The Divergent Roles of MicroRNAs in Endothelial-Driven Transdifferentiation and Calcification of Aortic Stenosis

Y. Sheng (Bonn)¹, Z. Li (Bonn)², H. Li (Bonn)³, A. M. Utami (Bonn)⁴, J. I. Muñoz-Manco (Bonn)⁵, P. R. Goody (Bonn)⁶, K. Wilhelm-Jüngling (Bonn)⁷, N. Gerdes (Düsseldorf)⁸, S. Zimmer (Bonn)⁶, F. Bakhtiary (Bonn)⁹, G. Nickenig (Bonn)⁶, M. R. Hosen (Bonn)^5
1Heart Center, Molecular Cardiology Med-II Bonn, Deutschland; ²Heart Center Bonn, Molecular Cardiology, Department of Internal Medicine II Department of Internal Medicine II, University Hospital Bonn Bonn, Deutschland; ³Heart Center Bonn, Molecular Cardiology, Department of Internal Medicine II, University Hospital Bonn, Venusberg-Campus 1, Department of Internal Medicine II, University Hospital Bonn Bonn, Deutschland; ⁴Heart Center Bonn, Molecular Cardiology, Department of Internal Medicine II, University Hospital Bonn, Department of Internal Medicine II, University Hospital Bonn Bonn, Deutschland; ⁵Heart Center, Molecular Cardilogy Internal Medicine-II Bonn, Deutschland; ⁶Universitätsklinikum Bonn Medizinische Klinik und Poliklinik II Bonn, Deutschland; ⁷Institute for Cardiovascular Sciences Endothelial Signaling and Metabolism Bonn, Deutschland; ⁸Universitätsklinikum Düsseldorf Klinik für Kardiologie, Pneumologie und Angiologie Düsseldorf, Deutschland; ⁹Universitätsklinikum Bonn Klinik und Poliklinik für Herzchirurgie Bonn, Deutschland

Background: Aortic stenosis (AS), the most prevalent valvular heart disease, is characterized by progressive calcification and endothelial dysfunction, with no pharmacological treatments available. Valvular endothelial cells (VECs) are pivotal in maintaining homeostasis, and their transition to a mesenchymal phenotype (EndMT) is a key driver of AS pathogenesis. MicroRNAs (miRNAs) are crucial post-transcriptional regulators, but the specific roles of VEC-enriched miRNAs in coordinating EndMT and calcification remain poorly defined. This study investigates the pathogenic contributions of miR-21, miR-92a, and miR-145, aiming to delineate their function in the calcific remodeling of the aortic valve.

Methods and Results: Human primary VECs and valvular interstitial cells (VICs) were transfected with miRNA inhibitors (anti-miR-21, anti-miR-92a, anti-miR-145) or a scrambled control. Efficient knockdown was confirmed by qRT-PCR. Functional assays demonstrated that inhibition of miR-21 and miR-92a significantly improved cell viability and reduced cytotoxicity (p<0.01). Furthermore, silencing these miRNAs attenuated EndMT, as shown by downregulation of α-SMA and SM22α, and robustly inhibited calcium deposition (Alizarin Red S staining, p<0.05). In scratch wound assays, anti-miR-21 and anti-miR-92a markedly impaired VEC migration, reducing wound closure to less than 50% of control levels within 10 hours. In contrast, inhibition of miR-145 produced a divergent phenotype: it enhanced VEC migration but concurrently promoted a potent pro-calcific response. This was evidenced by significant upregulation of osteogenic markers RUNX2 and ALPL and a substantial increase in calcium accumulation, indicating a critical loss of its endogenous protective function.

Conclusion: Our findings reveal a central regulatory axis in AS where miR-21 and miR-92a act as pathogenic drivers of EndMT and calcification, while miR-145 serves an essential protective, anti-calcific role. The contrasting effects of these miRNAs underscore the complexity of miRNA-mediated regulation in valvular pathophysiology. Ongoing research utilizing VEC-VIC co-culture models is focused on validating the intercellular transfer of these miRNAs via extracellular vesicles. This work identifies a promising miRNA signature with high potential for the development of novel RNA-based therapeutics aimed at halting the progression of aortic stenosis.