Treatment with the cytochrome P450 ω-hydroxylase inhibitor HET0016 attenuates cerebrovascular inflammation, oxidative stress and improves vasomotor function in spontaneously hypertensive rats

Abstract

Background and purpose: Hypertension increases cerebrovascular oxidative stress and inflammation and impairs vasomotor function. These pathological alterations lead to dysregulation of cerebral blood flow and exacerbate atherogenesis, increasing the morbidity of ischaemic cerebrovascular diseases and promoting vascular cognitive impairment. We aimed to test the hypothesis that increased production of the arachidonic acid metabolite 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) contributes to hypertension-induced cerebrovascular alterations.

Experimental approach: We treated male spontaneously hypertensive rats (SHR) with HET0016 (N-hydroxy-N'-(4-butyl-2-methylphenyl)-formamidine), an inhibitor of 20-HETE synthesis. In middle cerebral arteries (MCAs) of SHRs, we focused on vasomotor responses and end points that are highly relevant for cellular reactive oxygen species (ROS) production, inflammatory cytokine expression and NF-κB activation.

Key results: SHRs treated with HET0016 remained hypertensive (SHR + HET0016: 149 ± 8 mmHg, Wistar-Kyoto rat: 115 ± 4 mmHg; P < 0.05.), although their systolic blood pressure was decreased compared to untreated SHRs (191 ± 6 mmHg). In MCAs of SHRs, flow-induced constriction was increased, whereas ACh- and ATP-induced dilations were impaired. This functional impairment was reversed by treatment with HET0016. Treatment with HET0016 also significantly decreased oxidative stress in MCAs of SHRs (as shown by dihydroethidium staining and analysis of vascular 5-nitrotyrosine, 4-hydroxynonenal and carbonyl content) and inhibited cerebrovascular inflammation (shown by the reduced mRNA expression of TNFα, IL-1β and IL-6). Treatment of SHRs with HET0016 also attenuated vascular NF-κB activation. In vitro treatment with 20-HETE significantly increased vascular production of ROS and promoted NF-κB activation in cultured cerebromicrovascular endothelial cells.

Conclusions and implications: Taken together, treatment with HET0016 confers anti-oxidative and anti-inflammatory effects in the cerebral arteries of SHRs by disrupting 20-HETE-mediated autocrine/paracrine signalling pathways in the vascular wall. It is likely that HET0016-induced decreases in blood pressure also potentiate the cerebrovascular protective effects of the drug.

Figure 1

(A) Structure of HET0016. (B) The effect of HET0016 treatment (10 mg·kg⁻¹, 5 days i.p.) on blood pressure in control Wistar-Kyoto (n = 6) and spontaneously hypertensive rats (n = 6). Data are mean ± SEM *P < 0.05 versus WKY; ^#P < 0.05 versus SHR.

Figure 2

Inhibition of 20-HETE production improves vascular function in MCA of SHR. (A) Flow-induced constriction in MCAs isolated from control WKY rats, SHRs, WKYs and SHRs treated with HET0016 (SHR + HET0016; 10 mg·kg⁻¹·per day, i.p., n = 6 for each data point). Intraluminal flow was controlled by creating a pressure gradient through the vessel (see Methods). (B, above): Treatment with the TP receptor antagonist SQ29,548 (1 μmol·L⁻¹) inhibits flow-induced constriction in MCAs of SHRs (n = 7). Data are mean ± SEM *P < 0.05 versus control; ^#P < 0.05 versus untreated. (B, below): U46619 (10⁻⁷ mol·L⁻¹), a thromboxane A₂ analogue elicits comparable constriction in MCAs of WKY and SHR (n = 7). ACh- (C) and ATP (D)-induced dilations of MCAs isolated from control WKY rats and SHRs and WKYs and SHRs treated with HET0016 (n = 6 for each data point). Data are mean ± SEM *P < 0.05 versus control; ^#P < 0.05 versus untreated.

Figure 3

In vivo treatment with HET0016 attenuates oxidative stress in MCA of SHR. (A) Representative images showing red nuclear DHE fluorescence, representing cellular O₂^.- production, in sections of MCAs of control WKY rats, SHRs and treated WKY and SHRs with HET0016 (10 mg·kg⁻¹·per day, i.p.). (B) Summary data for nuclear DHE fluorescence intensities (n = 6 for each group). (C) Summary data showing the effect of 20-HETE (10⁻⁷ mol·L⁻¹) on production of ROS of vascular ring preparations (n = 7) in the absence and presence of NADPH oxidase inhibitor apocynin (3 × 10⁻⁴ mol·L⁻¹). Data are mean ± SEM *P < 0.05 versus control; ^#P < 0.05 versus untreated.

Figure 4

In vivo treatment with HET0016 attenuates vascular oxidative/nitrosative damage in SHRs. (A)–(C) show the effects of treatment with HET0016 on markers of vascular oxidative/nitrosative stress in SHRs, including 5-NT (a marker for peroxynitrite action, C), 4-HNE (a marker of lipid peroxidation, D) and carbonyl derivatives of protein oxidation (E). Data are mean ± SEM *P < 0.05 versus Control; ^#P < 0.05 versus untreated (n = 6 for each data point).

Figure 5

In vivo treatment with HET0016 attenuates vascular NF-κB activation in SHR. (A) shows NF-κB binding activity in nuclear extracts from carotid arteries isolated from control WKY rats, SHRs and SHRs treated with HET0016 (SHR + HET0016; 10 mg·kg⁻¹·per day, i.p., n = 6 for each data point). (B) effect of 20-HETE (10⁻⁷ mol·L⁻¹) on NF-κB activation measured by dual-luciferase reporter assay in cultured rat cerebromicrovascular endothelial cells (CMVEC) in the absence and presence of TP receptor blocker SQ 29548 (10⁻⁶ mol·L⁻¹). Data are mean ± SEM *P < 0.05 versus control; ^#P < 0.05 versus untreated.

Figure 6

In vivo treatment with HET0016 attenuates inflammation in MCA of SHR. Quantitative real-time RT-PCR data showing mRNA expression of TNF-α (A), IL-1β (B), IL-6 (C) and iNOS (D) in MCAs isolated from control WKY rats, SHRs and SHRs treated with HET0016 (SHR + HET0016; 10 mg·kg⁻¹·per 5 days, i.p., n = 6 for each data point). Data are mean ± SEM *P < 0.05 versus control; ^#P < 0.05 versus untreated. (E) Scheme depicting the proposed mechanisms underlying the cerebrovascular protective effects of HET0016 in hypertension. Accordingly, in the wall of cerebral arteries, hypertension increases 20-HETE production, which results in increased flow-induced constriction and promotes endothelial dysfunction via enhancing vascular oxidative stress. Increased oxidative stress also promotes NF-κB activation and vascular inflammation, exacerbating the development of cerebrovascular diseases. HET0016 inhibits 20-HETE synthesis in the vascular wall and thereby confers anti-oxidative and anti-inflammatory cerebrovascular protective effects in hypertension. HET0016 also decreases blood pressure likely via its renal action, which is also likely to contribute indirectly to its cerebrovascular protective effects.