Abstract Body: Introduction: Galectin-3 (Gal3), a β-galactoside-binding lectin, is a well-established inflammatory biomarker for cardiovascular disease (CVD), showing marked elevation during atherosclerosis and myocardial infarction. Despite this clinical recognition, the specific molecular mechanisms and intracellular functions of Gal3 within macrophages—and how these processes drive atherogenesis—remain poorly understood.

Methods: We analyzed single-cell RNA sequencing data to assess Lgals3 expression during plaque progression, specifically in the myeloid lineage. To explore Gal3’s role in the lysosomal damage response, primary macrophages were treated with cholesterol crystals and LLOMe to induce membrane rupture. Autophagy was assessed via LC3 puncta and TFEB nuclear localization; apoptosis and inflammasome activation were measured by caspase-3/7 assays and IL-1β secretion. Gal3-null bone marrow was transplanted into LDLR-null mice on a Western diet to analyze lesion size and plaque composition.

Results: Lgals3 expression correlates strongly with lysosomal gene networks. Lysosomal membrane damage triggers robust Gal3 recruitment to lysosomes via recognition of exposed carbohydrate moieties on proteins like Lamp1. In Gal3-deficient macrophages, this repair mechanism is impaired, leading to decreased autophagy/lysophagy, reduced TFEB activation, accumulation of dysfunctional lysosomes, increased apoptosis, and NLRP3 inflammasome/IL-1β activation. In vivo, LDLR-null mice transplanted with Gal3−/− bone marrow developed larger lesions and altered plaque composition. We discovered that NLRP3 inflammasome activation triggers Gal3 secretion, depleting intracellular stores and limiting its protective functions. Notably, while serum Gal3 is elevated in myocardial infarction patients, intracellular Gal3 in monocytes is reduced. This suggests the ratio of extracellular to intracellular Gal3 is a superior predictive marker for CVD outcomes compared to extracellular levels alone.

Conclusion: Our findings reveal a protective intracellular role for Galectin-3 in the lysosomal damage response of atherosclerotic macrophages, challenging the conventional view of Gal3 solely as a secreted biomarker. We identify a novel mechanism of Gal3 release and demonstrate that accounting for its dual extracellular/intracellular roles, specifically through the extracellular-to-intracellular Gal3 ratio, significantly enhances the prediction of cardiovascular disease risk and patient outcomes.