LGALS3 induces ferroptoisis via interaction of autophagy in CKD vascular calcification

Vascular calcification (VC) is prevalent in patients with chronic kidney disease (CKD) and represents a major cause of cardiovascular disease in this population. However, its fundamental mechanisms remain unclear, and effective interventions are lacking. Our previous studies revealed that autophagy and ferroptosis exert synergistic effects on vascular calcification by regulating osteoblastic trans-differentiation in vascular smooth muscle cells (VSMCs). Damage-associated molecular patterns (DAMPs) were increased by CKD vascular calcification involving ferroptosis and autophagy. However, it remains unclear about the role and mechanism of DAMPs in CKD vascular calcification involving autophagy and ferroptosis.  

We established 5/6 nephrectomy CKD rat model and induced vascular calcification by feeding a high-phosphorus diet for 12 weeks. Aortic samples were collected for transcriptome sequencing, and differentially expressed genes were screened based on |LogFC| ≥ 1 and P < 0.05. We cross-analyzed these differentially expressed genes with iron death-related gene sets from FerrDb, GSEA, and Genecards databases; autophagy-related gene sets from HAMdb, GSEA, and Genecards; and DAMP-related genes obtained from literature. A protein-protein interaction (PPI) network was constructed to identify key DAMP-associated genes involved in autophagy and iron death during CKD vascular calcification. Concurrently, we conducted in vivo and in vitro experiments. In rat models, we assessed the expression of LGALS3 levels, ferroptosis associated molecules, and autophagy associated molecules in vascular tissues in CKD vascular calcification. We also established β-glycerophosphate induced vascular smooth muscle cell calcification models to validate the in vivo findings.  

Bioinformatics analysis and in vivo experiments indicated that the DAMP-associated molecule LGALS3 was upregulated in calcified aortic samples. LGALS3 emerged as a key molecule potentially involved in regulating ferroptosis and autophagy in CKD vascular calcification. In vivo experiments demonstrated significantly increased LGALS3 expression in calcified rat vascular tissues, accompanied by autophagy and ferroptosis. In vitro experiments demonstrated that LGALS3, as an autophagy and ferroptosis associated molecule, was significantly expressed in calcified VSMCs. Knockdown of LGALS3 markedly attenuated vascular calcification and reduced the expression of the autophagy associated molecule LC3II/I. It was concurrently revealed decreased lipid peroxidation accumulation and restored levels of the ferroptosis associated molecule GPX4. Activation of LGALS3 significantly induced the autophagic flux in VSMCs by GFP-RFP-LC3 transfection. Treatment with autophagy inhibitors in LGALS3-overexpressing VSMCs reversed ferroptosis in calcific VSMCs. 

Our study provides compelling evidence that LGALS3 plays a crucial role in ferroptosis during vascular calcification in CKD. This process critically depends on LGALS3-induced autophagy. These findings reveal a novel molecular pathway involving LGALS3, autophagy, and ferroptosis in the pathogenesis of CKD vascular calcification. It indicates that potential role of LGALS3-mediated autophagy-ferroptosis interaction against CKD vascular calcification.