Sex- and isoform-specific mechanism of neuroprotection by transgenic expression of P450 epoxygenase in vascular endothelium

Cytochrome P450 epoxygenases (CYP) metabolize arachidonic acid to epoxyeicosatrienoic acids (EETs), which exhibit vasodilatory, anti-inflammatory and neuroprotective actions in experimental cerebral ischemia. We evaluated the effect of endothelial-specific CYP overexpression on cerebral blood flow, inflammatory cytokine expression and tissue infarction after focal cerebral ischemia in transgenic mice

Mechanism of protection by soluble epoxide hydrolase inhibition in type 2 diabetic stroke.

Inhibition of soluble epoxide hydrolase (sEH) is a potential target of therapy for ischemic injury. sEH metabolizes neuroprotective epoxyeicosatrienoic acids (EETs). We recently demonstrated that sEH inhibition reduces infarct size after middle cerebral artery occlusion (MCAO) in type 1 diabetic mice. We hypothesized that inhibition of sEH would protect against ischemic injury in type 2 diabetic mice. Type 2 diabetes was produced by combined high-fat diet, nicotinamide and streptozotocin in male mice. Diabetic and control mice were treated with vehicle or the sEH inhibitor t-AUCB then subjected to 60-min MCAO. Compared to chow-fed mice, high fat diet-fed mice exhibited an upregulation of sEH mRNA and protein in brain, but no differences in brain EETs levels were observed between groups. Type 2 diabetic mice had increased blood glucose levels at baseline and throughout ischemia, decreased laser-Doppler perfusion of the MCA territory after reperfusion, and sustained larger cortical infarcts compared to control mice. t-AUCB decreased fasting glucose levels at baseline and throughout ischemia, improved cortical perfusion after MCAO and significantly reduced infarct size in diabetic mice. We conclude that sEH inhibition, as a preventative treatment, improves glycemic status, post-ischemic reperfusion in the ischemic territory, and stroke outcome in type 2 diabetic mice

2 mg/kg t-AUCB prevented reduced infarct size in control mice following MCAO.

<p>Control mice were treated with the sEH inhibitor t-AUCB (1 mg/kg or 2 mg/kg i.p. daily, 7 days) or vehicle and subjected to middle cerebral artery occlusion (60 min). Brains were harvested at 24 h post-MCAO, and infarct size measured by TTC staining and corrected for edema as described in methods. Infarct size is shown for separately for cortex, striatum, and total hemisphere. *p<0.05 vs. Control Vehicle of same brain region via 2-way ANOVA post-hoc test, #p<0.01 vs. Control Vehicle Hemisphere via t-test only, N = 7 per group.</p

Pre-diabetic mice have increased body weight, increased insulin levels and decreased glucose tolerance.

<p>A) Body weight B) serum insulin levels, and C) blood glucose during i.p. glucose tolerance test (GTT) were measured in mice fed a low fat (Chow; n = 5) or high fat (n = 4) diet for 15 weeks. Mice were fasted overnight prior to the GTT. Glucose (2 g/kg body weight) was injected i.p. at time 0. D) Area under the curve from GTT test results was calculated for each treatment group. *p<0.001 vs. Chow.</p

sEH inhibition decreased glucose levels in type 2 diabetic mice before, during, and after ischemia.

<p>Control and HFD+STZ/NA mice were treated with the sEH inhibitor t-AUCB (1 mg/kg i.p. daily, 7 days) or vehicle and subjected to middle cerebral artery occlusion (60 min). Blood glucose levels were measured at baseline, 30 min into the occlusion, and 30 min post-reperfusion. *p<0.001 vs. Control Vehicle, #p<0.01 vs. HFD+STZ/NA Vehicle. ∧p<0.05 vs. baseline of same treatment group. N = 4–5 per group.</p

Baseline Characteristics of Type 2 Diabetic Mice and Controls.

<p>Age, weight, and body temperature, monitored via rectal temperature probe, were measured in control and HFD+STZ/NA mice treated with vehicle or t-AUCB treatment (n = 11–16 mice per group).</p

High fat diet increases sEH expression in brain.

<p>Mice were treated with a high fat (60% fat) or chow diet (13% fat) for 15 weeks. A) EPHX2 mRNA expression was measured via qPCR in cortex. EPHX2 expression was normalized to 18S expression. *p<0.05 vs. Chow, n = 7 per group. B) sEH protein expression was measured via Western blot in brain. sEH protein expression was normalized to beta actin protein expression. *p<0.01 vs. Chow, n = 4 per group.</p

sEH inhibition decreased fasting glucose levels in type 2 diabetic mice.

<p>Fasting blood glucose levels were obtained from control and HFD+STZ/NA mice before (A) and after (B) treatment with the sEH inhibitor t-AUCB (1 mg/kg i.p. daily, 7 days) or vehicle. C) Serum insulin levels were also measured in t-AUCB or vehicle treated mice 24 h after ischemia. *p<0.001 vs. Control Vehicle, #p<0.05 vs. HFD+STZ/NA Vehicle. A) n = 20–22 per group. B) n = 5–8 per group. C) n = 2–6 per group.</p

Sex- and isoform-specific mechanism of neuroprotection by transgenic expression of P450 epoxygenase in vascular endothelium

OBJECTIVE: Cytochrome P450 epoxygenases (CYP) metabolize arachidonic acid to epoxyeicosatrienoic acids (EETs), which exhibit vasodilatory, anti-inflammatory and neuroprotective actions in experimental cerebral ischemia. We evaluated the effect of endothelial-specific CYP overexpression on cerebral blood flow, inflammatory cytokine expression and tissue infarction after focal cerebral ischemia in transgenic mice. APPROACH AND RESULTS: Male and female wild-type and transgenic mice overexpressing either human CYP2J2 or CYP2C8 epoxygenases in vascular endothelium under control of the Tie2 promoter (Tie2-CYP2J2 and Tie2-CYP2C8) were subjected to 60-minutes middle cerebral artery occlusion (MCAO). Microvascular cortical perfusion was monitored during vascular occlusion and reperfusion using laser-Doppler flowmetry and optical imaging. Infarct size and inflammatory cytokines were measured at 24 hours of reperfusion by TTC and real-time quantitative PCR, respectively. Infarct size was significantly reduced in both Tie2-CYP2J2 and Tie2-CYP2C8 transgenic male mice compared to corresponding WT male mice (n=10 per group, p < 0.05). Tie2-CYP2J2, but not Tie2-CYP2C8 male mice maintained higher blood flow during MCAO; however, both Tie2-CYP2J2 and Tie2-CYP2C8 had lower inflammatory cytokine expression after ischemia compared to corresponding WT males (n=10 per group for CBF and n=3 for cytokines, p<0.05). In females, a reduction in infarct was observed in the caudate-putamen, but not in the cortex or hemisphere as a whole and no differences were observed in blood flow between female transgenic and WT mice (n=10 per group). CONCLUSIONS: Overexpression of CYP epoxygenases in vascular endothelial cells protects against experimental cerebral ischemia in male mice. The mechanism of protection is in part linked to enhanced blood flow and suppression of inflammation, and is both sex- and CYP isoform-specific