Soluble epoxide hydrolase inhibitors reduce the development of atherosclerosis in apolipoprotein e-knockout mouse model

Abstract

To determine whether sEH inhibitors influence atherosclerotic lesion formation, we used an established murine model of accelerated atherogenesis, ApoE knockout (-/-) mice. The sEH inhibitor, 1-adamantan-3-(5-(2-(2-ethylethoxy)ethoxy)pentyl)urea (AEPU) was delivered in drinking water. All animals were fed an atherogenic diet while simultaneously infused with angiotensin II by osmotic minipump to induce atherosclerosis. In AEPU-treated animals, there was a 53% reduction in atherosclerotic lesions in the descending aortae as compared to control aortae. AEPU and its major metabolites were detected in the plasma of animals which received it. As expected from the inhibition of sEH, a significant increase in linoleic and arachidonic acid epoxides, as well as an increase in individual 11,12-EET/DHET and 14,15-EET/DHET ratios, were observed. The reduction in atherosclerotic lesion area was inversely correlated with 11,12- and 14,15- EET/DHET ratios, suggesting that the reduction corresponds to the inhibition of sEH. Our data suggest that orally-available sEH inhibitors may be useful in the treatment of patients with atherosclerotic cardiovascular disease.

Figure 1. AEPU decreases plaque formation in ApoE knockout mice

A. Representative descending aorta in AEPU-treated and control mice visualized in situ prior to staining (1 and 2, control mice; 3 and 4, AEPU-treated mice) B. Representative descending aortae in AEPU-fed and control mice shown en face after staining with Sudan IV (1 and 2, control mice; 3 and 4, AEPU treated mice) shows decreased lesion area. C. Quantitative measurement of data in B from all mice. Control group n=6, AEPU treated group n=4. The data are shown as mean ±SEM (^*:p<0.05).

Figure 2. Mice treated with AEPU show increased plasma levels of P450 epoxygenase metabolites

A. Key epoxygenase metabolites. 14,15-EET and 9,10- EpOME were significantly higher in the AEPU-treated group, whereas 11,12-EET and 12,13-EpOME lost the significance due to the variation in the group. B. Ratios of lipid epoxides to their corresponding product diols showing the statistically significant differences of EpOME/DiHOME and total epoxide/ diol ratios between the control and AEPU treated groups. Levels of metabolites are expressed as percent of corresponding control animals; non-parametric univariate statistics was performed to compare experimental and control animals. The data are shown as mean ±SEM (^*: p<0.05).

Figure 3. P450 epoxygenase metabolites correlate inversely with plaque area

Plasma levels of arachidonic acid (20:4) and linoleic acid (18:2) metabolites are correlated with aortic plaque area for each mouse. The correlation was calculated using Pearson correlation test. The epoxides arising from arachidonic acid (20:4), 11,12- and 14,15-EET/DHET (circles) showing a strong correlation, epoxides arising from linoleic acid (18:2), 9,10- and 12,13- EpOME/DiHOME (squares) showing a moderate correlation and the total epoxide/diol ratio (diamonds) showing a strong correlation with the aortic plaque area are shown.

Figure 4. AEPU Metabolism

AEPU was incubated with a rat S9 liver preparation with a NADPH generating system (there was no metabolism in the absence of NADPH). At time points of 0, 20, 40, and 60 minutes aliquots of the incubation were monitored for AEPU and major metabolites by LC-MS and an in vitro enzyme inhibition assay was run to determine the IC₅₀ value of the material in units based on AEPU using the affinity purified recombinant murine enzyme. Theoretical IC₅₀s are also indicated with *. These values were calculated by multiplying the initial rat IC₅₀ for rat enzyme (IC₅₀ rat: 6 nM) with the fold decrease in AEPU amount.