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Arachidonic acid, a polyunsaturated fatty acid, can be metabolized to cardioprotective epoxyeicosatrienoic acids (EETs) by cytochrome P450 epoxygenases, which are subsequently hydrolyzed to less bioactive dihydroxyeicosatrienoic acids by soluble epoxide hydrolase (sEH). To study the effects of pharmacological inhibitor of sEH (sEHi), C57BL6 mice hearts were perfused in Langendorff mode for 40min of baseline and subjected to 30min of global no-flow ischemia followed by 40 min of reperfusion. Hearts were perfused with the sEHi, trans-4-[4-(3-adamantan-1-y1-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB; 0.05 µM, 0.1 µM, 0.5 µM and 1 µM). To study the mechanism(s), hearts were perfused with 0.1 µM t-AUCB in the presence or absence of putative EET receptor antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE, 10 µM) or phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin (200 nM) or LY294002 (5 µM). Infarct size was determined at the end of 2 h reperfusion by TTC staining. Inhibition of sEH by t-AUCB significantly improved postischemic left ventricular developed pressure (LVDP) recovery and reduced the infarct size following ischemia-reperfusion, as compared to control hearts. Perfusion with 14,15-EEZE, wortmannin or LY294002 prior to ischemia abolished the cardioprotective phenotype; however, co-perfusion of both t-AUCB and 11,12-EET did not result in an additive effect on improved LVDP recovery. Together, our data suggest that pharmacological inhibition of sEH by t-AUCB is cardioprotective.
Arachidonic acid, is a polyunsaturated fatty acid that is present in the phospholipids of cell membranes which can be released into cytosol in response to stressors such as ischemia.1,2 Released free AA can then be metabolized by cyclooxygenases, lipoxygenases, and cytochrome P450 (CYP) monooxygenases.1,3 Biologically active regioisomers, epoxyeicosatrienoic acids (5,6-, 8,9-, 11,12-, and 14,15-EET) are products by CYP monooxygenases.2,3 EETs can be reincorporated into phospholipid membranes or metabolized to smaller reactive epoxides by β-oxidation.4,5 However, the predominant pathway of EET metabolism is conversion to the less active vicinal diols, dihydroxyeicosatrienoic acids (5,6-, 8,9-, 11,12-, and 14,15-DHET), by soluble epoxide hydrolases (sEH).4,5
EETs act as important cellular lipid mediators in the cardiovascular, renal and nervous systems.3,6–8 EETs have been shown to have protective effects against ischemia reperfusion injury.1,9–14 Our current understanding of the cardioprotective mechanism(s) of EETs suggest involvement of signaling pathways including phosphoinositide 3-kinase (PI3K) – Akt, increased secretion of cardiac hormones, and activation of cardiac ion channels such as ATP-sensitive K+ channels.1,9,10,12,13 Recent evidence indicates that PI3K/Akt or natriuretic peptide pathways activated by EETs converge onto the mitochondria thereby limiting mitochondrial damage from ischemia reperfusion injury.10,12
Previously we demonstrated that targeted deletion of sEH gene is protective against ischemia reperfusion injury.1,10 Recently, Motoki et al. has reported pharmacological inhibition of sEH with 12-(3-adamantan-1-yl-ureido)-dodecanoic acid n-butyl ester (AUDA-nBE) was cardioprotective.15 In the present study we report cardioprotective effects of more potent, water soluble and metabolically stable sEH inhibitor (sEHi), trans-4-[4-(3-adamantan-1-y1-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB) than AUDA-nBE. Moreover, our data demonstrate marked reduction in infarction and improved contractile function at nanomolar concentrations which involved the PI3K pathway.
All experiments used male and female mice aged 3–5 months, weighing 22–33 g and were treated in accordance with the guidelines of Health Science Laboratory Animal Services (HSLAS), University of Alberta. C57BL6 mice were purchased from Charles River Laboratories (Pointe Claire, PQ). A colony of mice with targeted disruption of the Ephx2 gene (sEH null) and backcrossed onto a C57BL6 genetic background for more than 7 additional generations, is maintained at the University of Alberta.
EETs were a kind gift from Dr. J. R. Falck (University of Texas Southwestern Medical Center, Dallas, TX). Stock solutions (5 mM) were dissolved in 100% ethanol and working solutions were diluted in perfusion buffer (1 µM). The sEHi, trans-4-[4-(3-adamantan-1-y1-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB), was synthesized in the laboratory of Dr. Bruce Hammock (UC Davis) and dissolved in DMSO to make 10 mM stock solution. The putative EET receptor antagonist, 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) was a kind gift from Dr. J. R. Falck and dissolved in 100% ethanol to make 5 mM stock solution. PI3K inhibitors, Wortmannin (Sigma-Aldrich, Oakville, ON) and LY294002 (Cell Signaling Technology, Inc., Danvers, MA), were dissolved in DMSO to make 5 mM and 50 mM stock solutions, respectively.
Hearts were perfused in the Langendorff mode as previously published.1,10 Briefly, hearts from age/sex-matched mice were perfused in a retrograde fashion at constant pressure (90 cmH2O) with continuously aerated (95%O2/5%CO2) Krebs-Henseleit buffer at 37°C. Hearts were perfused with buffer for 40 min of stabilization period and then subjected to 30 min global no-flow ischemia, followed by 40 min reperfusion. To determine t-AUCB dose-response, hearts from C57Bl6 mice were perfused with increasing concentrations of t-AUCB (0, 0.05, 0.1, 0.5 or 1µM). For some experiments, hearts were perfused for 40 min baseline, subjected to 30 min ischemia and then perfused with 0.1 µM t-AUCB in the presence or absence of putative EET receptor antagonist 14,15-EEZE (10 µM), PI3K inhibitors wortmannin (200 nM) or LY294002 (5 µM). The percentage of left ventricular developed pressure (%LVDP) at 40 min of reperfusion (R40), as compared to baseline LVDP, was taken as a marker for recovery of contractile function. After 40 min of reperfusion, hearts were immediately frozen and stored below −20°C.
To determine the amount of infarction, following 40 min of stabilization period and 30 min global no-flow ischemia, hearts were reperfused for 2 h. After 2 h reperfusion, hearts were perfused with 1% solution of 2,3,5-triphenyltetrazolium chloride (TTC) dissolved in Krebs-Henseleit buffer at 37 °C for 10 min, then fixed in formalin and cut into thin cross-sectional slices. The area of infarction was quantified by measuring stained (red, live tissue) and unstained (white, necrotic) regions using Image J (NIH, USA).
Values expressed as mean ± standard error of mean (SEM). Statistical significance was determined by the unpaired Student’s t-test and one-way ANOVA followed by Newman-Keuls and Duncan’s tests to assess differences between groups. Values were considered significant if p < 0.05.
Recent evidence suggests that sEH is a good target in the prevention of ischemia reperfusion injury.1,10,15 To examine the effects of pharmacological inhibition of sEH on ischemia reperfusion injury, we first performed a dose-response study. We perfused the WT mouse hearts with 0, 0.05, 0.1, 0.5 and 1 µM of t-AUCB and monitored LVDP for postischemic functional recovery. No significant differences in baseline contractile function were observed between the groups, except LVDP in 1 µM t-AUCB treated hearts, during aerobic baseline perfusion (Table 1). Hearts perfused with t-AUCB had significantly improved postischemic recovery of LVDP compared to control mice (Fig 1b). The improved postischemic recovery followed a bell-shaped response, with the most improved functional recovery occurring at the mid-dose (0.1 µM). Therefore, 0.1 µM t-AUCB was used for further study the cardioprotective effects.
To further assess the cardioprotective effects of t-AUCB, we analyzed the infarct size following 2 h reperfusion in t-AUCB treated hearts and vehicle control hearts. Significant decrease in infarct size was observed in the hearts treated with 0.1 µM t-AUCB compared to vehicle controls (Fig 1c). Consistent with 40 min reperfusion, postischemic functional recovery remained significantly higher at 2 h reperfusion in t-AUCB treated hearts compared to vehicle controls (LVDP=43.3±13.3% vs. 21.2±1.8%).
To compare the cardioprotective effects of genetic and pharmacological inhibition of sEH, we perfused WT mice with 0.1 µM t-AUCB and sEH null mice with vehicle. Significant increase in postischemic functional recovery of sEH null (LVDP= 47.3±6.2%) and t-AUCB perfused WT (LVDP= 48.0±7.2%) was observed following ischemia reperfusion protocol, compared to vehicle controls (LVDP= 22.4±1.7%) and WT (LVDP= 19.7±3.8) (Fig 2a, Table 2). No significant differences were observed in cardiac parameters during baseline (Table 2). Consistent with LVDP, rate of contraction and relaxation were significantly higher in sEH null hearts and t-AUCB treated hearts following ischemia reperfusion (Table 2). No significant differences were observed in heart rate during baseline or following ischemia reperfusion (Table 2). The improved function was evident within 30 min of reperfusion following treatment with 0.1 µM t-AUCB and sEH null mice, which persisted throughout the recovery period (Fig 2b).
As previously reported, targeted deletion or pharmacological inhibition of sEH leads to increase in intracellular EET levels which can further produce cardioprotective effects following ischemia reperfusion injury.1 To confirm the EET mediated effects of t-AUCB, we perfused the WT hearts with 0.1 µM t-AUCB in presence or absence of putative pan-EET receptor antagonist 14, 15-EEZE (10 µM). 14,15-EEZE did not affect the baseline function of the WT mice hearts (Data not shown). However, postischemic functional recovery of t-AUCB treated hearts (LVDP= 49.9 ± 7.5%) was completely abolished in the hearts co-perfused with 14,15-EEZE (LVDP= 13.3±2.5%) (Fig 3a). To assess the effects of exogenous EET cardioprotective effects of t-AUCB, we perfused the WT hearts with t-AUCB in presence or absence of 11,12-EET (1 µM) and 14,15-EET (1 µM). Interestingly, no additive effects of exogenous EET was observed on t-AUCB treated hearts (Fig 3b). These data suggests that maximum recovery was achieved by treating the hearts with 0.1 µM t-AUCB and therefore addition of exogenous EET does not show any additional effect on functional recovery.
To determine the role of the PI3K signaling cascade in t-AUCB mediated cardioprotective response, we performed isolated perfused heart experiments in the presence or absence of the PI3K inhibitors wortmannin or LY294002. Neither inhibitor had a significant effect on baseline LVDP (data not shown). Perfusion with either wortmannin or LY294002 for 5 min prior to ischemia had no effect on postischemic LVDP recovery in vehicle controls; however, both inhibitors significantly reduced the improved postischemic functional recovery in t-AUCB treated hearts (Fig 4). Thus, percent LVDP recovery at 40 min reperfusion was comparable in both the groups after treatment with either wortmannin or LY294002 (Fig 4). These data suggest the involvement of the PI3K cascade in the cardioprotective effects of pharmacological inhibition of sEH.
In this paper, we report improved postischemic contractile function and reduced infarct size in isolated mice hearts perfused with a novel water-soluble and potent pharmacological sEHi, t-AUCB. The effects of t-AUCB were attenuated by the putative EET-receptor antagonist 14,15-EEZE and inhibition of PI3K. Taken together, our data suggest that pharmacological inhibition of sEH by t-AUCB can be protective against ischemia reperfusion injury and the effects are mediated through EETs and PI3K pathway.
sEH is a bifunctional enzyme with C-terminus hydrolase activity and N-terminus phosphatase activity.5,16 While the functional effects of phosphatase are not well known, much evidence implicates the importance of its hydrolase activity in cardiovascular diseases.1,5,11,15,17,18 Two different approaches have been used to study the effects of sEH on cardiovascular system: genetic modification and pharmacological inhibition.1,11,15 Genetic modulation by targeted deletion of Ephx2 gene results in lack of sEH expression and reported to produce anti-arrhythmic, antihypertensive and cardioprotective effects.1,19 On the other hand, sEHi causes inhibition of hydrolase activity exerting similar protective effects in the cardiovascular system.11,15,20,21 Consistent with the previous observations, the data in this manuscript indicate that both genetic and chemical knockout of sEH lead to a dramatic improvement on the cardiac parameters measure. However, neither approach resulted in full return to normal function indicating that additional approaches could be helpful in restoring cardiac function. The fact that both the chemical and genetic knockout experiments resulted in similar improvements in function indicates that a near maximum effect for a sEHi was reached with t-AUCB in this system.
Small molecule inhibitors are valuable tools to study the role of sEH in the pathophysiology of cardiovascular diseases. The first generation of sEHi were epoxide-containing compounds that turned out to be substrates of EH metabolism.16 These compounds tended to only have a transient inhibitory effect in vitro and were not effective in vivo.16,22 EH inhibitory properties were shown with urea, amide and carbamate functionalities as the central pharmacophore. The ureas were in general the most active with similar R groups, but amide and carbamate derivatives with optimized substituents can be as active as ureas.16 The next generation of sEHi demonstrated that 1,3-Disubstituted urea derivatives were stable and more potent sEHi than previous compounds.16,23 t-AUCB has a lipophilic adamantane on the N of the urea and a bicyclic system with a polar ether and acid on the N' position which dramatically increase water solubility.24–26 Formation of hydrogen bonds and salt bridges between urea functionality and active cites of sEH results in inhibition of hydrolase activity.16 sEH has a high Vmax and low KM for endogenous epoxides of arachidonic and linoleic acid which results in diminished biological activity. Emerging evidence demonstrates that sEHi are potential therapeutic compounds for the treatment of several diseases. cis-isomer of t-AUCB (cic-4-[4-(3-adamantan-1-y1-ureido)-cyclohexyloxy]-benzoic acid; c-AUCB) has recently been demonstrated to attenuate monocrotalin induced pulmonary hypertension in rats.27 Xu et al. has reported importance of sEH inhibition prevented and reversed cardiac hypertrophy using 1-adamantan-3-(5-(2-(2-ethylethoxy)ethoxy)pentyl)urea, AEPU.28 Similarly, 1-(1-methanesulfonyl-piperidin-4-yl)-3-(4-trifluoromethoxy-phenyl)-urea (TUPS) can block angiotensin-II induced cardiac hypertrophy.20 1,3-Disubstituted urea sEHi such as AUDA and AUDA-BE have demonstrated protective effects against ischemia reperfusion injury in the micromolar range.11,15 In the present study, t-AUCB exerted cardioprotective effects by inhibiting sEH activity at nanomolar concentrations. Apart from urea based sEHi, non-urea derivatives of potent sEHi have also been reported; however, no study has demonstrated in vivo effectiveness of these compounds.29,30
Cardioprotective effects towards ischemia reperfusion injury by sEHi compounds have only been demonstrated with AUDA and AUDA-BE.11,15 These sEHi compounds have several drawbacks which include limited oral bioavailability, instability due to rapid metabolism and poor physical properties such as low water solubility.18 With very careful formulation AUDA and its salts and esters can be orally administered for in vivo use, but the new generation of sEHi such as t-AUCB are dramatically easier to administer and have longer half lives.5,11,15,18,31 t-AUCB (IC50 = 1.3 nM) is a more potent inhibitor of sEH than AUDA (IC50 = 3 nM) and AUDA-nBE (IC50 = 7 nM), while it is equipotent to TPAU (IC50 = 1.1 nM) and c-AUCB (IC50 = 0.89 nM).26 Liu et al. compared AUDA-BE to t-AUCB for total epoxide and diols in plasma of LPS treated mice. Both, AUDA-BE and t-AUCB, significantly reduced DHET formation increasing the epoxide to diol ratio. However, t-AUCB was able to increase the EET/DHET ratio to similar levels of AUDA-BE at a 100 times lower dose.24 t-AUCB has better oral bioavailability and metabolic stability as higher area under concentration time curve (AUCt) and longer elimination half life (t1/2) (AUCt = 155 µM/L; t1/2 > 1400 min) compared to AUDA (AUCt = 24 µM/L; t1/2 = 575 min) and AUDA-BE (AUCt = 16 µM/L; t1/2 = 260 min).18,24,26,31 Similarly, pharmacokinetic studies done in canine model revealed that t-AUCB was found in the circulation for 1 day following oral administration.18 The improved physical and pharmacokinetic properties of t-AUCB make it an attractive therapeutic agent and experimental probe for increasing beneficial EETs.
Inhibition of sEH decreases metabolism of EET and thereby exerts protective effects against injury incurred following myocardial ischemia reperfusion and ischemic stroke.1,11,15,17 Increased EETs in the heart activate various cardioprotective pathways leading to improved contractile function and reduced damage to the heart.1,11,13 The cardioprotective effects of EETs and sEHi can be blocked by simultaneous treatment with putative EET receptor antagonist, 14,15-EEZE.1,9–11 Consistent with these observations, our data suggest EETs mediate the protective effects of t-AUCB, as the improved post ischemic functional recovery was abolished by co-perfusion with 14,15-EEZE. It was surprising that addition of EET to this system did not further increase cardiac function. This may be the result of sufficient amounts of EET, other omega-6 or 3 epoxides free or esterified in cardiac tissue, or the synthesis rate of epoxides is high enough that supplementation is not needed. Recent evidence suggests that EETs can activate PI3K/Akt signaling pathway which leads to inhibition of GSK-3β and reduces the damage to mitochondria.1,10 Dhanasekaran et al. reported EET-mediated activation of multiple antiapoptotic targets through PI3K/Akt survival signaling.31 Moreover, inhibition of PI3K pathway by wortmannin or LY294002, abolished the improved postischemic functional recovery of sEH null mice.1 Similarly, our results show significant reduction in postischemic functional recovery of hearts co-perfused with t-AUCB and PI3K inhibitor wortmannin or LY294003, which confirms the involvement of PI3K survival signaling.
In conclusion, we report improved postischemic contractile function and reduced infarct size by treatment with a sEHi, t-AUCB. Moreover, the cardioprotective effect was attributed to EETs and the PI3K survival pathway. The increased potency and water solubility of t-AUCB over other sEHi suggest this compound may serve as a potential therapeutic agent in myocardial ischemia reperfusion injury and potentially a valuable drug for treatment ischemic heart diseases.
FUNDING: JMS is the recipient of a New Investigator Award from the Heart and Stroke Foundation of Canada and a Health Scholar Award from the Alberta Heritage Foundation for Medical Research. This work was supported by Canadian Institutes of Health Research Grant (JMS MOP79465). The work was supported in part by NIEHS 37 ES02710 (BDH) and R01 ES013933 (BDH).
CONFLICT OF INTEREST: BDH founded Arete Therapeutics to move sEH inhibitors to the clinic. SHH and BDH are authors of University of California patents in the area.