Anti-inflammatory ω-3 endocannabinoid epoxides

Abstract

Clinical studies suggest that diets rich in ω-3 polyunsaturated fatty acids (PUFAs) provide beneficial anti-inflammatory effects, in part through their conversion to bioactive metabolites. Here we report on the endogenous production of a previously unknown class of ω-3 PUFA-derived lipid metabolites that originate from the crosstalk between endocannabinoid and cytochrome P450 (CYP) epoxygenase metabolic pathways. The ω-3 endocannabinoid epoxides are derived from docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) to form epoxyeicosatetraenoic acid-ethanolamide (EEQ-EA) and epoxydocosapentaenoic acid-ethanolamide (EDP-EA), respectively. Both EEQ-EAs and EDP-EAs are endogenously present in rat brain and peripheral organs as determined via targeted lipidomics methods. These metabolites were directly produced by direct epoxygenation of the ω-3 endocannabinoids, docosahexanoyl ethanolamide (DHEA) and eicosapentaenoyl ethanolamide (EPEA) by activated BV-2 microglial cells, and by human CYP2J2. Neuroinflammation studies revealed that the terminal epoxides 17,18-EEQ-EA and 19,20-EDP-EA dose-dependently abated proinflammatory IL-6 cytokines while increasing anti-inflammatory IL-10 cytokines, in part through cannabinoid receptor-2 activation. Furthermore the ω-3 endocannabinoid epoxides 17,18-EEQ-EA and 19,20-EDP-EA exerted antiangiogenic effects in human microvascular endothelial cells (HMVEC) and vasodilatory actions on bovine coronary arteries and reciprocally regulated platelet aggregation in washed human platelets. Taken together, the ω-3 endocannabinoid epoxides' physiological effects are mediated through both endocannabinoid and epoxyeicosanoid signaling pathways. In summary, the ω-3 endocannabinoid epoxides are found at concentrations comparable to those of other endocannabinoids and are expected to play critical roles during inflammation in vivo; thus their identification may aid in the development of therapeutics for neuroinflammatory and cerebrovascular diseases.

Keywords: cytochrome P450; endocannabinoid; epoxyeicosatrienoic acids; epoxygenase; neuroinflammation.

Fig. 1.

Overview of the CYP epoxygenase-mediated metabolism of endocannabinoids. Both ω-6 and ω-3 dietary fatty acids are stored in plasma membrane and can be converted to the ω-6 and ω-3 endocannabinoids AEA, DHEA, and EPEA. AEA, EPEA, and DHEA are substrates for CYP epoxygenases. The metabolism of AEA produces EET-EA metabolites (the 14,15-EET-EA regioisomer is shown). The metabolism of EPEA and DHEA by CYP epoxygenases leads to the formation of EEQ-EA and EDP-EA, respectively. The terminal endocannabinoid epoxide regioisomer is shown, and other possible epoxides at each double bond are denoted by the numbering system.

Fig. 2.

Endogenous levels of EEQ-EA and EDP-EA regioisomers in rat tissues and their production in BV-2 microglial cells. (A) Authentic standards were used for the development of a LC-MS/MS method in the separation and quantitation of EEQ-EA, EDP-EA, AEA, EPEA, and DHEA lipid mediators using MS/MS fragments and retention times unique to each lipid class. (B and C) Lipid metabolites were extracted and analyzed from pooled Sprague–Dawley rat brain (n = 3) (B) and peripheral organs (C). (D and E) The capacity of LPS-activated BV-2 microglia cells to convert EPEA directly to EEQ-EA regioisomers (D) and DHEA into EDP-EA regioisomers (E) was examined in the absence and presence of the CYP inhibitor ketoconazole (0.5 µM).

Fig. 3.

Direct enzymatic production of EEQ-EA and EDP-EA regioisomers and hydrolysis by soluble epoxide hydrolase. Brain microsomes were incubated with substrate in the presence of CPR and NADPH to measure the capacity of endoplasmic reticulum epoxygenases to directly epoxygenate either (A) EPEA (40 μM), (B) DHEA (40 μM), or (C) EPEA (40 μM) + DHEA (40 μM). (D) CYP2J2-CPR was incorporated into nanodiscs. The membrane scaffold protein (cyan) surrounds a lipid bilayer (aquamarine with gold phospholipid head groups) in which both CYP2J2 (dark blue) and CPR (gray) are incorporated. The regioselectivity and kinetics of EPEA and DHEA metabolism by human CYP2J2-CPR nanodiscs was performed leading to the epoxidation of EPEA to EEQ-EA or DHEA to EDP-EA. The production of the total product and each specific EEQ-EA and EDP-EA regioisomers were fit to Michaelis–Menten kinetics with the calculated K_m, V_max and V_max/K_m values listed in the adjacent table. (E) Schematic of soluble epoxide hydrolase (sEH) hydrolyzing 17,18-EEQ-EA and 19,20-EDP-EA. (F–G) Incubations containing sEH ([E] _final = 6 nM) in sodium phosphate (100 mM, pH 7.4), bovine serum albumin (0.1 mg/ml) and (F) 17,18-EEQ-EA or (G) 19,20-EDP-EA were performed at 37 °C to measure conversion of epoxides to the corresponding vicinal diols using LC-MS/MS. Enzyme kinetics were estimated using incremental increases of substrate for the generation of kinetic curves that were fitted to the Michaelis–Menten equation for calculation of V_max and K_m. Incubations were performed in triplicate or greater and kinetic parameters were calculated using Origin Pro.

Fig. 4.

Effects of 17,18-EEQ-EA and 19,20-EDP-EA on LPS-stimulated BV-2 microglial cells and signaling properties. (A and B) In dose-response studies, BV-2 microglial cells were pretreated with 17,18-EEQ-EA (A) or 19,20-EDP-EA (B) for 4 h followed by LPS (25 ng/mL) stimulation. The culture medium was collected after 24 h and was analyzed for the proinflammatory cytokines IL-6 and NO and the anti-inflammatory cytokine IL-10. LDH production was measured to assess cell toxicity in LPS in stimulated BV-2 microglia (n = 6). (C and D) The potential targets of 17,18-EEQ-EA (C) and 19,20-EDP-EA (D) were studied using AM630 (a CB2-specific inhibitor) and GW9662 (a PPARγ-specific inhibitor) to gauge the reversal of the anti-inflammatory effects by monitoring nitrite production (n = 6). (E–G) Dose–response curves were generated by monitoring the relative luminescence of cannabinoid receptor 1 (CNR1) and cannabinoid receptor 2 (CNR2) PRESTO-Tango gene-transfected HTLA cells as described in SI Appendix, SI Materials and Methods for 17,18-EEQ-EA, EPEA, and CP 55940 (E), 19,20-EDP-EA, DHEA, and CP 55940 (F), and AEA, 2-AG, and CP 55940 (G). Values shown are the mean ± SEM of experiments performed multiple times (n = 3–7). *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 5.

Biological effects of 17,18-EEQ-EA and 19,20-EDP-EA on platelet aggregation, vasculature tension, and angiogenesis. The physiological functions of 17,18-EEQ-EA and 19.20-EDP-EA regioisomers were examined to characterize their effects on platelet aggregation, vasodilation, and angiogenesis. (A and B) In whole human blood, 17,18-EEQ-EA dose-dependently inhibited AA-induced platelet aggregation (n = 4) (A), whereas 19,20-EDP-EA induced platelet aggregation (B) under stirring conditions in the absence of a platelet agonist (n = 7). (C and D) Dose-dependent relaxation of bovine coronary arteries preconstricted with U-46619 (40 nM) was measured for 17,18-EEQ-EA and 17,18-EEQ (C) and for 19,20-EDP and 19,20-EDP-EA (D) to calculate the ED₅₀. (E) 17,18-EEQ-EA and 19,20-EDP-EA were assessed for their ability to inhibit VEGF-promoted angiogenesis in HMVECs plated on Matrigel (n =7). Compounds were studied in parallel with each of their epoxide and ethanolamide parent compounds. All values are means ± SE; *P < 0.05, **P < 0.01, and ***P < 0.001.