Silencing CCL5 suppresses ferroptosis to alleviate calcific aortic valve disease through chemokine pathway inhibition

Background: Calcific aortic valve disease (CAVD) involves pathological mineralization, but the roles of chemokine signaling and Ferroptosis remain unclear. This study investigated the regulatory function of C-C motif chemokine ligand 5 (CCL5) in CAVD progression via the chemokine pathway and Ferroptosis.

Methods: Bioinformatics analysis and single-cell RNA Sequencing analysis were performed to identify hub genes and potential cell types. Human aortic valve interstitial cells (VICs) were treated with osteogenic medium (OM) to induce calcification. apoE^-/- mice were induced by a high-fat diet in vivo. Calcification, oxidative stress, and Ferroptosis markers were assessed by pathological staining, enzyme-linked immunosorbent assay, and Western blot, respectively. Ferroptosis was modulated using Ferrostatin-1 (inhibitor) or Erastin (inducer), and chemokine signaling was activated with the CXC motif Chemokine Receptor 4 (CXCR4) agonist ATI-2341 TFA.

Results: CCL5 was identified as a key hub gene in CAVD. Knockdown of CCL5 significantly attenuated OM-induced VICs calcification, osteogenic differentiation, oxidative stress, and Ferroptosis. Similar protective effects were observed in vivo, with reduced valve thickening and calcification in apoE^-/- mice. Ferroptosis inhibition mirrored these effects, while its induction reversed CCL5-knockdown benefits. Furthermore, chemokine signaling pathway was screened as the downstream pathway of CCL5. Mechanistically, CCL5 knockdown suppressed CXCR4/CXCL12 expression. Activating chemokine signaling with TFA abolished the protective effects of CCL5 silencing on calcification, Ferroptosis, and oxidative stress in vitro and in vivo.

Conclusion: CCL5 promoted CAVD progression by activating the chemokine signaling pathway to induce Ferroptosis. Targeting CCL5 may offer a novel therapeutic strategy for CAVD.