Hsing Ju Tsai

Prevention and reversal of cardiac hypertrophy by soluble epoxide hydrolase inhibitors

Sustained cardiac hypertrophy represents one of the most common causes leading to cardiac failure. There is emerging evidence to implicate the involvement of NF-κB in the development of cardiac hypertrophy. However, several critical questions remain unanswered. We tested the use of soluble epoxide hydrolase (sEH) inhibitors as a means to enhance the biological activities of epoxyeicosatrienoic acids (EETs) to treat cardiac hypertrophy. sEH catalyzes the conversion of EETs to form the corresponding dihydroxyeicosatrienoic acids. Previous data have suggested that EETs may inhibit the activation of NF-κB-mediated gene transcription. We directly demonstrate the beneficial effects of several potent sEH inhibitors (sEHIs) in cardiac hypertrophy. Specifically, we show that sEHIs can prevent the development of cardiac hypertrophy using a murine model of pressure-induced cardiac hypertrophy. In addition, sEHIs reverse the preestablished cardiac hypertrophy caused by chronic pressure overload. We further demonstrate that these compounds potently block the NF-κB activation in cardiac myocytes. Moreover, by using in vivo electrophysiologic recordings, our study shows a beneficial effect of the compounds in the prevention of cardiac arrhythmias that occur in association with cardiac hypertrophy. We conclude that the use of sEHIs to increase the level of the endogenous lipid epoxides such as EETs may represent a viable and completely unexplored avenue to reduce cardiac hypertrophy by blocking NF-κB activation.

Soluble epoxide hydrolase: a novel therapeutic target in stroke

The P450 eicosanoids epoxyeicosatrienoic acids (EETs) are produced in brain and perform important biological functions, including protection from ischemic injury. The beneficial effect of EETs, however, is limited by their metabolism via soluble epoxide hydrolase (sEH). We tested the hypothesis that sEH inhibition is protective against ischemic brain damage in vivo by a mechanism linked to enhanced cerebral blood flow (CBF). We determined expression and distribution of sEH immunoreactivity (IR) in brain, and examined the effect of sEH inhibitor 12-(3-adamantan-1-yl-ureido)-dodecanoic acid butyl ester (AUDA-BE) on CBF and infarct size after experimental stroke in mice. Mice were administered a single intraperitoneal injection of AUDA-BE (10 mg/kg) or vehicle at 30 mins before 2-h middle cerebral artery occlusion (MCAO) or at reperfusion, in the presence and absence of P450 epoxygenase inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl) hexanamide (MS-PPOH). Immunoreactivity for sEH was detected in vascular and non-vascular brain compartments, with predominant expression in neuronal cell bodies and processes. 12-(3-Adamantan-1-yl-ureido)-dodecanoic acid butyl ester was detected in plasma and brain for up to 24 h after intraperitoneal injection, which was associated with inhibition of sEH activity in brain tissue. Finally, AUDA-BE significantly reduced infarct size at 24 h after MCAO, which was prevented by MS-PPOH. However, regional CBF rates measured by iodoantipyrine (IAP) autoradiography at end ischemia revealed no differences between AUDA-BE- and vehicle-treated mice. The findings suggest that sEH inhibition is protective against ischemic injury by non-vascular mechanisms, and that sEH may serve as a therapeutic target in stroke.

Naturally occurring monoepoxides of eicosapentaenoic acid and docosahexaenoic acid are bioactive antihyperalgesic lipids

Beneficial physiological effects of long-chain n-3 polyunsaturated fatty acids are widely accepted but the mechanism(s) by which these fatty acids act remains unclear. Herein, we report the presence, distribution, and regulation of the levels of n-3 epoxy-fatty acids by soluble epoxide hydrolase (sEH) and a direct antinociceptive role of n-3 epoxy-fatty acids, specifically those originating from docosahexaenoic acid (DHA). The monoepoxides of the C18:1 to C22:6 fatty acids in both the n-6 and n-3 series were prepared and the individual regioisomers purified. The kinetic constants of the hydrolysis of the pure regioisomers by sEH were measured. Surprisingly, the best substrates are the mid-chain DHA epoxides. We also demonstrate that the DHA epoxides are present in considerable amounts in the rat central nervous system. Furthermore, using an animal model of pain associated with inflammation, we show that DHA epoxides, but neither the parent fatty acid nor the corresponding diols, selectively modulate nociceptive pathophysiology. Our findings support an important function of epoxy-fatty acids in the n-3 series in modulating nociceptive signaling. Consequently, the DHA and eicosapentaenoic acid epoxides may be responsible for some of the beneficial effects associated with dietary n-3 fatty acid intake.

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

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.

Soluble epoxide hydrolase deficiency alters pancreatic islet size and improves glucose homeostasis in a model of insulin resistance

Visceral obesity has been defined as an important element of the metabolic syndrome and contributes to the development of insulin resistance and cardiovascular disease. Increasing endogenous levels of epoxyeicosatrienoic acids (EETs) are known for their analgesic, antihypertensive, and antiinflammatory effects. The availability of EETs is limited primarily by the soluble epoxide hydrolase (sEH, EPHX2), which metabolizes EETs to their less active diols. In this study, we tested the hypothesis that EETs are involved in glucose regulation and in retarding the development of insulin resistance. To address the role of EETs in regulating glucose homeostasis and insulin signaling, we used mice with targeted gene deletion of sEH (Ephx2-null mice) and a subsequent study with a selective sEH inhibitor. When wild-type mice are fed a high fat diet, insulin resistance develops. However, knockout or inhibition of sEH activity resulted in a significant decrease in plasma glucose. These findings are characterized by enhancement of tyrosyl phosphorylation of the insulin receptor, insulin receptor substrate 1, and their downstream cascade. In addition, pancreatic islets were larger when sEH was disrupted. This effect was associated with an increase in vasculature. These observations were supported by pharmacological inhibition of sEH. These data suggest that an increase in EETs due to sEH-gene knockout leads to an increase in the size of islets and improved insulin signaling and sensitivity.

Soluble Epoxide Inhibition Is Protective Against Cerebral Ischemia via Vascular and Neural Protection

Inhibition of soluble epoxide hydrolase (SEH), the enzyme responsible for degradation of vasoactive epoxides, protects against cerebral ischemia in rats. However, the molecular and biological mechanisms that confer protection in normotension and hypertension remain unclear. Here we show that 6 weeks of SEH inhibition via 2 mg/day of 12-(3-adamantan-1-yl-ureido) dodecanoic acid (AUDA) in spontaneously hypertensive stroke-prone (SHRSP) rats protects against cerebral ischemia induced by middle cerebral artery occlusion, reducing percent hemispheric infarct and neurodeficit score without decreasing blood pressure. This level of cerebral protection was similar to that of the angiotensin-converting enzyme inhibitor, enalapril, which significantly lowered blood pressure. SEH inhibition is also protective in normotensive Wistar-Kyoto (WKY) rats, reducing both hemispheric infarct and neurodeficit score. In SHRSP rats, SEH inhibition reduced wall-to-lumen ratio and collagen deposition and increased cerebral microvessel density, although AUDA did not alter middle cerebral artery structure or microvessel density in WKY rats. An apoptosis mRNA expression microarray of brain tissues from AUDA-treated rats revealed that AUDA modulates gene expression of mediators involved in the regulation of apoptosis in neural tissues of both WKY and SHRSP rats. Hence, we conclude that chronic SEH inhibition protects against cerebral ischemia via vascular protection in SHRSP rats and neural protection in both the SHRSP and WKY rats, indicating that SEH inhibition has broad pharmacological potential for treating ischemic stroke.

Opposite Regulation of Cholesterol Levels by the Phosphatase and Hydrolase Domains of Soluble Epoxide Hydrolase

Soluble epoxide hydrolase (sEH) is a bifunctional enzyme with two catalytic domains: a C-terminal epoxide hydrolase domain and an N-terminal phosphatase domain. Epidemiology and animal studies have attributed a variety of cardiovascular and anti-inflammatory effects to the C-terminal epoxide hydrolase domain. The recent association of sEH with cholesterol-related disorders, peroxisome proliferator-activated receptor activity, and the isoprenoid/cholesterol biosynthesis pathway additionally suggest a role of sEH in regulating cholesterol metabolism. Here we used sEH knock-out (sEH-KO) mice and transfected HepG2 cells to evaluate the phosphatase and hydrolase domains in regulating cholesterol levels. In sEH-KO male mice we found a ∼25% decrease in plasma total cholesterol as compared with wild type (sEH-WT) male mice. Consistent with plasma cholesterol levels, liver expression of HMG-CoA reductase was found to be ∼2-fold lower in sEH-KO male mice. Additionally, HepG2 cells stably expressing human sEH with phosphatase only or hydrolase only activity demonstrate independent and opposite roles of the two sEH domains. Whereas the phosphatase domain elevated cholesterol levels, the hydrolase domain lowered cholesterol levels. Hydrolase inhibitor treatment in sEH-WT male and female mice as well as HepG2 cells expressing human sEH resulted in higher cholesterol levels, thus mimicking the effect of expressing the phosphatase domain in HepG2 cells. In conclusion, we show that sEH regulates cholesterol levels in vivo and in vitro, and we propose the phosphatase domain as a potential therapeutic target in hypercholesterolemia-related disorders.

Alteration in plasma testosterone levels in male mice lacking soluble epoxide hydrolase

Soluble epoxide hydrolase (Ephx2, sEH) is a bifunctional enzyme with COOH-terminal hydrolase and NH(2)-terminal phosphatase activities. sEH converts epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids (DHETs), and the phosphatase activity is suggested to be involved in cholesterol metabolism. EETs participate in a wide range of biological functions, including regulation of vascular tone, renal tubular transport, cardiac contractility, and inflammation. Inhibition of sEH is a potential approach for enhancing the biological activity of EETs. Therefore, disruption of sEH activity is becoming an attractive therapeutic target for both cardiovascular and inflammatory diseases. To define the physiological role of sEH, we characterized a knockout mouse colony lacking expression of the Ephx2 gene. Lack of sEH enzyme is characterized by elevation of EET to DHET ratios in both the linoleate and arachidonate series in plasma and tissues of both female and male mice. In male mice, this lack of expression was also associated with decreased plasma testosterone levels, sperm count, and testicular size. However, this genotype was still able to sire litters. Plasma cholesterol levels also declined in this genotype. Behavior tests such as anxiety-like behavior and hedonic response were also examined in Ephx2-null and WT mice, as all can be related to hormonal changes. Null mice showed a level of anxiety with a decreased hedonic response. In conclusion, this study provides a broad biochemical, physiological, and behavioral characterization of the Ephx2-null mouse colony and suggests a mechanism by which sEH and its substrates may regulate circulating levels of testosterone through cholesterol biosynthesis and metabolism.

Soluble epoxide hydrolase inhibition modulates vascular remodeling

The soluble epoxide hydrolase enzyme (SEH) and vascular remodeling are associated with cardiovascular disease. Although inhibition of SEH prevents smooth muscle cell proliferation in vitro, the effects of SEH inhibition on vascular remodeling in vivo and mechanisms of these effects remain unclear. Herein we determined the effects of SEH antagonism in an endothelium intact model of vascular remodeling induced by flow reduction and an endothelium denuded model of vascular injury. We demonstrated that chronic treatment of spontaneously hypertensive stroke-prone rats with 12-(3-adamantan-1-yl-ureido) dodecanoic acid, an inhibitor of SEH, improved the increment of inward remodeling induced by common carotid ligation to a level that was comparable with normotensive Wistar Kyoto rats. Similarly, mice with deletion of the gene responsible for the production of the SEH enzyme (Ephx2(-/-)) demonstrated enhanced inward vascular remodeling induced by carotid ligation. However, the hyperplastic response induced by vascular injury that denudes the endothelium was unabated by SEH inhibition or Ephx2 gene deletion. These results suggest that SEH inhibition or Ephx2 gene deletion antagonizes neointimal formation in vivo by mechanisms that are endothelium dependent. Thus SEH inhibition may have therapeutic potential for flow-induced remodeling and neointimal formation.

Pharmacokinetics and in vivo potency of soluble epoxide hydrolase inhibitors in cynomolgus monkeys.

BACKGROUND AND PURPOSE Soluble epoxide hydrolase inhibitors (sEHIs) possess anti‐inflammatory, antiatherosclerotic, antihypertensive and analgesic properties. The pharmacokinetics (PK) and pharmacodynamics in terms of inhibitory potency of sEHIs were assessed in non‐human primates (NHPs). Development of a sEHI for use in NHPs will facilitate investigations on the role of sEH in numerous chronic inflammatory conditions.