Acute vascular and cardiac effects of lenvatinib in mice

Abstract

Background: Tyrosine kinase inhibitors (TKIs) targeting vascular endothelial growth factor (VEGF) receptor signalling are used in cancer therapy to inhibit angiogenesis. Unfortunately, VEGF inhibitors are known to induce severe hypertension in patients. This study aimed to elucidate the impact of the TKI lenvatinib on blood pressure, arterial stiffness, vascular reactivity, as well as cardiac function in a short-term murine model to shed light on potential contributors to cardiovascular (CV) toxicities associated with VEGF inhibition.

Methods: Male C57BL/6J mice were randomly divided into 2 cohorts, either treated for 4 days with lenvatinib 4 mg/kg/day or 40% hydroxypropyl β-cyclodextrin as control. In an additional study, mice were subjected to a 4-day treatment followed by a 4-day wash-out, with echocardiography and blood pressure measurements performed on day 2 and 7. Subsequently, ex vivo vascular reactivity of thoracic aortic segments was determined.

Results: Lenvatinib induced hypertension and arterial stiffness (i.e., increased pulse wave velocity), starting from day 2 of treatment. Further, left ventricular ejection fraction was reduced and the ventricle dilated upon treatment. Lenvatinib induced neither endothelial dysfunction nor impaired vascular smooth muscle cell reactivity to nitric oxide (NO). Interestingly, lenvatinib demonstrated a concentration-dependent increase in ATP-mediated relaxation. In addition, after the 4-day wash-out period, lenvatinib-treated mice did not show complete remission of hypertension. However, arterial stiffness, ATP-mediated relaxation and cardiac adaptation were recovered.

Conclusion: This comprehensive investigation provides valuable insights into the interplay between VEGF inhibition, vascular function and cardiac outcomes, emphasising the need for nuanced understanding and further exploration of the differential effects of lenvatinib on the CV system. Additionally, the study proposes a synergistic formation between VEGF and ATP, indicating an enhanced response via P2Yx receptor signalling.

Keywords: ATP; Cardiovascular toxicity; Hypertension; Lenvatinib; TKIs; VEGFR.

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/dt_min = peak rate of pressure decreases in the ventricle; (C) Tau = isovolumetric relaxation constant; (D) ESP = end-systolic pressure; (E) dP/dt_max = 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)