Acute vascular and cardiac effects of lenvatinib in mice

Fig. 1. Mice treatment plan and experimental procedure. Animals were divided into two cohorts. Both cohorts received daily treatments of either 4 mg/kg lenvatinib or 40% (2-Hydroxypropyl)-β-cyclodextrin (vehicle) for 4 days. Echocardiography was performed on day 2 for both cohorts. Cohort 2 had additional blood pressure measurements on day 2. On day 4, cohort 1 underwent terminal in vivo LVP measurements and ex vivo vascular reactivity assessments. During wash-out, in vivo echocardiography and blood pressure measurements were done on day 7, and ex vivo vascular reactivity was assessed on day 8. Dashed line indicated in vivo measurements; Solid line indicate terminal experiments. HPβCD = 40% (2-Hydroxypropyl)-β-cyclodextrin; LVP = left ventricular pressure

Fig. 2. Blood pressure changes in lenvatinib-treated mice on day 2 and 7. Lenvatinib increased both systolic and diastolic blood pressure in mice. A SBP = systolic blood pressure; (B) DBP = diastolic blood pressure; Data are represented as mean ± SEM. N = 8 per cohort. Two-way ANOVA was conducted for between-group comparison (*p < 0.05)

Fig. 3. Blood pressure changes and pressure dependency of Peterson’s elastic modulus in lenvatinib-treated mice. Lenvatinib increased both systolic and diastolic blood pressure in mice. (A) PWV = pulse wave velocity; (B) PP = pulse pressure. Pressure dependency of Peterson’s elastic modulus (EP) was measured after stimulation with 2 μM PE in the presence of 300 μM L-NAME (C) or after relaxation with 2 μM DEANO (D). Data are represented as mean ± SEM. N = 7–8 per group. Two-way ANOVA was conducted for between-group comparison (****p < 0.0001)

Fig. 4. Relaxation of thoracic aortic segments and aortic mRNA expression of P2Y2 receptor and, P2Y6 receptor in lenvatinib-treated mice on day 4 and 8. Following maximal contraction with PE, VSMC relaxation was assessed in response to increasing concentrations of ATP (A&C), or DEANO (B&D). Lenvatinib increased both receptor mRNA expression on day 2 but not on day 8 (E&F). Vascular mRNA expression is normalised to the internal reference gene β-actin and is expressed relative to the vehicle-treated group. ATP = adenosine triphosphate; DEANO = diethylamine NONOate; dr = dose–response. N = 6 per group. Data are mean ± SEM. Data were analysed using a Two-way ANOVA with Dunnett’s post hoc test was performed (*p < 0.05, **p < 0.01, ***p < 0.001)

Fig. 5. Echocardiography in lenvatinib-treated mice on day 2 (cohort 1 & 2) and 7 (cohort 2). Lenvatinib caused a reversible reduction in LVEF. A LVEF of mice from cohort 1 was measured on day 2. B LVEF in mice from cohort 2 on both examination days. LVEF = left ventricular ejection fraction. Data is represented as mean ± SEM. N = 8 per cohort. A Mann–Whitney test (B) Two-way ANOVA (***p < 0.001, ****p < 0.0001)

Fig. 6. Indices of diastolic (A, B, C) and systolic function (D, E) derived from LVP analysis in lenvatinib-treated mice. (A) EDP = end-diastolic pressure; (B) dP/dtmin = peak rate of pressure decreases in the ventricle; (C) Tau = isovolumetric relaxation constant; (D) ESP = end-systolic pressure; (E) dP/dtmax = peak rate of pressure rises in the ventricle; Data points are means; vertical bars represent SEM. N = 10 for lenvatinib and N = 10 for control. Unpaired t-test with Welch’s correction was performed for between-group comparison

Fig. 7. Laminin immunohistochemical staining of cardiac tissue to quantify cardiomyocyte cross-sectional area. Representative images of cardiomyocyte cross-sectional area following 4-day treatment with vehicle (40% HPβCD) (A, B), and lenvatinib (4 mg/kg) (C, D). Scale bar = 50 µm. E Average of 100 analysed cardiomyocyte cross-sectional area per mouse. CM = cardiomyocyte. Data points are means; error bars represent SEM. Mann–Whitney test was performed (***p < 0.001)