Effect of Soluble Epoxide Hydrolase on the Modulation of Coronary Reactive Hyperemia: Role of Oxylipins and PPARγ

Abstract

Coronary reactive hyperemia (CRH) is a physiological response to ischemic insult that prevents the potential harm associated with an interruption of blood supply. The relationship between the pharmacologic inhibition of soluble epoxide hydrolase (sEH) and CRH response to a brief ischemia is not known. sEH is involved in the main catabolic pathway of epoxyeicosatrienoic acids (EETs), which are converted into dihydroxyeicosatrienoic acids (DHETs). EETs protect against ischemia/reperfusion injury and have numerous beneficial physiological effects. We hypothesized that inhibition of sEH by t-AUCB enhances CRH in isolated mouse hearts through changing the oxylipin profiles, including an increase in EETs/DHETs ratio. Compared to controls, t-AUCB-treated mice had increased CRH, including repayment volume (RV), repayment duration, and repayment/debt ratio (p < 0.05). Treatment with t-AUCB significantly changed oxylipin profiles, including an increase in EET/DHET ratio, increase in EpOME/DiHOME ratio, increase in the levels of HODEs, decrease in the levels of mid-chain HETEs, and decrease in prostanoids (p < 0.05). Treatment with MS-PPOH (CYP epoxygenase inhibitor) reduced CRH, including RV (p < 0.05). Involvement of PPARγ in the modulation of CRH was demonstrated using a PPARγ-antagonist (T0070907) and a PPARγ-agonist (rosiglitazone). T0070907 reduced CRH (p < 0.05), whereas rosiglitazone enhanced CRH (p < 0.05) in isolated mouse hearts compared to the non-treated. These data demonstrate that sEH inhibition enhances, whereas CYP epoxygenases-inhibition attenuates CRH, PPARγ mediate CRH downstream of the CYP epoxygenases-EET pathway, and the changes in oxylipin profiles associated with sEH-inhibition collectively contributed to the enhanced CRH.

Fig 1. Comparison of coronary reactive hyperemia (CRH) before (WT) and after (t-AUCB-treated WT) infusion of t-AUCB.

(A) tracing depicting coronary flow (CF) changes at baseline (before CRH) and after CRH was induced by 15-second no-flow ischemia in WT (continuous line) and WT + t-AUCB (dashed line). Repayment volume (B), repayment duration (C), repayment/debt ratio (D), and peak hyperemic flow (E) increased in t-AUCB-treated WT versus WT mice (p < 0.05). * p < 0.05 versus WT. n = 8.

Fig 2. Effect of sEH-inhibitor, t-AUCB, on coronary reactive hyperemia (CRH) in sEH-null (sEH^–/–) mice.

t-AUCB did not (p > 0.05) affect CRH in sEH^–/–mice, as evident in the unchanged repayment volume (A), repayment/debt ratio (B), and repayment duration (C) (p = 0.690). n = 6.

Fig 3. LC–MS/MS analysis for 14, 15–EET, 14, 15–DHET, and 11,12–DHET levels in WT and t-AUCB-treated WT mouse heart perfusates at baseline (pre-ischemia) and directly after 15-second ischemia (post-ischemia).

(A) At baseline and post-ischemia levels of 14, 15–EET had an increasing trend in t-AUCB-treated WT versus WT mice, but this trend was not significant. (B) 14, 15–DHET levels decreased at baseline and post-ischemia in t-AUCB-treated WT versus WT (p < 0.0001). (C) The 14, 15–EET/14, 15–DHET ratio increased (p < 0.05) in t-AUCB-treated WT versus WT mice at baseline (by 96%) and post-ischemia (by 173%). (D) 11, 12–DHET levels decreased (p < 0.001) at baseline and post-ischemia in t-AUCB-treated-WT versus WT mice. There was no difference in 14,15-EET, 14,15-DHET, and 11,12-DHET levels pre- and post-ischemia within each group. * p < 0.05 versus baseline WT. # p < 0.05 versus post-ischemia WT. n = 8.

Fig 4. LC–MS/MS analysis of 5-, 11-, 12- and 15-HETE levels in WT and t-AUCB-treated WT mouse heart perfusates at baseline (pre-ischemia) and post-ischemia.

In WT mice, 5-, 11-, 12- and 15-HETE levels decreased post-ischemia compared to baseline, but this was only significant for 5-HETE (A), 11-HETE (B), and 15-HETE (D) (p < 0.05). The same mid-chain HETEs had a decreasing trend post-ischemia compared to baseline in t-AUCB-treated WT mice, but this was not significant (p > 0.05, A-D). Treatment with t-AUCB decreased HETE levels in WT mice, which was significant for 5-HETE (A), 11-HETE (B) and 15-HETE (D) levels at baseline (p < 0.05), and in 11-HETE levels post-ischemia (p < 0.05, B). * p < 0.05 versus baseline WT. # p < 0.05 versus post-ischemia WT. n = 8.

Fig 5. LC–MS/MS analysis of EpOME and DiHOME levels and the EpOME/DiHOME ratio in WT and t-AUCB-treated-WT mouse heart perfusates at baseline (pre-ischemia) and post-ischemia.

(A) 9,10- and 12,13-EpOME levels had an increasing trend at baseline and post-ischemia in t-AUCB-treated-WT versus WT mice, but this was not significant (p > 0.05). Neither EpOME was significantly changed post-ischemia compared to baseline in both groups. (B) 9,10- and 12,13-DiHOME levels decreased at baseline and post-ischemia in t-AUCB-treated-WT versus WT mice (p < 0.001). (C) The EpOME/DiHOME ratio increased in t-AUCB-treated versus WT mice at baseline and post-ischemia (p < 0.0001). The measured EpOME and DiHOME levels and EpOME/DiHOME ratio did not change post ischemia versus baseline within the same group (p > 0.05, A-C). * p < 0.05 versus baseline WT. # p < 0.05 versus post-ischemia WT. n = 8.

Fig 6. LC–MS/MS analysis of HODEs in WT and t-AUCB-treated WT mouse heart perfusates at baseline (pre-ischemia) and post-ischemia.

9- and 13-HODE increased in t-AUCB-treated WT versus WT mice at baseline and post-ischemia (p < 0.05). Neither HODE changed post-ischemia versus baseline within the same group (p > 0.05). * p < 0.05 versus baseline WT. # p < 0.05 versus post-ischemia WT. n = 8.

Fig 7. LC–MS/MS analysis of 6-keto-PG-F_1α, PG-F_2α, thromboxane B₂, PG-D₂, and PG-E₂ in WT and t-AUCB-treated WT mouse heart perfusates at baseline (pre-ischemia) and post-ischemia.

Infusion of t-AUCB decreased 6-keto-PG-F_1α (A), PG-F_2α, (B), thromboxane B₂ (C), PG-D₂ (D), and PG-E₂ (E), at baseline and post-ischemia (p < 0.05). Compared to baseline WT, post-ischemia WT levels were decreased for thromboxane B₂, and PG-D₂, but were significant (p < 0.05) only for thromboxane B₂ (C). * p < 0.05 versus baseline WT. # p < 0.05 versus post-ischemia WT. n = 8.

Fig 8. Effect of CYP-epoxygenase inhibitor (MS-PPOH, 1 μM) on coronary reactive hyperemia (CRH) in wild type (WT) mice.

Each WT isolated mouse heart was used as its own control. (A) tracing depicting coronary flow changes at baseline (before CRH) and after CRH was induced by 15-second no-flow ischemia in WT (continuous line) and MS-PPOH-treated WT (dashed line) mice. Repayment volume (B), baseline CF (E), PHF (F), and LVDP (G) decreased (p < 0.05) in MS-PPOH-treated WT versus WT mice. Repayment/debt ratio (C) increased (p < 0.05), whereas repayment duration (D) did not change (p > 0.05) after in MS-PPOH-treated WT versus WT mice. * p < 0.05 versus WT. n = 8.

Fig 9. Effect of T0070907, PPARγ-antagonist (10 μM) on coronary reactive hyperemia (CRH) in wild type (WT) mice.

Each WT isolated mouse heart was used as its own control. Infusion of T0070907 into WT mice hearts attenuated CRH. (A) Repayment volume decreased (p < 0.05), as did RD (B) (p < 0.05) and baseline CF (D) (p < 0.05). Repayment/debt ratio (C) had a decreasing trend, but was not significant (p > 0.05). * p < 0.05 versus WT. n = 8.

Fig 10. Effect of T0070907, PPARγ-antagonist (10 μM) on t-AUCB-enhanced CRH in WT in wild type (WT) mice.

Each WT isolated mouse heart was used as its own control. Inhibition of sEH by t-AUCB enhanced CRH in WT mice. Infusion of t-AUCB (10 μM) increased repayment volume (RV) (p < 0.05, A), repayment/debt ratio (p < 0.05, B), and repayment duration (RD) in WT mice (p < 0.05, C). The t-AUCB-enhanced CRH was attenuated by T0070907. T0070907 decreased RV (A), R/D ratio (B), and RD (C) in t-AUCB-treated-WT mice (p < 0.05). * p < 0.05 versus WT. n = 6.

Fig 11. Comparison of CRH in WT before and after infusion of rosiglitazone (PPARγ-agonist, 10 μM).

Each isolated heart was used as its own control. Repayment volume (A), repayment duration (B), repayment/debt ratio (C), and baseline CF (D) increased after rosiglitazone administration (p < 0.05). * p < 0.05 versus WT. n = 8.

Fig 12. A schematic diagram comparing the oxylipin changes observed in response to a brief ischemia and their possible impact on coronary reactive hyperemia (CRH) between WT and t-AUCB-treated WT mice.

Treatment with t-AUCB enhanced CRH possibly through increased EET/DHET ratio, increased 13-HODE, increased EpOME/DiHOME ratio, decreased mid-chain HETEs, and PPARγ activation.