Oscillatory shear stress induced extracellular vesicles mediate valvular endothelial-interstitial cell crosstalk exacerbating aortic valve calcification [RIP-seq]

Calcific aortic valve disease (CAVD) involves pathological crosstalk between human valvular endothelial cells (hVECs) and interstitial cells (hVICs), yet the role of this communication in pathogenesis remains poorly defined. Here, we identify a novel extracellular vesicle-derived circRNA, CircILRUN, originating from hVECs and demonstrate its critical function in promoting hVIC osteogenesis and CAVD progression in vitro and in vivo. CircILRUN expression is significantly elevated in extracellular vesicles from hVECs under oscillatory shear stress. Its deletion markedly attenuates aortic valve calcification (AVC) in mice, reducing valve thickness, calcium deposition, and improving echocardiographic parameters, findings corroborated in osteogenic medium-induced hVICs. Through unbiased protein-RNA screening and biochemical validation, we identify N-acetyltransferase 10 (NAT10) as a key target of CircILRUN. Nat10 depletion also effectively reduces AVC in vivo. Mechanistically, CircILRUN acts as a molecular scaffold facilitating the interaction between NAT10 and the deubiquitinase USP11, thereby stabilizing NAT10 protein. Integrated transcriptomic and ac4C epitranscriptomic profiling reveals that CircILRUN promotes NAT10-mediated acetylation of CD36 mRNA, stabilizing CD36 and accelerating AVC progression. Collectively, our findings establish an extracellular vesicle-mediated circILRUN–NAT10–CD36 axis driving CAVD pathogenesis and identify both circILRUN and NAT10 as promising therapeutic targets CircRNAs expression in human valvular endothelial cell-derived exosomes (hVECs-Exos) were measured after exposure to oscillatory shear stress (OSS) and laminar shear stress (LSS), respectively. Three independent experiments were performed at each group (OSS and LSS) using different donors for each experiment.