Selective improvement of pulmonary arterial hypertension with a dual ETA/ETBreceptors antagonist in the apolipoprotein E−/−model of PAH and atherosclerosis

Figures

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  Fig. 1.. Macitentan treatment reduces hemodynamic indices of PAH in HFD-fed ApoE−/− mice. Bar graphs show (a) pulmonary artery acceleration time (PA-AT), (b) RV systolic pressure (RVSP), (c) RV arterial elastane (RVEa), (d) RV dP/dtmax in mm Hg/s, (e) cardiac output, (f) mean aortic pressure (mAoP), (g) LV end-diastolic pressure (LVeDP). (a, f) Measures were made using Vevo 770 echocardiography v3.0. All other measures were made using closed chest cardiac pressure volume catheterization. Bars represent mean ± SEM, n = 6–20, individual animals represented by dots. (a–g) Statistical differences between chow-fed ApoE−/−, HFD-fed, and HFD-fed + macitentan were assessed by Kruskal–Wallis analysis of variance with Dunn’s multiple comparison post-hoc test; *P < 0.05, **P < 0.01.

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  Fig. 2.. Macitentan treatment reduces pulmonary vascular remodeling in HFD-fed ApoE−/− mice. Bar charts (a, b) illustrating the degree of medial wall thickening (identified by SMA) as a ratio of total vessel size (SMA / CSA) in <50 µm arteriole (a) and small to medium pulmonary arteries (b). (c, d) The percentage of muscularized and non-muscularized arterioles (c) and arteries (d). Representative images of ABEVG and SMA-stained tissue (e); ABEVG and SMA-stained images of pulmonary arteries from ApoE−/− mice that were fed either chow (top), HFD with no treatment (middle), and HFD with macitentan (bottom). Scale bar represents 100 µm. (a, b) Bars represent mean ± SEM, n = 7. Kruskal–Wallis analysis of variance; *P < 0.05, ***P < 0. (c, d) Stars represent level of statistical significance when compared with chow-fed ApoE−/− mice; ****P < 0.0001. Post-hoc analysis shows statistical differences between macitentan-treated and treatment-naïve HFD-fed mice (P < 0.0001).

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  Fig. 3.. Macitentan treatment improves vascular function in thoracic aortae. Graphs show cumulative dose response curves to increasing concentrations of acetylcholine (a) or sodium nitroprusside (b) in thoracic aortae pre-contracted with 10 µM phenylephrine. Data are expressed as mean ± SEM and presented as a % of endothelium-dependent (a) or -independent (b) relaxation, n = 4. *P < 0.05, ***P < 0.001. Statistical differences between treated and untreated HFD-fed mice were assessed by two-way analysis of variance with Bonferroni post-hoc analysis.

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  Fig. 4.. Macitentan treatment does not alter the composition or atherosclerotic burden in HFD-fed ApoE−/− mice. Bar charts show ORO stain as a % of total aortic area (a), and within the aortic arch as a % of total arch area (b). (c) Representative images of ORO in control and macitentan-treated mice. (d) Bar charts show the ratio of atherosclerotic lesion area within the brachiocephalic artery normalized to cross-sectional area, and (d) the % α-SMA positive tissue within the brachiocephalic sections. (f) The ratio of lesion area to cross-sectional area within the aortic root sinus, (g) % collagen content, and (h) % α-SMA positive tissue in aortic root sections. (i) Representative aortic root histological sections from control and macitentan-treated HFD-fed ApoE−/− mice stained with ABEVG, martius scarlet blue (collagen), and α-SMA. Bars represent mean ± SEM. Statistical differences between sections from treated and untreated mice were assessed by Mann–Whitney U test, n = 11–12. Individual mice are represented by dots. *P < 0.05. Statistical differences between groups were measured by Mann–Whitney U test.

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  Fig. 5.. Macitentan treatment induces changes in inflammatory plasma cytokines in HFD-fed ApoE−/− mice. Bar charts show the plasma concentration of (a) IL-6, (b) leptin, (c) IL-10, (d) IL-1β, (e) MCP-1, (f) TNF-α, (g) HGF, (h) insulin, (i) IP-10, (j) SDF-1, and (k) CRP. Bar graphs represent mean ± SEM, n = 4–9 with individual animals represented by dots. *P < 0.05. Statistical differences were assessed by Mann–Whitney U test.