Rationale: Intraplaque hemorrhage (IPH) drives atherosclerosis through the dual metabolic stresses of cholesterol-enriched erythrocyte membranes and pro-oxidant heme/iron. When clearing tissue hemorrhage, macrophages are typically seen storing either iron or lipid. We have recently defined hemorrhage-associated macrophages (HA-mac) as a plaque macrophage population that responds adaptively to IPH.Objective: This study aimed to define the key transcription factor(s) involved in HO-1 induction by heme. Methods and Results:To address this question, we used microarray analysis and transfection with siRNA and plasmids. To maintain physiological relevance, we focused on human blood-derived monocytes. We found that heme stimulates monocytes through induction of activating transcription factor 1 (ATF-1). ATF-1 coinduces heme oxygenase-1 (HO-1) and Liver X receptor beta (LXR-␤). Heme-induced HO-1 and LXR-␤ were suppressed by knockdown of ATF-1, and HO-1 and LXR-␤ were induced by ATF-1 transfection. ATF-1 required phosphorylation for full functional activity. Expression of LXR-␤ in turn led to induction of other genes central to cholesterol efflux, such as LXR-␣ and ABCA1. This heme-directed state was distinct from known macrophage states (M1, M2, Mox) and, following the same format, we have designated them Mhem. Key Words: macrophages Ⅲ atherosclerosis Ⅲ heme oxygenase-1 Ⅲ lipids Ⅲ activating transcription factor-1 A therosclerosis is an inflammatory disease of large artery walls, driven mainly by lipid peroxidant stress from oxidized low-density lipoprotein (OxLDL). 1 Intraplaque hemorrhage (IPH) is particularly important in promoting both atherosclerotic lesion progression and destabilization. 2 Thus, erythrocytes provide a potent combination of cholesterolenriched membrane lipids and heme-iron, which together pose a serious metabolic challenge in a pathology largely driven by oxidized cholesterols. 2 Along with intracranial hemorrhage, IPH is one of the most important examples of tissue damage due to extravasated blood. 2 Heme-iron is an effective peroxidant catalyst, through hydrogen peroxide coordination and Fenton chemistry 3 ; cholesterol is modified by peroxidation to potently cytotoxic and inflammatory oxysterols (5Ј, 6Ј-epoxycholesterol, 7Ј-ketocholesterol, and 21Ј-keto-cholesterol), 4 and macrophages are abundant in atherosclerosis and generate hydrogen peroxide when activated. The combination of cholesterol loading, heme/iron loading, and macrophage activation would therefore promote lipid peroxidation. 5 How monocytes entering plaques differentiate adaptively to clear hemorrhage-related iron and lipid may therefore be a key transcriptional decision in atherosclerosis. Conclusions:Heme oxygenase-1 (HO-1) is a vital enzyme for iron homeostasis and protection from oxidant stress. It catalyzes pro-oxidant heme and generates biliverdin, free iron and carbon monoxide as reaction products. 6 HO-1 activity also stimulates upregulation of ferritin genes, leading to the safe chelation of iron. 6 Biliverdin is pro...

Key Points This study has identified a novel mechanism by which TF expression is posttranscriptionally regulated in macrophages. The mechanism involves the control of mRNA stability by a cooperation between PARP-14 and TTP.

Antiendothelial cell antibodies (AECAs) are commonly detectable in diseases associated with vascular injury, including systemic lupus erythematosus (SLE), systemic sclerosis, Takayasu arteritis, Wegener granulomatosis, Behç et syndrome, and transplant arteriosclerosis. Here, we explore the hypothesis that these antibodies might augment polymorphonuclear leukocyte (PMN) adhesion to endothelium in inflammation. Initially, we established that a mouse IgG mAb bound to endothelial cells (ECs) significantly increased PMN adhesion to cytokine-stimulated endothelium in an Fc␥RIIa-dependent manner. Neutralizing antibodies, and adenoviral transduction of resting ECs, demonstrated that the combination of Eselectin, CXCR1/2, and ␤ 2 integrins is both necessary and sufficient for this process. We observed an identical mechanism using AECA IgG isolated directly from patients with SLE. Assembled immune complexes also enhanced PMN adhesion to endothelium, but, in contrast to adhesion because of AECAs, this process did not require CXCR1/2, was not inhibited by pertussis toxin, and was Fc␥RIIIb rather than Fc␥RIIa dependent. IntroductionThe recruitment of circulating polymorphonuclear leukocytes (PMNs) to sites of inflammation is mediated by a series of adhesion and activation steps, known as the "adhesion cascade." 1 Thus, selectins are largely responsible for initial tethering of flowing PMNs to endothelium and also mediate subsequent PMN rolling on the endothelial surface. Activation of rolling PMNs leads to leukocyte arrest via modulation of the affinity and avidity of ␤ 2 integrins, with LFA-1 (CD11a/CD18) acting primarily to slow rolling and to promote arrest, and Mac-1 (CD11b/CD18) acting to stabilize adhesion. [2][3][4][5] The capacity of endothelial cells (ECs) to support these interactions with PMNs is stimulated by cytokines such as TNF␣ and IL-1␤, which induce expression of a large number of adhesion molecules, chemottractant, and other proinflammatory genes, including E-selectin, chemokines (eg, IL-8), and ICAM-1. 6,7 Antibodies that react with the surface of vascular endothelial cells (antiendothelial cell antibodies, AECAs) are found in a variety of diseases associated with vascular injury, including systemic lupus erythematosus (SLE), systemic sclerosis, Takayasu arteritis, Wegener granulomatosis, Behçets syndrome, and transplant arteriosclerosis, 8,9 A number of mechanisms have been proposed whereby IgG binding to ECs may exert pathogenic effects, including the induction of EC inflammatory activation and thrombogenicity, the stimulation of leukocyte free-radical production and cellular cytotoxicity, and the induction of EC apoptosis. [10][11][12][13][14] Interactions between circulating human PMNs and immune complexes are mediated via 2 low-affinity Fc␥ receptors, Fc␥RIIa (CD32a) and Fc␥RIIIb (CD16b), which are both thought to form homodimers and have distinct membrane-anchoring and -signaling capacities. [15][16][17][18][19] The cytoplasmic domain of Fc␥RIIa has a specialized immunoreceptor tyrosine-based activ...

Immune reconstitutions (hu-PBL-SCID mice) resulting from adoptive transfer of human peripheral blood mononuclear cells into 1800 C.B-17 scid-/scid-mice were characterized. Over 90% of reconstitutions were successful as evidenced by human immunoglobulin production. Variability was noted with donor, cell number, and cell type. Human cells (T lymphocytes, few B cells) could be recovered by 5 days after engraftment. High levels of soluble CD8 and interleukin-2 receptors were detected in sera of hu-PBL-SCID mice. Cells recovered from 17 mice proliferated in response to antigens to which the donor had been primed; responses to nonboosted antigen also increased in some animals. After reconstitution, lymphocytes were found in the spleen and lymph nodes without full restoration of normal architecture. The hu-PBL-SCID mouse shows promise as a model system for a variety of immunologic studies. The inherent variation in the system must be minimized for appropriate use of the model.

AimsWe aimed to determine whether the levels of total serum IgM and IgG, together with specific antibodies against malondialdehyde-conjugated low-density lipoprotein (MDA-LDL), can improve cardiovascular risk discrimination.Methods and ResultsThe Anglo-Scandinavian Cardiac Outcomes Trial (ASCOT) randomized 9098 patients in the UK and Ireland into the Blood Pressure-Lowering Arm. 485 patients that had cardiovascular (CV) events over 5.5 years were age and sex matched with 1367 controls. Higher baseline total serum IgG, and to a lesser extent IgM, were associated with decreased risk of CV events (IgG odds ratio (OR) per one standard deviation (SD) 0.80 [95% confidence interval, CI 0.72,0.89], p < 0.0001; IgM 0.83[0.75,0.93], p = 0.001), and particularly events due to coronary heart disease (CHD) (IgG OR 0.66 (0.57,0.76); p < 0.0001, IgM OR 0.81 (0.71,0.93); p = 0.002). The association persisted after adjustment for a basic model with variables in the Framingham Risk Score (FRS) as well as following inclusion of C-reactive protein (CRP) and N-terminal pro-B-type natriuretic peptide (NtProBNP). IgG and IgM antibodies against MDA-LDL were also associated with CV events but their significance was lost following adjustment for total serum IgG and IgM respectively. The area under the receiver operator curve for CV events was improved from the basic risk model when adding in total serum IgG, and there was improvement in continuous and categorical net reclassification (17.6% and 7.5% respectively) as well as in the integrated discrimination index.ConclusionHigh total serum IgG levels are an independent predictor of freedom from adverse cardiovascular events, particularly those attributed to CHD, in patients with hypertension.

Objective-Intraplaque hemorrhage (IPH) is an important progression event in advanced atherosclerosis, in large part because of the delivery of prooxidant hemoglobin in erythrocytes. We have previously defined a novel macrophage phenotype (hemorrhage-associated-mac) in human advanced plaques with IPH. These may be atheroprotective in view of raised heme oxygenase 1 (HO-1), CD163, and interleukin-10 expression and suppressed oxidative stress. Methods and Results-We have used a combination of small interfering RNA and pharmacological reagents, protein analysis, and oxidative stress measurements to dissect the pathway leading to the development of this phenotype. We found that erythrocytes, hemoglobin, or purified heme similarly induced CD163 and suppressed human leukocyte antigen and reactive oxygen species. HO-1 was required for the development of each of these features. Challenge of macrophages with purified heme provoked nuclear translocation of Nrf2, and Nrf2 small interfering RNA resulted in significant inhibition of the ability of heme to induce HO-1 protein. Furthermore, tert-butyl-hydroquinone, which activates Nrf2, upregulated CD163, suppressed human leukocyte antigen, and induced interleukin-10, further supporting a role for Nrf2-mediated signaling. However, an inducible protein transactivator is also probably necessary, as heme-induced HO-1 mRNA expression was fully inhibited by the protein synthesis inhibitor cycloheximide. Conclusion-Our

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.

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