Abstract 3066: Pro-Atherogenic Macrophages Induce The Upregulation Of MLKL And Cell Death In Vascular Smooth Muscle Cells During Atherogenic Stimulation

Abstract

Background: A hallmark of advanced, rupture-prone atherosclerotic plaques is the presence of a necrotic core - a dense mass of pro-inflammatory and cellular debris. Traditionally, it has been thought that most of the foam cells within this core are derived from macrophages. However, recent advances in single cell technologies have revealed that vascular smooth muscle cells have the capacity to transdifferentiate into "macrophage-like foam cells", accounting for almost half of the cell content in advanced plaques. Our lab and others have discovered that a primary cause of the necrotic core is necroptotic cell death, where the mixed lineage kinase domain like pseudokinase MLKL is a key executioner of this process. While the contribution of necroptotic macrophage-derived foam cells to atherosclerotic lesion growth and instability has been fairly well-studied, the role of necroptotic vascular smooth muscle cell-derived foam cells in this process remains unclear. Hypothesis: MLKL regulates cell death differently in macrophages and vascular smooth muscle cells when challenged with pro-atherogenic stimuli. Methods and Results: Bone marrow-derived macrophages (BMDMs) and aortic vascular smooth muscle cells (VSMCs) were isolated from wildtype mice and treated with pro-atherogenic stimuli in the presence or absence of apoptotic and necroptotic inhibitors, zVAD and Necrostatin-1 (Nec1). BMDMs underwent cell death in response to treatment with oxLDL or LPS plus zVAD within 24 hours (as measured by LDH release and SYTOX assays) while VSMCs survive up to 72 hours. However, when VSMCs are treated with BMDM conditioned media in addition to pro-atherogenic stimuli, VSMCs begin to show higher relative rates of cell death compared to controls. Confocal microscopy and Western blots revealed that VSMCs treated with cell death ligands in conjunction with BMDM conditioned media showed an increase in relative expression of total and pMLKL- a hallmark of necroptosis, compared to VSMCs without conditioned media. Conclusions: Overall, this work suggests VSMCs are inherently resistant to necroptosis unless treated with conditioned media from BMDMs, which may suggest that macrophages have the capacity to prime necroptosis in VSMCs, likely via secreted factors.