Abstract Body (Do not enter title and authors here): Introduction: Myxomatous mitral valve degeneration (MMVD) is a prevalent cause of mitral valve regurgitation in developed countries, yet effective medical therapies targeting this condition remain lacking. Although the inflammatory processes associated with mitral valve degeneration are well established, the specific mechanisms by which immune cells contribute to extracellular matrix (ECM) remodeling in MMVD remain poorly understood.

Methods: Single-cell RNA sequencing performed on human myxomatous mitral valves was analyzed to map the immune cell landscape of MMVD. Both human and mouse specimens were analyzed to confirm macrophage-specific alterations and the expression of legumain (LGMN), a protease implicated in ECM remodeling. Additionally, in vivo studies using macrophage-specific LGMN-deficient mice were conducted to further investigate the functional role of LGMN in macrophage-mediated ECM changes. A macrophage and valvular interstitial cell co-culture system was employed to examine cellular interactions in vitro.
Results: An association of MMVD with Marfan syndrome resulting from pathogenic FBN1 variants supports the use of fibrillin-1 deficient mice to investigate mechanism for MMVD. Fbn1 ^C1041G/+ mice developed severe myxomatous mitral valve disease. Using single-cell RNA sequencing, we mapped the immune cell landscape in myxomatous mitral valves (Figure 1A). Our findings revealed a significant increase in a subset of Lgmn+ macrophages in the myxomatous mitral valves (Figure 1B), which was confirmed by immunofluorescence staining in both mouse and human specimens (Figure 2). Analysis of cell-cell interactions revealed that macrophages exhibit strong interactions with valvular interstitial cells (Figure 3A). Additionally, LGMN amplified the TGF-β-mediated transition of fibroblasts into myofibroblasts (Figure 3B). Furthermore, macrophage-specific deficiency of legumain (Lgmn) resulted in reduced ECM remodeling and stabilization of mitral valve degeneration (Figure 3C).
Conclusion: This study demonstrated that macrophage derived LGMN promote myxomatous mitral valve degeneration by enhancing the extracellular matrix remodelling via macrophage-valvular interstitial cell interaction and provided a novel target for the treatment of MMVD.