Rationale: A hallmark of chronic inflammatory disorders is persistence of pro-inflammatory macrophages in diseased tissues. In atherosclerosis this is associated with dyslipidemia and oxidative stress, but mechanisms linking these phenomena to macrophage activation remain incompletely understood. Objective: To investigate mechanisms linking dyslipidemia, oxidative stress and macrophage activation through modulation of immunometabolism, and to explore therapeutic potential targeting specific metabolic pathways. Methods and Results: Using a combination of biochemical, immunological, and ex vivo cell metabolic studies, we report that CD36 mediates a mitochondrial metabolic switch from oxidative phosphorylation to superoxide production in response to its ligand, oxLDL. Mitochondrial-specific inhibition of superoxide inhibited oxLDL-induced NF-κB activation and inflammatory cytokine generation. RNAseq, flow cytometry, 3H-labeled palmitic acid uptake, lipidomic analysis, confocal and EM imaging, and functional energetics revealed that oxLDL upregulated effectors of long-chain fatty acid (FA) uptake and mitochondrial import, while downregulating FA oxidation and inhibiting ATP5A, an electron transport chain (ETC) component. The combined effect is long-chain FA accumulation, alteration of mitochondrial structure and function, repurposing of the ETC to superoxide production, and NF-κB activation. Apoe null mice challenged with high fat diet showed similar metabolic changes in circulating Ly6C+ monocytes and peritoneal macrophages, along with increased CD36 expression. Moreover, mitochondrial ROS was positively correlated with CD36 expression in aortic lesional macrophages. Conclusions: These findings reveal that oxLDL/CD36 signaling in macrophages links dys-regulated FA metabolism to oxidative stress from the mitochondria, which drives chronic inflammation. Thus, targeting to CD36 and its downstream effectors may serve as potential new strategies against chronic inflammatory diseases such as atherosclerosis.

Atherothrombosis is a process mediated by dysregulated platelet activation that can cause life-threatening complications and is the leading cause of death by cardiovascular disease. Platelet reactivity in hyperlipidemic conditions is enhanced when platelet scavenger receptor CD36 recognizes oxidized lipids in oxidized low-density lipoprotein (oxLDL) particles, a process that induces an overt prothrombotic phenotype. The mechanisms by which CD36 promotes platelet activation and thrombosis remain incompletely defined. In this study, we identify a mechanism for CD36 to promote thrombosis by increasing activation of MAPK extracellular signal-regulated kinase 5 (ERK5), a protein kinase known to be exquisitely sensitive to redox stress, through a signaling pathway requiring Src kinases, NADPH oxidase, superoxide radical anion, and hydrogen peroxide. Pharmacologic inhibitors of ERK5 blunted platelet activation and aggregation in response to oxLDL and targeted genetic deletion of ERK5 in murine platelets prevented oxLDL-induced platelet deposition on immobilized collagen in response to arterial shear. Importantly, in vivo thrombosis experiments after bone marrow transplantation from platelet-specific ERK5 null mice into hyperlipidemic apolipoprotein E null mice showed decreased platelet accumulation and increased thrombosis times compared with mice transplanted with ERK5 expressing control bone marrows. These findings suggest that atherogenic conditions critically regulate platelet CD36 signaling by increasing superoxide radical anion and hydrogen peroxide through a mechanism that promotes activation of MAPK ERK5.

One characteristic of atherosclerosis is the accumulation of lipid-laden macrophage foam cells in the arterial wall. We have previously shown that the binding of oxidized LDL (oxLDL) to the scavenger receptor CD36 activates the kinase Lyn, initiating a cascade that inhibits macrophage migration and is necessary for foam cell generation. Here, we identified the plasma membrane ion transporter Na/K-ATPase as a key component in the macrophage oxLDL-CD36 signaling axis. Using peritoneal macrophages isolated from Atp1a1 heterozygous or Cd36 null mice, we demonstrated that CD36 recruited a Na/K-ATPase-Lyn complex for Lyn activation in response to oxLDL. Macrophages deficient in the α1 Na/K-ATPase catalytic subunit did not respond to activation of CD36, showing attenuated oxLDL uptake and foam cell formation, and oxLDL failed to inhibit migration of these macrophages. Furthermore, Apoe-null mice, which are a model of atherosclerosis, were protected from diet-induced atherosclerosis by global deletion of a single allele encoding the α1 Na+/K+-ATPase subunit or reconstitution with macrophages that lacked an allele encoding the α1 Na+/K+-ATPase subunit.. These findings identify Na/K-ATPase as a potential target for preventing or treating anti-atherosclerotic therapy.