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.
Highlights
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
The authentic standards of epoxyeicosatetraenoic acid-ethanolamide (EEQ-EA) and epoxydocosapentaenoic acid-ethanolamide (EDP-EA) regioisomers were prepared in a two-step synthesis process and were purified using HPLC as reported in Experimental Procedures
The values of 17,18-EEQ-EA were relatively consistent among tissues from brain, spleen, heart, liver, and kidney, with levels varying from 60 to 90 pmol/g; the values of 19,20-EDP-EA were more variable, with levels in brain, spleen, heart, liver, and kidney measured at 70–400 pmol/g (Fig. 2C and SI Appendix, Table ST1)
Summary
CYP2J2 (Fig. 1) [21]. It was further demonstrated that one of the EET-EA regioisomers selectively binds CB2 with 1,000-fold greater affinity than CB1 [22]. Anti-inflammatory effects are common to both motifs, and, given the presence of these metabolites in the brain, we explored their potential role in a model of neuroinflammation using microglial cells Because these epoxides are widely distributed in the periphery and because ω-3 FA epoxides are known to be vasoactive, we studied the “epoxide-like” property of these molecules by measuring vasodilation in bovine coronary artery, angiogenesis in human microvascular endothelial cells (HMVECs), and platelet aggregation in washed human platelets. Taking these findings together, we identify a class of bioactive lipids that we termed “ω-3 endocannabinoid epoxides,” EDP-EA and EEQ-EA. These metabolites provide targets for the development of therapeutics in the ongoing search for nonaddictive, analgesic, anti-inflammatory, and vasodilatory molecules
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