Extracellular Vesicles Released after Doxorubicin Treatment in Rats Protect Cardiomyocytes from Oxidative Damage and Induce Pro-Inflammatory Gene Expression in Macrophages

Abstract

Doxorubicin (DOXO)-induced cardiomyopathy (DIC) is a lethal complication in cancer patients. Major mechanisms of DIC involve oxidative stress in cardiomyocytes and hyperactivated immune response. Extracellular vesicles (EVs) mediate cell–cell communication during oxidative stress. However, functions of circulating EVs released after chronic DOXO exposure on cardiomyocytes and immune cells are still obscured. Herein, we developed a DIC in vivo model using male Wistar rats injected with 3 mg/kg DOXO for 6 doses within 30 days (18 mg/kg cumulative dose). One month after the last injection, the rats developed cardiotoxicity evidenced by increased BCL2-associated X protein and cleaved caspase-3 in heart tissues, along with N-terminal pro B-type natriuretic peptide in sera. Serum EVs were isolated by size exclusion chromatography. EV functions on H9c2 cardiomyocytes and NR8383 macrophages were evaluated. EVs from DOXO-treated rats (DOXO_EVs) attenuated ROS production via increased glutathione peroxidase-1 and catalase gene expression, and reduced hydrogen peroxide-induced cell death in cardiomyocytes. In contrast, DOXO_EVs induced ROS production, interleukin-6, and tumor necrosis factor-alpha, while suppressing arginase-1 gene expression in macrophages. These results suggested the pleiotropic roles of EVs against DIC, which highlight the potential role of EV-based therapy for DIC with a concern of its adverse effect on immune response.

Potential roles of 4HNE-adducted protein in serum extracellular vesicles as an early indicator of oxidative response against doxorubicin-induced cardiomyopathy in rats

“…The positive correlation of 4HNE adduction in serum EVs with Cat and Gpx1 gene expression in heart tissues is consistent with our previous in vitro study finding, which demonstrated that EVs extracted from serum of DOXO-treated animals at the endpoint effectively reduced ROS formation of H9c2 cardiomyocytes via increased Cat and Gpx1 gene expression and reduced hydrogen peroxide-induced cell death ( Yarana et al, 2022 ). Further animal studies should be conducted to increase the protective impact of EVs on cardiac function by pretreating animals with DOXO_EVs prior to DOXO administration and determining whether these EV-treated rats had improved cardiac functions compared to those who did not get EV pretreatment.…”

“…1 A ). Using this protocol, we previously reported that all rats developed cardiomyopathy, as evidenced by a decrease in cardiac functions, the development of cardiac sympathovagal imbalance, and an increase in pro-apoptotic proteins in the left ventricles ( Yarana et al, 2022 ). In this study, serum NT-proBNP in the NSS group significantly increased over time beginning at day 9 post treatment.…”

“…Despite the absence of a universally accepted model for chronic cardiotoxicity of DOXO in rats, commonly used long-term treatment models with DOXO 1–5 mg/kg once weekly for 2–12 weeks at a cumulative dose of 3–25 mg/kg are found suitable for evaluating chronic cardiotoxicity ( Podyacheva et al, 2021 ). Our research team developed a rat model of chronic DOXO treatment at the dosage of 3 mg/kg every four days for three times, followed by once per week for three weeks, totaling 18 mg/kg, and discovered that 30 days after the last treatment (60 days after the treatment's initiation), the rats had impaired cardiac function ( Arinno et al, 2021 , Chunchai et al, 2022 , Khuanjing et al, 2021 , Maneechote et al, 2022 , Yarana et al, 2022 ). Using the same treatment protocol, the current study demonstrated that the levels of NT-proBNP in the DOXO group were substantially higher than those in the NSS group at day 30 and 60 after beginning treatment, indicating the development of cardiotoxic effects.…”

“…We chose the dose of 3 mg/kg per dose, a total of 18 mg/kg, and a one-month recovery period after the last treatment based on previous research showing that at this time point, rats treated with DOXO based on this protocol had impaired cardiac function ( Arinno et al, 2021 , Chunchai et al, 2022 , Khuanjing et al, 2021 , Maneechote et al, 2022 , Yarana et al, 2022 ). This impairment of cardiac function is a crucial aspect to be investigated in our study.…”

Challenges and Opportunities for Extracellular Vesicles in Clinical Oncology Therapy

“…In recent years, EVs have been found in a variety of physiological fluids including blood, urine, saliva, breast milk, amniotic fluid, ascites, cerebrospinal fluid, bile, and semen [ 8 , 9 , 10 , 11 , 12 ]. More importantly, EVs can be directly isolated from physiological fluid in solid organs including the liver, heart, brain, and others [ 13 , 14 , 15 , 16 , 17 ]. Originally, the majority of studies focused on clinical diagnosis; however, in recent years, EVs have shown a bright future as an excellent therapeutic vehicle for treating diseases, especially cancer.…”