Wenri Zhang

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.

Soluble Epoxide Hydrolase Gene Deletion Is Protective Against Experimental Cerebral Ischemia

Background and Purpose— Cytochrome P450 epoxygenase metabolizes arachidonic acid to epoxyeicosatrienoic acids (EETs). EETs are produced in the brain and perform important biological functions, including vasodilation and neuroprotection. However, EETs are rapidly metabolized via soluble epoxide hydrolase (sEH) to dihydroxyeicosatrienoic acids (DHETs). We tested the hypothesis that sEH gene deletion is protective against focal cerebral ischemia through enhanced collateral blood flow. Methods— sEH knockout (sEHKO) mice with and without EETs antagonist 14, 15 epoxyeicosa-5(Z)-enoic acid (EEZE) were subjected to 2-hour middle cerebral artery occlusion (MCAO), and infarct size was measured at 24 hours of reperfusion and compared to wild-type (WT) mice. Local CBF rates were measured at the end of MCAO using iodoantipyrine (IAP) autoradiography, sEH protein was analyzed by Western blot and immunohistochemistry, and hydrolase activity and levels of EETs/DHETs were measured in brain and plasma using LC-MS/MS and ELISA, respectively. Results— sEH immunoreactivity was detected in WT, but not sEHKO mouse brain, and was localized to vascular and nonvascular cells. 14,15-DHET was abundantly present in WT, but virtually absent in sEHKO mouse plasma. However, hydrolase activity and free 14,15-EET in brain tissue were not different between WT and sEHKO mice. Infarct size was significantly smaller, whereas regional cerebral blood flow rates were significantly higher in sEHKO compared to WT mice. Infarct size reduction was recapitulated by 14,15-EET infusion. However, 14,15-EEZE did not alter infarct size in sEHKO mice. Conclusions— sEH gene deletion is protective against ischemic stroke by a vascular mechanism linked to reduced hydration of circulating EETs.

Role of cocaine- and amphetamine-regulated transcript in estradiol-mediated neuroprotection.

Estrogen reduces brain injury after experimental cerebral ischemia in part through a genomic mechanism of action. Using DNA microarrays, we analyzed the genomic response of the brain to estradiol, and we identified a transcript, cocaine- and amphetamine-regulated transcript (CART), that is highly induced in the cerebral cortex by estradiol under ischemic conditions. Using in vitro and in vivo models of neural injury, we confirmed and characterized CART mRNA and protein up-regulation by estradiol in surviving neurons, and we demonstrated that i.v. administration of a rat CART peptide is protective against ischemic brain injury in vivo. We further demonstrated binding of cAMP response element (CRE)-binding protein to a CART promoter CRE site in ischemic brain and rapid activation by CART of ERK in primary cultured cortical neurons. The findings suggest that CART is an important player in estrogen-mediated neuroprotection and a potential therapeutic agent for stroke and other neurodegenerative diseases.

Soluble epoxide hydrolase: Regulation by estrogen and role in the inflammatory response to cerebral ischemia

The protection from ischemic brain injury enjoyed by females is linked to the female sex hormone 17beta-estradiol. We tested the hypothesis that neuroprotection by estradiol entails the prevention of ischemia-induced inflammatory response, through suppression of the P450 eicosanoids-metabolizing enzyme soluble epoxide hydrolase (sEH). Ovariectomized female rats with and without estradiol replacement underwent 2-hour middle cerebral artery occlusion (MCAO). SEH expression was determined using Western blot, and inflammatory cytokine mRNA levels were measured at 6, 24 and 48 hours after MCAO. Cytokine mRNA was also measured in sEH-knockout mice, and in rats treated with sEH inhibitors. Estradiol reduced basal and post-ischemic sEH expression. MCAO strongly induced mRNA levels of tumor necrosis factor-alpha, interleukin 6, and interleukin 1beta, which was attenuated in sEH-knockouts, but not by sEH inhibitors. Estradiol replacement exhibited a bimodal effect on cytokine mRNA, with increased early and reduced delayed expression. While estradiol suppresses cerebral sEH expression, and sEH suppression diminishes inflammation after MCAO, our findings suggest that the effect of estrogen on inflammation is complex, and only partially explained by sEH suppression.

Role of soluble epoxide hydrolase in the sex-specific vascular response to cerebral ischemia

Soluble epoxide hydrolase (sEH), a key enzyme in the metabolism of vasodilator eicosanoids called epoxyeicosatrienoic acids (EETs), is sexually dimorphic and suppressed by estrogen. We determined if the sex difference in blood flow during focal cerebral ischemia is linked to sEH. Soluble epoxide hydrolase expression in brain, hydrolase activity in cerebral vessels, and plasma 14,15-dihydroxyeicosatrienoic acid (14,15-DHET) were determined in male and female wild-type (WT) and sEH knockout (sEHKO) mice. Male, female, and ovariectomized female WT and sEHKO mice were subjected to 2-h middle cerebral artery occlusion (MCAO) and infarct size was measured at 24 h of reperfusion. Laser—Doppler cortical perfusion during MCAO was compared among groups and differences in cortical blood flow rates were confirmed using in vivo quantitative optical microangiography. Cerebrovascular expression and activity of sEH and plasma 14,15-DHET were lower in WT female than male mice, and blood flow during MCAO was higher and infarct size was smaller in WT female compared with male mice. Sex differences in cerebral blood flow and ischemic damage were abolished after ovariectomy and were absent in sEHKO mice. We conclude that sEH is an important mechanism underlying sex-linked differences in blood flow and brain damage after cerebral ischemia.

Estrogen Is Renoprotective via a Nonreceptor-dependent Mechanism after Cardiac Arrest In Vivo

Background:Severe ischemia induces renal injury less frequently in women than men. In this study, cardiac arrest and cardiopulmonary resuscitation were used to assess whether estradiol is renoprotective via an estrogen receptor (ER)-dependent mechanism. Materials and Methods:Male and female C57BL/6 and ER gene-deleted mice underwent 10 min of cardiac arrest followed by cardiopulmonary resuscitation. Serum chemistries and renal stereology were measured 24 h after arrest. Results:Estrogen did not affect mean arterial pressure, regional renal cortical blood flow, and arterial blood gases. Hence, female kidneys were protected (mean ± SEM: blood urea nitrogen, 65 ± 21 vs.149 ± 27 mg/dl, P = 0.04; creatinine, 0.14 ± 0.05 vs. 0.73 ± 0.16 mg/dl, P = 0.01; volume of necrotic tubules, 7 ± 1% vs. 10 ± 0%, P = 0.04). Estrogen also reduced renal injury. In intact females (n = 5), ovariectomized/vehicle-treated (n = 8), and ovariectomized/estrogen-treated (n = 8) animals, blood urea nitrogen was 65 ± 21, 166 ± 28, and 50 ± 14 mg/dl (P = 0.002); creatinine was 0.14 ± 0.05, 0.74 ± 0.26, and 0.23 ± 0.27 mg/dl (P = 0.014); necrotic tubules were 2.5 ± 0.25%, 12.0 ± 1.9%, and 5.0 ± 1.6% (P = 0.004), respectively. In ER-α and ER-β gene-deleted mice and controls estradiol-reduced functional injury (blood urea nitrogen: estradiol 117 ± 71, vehicle 167 ± 56, P = 0.007; creatinine: estradiol 0.5 ± 0.5, vehicle 1.0 ± 0.4, P = 0.013), but the effect of estradiol was not different between ER-α or ER-β gene-deleted mice. Adding ICI 182,780 to estradiol did not alter injury. Conclusions:In women, kidneys were protected from cardiac arrest through estrogen. Estradiol-mediated renoprotection was not affected by ER deletion or blockade. Estradiol is renoprotective after cardiac arrest. The results indicate that estradiol renoprotection is ER-α and ER-β independent.

Role of Salt-Induced Kinase 1 in Androgen Neuroprotection against Cerebral Ischemia

Androgens within physiological ranges protect castrated male mice from cerebral ischemic injury. Yet, underlying mechanisms are unclear. Here, we report that, after middle cerebral artery occlusion (MCAO), salt-induced kinase 1 (SIK1) was induced by a potent androgen—dihydrotestosterone (DHT) at protective doses. To investigate whether SIK1 contributes to DHT neuroprotection after cerebral ischemia, we constructed lentivirus-expressing small interference RNA (siRNA) against SIK1. The SIK1 knockdown by siRNA exacerbated oxygen–glucose deprivation (OGD)-induced cell death in primary cortical neurons, suggesting that SIK1 is an endogenous neuroprotective gene against cerebral ischemia. Furthermore, lentivirus-mediated SIK1 knockdown increased both cortical and striatal infarct sizes in castrated mice treated with a protective dose of DHT. Earlier studies show that SIK1 inhibits histone deacetylase (HDAC) activities by acting as a class IIa HDAC kinase. We observed that SIK1 knockdown decreased histone H3 acetylation in primary neurons. The SIK1 siRNA also exacerbated OGD-induced neuronal death in the presence of trichostatin A (TSA), an HDAC inhibitor, and decreased histone H3 acetylation at 4 hours reoxygenation in TSA-treated neurons. Finally, we showed that DHT at protective doses prevented ischemia-induced histone deacetylation after MCAO. Our finding suggests that SIK1 contributes to neuroprotection by androgens within physiological ranges by inhibiting histone deacetylation.

Role of soluble epoxide hydrolase in exacerbation of stroke by streptozotocin-induced type 1 diabetes mellitus

Hyperglycemia worsens stroke, yet rigorous glycemic control does not improve neurologic outcome. An alternative is to target downstream molecular mediator(s) triggered by hyperglycemia but independent of prevailing glycemia. Soluble epoxide hydrolase (sEH) is a potential mediator of injury via its metabolism of neuroprotective epoxyeicosatrienoic acids (EETs). We tested whether hyperglycemia exacerbates cerebral injury by upregulating sEH and decreasing brain EET levels. Type 1 diabetes mellitus was modeled by streptozotocin (STZ;50 mg/kg per day intraperitoneally, 5 days) in male mice. At 4 weeks, STZ-treated and control mice underwent 45-minute middle cerebral artery occlusion (MCAO) with or without sEH blockade by trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB;1 mg/kg intraperitoneally daily for 6 days before MCAO). The STZ-treated mice had increased sEH mRNA expression in cerebral vessels and decreased EET concentrations in brain. There was no difference in cortical perfusion between groups. The STZ-treated mice sustained larger brain infarct than controls. Pretreatment with t-AUCB eliminated the difference in infarct size and EETs concentration between STZ-treated mice and controls, without altering glycemia. We conclude that type 1 diabetes mellitus upregulates sEH mRNA and decreases concentrations of neuroprotective EETs within the brain, leading to worse stroke outcome. The data indicate that sEH antagonism may be beneficial in the setting of hyperglycemic stroke.

Role of endothelial soluble epoxide hydrolase in cerebrovascular function and ischemic injury.

Soluble Epoxide Hydrolase (sEH) is a key enzyme in the metabolism and termination of action of epoxyeicosatrienoic acids, derivatives of arachidonic acid, which are protective against ischemic stroke. Mice lacking sEH globally are protected from injury following stroke; however, little is known about the role of endothelial sEH in brain ischemia. We generated transgenic mice with endothelial-specific expression of human sEH (Tie2-hsEH), and assessed the effect of transgenic overexpression of endothelial sEH on endothelium-dependent vascular reactivity and ischemic injury following middle cerebral artery occlusion (MCAO). Compared to wild-type, male Tie2-hsEH mice exhibited impaired vasodilation in response to stimulation with 1 µM acetylcholine as assessed by laser-Doppler perfusion monitoring in an in-vivo cranial window preparation. No difference in infarct size was observed between wild-type and Tie2-hsEH male mice. In females, however, Tie2-hsEH mice sustained larger infarcts in striatum, but not cortex, compared to wild-type mice. Sex difference in ischemic injury was maintained in the cortex of Tie2-hsEH mice. In the striatum, expression of Tie2-hsEH resulted in a sex difference, with larger infarct in females than males. These findings demonstrate that transgenic expression of sEH in endothelium results in impaired endothelium-dependent vasodilation in the cerebral circulation, and that females are more susceptible to enhanced ischemic damage as a result of increased endothelial sEH than males, especially in end-arteriolar striatal region.

High fat diet-induced diabetes in mice exacerbates cognitive deficit due to chronic hypoperfusion:

Diabetes causes endothelial dysfunction and increases the risk of vascular cognitive impairment. However, it is unknown whether diabetes causes cognitive impairment due to reductions in cerebral blood flow or through independent effects on neuronal function and cognition. We addressed this using right unilateral common carotid artery occlusion to model vascular cognitive impairment and long-term high-fat diet to model type 2 diabetes in mice. Cognition was assessed using novel object recognition task, Morris water maze, and contextual and cued fear conditioning. Cerebral blood flow was assessed using arterial spin labeling magnetic resonance imaging. Vascular cognitive impairment mice showed cognitive deficit in the novel object recognition task, decreased cerebral blood flow in the right hemisphere, and increased glial activation in white matter and hippocampus. Mice fed a high-fat diet displayed deficits in the novel object recognition task, Morris water maze and fear conditioning tasks and neuronal loss, but no impairments in cerebral blood flow. Compared to vascular cognitive impairment mice fed a low fat diet, vascular cognitive impairment mice fed a high-fat diet exhibited reduced cued fear memory, increased deficit in the Morris water maze, neuronal loss, glial activation, and global decrease in cerebral blood flow. We conclude that high-fat diet and chronic hypoperfusion impair cognitive function by different mechanisms, although they share commons features, and that high-fat diet exacerbates vascular cognitive impairment pathology.