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Objectives: This study sought to examine the effect of oral metformin (Mf) therapy on endothelialization in the setting of drug-eluting stents (DES)., Background: Mf is a commonly used therapy in diabetic patients receiving DES. Mf and locally eluted mammalian target of rapamycin (mTOR) inhibitors used in DES have convergent molecular signaling; however, the impact of this drug interaction on stent endothelialization is unknown., Methods: We examined human endothelial aortic cells (HAECs) and a rabbit model of stenting to determine points on molecular convergence between these 2 agents and their impact on stent endothelialization., Results: Western blotting of HAECs treated with Mf and the mTOR inhibitor sirolimus and 14-day rabbit iliacs treated with the combination of zotarolimus-eluting stents (ZES) and oral Mf demonstrated greater inhibition of S6 kinase (S6K), a downstream effector of mTOR complex 1, than either treatment alone. HAEC proliferation was significantly inhibited by Mf or sirolimus treatments alone and further reduced when they were combined. Knockdown of S6K via short interfering RNA in HAECs impaired cell proliferation via a cyclin D1-dependent mechanism, whereas its overexpression rescued the antiproliferative effects of both agents. Last, endothelialization and endothelial cell proliferation at 14 days were assessed in rabbits receiving ZES or bare-metal stents and Mf or placebo by scanning electron microscopy and bromodeoxyuridine/CD31 labeling, respectively. Both endpoints were inhibited by ZES treatment alone and were further reduced by the combination of Mf and ZES., Conclusions: Significant convergence of signaling occurs between Mf and locally delivered mTOR inhibitors at S6K. This further impairs endothelial recovery/proliferation via an S6K-dependent mechanism. Patients receiving Mf in combination with stents that elute mTOR inhibitors are potentially at increased risk of delayed endothelial healing and stent thrombosis., (Copyright © 2013 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Objectives: The purpose of this study was to examine selective macrophage differentiation occurring in areas of intraplaque hemorrhage in human atherosclerosis., Background: Macrophage subsets are recognized in atherosclerosis, but the stimulus for and importance of differentiation programs remain unknown., Methods: We used freshly isolated human monocytes, a rabbit model, and human atherosclerotic plaques to analyze macrophage differentiation in response to hemorrhage., Results: Macrophages characterized by high expression of both mannose and CD163 receptors preferentially exist in atherosclerotic lesions at sites of intraplaque hemorrhage. These hemoglobin (Hb)-stimulated macrophages, M(Hb), are devoid of neutral lipids typical of foam cells. In vivo modeling of hemorrhage in the rabbit model demonstrated that sponges exposed to red cells showed an increase in mannose receptor-positive macrophages only when these cells contained Hb. Cultured human monocytes exposed to Hb:haptoglobin complexes, but not interleukin-4, expressed the M(Hb) phenotype and were characterized by their resistance to cholesterol loading and up-regulation of ATP-binding cassette (ABC) transporters. M(Hb) demonstrated increased ferroportin expression, reduced intracellular iron, and reactive oxygen species (ROS). Degradation of ferroportin using hepcidin increased ROS and inhibited ABCA1 expression and cholesterol efflux to apolipoprotein A-I, suggesting reduced ROS triggers these effects. Knockdown of liver X receptor alpha (LXRα) inhibited ABC transporter expression in M(Hb) and macrophages differentiated in the antioxidant superoxide dismutase. Last, LXRα luciferase reporter activity was increased in M(Hb) and significantly reduced by overnight treatment with hepcidin. Collectively, these data suggest that reduced ROS triggers LXRα activation and macrophage reverse cholesterol transport., Conclusions: Hb is a stimulus for macrophage differentiation in human atherosclerotic plaques. A decrease in macrophage intracellular iron plays an important role in this nonfoam cell phenotype by reducing ROS, which drives transcription of ABC transporters through activation of LXRα. Reduction of macrophage intracellular iron may be a promising avenue to increase macrophage reverse cholesterol transport., (Copyright © 2012 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)