Doxorubicin Impairs Smooth Muscle Cell Contraction: Novel Insights in Vascular Toxicity

Abstract

Clinical and animal studies have demonstrated that chemotherapeutic doxorubicin (DOX) increases arterial stiffness, a predictor of cardiovascular risk. Despite consensus about DOX-impaired endothelium-dependent vasodilation as a contributing mechanism, some studies have reported conflicting results on vascular smooth muscle cell (VSMC) function after DOX treatment. The present study aimed to investigate the effects of DOX on VSMC function. To this end, mice received a single injection of 4 mg DOX/kg, or mouse aortic segments were treated ex vivo with 1 μM DOX, followed by vascular reactivity evaluation 16 h later. Phenylephrine (PE)-induced VSMC contraction was decreased after DOX treatment. DOX did not affect the transient PE contraction dependent on Ca²⁺ release from the sarcoplasmic reticulum (0 mM Ca²⁺), but it reduced the subsequent tonic phase characterised by Ca²⁺ influx. These findings were supported by similar angiotensin II and attenuated endothelin-1 contractions. The involvement of voltage-gated Ca²⁺ channels in DOX-decreased contraction was excluded by using levcromakalim and diltiazem in PE-induced contraction and corroborated by similar K⁺ and serotonin contractions. Despite the evaluation of multiple blockers of transient receptor potential channels, the exact mechanism for DOX-decreased VSMC contraction remains elusive. Surprisingly, DOX reduced ex vivo but not in vivo arterial stiffness, highlighting the importance of appropriate timing for evaluating arterial stiffness in DOX-treated patients.

Keywords: arterial stiffness; cardio-oncology; doxorubicin; endothelial dysfunction; non-selective cation channel; vascular smooth muscle cell contraction.

Figure 1

VSMC contraction and relaxation in aortic segments, 16 h after in vivo DOX administration. PE-induced contraction was decreased in aortic segments after in vivo DOX treatment (A). The PE-elicited contraction response was reduced after DOX treatment, which suggests perturbed tonic contraction (B). The increase in force between 800 s and 100 s was lower in the DOX group, indicating that DOX impairs Ca²⁺ influx (C). The DOX-induced reduction in contraction persisted under L-NAME conditions (D). DOX impaired ACh-induced relaxation, but relaxation of aortic segments in response to DEANO did not change (E). For (A,B,E), repeated measures two-way ANOVA with Šidàk’s multiple comparisons test. For (C), unpaired t-test. For (D), two-way ANOVA with Tukey’s multiple comparisons test. *, **, ***, **** p < 0.05, 0.01, 0.001, 0.0001; n = 6 for each group.

Figure 2

VSMC contraction and relaxation in aortic segments after 16 h of ex vivo DOX treatment. Following 16 h of ex vivo DOX treatment, VSMC contraction was decreased in response to PE (A,B). In the DOX-treated aortic segments, there was a lower increase in force between 800 s and 100 s (C). The DOX-induced decrease in contraction was still observed in the presence of L-NAME (D). ACh-induced relaxation, but not DEANO-induced relaxation, was impaired in response to DOX (E). For (A,B,E), repeated measures two-way ANOVA with Šidàk’s multiple comparisons test. For (C), unpaired t-test. For (D), two-way ANOVA with Tukey’s multiple comparisons test. *, **, ***, **** p < 0.05, 0.01, 0.001, 0.0001; n = 8 for vehicle group and n = 9 for DOX group.

Figure 3

Contraction of aortic segments mediated by Ca²⁺ release from the sarcoplasmic reticulum and Ca²⁺ entry through VGCCs and NSCCs after 16 h of ex vivo DOX treatment. (A) Contraction through Ca²⁺ release from the sarcoplasmic reticulum in response to PE (2 μM) in the absence of extracellular Ca²⁺ (0 mM Ca²⁺). (B) Addition of 3.5 mM Ca²⁺ after 0 mM Ca²⁺ PE-elicited contraction. (C) Addition of 35 μM diltiazem following stable CaCl₂-mediated contraction. (D) Dose–response of K⁺-elicited contraction (10, 20, 30, 40 and 50 mM). (E) PE-induced contraction (with L-NAME) under levcromakalim (1 μM). DOX did not change contraction through Ca²⁺ release from the sarcoplasmic reticulum, nor Ca²⁺ uptake and Ca²⁺ efflux after 16 h of ex vivo DOX treatment (A), but VSMC contraction was decreased in the DOX-treated group after CaCl₂ addition (B). There was no difference in VSMC relaxation between the groups after blocking of VGCCs with diltiazem (C), nor in the contraction magnitude in response to different K⁺ doses (D). DOX decreased PE-induced contraction (with L-NAME) in the presence of levcromakalim (E). For (A,B,D,E), repeated measures two-way ANOVA with Šidàk’s multiple comparisons test. For (C), unpaired t-test. *, ***, **** p < 0.05, 0.001, 0.0001; p > 0.05 in (A,C,D); (A–C) n = 10 in each group; (D,E) n = 6 in each group.

Figure 4

Evaluation of the involvement of members of the NSCC family in decreased PE-induced contraction in aortic segments after 16 h of ex vivo DOX treatment. (A) PE-induced contraction (2 μM, with L-NAME) under diltiazem (35 μM). (B,C) Absolute and relative decline in PE-induced contraction (with L-NAME and diltiazem) under different NSCC-blocker conditions. DOX decreased PE-induced contraction (with L-NAME) in the presence of diltiazem (A). In addition, the DOX-treated group did not display a significant change in VSMC contraction in absolute values (B) but showed a greater decline in VSMC contraction when expressed relative to its preceding contraction magnitude under tranilast (100 μM), SKF-96365 (10 μM), 2-APB (20 μM) and 9-phenantrol (1 μM) (C). For (A), repeated measures two-way ANOVA with Šidàk’s multiple comparisons test. For (B,C), unpaired t-test for each NSCC blocking condition. *, **, ***, **** p < 0.05, 0.01, 0.001, 0.0001; p > 0.05 in (B); n = 6 in each group.

Figure 5

Contraction of aortic segments with endothelin-1, angiotensin II, serotonin and K⁺ after 16 h of ex vivo DOX treatment. DOX decreased VSMC contraction in response to 0.25 μM endothelin-1 (A) but not after stimulation with 2 μM angiotensin II (B), 2 μM serotonin (C) and 50 mM K⁺ (D). For (A–D), repeated measures two-way ANOVA with Šidàk’s multiple comparisons test. *, *** p < 0.05, 0.001; p > 0.05 in (B–D); For (A–C), n = 6 in each group; for (D), n = 10 in each group.

Figure 6

Evaluation of in and ex vivo aortic stiffness, 16 h after in vivo DOX administration. (A–C) Ex vivo evaluation of arterial stiffness with ROTSAC set-up. (D,E) In vivo evaluation of arterial stiffness with ultrasound imaging (D) and tonometry (E). (F) Systolic blood pressure measurements. (G) Diastolic blood pressure measurements. (H) Evaluation of LVEF with ultrasound imaging. Ep was lower in the DOX-treated group in a pressure-dependent way under Krebs Ringer conditions (A), but the effect was less pronounced in the presence of PE with L-NAME (B). Ep in response to DEANO was lower after DOX treatment (C). aaPWV (D), cfPWV (E), systolic blood pressure (F), diastolic blood pressure (G) and LVEF (H) were not affected after DOX treatment. For (A–H), repeated measures two-way ANOVA with Šidàk’s multiple comparisons test. *, **** p < 0.05, 0.0001; n = 6 for each group.

Figure 7

Schematic representation of the PE-elicited VSMC contraction response, including the compounds used to delineate the mechanisms of reduced VSMC contraction after DOX treatment. VSMC contraction through stimulation of α1-adrenergic receptors (α1R) with PE is characterised by two components—namely, a fast transient contraction (event 1) and a concomitant tonic contraction (event 2) [26]. Event 1: The fast transient contraction is determined by inositol trisphosphate (IP₃)-mediated release of Ca²⁺ from the sarcoplasmic reticulum through IP₃ receptors (IP₃R) [26]. Event 2: The concomitant tonic contraction is determined by Ca²⁺ influx via VGCCs and NSCCs [26]. In both phases, cytoplasmic Ca²⁺ content increases, thereby forming a Ca²⁺–calmodulin complex [31], which activates the myosin light chain kinase, promotes the binding of actin to myosin and thus initiates contraction [31]. Production of NO in ECs inhibits Ca²⁺ release from the sarcoplasmic reticulum and VGCC- and NSCC-mediated Ca²⁺ influx in a cyclic guanosine monophosphate (cGMP)-dependent way, thereby continuously diminishing contraction [30]. This eNOS-mediated NO production can be inhibited by L-NAME. A 0 mM Ca²⁺ Krebs environment prevents Ca²⁺ influx, which results in exclusively IP₃-mediated contraction. Diltiazem actively inhibits VGCCs, thereby inhibiting VGCC-mediated Ca²⁺ influx. Levcromakalim activates ATP-dependent K⁺ channels (K⁺C), resulting in K⁺ efflux, thereby causing a hyperpolarisation (event *) of the resting membrane potential and consequently deactivating VGCCs. In the presence of 50 mM K⁺, depolarisation will occur (event **), which will promote VGCC-mediated Ca²⁺ influx and subsequently leads to VSMC contraction. DEANO acts as an exogenous NO donor and inhibits VGCCs and NSCCs in a manner similar to NO. Members of the diverse family of NSCCs are TRPV, TRPM, STIM1-Orai1, channels, which can be inhibited with tranilast, SKF-96365, 2-APB and 9-phenantrol. DOX may interfere with these channels through an unclear mechanism. Red lines represent inhibition or deactivation, while green lines correspond to activation or stimulation.