Fenofibrate, but not eicosapentanoic acid, attenuates hyperlipidaemia-induced endothelial dysfunction and elevated vascular endocannabinoid levels in ApoE-/- mice

Lisa Michel1, Sarah Walsh1, Sharon Wood2, Baukje De Roos2, Helen Vosper1, Marie Goua1. 1The Robert Gordon University, School of Pharmacy and Life Sciences, Aberdeen, AB10 1FR, UK, 2The Rowett Institute of Nutrition and Health, Aberdeen, AB21 9SB, UK.

The omega-3 fatty acids (FA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have been shown to have anti-atherosclerotic actions such as structural stabilisation of lesions1 and modification of the inflammatory response2,3. There is evidence that increased dietary intake of omega-3 FA’s results in elevated levels of endocannabinoids4 (EC’s) and, since synthetic cannabinoid agonists have been shown to exert anti-atherosclerotic effects5, we explored whether a link exists between the EC system and the beneficial effects of omega-3 FA’s in the setting of hyperlipidaemia.

A dietary intervention study was undertaken in which 5 week-old male apolipoprotein E knockout (ApoE-/-) mice were fed a western diet containing a high fat/high cholesterol base (16% & 0.25% w/w respectively), either alone (Control; n = 10), or supplemented with either 3% EPA oil (70% concentrate; n = 10) or 0.04% fenofibrate (n = 10) for 24 weeks. Age-matched male C57BL/6J mice, fed regular chow, were employed as wild-type controls. Following completion of the dietary intervention period the mice (26g-35g) were anaesthetised with ketamine and xylazine (120mg kg-1 i.p. & 16mg kg-1 i.p. respectively) supplemented with 50µl 25g-1 i.p. of the ketamine and xylazine mixture as required. The left carotid artery and right jugular vein were cannulated for mean arterial blood pressure (MABP) measurement drug administration, respectively. Respiration was maintained at 120 strokes/minute on a small animal ventilator. Heart rate (HR) was calculated from a Lead I electrocardiogram recorded from subcutaneous limb leads. Following a 10 minute stabilisation period, mice were administered a bolus dose of vehicle (saline) followed (at regular intervals) by increasing doses of either bradykinin (BK; 3-300µg kg-1) or sodium nitroprusside (SNP; 3-300µg kg-1). MABP and HR responses were calculated as area under the curve for the entire depressor response (expressed as arbitrary units; AU). Following completion of the in vivo protocol, mice were euthanized via an overdose of anaesthetic and the brain, heart, and thoracic aorta were removed and flash frozen for measurement of endocannabinoid (anandamide (AEA) and 2-AG) levels using liquid chromatography-mass spectrometry (LC-MS). Plasma was collected for measurement of lipid profiles. Data are expressed as mean±SEM and were statistically analysed using either an ANOVA with Dunnett’s pot-hoc test or a two-way ANOVA with Bonferroni post-hoc test.

In control ApoE-/- mice AEA levels were significantly elevated in brain (70.4±12.7pmol/g tissue vs. 34.9±3.2pmol/g tissue in C57BL/6 mice; P<0.01), heart (22.1±2.5pmol/g tissue vs. 11.6±0.6pmol/g tissue in C57BL/6 mice; P<0.01) and vascular (15.1±1.8pmol/g tissue vs. 5.7±0.6pmol/g tissue in C57BL/6 mice; P<0.01) tissue. EPA supplementation significantly decreased AEA levels in brain (32.2±5.2pmol/g tissue vs control ApoE-/- mice; P<0.01) and heart (2.8±0.8pmol/g tissue vs control ApoE-/- mice; P<0.01) tissue, while fibrate supplementation significantly reduced AEA levels in heart (14.5±1.6pmol/g tissue vs control ApoE-/- mice; P<0.01) and vascular tissue (6.8±1.5pmol/g tissue vs control ApoE-/- mice; P<0.05). In terms of endothelial function, at 300µg kg-1, the BK induced depressor response was significantly reduced in the ApoE-/- control group compared with C57BL/6 mice (4530±946 AU vs 7903±1202 AU; P<0.05), an effect that was reversed in the fibrate group (7471±395 AU; P<0.05), but not the EPA group (5047±2197 AU).

Induction of hyperlipidaemia markedly increases tissue endocannabinoid levels and reduces endothelial function. While both fibrate and EPA supplementation had similar beneficial effects on lipid profiles, fenofibrate alone improved endothelial function and vascular EC levels, suggesting a possible link between endocannabinoids and hyperlipidaemia-induced endothelial dysfunction.

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