Pro-Atherogenic Macrophages Induce Upregulation of MLKL and Cell Death in Vascular Smooth Muscle Cells During Atherogenic Stimulation

Abstract

Background: A hallmark of advanced, rupture-prone atherosclerotic lesions 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 necroptosis in macrophages and macrophage-derived foam cells to plaque growth and instability has been fairly-well studied, the role of necroptosis in vascular smooth muscle cells in this process remains unclear. Objective: This work explores the role of MLKL in regulating necroptosis of macrophages and vascular smooth muscle cells when challenged with pro-inflammatory and pro-atherogenic stimuli. Hypothesis: MLKL regulates cell death differently in macrophages and vascular smooth muscle cells when challenged with pro-inflammatory or pro-atherogenic stimuli. Methods and Results: Bone marrow-derived macrophages (BMDMs) and aortic vascular smooth muscle cells (VSMCs) were isolated from wildtype and MLKL-/- mice and treated with pro-atherogenic stimuli in the presence or absence of apoptosis and necroptosis inhibitors, zVAD and Necrostatin-1 (Nec1). BMDMs underwent cell death in response to treatment within 3-6 hours (as measured by LDH release and SYTOX assays), while VSMCs survive up to 18-24 hours. However, when treated with BMDM conditioned media, VSMCs begin to show higher rates of relative cell death compared to controls, particularly in response to treatment with oxLDL. Confocal microscopy and Western blots revealed that VSMCs treated with oxLDL in addition to BMDM conditioned media show an increase in relative expression of total and phosphorylated-MLKL – a key hallmark of necroptosis – compared to VSMCs without conditioned media. Moreover, these experiments implicate STAT1 and IRF1 as possible transcriptional regulators of MLKL expression in VSMCs treated in BMDM conditioned media. Preliminary staining of atherosclerotic lesions in mice may suggest that MLKL expression is more abundant within macrophages, and upregulation of MLKL expression in VSMCs may be associated with phenotypic switching. Conclusion: 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 through secreted factors.