TET-dependent GDF7 hypomethylation impairs aqueous humor outflow and serves as a potential therapeutic target in glaucoma

Abstract

Glaucoma is the leading cause of irreversible vision loss, affecting more than 70 million individuals worldwide. Circulatory disturbances of aqueous humor (AH) have long been central pathological contributors to glaucomatous lesions. Thus, targeting the AH outflow is a promising approach to treat glaucoma. However, the epigenetic mechanisms initiating AH outflow disorders and the targeted treatments remain to be developed. Studying glaucoma patients, we identified GDF7 (growth differentiation factor 7) hypomethylation as a crucial event in the onset of AH outflow disorders. Regarding the underlying mechanism, the hypomethylated GDF7 promoter was responsible for the increased GDF7 production and secretion in primary open-angle glaucoma (POAG). Excessive GDF7 protein promoted trabecular meshwork (TM) fibrosis through bone morphogenetic protein receptor type 2 (BMPR2)/Smad signaling and upregulated pro-fibrotic genes, α-smooth muscle actin (α-SMA) and fibronectin (FN). GDF7 protein expression formed a positive feedback loop in glaucomatous TM (GTM). This positive feedback loop was dependent on the activated TET (ten-eleven translocation) enzyme, which kept the GDF7 promoter region hypomethylated. The phenotypic transition in TM fortified the AH outflow resistance, thus elevating the intraocular pressure (IOP) and attenuating the nerve fiber layer. This methylation-dependent mechanism is also confirmed by a machine-learning model in silico with a specificity of 84.38% and a sensitivity of 89.38%. In rhesus monkeys, we developed GDF7 neutralization therapy to inhibit TM fibrosis and consequent AH outflow resistance that contributes to glaucoma. The neutralization therapy achieved high-efficiency control of the IOP (from 21.3 ± 0.3 to 17.6 ± 0.2 mmHg), a three-fold improvement in the outflow facility (from 0.1 to 0.3 μL/min · mmHg), and protection of nerve fibers. This study provides new insights into the epigenetic mechanism of glaucoma and proposes an innovative GDF7 neutralization therapy as a promising intervention.

Keywords: DNA methylation; computational modeling; fibrosis; glaucoma; neutralizing antibody; trabecular meshwork.

Graphical abstract

Figure 1

GDF7 hypomethylation was the crucial event in glaucoma (A) Diagram of aberrantly methylated regions in primary open angle glaucoma (POAG). From the outside in, the first layer presents the chromosomal information; the second and third layers present the different methylation sites in POAG patients and healthy controls (CONs), respectively; and the fourth and fifth layers present aberrantly methylated promoters and untranslated regions (UTRs) in POAG patients. (B) The top 20 differentially methylated sites in glaucomatous trabecular meshwork (GTM) samples (presented in reference name). As normalized to the controls, the relative methylation levels of the target regions were represented in the pseudocolor (n = 8 per group). (C) The disrupted methylation genes in POAG patients enriched in seven biological pathways as analyzed by Kyoto Encyclopedia of Genes and Genomes (KEGG). (D) The dysregulated methylation levels of five candidates were confirmed by bisulfite sequencing PCR (BSP) in trabecular meshwork (TM) samples and cells (n = 3 per group). (E) Decrease in methylation level of the GDF7 promoter was confirmed in eight GTM samples compared to healthy controls by BSP (n = 8 per group). (F) Ninety GTM samples were obtained from trabeculectomy (clinical information in Table S2) to validate the expression of GDF7 by quantitative real-time PCR and ELISA. Note: the readouts were normalized to the eight normal TM (NTM), due to the limitation of the donations we can get. (G) The GDF7 methylation level in response to 5-aza-2′-deoxycytidine (DAC) treatment was tested by BSP, and the change in GDF7 mRNA level was measured by real-time PCR in TM cells (n = 3 per group). (H) Correlation analysis between GDF7 methylation level and clinical manifestations in POAG patients (n = 8). The data represent the mean ± SD. Compared with NTM samples: ∗p < 0.05, ∗∗p < 0.01. Compared with NTM cells: #p < 0.05, ##p < 0.01. NC, normal control; IOP, intraocular pressure; CDR, cup/disc ratio; RNFL, retinal nerve fiber layer.

Figure 2

Excessive GDF7 protein-promoted fibrosis in cultured TM cells (A) In cultured TM cells, the expression level of fibrosis-related markers was presented with immunofluorescence, and the collagen accumulation in TM cells was presented by Masson stain (n = 3 per group). (B) The mRNA levels of N-cadherin (N-cad), α-smooth muscle actin (α-SMA), and fibronectin (FN) were tested in recombinant human GDF7 (rhGDF7)-treated TM cells by quantitative real-time PCR assay (n = 3 per group). (C and D) As presented in western blot results, the protein levels of N-cad, α-SMA, and FN were measured in NTM cells with or without rhGDF7 treatment (n = 3 per group). Scale bars, 20 μm. The data represent the mean ± SD. Compared with NTM cells: ∗∗p < 0.01. Compared with rhGDF7 group: #p < 0.05, ##p < 0.01. nGDF7, GDF7 neutralizing antibody; Col I, collagen I.

Figure 3

Ten-eleven translocation (TET) activation promoted glaucomatous GDF7 production (A) The activity of the TET enzyme was measured in nucleus extracts from TM samples/cells by chemiluminescence assay (n = 3 per group). (B) BSP assay was recruited to measure the methylation level of the GDF7 promoter in response to dimethyloxallyl glycine (DMOG) in NTM and GTM cells (n = 3 per group). (C) All of the RNA fragments that bond to the ETS1 protein were pulled down by ChIP and then tested by qPCR. The amplification of different regions in the GDF7 promoter was conducted with specially designed primers (details in Table S7; n = 3 per group). (D) Luciferase activity was measured to indicate the binding affinity between the GDF7 promoter and ETS1 (n = 3 per group). (E) The binding between ETS1 and the GDF7 promoter from NTM or GTM cells was tested by luciferase reporter assay (n = 3 per group). (F) As measured by ChIP-qPCR, the binding between the ETS1 protein and the GDF7 promoter presented in GTM cells compared with NTM cells in response to DMOG (n = 3 per group). (G) The transcription activity of the GDF7 promoter region in NTM cells and GTM was tested by the luciferase reporter assay. The transcription activity disturbance in response to DMOG was also measured (n = 3 per group). (H) GDF7 secretion was tested in conditioned medium by ELISA. GDF7 secretion was dramatically increased in GTM cells, which was effectively prevented by DMOG treatment (n = 3 per group). The data were represented as mean ± SD. Compared with NTM samples: ∗∗p < 0.01. Compared with NTM cells: ##p < 0.01. Compared with vector or scramble: @p < 0.05, @@p < 0.01. Compared with GTM cells: $p < 0.05, $$p < 0.01. siETS1, siRNA of ETS1.

Figure 4

DMOG broke the positive feedback loop of GDF7 expression (A) The conditioned medium from P3 cells was collected and measured by ELISA. The GDF7 protein concentration was presented (n = 3 per group). (B) The methylation level of the GDF7 promoter and mRNA level of the GDF7 gene were measured by BSP and RT-PCR, respectively, in P3 cells (n = 3 per group). (C) The expression of pro-fibrotic genes, α-SMA and FN, and the level of N-cad were presented by immunofluorescence labeling in P3 cells (n = 3 per group). Scale bars, 20 μm. The data were represented as mean ± SD. Compared with NTM cells: ∗∗p < 0.01. Compared with P3 cells: #p < 0.05, ##p < 0.01. Compared with siRNA of GDF7 (siGDF7)-treated P3 cells: $$p < 0.01.

Figure 5

GDF7 promoted fibrosis through the BMPR2/Smad1, -5, and -9 pathway (A) Fibrosis was determined by immunofluorescence labeling with N-cad, α-SMA, and FN together with Masson staining in cultured TM cells. (B) The mRNA levels of GDF7 and fibrosis-related markers N-cad, α-SMA, and FN were tested by quantitative real-time PCR in four groups of TM cells (n = 3 per group). (C and D) The ratio between phosphorylated and total Smad1, -5, and -9 and Smad4 protein was measured and analyzed by immunoblot in TM cells (n = 3 per group). Scale bars, 20 μm. The data were represented as mean ± SD. Compared with rhGDF7 group: ∗p < 0.05, ∗∗p < 0.01. Compared with NTM cells: #p < 0.05.

Figure 6

Validation of GDF7 methylation in POAG pathogenesis with the ANN model (A) Study pipeline for agent training, validation, and testing. Methylation levels of GDF7, BMPR2, Smad1, Smad5, Smad9, and Smad4 were enrolled in the training library to predict four binary POAG-related outcomes (high IOP, attenuation of RNFL thickness, high CDR, and visual field [VF] defects). (B) The ANN-based model was used to predict the four binary POAG-related outcomes. The accuracy of the model was presented by area under the curve (AUC). (C and D) Summary of the confusion matrices in the four outcome networks was displayed. (E) The contribution of input indexes (methylation levels of GDF7, Smad1, Smad5, Smad9, and Smad4) in predicting clinical outcomes related to POAG was calculated. TP, true positive; TN, true negative; FP, false positive; FN, false negative.

Figure 7

GDF7 neutralization protected TM function in rhesus monkeys (A) Simplified workflow of animal experiment. In brief, rhGDF7 was delivered into the anterior chamber three times on the first days of weeks 0, 2, and 4. The nGDF7 was injected into the anterior chamber for three times on the first days of weeks 10, 12, and 14. Ophthalmic examinations were conducted every 2 weeks throughout the entire process. The TM samples were harvested on the end of week 28. (B) The aqueous humor (AH) outflow facility was measured throughout the whole experiment. (C) Curves presented the IOP change in response to rhGDF7 delivery or/and nGDF7 treatment in monkey eyes (n = 4 per group). (D) Immunofluorescence labeling with N-cad, α-SMA, and FN was conducted in monkey TM tissues, and collagen accumulation was determined by Masson trichrome staining (n = 4 per group). Scale bars, 20 μm.

Figure 8

Proposed model for the methylation-dependent pathology in POAG Hypomethylation in the GDF7 gene gave rise to the increased GDF7 mRNA transcription via facilitating transcription factors binding to open chromatin. The TET enzyme sustained GDF7 overexpression by keeping the promoter region hypomethylated and open. Excessive GDF7 proteins activated BMPR2 and phosphorylated the downstream effectors, Smad1, -5, and -9 and Smad4. The phosphorylated Smad complex translocated into the nucleus and further promoted the overexpression of GDF7 and fibrosis markers, including Col I, α-SMA, and FN. Meanwhile, the expression of N-cad was suppressed, which remains to be explored in further studies. The imbalance between these proteins triggered TM fibrosis, which leads to the function failure in TM and ended up in IOP elevation. The accumulated GDF7 protein also activated the TET enzyme via an unknown mechanism, thus forming a positive feedback loop. Based on the aforementioned mechanism, the nGDF7 could impede the pro-fibrotic effect of GDF7. Therefore, GDF7 neutralization halts the progression toward fibrosis and maintains a normal AH through the TM.