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. Author manuscript; available in PMC: 2022 Oct 6.
Published in final edited form as: Metabolism. 2022 Jul 19;134:155266. doi: 10.1016/j.metabol.2022.155266

Cytochrome P450 oxidase 2J inhibition suppresses choroidal neovascularization in mice

Yan Gong a,b,1, Yohei Tomita b,1, Matthew L Edin c, Anli Ren a,d, Minji Ko b, Jay Yang b, Edward Bull b, Darryl C Zeldin c, Ann Hellström e, Zhongjie Fu b, Lois EH Smith b,*
PMCID: PMC9535696  NIHMSID: NIHMS1834402  PMID: 35868524

Abstract

Introduction:

Choroidal neovascularization (CNV) in age-related macular degeneration (AMD) leads to blindness. It has been widely reported that increased intake of ω–3 long-chain polyunsaturated fatty acids (LCPUFA) diets reduce CNV. Of the three major pathways metabolizing ω–3 (and ω–6 LCPUFA), the cyclooxygenase and lipoxygenase pathways generally produce pro-angiogenic metabolites from ω–6 LCPUFA and anti-angiogenic ones from ω–3 LCPUFA. Howevehr, cytochrome P450 oxidase (CPY) 2C produces pro-angiogenic metabolites from both ω–6 and ω–3 LCPUFA. The effects of CYP2J2 products on ocular neovascularization are still unknown. Understanding how each metabolic pathway affects the protective effect of ω–3 LCPUFA on retinal neovascularization may lead to therapeutic interventions.

Objectives:

To investigate the effects of LCPUFA metabolites through CYP2J2 pathway and CYP2J2 regulation on CNV both in vivo and ex vivo.

Methods:

The impact of CYP2J2 overexpression and inhibition on neovascularization in the laser-induced CNV mouse model was assessed. The plasma levels of CYP2J2 metabolites were measured by liquid chromatography and tandem mass spectroscopy. The choroidal explant sprouting assay was used to investigate the effects of CYP2J2 inhibition and specific LCPUFA CYP2J2 metabolites on angiogenesis ex vivo.

Results:

CNV was exacerbated in Tie2-Cre CYP2J2-overexpressing mice and was associated with increased levels of plasma docosahexaenoic acids. Inhibiting CYP2J2 activity with flunarizine decreased CNV in both ω–6 and ω–3 LCPUFA-fed wild-type mice. In Tie2-Cre CYP2J2-overexpressing mice, flunarizine suppressed CNV by 33 % and 36 % in ω–6, ω–3 LCPUFA diets, respectively, and reduced plasma levels of CYP2J2 metabolites. The proangiogenic role of CYP2J2 was corroborated in the choroidal explant sprouting assay. Flunarizine attenuated ex vivo choroidal sprouting, and 19,20-EDP, a ω–3 LCPUFA CYP2J2 metabolite, increased sprouting. The combined inhibition of CYP2J2 with flunarizine and CYP2C8 with montelukast further enhanced CNV suppression via tumor necrosis factor-α suppression.

Conclusions:

CYP2J2 inhibition augmented the inhibitory effect of ω–3 LCPUFA on CNV. Flunarizine suppressed pathological choroidal angiogenesis, and co-treatment with montelukast inhibiting CYP2C8 further enhanced the effect. CYP2 inhibition might be a viable approach to suppress CNV in AMD.

Keywords: Cytochrome P450 oxidase 2J, Long-chain polyunsaturated fatty acid, Lipid metabolism, Choroidal neovascularization, Age-related macular degeneration, Tumor necrosis factor-α

Graphical Abstract

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1. Introduction

Age-related macular degeneration (AMD) is a blinding eye disease that leads to progressive vision loss in the elderly [1]. The overall prevalence of AMD is 16 % in Europe, and the number of AMD patients globally is expected to reach 288 million by 2040 [2,3]. With aging, retinal pigment epithelial (RPE) cells lose digestive and phagocytic capacity, and residual material from disc membrane digestion is deposited on Bruch’s membrane, resulting in drusen formation [4]. Choroidal neovascularization (CNV) in AMD forms from choroidal vessels and extends through ruptured Bruch’s membrane and the RPE into the photoreceptor layer in the retina which is normally avascular [5,6].

The development and pathogenesis of AMD is becoming better characterized [7,8], but options for therapeutic intervention are still limited [9–13]. Intravitreal injection of anti-vascular endothelial growth factor (VEGF) drugs is widely used [14,15]. However, long-term suppression of VEGF may lead to central retinal atrophy and loss of visual acuity [16,17], or damage ganglion and RPE cell function [18,19]. Therefore, we need to better understand the role of other factors that modulate CNV.

Altered lipid intake and metabolism is associated with many age-related disorders, including obesity, atherosclerosis, cancer, and AMD [20–22]. Genome-wide association meta-analysis suggests that long-chain polyunsaturated fatty acids (LCPUFA) (and their metabolites) contribute to both early and late AMD [23]. In healthy individuals above age 55, 16.7 % of retinal lipids are ω–6 LCPUFA, and 16.2 % are ω–3 LCPUFA [24]. The Age-Related Eye Disease Study (AREDS) suggests that the patients with higher fish consumption (and assumed higher ω–3 LCPUFA intake) are 30 % less likely to develop AMD (with or without CNV) [25–27]. However, some clinical trials do not show a protective effect of pure ω–3 LCPUFA intake on AMD [28].

To better understand the mechanisms underlying ω–3 LCPUFA suppression of CNV, we fed mice ω–3 LCPUFA-enriched versus ω–6 LCPUFA-enriched (control) diets that were otherwise matched, then examined the effects of blocking or enhancing downstream metabolites that might mediate CNV progression. Both ω–6 and ω–3 LCPUFA are oxidized by three major pathways, lipoxygenase (LOX), cyclooxygenase (COX), and cytochrome P450 oxidase (CYP) [29]. While the major ω–6 LCPUFA metabolites through COX and LOX pathways increase ocular CNV [30,31], the major ω–3 LCPUFA metabolites through COX and LOX pathways suppress ocular neovascularization and preserve retinal functions against AMD [32–34]. Therefore, because COX and LOX produce pro-angiogenic metabolites from ω–6 LCPUFA and anti-angiogenic metabolites from ω–3 LCPUFA, blocking COX and LOX in individuals on a normal diet containing both lipids will not enhance the ω–3 LCPUFA dietary protective effect. Thus, the effects of CYP products from ω–3 and ω–6 LCPUFA on ocular neovascularization need to be further explored.

Our previous studies suggest that inhibition of CYP2C8 increases the inhibitory effect of ω–3 LCPUFA on pathological retinal and choroidal neovascularization [35]. The CYP2C8 inhibitors fenofibrate and montelukast decrease the levels of ω–3 LCPUFA product 19,20-epoxydocosapentaenoic acids (19,20-EDP), and suppress CNV both in the CNV mouse model in vivo and in the choroidal sprouting assay ex vivo [36,37]. CYP2J, a separate subfamily of CYP2, actively metabolizes ω–3 and ω–6 LCPUFA to bioactive eicosanoids. CYP2J2 is detected in cardiac myocytes, pancreas, lung, kidney, and small intestine [38,39], and is more highly expressed than CYP2C8 in the vascular endothelium in humans [40]. However, the role of CYP2J2 in ocular neovascularization has not been explored. Since CYP inhibition decreases pro-angiogenic metabolites from both ω–3 LCPUFA and ω–6 LCPUFA, it might be an attractive option for AMD treatment.

In this study, we investigated if CYP2J2 inhibition might decrease CNV and therefore further enhance the ω–3 LCPUFA protection against CNV. In addition, we investigated the impact of dual inhibition of CYP2J2 with flunarizine and CYP2C8 with montelukast on CNV in a mouse model of CNV.

2. Materials and methods

2.1. Mice

Wild type (WT) C57BL/6J mice were obtained from Jackson Laboratory (Bar Harbor, ME). Tie2-Cre; CYP2J2-overexpressing transgenic (CYP2J2 TG) mice were developed on a C57BL/6 background as described [41,42]. Omega-6 LCPUFA, arachidonic acids (AA), ω–3 LCPUFA, docosahexaenoic acids (DHA), and eicosapentaenoic acids (EPA) were sourced from DSM Nutritional Products (TE Heerlen, Netherlands) and processed into rodent feed by Research Diets (New Brunswick, NJ). The mice were fed the designed rodent diets (Diet composition Supplemental Table 1) consisting of safflower oil containing either ω–6 LCPUFA (AA) and no ω–3 LCPUFA, or ω–3 LCPUFA (EPA and DHA) and no ω–6 LCPUFA 7 days before laser photocoagulation. The mice were randomly grouped into different treatment conditions, and both genders were used. All mice were housed in a 12-h light-dark cycle. All animal experiments were approved by the Institutional Animal Care and Use Committee at Boston Children’s Hospital, adhered to Animal Research Reporting of In Vivo Experiments (ARRIVE) guidelines, and complied with the Association for Research in Vision and Ophthalmology Statement.

2.2. Laser-induced CNV

We induced CNV in 6-to-8-week-old male and female mouse retinas using an image-guided laser system (Micron IV, Phoenix, Pleasanton, CA) according to our previously reported protocol [43,44]. We generated four laser burns at equal distances from the optic nerve head in each eye with a green Argon laser pulse of 532 nm wavelength, 50 μm diameter, 70 ms duration, and 240 mW power. After the procedure, the mice were monitored daily, and no pain response was observed. We treated CYP2J2 TG and littermate WT CNV mice with flunarizine (i.p. 10 mg/kg), montelukast (i.p. 1 mg/kg), or vehicle from D0 to D6 after the laser induction of CNV. Eyes were isolated 7 days following photocoagulation and fixed with 4 % paraformaldehyde (PFA). We dissected the posterior eyecups consisting of the RPE/choroid/sclera and then permeabilized them with Triton X-100 (0.1 %, Thermo Fisher, Waltham, MA). The samples were then incubated overnight in Isolectin (GS-IB4, 10 μg/ml, I21411, Thermo Fisher) to stain the CNV lesions. After whole-mounting with the scleral side facing down, the lesion area was visualized with a fluorescent microscope (AxioObserver Z1, Zeiss, Jena, Germany) at 200× magnification and quantified with ImageJ (National Institute of Health) by investigators blind to the sample identities. CNV area quantification is per lesion with one biological replicate with four technical replicates each. Exclusion criteria were followed as in previous publications [43].

2.3. Liquid chromatography and tandem mass spectroscopy

Plasma levels of CYP2J2 metabolic products in mice were determined by liquid chromatography and tandem mass spectroscopy after liquid/liquid extraction with ethyl acetate and passage through phospholipid removal columns, similar to previous reports [45]. We performed online liquid chromatography of extracted samples using capillary high-performance liquid chromatography (1200 Series, Agilent, Santa Clara, CA). Analytes were separated using a Luna column (5 m, 150 × 1 mm, Phenomenex, Torrance, CA) and quantified on an MDS Sciex API 3000 equipped with a TucolonSpray source (Applied Biosystems, Foster City, CA). The assay was designed to examine prevalent CYP, LOX, and COX-derived metabolites of both ω–6 and ω–3 LCPUFA.

2.4. Aortic ring assay

Aortae from 4-to-6-week-old C57BL/6J mice were dissected, cut into approximately 1 mm thick rings, embedded in growth factor-reduced Matrigel (354230, Corning, Corning, NY), and then cultured in complete medium (420–500, Cell Systems, Kirkland, WA) supplemented with penicillin-streptomycin (Thermo Fisher) and Culture Boost (Cell Systems). Flunarizine (20 μg/ml) and 19,20-EDP (1 μM, 10175, Cayman, Ann Arbor, MI) were added to the medium 48 h after embedding, and the medium was changed every other day. Images were taken six days after plating. The aortic sprouting areas were quantified using ImageJ with a semi-automated macro plugin for vessel sprout quantification, as previously reported [35,46].

2.5. Choroidal explant sprouting assay

RPE/choroid/sclera complexes (choroidal explant) from 3-week-old C57BL/6J mice were dissected and cut into approximately 1 × 1 mm2 pieces and then embedded into Matrigel and cultured as we described above (2.4). Flunarizine (20 μg/ml) and 19,20-EDP (1 μM) were added to the medium 48 h after embedding. Images of explants were taken 6 days after plating. As previously reported, the choroidal sprouting areas were quantified using ImageJ with a semi-automated macro plugin for vessel sprout quantification [46,47].

2.6. Real-time PCR

We performed quantitative real-time PCR (qPCR) as previously described [48,49]. RNA from retina and choroid complex was extracted using a PureLink® RNA Mini Kit (12,183,025; Invitrogen, Grand Island, NY), and cDNA was synthesized using an iScript™ cDNA synthesis kit (1708841; BioRad, Hercules, CA). qPCR was performed using the following primers: Tnf-a: Fw 5′-CATCTTCTCAAAATTCGAGTGACAA-3′, Re 5′-TGGGAGTAGACAAGGTACAACCC-3′.

Gene expression was quantified using an Applied Biosystems 7300 Sequence Detection System (Thermo Fisher) with a 2× SYBR Green Master Mix Kit (B21202; Bimake, Houston, TX). Gene expression was calculated relative to the housekeeping gene 18s (Fw 5′-ACGGAAGGGCACCACCAGGA-3′, Re 5′-CACCACCACCCACGGAATCG-3′) using the ΔΔCt method. The relative mRNA levels are presented as the fold change between Tnf-a and 18s versus vehicle-treated mice.

2.7. Statistical analysis

Student’s unpaired two-tailed t-test was used to compare between two groups. Two-way ANOVA was used to analyze more than two groups, and Tukey’s post hoc test was used to correct multiple comparisons using Prism v9.0 (GraphPad, San Diego, CA). Data are presented as mean ± standard error of the mean (SEM). P < 0.05 is the threshold for statistical significance. We performed more than three repeats for in vitro experiments and more than five repeats for ex vivo experiments. We used 20 mice to measure the lesion area of laser-CNV (each group; control, treatment, TG, WT). For qPCR, we used 11 mice for control and nine mice for treatment that had undergone laser CNV.

3. Results

3.1. CYP2J2 overexpression increased CNV lesion size in mice fed either ω–6 or ω–3 LCPUFA-enriched diets

To investigate the role of CYP2J2, we induced CNV in CYP2J2 TG and littermate WT mice controls, fed ω–6 or ω–3 LCPUFA-enriched diets (Fig. 1A). In WT mice, ω–3 vs. ω–6 LCPUFA diets decreased CNV, which is in line with our previous findings [35]. In CYP2J2 TG vs. WT mice, there was a 52 % and 26 % (ω–3 and ω–6 LCPUFA diets, respectively) increase in CNV lesion area, suggesting that activating the CYP2J2 pathway exacerbated CNV induction with both ω–6 and ω–3 LCPUFA diets (n = 41–67 laser lesions, both sexes; Fig. 1B). Interestingly, the ω–3 vs. ω–6 LCPUFA protection against CNV was no longer observed with overexpression of CYP2J2 in CYP2J2TG mice, suggesting that inhibition of CYP2J2 might enhance ω–3 suppression of CNV.

Fig. 1.

Fig. 1.

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CYP2J2 overexpression increased CNV and increased plasma levels of 19,20-EDP. (A) Representative images of CNV lesion areas in WT and CYP2J2 TG mice on ω–6 or ω–3 LCPUFA feed. (B) The CNV lesion areas were significantly larger in CYP2J2 TG mice than in WT. WT; wild type, TG; Tie2-Cre; CYP2J2-overexpressing mice. Scale bar, 200 μm. n = 41–67; *, P < 0.05; ***, P < 0.001.

3.2. Inhibition of CYP2J2 activity suppressed CNV

To investigate if CYP2J2 inhibition suppressed choroidal angiogenesis, we treated CYP2J2 TG and littermate WT CNV mice with flunarizine to inhibit CYP2J2 after laser CNV induction (Fig. 2A). Flunarizine treatment reduced CNV lesion area in both WT and CYP2J2 TG mice fed either a ω–6 LCPUFA or a ω–6 LCPUFA diet; ω–6 LCPUFA diet (WT, 15 %, P < 0.01 and TG 33 %, P < 0.001, n = 26–45 laser lesions); ω–3 LCPUFA diet (WT, 21 %, P < 0.05 and TG 36 %, P < 0.001, n = 49–66 laser lesions). Both sexes were used (Fig. 2B, C). These results suggest that CYP2J2 metabolites from both ω–6 and ω–3 promote CNV.

Fig. 2.

Fig. 2.

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Inhibition of CYP2J2 activity decreased CNV. (A) Representative images of CNV lesion areas in WT and CYP2J2 TG mice with or without flunarizine treatment and fed ω–6 or ω–3 LCPUFA diets. (B, C) Flunarizine treatment significantly reduced CNV lesion area in both WT and CYP2J2 TG mice on both ω–6 (B, n = 26–45) and ω–3 (C, n = 49–66) LCPUFA feed. WT; wild type, TG; Tie2-Cre (CYP2J2-overexpressing mice). Scale bar, 200 μm. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

3.3. The effects of CYP2J on metabolic products with ω–3 and ω–6 LCPUFA diets

We then examined the plasma levels of AA and DHA CYP metabolites, 19,20-EDP and 14,15-EET, respectively (Fig. 3), which exhibit proangiogenic effects on pathological retinal and choroidal neovascularization as we previously reported [35,50]. The plasma levels of 19,20-EDP and 14,15-EET were higher in CYP2J2 TG mice than WT mice fed a ω–3 but not a ω–6 LCPUFA diet (n = 5 mice, each group, both sexes, Fig. 3). The plasma levels of 19,20-EDP and 14,15-EET were comparable to CYP2J2 TG and WT mice fed a ω–6 LCPUFA diet (Fig. 3). The plasma level of 14,15-DHET, the soluble epoxide hydrolase (sEH) product from 14,15-EET, was higher in CYP2J2 TG mice than WT mice fed a ω–6 LCPUFA diet (Fig. 3, bottom).

Fig. 3.

Fig. 3.

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Schematic diagram of CYP2C/CYP2J and sEH products from ω–6 and ω–3 LCPUFA. The plasma levels of 19,20-EDP and 14,15-EET in WT and CYP2J2 TG mice with or without flunarizine treatment and on ω–6 or ω–3 LCPUFA feed. WT; wild type, TG; Tie2-Cre; CYP2J2-overexpressing mice. sEH; soluble epoxide hydrolase, EET; epoxydocosapentaenoic acids, EDP; docosahexaenoic acids, DHET; dihydroxyeicosatrienoic acids, n = 5 each group; *, P < 0.05; **, P < 0.01.

Flunarizine reduced plasma levels of 19,20-EDP in mice fed a ω–3 LCPUFA diet and reduced plasma levels of 14,15-EET in both CYP2J2 TG and WT mice fed a ω–6 LCPUFA diet (n = 5, both sexes; Fig. 3). Comparable 19,20-EDP and 14,15-EET levels were observed in flunarizine-treated CYP2J2 TG vs. WT mice fed ω–6 or ω–3 LCPUFA diet (Fig. 3). We also observed that flunarizine reduced 14,15-EET blood levels in mice fed a ω–3 LCPUFA diet, which possibly resulted from the incomplete depletion of AA after the 7-day dietary intervention (Fig. 3). We have previously reported that in mice with 17-day dietary ω–3 LCPUFA supplementation, retinal AA is still present [51].

3.4. 19,20-EDP reversed the inhibition of flunarizine on angiogenesis ex vivo

We further evaluated the effects of CYP2J2 inhibition on angiogenesis using the ex vivo aortic ring and choroid sprouting assays (Fig. 4A, B). Pieces of the aortic ring or sclera/choroid with attached RPE were dissected from WT and CYP2J2 TG littermate mice. Increased sprouting was observed in CYP2J2 TG vs. WT mouse explants (Fig. 4C, D), in line with increased CNV in CYP2J2 TG observed in vivo. Flunarizine treatment attenuated the sprouting in both CYP2J2 TG and WT mouse explants, in line with the treatment reduction of CNV observed in vivo. Adding 19,20-EDP to the explant abolished the suppressive effects of flunarizine on sprouting, further confirming the pro-angiogenic role of 19,20-EDP in choroidal vessel growth.

Fig. 4.

Fig. 4.

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19,20-EDP abolished inhibition of angiogenesis by flunarizine ex vivo. (A) Representative images of aortic ring sprouting from WT and CYP2J2 TG mice with or without flunarizine and EDP treatment. (B) Representative images of choroid explant sprouting from WT and CYP2J2 TG mice with or without flunarizine and EDP treatment. (C) 19,20-EDP abolished aortic ring sprouting inhibited by flunarizine. (n = 5, each explant) (D) 19,20-EDP rescued choroid explant sprouting inhibition by flunarizine (n = 6, each explant), WT; wild type, TG; Tie2-Cre; CYP2J2-overexpressing mice. Scale bar, 1 mm. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

3.5. Dual inhibition of CYP2J2 and CYP2C8 synergistically inhibited CNV via tumor necrosis factor (TNF)α suppression

We have previously reported that inhibition of CYP2C8 (another important enzyme in the CYP pathway metabolism of omega 3,6 LCPUFA) with montelukast suppresses pathological angiogenesis in the retina and choroid [35]. We investigated if there were synergistic effects of flunarizine inhibition of CYP2J2 and montelukast inhibition of CYP2C8 on CNV. C57BL/6J (WT) mice were co-treated with flunarizine (i.p. 10 mg/kg) and montelukast (i.p. 1 mg/kg) from D0 to D6 after laser treatment (Fig. 5A). The body weight was unchanged among these groups before and after treatment suggesting no toxicity (Fig. S1). Dual treatment with flunarizine and montelukast led to a further reduction of CNV lesion area compared to flunarizine or montelukast treatment alone (n = 58–66, *P < 0.05, Fig. 5B). To address the mechanism underlying CNV suppression, we focused on the contribution of inflammatory cytokines to CNV lesion formation. We found that dual therapy suppressed the gene expression levels of Tnf-α mRNA expression compared with vehicle in retina/choroid/RPE complex (n = 9–11, *P < 0.05; Fig. 5C).

Fig. 5.

Fig. 5.

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Inhibition of both CYP2J2 and CYP2C8 had synergistic effects on CNV inhibition. (A) Representative images of CNV lesion areas in WT mice with both flunarizine and montelukast treatment on normal feed. (B) Double treatment of flunarizine and montelukast further reduced CNV lesion areas than a single treatment. Scale bar, 200 μm. n = 58–66; *, P < 0.05. (C) qRT-PCR analysis of Vegfa, Il-1β, Tnfα, and Il-6 mRNA expression level in double treatment vs. control (n = 9–11 mice in each group).

4. Discussion

The current study showed that products from CYP2J2 metabolism of both ω–3 and ω–6 LCPUFA induced angiogenesis in vivo and ex vivo. Inhibition of CYP2J2 with flunarizine suppressed pathological choroidal vessel growth. Interestingly, inhibition of both CYP2 enzymes, CYP2J2 and CYP2C8 together enhanced CNV suppression associated with decreasing inflammatory cytokine expression (Graphical abstract).

Activation of the CYP metabolic pathway increases laser-induced CNV. We have shown that CYP2C8 ω–3 and ω–6 LCPUFA metabolites 19,20-EDP and 14,15-EET induce CNV and that pharmacological inhibition using the CYP2C8 inhibitors montelukast and fenofibrate suppresses CNV in vivo and choroidal angiogenesis in ex vivo [36,37]. In the present study, we found that CYP2J2-mediated LCPUFA metabolism also contributed to increased levels of 19,20-EDP and increased CNV formation. In addition to 19,20-EDP, we also observed increased 14,15-EET levels in mice fed a ω–3 LCPUFA diet. The metabolite 14,15-EET induces angiogenesis in part by regulating Src-dependent and STAT-3-mediated VEGF expression [52]. 14,15-EET also induces the growth of dormant micro-metastases by triggering neutrophilic infiltration [53]. Although we did not observe significant changes in 14,15-EET levels in mice fed the ω–6 LCPUFA diet, the levels of 14,15-DHET, the sEH product from 14,15-EET increased (Fig. 3). In vitro 14,15-DHET induces tubule formation in human umbilical vein endothelial cells [54], suggesting a potential contribution of 14,15-DHET to vessel growth. Furthermore, levels of 5-hydroxyeicosatetraenoic acid (5-HETE), an ω–6 LCPUFA (AA) LOX metabolite, were increased in the CYP2J2 TG mice fed the ω–6 LCPUFA diet (Fig. S2). 5-HETE levels are increased in vitreous humor of patients with diabetic retinopathy, and 5-HETE is an essential inflammatory mediator in lung diseases [55,56]. Therefore, we speculate that the increased levels of both 14,15-DHET and 5-HETE in the CYP2J2 TG mice on the ω–6 LCPUFA diet could contribute at least in part to increased CNV lesion formation.

We found that the plasma level of 14,15-DHET was higher in CYP2J2 TG mice than in WT mice fed a ω–6 LCPUFA diet (Fig. 3 bottom). The level of 14,15-DHET is three-fold higher than that of 14,15-EET in TG mice. There may be higher sEH levels or higher activity in those mice, suggesting a faster conversion of EET to DHET. Further studies are needed to clarify the issue.

Flunarizine treatment decreased CNV formation and reduced CYP2J2 overexpression increased plasma levels of 19,20-EDP and 14,15-EET (Figs. 2, 3), confirming that flunarizine inhibits CYP2J2 activity. We further examined how CYP2J2 impacts ω–3 LCPUFA inhibition of angiogenesis using ex vivo explants co-treated with flunarizine and 19, 20-EDP. Flunarizine suppressed explant sprouting and adding 19,20-EDP abolished this suppression. These findings suggest that ω–3 LCPUFA, metabolized through CYP2J2, generate pro-angiogenic 19,20-EDP to promote choroidal angiogenesis. However, further validation is still needed to better understand the role of CYP2J2 in ocular angiogenesis. In a recent finding, overexpression of CYP2J2 was shown to inhibit cell viability and angiogenesis in hypoxia-induced retinal vascular endothelial cells in vitro [57]. Therefore, a tissue-specific, role of CYP2J2 should be characterized. Furthermore, the combination therapy of CYP2J2 and CYP2C8 inhibitors suppressed the mRNA expression levels of Tnf-α in the retina, choroid, and RPE complex. Several studies show that the expression level of TNF-α is increased in laser CNV vs. non-laser controls [58,59]. It was reported previously that 19,20-EDP significantly increases TNF-α in IL-1B treated Muller glial cells [60]. Thus, it is assumed that the dual therapy of CYP2J2 and CYP2C8 inhibitor suppresses the CNV lesions by inhibiting TNF-α through regulation of CYP2 metabolites.

The current study has limitations. First, although the laser-CNV mouse model mimics the CNV aspect of AMD, it does not mimic aging. Nor do mice or any AMD models have a macula. Second, we injected flunarizine i.p., although it is usually administered orally to humans. We need to consider drug delivery in future studies, including intravitreal injection and oral gavage. Finally, further investigation is needed to understand better the molecular mechanisms underlying CYP2J’s role in CNV development. In particular, further research is needed to clarify how CYP2J2 metabolites (likely in addition to EDP and EET) function in pathological ocular angiogenesis.

In conclusion, we report that inhibition of CYP2J2 enhanced dietary ω–3 LCPUFA suppression of pathological choroidal angiogenesis. There has been little published on the role of CYP2J2 in CNV. More interestingly, we found that the combination of CYP2J2 and CYP2C8 inhibition further suppressed CNV formation, possibly through decreasing TNF-α, which is known to promote laser-induced CNV formation [61]. CYP2 inhibition may be a viable approach to treat CNV in AMD. Montelukast and flunarizine have been approved for use in other diseases and are widely used in several countries. Clinical trials or even retrospective studies on AMD patients could further elucidate the role of CYP2J2 and CYP2C8 in pathological ocular angiogenesis.

Supplementary Material

supplementary material

Funding sources

This work was supported by the National Institutes of Health (R24EY024868, R01EY017017, and R01EY030904 to LEHS, R01EY032492 to ZF), BCH IDDRC (1U54HD090255); and the Massachusetts Lions Eye Research Fund to LEHS and ZF, National Natural Science Foundation of China (81800429 to YG), Young and Middle-Aged Medical Backbone Talents of Wuhan (WHQG201902 to YG), Application Foundation Frontiers Project of Wuhan (2020020601012221 to YG) and Medical Science and Technology Innovation Platform Construction Support Project of Zhongnan Hospital of Wuhan University (PTXM2022009 to YG). The Alcon Research Institute, and Bert M. Glaser, MD Award to YT. The funders had no involvement in the study design, collection, analysis, interpretation of data, or report writing.

Abbreviations:

AA

arachidonic acid

AMD

age-related macular degeneration

AREDS

Age-Related Eye Disease Study

ARRIVE

Animal Research: Reporting of In Vivo Experiments

CNV

choroidal neovascularization

COX

cyclooxygenase

CYP

cytochrome P450 oxidase

DHA

docosahexaenoic acids

DHET

dihydroxyeicosatrienoic acids

EDP

epoxydocosapentaenoic acids

EET

epoxyeicosatrienoic acids

EPA

eicosapentaenoic acids

LCPUFA

long-chain polyunsaturated fatty acids

LOX

lipoxygenase

RPE

retinal pigment epithelium

sEH

soluble epoxide hydrolase

SEM

standard error of the mean

TNF

tumor necrosis factor

VEGF

vascular endothelial growth factor

WT

wild type

Footnotes

Supplementary data to this article can be found online at https://doi.org/10.1016/j.metabol.2022.155266.

CRediT authorship contribution statement

Yan Gong: Conceptualization, Conducted research, Analyzed data, Writing - Original draft preparation, Funding acquisition

Yohei Tomita: Conceptualization, Conducted research, Analyzed data, Writing - Original draft preparation, Funding acquisition

Matthew L Edin: Conducted research, Writing - Original draft preparation

Anli Ren: Conducted research, Writing - Reviewing and editing

Minji Ko: Conducted research, Writing - Reviewing and editing

Jay Yang: Conducted research, Writing - Reviewing and editing

Edward Bull: Conducted research, Writing - Reviewing and editing

Darryl C Zeldin: Analyzed data, Writing - Reviewing and editing

Ann Hellström: Conceptualization, Writing - Reviewing and editing

Zhongjie Fu: Conceptualization, Analyzed data, Writing - Reviewing and editing, Funding acquisition

Lois EH Smith: Conceptualization, Writing - Reviewing and editing, Supervision, Funding acquisition.

Declaration of competing interest

All authors have no conflicts of interest.

Data availability

Data described in the manuscript will be available upon request pending application and approval.

References

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Supplementary Materials

supplementary material
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