ANGPTL7, a therapeutic target for increased intraocular pressure and glaucoma | Communications Biology – Nature.com

Ethics approval and informed consent

All participants provided informed consent, and studies were approved by the individual IRBs at the respective institutions. UK Biobank has approval from the North West Multi-center Research Ethics Committee (MREC), which covers the UK. It also sought the approval in England and Wales from the Patient Information Advisory Group (PIAG) for gaining access to information that would allow it to invite people to participate. The DiscovEHR study was approved by the Institutional Review Board (IRB) at Geisinger. The BioMe Biobank is an ongoing research biorepository approved by the Icahn School of Medicine at Mount Sinais IRB. The Ethical Committee at Lund University approved the Malmo Diet and Cancer Study (LU 5190) and all the participants provided a written informed consent. The CGPS study (H-KF-01-144/01) was approved by the Ethics Committee of the Capital Region and from the Danish Data Protection Agency. Research at Estonian Biobank is regulated by Human Gene Research Act and all participants have signed a broad informed consent. IRB approval for current study was granted by Research Ethics Committee of University of Tartu, approval nr 236/T-23. For the POAAGG study, approval to enroll and to recontact subjects was obtained from the University of Pennsylvania IRB. The Finngen Biobank was evaluated and approved by the Coordinating Ethics Committee of the Helsinki and Uusimaa Hospital District.

Association with IOP was tested on a total of 101,678 individuals and 27,529 individuals of European ancestry from the United Kingdom Biobank (UKB) and the MyCode Community Health Initiative cohort from Geisinger Health System (GHS), respectively. The UKB is a population-based cohort study of people aged between 40 and 69 years recruited through 22 testing centers in the UK between 2006 and 201040. The GHS MyCode study is a health system-based cohort of patients from Central and Eastern Pennsylvania (USA) recruited in 2007201941. For IOP association tests in African ancestry individuals, we included 4114 individuals from UKB and 3167 individuals from the Primary Open Angle African-American Glaucoma Genetics (POAAGG) study conducted at the University of Pennsylvania Perelman School of Medicine42. We excluded all participants with a glaucoma diagnosis code (ICD-10 H40) or self-reported glaucoma (UKB field IDs: 6148 and 20002) from IOP analyses.

Association of ANGPTL7 variants with glaucoma was tested in 8 studies: UKB, GHS, Mt. Sinai BioMe cohort (SINAI), the Malm Diet and Cancer study (MALMO)43, the Estonia Biobank (EstBB)44, The Trndelag Heath Study (HUNT)45, FinnGen, a study from Finland, and the Copenhagen General Population Study and the Copenhagen City Heart Study (CGPS-CCHS)46. We had, in total, up to 40,042 cases (UKB: 12,377, GHS: 8032, SINAI: 409, MALMO: 2395, EstBB: 7629, HUNT: 3874; CPGS-CCHS: 1863; FinnGen: 3463) and 947,782 controls of European ancestry, and 5153 cases (UKB: 448, POAAGG: 3444, SINAI: 1261) and 21,650 controls of African ancestry in glaucoma analyses.

IOP in UKB was measured in each eye using the Ocular Response Analyzer (Reichert Corp., Buffalo, New York). Participants were excluded from this test if they reported having eye surgery in the preceding 4 weeks or having an eye infection. The Ocular Response Analyzer calculates two forms of IOP, a Goldmann-correlated IOP (IOPg) and a corneal-compensated IOP (IOPcc). IOPg most closely approximates the IOP measured by the Goldmann Applanation Tonometer(GAT), which has been the gold standard for measuring IOP, while IOPcc provides a measure of IOP that is adjusted to remove the influence of corneal biomechanics47. For this study, we focused on IOPg as this measurement is the most comparable to IOP measurements in other cohorts, and herein IOPg will be referred to as IOP. IOP in POAAGG was measured using a GAT. In GHS, IOP measurements were obtained from several instruments including GAT, Tono-pen and I-Care, which are correlated with IOPg readings from the Ocular Response Analyzer48. For GHS individuals who were not prescribed any IOP medications, we used the median of all IOP measurements available. For individuals who had an IOP medication prescribed, we used the median of IOP measurements available preceding the start date for IOP medications (if available). Individuals for whom we did not have non-medicated IOP values were excluded from the IOP genetic analyses. For association analyses of IOP, we excluded individuals with: (1) a glaucoma diagnosis; (2) IOP measures that were more than 5 standard deviations away from the mean; (3) more than a 10-mmHg difference between both eyes. We derived a mean IOP measure between both eyes for each individual. IOP of only one eye was used in instances where IOP measures for both eyes were not available.

Details on glaucoma definition in each cohort are given in the Supplementary Methods. In brief, glaucoma cases in GHS, SINAI, MALMO, HUNT, EstBB, FinnGen (v.R3) and CGPS-CCHS were defined by the presence of an ICD-10 H40 diagnosis code in either outpatient or inpatient electronic health records. In UKB, glaucoma cases were defined as individuals with either an ICD-10 H40 diagnosis or self-reported glaucoma (UKB field ID: 6148 or 20002). In the POAAGG cohort, glaucoma cases and controls were classified based on an ophthalmic examination by glaucoma specialists, and glaucoma suspects were also included in the cases42.

High coverage whole exome sequencing and genotyping was performed at the Regeneron Genetics Center49,50 as described in Supplementary Methods. We estimated the association with IOP and glaucoma of genetic variants or their gene burden using REGENIE v1.0.4351 (UKB, GHS, MALMO, SINAI), SAIGE52 (HUNT, EstBB, FinnGen) or logistic regression (CGPS-CCHS). Analyses were adjusted for age, age2, sex, an age-by-sex interaction term, experimental batch-related covariates, and genetic principal components, where appropriate. Cohort-specific statistical analysis details are provided in Supplementary Methods. Results across cohorts were pooled using inverse-variance weighted meta-analysis. Details on the PheWAS analysis conducted in UKB and GHS are provided in Supplementary Methods. Western blotting and ELISA analyses were repeated on three independent biological replicates and data are presented as meanSEM. Technical replicates (n=3) were run for the ELISA analysis. P values were calculated by one-way ANOVA with Tukeys multiple comparison test for multiple groups analysis (Supplementary Data1). A total of 12 eyes were used to test the effect of increasing mAngptl7 levels in mouse eyes and Students t test was used to calculate the significance of the resulting change in IOP. The IOP was measured on 33 WT, 41 Angptl7 KO and 15 Angptl7 Het mice and conventional outflow facility was measured on 4 WT and 7 Angptl7 KO mice. Unpaired Students t-test was used to calculate the statistical significance of the results between the different genotypes. For in vivo siRNA knockdown of mAngptl7, we used 8, 6, 6 and 5 mouse eyes for siRNA#3, siRNA#5, PBS-treated and Nave controls, respectively. Statistical significance was calculated using one-way ANOVA with Dunnetts post hoc analysis (Supplementary Data1).

HEK293 cells, derived within Regeneron, were cultured in DMEM media 4.5g/L D-Glucose, (+) L-Glutamine, () Sodium Phosphate, () Sodium Pyruvate supplemented with 10% FBS and 1% Penicillin-Streptomycin-Glutamine (Invitrogen), at 37C in a humidified atmosphere under 5% CO2. The day before transfection, HEK293 cells were seeded in OptiMEM supplemented with 10% FBS. After 24h, the cells were transfected with FuGENE 6, and 10g of pcDNA 3.1(+) encoding the following proteins: ANGPTL7 WT, Gln175His, Arg177* and Trp188*. After 24h, the media was changed with 2% FBS OptiMEM. The following day, the cells were collected in RIPA buffer, supplemented with protease and phosphatase inhibitors (BRAND) or TRIzol reagent (Invitrogen) for protein and RNA analysis, respectively. The supernatants were transferred to an Eppendorf tube and immediately flash frozen for downstream protein analysis. Western blot analysis was performed using a rabbit polyclonal antibody against ANGPTL7 at 1:1000 dilution (10396-1-AP ProteinTech), using standard procedures. ANGPTL7 was quantified by ELISA according to manufacturers instructions (LS-F50425 Life Sciences). The cell lysates were diluted 1:1000. The supernatants were diluted 1:10,000. The ELISA plate was read at 450nm via SpectraMax M4 plate reader (Molecular Devices).

Total RNA was extracted using TRIzol reagent (Invitrogen) and RNeasy kit (Qiagen) according to manufacturers instructions and treated with RNase-free DNase I (Promega). cDNA was synthesized using Superscript VILO cDNA synthesis kit (Invitrogen). Taqman analysis was performed using TaqMan Fast Advanced Master Mix (Applied Biosystems) in a QuantStudio 6 Flex (Applied Biosystems) and commercially available primers and probes for ANGPTL7 (Hs00221727Applied Biosystems) and GAPDH (Hs02786624_g1Applied Biosystems).

All animal protocols were approved by the Institutional Animal Care and Use Committee in accordance with the Regenerons Institutional Animal Care and Use Committee (IACUC) and the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. Angptl7/ mice, on 63% C57BL/6NTac and 37% 129SvEvTac background, were generated by Regeneron Pharmaceuticals using the VelociMouse technology53. Heterozygous mice (Angptl7+/) were bred to generate age-matched wild-type, het and KO littermates that were used for experimentation at 3-4 months of age (mixed gender). Ocular anatomy in these mice was characterized using optical coherence tomography. Detailed methods on generation and characterization of KO mice are provided in Supplementary Methods. For in vivo siRNA experiments, we used C57BL/6J male mice, 3-4 months old, from Jackson Labs.

Mice were anesthetized and IOP was measured in both eyes using a TonoLab rebound tonometer (Colonial Medical Supply, Franconia, NH) before the start of Angptl7 injection and every day afterwards for six days54,55,56. When testing Angptl7 siRNAs, IOPs were measured in each eye before then start of experiment and then every week until end of study. IOP measurements for both eyes were completed within 35min. Six correct single measurements were done on each eye to generate one IOP reading. We took five IOP readings for each eye and used the average of those readings at each time-point.

Aqueous humor outflow facility (C) was measured by using our constant flow infusion technique in live mice55,56,57,58. Mice were anesthetized by using a 100/10mg/kg ketamine/xylazine cocktail. A quarter to half of this dose was administered for maintenance of anesthesia as necessary. One to two drops of proparacaine HCl (0.5%) (Bausch+Lomb) were applied topically to both eyes for corneal anesthesia. The anterior chambers of both eyes were cannulated by using a 30-gauge needle inserted through the cornea 12mm anteriorly to the limbus and pushed across the anterior chamber to a point in the chamber angle opposite to the point of cannulation, taking care not to touch the iris, anterior lens capsule epithelium, or corneal endothelium. Each cannulating needle was connected to a previously calibrated (sphygmomanometer, Diagnostix 700; American Diagnostic Corporation, Hauppauge, NY, USA) flow-through BLPR-2 pressure transducer (World Precision Instruments [WPI], Sarasota, FL, USA) for continuous determination of pressure within the perfusion system. A drop of genteal (Alcon) was also administered to each eye to prevent corneal drying. The opposing ends of the pressure transducer were connected via further tubing to a 1ml syringe loaded into a microdialysis infusion pump (SP200I Syringe Pump; WPI). The tubing, transducer, and syringe were all filled with sterile DPBS (Gibco). Signals from each pressure transducer were passed via a TBM4M Bridge Amplifier (WPI) and a Lab-Trax Analog-to-Digital Converter (WPI) to a computer for display on a virtual chart recorder (LabScribe2 software; WPI). Eyes were initially infused at a flow rate of 0.1 l/min. When pressures stabilized within 1030min, pressure measurements were recorded over a 5-min period, and then flow rates were increased sequentially to 0.2, 0.3, 0.4, and 0.5l/min. Three stabilized pressures at 3-minute intervals at each flow rate were recorded. C in each eye of each animal was calculated as the reciprocal of the slope of a plot of mean stabilized pressure as ordinate against flow rate as abscissa.

A 33-gauge needle with a glass microsyringe (5-uL volume; Hamilton Company) was used for injections of Angptl7 protein/siRNA into mice eyes. For intravitreal injections, the eye was proptosed, and the needle was inserted through the equatorial sclera and into the vitreous chamber at an angle of approximately 45 degrees, taking care to avoid touching the posterior part of the lens or the retina. Angptl7 protein (catalog# 4960-AN-025; R&D Systems, Minneapolis, MN) or siRNA (from Alnylam Pharmaceuticals, Supplementary Methods) or PBS (1uL) was injected into the vitreous over the course of 1minute. The needle was then left in place for a further 45s (to facilitate mixing), before being rapidly withdrawn. siRNA sequences for all six probes tested are provided in Table2. Before and during intracameral injections of Angptl7 protein, mice were anesthetized with isoflurane (2.5%) containing oxygen (0.8L/min). For topical anesthesia, both eyes received one to two drops of 0.5% proparacaine HCl (Akorn Inc.). Each eye was proptosed and the needle was inserted through the cornea just above the limbal region and into the anterior chamber at an angle parallel to the cornea, taking care to avoid touching the iris, anterior lens capsule epithelium, or corneal endothelium. Up to 1L of Angptl7 protein or PBS was injected into each eye over a 30-s period before the needle was withdrawn. Only one injection was administered at day 0.

Further information on research design is available in theNature Research Reporting Summary linked to this article.

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ANGPTL7, a therapeutic target for increased intraocular pressure and glaucoma | Communications Biology - Nature.com