Hypoxia-Inducible Factor Prolyl Hydroxylase EGLN3 Stabilizes Atherosclerotic Plaques in ApoE-/- Mice Independently of Its Catalytic Activity.
BACKGROUND The NLRP3 (nucleotide-binding oligomerization domain, leucine-rich repeat-containing receptor family pyrin domain-containing 3) inflammasome plays a causal role in driving atherosclerosis. HIF (hypoxia-inducible factor) prolyl hydroxylase EGLN3 (Caenorhabditis elegans gene egl-9 [EGLN] 3) inhibits NFκB (nuclear factor kappa B) signaling independently of its hydroxylase activity. This study determined the role and mechanism of EGLN3 prolyl hydroxylase in regulating NLRP3 inflammasome activation, vascular inflammation, and the development of atherosclerosis. METHODS ApoE-/- mice deficient for global EGLN3, macrophage EGLN3, or EGLN3 hydroxylase activity were used to explore the role of EGLN3 and its activity in atherosclerosis. MCC950 was applied to antagonize the NLRP3 inflammasome in mice. Various cellular and molecular methods were used for mechanistic studies. RESULTS EGLN3 elimination promotes the production of mitochondrial reactive oxygen species and the release of mitochondrial DNA in macrophages. EGLN3 ablation enhances NLRP3 inflammasome assembly and promotes GSDMD (gasdermin D) cleavage, IL (interleukin) 1β production, and pyroptosis of macrophages. TRAF6 (TNF receptor-associated factor 6) catalyzes lysine (K) 63-linked NLRP3 ubiquitination and potentiates NLRP3 inflammasome assembly and activation, which can be antagonized by EGLN3. EGLN3 also blunts K63-polyubiquitination of TRAF6. EGLN3 deficiency enhances lipid uptake through increasing CD36 expression and impedes cholesterol efflux by declining ABCA1 (ATP-binding cassette transporter A1) expression, thereby boosting macrophage foam cell formation. EGLN3 depletion restrains MerTK expression and efferocytosis of macrophages. Inhibition of the NLRP3 inflammasome counteracts the impacts of EGLN3 deficiency on foam cell formation and efferocytosis. Global and macrophage-specific ablation of EGLN3 exacerbates vascular inflammation and instability of the atherosclerotic plaques in ApoE-/- mice. Pharmacological inhibition of the NLRP3 inflammasome ameliorates the detrimental effect of EGLN3 deficiency on vascular inflammation and the formation and stability of the plaques. Genetic inactivation of EGLN3 activity retains its ability to restrict NLRP3 inflammasome activation and atherosclerosis development. CONCLUSIONS EGLN3 inhibits NLRP3 inflammasome assembly and activation through antagonizing TRAF6-induced NLRP3 ubiquitination and activation. EGLN3 suppresses vascular inflammation and atherosclerosis by modulating pyroptosis, foam cell formation, and efferocytosis of macrophages through targeting the NLRP3 inflammasome. EGLN3 protects against atherosclerosis independently of its hydroxylase activity.