Comment on: Prieto-Vila M, Yoshioka Y, Kuriyama N, Okamura A, Yamamoto Y, Muranaka A, et al. Adult cardiomyocytes-derived EVs for the treatment of cardiac fibrosis. J Extracell Vesicles. 2024;13(7): e12461. https://doi.org/10.1002/jev2.12461.

Cardiac fibrosis is a major pathological process that contributes to the development and progression of various cardiovascular diseases (CVDs), leading to impaired cardiac function and ultimately heart failure [1]. Despite remarkable advancements in contemporary medical interventions, there remains an unmet need for therapeutic strategies capable of directly addressing and reversing the underlying fibrotic alterations within the myocardium [2]. In this context, the paper by Prieto-Vila et al. recently published in the Journal of Extracellular Vesicles unveils an innovative and promising approach using extracellular vesicles (EVs) derived from adult human cardiomyocytes (CMs) as a potential therapeutic strategy for cardiac fibrosis [3].

The research team first addressed a critical challenge in the field of cardiac healthy and repair—the limited availability and lifespan of adult CMs in vitro. By utilizing the ROCK inhibitor Y-27632, they were able to establish an extended culture system for adult human CMs, which enabled the collection of a substantial quantity of EVs without compromising the cellular integrity or inducing any malignant phenotype. This technical advancement is a significant step forward, as it may overcome a major hurdle in the production of CM-derived EVs for therapeutic applications.

Subsequently, the researchers conducted a comprehensive analysis of the cargo and functional properties of the CM-derived EVs. Through extensive in vitro experiments, they demonstrated that these EVs effectively reduced the activation of cardiac fibroblasts and the deposition of extracellular matrix components, such as fibronectin and collagen, in the presence of the pro-fibrotic cytokine transforming growth factor-β (TGF-β). Notably, the antifibrotic effects of CM-derived EVs were superior to those of EVs obtained from alternative cell sources, underscoring the importance of the specific cargo within the CM-derived EVs.

To elucidate the underlying molecular mechanisms, the investigators performed comprehensive RNA sequencing and pathway analysis of the EV cargo, unveiling an enrichment of antifibrotic microRNAs and a diverse array of cardiac-specific microRNAs. The affected pathways encompass MAPK, mTOR, JAK/STAT, TGF-β, and PI3K/Akt signaling axes. This intricate combination of regulatory molecules appears to be more instrumental in orchestrating overall cardiac recovery.

The findings of this study highlight the distinctive advantages of using adult human CMs as a source of EVs for mitigating cardiac fibrosis. The specialized cargo of the CM-derived EVs, enriched in antifibrotic and cardioprotective microRNAs, emerges as a critical determinant of their superior therapeutic efficacy compared to EVs derived from alternative cell types. This specificity is of paramount importance, as it portends a targeted approach to address the underlying pathological processes within the compromised myocardium.

From a methodological perspective, the authors have provided a detailed and well-designed experimental approach, including the establishment of an extended adult human CM culture system, rigorous in vitro and in vivo assessments, and the thorough characterization of the EVs cargo and its functional consequences. The EVs isolation protocol employed a sophisticated multi-step approach, including cell culture conditioning, differential centrifugation, ultracentrifugation, size exclusion chromatography, and ultrafiltration. The subsequent characterization utilized a set of cutting-edge techniques: nanoparticle tracking analysis (NTA) for particle quantification, immunoblotting for marker detection, and the highly sensitive ExoScreen assay for precise quantitative expression analysis of EV markers, further strengthening the rigor and depth of the study. However, further investigations in additional disease models and a more comprehensive assessment of the cardiac regenerative potential of adult CM-derived EVs would be valuable.

In conclusion, the study by Prieto-Vila and colleagues represents a significant advancement in the field of cardiac protection, demonstrating the therapeutic potential of CM-derived EVs for the treatment of cardiac fibrosis. The innovative approach to establish an extended culture system for adult human CMs and the subsequent characterization of the unique cargo and functional properties of the CM-derived EVs offer a promising avenue for developing precision therapies in cardiovascular medicine.