Abstract Body (Do not enter title and authors here): Background
Calcific aortic valve disease (CAVD) is a progressive condition that culminates in severe aortic stenosis, for which no effective pharmacological therapies currently exist. Intraleaflet hemorrhage ensuing valvular iron overload induces ferroptotic cell death and is associated with CAVD development and severity.
Objective
To identify druggable molecular pathways involved in ferroptosis for alleviating CAVD progression and to deploy their translational relevance in a large population-based cohort.
Method
Aortic valves from 136 patients were analyzed using bulk transcriptomics, histology, immunohistochemistry, and primary valvular interstitial cell (VIC) cultures for mechanistic studies. In vivo relevance was assessed using ferroptosis induced hyperlipidemic mice. Clinical applicability was examined in 4,874 participants of Swedish CArdioPulmonary bioImage Study (SCAPIS) cohort.
Result
Among 2,002 differentially expressed genes between healthy and calcified aortic valve tissue derived from N=74 aortic stenosis patients, 54 ferroptosis-related genes were identified with fatty acid peroxidation as the dominating valvular ferroptosis pathways. Of these, 5-lipoxygenase emerged as central hub along with the 5-lipoxygenase-acyl-CoA synthetase long-chain family member 4 (ACSL4), which were also spatially coupled to sites of intraleaflet hemorrhage. In vitro ferroptosis induction led to a 2.4-fold increase (SEM = ± 0.42) fatty acid peroxidation (p<0.05) and a 3-fold reduction (SEM = ± 0.02) VIC viability (p<0.05), which was significantly reversed by pharmacological inhibition of 5-lipoxygenase with zileuton (1 uM) and ASCL4 with rosiglitazonre (10 mM), independently of lipid mediator and PPAR signaling. An in vivo ferroptosis model by administrating doxorubicin to hyperlipidemic mice increased aortic valve calcification and significantly correlated with concomitant ACSL4 overexpression (R=0.54, p=0.05). In SCAPIS cohort, plasma AA levels independently predicted aortic valve calcification (OR=1.04, q=0.049) and correlated with the ferroptosis biomarker HMOX1 (p<0.05), providing a key clinical coupling to an AA-ferroptosis interplay in CAVD pathogenesis.
Conclusion
The 5-lipoxygenase-ACSL4-AA pathway represents a central, pharmacologically targetable mechanism driving ferroptosis and CAVD. These findings support the potential for clinical repurposing of approved 5-lipoxygenase and ACSL4 inhibitors as a novel therapeutic strategy for CAVD.