Reduced coronary reactive hyperemia in mice was reversed by the soluble epoxide hydrolase inhibitor (t-AUCB): Role of adenosine A2A receptor and plasma oxylipins

Abstract

Coronary reactive hyperemia (CRH) protects the heart against ischemia. Adenosine A_2AAR-deficient (A_2AAR^-/-) mice have increased expression of soluble epoxide hydrolase (sEH); the enzyme responsible for breaking down the cardioprotective epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids (DHETs). sEH-inhibition enhances CRH, increases EETs, and modulates oxylipin profiles. We investigated the changes of oxylipins and their impact on CRH in A_2AAR^-/- and wild type (WT) mice. We hypothesized that the attenuated CRH in A_2AAR^-/- mice is mediated by changes in oxylipin profiles, and that it can be reversed by either sEH- or ω-hydroxylases-inhibition. Compared to WT mice, A_2AAR^-/- mice had attenuated CRH and changed oxylipin profiles, which were consistent between plasma and heart perfusate samples, including decreased EET/DHET ratios, and increased hydroxyeicosatetraenoic acids (HETEs). Plasma oxylipns in A_2AAR^-/- mice indicated an increased proinflammatory state including increased ω-terminal HETEs, decreased epoxyoctadecaenoic/dihydroxyoctadecaenoic acids (EpOMEs/DiHOMEs) ratios, increased 9-hydroxyoctadecadienoic acid, and increased prostanoids. Inhibition of either sEH or ω-hydroxylases reversed the reduced CRH in A_2AAR^-/- mice. In WT and sEH^-/- mice, blocking A_2AAR decreased CRH. These data demonstrate that A_2AAR-deletion was associated with changes in oxylipin profiles, which may contribute to the attenuated CRH. Also, inhibition of sEH and ω-hydroxylases reversed the reduction in CRH.

Keywords: Adenosine A(2A) receptor; Coronary reactive hyperemia; Heart perfusate oxylipins; Plasma oxylipins; Soluble epoxide hydrolase; ω-hydroxylases.

Fig 1. Effect of the selective A_2AAR–antagonist (SCH-58261, 0.1 μM) on coronary reactive hyperemia (CRH) in WT and sEH^−/− mice

The selective A_2AAR–antagonist, SCH-58261, decreased CRH in both WT and sEH^−/− mice. Repayment volume (A), repayment/debt ratio (B), and repayment duration (C) were more increased in sEH^−/− compared to WT mice. They, and baseline CF (D), were decreased by SCH-58261 in both WT and sEH^−/− mice. No significant difference between SCH-58261–treated WT and SCH-58261–treated sEH^−/− mice in the above–mentioned parameters was observed. Baseline CF (D), LVPD (E), and HR (F) were not different between and within the two groups. * P < 0.05 versus untreated WT. # P < 0.05 versus SCH-58261–treated WT. n = 8 per group.

Fig 2. Comparison of coronary reactive hyperemia (CRH) between WT and A_2AAR^−/− mice

Repayment volume (A), repayment/debt ratio (B), and repayment duration (C), were decreased in A_2AAR^−/− compared to WT mice (P < 0.05). Baseline CF (D), LVPD (E), and HR (F) were not different between the two groups. * P < 0.05 versus A_2AAR^−/−. n = 8 per group.

Fig 3. Effect of the selective sEH-inhibitor (t-AUCB, 10 μM) on coronary reactive hyperemia (CRH) in A_2AAR^−/− mice

Repayment volume (A), repayment/debt ratio (B), and repayment duration (C), were enhanced in A_2AAR^−/− mice by t-AUCB (P < 0.05). Baseline CF (D), LVPD (E), and HR (F) were not different between the two groups. * P < 0.05 versus A_2AAR^−/−. n = 10 per group.

Fig 4. Effect of the ω-hydroxylases-inhibitor (DDMS, 1 μM) on coronary reactive hyperemia (CRH) in A_2AAR^−/− mice

Repayment volume (A), repayment/debt ratio (B), and repayment duration (C), were enhanced in A_2AAR^−/− mice by DDMS (P < 0.05). Baseline CF (D), LVPD (E), and HR (F) were not different between the two groups. * P < 0.05 versus A_2AAR^−/−. n = 6 per group.

Fig 5. LC–MS/MS analysis for DHETs (11, 12–, and 14, 15–DHETs) levels in WT and A_2AAR^−/− mouse heart perfusate at baseline and post-ischemia

Both 11,12-, and 14,15-DHETs were increased in A_2AAR^−/− vs. WT mice at baseline and post-ischemia. Only 11,12-DHET's increase (A) was statistically significant. * P < 0.05 versus WT. n = 6 per group.

Fig 6. LC–MS/MS analysis for EETs (8, 9–, 11, 12–, and 14, 15–) and DHETs (8, 9–, 11, 12–, and 14, 15–) levels in WT and A_2AAR^−/− mouse plasma

Plasma 8,9-EET (A), 11,12-EET (B), and 14,15-EET (C) were not different, whereas, 8,9-DHET (D), 11,12- DHET (E), and 14,15- DHET (F) were increased in A_2AAR ^−/− compared to WT mice. As a result, 8,9- (G), 11,12- (H), and 14,15- (I) EET/DHET ratios decreased in A_2AAR^−/− compared to WT mice. * P < 0.05 versus WT. n = 10 per group.

Fig 7. LC–MS/MS analysis for mid-chain HETE (5-, 11-, 12-, and 15-HETE) levels in WT and A_2AAR^−/− mouse heart perfusate at baseline and post-ischemia

Baseline and post-ischemic levels of 5- (A), 11- (B), 12- (C), and 15- (D) HETE were increased in A_2AAR^−/− compared to WT mice, and was significant for 11-, 12-, and 15-HETEs. * P < 0.05 versus WT. n = 6 per group.

Fig 8. LC–MS/MS analysis for mid-chain HETE (5-, 11-, 12-, and 15-HETE) levels in WT and A_2AAR−/− mouse plasma

Plasma 5- (A), 11- (B), and 15- (D) HETEs, but not 12- (C) HETE, were significantly increased in A_2AAR^−/− compared to WT mice. * P < 0.05 versus WT. n = 9 per group.

Fig 9. LC–MS/MS analysis for ω–Terminal HETEs (19- and 20-HETE) levels in WT and A_2AAR−/− mouse plasma

Plasma 19-HETE (A) and 20-HETE (B) were increased in A_2AAR^−/− compared to WT mice, but only 20-HETE was statistically significant. * P < 0.05 versus WT. n = 9 per group.

Fig 10. LC-MS/MS analysis for prostanoids (6-keto-PG-F_1α, PG-F_2α, PG-D₂, PG-E₂, and T×B₂) levels in WT and A_2AAR^−/− mouse plasma

Plasma 6-keto-PG-F_1α (A), PG-F_2α (B), PG-E₂ (C), PG-D₂ (D), and T×B₂ (E) were increased in A_2AAR^−/− compared to WT mice, but were significant for PG-F_2α (A), PG-E₂ (C), and T×B₂ (E). * P < 0.05 versus WT. n = 9 per group.

Fig 11. LC–MS/MS analysis for EpOME and DiHOME levels in WT and A_2AAR−/− mouse plasma

Compared to WT, A_2AAR^−/− mice had similar levels of plasma 9,10- (A) and 12,13-EpOMEs (D), increased 9,10- (B) and 12,13-DiHOMEs (E), and as a result, decreased 9,10- (C) and 12,13- EpOME/DiHOME ratios (F). * P < 0.05 versus WT. n = 9 per group.

Fig 12. LC–MS/MS analysis for HODE levels in WT and A_2AAR^−/− mouse plasma

Plasma 9-HODE (A), but not 13-HODE (B), was increased in A_2AAR^−/− compared to WT. * P < 0.05 versus WT. n = 9 per group.

Fig 13. A schematic diagram comparing the changes in heart perfusate and plasma oxylipin profiles observed in response to A_2AAR-deletion as well as their possible effect on coronary reactive hyperemia (CRH) in WT and A_2AAR^−/− mice

The observed changes in the measured heart perfusate and plasma oxylipin profiles collectively resulted in attenuated CRH in A_2AAR^−/− compared to WT mice. These changes included increased plasma 20-HETE, increased heart perfusate and plasma mid-chain HETEs, decreased heart perfusate and plasma EET/DHET ratio, increased plasma 9-HODE, and decreased plasma EpOME/DiHOME ratio. It is not clear what impact the increased prostanoid levels has on CRH, but it may, along with the other changes in oxylipins, contribute to the increased proinflammatory state in A_2AAR^−/− mice.