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Robert M-C, Arafat SN, Spurr-Michaud S, Chodosh J, Dohlman CH, Gipson IK. Tear Matrix Metalloproteinases and Myeloperoxidase Levels in Patients With Boston Keratoprosthesis Type I. Cornea 2016;35(7):1008-14.Abstract

PURPOSE: To investigate the tear levels of matrix metalloproteinases (MMPs), myeloperoxidase (MPO), and tissue inhibitor of metalloproteinase-1 in eyes after Boston keratoprosthesis type I (B-KPro) implantation and to correlate these markers with the established B-KPro prognostic categories. METHODS: Tear washes were collected from 40 patients (7 with autoimmune disease, 2 with chemical burn, and 31 with other noncicatrizing diagnoses). Tear levels of MMPs, MPO, and tissue inhibitor of metalloproteinase-1 were quantified using multianalyte bead-based enzyme-linked immunosorbent assays. The total MMP activity was determined using a fluorimetric assay. The analytes were compared to the underlying diagnosis and other clinical factors. RESULTS: The MMP-8, MMP-9, and MPO levels were markedly elevated in the eyes with B-KPro (80 ± 31, 291 ± 77, and 244 ± 33 pg/μg, respectively). Chemical burn was associated with significantly higher tear MMP-8 (474 ± 376 pg/μg) and MMP-9 levels (1300 ± 635 pg/μg) compared with noncicatrizing diseases (MMP-8: 41 ± 15 pg/μg, P = 0.02 and MMP-9: 196 ± 57 pg/μg, P = 0.02) and higher MMP-9 levels compared with autoimmune diseases (MMP-8: 96 ± 65 pg/μg, P = 0.21 and MMP-9: 306 ± 196 pg/μg, P = 0.04). Similar analyte levels were observed in the B-KPro eye and the contralateral non-B-KPro eye of patients with bilateral diseases. MMP-8, MMP-9, and total MMP activities correlated strongly with each other. CONCLUSIONS: In the eyes with B-KPro, tear MMP-8 and MMP-9 levels seem to be related to the underlying ocular surface pathology and not significantly influenced by the presence of the prosthesis.

Abusamra K, Oray M, Ebrahimiadib N, Lee S, Anesi S, Foster SC. Intraocular Lymphoma: Descriptive Data of 26 Patients Including Clinico-pathologic Features, Vitreous Findings, and Treatment Outcomes. Ocul Immunol Inflamm 2016;:1-6.Abstract

PURPOSE: To describe clinical manifestations, diagnostic approaches, therapy, and outcomes of biopsy-proven intraocular lymphoma. METHODS: Review of tertiary referral center records between 2005 and 2015. RESULTS: A total of 51 eyes of 26 patients were included; mean age of onset was 60.42 years. Common ocular complaints included floaters (42%) and blurred vision (35%); 62% of patients had ocular and central nervous system involvement; 11% had systemic lymphoma; and 27% had only ocular involvement. Vitreous analysis was positive for malignant cells in 77% of patients on initial biopsy, and in 100% of patients on repeat biopsy. In total, 20/26 patients received systemic and topical treatment before IOL diagnosis was made; 25 patients received intravitreal methotrexate and/or rituximab; one patient received intracameral rituximab. All patients achieved remission by their final visit. CONCLUSIONS: Intraocular lymphoma often masquerades as intraocular inflammation, resulting in delayed or misdiagnosis with subsequent inappropriate management. Optimal therapy is a challenge for oncologists and ophthalmologists.

Ma G, Duan Y, Huang X, Qian CX, Chee Y, Mukai S, Cui J, Samad A, Matsubara JA, Kazlauskas A, D'Amore PA, Gu S, Lei H. Prevention of Proliferative Vitreoretinopathy by Suppression of Phosphatidylinositol 5-Phosphate 4-Kinases. Invest Ophthalmol Vis Sci 2016;57(8):3935-43.Abstract

PURPOSE: Previous studies have shown that vitreous stimulates degradation of the tumor suppressor protein p53 and that knockdown of phosphatidylinositol 5-phosphate 4-kinases (PI5P4Kα and -β) abrogates proliferation of p53-deficient cells. The purpose of this study was to determine whether vitreous stimulated expression of PI5P4Kα and -β and whether suppression of PI5P4Kα and -β would inhibit vitreous-induced cellular responses and experimental proliferative vitreoretinopathy (PVR). METHODS: PI5P4Kα and -β encoded by PIP4K2A and 2B, respectively, in human ARPE-19 cells were knocked down by stably expressing short hairpin (sh)RNA directed at human PIP4K2A and -2B. In addition, we rescued expression of PI5P4Kα and -β by re-expressing mouse PIP4K2A and -2B in the PI5P4Kα and -β knocked-down ARPE-19 cells. Expression of PI5P4Kα and -β was determined by Western blot and immunofluorescence. The following cellular responses were monitored: cell proliferation, survival, migration, and contraction. Moreover, the cell potential of inducing PVR was examined in a rabbit model of PVR effected by intravitreal cell injection. RESULTS: We found that vitreous enhanced expression of PI5P4Kα and -β in RPE cells and that knocking down PI5P4Kα and -β abrogated vitreous-stimulated cell proliferation, survival, migration, and contraction. Re-expression of mouse PIP4Kα and -β in the human PI5P4Kα and -β knocked-down cells recovered the loss of vitreous-induced cell contraction. Importantly, suppression of PI5P4Kα and -β abrogated the pathogenesis of PVR induced by intravitreal cell injection in rabbits. Moreover, we revealed that expression of PI5P4Kα and -β was abundant in epiretinal membranes from PVR grade C patients. CONCLUSIONS: The findings from this study indicate that PI5P4Kα and -β could be novel therapeutic targets for the treatment of PVR.

Joshi AD, Andersson C, Buch S, Stender S, Noordam R, Weng L-C, Weeke PE, Auer PL, Boehm B, Chen C, Choi H, Curhan G, Denny JC, De Vivo I, Eicher JD, Ellinghaus D, Folsom AR, Fuchs C, Gala M, Haessler J, Hofman A, Hu F, Hunter DJ, Janssen HLA, Kang JH, Kooperberg C, Kraft P, Kratzer W, Lieb W, Lutsey PL, Darwish Murad S, Nordestgaard BG, Pasquale LR, Reiner AP, Ridker PM, Rimm E, Rose LM, Shaffer CM, Schafmayer C, Tamimi RM, Uitterlinden AG, Völker U, Völzke H, Wakabayashi Y, Wiggs JL, Zhu J, Roden DM, Stricker BH, Tang W, Teumer A, Hampe J, Tybjærg-Hansen A, Chasman DI, Chan AT, Johnson AD. Four Susceptibility Loci for Gallstone Disease Identified in a Meta-analysis of Genome-Wide Association Studies. Gastroenterology 2016;151(2):351-363.e28.Abstract

BACKGROUND & AIMS: A genome-wide association study (GWAS) of 280 cases identified the hepatic cholesterol transporter ABCG8 as a locus associated with risk for gallstone disease, but findings have not been reported from any other GWAS of this phenotype. We performed a large-scale, meta-analysis of GWASs of individuals of European ancestry with available prior genotype data, to identify additional genetic risk factors for gallstone disease. METHODS: We obtained per-allele odds ratio (OR) and standard error estimates using age- and sex-adjusted logistic regression models within each of the 10 discovery studies (8720 cases and 55,152 controls). We performed an inverse variance weighted, fixed-effects meta-analysis of study-specific estimates to identify single-nucleotide polymorphisms that were associated independently with gallstone disease. Associations were replicated in 6489 cases and 62,797 controls. RESULTS: We observed independent associations for 2 single-nucleotide polymorphisms at the ABCG8 locus: rs11887534 (OR, 1.69; 95% confidence interval [CI], 1.54-1.86; P = 2.44 × 10(-60)) and rs4245791 (OR, 1.27; P = 1.90 × 10(-34)). We also identified and/or replicated associations for rs9843304 in TM4SF4 (OR, 1.12; 95% CI, 1.08-1.16; P = 6.09 × 10(-11)), rs2547231 in SULT2A1 (encodes a sulfoconjugation enzyme that acts on hydroxysteroids and cholesterol-derived sterol bile acids) (OR, 1.17; 95% CI, 1.12-1.21; P = 2.24 × 10(-10)), rs1260326 in glucokinase regulatory protein (OR, 1.12; 95% CI, 1.07-1.17; P = 2.55 × 10(-10)), and rs6471717 near CYP7A1 (encodes an enzyme that catalyzes conversion of cholesterol to primary bile acids) (OR, 1.11; 95% CI, 1.08-1.15; P = 8.84 × 10(-9)). Among individuals of African American and Hispanic American ancestry, rs11887534 and rs4245791 were associated positively with gallstone disease risk, whereas the association for the rs1260326 variant was inverse. CONCLUSIONS: In this large-scale GWAS of gallstone disease, we identified 4 loci in genes that have putative functions in cholesterol metabolism and transport, and sulfonylation of bile acids or hydroxysteroids.

Igo RP, Cooke Bailey JN, Romm J, Haines JL, Wiggs JL. Quality Control for the Illumina HumanExome BeadChip. Curr Protoc Hum Genet 2016;90:2.14.1-2.14.16.Abstract

The Illumina HumanExome BeadChip and other exome-based genotyping arrays offer inexpensive genotyping of some 240,000 mostly nonsynonymous coding variants across the human genome. The HumanExome chip, with its highly non-uniform distribution of markers and emphasis on rare coding variants, presents some unique challenges for quality control (QC) and data cleaning. Here, we describe QC procedures for HumanExome data, with examples of challenges specific to exome arrays from our experience cleaning a data set of ∼7,500 samples from the NEIGHBORHOOD Consortium. We focus on standard procedures for QC of genome-wide array data including genotype calling, sex verification, sample identity verification, relationship checking, and population structure that are complicated by the HumanExome panel's enrichment in rare, exonic variation. © 2016 by John Wiley & Sons, Inc.

Gong Y, Fu Z, Edin ML, Liu C-H, Wang Z, Shao Z, Fredrick TW, Saba NJ, Morss PC, Burnim SB, Meng SS, Lih FB, Stephen Lee KS, Moran EP, SanGiovanni JP, Hellström A, Hammock BD, Zeldin DC, Smith LEH. Cytochrome P450 Oxidase 2C Inhibition Adds to ω-3 Long-Chain Polyunsaturated Fatty Acids Protection Against Retinal and Choroidal Neovascularization. Arterioscler Thromb Vasc Biol 2016;36(9):1919-27.Abstract

OBJECTIVE: Pathological ocular neovascularization is a major cause of blindness. Increased dietary intake of ω-3 long-chain polyunsaturated fatty acids (LCPUFA) reduces retinal neovascularization and choroidal neovascularization (CNV), but ω-3 LCPUFA metabolites of a major metabolizing pathway, cytochrome P450 oxidase (CYP) 2C, promote ocular pathological angiogenesis. We hypothesized that inhibition of CYP2C activity will add to the protective effects of ω-3 LCPUFA on neovascular eye diseases. APPROACH AND RESULTS: The mouse models of oxygen-induced retinopathy and laser-induced CNV were used to investigate pathological angiogenesis in the retina and choroid, respectively. The plasma levels of ω-3 LCPUFA metabolites of CYP2C were determined by mass spectroscopy. Aortic ring and choroidal explant sprouting assays were used to investigate the effects of CYP2C inhibition and ω-3 LCPUFA-derived CYP2C metabolic products on angiogenesis ex vivo. We found that inhibition of CYP2C activity by montelukast added to the protective effects of ω-3 LCPUFA on retinal neovascularization and CNV by 30% and 20%, respectively. In CYP2C8-overexpressing mice fed a ω-3 LCPUFA diet, montelukast suppressed retinal neovascularization and CNV by 36% and 39% and reduced the plasma levels of CYP2C8 products. Soluble epoxide hydrolase inhibition, which blocks breakdown and inactivation of CYP2C ω-3 LCPUFA-derived active metabolites, increased oxygen-induced retinopathy and CNV in vivo. Exposure to selected ω-3 LCPUFA metabolites of CYP2C significantly reversed the suppression of both angiogenesis ex vivo and endothelial cell functions in vitro by the CYP2C inhibitor montelukast. CONCLUSIONS: Inhibition of CYP2C activity adds to the protective effects of ω-3 LCPUFA on pathological retinal neovascularization and CNV.

Wu N, Wang Y, Yang L, Cho K-S. Signaling Networks of Retinal Ganglion Cell Formation and the Potential Application of Stem Cell-Based Therapy in Retinal Degenerative Diseases. Hum Gene Ther 2016;27(8):609-620.Abstract

Retinal degenerative diseases such as age-related macular degeneration, retinitis pigmentosa, and glaucoma result in permanent loss of retinal neurons and vision. Stem cell therapy could be a novel treatment strategy to restore visual function. In an ideal situation, a homogenous population of stem cell-derived retinal neurons with high purity is used for replacement therapy. Thus, it is crucial to elucidate the molecular mechanisms that regulate the development of retinal progenitor cells and subsequent generation of specific retinal neurons. Here, recent findings concerning the intrinsic and extrinsic factors that regulate retinal progenitor cell maintenance and differentiation are summarized, especially transcriptional factors and extrinsic signals. Understanding these mechanisms is indispensable because they have potential clinical applications, chiefly the generation of specific retinal cells such as retinal ganglion cells to treat glaucoma and other optic neuropathy diseases.

Duan Y, Ma G, Huang X, D'Amore PA, Zhang F, Lei H. The Clustered, Regularly Interspaced, Short Palindromic Repeats-associated Endonuclease 9 (CRISPR/Cas9)-created MDM2 T309G Mutation Enhances Vitreous-induced Expression of MDM2 and Proliferation and Survival of Cells. J Biol Chem 2016;291(31):16339-47.Abstract

The G309 allele of SNPs in the mouse double minute (MDM2) promoter locus is associated with a higher risk of cancer and proliferative vitreoretinopathy (PVR), but whether SNP G309 contributes to the pathogenesis of PVR is to date unknown. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease (Cas) 9 from Streptococcus pyogenes (SpCas9) can be harnessed to manipulate a single or multiple nucleotides in mammalian cells. Here we delivered SpCas9 and guide RNAs using dual adeno-associated virus-derived vectors to target the MDM2 genomic locus together with a homologous repair template for creating the mutation of MDM2 T309G in human primary retinal pigment epithelial (hPRPE) cells whose genotype is MDM2 T309T. The next-generation sequencing results indicated that there was 42.51% MDM2 G309 in the edited hPRPE cells using adeno-associated viral CRISPR/Cas9. Our data showed that vitreous induced an increase in MDM2 and subsequent attenuation of p53 expression in MDM2 T309G hPRPE cells. Furthermore, our experimental results demonstrated that MDM2 T309G in hPRPE cells enhanced vitreous-induced cell proliferation and survival, suggesting that this SNP contributes to the pathogenesis of PVR.

Tian B, Maidana DE, Dib B, Miller JB, Bouzika P, Miller JW, Vavvas DG, Lin H. miR-17-3p Exacerbates Oxidative Damage in Human Retinal Pigment Epithelial Cells. PLoS One 2016;11(8):e0160887.Abstract

Oxidative stress has been shown to contribute to the development of age-related macular degeneration (AMD). MicroRNAs (miRNA) are small non-coding RNA molecules that function in RNA silencing and post-transcriptional regulation of gene expression. We showed miR-17-3p to be elevated in macular RPE cells from AMD patients and in ARPE-19 cells under oxidative stress. Transfection of miR-17-3p mimic in ARPE-19 induced cell death and exacerbated oxidative lethality that was alleviated by miR-17-3p inhibitor. The expression of antioxidant enzymes manganese superoxide dismutase (MnSOD) and thioredoxin reductase-2 (TrxR2) were suppressed by miR-17-3p mimic and reversed by miR-17-3p inhibitor. These results suggest miR-17-3p aggravates oxidative damage-induced cell death in human RPE cells, while miR-17-3p inhibitor acts as a potential protector against oxidative stress by regulating the expression of antioxidant enzymes.

Zhu H, Alt C, Webb RH, Melki S, Kochevar IE. Corneal Crosslinking With Rose Bengal and Green Light: Efficacy and Safety Evaluation. Cornea 2016;35(9):1234-41.Abstract

PURPOSE: To evaluate crosslinking of cornea in vivo using green light activation of Rose Bengal (RGX) and assess potential damaging effects of the green light on retina and iris. METHODS: Corneas of Dutch belted rabbits were de-epithelialized, then stained with Rose Bengal and exposed to green light, or not further treated. Corneal stiffness was measured by uniaxial tensiometry. Re-epithelialization was assessed by fluorescein fluorescence. Keratocytes were counted on hematoxylin and eosin (H&E)-stained sections, and iris cell damage was assessed by lactate dehydrogenase staining. Thermal effects on the blood-retinal barrier (BRB) were assessed by fluorescein angiography and those on photoreceptors, retinal pigment epithelium (RPE), and choriocapillaris by light microscopy and transmission electron microscopy. RESULTS: RGX (10-min irradiation; 150 J/cm) increased corneal stiffness 1.9-fold on day 1 (1.25 ± 0.21 vs. 2.38 ± 0.59 N/mm; P = 0.036) and 2.8-fold compared with controls on day 28 (1.70 ± 0.74 vs. 4.95 ± 1.86 N/mm; P = 0.003). Keratocytes decreased only in the anterior stroma on day 1 (24.0 ± 3.0 vs. 3.67 ± 4.73, P = 0.003) and recovered by day 28 (37.7 ± 8.9 vs. 34.5 ± 2.4, P = 0.51). Iris cells were not thermally damaged. No evidence of BRB breakdown was detected on days 1 or 28. Retina from RGX-treated eyes seemed normal with RPE cells showing intact nuclei shielded apically by melanosomes, morphologically intact photoreceptor outer segments, normal outer nuclear layer thickness, and choriocapillaris containing intact erythrocytes. CONCLUSIONS: The substantial corneal stiffening produced by RGX together with the lack of significant effects on keratocytes and no evidence for retina or iris damage suggest that RGX-initiated corneal crosslinking may be a safe, rapid, and effective treatment.

Shieh E, Lee R, Que C, Srinivasan V, Guo R, DeLuna R, Pandit S, Simavli H, Seevaratnam R, Tsikata E, de Boer J, Chen TC. Diagnostic Performance of a Novel Three-Dimensional Neuroretinal Rim Parameter for Glaucoma Using High-Density Volume Scans. Am J Ophthalmol 2016;169:168-78.Abstract

PURPOSE: To evaluate the diagnostic performance of a 3-dimensional (3D) neuroretinal rim parameter, the minimum distance band (MDB), using optical coherence tomography (OCT) high-density volume scans for open-angle glaucoma. DESIGN: Reliability analysis. METHODS: setting: Institutional. STUDY POPULATION: Total of 163 patients (105 glaucoma and 58 healthy subjects). OBSERVATION PROCEDURES: One eye of each patient was included. MDB and retinal nerve fiber layer (RNFL) thickness values were determined for 4 quadrants and 4 sectors using a spectral-domain OCT device. MAIN OUTCOME MEASURES: Area under the receiver operating characteristic curve (AUROC) values, sensitivities, specificities, and positive and negative predictive values. RESULTS: The best AUROC values of 3D MDB thickness for glaucoma and early glaucoma were for the overall globe (0.969, 0.952), followed by the inferior quadrant (0.966, 0.949) and inferior-temporal sector (0.966, 0.944), and then followed by the superior-temporal sector (0.964, 0.932) and superior quadrant (0.962, 0.924). All 3D MDB thickness AUROC values were higher than those of 2D RNFL thickness. Pairwise comparisons showed that the diagnostic performance of the 3D MDB parameter was significantly better than 2D RNFL thickness only for the nasal quadrant and inferior-nasal and superior-nasal sectors (P = .023-.049). Combining 3D MDB with 2D RNFL parameters provided significantly better diagnostic performance (AUROC 0.984) than most single MDB parameters and all single RNFL parameters. CONCLUSIONS: Compared with the 2D RNFL thickness parameter, the 3D MDB neuroretinal rim thickness parameter had uniformly equal or better diagnostic performance for glaucoma in all regions and was significantly better in the nasal region.

Ebrahimiadib N, Modjtahedi BS, Roohipoor R, Anesi SD, Foster SC. Successful Treatment Strategies in Granulomatosis With Polyangiitis-Associated Peripheral Ulcerative Keratitis. Cornea 2016;35(11):1459-1465.Abstract

PURPOSE: Management of granulomatosis with polyangiitis (GPA)-associated peripheral ulcerative keratitis (PUK) is challenging and lacks definite guidelines. We aimed to summarize our treatment and outcome experience with patients with GPA-PUK. METHODS: The Massachusetts Eye Research and Surgery Institution patient database was searched from 2005 to 2015 to identify patients with diagnosis of PUK who suffered from GPA. Individual patient histories were examined, and treatment strategies and outcomes were summarized. RESULTS: There were 16 patients who started treatment with a mean duration follow-up of 64 months (range: 12-110 mo). Rituximab and cyclophosphamide, either alone or in combination with other agents, were the most successful agents in controlling inflammation. Rituximab was administered in 11 patients with remission being achieved in all. Cyclophosphamide successfully controlled inflammation in 50% (5/10). Two of the patients (2/5, 40%) who had achieved initial control on cyclophosphamide had flares of their PUK. Two of 11 (18%) patients on rituximab had flares of scleritis and orbital inflammation but not PUK. Two patients, one in each treatment group, stopped treatment after achieving remission after 6 months of therapy but suffered disease recurrence within 2 months of treatment cessation. CONCLUSIONS: Rituximab achieved a high rate of disease control in PUK patients with GPA and is the preferred agent in halting disease progression.

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