Weiwei Wang,1,* Ying Li,1,* Cheng Zhang,2,* Haoyang Zhou,1 Chunyu Li,3 Rong Cheng,1 Xufeng Chen,1 Yanan Pu,4 Yan Chen1,5,6 

1Department of Emergency and Critical Care Medicine, The First Affiliated Hospital of Nanjing Medical University, Nanjing, 210029, People’s Republic of China; 2Long Jiang Central Laboratory, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, 210029, People’s Republic of China; 3Long Jiang Intensive Care Unit, The First Affiliated Hospital of Nanjing Medical University, Nanjing, 210029, People’s Republic of China; 4Department of Clinical Laboratory, Nanjing Chest Hospital, Affiliated Nanjing Brain Hospital, Nanjing Medical University, Nanjing, 210029, People’s Republic of China; 5Department of Emergency and Critical Care Medicine, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou Municipal Hospital, Gusu School, Nanjing Medical University, Suzhou, 215000, People’s Republic of China; 6Department of Emergency Management, School of Health Policy & Management, Nanjing Medical University, Nanjing, 211166, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Yan Chen; Yanan Pu, Email chenyandoc@njmu.edu.cn; pyn342626@njmu.edu.cn

Purpose: Cardiac fibrosis, a key contributor to ventricular pathologic remodeling and heart failure, currently lacks effective therapeutic approaches.
Patients and Methods: Small extracellular vesicles from young healthy human plasma (Young-sEVs) were characterized via protein marker, transmission electron microscopy, and nanoparticle tracking analysis, then applied in cellular models and mouse models of cardiac fibrosis. Western blotting and qRT-PCR were used to identify protective signaling pathways in cardiac fibroblasts (CFs).
Results: Young-sEVs significantly inhibited cardiac fibrosis and subsequent cardiac dysfunction post-myocardial infarction (MI) in mice. The main findings included that echocardiographic assessments four weeks post-MI indicated that Young-sEVs improved left ventricular ejection fraction (LVEF) and fractional shortening (LVFS), and reduced left ventricular internal diameter in diastole (LVIDd) and systole (LVIDs). Treatment with Young-sEVs also decreased Masson-positive fibroblast areas and collagen synthesis in cardiac tissue. However, sEVs from the old control group did not achieve the above effect. Consistent with in vivo results, Young-sEVs could also inhibit the proliferation, migration, and collagen synthesis of CFs in the TGF-β 1-induced cellular fibrosis model. High-throughput microRNA (miRNA) sequencing and qRT-PCR analysis revealed that miR-664a-3p was abundant in Young-sEVs. The high expression of miR-664a-3p significantly inhibited the proliferation, migration, and collagen synthesis of TGF-β 1-induced CFs. However, suppressing the expression of miR-664a-3p in Young-sEVs eliminated their therapeutic effect on cardiac fibrosis in mice. Further studies confirmed SMAD4 as a direct downstream target of miR-664a-3p, whose overexpression could reverse the anti-fibrotic effects of miR-664a-3p.
Conclusion: In summary, these findings firstly revealed that Young-sEVs could directly bind to the 3′-untranslated region of SMAD4 mRNA through miR-664a-3p, thereby inhibiting the TGF-β/SMAD4 signaling pathway to protect heart from fibrosis and improve cardiac function. Considering the ease of obtaining plasma-derived sEVs, our study offers a promising therapeutic strategy for heart failure, with the potential for rapid clinical translation in the near future.

Keywords: extracellular vesicles, young human plasma, cardiac fibrosis, MiR-664a-3p, SMAD4