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-min 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 24h 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.

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.

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.

gammaA/gamma' fibrinogen inhibits thrombin-induced platelet aggregation.

The minor gammaA/gamma' fibrinogen isoform contains a high affinity binding site for thrombin exosite II that is lacking in the major gammaA/gammaA fibrinogen isoform. We therefore investigated the biological consequences of the gamma' chain binding to thrombin. Thrombin-induced platelet aggregation was inhibited by gammaA/gamma' fibrinogen. Carboxyl terminal peptide fragment gamma'410-427 from the gamma' chain was also inhibitory, with an IC(50) of approximately 200 microM in whole plasma. Deletion of the peptide from either the amino or carboxyl end significantly decreased inhibition. In contrast to thrombin-induced platelet aggregation, aggregation induced by epinephrine, ADP, arachidonic acid, or SFLLRN peptide showed little inhibition by the gamma' peptide. The inhibition of thrombin-induced platelet aggregation was not due to direct inhibition of the thrombin active site, since cleavage of a small peptidyl substrate was 91% of normal even in the presence of 1 mM gamma'410-427. The gamma'410-427 peptide blocked platelet adhesion to immobilized thrombin under both static and flow conditions, blocked soluble thrombin binding to platelet GPIbalpha, and inhibited PAR1 cleavage by thrombin. These results suggest that the gamma' chain of fibrinogen inhibits thrombin-induced platelet aggregation by binding to thrombin exosite II. Thrombin that is bound to the gamma' chain is thereby prevented from activating platelets, while retaining its amidolytic activity.