Dexrazoxane does not mitigate early vascular toxicity induced by doxorubicin in mice.

Apart from cardiotoxicity, the chemotherapeutic agent doxorubicin (DOX) provokes acute and long-term vascular toxicity. Dexrazoxane (DEXRA) is an effective drug for treatment of DOX-induced cardiotoxicity, yet it remains currently unknown whether DEXRA prevents vascular toxicity associated with DOX. Accordingly, the present study aimed to evaluate the protective potential of DEXRA against DOX-related vascular toxicity in a previously-established in vivo and ex vivo model of vascular dysfunction induced by 16 hour (h) DOX exposure. Vascular function was evaluated in the thoracic aorta in organ baths, 16h after administration of DOX (4 mg/kg) or DOX with DEXRA (40 mg/kg) to male C57BL6/J mice. In parallel, vascular reactivity was evaluated after ex vivo incubation (16h) of murine aortic segments with DOX (1 µM) or DOX with DEXRA (10 µM). In both in vivo and ex vivo experiments, DOX impaired acetylcholine-stimulated endothelium-dependent vasodilation. In the ex vivo setting, DOX additionally attenuated phenylephrine-elicited vascular smooth muscle cell (VSMC) contraction. Importantly, DEXRA failed to prevent DOX-induced endothelial dysfunction and hypocontraction. Furthermore, RT-qPCR and Western blotting showed that DOX decreased the protein levels of topoisomerase-IIß (TOP-IIß), a key target of DEXRA, in the heart, but not in the aorta. Additionally, the effect of N-acetylcysteine (NAC, 10 µM), a reactive oxygen species (ROS) scavenger, was evaluated ex vivo. NAC did not prevent DOX-induced impairment of acetylcholine-stimulated vasodilation. In conclusion, our results show that DEXRA fails to prevent vascular toxicity resulting from 16h DOX treatment. This may relate to DOX provoking vascular toxicity in a ROS- and TOP-IIß-independent way, at least in the evaluated acute setting. However, it is important to mention that these findings only apply to the acute (16h) treatment period, and further research is warranted to delineate the therapeutic potential of DEXRA against vascular toxicity associated with longer-term repetitive DOX dosing.

Doxorubicin-induced cardiovascular toxicity: a longitudinal evaluation of functional and molecular markers.

AIMS: Apart from cardiotoxicity, the chemotherapeutic doxorubicin (DOX) induces vascular toxicity, represented by arterial stiffness and endothelial dysfunction. Both parameters are of interest for cardiovascular risk stratification as they are independent predictors of future cardiovascular events in the general population. However, the time course of DOX-induced cardiovascular toxicity remains unclear. Moreover, current biomarkers for cardiovascular toxicity prove insufficient. Here, we longitudinally evaluated functional and molecular markers of DOX-induced cardiovascular toxicity in a murine model. Molecular markers were further validated in patient plasma. METHODS AND RESULTS: DOX (4 mg/kg) or saline (vehicle) was administered intra-peritoneally to young, male mice weekly for 6 weeks. In vivo cardiovascular function and ex vivo arterial stiffness and vascular reactivity were evaluated at baseline, during DOX therapy (Weeks 2 and 4) and after therapy cessation (Weeks 6, 9, and 15). Left ventricular ejection fraction (LVEF) declined from Week 4 in the DOX group. DOX increased arterial stiffness in vivo and ex vivo at Week 2, which reverted thereafter. Importantly, DOX-induced arterial stiffness preceded reduced LVEF. Further, DOX impaired endothelium-dependent vasodilation at Weeks 2 and 6, which recovered at Weeks 9 and 15. Conversely, contraction with phenylephrine was consistently higher in the DOX-treated group. Furthermore, proteomic analysis on aortic tissue identified increased thrombospondin-1 (THBS1) and alpha-1-antichymotrypsin (SERPINA3) at Weeks 2 and 6. Up-regulated THBS1 and SERPINA3 persisted during follow-up. Finally, THBS1 and SERPINA3 were quantified in plasma of patients. Cancer survivors with anthracycline-induced cardiotoxicity (AICT; LVEF < 50%) showed elevated THBS1 and SERPINA3 levels compared with age-matched control patients (LVEF ≥ 60%). CONCLUSIONS: DOX increased arterial stiffness and impaired endothelial function, which both preceded reduced LVEF. Vascular dysfunction restored after DOX therapy cessation, whereas cardiac dysfunction persisted. Further, we identified SERPINA3 and THBS1 as promising biomarkers of DOX-induced cardiovascular toxicity, which were confirmed in AICT patients.

Cardiac electrophysiology studies in mice via the transjugular route: a comprehensive practical guide.

Cardiac arrhythmias are associated with cardiovascular morbidity and mortality. Cardiac electrophysiology studies (EPS) use intracardiac catheter recording and stimulation for profound evaluation of the heart's electrical properties. The main clinical application is investigation and treatment of rhythm disorders. These techniques have been translated to the murine setting to open opportunities for detailed evaluation of the impact of different characteristics (including genetics) and interventions on cardiac electrophysiology and -pathology. Currently, a detailed description of the technique of murine transjugular EPS (which is the standard route of catheter introduction) is lacking. This article provides detailed information on EPS in mice via the transjugular route. This includes catheter placement, stimulation protocols, intracardiac tracing interpretation, artifact reduction, and surface ECG recording. In addition, reference values as obtained in C57BL/6N mice are presented for common electrophysiological parameters. This detailed methodological description aims to increase accessibility and standardization of EPS in mice. Ultimately, also human research and patient care may benefit from translation of the knowledge obtained in preclinical models using this technique.NEW & NOTEWORTHY Electrophysiology studies (EPS) allow in-depth evaluation of cardiac electrophysiology and -pathology. These techniques have been adapted to the murine setting for (translational) studies, mainly focusing on arrhythmogenesis. Despite the frequent application of EPS via the transjugular route, a thorough description of the technique is currently lacking. This article aims to function as a comprehensive guide, also elaborating (for the first time) on nonsurgical aspects such as catheter positioning, tracing artifacts, stimulation protocols, and reference values.

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

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.

Toll-Like Receptors: Are They Taking a Toll on the Heart in Viral Myocarditis?

Myocarditis is an inflammatory disease of the heart with viral infections being the most common aetiology. Its complex biology remains poorly understood and its clinical management is one of the most challenging in the field of cardiology. Toll-like receptors (TLRs), a family of evolutionarily conserved pattern recognition receptors, are increasingly known to be implicated in the pathophysiology of viral myocarditis. Their central role in innate and adaptive immune responses, and in the inflammatory reaction that ensues, indeed makes them prime candidates to profoundly affect every stage of the disease process. This review describes the pathogenesis and pathophysiology of viral myocarditis, and scrutinises the role of TLRs in every phase. We conclude with directions for future research in this field.

A systematic literature review on the effects of exercise on human Toll-like receptor expression.

BACKGROUND: Toll-like receptors (TLRs) are a family of transmembrane pattern recognition receptors that are mainly expressed on immune cells. Recognition of various exogenous and endogenous molecular patterns activates the TLR signalling cascade, which orchestrates an inflammatory immune response. Dysfunctional immune responses, including aberrant TLR signalling, are increasingly implicated in the associations between sedentarism, chronic low-grade systemic inflammation and various non-communicable diseases. Conversely, exercise exerts anti-inflammatory effects, which could be conferred through its immunomodulatory properties, potentially affecting TLRs. This study aims to systematically review the effects of exercise on human TLR expression. METHOD: A systematic literature search of Pubmed, Embase, The Cochrane Library and SPORTDiscus for articles addressing the impact of exercise (as isolated intervention) on TLRs in humans was conducted, ending in February 2020. RESULTS: A total of 66 articles were included. The publications were categorised according to exercise modality and duration: acute resistance exercise (4 studies), acute aerobic exercise (26 studies), resistance training program (9 studies), aerobic training program (16 studies), combined (i.e. resistance and aerobic) training program (8 studies) and chronic exercise not otherwise classifiable (9 studies). Five articles investigated more than one of the aforementioned exercise categories. Several trends could be discerned with regard to the TLR response in the different exercise categories. Acute resistance exercise seemed to elicit TLR upregulation, whereas acute aerobic exercise had less activating potential with the majority of responses being neutral or, especially in healthy participants, downregulatory. Chronic resistance and combined exercise programs predominantly resulted in unaltered or decreased TLR levels. In the chronic aerobic exercise category, mixed effects were observed, but the majority of measurements demonstrated unchanged TLR expression. CONCLUSION: Currently published research supports an interplay between exercise and TLR signalling, which seems to depend on the characteristics of the exercise. However, there was large heterogeneity in the study designs and methodologies. Therefore, additional research is required to further corroborate these findings, to define its pathophysiological implications and to elucidate the mechanism(s) linking exercise to TLR signalling.

A reliable set of reference genes to normalize oxygen-dependent cytoglobin gene expression levels in melanoma.

Cytoglobin (CYGB) is a ubiquitously expressed protein with a protective role against oxidative stress, fibrosis and tumor growth, shown to be transcriptionally regulated under hypoxic conditions. Hypoxia-inducible CYGB expression is observed in several cancer cell lines and particularly in various melanoma-derived cell lines. However, reliable detection of hypoxia-inducible mRNA levels by qPCR depends on the critical choice of suitable reference genes for accurate normalization. Limited evidence exists to support selection of the commonly used reference genes in hypoxic models of melanoma. This study aimed to select the optimal reference genes to study CYGB expression levels in melanoma cell lines exposed to hypoxic conditions (0.2% O2) and to the HIF prolyl hydroxylase inhibitor roxadustat (FG-4592). The expression levels of candidate genes were assessed by qPCR and the stability of genes was evaluated using the geNorm and NormFinder algorithms. Our results display that B2M and YWHAZ represent the most optimal reference genes to reliably quantify hypoxia-inducible CYGB expression in melanoma cell lines. We further validate hypoxia-inducible CYGB expression on protein level and by using CYGB promoter-driven luciferase reporter assays in melanoma cell lines.

Doxorubicin induces arterial stiffness: A comprehensive in vivo and ex vivo evaluation of vascular toxicity in mice.

Arterial stiffness is an important predictor of cardiovascular risk. Clinical studies have demonstrated that arterial stiffness increases in cancer patients treated with the chemotherapeutic doxorubicin (DOX). However, the mechanisms of DOX-induced arterial stiffness remain largely unknown. This study aimed to evaluate artery stiffening in DOX-treated mice using in vivo and ex vivo techniques. Male C57BL/6J mice were treated for 2 weeks with 2 mg/kg (low dose) or 4 mg/kg (high dose) of DOX weekly. Arterial stiffness was assessed in vivo with ultrasound imaging (abdominal aorta pulse wave velocity (aaPWV)) and applanation tonometry (carotid-femoral PWV) combined with ex vivo vascular stiffness and reactivity evaluation. The high dose increased aaPWV, while cfPWV did not reach statistical significance. Phenylephrine (PE)-contracted aortic segments showed a higher Peterson's modulus (Ep) in the high dose group, while Ep did not differ when vascular smooth muscle cells (VSMCs) were relaxed by a NO donor (DEANO). In addition, aortic rings of DOX-treated mice showed increased PE contraction, decreased basal nitric oxide (NO) index and impaired acetylcholine-induced endothelium-dependent relaxation. DOX treatment contributed to endothelial cell loss and reduced endothelial nitric oxide synthase (eNOS) expression in the aorta. In conclusion, we have replicated DOX-induced arterial stiffness in a murine model and this aortic stiffness is driven by impaired endothelial function, contributing to increased vascular tone.