Extracellular vesicle-enriched secretome of adipose-derived stem cells upregulates clusterin to alleviate doxorubicin-induced apoptosis in cardiomyocytes

Abstract

Doxorubicin (DOX) is a potent chemotherapeutic widely used against various cancers, but its clinical application is limited by DOX-induced cardiotoxicity (DIC). This study explored the cardioprotective potential of extracellular vesicle-enriched secretome derived from adipose stem cells (EVSASC) in mitigating DOX-induced apoptosis in cardiomyocytes. Adipose-derived stem cells were cultured, and their conditioned medium and extraceullular vesicles were isolated and characterized according to the Minimal Information for Studies of Extracellular Vesicles 2023 guidelines. HL-1 cardiomyocytes were pretreated with EVSASC before exposure to 1 µM DOX. Cell viability was assessed via the cell counting kit-8 assay, while apoptosis markers and survival mediators were evaluated through Western blotting. RNA sequencing identified differentially expressed genes, including clusterin (Clu), which was further quantified using an enzyme-linked immunosorbent assay. The functional role of clusterin was validated through siRNA-mediated knockdown. EVSASC significantly improved cell viability in DOX-exposed cardiomyocytes and reduced the cleaved caspase-3 to procaspase-3 ratio. Clusterin expression was highest in EVSASC-treated cells, and its knockdown markedly increased caspase-3 cleavage, confirming its pivotal role in cardioprotection. Moreover, EVSASC enhanced the phosphorylation of AKT, Bcl2-associated agonist of cell death, and glycogen synthase kinase-3β, implicating PI3K/AKT pathway activation in clusterin upregulation and anti-apoptotic effects. These findings demonstrate that EVSASC mitigates DOX-induced apoptosis in cardiomyocytes through clusterin upregulation and PI3K/AKT pathway activation. Clusterin is identified as a potential biomarker for evaluating EVSASC efficacy. While EVSASC shows promise as a cardioprotective strategy against DIC, further studies are needed to optimize its therapeutic safety by addressing potential oncogenic risks.