Abstract
Clonal hematopoiesis of indeterminate potential (CHIP), defined as presence of somatic mutations in the blood in genes described to be affected in hematologic cancers, most commonly in TET2 and DNTM3A, with a variant allele frequency of 2%, is an inherent consequence of aging and is associated with atherosclerotic cardiovascular disease. Tet2-mutant cells contribute to atherosclerosis by promoting inflammatory cytokine secretion from macrophages (Fuster et al., Science 2017). Macrophages are central to the initiation and progression of atherosclerosis, regulating inflammation and lipid homeostasis. At the arterial wall, excessive lipid uptake combined with impaired lipid processing via autophagy transforms macrophages into lipid-laden foam cells (Fayad et al., JACC 2018). These foamy macrophages constitute the majority of cells within early atherosclerotic plaques and contribute to plaque necrotic core formation (Hilgendorf et al., ATVB 2014). However, the precise mechanism by which TET2 loss directly impacts macrophage foam cell formation in Tet2-deficiency–associated atherosclerosis remains unclear.
To elucidate the role of TET2 in regulating foam cell formation, bone marrow-derived macrophages (BMDMs) or peritoneal macrophages (PMs) were isolated from wild-type (WT) or Tet2-/- (KO) mice. We found that Tet2 KO macrophages exhibited augmented transformation into foamy macrophages. To assess foam cell formation, PMs from WT or TET2 KO mice were stimulated with oxidized low-density lipoproteins (oxLDL) for 24-hours, followed by Oil red O to detect neutral lipid droplets. KO PMs had an increased number of Oil red O+ vesicles per cell compared to WT PMs. Further, KO BMDMs had increased median fluorescence intensity of DiI-labeled oxLDL. These findings suggest Tet2 deficiency in macrophages results in inefficient autophagic processing of oxLDL, promoting lipid-rich foam cell transformation.
To assess whether impaired autophagy underlies increased lipid accumulation in Tet2-deficient macrophages, we examined lysosomal function and autophagic flux in TET2 KO macrophages. Prior studies have demonstrated that the functional status of lysosomes is critical for mediating the degradation of lipid-rich autophagosomes. Lysosomal degradation of lipid-rich autophagosomes is critically dependent on vacuolar ATPases, which drive lysosomal acidification during autophagosome to lysosome fusion and enable breakdown of intracellular lipids. We found that ATP6V0D2, a critical vacuolar ATPase, is downregulated in TET2 KO BMDMs following oxLDL stimulation. Further, treatment with cholesterol crystals, a hallmark of atherosclerotic plaques, reduced LysoTracker signal intensity in TET2 KO BMDMs, suggesting that TET2 KO BMDMs have dysfunctional lysosomal function in response to various forms of cholesterol. Using p62/SQSTM1, a chaperone responsible for selective autophagy, as a marker of autophagic status, we observed increased p62 puncta in oxLDL-stimulated TET2 KO BMDMs, indicating decreased autophagic flux. Interestingly, induction of autophagy with rapamycin rescued TET2 KO BMDMs from lipid-rich foam cell transformation.
Together, our in vitro findings highlight a role for TET2 in regulating foam cell formation and provide mechanistic insight into how TET2 loss in HSCs and their mature myeloid progeny drive atherosclerosis. Specifically, TET2 deficiency in macrophages impaired autophagic processing of oxidized cholesterol, leading to increased lipid accumulation. This dysfunction may contribute to atherosclerotic plaque development and increase risk of plaque rupture (Yalcinkaya et al., Circulation 2023). Future studies investigating into enhancing autophagy and lysosomal function to reverse foam cell formation in the setting of TET2 loss may unveil novel therapeutic strategies to combat cardiovascular disease progression in ageing individuals with TET2 mutations.
