SMAD3 silencing induces Epithelial-to-Mesenchymal Transition (EMT) towards cardiac pericyte progenitors with proangiogenic potential in hiPSC-derived epicardial cells

Abstract

Abstract Background The epicardium is only a single mesothelial layer and "quiescent" in the adult heart. However, once the heart is injured, the epicardium reactivates and acts as a key for cardiac regeneration through epithelium-to-mesenchymal transition (EMT). While the canonical TGF-β-SMAD pathway plays a central role in regulating EMT, SMAD3 functions independent of this pathway in epicardium remain unknown. Purpose The purpose of this study is to investigate the effects of SMAD3 downregulation on EMT in epicardial cells. Methods First, we differentiated human iPSC-derived epicardial cells (EPI cells) by mimicking the process of cardiac mesoderm development. Next, siRNA targeting SMAD3 was transfected into EPI cells to silence SMAD3. We performed qRT-PCR, western blotting, immunocytochemistry, flow cytometry, transcriptional analysis, endothelial tube formation assay, and paracrine experiments using FUCCI (fluorescent ubiquitination-based cell cycle indicator) system to evaluate the phenotype of EPI cells with silenced SMAD3. Results We found that SMAD3 silencing in EPI cells resulted in loss of epicardial identity and upregulation of multiple EMT markers. Additionally, NG2 and ENG (CD105), both cardiac pericyte markers, were highly expressed in SMAD3-silenced EPI cells. RNA-sequence also revealed that SMAD3 silencing in EPI cells induced EMT facilitated towards cardiac pericyte progenitors with proangiogenic potential. In endothelial tube formation assay, SMAD3-silenced EPI cells demonstrated enhanced tube formation compared to the control (1.48-fold increase in the number of junctions, n=3; p<0.0001), which indicated their angiogenic potential. At last, we revealed the paracrine effect of SMAD3-silenced EPI cells to enhance the proliferative reactivation in iPSC-derived cardiomyocytes by showing upregulation in CDK6/CCND1 axis and FUCCI-green. Conclusion Our findings demonstrate a new role of SMAD3 biology in the human epicardium, specifically in the generation of cardiac pericyte progenitor cells that contribute to better responses in angiogenesis of the heart microvasculature enabling the possibility to continue exploiting epicardial biology for effective cardiac regeneration.