Inhibition of soluble epoxide hydrolase prevents diabetic retinopathy

Abstract

Diabetic retinopathy is an important cause of blindness in adults, and is characterized by progressive loss of vascular cells and slow dissolution of inter-vascular junctions, which result in vascular leakage and retinal oedema. Later stages of the disease are characterized by inflammatory cell infiltration, tissue destruction and neovascularization. Here we identify soluble epoxide hydrolase (sEH) as a key enzyme that initiates pericyte loss and breakdown of endothelial barrier function by generating the diol 19,20-dihydroxydocosapentaenoic acid, derived from docosahexaenoic acid. The expression of sEH and the accumulation of 19,20-dihydroxydocosapentaenoic acid were increased in diabetic mouse retinas and in the retinas and vitreous humour of patients with diabetes. Mechanistically, the diol targeted the cell membrane to alter the localization of cholesterol-binding proteins, and prevented the association of presenilin 1 with N-cadherin and VE-cadherin, thereby compromising pericyte-endothelial cell interactions and inter-endothelial cell junctions. Treating diabetic mice with a specific sEH inhibitor prevented the pericyte loss and vascular permeability that are characteristic of non-proliferative diabetic retinopathy. Conversely, overexpression of sEH in the retinal Müller glial cells of non-diabetic mice resulted in similar vessel abnormalities to those seen in diabetic mice with retinopathy. Thus, increased expression of sEH is a key determinant in the pathogenesis of diabetic retinopathy, and inhibition of sEH can prevent progression of the disease.

Extended Data Figure 1. Characterization of Ins2^Akita mice

a–d, Wild-type and Ins2^Akita littermates were treated with vehicle (Veh; 0.3% ethanol) or the sEH inhibitor (sEH-I) from the age of 6 weeks. (a) Body weight and (b) fasting blood glucose were recorded at monthly intervals. (c) Systolic blood pressure (SBP) and (d) heart rate (HR) were assessed at the age of 12 months. e, sEH (red) and glutamine synthetase (GS; green) in retinas from 12 month old Ins2^Akita mice and their wild-type littermates. Glial fibrillary acidic protein (GFAP; blue). Retinas from sEH^−/− mice were used to demonstrate the specificity of the sEH antibody used. f, Retinal digest preparations from 3 month old Ins2^Akita mice and their non-diabetic wild-type littermates; Acellular capillaries are marked by arrows. g–j, Quantitative retinal morphometry images shown in f; i.e., endothelial cells (EC, g), pericytes (PC, h), extravascular pericytes (ev-PC, i) and acellular capillaries (ac-Cap, j). k, Quantification of leaked FITC-BSA. l, Retinal digest preparations from 6 month old Ins2^Akita mice and their non-diabetic wild-type littermates. Acellular capillaries are marked by arrows. m–p, Quantitative retinal morphometry of images in l. q, Quantification of leaked FITC-BSA. Scale bars 50 μm. n = 4 (a–b), 6 (e–q) or 8 (c–d) mice per group. Data are mean ± s.e.m. P values determined by 2way ANOVA (a–d), or Student’s two-tailed t-test (g–k, m–q).

Extended Data Figure 2. Epoxide and diol profile in retinas from wild-type and Ins2^Akita mice and sEH expression in high fat-fed mice

a, Effect of diabetes and sEH inhibition on metabolites of polyunsaturated fatty acids in 12 month old wild-type (WT) and Ins2^Akita (Akita) mice treated with either vehicle (Veh; 0.3% ethanol) or sEH inhibitor (sEH-I). EET, epoxyeicosatrienoic acid; DHET, dihydroxyeicosatrienoic acid; HODE, hydroxyoctadecadienoic acid; DiHOME, dihydroxyoctadecenoic acid; EpOME, epoxyoctadecenoic acid; EpETE, epoxyeicosatetraenoic acid; DiHETE, dihydroxyeicosatetraenoic acid; HEPE, hydroxyeicosapentaenoic acid; EDP, epoxydocosapentaenoic acid; DHDP, dihydroxydocosapentaenoic acid; nd = not detectable. n = 6 biologically independent samples per group. b, Expression of epoxide hydrolase (Ephx) genes in retinas from 12 month old wild-type (WT) and Ins2^Akita (Akita) littermates. c, Retinal sEH expression in wild-type mice given a normal diet (ND, n = 6) or a high fat, high carbohydrate (HFHC, n = 8) diet for 20 weeks. For gel source data, see Supplementary Fig. 1. d, sEH activity in retinas from mice given a normal diet (ND, n = 6) or a high fat, high carbohydrate (HFHC, n = 8) diet. e, Retinal digest preparations from animals fed a normal diet (ND) or a high fat, high carbohydrate (HFHC) diet for 20 weeks. Acellular capillaries are marked by arrows. f–i, Quantitative retinal morphometry of the images shown in e; i.e., endothelial cells (EC, f), pericytes (PC, g), extravascular pericytes (ev-PC, h) and acellular capillaries (ac-Cap, i). n = 6 mice per group (b, e–i). Data expressed as mean ± s.e.m. P values determined by 1way ANOVA (a), or Student’s two-tailed t-test (b–d, f–i).

Extended Data Figure 3. Growth factor expression in the retina and lipid raft preparations

a–m, Growth factor expression in retinas from wild-type (WT) and Ins2^Akita (Akita) littermates treated with vehicle (Veh; 0.3% ethanol) or the sEH inhibitor (sEH-I). n = 6 biologically independent samples per group. n–o, Endothelial cells and pericytes were co-cultured and treated with solvent, 19,20-EDP or 19,20-DHDP in the presence of sEH inhibitor. Lipid rafts (LR) were isolated by gradient centrifugation (see Methods) and the distribution of (n) VE-cadherin and (o) N-cadherin assessed by Western blotting. Flotillin-1 (Flot1) was included as a marker of lipid raft enriched fractions. Similar results were obtained in 3 additional cell batches. p–q, Co-precipitation of p120 with (p) N-cadherin and (q) VE-cadherin in endothelial cell-pericyte co-cultures after treatment with solvent, 19,20-EDP or 19,20-DHDP. VEGF was included as a positive control. Similar results were obtained in 3 additional experiments. r, VE-cadherin phosphorylation in endothelial cells treated with solvent, 19,20-EDP, 19,20-DHDP or VEGF for 5 or 15 minutes. For gel source data, see Supplementary Fig. 1. Comparable results were obtained in 3 additional experiments. Data expressed as mean ± s.e.m. P values determined by 2way ANOVA.

Extended Data Figure 4. Mural cell coverage and N-cadherin localization in endothelial cell-pericyte co-cultures

a, Smooth muscle actin (α-SMA) showing mural cell coverage of 20 μm, 15 μm and 10 μm diameter retinal vessels; bar = 50 mm. Comparable results were obtained in retinas from 5 additional animals in each group. b, Permeability of human endothelial cells to dextrans of different molecular mass; n = 6 independent cell preparations. c, VE-cadherin and N-cadherin expression in endothelial cells (EC), pericytes (PC) or endothelial-pericyte co-cultures (EC+PC). Similar results were obtained in 3 different cell batches. For gel source data, see Supplementary Fig. 1. d, Cartoon showing the filter-based co-culture system studied. e, VE-cadherin (red), N-cadherin (green), desmin (cyan) and DAPI (grey) on transwell membranes with endothelial-pericyte co-cultures; bar = 5 μm in top view and 2 μm in side views. Similar observations were made in 2 additional experiments. f–g, Internalized N-cadherin (yellow) in pericytes treated with solvent, 19,20-EDP or 19,20-DHDP in the presence of sEH inhibitor; n = 5 independent experiments; bar = 10 μm. Data expressed as mean ± s.e.m. P values determined by 1way ANOVA.

Extended Data Figure 5. Association of cholesterol with cadherins and PS1

a, Experimental pipeline for studying the association of proteins with cholesterol in Fig. 3n–o. b, Reverse immunoprecipitation for studying the association of proteins with cholesterol. Note the difference in step 5 and 6 from experimental procedure in a. c–e, Representative Li-cor system images. Proteins clicked with cholesterol were visualized in green; i.e. (c) N-cadherin, (d) VE-cadherin and (e) presenilin1 (PS1-F: full length; PS1-N: N-terminal fragment) were detected on the same membrane and visualized in red. Comparable results were observed in 3 additional experiments.

Extended Data Figure 6. ¹H MAS NMR spectra from native brain membranes and epoxide-diol profile of retinas 14 days after adenovirus injection

a, ¹H MAS NMR spectra of membranes from wild-type and a sEH^−/− mouse brains. At low temperatures ordered phases are predominant. They are characterized by a high chemical shift anisotropy and strong dipolar couplings resulting in broad components with low spectral resolution and pronounced spinning sidebands. With increasing temperatures narrow components caused by fast axial motions and gauche-trans isomerization of the acyl chains dominate the centerband of the ¹H MAS NMR spectrum. Therefore, the centerband CH₂ resonance is a qualitative indicator of membrane dynamics and lipid order. b, Fatty acid epoxide and diol levels in retinas from wild-type animals 14 days after administration of adenovirus encoding GFP, wild-type sEH (sEH) or a sEH mutant without epoxide hydrolase activity (ΔEH), some of animals were treated with the sEH inhibitor (sEH+I) immediately after virus injection. EET, epoxyeicosatrienoic acid; DHET, dihydroxyeicosatrienoic acid; HODE, hydroxyoctadecadienoic acid; DiHOME, dihydroxyoctadecenoic acid; EpOME, epoxyoctadecenoic acid; EDP, epoxydocosapentaenoic acid; DHDP, dihydroxydocosapentaenoic acid. nd = not detectable; n = 6 biologically independent samples per group, Data expressed as mean ± s.e.m. P values determined by 1way ANOVA.

Extended Data Figure 7. sEH-induced retinopathy

Desmin staining of non-diabetic wild-type retinas 14 days after administration of adenoviruses encoding GFP, wild-type sEH (sEH^WT) or a sEH mutant without epoxide hydrolase activity (sEH^ΔEH). Some mice were treated with the sEH inhibitor (sEH-I). Arrows indicate desmin negative regions along a retinal artery. Comparable observations were made using 5 additional animals in each group.

Extended Data Figure 8. Retinopathy induced by AAV-mediated sEH overexpression in non-diabetic mice

a, sEH (red) in retinas from wild-type mice 3 months after intravitreal injection of 10¹⁰ genomic copies of AAV-RLBP1-sEH^WT (with myc-tag, green). b, Retinal digest preparations from wild-type mice 3 months after intravitreal injection of 10¹⁰ genomic copies of AAV-RLBP1-GFP (CTL) or AAV-BLBP1-sEH^WT. sEH inhibitor (sEH-I) treatment began directly after AAV injection. Acellular capillaries are marked by arrows, Bar = 50 μm. c–f, Quantitative retinal morphometry of the images shown in b; i.e. endothelial cells (EC, c), pericytes (PC, d), extravascular pericytes (ev-PC, e) and acellular capillaries (ac-Cap, f). g, Desmin (red) and CD31 (cyan) in retinas from wild-type mice 3 months after intravitreal injection of AAVs. Scale bars = 50 μm. n = 6 (a, g) or 8 (b–f) mice per group. Data expressed as mean ± s.e.m. P values determined by 1way ANOVA.

Fig. 1. sEH expression in retinas from diabetic mice and humans

(a) sEH (red) and aquaporin 4 (green) in retinas from 12 month old mice. (b) Time course of retinal sEH expression. For gel source data, see Supplementary Fig. 1. (c) sEH activity in murine retinas. (d) sEH (red), glutamine synthetase (green), glial fibrillary acidic protein (blue) and DAPI (white) in retinas from patients with no diabetic retinopathy (non-DR, n = 6), mild non proliferative diabetic retinopathy (NPDR, n = 7) or severe NPDR (n = 6). (e–f) Quantification of GFAP and sEH from sections shown in d. (g) 19,20-EDP/DHDP in vitreous humor from individuals with non-diabetic retinopathy (Non-DR, n = 14) or diabetic retinopathy (DR, n = 17). (h) Retinal levels of 19,20-EDP/DHDP in 12 month old mice treated with vehicle or sEH inhibitor (sEH-I). Scale bar 50μm. n = 5 (b) or 6 (a, c, h) biologically independent samples per group. Data are mean ± s.e.m. P values determined by 2way ANOVA (b), Student’s two-tailed t-test (c, g) or 1way ANOVA (e–f, h).

Fig. 2. Consequences of sEH inhibitor treatment on diabetic retinopathy in 12 month old mice

(a) Retinal digest preparations; acellular capillaries are marked by arrows. (b–e) Quantitative retinal morphometry of images in a; i.e., endothelial cells (EC, b), pericytes (PC, c), extravascular pericytes (Ev-PC, d), and acellular capillaries (ac-Cap, e). (f) Fluorescent images of FITC-BSA 1 hour after injection. (g) Quantification of leaked FITC-BSA. (h) N- and VE-cadherin expression in retinas from wild-type and Ins2^Akita littermates treated with or without sEH inhibitor (sEH-I). For gel source data, see Supplementary Fig. 1. (i) N-cadherin (green) and desmin (red) in the primary vascular layer and second capillary layer. (j) VE-cadherin in the retinal vasculature. Scale bar 20μm. n = 5 (f–h) or 6 (a–e, i, j) mice per group. Data are mean ± s.e.m. P values determined by 2way ANOVA.

Fig. 3. Effect of 19,20-DHDP on endothelial cell permeability and pericyte migration

(a) VE-cadherin in endothelial cells treated with solvent, 19,20-EDP/DHDP or VEGF. Arrowheads indicate the discontinuous VE-cadherin pattern. (b) Permeability of human endothelial cells to dextran with different molecular masses. (c) Transendothelial electrical resistance (TEER) in murine brain endothelial cells. (d–e) Internalized VE-cadherin (yellow) in endothelial cells. (f) Surface and internalised VE-cadherin in endothelial cells. (g) N- and VE-cadherin expression in endothelial-pericyte co-cultures. (h) Pericyte mobility on endothelial cells after N-cadherin downregulation (N-si), N-cadherin overexpression (N-OV), or treatment with EDP/DHDP; n = 120 cells per group. (i–j) Internalized N-cadherin (yellow) in human pericytes. (k) Migrating pericytes (arrowheads) in ex vivo retinal whole mounts. (l) Association of PS1 (PS1-F: full length; PS1-N: N-terminal fragment) with VE- and N-cadherin immunoprecipitated from endothelial cell-pericyte co-cultures. (m) VE-cadherin (green), PS1 (red) and N-cadherin (blue) in retinas from 12 month old mice treated with vehicle or sEH inhibitor (sEH-I). Arrows indicate disrupted VE-cadherin pattern. (n) Co-precipitation of PS1 with cholesterol from endothelial cell-pericyte co-cultures. (o) Co-precipitation of VE- and N-cadherins with cholesterol from endothelial cell-pericyte co-cultures. (p) CH₂ resonance centerband intensities of ¹H MAS NMR temperature scans of isolated brain plasma membranes from wild-type and sEH^−/− mice exposed to 19,20-EDP (100 μmol/L) or 19,20-DHDP (10 or 100 μmol/L). For gel source data, see Supplementary Fig. 1. Scale bars 20μm. n = 4 (a, f–h, l, n–o), 5 (i–j), 6 (b, d–e), 8 (c) independent experiments, or 6 retinas (k, m) per group. Data are mean ± s.e.m. P values determined by 2way ANOVA (b) or 1way ANOVA (c, e–f, h, j–k).

Fig. 4. sEH-induced retinopathy in non-diabetic mice

(a) sEH (red) in wild-type mice retinas 7 days after intravitreal injection of GFP track adenoviral-sEH (sEH^WT). (b) Retinal sEH activity 14 days after intravitreal injection of adenoviruses encoding GFP, sEH^WT or the sEH^ΔEH mutant. (c) 19,20-EDP/DHDP levels in retinas 14 days after adenoviral injection to animals receiving vehicle or the sEH inhibitor (sEH-I). (d) Retinal digest preparations 14 days after adenovirus injection with or without sEH inhibitor (+I) treatment. Acellular capillaries are marked by arrowheads. (e) Vascular permeability (titrc-dextrin; red) 14 days after virus administration. (f–i) Quantitative retinal morphometry of images in d; i.e., endothelial cells (EC, f), pericytes (PC, g), extravascular pericytes (Ev-PC, h), and acellular capillaries (ac-Cap, i); n = 6 (GFP), 9 (sEH^WT) or 8 (sEH^WT+I, sEH^ΔEH) mice per group. (j) Quantification of leaked Titrc-dextrin. (k) N-cadherin (cyan) and desmin (red) in retinas treated with adenoviruses. Note the perivascular desmin positive protrusions that mark extravascular pericytes in sEH^WT treated retinas. (l) VE-cadherin (green) and collagen IV (red) in retinas 14 days after adenovirus injection. Capillaries with abnormal VE-cadherin indicated with arrowheads. Scale bars 50μm. n = 5 (b), 6 (e, j–l) mice or 6 biologically independent samples per group (c). Data are mean ± s.e.m. P values determined by 1way ANOVA.