Doxorubicin-induced vasotoxicity in coronary vessels

: Investigating the G-protein coupled receptor mediated vasoconstriction and the effect of the MEK 1/2 inhibitor U0126

Abstract

Doxorubicin is one of the most efficient chemotherapeutic drugs used for various cancers, however, therapy with doxorubicin is associated with cardiotoxicity. The effect of doxorubicin on the vasculature remains an uncharted research area. Vascular injury can lead to an increased G-protein coupled receptor (GPCR) mediated vasoconstriction, such as through endothelin receptors subtype A (ETA) and B (ETB), 5-Hydroxytryptamine 1B (5-HT1B) receptor and thromboxane prostanoid (TP) receptor. Furthermore, the expression of some GPCRs is regulated through the MEK 1/2 pathway. This study used an organ culture model to investigate the GPCR-mediated vasocontractility in 0.5 µM doxorubicin-treated left anterior descending artery (LAD) from Sprague-Dawley rats by wiremyography. The involvement of the MEK/ERK 1/2 pathway was investigated by co-incubation withU0126 (5 µM). The following GPCR agonists were used to obtain the individual dose-response curves: ETB agonist sarafotoxin 6c (S6c), ETA and ETB agonist endothelin-1 (ET-1), 5-HT1B agonist 5-carboxamidotryptamine (5-CT), and TP agonist U46619. The specificity of each agonist was verified by the application of specific antagonists. The GPCR mRNA levels and GPCR expression and localisation on LAD arteries were investigated by Real-Time PCR and Immunohistochemistry. In addition, the circulating plasma miRNA levels in cancer patients treated with anthracycline chemotherapy includingdoxorubicin were studied to assess the expression pattern of circulating miRNAs, with particular focus on miRNAs associated with cardiac- and vascular- injury (collaborative project with Prof M J Walker, UCL). The miRNA expression levels in cancer patient plasma samples were investigated with TaqMan array card technology and individual RT-qPCR verification.Application of doxorubicin altered the LAD vasoconstriction through GPCRs ETB, 5-HT1B and TP, and the molecular assessment revealed both transcriptional and translational mechanisms were involved. Furthermore, co-incubation with U0126 highlighted that the MEK/ERK 1/2 pathway plays a key role during the doxorubicin-altered GPCRs-mediated vasoconstriction. Analysis of the cancer patient plasma samples pre- and post- anthracycline chemotherapy revealed candidate vascular injuryassociated miRNAs. Profiling of pooled plasma samples from the miRNA array study showed a significantly altered expression of 174 miRNAs as a result of anthracycline chemotherapy. From those altered miRNAs, five vascular injury associated miRNAs were selected and re-analysed on individual cancer patients samples to investigate specificity towards patients that developed cardiac dysfunction, which revealed two vascular injury associated miRNAs, miR-126 and miR-320a.In conclusion this study shows for the first time that doxorubicin mediated its vasotoxic and cardiotoxic adverse effect on coronary vessels by targeting specific GPCRs through the MEK/ERK 1/2 pathway, and that doxorubicin-treatment of cancer patients alters key circulating vascular injuryassociated miRNAs. Overall, this study has improved the understanding of doxorubicin-mediated cardiovascular adverse effects and the vascular and intracellular mechanisms involved

Date of Award	2023
Original language	English
Awarding Institution	Coventry University
Supervisor	Hardip Sandhu (Supervisor), Helen Maddock (Supervisor) & Mark Wheatley (Supervisor)