Mitochondrial extracellular vesicles promote aortic valve calcification via carnitine O-octanoyltransferase

ABSTRACT: Calcific aortic valve disease (CAVD) is a leading cause of cardiovascular morbidity and mortality with no effective medical therapy. We previously identified carnitine O-octanoyltransferase (CROT) as a mediator of mitochondrial dysfunction and vascular calcification. However, its role in CAVD remains unexplored. Here, we investigate whether CROT contributes to CAVD progression by promoting the release of pro-calcific mitochondria-derived extracellular vesicles (mitoEVs). Methods and Results: Human valvular interstitial cells (VICs) were isolated from aortic valves obtained from patients with aortic stenosis (AS; n=34 donors). VICs were cultured in osteogenic medium (OM) to induce calcification, which was evaluated by Alizarin Red staining. Proteomic analysis was performed to identify differentially abundant proteins between VICs cultured in normal medium (NM) and OM, with the goal of elucidating mechanisms linking CROT to VIC calcification. Mitochondrial function was assessed using the Seahorse XF Analyzer to measure oxygen consumption rate (OCR), and mitochondrial morphology was examined by MitoTracker staining. Extracellular vesicles (EVs) were isolated from VIC-conditioned media by ultracentrifugation and characterized for particle size and concentration using NanoSight Pro. In vivo, AS was induced in C57BL/6J, Ldlr⁻/⁻ Crot (Sham, n=12), Ldlr-/- Crot+/+ (Aortic valve wire injury; AVWI, n=19) and Ldlr-/- Crot-/- (AVWI, n=23). Disease progression was monitored by echocardiography, and aortic valve calcification was visualized using OsteoSense 680EX near-infrared fluorescence-based molecular imaging. OM–induced calcification in VICs was reduced by siRNA-mediated CROT silencing (siCROT; p<0.05). Proteomic analysis revealed that mitochondrial-associated proteins were markedly altered following siCROT silencing. Examination of mitochondrial morphology showed that OM culture induced marked mitochondrial fragmentation, which was restored by siCROT treatment. Western blotting and flow cytometry analyses indicated that OM-induced mitochondrial fragmentation promotes the release of mitoEVs from VICs, which in turn contributes to calcification. Immunofluorescence revealed mitoEVs within the extracellular space, colocalizing with calcification. In vivo, CROT deficiency attenuated the progression of AS in AVWI mouse model by reducing valvular calcification. Conclusion: Osteogenic stimulation disrupts mitochondrial dynamics in VICs, promoting the release of pro-calcific mitoEVs. This study identifies CROT as a key regulator of this process, where CROT inhibition restores mitochondrial homeostasis and suppresses VIC calcification. These findings highlight CROT as a promising therapeutic target for the treatment of CAVD.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Fibroblast, Aortic Valve

SUBMITTER: Sasha Singh  

LAB HEAD: Elena Aikawa

PROVIDER: PXD072508 | Pride | 2026-05-13

REPOSITORIES: Pride