Objectives 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 remains 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 hemoglobin (Hb). Cultured human monocytes exposed to hemoglobin:haptoglobin complexes (Hb:Hp), but not IL-4, expressed the M(Hb) phenotype and were characterized by their resistance to cholesterol loading and upregulation of ABC transporters. M(Hb) demonstrated increased ferroportin (FPN) expression, reduced intracellular iron, and reactive oxygen species (ROS). Degradation of FPN using hepcidin increased ROS, inhibited ABCA1 expression, and cholesterol efflux to ApoAI, 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 anti-oxidant superoxide dismutase. Lastly, liver X receptor α (LXR) luciferase reporter activity was increased in M(Hb) and significantly reduced by overnight treatment with hepcidin. Collectively, these data suggest reduced ROS triggers LXRα activation and macrophage reverse cholesterol transport (RCT). Conclusions Hb is a stimulus for macrophage differentiation in human atherosclerotic plaques. A reduction of macrophage intracellular iron plays an important role in this non- foam 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 RCT.

Macrophages are an essential component of the immune response to ischaemic injury and play an important role in promoting inflammation and its resolution, which is necessary for tissue repair. The type I transmembrane glycoprotein CD163 is exclusively expressed on macrophages, where it acts as a receptor for haemoglobin:haptoglobin complexes. An extracellular portion of CD163 circulates in the blood as a soluble protein, for which no physiological function has so far been described. Here we show that during ischaemia, soluble CD163 functions as a decoy receptor for TWEAK, a secreted pro-inflammatory cytokine of the tumour necrosis factor family, to regulate TWEAK-induced activation of canonical nuclear factor-κB (NF-κB) and Notch signalling necessary for myogenic progenitor cell proliferation. Mice with deletion of CD163 have transiently elevated levels of TWEAK, which stimulate muscle satellite cell proliferation and tissue regeneration in their ischaemic and non-ischaemic limbs. These results reveal a role for soluble CD163 in regulating muscle regeneration after ischaemic injury.

Coronary artery disease is a leading cause of death and disability worldwide with contemporary treatment strategies employing both optimal medical therapy and catheter based percutaneous coronary intervention (PCI) with drug eluting stents (DES). While DES have dramatically reduced restenosis rates, their use has been associated with an increased risk of late stent thrombosis and accelerated neointimal atherosclerosis (i.e. “neoatherosclerosis”) both major contributors to late stent failure. The underlying substrate of late DES failure is likely related to vascular endothelial dysfunction such as poor endothelial regrowth and barrier function (i.e. “endothelial healing”). Initial concerns with 1st generation DES have lead to improvements in mechanical and biologic properties of current 2nd generation DES, which inhibit endothelial regrowth to a lesser extent, lessening late stent failure and resulting in an overall improved safety profile. Current guidelines recommend duration of at least one year of dual anti-platelet therapy with aspirin and a thienopyridine agent such as clopidogrel or prasugrel as sufficient to prevent late thrombotic complications. Recent studies, however, suggest a shorter duration of dual anti-platelet therapy may be equally as safe and efficacious in preventing stent thrombosis with newer generation DES. However, higher risk populations such as patients receiving 1st generation DES or those with increased risk for future ischemic events may benefit from a longer duration (i.e. 30 months) of DAPT to prevent major cardiovascular events with the caveat that such an approach may be associated with an increased risk for bleeding. This review examines the vascular responses to 1st and second generation DES and recent clinical trials examining DAPT duration.

Iron is an essential mineral needed for normal physiologic processes. While its function in oxygen transport and other important physiologic processes is well known, less is understood about its role in inflammatory diseases such as atherosclerosis. Existing paradigms suggest iron as a driver of atherosclerosis through its actions as a pro-oxidant capable of causing lipid oxidation and tissue damage. Recently we and others have identified hemoglobin (Hb) derived iron as an important factor in determining macrophage differentiation and function in areas of intraplaque hemorrhage within human atherosclerosis. Hb associated macrophages, M(Hb), are distinct from traditional macrophage foam cells because they do not contain large amounts of lipid or inflammatory cytokines, are characterized by high levels of expression of mannose receptor (CD206) and CD163 in addition to producing anti-inflammatory cytokines such as IL-10. Despite the well-known role of iron as an catalyst capable of producing lipid peroxidation through generation of reactive oxygen species (ROS) such as hydroxyl radical, we and others have shown that macrophages in areas of intraplaque hemorrhage demonstrate reduced intracellular iron and ROS which triggers production of anti-inflammatory cytokines as well as genes involved in cholesterol efflux. These data suggest that manipulation of macrophage iron itself may be a promising pharmacologic target for atherosclerosis prevention through its effects on macrophage inflammation and lipid metabolism. In this review we will summarize the current understanding of iron as it relates to plaque inflammation and discuss how further exploration of this subject may lead to new therapies for atherosclerosis.

Objective Sirolimus (SRL) is an immunosuppressant drug used to prevent rejection in organ transplantation and neointimal hyperplasia when delivered from drug eluting stents (DES). Major side effects of SRL include edema and local collection of intimal lipid deposits at the DES site suggesting that SRL impairs endothelial barrier function (EBF). The aim of this study was to address the role of SRL on impaired EBF and the potential mechanisms involved. Approach and Results Cultured human aortic endothelial cells (HAEC) and intact human and mouse endothelium was examined to determine the effect of SRL, which binds FKBP12.6 to inhibit the mammalian target of rapamycin (mTOR), on EBF. EBF, measured by transendothelial electrical resistance (TEER), was impaired in HAEC when treated with SRL or siRNA for FKBP12.6 and reversed when pretreated with ryanodine, a stabilizer of RyR2 intracellular calcium release channels. Intracellular calcium increased in HAEC treated with SRL and normalized with ryanodine pretreatment. SRL treated HAEC demonstrated increases in PKCα phosphorylation, a calcium sensitive serine/threonine kinase important in VE cadherin barrier function through its interaction with p120-catenin (p120). Immunostaining of HAEC, human coronary and mouse aortic endothelium showed disruption of p120-VE cadherin interaction treated with SRL. SRL impairment of HAEC EBF was reduced with PKCα siRNA. Mice treated with SRL demonstrated increased vascular permeability by Evans blue albumin extravasation (EBAE) in the lungs, heart and aorta. Conclusions SRL-FKBP12.6 impairs EBF by activation of PKCα and downstream disruption of the p120-VE cadherin in vascular endothelium. These data suggest this mechanism may be an important contributor of SRL side effects related to impaired EBF.