Scleral HIF-1α is a prominent regulatory candidate for genetic and environmental interactions in human myopia pathogenesis

Abstract

Background: Myopia is a good model for understanding the interaction between genetics and environmental stimuli. Here we dissect the biological processes affecting myopia progression.

Methods: Human Genetic Analyses: (1) gene set analysis (GSA) of new genome wide association study (GWAS) data for 593 individuals with high myopia (refraction ≤ -6 diopters [D]); (2) over-representation analysis (ORA) of 196 genes with de novo mutations, identified by whole genome sequencing of 45 high-myopia trio families, and (3) ORA of 284 previously reported myopia risk genes. Contributions of the enriched signaling pathways in mediating the genetic and environmental interactions during myopia development were investigated in vivo and in vitro.

Results: All three genetic analyses showed significant enrichment of four KEGG signaling pathways, including amphetamine addiction, extracellular matrix (ECM) receptor interaction, neuroactive ligand-receptor interaction, and regulation of actin cytoskeleton pathways. In individuals with extremely high myopia (refraction ≤ -10 D), the GSA of GWAS data revealed significant enrichment of the HIF-1α signaling pathway. Using human scleral fibroblasts, silencing the key nodal genes within protein-protein interaction networks for the enriched pathways antagonized the hypoxia-induced increase in myofibroblast transdifferentiation. In mice, scleral HIF-1α downregulation led to hyperopia, whereas upregulation resulted in myopia. In human subjects, near work, a risk factor for myopia, significantly decreased choroidal blood perfusion, which might cause scleral hypoxia.

Interpretation: Our study implicated the HIF-1α signaling pathway in promoting human myopia through mediating interactions between genetic and environmental factors.

Funding: National Natural Science Foundation of China grants; Natural Science Foundation of Zhejiang Province.

Keywords: Genetic and environmental interactions; HIF-1α; Myopia; Myopia risk genes; Near work; Sclera.

Fig. 1

Study design for discovering genetically directed biological processes underlying myopia development. (a) Three different genetic analyses were performed, including GSA of GWAS data obtained from the Wenzhou study, whole genome sequencing for 45 trio families of high myopia to identify functional de novo mutations, and an assemblage of 284 previously reported myopia risk genes. (b) To determine the enrichment of these genetic factors in biological processes, we applied GSA to detect the enrichment of minor effects of genetic polymorphisms in all 321 human regulatory KEGG pathways. Furthermore, we expanded the KEGG pathways by combining the protein-protein interaction (PPI) evidence and constructed the KEGG-PPI networks with the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database. Then, we also applied the ORA strategy to detect the over-representation of 196 genes with functional DNMs and 284 myopia risk genes in KEGG-PPI networks. (c) Numbers in overlapping regions indicate the number of KEGG pathways in common, between and among the analyzed groups. Among all significant gene sets (P<0.05), the same four KEGG pathways were identified as being significant, by analyses of three different data sets: Wenzhou, GWAS data in Wenzhou; DNM196, 196 genes with functional DNMs; and GWAS284, 284 previously reported myopia risk genes. GSA: gene set analysis; GWAS: genome wide association; PPI: protein-protein interaction; ORA: over-represatation analysis.

Fig. 2

Protein-protein interaction (PPI) network constructed for myopia-associated genes in candidate pathways. (a) Myopia risk genes were identified by previous genome-wide association study (GWAS) results. (b) Genes with de novo mutations in whole-genome sequencing of 45 trio families of high myopia. (c) Genes with P-values <0.05 in gene set analysis (GSA) results for three candidate pathways in the Wenzhou and Guangzhou studies. The networks were constructed based on interactions extracted from the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database (https://string-db.org/) and visualized using Cytoscape (https://cytoscape.org/).

Fig. 3

Differential expression of the key nodal genes in the three KEGG-PPI networks in the scleras of FD-T and FD-F eyes after two days of monocular form deprivation, and in non-treated control (NC) mice. Panels a-d represent the relative mRNA expression levels for different sets of genes. Expression levels were normalized to levels in the NC group. Each data point represents an independent mouse. * P<0.05, ** P<0.01, *** P<0.001; one-way ANOVA with Bonferroni's post hoc tests (for normally distributed data), Kruskal-Wallis non-parametric test with Dunn's post hoc test (for non-normally distributed data). The detailed statistical data, including the estimated differences, the appropriate 95% confidence intervals for the differences, and P-values were given in Supplementary Table 17.

Fig. 4

Loss of GAS6 function blunts on hypoxia-dependent events in HSFs. (a) HSFs transfected with siRNAs targeting GAS6 were treated with 1% O₂, and protein levels of HIF-1A, GAS6, COL1Α1, paxillin, and α-SMA proteins were analyzed by western blotting. (b-f) The bar graphs represent relative protein levels of HIF-1A (b), GAS6 (c), COL1Α1 (d), paxillin (e), and α-SMA (f), respectively (n=3). Data are expressed as means ± standard error of the means. Mock: blank control (transfection reagent only); NC, normal control (cells transfected with scrambled normal control siRNA). α-tubulin was the loading control. **P<0.05; ***P<0.001; two-way ANOVA with Bonferroni's post hoc tests. The detailed statistical data, including the estimated differences, the appropriate 95% confidence intervals for the differences, and P-values were given in Supplementary Table 17.

Fig. 5

Scleral HIF-1α knock-down shifts refraction towards hyperopia in normal eyes under a normal viewing environment. The right eye of each Hif-1α^fl/fl mouse (Right) was treated with AAV8-Cre or AAV8-Vector Control; the left eye (Left) served as untreated fellow control. Data from age-matched Hif-1α^fl/fl mice that received no injections (Control) are shown for comparison. (a) Levels of scleral HIF-1α, COL1α1, and α-SMA proteins, were determined by western blot analysis four weeks after AAV8 injection; α-tubulin was the loading control. (b) Densitometric quantification of western blot results. Each data point represents one scleral sample pooled from two mice. (c) Refraction of Left and Right eyes in the three groups before and two weeks and four weeks after AAV8 injection. Box-plot diagrams showing the distribution of the data. Lines within the boxes indicate medians; bars, range; black dots, outliers. (d-f) Inter-ocular differences in refraction (d), AL (e), and VCD (f). Data are expressed as means ± standard errors of the mean, except as indicated otherwise (c). Data in b were assessed by one-way ANOVA with Bonferroni's post hoc tests; n=3 and 4 for AAV8-Vector Control and AAV8-Cre respectively. Data in c-f were assessed by two-way RM-ANOVA with Bonferroni's post hoc tests; n=10, 13, and 16 mice for Control, AAV8-Vector Control, and AAV8-Cre respectively. *P<0.05, **P<0.01. The detailed statistical data, including the estimated differences, the appropriate 95% confidence intervals for the differences, and P-values were given in Supplementary Table 17. D, diopter.

Fig. 6

Scleral HIF-1α knock-down inhibits myopia development in form deprived eyes. The sub-Tenon's space in the right eye of each Hif-1α^fl/fl mouse was injected with AAV8-Cre or AAV8-Vector Control. After one week, monocular form deprivation (FD) was imposed for two additional weeks in the right eye (FD-T), while the left eye served as non-FD fellow-eye control (FD-F). A separate group of form deprived, non-injected eyes served as uninjected FD-only controls (Control+FD). (a) Western blot analysis of scleral levels of proteins HIF-1α, α-SMA, and COL1α1. α-tubulin was the loading control. (b) Densitometric quantification of western blot results Each data point represents one scleral sample pooled from two mice. (c) Refraction of FD-T and FD-F eyes in the three groups. (d-f) Interocular differences (FD-T eye minus FD-F eye) in refraction (d), AL (e), and VCD (f), before and after two weeks of FD. Data are expressed as means ± standard error of the means (b). Box-plot diagrams showing the distribution of the data; lines within the boxes indicate medians; bars, range; black dots, outliers (c-f). Data in b were assessed by one-way ANOVA with Bonferroni's post hoc test (for normally distributed data), or Kruskal-Wallis with Dunn's post hoc test (for non-normally distributed data); n=4 for each group. Data in c-f were assessed by two-way RM-ANOVA with Bonferroni's post hoc tests; n=11, 14, 19 mice, for Control, AAV8-Vector, and AAV8-Cre, respectively; *P<0.05, **P<0.01. The detailed statistical data, including the estimated differences, the appropriate 95% confidence intervals for the differences, and P-values were given in Supplementary Table 17. D, diopter.

Fig. 7

Effects of accommodation on choroidal thickness (ChT) and choroidal blood perfusion (ChBP). (a) Schematic diagram of OCTA measurement, under 0 D accommodative stimulus. The white symbol “+” on black background served as the fixation target, and depending upon viewing distance, provided either 0 D defocus, or 6 D hyperopic defocus, causing linked accommodative responses in both eyes. When the stimulus was presented to the right eye, ChT and ChBP of the left eye were examined by OCTA. (b) Schematic diagram of OCTA detection under a 6 D accommodative stimulus. When moving the fixation target, L1 did not move, but L2 moved together with the target. Meanwhile, the left eye turned inward, and the OCTA position was adjusted to maintain a clear, well-focused fundus image. L1: Badal lens, f=75 mm; L2: Badal lens, f=50 mm. (c) ChT and (d) ChBP after 0 D and 6 D accommodation stimuli. Data are expressed as mean ± standard error of the mean. n=16; ** P<0.01; paired two-tailed t-tests. The detailed statistical data, including the estimated differences, the appropriate 95% confidence intervals for the differences, and P-values were given in Supplementary Table 17. D, diopter.

Fig. 8

Recapitulation of findings and derived model describing how scleral hypoxia induces myopia. Previous known data are shown in blue font. ChT: Choroidal thickness; ChBP: Choroidal blood perfusion; DNMs: De novo mutations; ECM: Extracellular matrix; GSA: Gene set analysis; GWAS: Genome wide association; KEGG: Kyoto Encyclopedia of Genes and Genomes; ORA: Over-representation analysis; PPI: Protein-protein interaction; WGS: Whole genome sequencing; D: diopter (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).