The Long Noncoding RNA FGD5-AS1 Suppresses Endothelial-Mesenchymal Transition and Osteogenic Differentiation, Halting the Progression of Calcific Aortic Valve Disease

H. Li (Bonn)¹, Z. Li (Bonn)², A. M. Utami (Bonn)³, J. I. Muñoz-Manco (Bonn)⁴, P. R. Goody (Bonn)⁵, S. Zimmer (Bonn)⁵, F. Bakhtiary (Bonn)⁶, G. Nickenig (Bonn)⁵, M. R. Hosen (Bonn)^4
1Heart 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; 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, Department of Internal Medicine II, University Hospital Bonn Bonn, Deutschland; ⁴Heart Center, Molecular Cardilogy Internal Medicine-II Bonn, Deutschland; ⁵Universitätsklinikum Bonn Medizinische Klinik und Poliklinik II Bonn, Deutschland; ⁶Universitätsklinikum Bonn Klinik und Poliklinik für Herzchirurgie Bonn, Deutschland

Rationale: Calcific aortic valve disease (CAVD) is a prevalent condition with no pharmacological treatments, driven by endothelial-to-mesenchymal transition (EndMT) of valvular endothelial cells (VECs) and osteogenic differentiation of valvular interstitial cells (VICs). Our previous RNA sequencing identified the long non-coding RNA FGD5-AS1 as significantly downregulated in human calcified aortic valves, suggesting a potential protective role that remains functionally uncharacterized and represents a critical knowledge gap.

Methods and Results: We investigated the functional role of FGD5-AS1 using established in vitro models of CAVD. Primary human VECs and VICs were transfected with FGD5-AS1-targeting siRNA (siFGD5-AS1) or a scramble control (SCR). Pathological conditions were then induced: EndMT was stimulated in VECs using TNF-α or TGF-β1+IL-1β, while calcification was induced in VICs using osteogenic medium (OM) or phosphate-containing medium (PCM). Quantitative RT-PCR confirmed highly efficient FGD5-AS1 knockdown. Our results demonstrate a dual protective mechanism. In VICs, FGD5-AS1 silencing significantly upregulated the master osteogenic transcription factor RUNX2 under both OM and PCM stimulation, directly promoting a pro-calcific phenotype. Conversely, in VECs under EndMT-inducing conditions, FGD5-AS1 knockdown markedly downregulated endothelial nitric oxide synthase (NOS3), a critical guardian of endothelial homeostasis, thereby facilitating EndMT.

Conclusion and Future Directions: This study establishes FGD5-AS1 as a key suppressor of CAVD pathogenesis, acting dually to inhibit osteogenic differentiation in VICs and preserve endothelial integrity in VECs. Future work will validate these findings through independent replication and further investigate the underlying mechanisms by assessing the functional impact of FGD5-AS1 on processes such as tube formation, migration, cytotoxicity, proliferation (via LDH and MTT assays), and calcium deposition (via Alizarin Red staining). Subsequent mechanistic studies will identify direct molecular partners and downstream pathways, positioning FGD5-AS1 as a promising candidate for novel RNA-based therapeutic strategies.