Estudio de la capacidad terapeútica de las vesículas extracelulares procedentes de células estromales mesenquimales de la médula ósea sobre la cardiotoxicidad inducida por doxorubicina

Abstract

[EN] Background The use of anthracyclines increases the survival of some cancer patients, but at the same time it may also promote type I cardiotoxicity in some of them (1). Doxorubicin (dox) is one of the most used anthracyclines to treat a wide range of cancer types (2). Its mechanisms of action in the myocardium include the generation of oxygen reactive species (ROS), the alteration in mitochondrial function, the deregulation of calcium homeostasis and energy generation and the inhibition of topoisomerase II. In addition, its binding to DNA causes the alteration of gene expression and the synthesis of RNA and proteins, which ultimately cause apoptosis, loss of functional cardiomyocytes and irreversible myocardial damage. Currently, treatments to prevent or revert the damage caused by dox are not effective. So, as an alternative, cell therapy with stem cells is one of the areas of greatest scientific interest for its potential use in diseases, especially in those without effective treatment to date (3, 4). MSC exert

their regenerative, anti-inflammatory and immunomodulatory effects not only through direct cell- to-cell contact, but also secreting exosomes and microvesicles named as extracellular vesicles

(EV), which have a size ranging from 30 nm to 1 μm, that are capable of transferring biological molecules to neighbouring and distant cells and exert regulatory effects on them (5-8). Therefore, EV could have a potential therapeutic use in cell therapy programs. Hypothesis and objectives With this background, we wanted to develop an in vitro model of doxorubicin-induced acute myocardial damage (as the most paradigmatic cardiotoxic agent) establishing accurate biological variables that let us evaluate the therapeutic or preventive potential in a reliable way that, on this damage, extracellular vesicles derived from mesenchymal stem cells (MSC-EV) from the bone marrow of healthy donors may exert, after their eventual incorporation, and that allow us to analyse which key mechanisms of this potential beneficial effect could be. To analyse the therapeutic effect of bone marrow MSC-EV from healthy donors on cardiomyocytes in an in vitro model of doxorubicin-induced cardiotoxicity. More specifically we wanted to develop and establish an in vitro model of doxorubicin-induced cardiotoxicity and to evaluate if human bone marrow MSC-EV from healthy donors are able to incorporate into murine cardiomyocytes and to analyze the incorporation rate. Finally, we assessed the effects of EV incorporation into cardiomyocytes damaged by doxorubicin (comparing with control cardiomyocytes) and the mechanisms by which these effects could be induced. Methodology To identify and characterize murine cardiomyocytes (CM) from 1-3-day old C57/BL6 neonatal mice, hearts were removed and the ventricles digested with collagenase II to isolate and culture the cardiomyocytes. First, we tested the cardiomyocyte purity within the ventricle’s cell population at 24 hours in culture by flow cytometry (FC) with the specific antibodies for cardiomyocytes: anti-a-actinin, anti-troponin T and anti-CD309 (VEGFR-2), and with 7-AAD to evaluate their viability. We also identified murine primary cardiomyocytes by immunofluorescence (IF) labeling them with a-actinin. In order to establish the in vitro model of doxorubicin-induced damage, we performed studies of time-dependent response with 1 μM of doxorubicin in which we evaluated cell viability in order to test the differential response to different times (1, 3, 6, 12 and 24 hours) and thus be able to select the time in which the viability decreases to perform the subsequent co-culture experiments with extracellular vesicles. The release of cardiac troponin T (cTnT), as a biomarker of acute cardiac damage (9), into the culture medium by cardiomyocytes treated with 1 μM of doxorubicin was evaluated after 1, 3, 6, 12- and 24-hours post-treatment. We also evaluated the pulsations of primary cardiomyocytes after 24 hours of treatment. For the characterization and incorporation of MSC-EV into murine primary cardiomyocytes, MSC from 20 healthy donors were isolated from bone marrow and expanded until culture passage 6. MSC were characterised analysing by FC different antibodies: CD73, CD90, CD105, CD44, CD166, CD14, CD19, CD34, CD45 and HLA-DR. Their in vitro differentiation capability to adipocytes and osteoblasts was also evaluated in passage 3. We purified the EV-MSC from healthy donors released into the culture medium by ultracentrifugation and identified and characterized them by transmission electron microscopy (TEM) and nanoparticle-tracking analysis (NTA). To study if human MSC-derived EV were able to incorporate into murine cardiomyocytes, we added fluorescence dye-labelled MSC-EV to primary cardiomyocytes during different times (1, 3, 6 and 24 hours) to study the optimal incorporation time by FC and we confirmed the incorporation of MSC-EV by IF after 24 hours. Co-culture of MSC-EV with cardiomyocytes treated with doxorubicin was conducted to assess whether MSC-EV were able to attenuate doxorubicin-induced cardiac damage. We analysed the effect of MSC-EV incorporation into cardiomyocytes damaged with 1�M doxorubicin through the viability by MTT, luminescence (ATP production) and FC (Annexin V – 7-AAD) and cell death by luminescence (activity of caspases-3/7) and FC (Annexin V – 7- AAD). We also evaluated cTnT release by luminescence, the rate of beating, ROS production and DNA damage through FC and cellular response to stress through p21 expression by RT-PCR. Results We detected the presence of cardiomyocytes in the cell population with a mean of viability of 94,3% ± 4,4 at 24 hours. The viability of cardiomyocytes treated with 1 μM of doxorubicin decreased in a time- dependent manner, with the greatest reduction occurring at 24 hours compared to untreated

control cardiomyocytes (76,4% ± 8,6 viability versus 100%, respectively). Regarding the cTnT release, there were only statistical differences 24 hours post-treatment. At 24 hours after treatment with doxorubicin, a significant reduction in mean beats/min was observed versus baseline cardiomyocytes (42 ± 8,7 vs 85 ± 13,8). MSC characterization showed that: MSC adhered to a plastic surface with a characteristic fibroblast morphology; MSC expressed CD73, CD90, CD105, CD44 and CD166 and were negative for CD14, CD19, CD34, CD45 and HLA-DR; and MSC differentiated to the osteoblastic line, observing a polygonal morphology and positivity after the cytochemical labelling of alkaline phosphatase and they also differentiated to the adipogenic line, forming the lipid vacuoles stained through Oil-Red-O dye, according to the defining criteria of the International Society of Cell Therapy (ISCT) (10). TEM confirmed that MSC-EV had a rounded morphology and a characteristic size (an average size of 215 nm) and a concentration of 1,05·106 particles/mL was observed by NTA, which values are established by the International Society for Extracellular Vesicles (ISEV) (11). FC analysis revealed that EV were incorporated from the first hour of incubation (0.61%), and the highest rate of incorporation was observed after 24 hours (moment in which 21,5% ± 8,3 of the cardiomyocytes had incorporated EV). The incorporation of MSC-EV from healthy donors into cardiomyocytes after 24 hours by confocal microscopy was also confirmed by IF. In the studies of viability and death of murine primary cardiomyocytes by FC, doxorubicin treatment for 24 hours and also at 48 hours decreased the viability of murine cardiomyocytes in a statistically significant way. The presence of EV did not reverse this deleterious effect on doxorubicin-induced viability. From the point of view of apoptosis, doxorubicin for 24 hours does not significantly increase apoptosis, neither early nor late, while it does induce a significant increase in necrosis. The incorporation of EV into basal cardiomyocytes (without cardiotoxic treatment) or into treated cardiomyocytes did not cause significant changes in apoptosis or necrosis. With regard to the data at 48 hours after doxorubicin, the most relevant data is that the incorporation of EV into cardiomyocytes damaged with doxorubicin does significantly decrease cell necrosis, without relevant changes in the other parameters (similar to changes at 24 hours). In the study of cell viability using MTT, it is also observed that the incorporation of EV into cardiomyocytes damaged with doxorubicin did not reverse the reduction in cell viability induced by the drug. The same was shown by quantifying the intracellular amount of ATP present in cardiomyocytes at 24 hours and at 48 hours. After 24 hours of treatment with doxorubicin, a significant activation of caspases 3/7 was detected versus basal cardiomyocytes and the incorporation of EV from MSC into cardiomyocytes treated with doxorubicin did not reverse this effect. Both 24 hours and 48 hours after treatment with 1 μM of doxorubicin, there was a significant increase in the release of cardiac TnT to the culture medium. The addition of MSC-EV simultaneously with the drug significantly reduced this release of cardiac TnT at both times, even reaching values similar to those of undamaged cells. In basal cardiomyocytes, the mean value of the beating rate in vitro was 85,5 ± 13,8 beats

per minute, observing a significant reduction in cells treated with doxorubicin for 24 hours. MSC- EV incorporation into damaged cardiomyocytes reversed this effect significantly.

The damage induced in cardiomyocytes by doxorubicin induced a significant increase in ROS levels after 24 hours of treatment, and the incorporation of EV into damaged cells reversed this effect, significantly reducing the intracellular levels of ROS. In basal conditions, cardiomyocytes presented low percentages of double-stranded breaks in DNA. Treatment with doxorubicin caused a significant increase in DNA breaks, which was significantly reversed after the incorporation of MSC-EV together with the drug. Doxorubicin induced a significant increase in total DNA damage and the incorporation of MSC-EV significantly reduced DNA damage at 24 hours. Finally, we evaluated the gene expression of p21, related to cellular response to stress, and the results showed that treatment with 1 μM of doxorubicin stimulated p21 expression, and the incorporation of MSC-EV into damaged cardiomyocytes did not induce significant changes. Conclusions Regarding the development of an in vitro model of doxorubicin-induced cardiotoxicity: 1. The addition of doxorubicin (1 μM of doxorubicin for 24 hours) in the culture media containing neonatal murine cardiomyocytes induces acute cardiotoxicity in vitro decreasing viability and contractility and increasing cTnT release to the supernatant. This in vitro model may be employed to analyze drug-induced cardiotoxicity. Regarding the incorporation of BM-MSC-EV into doxorubicin-damaged cardiomyocytes: 2. BM-MSC-EV are able to incorporate into murine primary cardiomyocytes in a time dependent manner, with higher efficiency after 24 hours of incubation. Regarding the effects of EV incorporation into cardiomyocytes damaged by doxorubicin and the mechanisms by which these effects could be induced: 3. BM-MSC-EV incorporation do not prevent cell death and are insufficient to compensate for the loss of cardiomyocytes occurring during acute injury at 24 hours, while they do decrease necrosis after 48 hours of doxorubicin treatment. 4. Nevertheless, BM-MSC-EV incorporation contributes to reduce chemotherapy-induced cardiotoxicity by decreasing cTnT release and ROS production, improving cardiomyocytes’ contractility and attenuating DNA damage in our in vitro model.