Kenji Yamashiro

VEGF164-mediated Inflammation Is Required for Pathological, but Not Physiological, Ischemia-induced Retinal Neovascularization

Hypoxia-induced VEGF governs both physiological retinal vascular development and pathological retinal neovascularization. In the current paper, the mechanisms of physiological and pathological neovascularization are compared and contrasted. During pathological neovascularization, both the absolute and relative expression levels for VEGF164 increased to a greater degree than during physiological neovascularization. Furthermore, extensive leukocyte adhesion was observed at the leading edge of pathological, but not physiological, neovascularization. When a VEGF164-specific neutralizing aptamer was administered, it potently suppressed the leukocyte adhesion and pathological neovascularization, whereas it had little or no effect on physiological neovascularization. In parallel experiments, genetically altered VEGF164-deficient (VEGF120/188) mice exhibited no difference in physiological neovascularization when compared with wild-type (VEGF+/+) controls. In contrast, administration of a VEGFR-1/Fc fusion protein, which blocks all VEGF isoforms, led to significant suppression of both pathological and physiological neovascularization. In addition, the targeted inactivation of monocyte lineage cells with clodronate-liposomes led to the suppression of pathological neovascularization. Conversely, the blockade of T lymphocyte–mediated immune responses with an anti-CD2 antibody exacerbated pathological neovascularization. These data highlight important molecular and cellular differences between physiological and pathological retinal neovascularization. During pathological neovascularization, VEGF164 selectively induces inflammation and cellular immunity. These processes provide positive and negative angiogenic regulation, respectively. Together, new therapeutic approaches for selectively targeting pathological, but not physiological, retinal neovascularization are outlined.

Genetic variants near TIMP3 and high-density lipoprotein–associated loci influence susceptibility to age-related macular degeneration

We executed a genome-wide association scan for age-related macular degeneration (AMD) in 2,157 cases and 1,150 controls. Our results validate AMD susceptibility loci near CFH (P < 10−75), ARMS2 (P < 10−59), C2/CFB (P < 10−20), C3 (P < 10−9), and CFI (P < 10−6). We compared our top findings with the Tufts/Massachusetts General Hospital genome-wide association study of advanced AMD (821 cases, 1,709 controls) and genotyped 30 promising markers in additional individuals (up to 7,749 cases and 4,625 controls). With these data, we identified a susceptibility locus near TIMP3 (overall P = 1.1 × 10−11), a metalloproteinase involved in degradation of the extracellular matrix and previously implicated in early-onset maculopathy. In addition, our data revealed strong association signals with alleles at two loci (LIPC, P = 1.3 × 10−7; CETP, P = 7.4 × 10−7) that were previously associated with high-density lipoprotein cholesterol (HDL-c) levels in blood. Consistent with the hypothesis that HDL metabolism is associated with AMD pathogenesis, we also observed association with AMD of HDL-c—associated alleles near LPL (P = 3.0 × 10−3) and ABCA1 (P = 5.6 × 10−4). Multilocus analysis including all susceptibility loci showed that 329 of 331 individuals (99%) with the highest-risk genotypes were cases, and 85% of these had advanced AMD. Our studies extend the catalog of AMD associated loci, help identify individuals at high risk of disease, and provide clues about underlying cellular pathways that should eventually lead to new therapies.

Macular Choroidal Thickness and Volume in Normal Subjects Measured by Swept-Source Optical Coherence Tomography

PURPOSE To study the choroidal thickness in healthy subjects by swept-source optical coherence tomography (SS-OCT) at longer wavelength. METHODS The macular area of 31 eyes (31 healthy volunteers) was studied with an SS-OCT prototype system, which uses a tunable laser as a light source operated at 100,000 Hz A scan repetition rate in the 1-μm wavelength region. Three-dimensional volumetric measurement comprised of 512 × 128 A scans was acquired in 0.8 second. From a series of OCT images, a chroidal thickness map of the macular area was created by manual segmentation. To evaluate interoperator reproducibility, the choroidal thickness in each section from 10 subjects was determined independently by two observers. RESULTS SS-OCT at the 1-μm wavelength region allowed visualization of the fine structure of the choroid as well as that of the retina. Mean choroidal thickness and volume in the macula area were, respectively, 191.5 ± 74.2 μm and 5.411 ± 2.097 mm(3). The mean choroidal thickness of the outer nasal area was significantly thinner than that of all other areas (P < 0.05). The measurements by the two independent observers were significantly identical; the intraclass correlation coefficient in mean choroidal thickness was between 0.945 and 0.980 in each area. The macular choroidal thickness was significantly correlated with axial length after adjustment for age (P < 0.001), and with age after adjustment for axial length (P < 0.001). CONCLUSIONS SS-OCT system at 1 μm provides macular choroidal thickness maps and allows one to evaluate the choroidal thickness more accurately.

Lectin-like oxidized LDL receptor-1 is a cell-adhesion molecule involved in endotoxin-induced inflammation

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a major endothelial receptor for oxidized low-density lipoprotein, and is assumed to play a proatherogenic role in atherosclerosis. LOX-1 expression is induced by inflammatory cytokines as well as by proatherogenic stimuli. LOX-1 protein binds aged/apoptotic cells, activated platelets, and bacteria, suggesting that it may have diverse activities in vivo. Here, we reveal a role for LOX-1 in endotoxin-induced inflammation. In a model of endotoxemia, injection of a high dose of endotoxin into rats induced leukopenia within 1 h and death of the animals within 24 h. Preadministration of anti-LOX-1 antibody reduced the degree of leukopenia and completely rescued the animals, whereas control IgG did not. In a model of low-dose endotoxin-induced uveitis, anti-LOX-1 antibody efficiently suppressed leukocyte infiltration and protein exudation. In situ videomicroscopic analyses of leukocyte interactions with retinal veins revealed that anti-LOX-1 antibody reduced the number of rolling leukocytes and increased the velocity of rolling, suggesting that LOX-1 functions as a vascular tethering ligand. The ability of LOX-1 to capture leukocytes under physiologic shear was confirmed in an in vitro flow model. Thus, LOX-1 is an adhesion molecule involved in leukocyte recruitment and may represent an attractive target for modulation of endotoxin-induced inflammation.

Assessment of macular choroidal thickness by optical coherence tomography and angiographic changes in central serous chorioretinopathy.

OBJECTIVE To investigate the relationship between macular choroidal thickness measured by high-penetrating swept-source optical coherence tomography (SS-OCT) and angiographic findings in central serous chorioretinopathy (CSC). DESIGN Prospective cross-sectional case series. PARTICIPANTS AND CONTROLS Thirty-four patients with CSC (44 eyes) and 17 volunteer subjects (17 normal eyes). METHODS All subjects underwent a comprehensive ophthalmologic and SS-OCT prototype examination. All patients with CSC also underwent simultaneous fluorescein angiography (FA) and indocyanine green angiography (IA). Mean regional choroidal thickness measurements on the Early Treatment Diabetic Retinopathy Study (ETDRS) layout and squared sector grids were obtained by 3-dimensional raster scanning using SS-OCT. MAIN OUTCOME MEASURES Macular choroidal thickness and angiographic abnormalities. RESULTS Mean whole macular choroidal thickness in eyes with CSC (total, 329.3±83.0 μm; classic CSC, 326.9±83.1 μm; chronic CSC, 325.4±93.3 μm; and multifocal posterior pigment epitheliopathy, 359.0±15.5 μm) was greater than that in normal eyes (233.0±67.0 μm) (P < 0.001). In unilateral cases, mean whole macular choroidal thickness was greater in eyes with unilateral CSC than in unaffected fellow eyes (P=0.021). There was no significant difference in choroidal thickness between active eyes and resolved eyes in any of the ETDRS sectors. Mean choroidal thickness was greater in areas with leakage on FA than in areas without leakage (P=0.001). Mean choroidal thickness was greater in areas with choroidal vascular hyperpermeability and in areas with punctate hyperfluorescent spots on IA than in unaffected areas (P<0.001 for both). CONCLUSIONS Increased choroidal thickness was observed in the whole macular area of eyes with any of the CSC subtypes. Choroidal thickness was related to leakage from the retinal pigment epithelium, choroidal vascular hyperpermeability, and punctate hyperfluorescent lesions. These findings provide evidence that CSC may be caused by focally increased hydrostatic pressure in the choroid.

Choroidal Thickness, Vascular Hyperpermeability, and Complement Factor H in Age-Related Macular Degeneration and Polypoidal Choroidal Vasculopathy

PURPOSE To investigate the relationship between subfoveal choroidal thickness, choroidal vascular hyperpermeability, and complement factor H (CFH) gene polymorphism in typical age-related macular degeneration (AMD) and polypoidal choroidal vasculopathy (PCV). METHODS Fifty-eight patients with typical AMD and 63 patients with PCV underwent fluorescein angiography, indocyanine green angiography (IA), and spectral-domain optical coherence tomography (OCT) using enhanced depth imaging (EDI). Subfoveal choroidal thickness was measured using EDI-OCT images, and choroidal hyperpermeability was evaluated using late-phase IA images. The major AMD-associated single-nucleotide polymorphisms were genotyped in 86 patients. RESULTS Mean subfoveal choroidal thickness was significantly lower in eyes with typical AMD than that in eyes with PCV (P = 0.025). Subfoveal choroidal thickness was greater in eyes with choroidal hyperpermeability than that in eyes without it in typical AMD (P < 0.001) and PCV (P = 0.020), and in the fellow eyes of typical AMD (P < 0.001) and PCV (P = 0.027). In eyes without choroidal hyperpermeability, the mean subfoveal choroidal thickness was greater in PCV than that in typical AMD (P = 0.001). Choroidal thickness decreased after photodynamic therapy combined with intravitreal ranibizumab in typical AMD (P = 0.016) and PCV (P = 0.036). In eyes with PCV, the I62V polymorphism in the CFH gene contributed to choroidal thickness (P = 0.043). CONCLUSIONS Choroidal thickness is related to the AMD subtypes, choroidal hyperpermeability, and I62V CFH gene polymorphism. In eyes without choroidal hyperpermeability, EDI-OCT is useful as an auxiliary measure for differentiating typical AMD and PCV.

A Genome-Wide Association Analysis Identified a Novel Susceptible Locus for Pathological Myopia at 11q24.1

Myopia is one of the most common ocular disorders worldwide. Pathological myopia, also called high myopia, comprises 1% to 5% of the general population and is one of the leading causes of legal blindness in developed countries. To identify genetic determinants associated with pathological myopia in Japanese, we conducted a genome-wide association study, analyzing 411,777 SNPs with 830 cases and 1,911 general population controls in a two-stage design (297 cases and 934 controls in the first stage and 533 cases and 977 controls in the second stage). We selected 22 SNPs that showed P-values smaller than 10−4 in the first stage and tested them for association in the second stage. The meta-analysis combining the first and second stages identified an SNP, rs577948, at chromosome 11q24.1, which was associated with the disease (P = 2.22×10−7 and OR of 1.37 with 95% confidence interval: 1.21–1.54). Two genes, BLID and LOC399959, were identified within a 200-kb DNA encompassing rs577948. RT–PCR analysis demonstrated that both genes were expressed in human retinal tissue. Our results strongly suggest that the region at 11q24.1 is a novel susceptibility locus for pathological myopia in Japanese.

High-resolution imaging of resolved central serous chorioretinopathy using adaptive optics scanning laser ophthalmoscopy.

OBJECTIVE To compare pathologic changes in photoreceptors in eyes with resolved central serous chorioretinopathy (CSC) seen on high-resolution images obtained by adaptive optics scanning laser ophthalmoscopy (AO SLO) with visual acuity (VA) and findings on spectral-domain optical coherence tomography (SD OCT). DESIGN Observational case series. PARTICIPANTS Forty-five eyes of 38 patients with resolved CSC and 20 normal eyes of 20 volunteer subjects. METHODS All patients underwent a full ophthalmologic examination, SD OCT, and imaging with an original prototype AO SLO system fabricated using liquid crystal-on-silicon technology. MAIN OUTCOME MEASURES Cone mosaic patterns and cone density on AO SLO images and VA in eyes with CSC. RESULTS In normal eyes, AO SLO images showed a regular photoreceptor mosaic pattern and average cone densities 0.2, 0.5, and 1.0 mm from the central fovea of 67,900, 33,320, and 14,450 cones/mm(2). In eyes with CSC, cone densities were significantly lower at each distance from the central fovea (P = 0.009 at 0.2 mm, P = 0.007 at 0.5 mm, and P = 0.004 at 1.0 mm), and 2 distinct cone mosaic patterns were seen. Group 1 CSC eyes had regular cone mosaic patterns with small dark regions. Group 2 CSC eyes had irregular mosaic patterns with large dark regions. Compared with group 1, group 2 had significantly lower average cone density and worse average logarithm of the minimum angle of resolution (logMAR) VA (P<0.001). Mean cone density in eyes with disruptions in the photoreceptor inner and outer segment (IS/OS) junction or in the intermediate line on SD OCT images was significantly lower than that in eyes with an intact IS/OS junction or intermediate line (P<0.001 for both). Cone density 0.2 mm from the central fovea correlated with logMAR VA and mean foveal thickness (1-mm diameter area) measured on SD OCT images (P<0.001 for both). CONCLUSIONS Adaptive optics SLO images showed abnormal cone mosaic patterns and reduced cone densities in eyes with resolved CSC, and these abnormalities were associated with VA loss, suggesting that AO SLO is a useful means to detect and measure cone abnormalities associated with VA loss in these eyes.

Association between Foveal Photoreceptor Integrity and Visual Outcome in Neovascular Age-related Macular Degeneration

PURPOSE To evaluate the correlation between visual outcome and foveal photoreceptor integrity after successful treatment of eyes with neovascular age-related macular degeneration (AMD). DESIGN Retrospective chart review. METHODS We retrospectively studied the medical records of 51 eyes of 51 patients with neovascular AMD who were treated successfully with photodynamic therapy (PDT). All eyes were followed-up for more than 24 months after the initial treatment. Using spectral-domain optical coherence tomography, the status of the inner segment and outer segment (IS/OS) photoreceptor junction was assessed as a hallmark of the integrity of the foveal photoreceptor layer. RESULTS At the final visit, no eyes showed an exudative change. A complete or discontinuous IS/OS line was detected beneath the fovea in 8 (15.7%) and 25 (29.4%) eyes, respectively, whereas 28 (54.9%) had no IS/OS line. Eyes with a continuous or discontinuous IS/OS line beneath the fovea had better final visual acuity (VA) than did eyes without an IS/OS line (P < .001, respectively). Of the 51 eyes, 36 showed polypoidal choroidal vasculopathy (PCV), whereas 15 were diagnosed as having typical AMD without PCV. Visual outcome was significantly better in eyes with PCV (P = .026). Most eyes (13/15; 86.7%) with typical AMD had no IS/OS line at the final visit, whereas only 13 (36.1%) of the 36 eyes with PCV had no IS/OS line beneath the fovea. CONCLUSIONS Integrity of the photoreceptor layer beneath the fovea is associated with the final VA in neovascular AMD after successful PDT.

Simvastatin Inhibits Leukocyte Accumulation and Vascular Permeability in the Retinas of Rats with Streptozotocin-Induced Diabetes

Leukocytes play important roles in the pathogenesis of diabetic retinopathy. Recently, 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors have been reported to exert various effects in addition to their lipid-lowering ability. We investigated the effects of simvastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, on leukocyte-induced diabetic changes in retinas. Diabetes was induced in Long-Evans rats with streptozotocin, and simvastatin administration was begun immediately after the induction of diabetes. Two weeks of treatment with simvastatin suppressed significantly the number of leukocytes adhering to retinal vessel endothelium and the number of leukocytes accumulated in the retinal tissue by 72.9% and 41.0%, respectively (P < 0.01). The expression of intercellular adhesion molecule-1 (ICAM-1) and the CD18 (the common beta-chain of ICAM-1 ligands) were both suppressed with simvastatin. The amount of vascular endothelial growth factor in the retina was attenuated in the simvastatin-treated group. To evaluate the effects of simvastatin on leukocyte-induced endothelial cell damage, vascular permeability in the retina was measured with fluorescein-labeled dextran. Treatment with simvastatin markedly reduced retinal permeability (P = 0.014). This suggests that simvastatin attenuates leukocyte-endothelial cell interactions and subsequent blood-retinal barrier breakdown via suppression of vascular endothelial growth factor-induced ICAM-1 expression in the diabetic retina. Simvastatin may thus be useful in the prevention of diabetic retinopathy.