p

Arhgef15 Promotes Retinal Angiogenesis by Mediating VEGF-Induced Cdc42 Activation and Potentiating RhoJ Inactivation in Endothelial Cells

<div><h3>Background</h3><p>Drugs inhibiting vascular endothelial growth factor (VEGF) signaling are globally administered to suppress deregulated angiogenesis in a variety of eye diseases. However, anti-VEGF therapy potentially affects the normal functions of retinal neurons and glias which constitutively express VEGF receptor 2. Thus, it is desirable to identify novel drug targets which are exclusively expressed in endothelial cells (ECs). Here we attempted to identify an EC-specific Rho guanine nucleotide exchange factor (GEF) and evaluate its role in retinal angiogenesis.</p> <h3>Methodology/Principal Findings</h3><p>By exploiting fluorescence-activated cell sorting and microarray analyses in conjunction with in silico bioinformatics analyses, we comprehensively identified endothelial genes in angiogenic retinal vessels of postnatal mice. Of 9 RhoGEFs which were highly expressed in retinal ECs, we show that Arhgef15 acted as an EC-specific GEF to mediate VEGF-induced Cdc42 activation and potentiated RhoJ inactivation, thereby promoting actin polymerization and cell motility. Disruption of the Arhgef15 gene led to delayed extension of vascular networks and subsequent reduction of total vessel areas in postnatal mouse retinas.</p> <h3>Conclusions/Significance</h3><p>Our study provides information useful to the development of new means of selectively manipulating angiogenesis without affecting homeostasis in un-targeted tissues; not only in eyes but also in various disease settings such as cancer.</p> </div

Arhgef15 facilitates actin polymerization and cell motility in ECs.

<p>(A) Phase-contrast (PhC) microscopy in cultured HUVECs at 72 h after siRNA transfection or 30 min after Sema3E stimulation. (B) The ratio of collapsing cells in cultured HUVECs. All experiments were repeated at least four times. (C) Confocal microscopy for phalloidin in cultured HUVECs. Note the actin depolymerization after transfection of siRNAs for Arhgef15 and Cdc42, and after Sema3E stimulation. (D) Quantification of the cell surface area in cultured HUVECs. The number of cells analyzed were: si-Ctrl, 151; si-GEF15, 170; si-CDC42, 151; Sema3E, 168. (E) Confocal microscopy for phalloidin in cultured HUVECs. Note the thickening of actin fibers at 24 h after transfection of plasmid vectors expressing Arhgef15 and Cdc42-CA, in contrast to the disruption of actin fibers by RhoJ-WT overexpression. (F) Scratch-wound assay in HUVECs transfected with siRNAs. Note the reduced EC motility by knockdown of Arhgef15 and Cdc42. (G) Quantification of the wound closure. <i>n</i> = 3 per group. (H) Tube formation assay in HUVECs transfected with siRNAs. Note the inhibition of capillary-like network formation by knockdown of Arhgef15 and Cdc42. (I) Quantification of the total tube length. <i>n</i> = 7 per group. Scale bar: 50 µm (A); 10 µm (C and E); 100 µm (F and H). Error bars represent SEM; **<i>P</i><0.01, ***<i>P</i><0.001.</p

Arhgef15 mediates VEGF-induced Cdc42 activation and potentiates RhoJ inactivation.

<p>(A) Ectopic expression of full-length Arhgef15 (GEF15) and its DH-PH truncated proteins activated endogenous Cdc42 in 293T cells. (B) VEGF-induced Cdc42 activation at 5 min was abolished by Arhgef15 knockdown in HUVECs. (C) Ectopic expression of GEF15 and its DH-PH proteins inactivated co-transfected RhoJ-WT in 293T cells. CA, constitutively-active; DN, dominant negative. In A–C, representative immunoblotting images of 3 independent experiments are shown. Error bars represent SEM; *<i>P</i><0.05, **<i>P</i><0.01. (D) A scheme xrepresenting signal transduction in ECs.</p

EC-specific expression of Arhgef15 in mouse retinas.

<p>(A–D) IHC for lacZ and PECAM-1 in whole-mount retinas (A, B, and D) and retinal cryo-sections (C) of <i>Arhgef15</i>^lacZ/lacZ mice at P5 (A–C) and P17 (D). Note the endothelial specificity of Arhgef15 expression in mouse retinas at early and late postnatal stages. At P17, endothelial expression of Arhgef15 in the superficial vascular layer was diminished in arteries but was maintained in veins. A, artery; V, vein. Scale bar: 200 µm (A); 20 µm (B and C); 50 µm (D).</p

Retardation of retinal vascular growth in <i>Arhgef15</i>-KO mouse.

<p>(A) Whole-mount IHC for PECAM-1 in P5 and P8 retinas. Circles represent the vascular margin of the <i>Arhgef</i>15^WT/WT retinas. (B) Quantification of the radius of the retinal vascular networks (P5, <i>n</i> = 6; P8, <i>n</i> = 4). (C) Morphometric analyses of vascular networks in P5 retinas (<i>n</i> = 6 per group). (D) Whole-mount IHC for PECAM-1 in P4 retinas. Circles represent the vascular margin of the <i>R26-Rhoj</i> retina. (E) Quantification of the radius of the retinal vascular networks at P4. Scale bar: 200 µm. Error bars represent SEM; *<i>P</i><0.05, **<i>P</i><0.01.</p