ANGPTL7, a therapeutic target for increased intraocular pressure and glaucoma

Abstract

Glaucoma is a leading cause of blindness. Current glaucoma medications work by lowering intraocular pressure (IOP), a risk factor for glaucoma, but most treatments do not directly target the pathological changes leading to increased IOP, which can manifest as medication resistance as disease progresses. To identify physiological modulators of IOP, we performed genome- and exome-wide association analysis in >129,000 individuals with IOP measurements and extended these findings to an analysis of glaucoma risk. We report the identification and functional characterization of rare coding variants (including loss-of-function variants) in ANGPTL7 associated with reduction in IOP and glaucoma protection. We validated the human genetics findings in mice by establishing that Angptl7 knockout mice have lower (~2 mmHg) basal IOP compared to wild-type, with a trend towards lower IOP also in heterozygotes. Conversely, increasing murine Angptl7 levels via injection into mouse eyes increases the IOP. We also show that acute Angptl7 silencing in adult mice lowers the IOP (~2-4 mmHg), reproducing the observations in knockout mice. Collectively, our data suggest that ANGPTL7 is important for IOP homeostasis and is amenable to therapeutic modulation to help maintain a healthy IOP that can prevent onset or slow the progression of glaucoma.

Fig. 1. An aggregate of rare (MAF < 1%) loss-of-function and missense variants in ANGPTL7 is associated with IOP.

a Association of an aggregate of 63 pLOF and deleterious (based on 5 prediction algorithms) missense variants in ANGPTL7 with reduced IOP in 129,207 individuals of European descent. b Missense and predicted loss-of-function (pLOF) variants in ANGPTL7 and IOP levels in individuals of European descent from UKB. The plots represent Goldmann-correlated IOP (IOPg; mean of both eyes) levels in carriers of 1 pLOF and 10 missense variants in ANGPTL7 that are predicted deleterious by five different algorithms and have at least five carriers amongst the 101,678 exome-sequenced individuals with IOP measurements in the UK Biobank. The median IOP level across carriers of all 49 pLOF and predicted-deleterious missense ANGPTL7 variants (14.64 mmHg) is indicated by the red line, and the median IOP in non-variant carriers (15.46 mmHg) is indicated by the blue line. Magenta diamonds mark the median IOP in carriers of each variant. Beneath the plots is the median and interquartile range (IQR) of IOP and the numbers of variant carriers diagnosed with glaucoma or controls in UKB (n = 385,040). GHS Geisinger DiscovEHR, UKB UK Biobank, MAF Minor allele frequency.

Fig. 2. Gln175His and Arg177* are major contributors to the gene burden association of ANGPTL7 with IOP.

Association of Gln175His (a) and Arg177* (c) variants in ANGPTL7 with IOP, effect measured in standard deviation units, in individuals of European descent. b, d Boxplots representing IOPg in the UK Biobank across genotypes. b Gln175His heterozygous and homozygous carriers have a 0.8-mmHg and 4.1-mmHg lower median IOPg, respectively, compared to non-carriers. d Arg177* heterozygous carriers have a 1.4-mmHg lower IOPg compared to non-carriers. GHS Geisinger DiscovEHR, UKB UK Biobank, MAF Minor allele frequency.

Fig. 3. Association of ANGPTL7 variants with glaucoma.

a Meta-analysis results for Gln175His with glaucoma across 8 different cohorts. b Cross-ancestry meta-analysis of Arg177* and Trp188* across 5 European (EUR) and 3 African (AFR) ancestry cohorts. The variants in the meta-analysis of EUR and AFR cohorts were Arg177* and Trp188*, respectively. GHS Geisinger DiscovEHR, UKB UK Biobank, SINAI Mt. Sinai Medical School BioMe Biobank, MALMO Malmö Diet and Cancer Study, EstBB the Estonia Biobank at the University of Tartu, HUNT the HUNT study from Nord-Trøndelag, CGPS-CCHS the Copenhagen General Population Study and the Copenhagen City Heart Study, POAAGG Primary Open Angle African-American Glaucoma Genetics, MAF Minor allele frequency.

Fig. 4. Expression analysis of ANGPTL7 Gln175His, Arg177*, and Trp188* in a HEK293 cell line.

a, b Western blotting shows intra- and extra-cellular protein levels of ANGPTL7 wild-type (WT), Gln175His, Arg177*, and Trp188*. c ELISA was run to quantify intra- and extra-cellular protein levels of ANGPTL7 WT, Gln175His, Arg177*, and Trp188* transiently transfected in HEK293 cells (whole-cell lysate was diluted 1:1,000; supernatant was diluted 1:10,000. Both whole-cell lysate and supernatant were normalized against the total amount of protein from the whole-cell lysate); ****= P < 1 × 10^–4 statistical difference from WT whole cell lysate; ^ = P < 0.05, ^^^ = P < 1 × 10^–4 statistical difference from WT supernatant. d Ratio of secreted versus intracellular ANGPTL7 WT, Gln175His, Arg177*, and Trp188* protein levels. Raw ANGPTL7 WT, Gln175His, Arg177*, and Trp188* protein levels were normalized to the whole-lysate protein concentration; **** = P < 1 × 10^-4 statistical difference from WT. Western blotting and ELISA analysis were repeated on three independent biological replicates. Technical replicates (n = 3) were run for the ELISA analysis. P values were calculated by one-way ANOVA with Tukey’s post hoc analysis. All data are presented as mean and error bars indicate the standard error of the mean (SEM). MWt molecular weight marker.

Fig. 5. ANGPTL7 expression in ocular tissues across species.

RNA-sequencing-based expression levels (measured in transcripts per million, TPM, and represented as median and interquartile range) are highest in cornea, trabecular meshwork (TM), and sclera in human (a), and African green monkey (b) eyes, and in cornea, TM, sclera, optic nerve head, and choroid/RPE in C57BL/6 J mouse eyes (c); In situ hybridization (RNAscope) shows ANGPTL7/Angptl7 (red) expression in TM, cornea and sclera in human (d) and murine (e) eyes. Scale bars represent 100 μm. DAPI staining (blue) counterstains cell nuclei. RPE retinal pigmented epithelium, CB ciliary body, SC Schlemm’s canal, CM ciliary muscle, AC anterior chamber, RGC retinal ganglion cell, INL inner nuclear layer, ONL outer nuclear layer.

Fig. 6. Increasing mAngptl7 levels in mouse eyes increases IOP.

Murine Angptl7 (mAngptl7) protein was injected into mouse eyes via intravitreal (a) or intracameral route (b), and IOP was measured over time. After an initial drop, IOP remained elevated for several days in mAngptl7-treated eyes compared to PBS treated (CTRL (PBS)) eyes. * = P < 0.05; ** = P < 0.01 statistical significance compared to PBS treatment. Statistical analyses were performed using Student’s t test.

Fig. 7. In situ characterization of Angptl7 mRNA in WT and Angptl7 KO mouse eyes.

Angptl7 mRNA was not expressed in any ocular tissue in Angptl7 KO mice whereas it was expressed in TM, cornea, and sclera of WT mice as shown by in situ hybridization (RNAscope). Brightfield images showing following probes. a Negative control: DapB. b Positive control: Ubc (red). c WT mice: Angptl7 (red), and (d) Angptl7 KO mice: Angptl7 (no signal). Scale bars represent 100 μm.

Fig. 8. Reducing mAngptl7 levels in mice lowers IOP.

a IOP was significantly lowered in Angptl7 KO compared to Het and WT mice (mean ± SEM; **** = P < 1 × 10^–4). b Comparison of conventional outflow facility (C) between KO and WT mice. In KO mice (n = 7), C was increased compared to WT mice (n = 4, P = 0.16, unpaired Student’s t test). Data are presented as mean ± SEM. c Intravitreal injection with 15 µg of Angptl7-siRNA significantly lowered IOP in two of six siRNAs tested (n = 6–8/group) compared to the PBS (n = 6) and Naïve (no injection, n = 5) groups and remained lowered throughout the end of the study. siRNAs 3 and 5 lowered IOP between 2 and 4 mmHg starting at week 2 compared to PBS-treated mice. * = P < 0.05, ** = P < 0.01, *** = P < 1 × 10⁻³, **** = P < 1 × 10⁻⁴ statistical difference between the mean IOP for siRNA #5 and PBS-treatment.^# = P < 0.05,^## = P < 0.01, ^### = P < 1 × 10⁻³, ^#### = P < 1 × 10⁻⁴ statistical difference between the mean IOP for siRNA #3 and PBS-treatment. d qPCR results from micro-dissected limbal ring showed the highest level of knockdown (>50%) of Angptl7 mRNA with siRNAs #3 and #5 compared to PBS-treated mice, which is highly consistent with the IOP lowering observed in mice injected with these two siRNAs (b). Data are presented as mean and error bars represent SEM.