Abstract Body (Do not enter title and authors here): Background: Calcific aortic valve stenosis (AS) affects approximately 3% of the general population and lacks pharmacological treatment. Proprotein-convertase-subtilisin/kexin-type9 (PCSK9) inhibition shows promise in attenuating calcium accumulation on human aortic valve interstitial cells (VIC), offering new therapeutic prospect to curb CAVS progression.
Hypothesis: Our investigation aims to clarify PCSK9 involvement in mechanisms driving the aortic leaflets calcification processes.
Methods: Human aortic valve leaflets were collected for histology and VIC isolation, followed by enzyme-linked immunosorbent assay (ELISA) and calcification assays. PCSK9 knockout (KO), overexpression (Tg), and wild-type (WT) C57BL/6J mice were fed standard or high-fat diet for 18 months. Before sacrifice, mice underwent Doppler echocardiography to assess haemodynamic changes. RNA sequencing was performed on AS human valve specimens from patients with high or low circulating PCSK9, and from those with tissue-specific PCSK9 differences. PCSK9-overexpressing immortalized VICs (PCSK9+iVICs) were generated using CRISPR/Cas9.
Results: During AS progression, tissue PCSK9 levels increased significantly, as confirmed by both immunohistochemistry (log2FC: sclerotic +1.8±0.7 and stenotic +2.7±0.5; ANOVA p=0.0008) and ELISA (FC: sclerotic +9.8±1.6 and stenotic +11.13±2.3; ANOVA p=0.0012). Under both normal and osteogenic conditions, sclerotic and stenotic human VICs exhibited significantly greater calcification than controls (p≤0.001). A stage-dependent correlation emerged between calcium and secreted PCSK9 (control: r_s=0.04, p=0.8; sclerotic: r_s=0.5, p=0.004; stenotic: r_s=0.6, p=0.0003).
In vivo, only PCSK9 KO mice had 10% lower peak aortic jet velocity, vs Tg and WT before sacrifice (p≤0.05), suggesting that PCSK9 expressed within the aortic valve could have a role in leaflet remodelling.
Ex vivo, RNA-seq of AS leaflets showed that PCSK9 tissue-level variation alters the valvular transcriptome to a greater extent than circulating PCSK9 (549 vs 58 differentially expressed genes, respectively; high vs low PCSK9 levels).
Finally, PCSK9+iVICs showed an increased calcification content starting at day 7 (p≤0.05) and increasing up to day 21 (p≤0.0001) under pro-osteogenic stimulus compared to WT iVICs.
Conclusion: PCSK9 is upregulated in AS, enhances VIC calcification, and drives valve remodeling. Tissue-level variation shows greater transcriptomic impact, suggesting a local role of PCSK9 in AS.