Megakaryocyte extracellular vesicles plays a role in doxorubicin induced cardiovascular damage

Abstract Introduction Doxorubicin-induced cardiovascular toxicity is a major complication of cancer therapy, contributing significantly to morbidity and mortality. Anthracyclines such as doxorubicin are widely used chemotherapeutics, but their cardiotoxic effects extend beyond myocardial injury to include vascular dysfunction. These effects involve endothelial damage, oxidative stress, and chronic inflammation, collectively impairing vascular integrity and cardiac function. Despite extensive clinical observations, the cellular and molecular pathways remain incompletely understood. Megakaryocytes, bone marrow precursors of platelets, are emerging as key players in vascular homeostasis and inflammatory signaling. They also produce extracellular vesicles (EVs), which transport bioactive molecules—proteins, lipids, and RNAs—that influence vascular and immune responses. We hypothesize that doxorubicin triggers a pro-inflammatory, dysfunctional phenotype in megakaryocytes, altering EV cargo and contributing to endothelial injury and cardiac dysfunction during anthracycline therapy. Materials and Methods Human megakaryocytic MEG-01 cells were treated with clinically relevant doxorubicin (0.2 µM, 3 days). Senescence was assessed by β-galactosidase staining and DNA damage by γH2AX immunofluorescence. Gene expression of senescence and inflammatory markers (p21, IL-6, MMP, VCAM-1) was analyzed by RT-qPCR. EVs were isolated by differential centrifugation and ultracentrifugation; size and concentration were determined by nanoparticle tracking analysis (NTA). RNA from EVs was analyzed for TGF-β1, IL-6, and TNFα by RT-qPCR. To assess functional relevance, fibroblasts were treated for one week with EVs from control and doxorubicin-treated MEG-01 cultures, then stained for α-smooth muscle actin (α-SMA) to evaluate myofibroblast activation. Results Doxorubicin-treated MEG-01 cells showed increased β-galactosidase-positive cells and γH2AX foci, confirming senescence and DNA damage. RT-qPCR revealed upregulation of VCAM-1, p21, and MMP9. EVs from treated cells carried elevated IL-6 and TGF-β1 transcripts, suggesting selective loading of pro-inflammatory cargo. When applied to fibroblasts, these EVs induced α-SMA expression and morphological changes consistent with a myofibroblast-like phenotype, indicating EV-mediated fibroblast activation. Conclusion Our results demonstrate that doxorubicin induces a senescence-associated, pro-inflammatory phenotype in megakaryocytes reflected in EV cargo. These EVs activate fibroblasts and promote a pro-fibrotic phenotype, revealing a novel mechanism by which chemotherapy-induced bone marrow stress may contribute to systemic vascular inflammation and tissue remodeling.For image description, please refer to the figure legend and surrounding text.