Ivabradine induces an atheroprotective gene expression profile in the endothelium of ApoE deficient mice before plaque formation

Atherosclerosis consists in a progressive accumulation of atheromatous plaques in medium and large caliber artery walls. Shear stress is generated by the friction of blood flow on the endothelium. Many studies have shown that plaques form in regions of the arterial tree, near bends and branches, characterized by low and oscillatory shear stress (disturbed), whereas linear regions exposed to unidirectional shear stress (laminar) are not susceptible to the plaques development. Hemodynamic studies have shown that the most atherogenic disturbed shear stress occurs in geometrically irregular regions during systolic phase, whereas, in the same regions, the magnitude of shear stress rises to less pathogenetic levels during diastole. The alteration of the disturbed shear stress during the cardiac cycle has a pivotal role in the context of atherosclerosis, since data from the literature indicate that an elevated heart rate stimulates the atherosclerotic plaques onset and progression. Ivabradine is a drug that reduces heart rate by selectively inhibiting the If current in the sinus node and numerous studies demonstrated that it limits atherosclerotic plaques formation in animal models. The mechanism of this protection is still unknown. It has been hypothesized that as heart rate decreases, following ivabradine treatment, the diastolic phase increases, thereby decreasing the exposure time of aortic arch endothelium to systolic flow, which is characterized by a more atherogenic low and oscillatory shear stress. The aim of this study was to determine the molecular effect of a short-term treatment with ivabradine in the initial steps of atherosclerosis development in an in vivo model of severe dyslipidemia (ApoE deficient-mouse), fed a standard diet. For this purpose, we performed a microarray analysis to determine the role of ivabradine in the modulation of gene expression in the endothelium of the aortic arch of ApoE -/- mice. Treatment induced changes in the expression of 930 transcripts. Shear stress-modulated MAPK signalling and sterol metabolic processes were among the most significantly affected pathways. We found upregulation of anti-inflammatory genes and downregulation of pro-apoptotic and pro-inflammatory genes, the majority of which were NF-kappaB- and/or AngII-regulated genes. Moreover, our results indicate that ivabradine modulates genes belonging to the Notch pathway, known to affect endothelial cells apoptosis and to be regulated by shear stress. Our in vitro flow experiments showed a sustained upregulation of Notch signalling by atheroprotective laminar shear stress. Our in vivo analysis on mice aorta confirms the activation of Notch signalling components by the atheroprotective laminar shear stress and provides the first in vivo evidence of differences in expression levels of Notch components between distinct regions of mouse aorta. In conclusion, this study provides strong evidence of ivabradine-mediated induction of a protective gene expression profile in the endothelium of aortic arch of ApoE-deficient mice. Among the identified pathways, Notch signalling modulation by ivabradine treatment may contribute this protection. Since numerous of these reported genes, including Notch, are shear stress modulated, our data are in agreement with the shared views that ivabradine, through heart rate reduction, modifies the shear stress on endothelium.