5'-AMP-activated protein kinase-activating transcription factor 1 cascade modulates human monocyte-derived macrophages to atheroprotective functions in response to heme or metformin.

OBJECTIVE: Intraplaque hemorrhage (IPH) is an important driver of the progression of atherosclerotic plaques. Recently, we characterized Mhem as a novel macrophage phenotype that limits the atherogenicity of IPH. Mhem are directed by activating transcription factor 1 (ATF1), which is activated by phosphorylation. A better understanding of the counteratherogenic ATF1-Mhem pathway may facilitate antiatherosclerotic therapies. APPROACH AND RESULTS: We tested the hypothesis that heme in pathologically relevant concentrations activates the ATF1-Mhem pathway via 5'-AMP-activated protein kinase (AMPK) in primary human monocyte-derived macrophages and mouse bone marrow macrophages. We found that heme (10 µmol/L) activates AMPK, and downstream ATF1-mediated coinduction of heme oxygenase and liver X receptor that characterize Mhem. Heme increased macrophage phospho-AMPK, phospho-ATF1, and its target genes, and these effects were inhibited by the AMPK antagonist dorsomorphin, or by AMPK-knockdown with small inhibitory ribonucleic acid. The AMPK-activating oral hypoglycemic agent metformin also induced and phosphorylated ATF1 at a clinically relevant concentration (10 µmol/L). Functional effects of heme and metformin were inhibited by AMPK-knockdown and included suppression of macrophage oxidative stress; increased cholesterol export; protection from foam-cell formation; and suppression of macrophage inflammatory activation (human leukocyte antigen type DR expression). CONCLUSIONS: Our data indicate that heme activates the ATF1 pathway in human macrophages via AMPK, and that a similar response occurs after treatment of cells with metformin. Our results suggest an in vitro mechanism that may explain the clinical evidence that metformin has vascular protective effects beyond its role in treating hyperglycemia.

Coronary intraplaque hemorrhage evokes a novel atheroprotective macrophage phenotype.

Intraplaque hemorrhage accelerates atherosclerosis via oxidant stress and contributes to lesion development and destabilization. Normally, macrophages scavenge hemoglobin-haptoglobin (HbHp) complexes via CD163, and this process provokes the secretion of the anti-inflammatory atheroprotective cytokine interleukin (IL)-10. We therefore tested the hypothesis that HbHp complexes may drive monocyte differentiation to an atheroprotective phenotype. Examination of the macrophage phenotype in hemorrhaged atherosclerotic plaques revealed a novel hemorrhage-associated macrophage population (HA-mac), defined by high levels of CD163, but low levels of human leukocyte antigen-DR. HA-mac contained more iron, a pro-oxidant catalyst, but paradoxically had less oxidative injury, measured by 8-oxo-guanosine content. Differentiating monocytes with HbHp complexes reproduced the CD163(high) human leukocyte antigen-DR(low) HA-mac phenotype in vitro. These in vitro HA-mac cells cleared Hb more quickly, and consistently showed less hydrogen peroxide release, highly reactive oxygen species and oxidant stress, and increased survival. Differentiation to HA-mac was prevented by neutralizing IL-10 antibodies, indicating that IL-10 mediates an autocrine feedback mechanism in this system. Nonlinear dynamic modeling showed that an IL-10/CD163-positive feedback loop drove a discrete HA-mac lineage. Simulations further indicated an all-or-none switch to HA-mac at threshold levels of HbHp, and this conversion was experimentally verified. These data demonstrate the creation of a novel atheroprotective (HA-mac) macrophage subpopulation in response to intraplaque hemorrhage and raise the possibility that therapeutically reproducing this macrophage phenotype may be cardio-protective in cases of atherosclerosis.