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Aims P-selectin is an activatable adhesion molecule on platelets promoting platelet aggregation, and platelet–leukocyte complex (PLC) formation. Increased numbers of PLC are circulating in the blood of patients shortly after acute myocardial infarction and predict adverse outcomes. These correlations led to speculations about whether PLC may represent novel therapeutic targets. We therefore set out to elucidate the pathomechanistic relevance of PLC in myocardial ischemia and reperfusion injury. Methods and results By generating P-selectin deficient bone marrow chimeric mice, the post-myocardial infarction surge in PLC numbers in blood was prevented. Yet, intravital microscopy, flow cytometry and immunohistochemical staining, echocardiography, and gene expression profiling showed unequivocally that leukocyte adhesion to the vessel wall, leukocyte infiltration, and myocardial damage post-infarction were not altered in response to the lack in PLC. Conclusion We conclude that myocardial infarction associated sterile inflammation triggers PLC formation, reminiscent of conserved immunothrombotic responses, but without PLC influencing myocardial ischemia and reperfusion injury in return. Our experimental data do not support a therapeutic concept of selectively targeting PLC formation in myocardial infarction.

MRI sensitivity for diagnosis and localization of early myocarditis is limited, although it is of central clinical interest. The aim of this project was to test a contrast agent targeting activated platelets consisting of microparticles of iron oxide (MPIO) conjugated to a single-chain antibody directed against ligand-induced binding sites (LIBS) of activated glycoprotein IIb/IIIa (= LIBS-MPIO). Myocarditis was induced by subcutaneous injection of an emulsion of porcine cardiac myosin and complete Freund’s adjuvant in mice. 3D 7 T in-vivo MRI showed focal signal effects in LIBS-MPIO injected mice 2 days after induction of myocarditis, whereas in control-MPIO injected mice no signal was detectable. Histology confirmed CD41-positive staining, indicating platelet involvement in myocarditis in mice as well as in human specimens with significantly higher LIBS-MPIO binding compared to control-MPIO in both species. Quantification of the myocardial MRI signal confirmed a signal decrease after LIBS-MPIO injection and significant less signal in comparison to control-MPIO injection. These data show, that platelets are involved in inflammation during the course of myocarditis in mice and humans. They can be imaged non-invasively with LIBS-MPIO by molecular MRI at an early time point of the inflammation in mice, which is a valuable approach for preclinical models and of interest for both diagnostic and prognostic purposes.

Objective Interferon regulatory factor (IRF) 5 is a transcription factor known for promoting M1 type macrophage polarization in vitro. Given the central role of inflammatory macrophages in promoting atherosclerotic plaque progression, we hypothesize that myeloid cell-specific deletion of IRF5 is protective against atherosclerosis. Methods Female Apoe–/–LysmCre/+Irf5fl/fl and Apoe−/−Irf5fl/fl mice were fed a high-cholesterol diet for three months. Atherosclerotic plaque size and compositions as well as inflammatory gene expression were analyzed. Mechanistically, IRF5-dependent bone marrow-derived macrophage cytokine profiles were tested under M1 and M2 polarizing conditions. Mixed bone marrow chimeras were generated to determine intrinsic IRF5-dependent effects on macrophage accumulation in atherosclerotic plaques. Results Myeloid cell-specific Irf5 deficiency blunted LPS/IFNγ-induced inflammatory gene expression in vitro and in the atherosclerotic aorta in vivo. While atherosclerotic lesion size was not reduced in myeloid cell-specific Irf5-deficient Apoe–/– mice, plaque composition was favorably altered, resembling a stable plaque phenotype with reduced macrophage and lipid contents, reduced inflammatory gene expression and increased collagen deposition alongside elevated Mertk and Tgfβ expression. Irf5-deficient macrophages, when directly competing with wild type macrophages in the same mouse, were less prone to accumulate in atherosclerotic lesion, independent of monocyte recruitment. Irf5-deficient monocytes, when exposed to oxidized low density lipoprotein, were less likely to differentiate into macrophage foam cells, and Irf5-deficient macrophages proliferated less in the plaque. Conclusion Our study provides genetic evidence that selectively altering macrophage polarization induces a stable plaque phenotype in mice., Highlights • IRF5 is widely expressed in inflammatory, foamy, and resident-like macrophages in murine and human plaque. • IRF5 expression in macrophages destabilizes the atherosclerotic plaque. • IRF5 promotes mono to macro differentiation, macrophage proliferation, and foam cell formation in the plaque environment.

Previous mouse studies have shown the increased presence of platelets in the myocardium during early stages of myocarditis and their selective detection by MRI. Here, we aimed to depict early myocarditis using molecular contrast-enhanced ultrasound of activated platelets, and to evaluate the impact of a P2Y12 receptor platelet inhibition. Experimental autoimmune myocarditis was induced in BALB/c mice by subcutaneous injection of porcine cardiac myosin and complete Freund adjuvant (CFA). Activated platelets were targeted with microbubbles (MB) coupled to a single-chain antibody that binds to the “ligand-induced binding sites” of the GPIIb/IIIa-receptor (=LIBS-MB). Alongside myocarditis induction, a group of mice received a daily dose of 100 g prasugrel for 1 month. Mice injected with myosin and CFA had a significantly deteriorated ejection fraction and histological inflammation on day 28 compared to mice only injected with myosin. Platelets infiltrated the myocardium before reduction in ejection fraction could be detected by echocardiography. No selective binding of the LIBS-MB contrast agent could be detected by either ultrasound or histology. Prasugrel therapy preserved ejection fraction and significantly reduced platelet aggregates in the myocardium compared to mice without prasugrel therapy. Therefore, P2Y12 inhibition could be a promising early therapeutic target in myocarditis, requiring further investigation.

Extracellular adenosine-5'-triphosphate (ATP) acts as an import signaling molecule mediating inflammation via purinergic P2 receptors. ATP binds to the purinergic receptor P2X

The acute respiratory distress syndrome (ARDS) is a life-threatening clinical condition. The number of ARDS cases has risen dramatically recently but specific treatment options are limited. ARDS is associated with an overshooting inflammatory response and neutrophils play a central role in its pathogenesis. Neutrophils express the integrin Mac-1 on their surface which adopts a resting and activated conformation depending on leukocyte activation. The aim of this study was to investigate the anti-inflammatory effects of the unique activation-specific anti-Mac-1 DARPin ‘F7’ in a mouse model of ARDS. ARDS was induced by intratracheal lipopolysaccharide (LPS) instillation and the acute (day 1–4) and chronic phase (day 5–10) were studied. After expression and purification, F7, a control DARPin and PBS, were applied daily via the intraperitoneal route. Survival and weight loss were recorded. Histological analysis of lung sections, flow cytometric leukocyte analysis of blood and bronchioalveolar lavage (BALF) were performed. Moreover, protein concentration and cytokine levels were determined in the BALF. Treatment with F7 improved survival and reduced weight loss significantly compared to treatment with the control DARPin or PBS. Neutrophil count in the BALF and peripheral blood were significantly reduced in mice treated with F7. Histology revealed significantly reduced pulmonary inflammation in the F7 treated group. Treatment with DARPin F7 inhibited neutrophil accumulation, reduced signs of local and systemic inflammation and improved survival in a mouse model of ARDS. F7 may be a novel anti-inflammatory drug candidate for the treatment of severe ARDS.

Emergency hematopoiesis is the driving force of the inflammatory response to myocardial infarction (MI). Increased proliferation of hematopoietic stem and progenitor cells (LSK) after MI enhances cell production in the bone marrow (BM) and replenishes leukocyte supply for local cell recruitment to the infarct. Decoding the regulation of the inflammatory cascade after MI may provide new avenues to improve post-MI remodeling. In this study, we describe the influence of adenosine diphosphate (ADP)-dependent P2Y

Statins induce plaque regression characterized by reduced macrophage content in humans, but the underlying mechanisms remain speculative. Studying the translational APOE*3-Leiden.CETP mouse model with a humanized lipoprotein metabolism, we find that systemic cholesterol lowering by oral atorvastatin or dietary restriction inhibits monocyte infiltration, and reverses macrophage accumulation in atherosclerotic plaques. Contrary to current believes, none of (1) reduced monocyte influx (studied by cell fate mapping in thorax-shielded irradiation bone marrow chimeras), (2) enhanced macrophage egress (studied by fluorescent bead labeling and transfer), or (3) atorvastatin accumulation in murine or human plaque (assessed by mass spectrometry) could adequately account for the observed loss in macrophage content in plaques that undergo phenotypic regression. Instead, suppression of local proliferation of macrophages dominates phenotypic plaque regression in response to cholesterol lowering: the lower the levels of serum LDL-cholesterol and lipid contents in murine aortic and human carotid artery plaques, the lower the rates of in situ macrophage proliferation. Our study identifies macrophage proliferation as the predominant turnover determinant and an attractive target for inducing plaque regression. Supplementary Information The online version contains supplementary material available at 10.1007/s00395-020-00838-4.

X-ray fluoroscopy is the gold standard for coronary diagnostics and intervention. Magnetic resonance imaging is a radiation-free alternative to x-ray with excellent soft tissue contrast in arbitrary slice orientation. Here, we assessed real-time MRI-guided coronary interventions from femoral access using newly designed MRI technologies. Six Goettingen minipigs were used to investigate coronary intervention using real-time MRI. Catheters were custom-designed and equipped with an active receive tip-coil to improve visibility and navigation capabilities. Using modified standard clinical 5 F catheters, intubation of the left coronary ostium was successful in all animals. For the purpose of MR-guided coronary interventions, a custom-designed 8 F catheter was used. In spite of the large catheter size, and therefore limited steerability, intubation of the left coronary ostium was successful in 3 of 6 animals within seconds. Thereafter, real-time guided implantation of a non-metallic vascular scaffold into coronary arteries was possible. This study demonstrates that real-time MRI-guided coronary catheterization and intervention via femoral access is possible without the use of any contrast agents or radiation, including placement of non-metallic vascular scaffolds into coronary arteries. Further development, especially in catheter and guidewire technology, will be required to drive forward routine MR-guided coronary interventions as an alternative to x-ray fluoroscopy.

Rationale: The coincidence of inflammation and metabolic derangements in obese adipose tissue has sparked the concept of met-inflammation. Previous observations, however, suggest that inflammatory pathways may not ultimately cause dysmetabolism. Objective: We have revisited the relationship between inflammation and metabolism by testing the role of TRAF (tumor necrosis receptor-associated factor)-1, an inhibitory adapter of inflammatory signaling of TNF (tumor necrosis factor)-a, IL (interleukin)-1ß, and TLRs (toll-like receptors). Methods and Results: Mice defcient for TRAF-1, which is expressed in obese adipocytes and adipose tissue lymphocytes, caused an expected hyperinflammatory phenotype in adipose tissue with enhanced adipokine and chemokine expression, increased leukocyte accumulation, and potentiated proinflammatory signaling in macrophages and adipocytes in a mouse model of diet-induced obesity. Unexpectedly, TRAF-1-/-mice were protected from metabolic derangements and adipocyte growth, failed to gain weight, and showed improved insulin resistance-an effect caused by increased lipid breakdown in adipocytes and UCP (uncoupling protein)-1-enabled thermogenesis. TRAF-1-dependent catabolic and proinflammatory cues were synergistically driven by ß3-adrenergic and inflammatory signaling and required the presence of both TRAF-1-defcient adipocytes and macrophages. In human obesity, TRAF-1-dependent genes were upregulated. Conclusions: Enhancing TRAF-1-dependent inflammatory pathways in a gain-of-function approach protected from metabolic derangements in diet-induced obesity. These fndings identify TRAF-1 as a regulator of dysmetabolism in mice and humans and question the pathogenic role of chronic inflammation in metabolism. Fil: Michel, Nathaly Anto. University of Freiburg. University Medical Center; Alemania Fil: Colberg, Christian. University of Freiburg. University Medical Center; Alemania Fil: Buscher, Konrad. La Jolla Institute for Allergy and Immunology; Estados Unidos Fil: Sommer, Björn. Universitat Erlangen-Nuremberg; Alemania Fil: Pramod, Akula Bala. La Jolla Institute for Allergy and Immunology; Estados Unidos Fil: Ehinger, Erik. La Jolla Institute for Allergy and Immunology; Estados Unidos Fil: Dufner, Bianca. University of Freiburg. University Medical Center; Alemania Fil: Hoppe, Natalie. University of Freiburg. University Medical Center; Alemania Fil: Pfeiffer, Katharina. University of Freiburg. University Medical Center; Alemania Fil: Marchini, Timoteo Oscar. University of Freiburg. University Medical Center; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Bioquímica y Medicina Molecular. Universidad de Buenos Aires. Facultad Medicina. Instituto de Bioquímica y Medicina Molecular; Argentina Fil: Willecke, Florian. University of Freiburg. University Medical Center; Alemania Fil: Stachon, Peter. University of Freiburg. University Medical Center; Alemania Fil: Hilgendorf, Ingo. University of Freiburg. University Medical Center; Alemania Fil: Heidt, Timo. University of Freiburg. University Medical Center; Alemania Fil: Von Zur Muhlen, Constantin. University of Freiburg. University Medical Center; Alemania Fil: Von Elverfeldt, Dominik. University of Freiburg. University Medical Center; Alemania Fil: Pfeifer, Dietmar. University of Freiburg; Alemania Fil: Schüle, Roland. University of Freiburg; Alemania Fil: Kintscher, Ulrich. University of Freiburg; Alemania Fil: Brachs, Sebastian. Center For Cardiovascular Research; Alemania Fil: Ley, Klaus. La Jolla Institute for Allergy and Immunology; Estados Unidos Fil: Bode, Christoph. University of Freiburg. University Medical Center; Alemania Fil: Zirlik, Andreas. University of Freiburg. University Medical Center; Alemania Fil: Wolf, Dennis. La Jolla Institute for Allergy and Immunology; Estados Unidos

Sterile inflammation of visceral fat, provoked by dying adipocytes, links the metabolic syndrome to cardiovascular disease. Danger-associated molecular patterns, such as adenosine triphosphate (ATP), are released by activated or dying cells and orchestrate leukocyte infiltration and inflammation via the purinergic receptor P2Y

Rationale: The mechanisms leading to an expanded neutrophil and monocyte supply after stroke are incompletely understood. Objective: To test the hypothesis that transient middle cerebral artery occlusion (tMCAO) in mice leads to activation of hematopoietic bone marrow stem cells. Methods and Results: Serial in vivo bioluminescence reporter gene imaging in mice with tMCAO revealed that bone marrow cell cycling peaked 4 days after stroke ( P P P P 3 adrenergic receptor, hematopoietic stem cells did not enter the cell cycle in increased numbers after tMCAO (naive control, 3.23±0.22; tMCAO, 3.74±0.33, P =0.51). Conclusions: Ischemic stroke activates hematopoietic stem cells via increased sympathetic tone, leading to a myeloid bias of hematopoiesis and higher bone marrow output of inflammatory Ly6C high monocytes and neutrophils.

Platelets can be found on the surface of inflamed and ruptured atherosclerotic plaques. Thus, targeting of activated platelets may allow for molecular imaging of vulnerable atherosclerotic lesions. We here investigated microbubbles (MB) functionalized with the selectin ligand sialyl Lewisa individually (MBsLea) or dually with sLea and an antibody targeting ligand-induced binding sites of the activated GPIIb/IIIa receptor (MBDual). Assessed by in vitro flow chamber, targeted MB exhibited increased adhesion to platelets as compared to MBControl. While MBsLea rolled slowly on the platelets’ surface, MBDual enhanced the percentage of firm adhesion. In vivo, MB were investigated by ultrasound in a model of ferric chloride induced non-occlusive carotid artery thrombosis. MBsLea and MBDual revealed a higher ultrasound mean acoustic intensity than MBControl (p Dual demonstrated no additional increase in mean signal intensity as compared to MBsLea. The degree of carotid artery stenosis on histology correlated well with the ultrasound acoustic intensity of targeted MB (p in vivo models.

Rationale : Macrophages populate the steady-state myocardium. Previously, all macrophages were thought to arise from monocytes; however, it emerged that, in several organs, tissue-resident macrophages may self-maintain through local proliferation. Objective : Our aim was to study the contribution of monocytes to cardiac-resident macrophages in steady state, after macrophage depletion in CD11b DTR/+ mice and in myocardial infarction. Methods and Results : Using in vivo fate mapping and flow cytometry, we estimated that during steady state the heart macrophage population turns over in ≈1 month. To explore the source of cardiac-resident macrophages, we joined the circulation of mice using parabiosis. After 6 weeks, we observed blood monocyte chimerism of 35.3±3.4%, whereas heart macrophages showed a much lower chimerism of 2.7±0.5% ( P DTR/+ mice, blood monocytes replenished heart macrophages. However, in the chronic phase after myocardial infarction, macrophages residing in the infarct were again independent from the blood monocyte pool, returning to the steady-state situation. Conclusions : In this study, we show differential contribution of monocytes to heart macrophages during steady state, after macrophage depletion or in the acute and chronic phase after myocardial infarction. We found that macrophages participate in the immunosurveillance of myocardial tissue. These data correspond with previous studies on tissue-resident macrophages and raise important questions on the fate and function of macrophages during the development of heart failure.

Early and reliable detection of pulmonary embolism (PE) is critical for improving patient morbidity and mortality. The desire for low-threshold screening for pulmonary embolism is contradicted by unfavorable radiation of currently used computed tomography or nuclear techniques, while standard magnetic resonance imaging still struggles to provide sufficient diagnostic sensitivity in the lung. In this study we evaluate a molecular-targeted contrast agent against activated platelets for non-invasive detection of murine pulmonary thromboembolism using magnetic resonance imaging. By intravenous injection of human thrombin, pulmonary thromboembolism were consistently induced as confirmed by immunohistochemistry of the lung. Magnetic resonance imaging after thrombin injection showed local tissue edema in "Equation missing" weighted images which co-localized with the histological presence of pulmonary thromboembolism. Furthermore, injection of a functionalized contrast agent targeting activated platelets provided sensitive evidence of focal accumulation of activated platelets within the edematous area, which, ex vivo, correlated well with the size of the pulmonary embolism. In summary, we here show delivery and specific binding of a functionalized molecular contrast agent against activated platelets for targeting pulmonary thromboembolism. Going forward, molecular imaging may provide new opportunities to increase sensitivity of magnetic resonance imaging for detection of pulmonary embolism.

Macrophages in the arterial intima sustain chronic inflammation during atherogenesis. Under hypercholesterolemic conditions murine Ly6Chigh monocytes surge in the blood and spleen, infiltrate nascent atherosclerotic plaques, and differentiate into macrophages that proliferate locally as disease progresses. Spleen tyrosine kinase (SYK) may participate in downstream signaling of various receptors that mediate these processes. We tested the effect of the SYK inhibitor fostamatinib on hypercholesterolemia-associated myelopoiesis and plaque formation in Apoe−/− mice during early and established atherosclerosis. Mice consuming a high cholesterol diet supplemented with fostamatinib for 8 weeks developed less atherosclerosis. Histologic and flow cytometric analysis of aortic tissue showed that fostamatinib reduced the content of Ly6Chigh monocytes and macrophages. SYK inhibition limited Ly6Chigh monocytosis through interference with GM-CSF/IL-3 stimulated myelopoiesis, attenuated cell adhesion to the intimal surface, and blocked M-CSF stimulated monocyte to macrophage differentiation. In Apoe−/− mice with established atherosclerosis, however, fostamatinib treatment did not limit macrophage accumulation or lesion progression despite a significant reduction in blood monocyte counts, as lesional macrophages continued to proliferate. Thus, inhibition of hypercholesterolemia-associated monocytosis, monocyte infiltration, and differentiation by SYK antagonism attenuates early atherogenesis but not established disease when local macrophage proliferation dominates lesion progression. Electronic supplementary material The online version of this article (doi:10.1007/s00395-016-0535-8) contains supplementary material, which is available to authorized users.

Rationale: Antibody-targeted delivery of imaging agents can enhance the sensitivity and accuracy of current imaging techniques. Similarly, homing of effector cells to disease sites increases the efficacy of regenerative cell therapy while reducing the number of cells required. Currently, targeting can be achieved via chemical conjugation to specific antibodies, which typically results in the loss of antibody functionality and in severe cell damage. An ideal conjugation technique should ensure retention of antigen-binding activity and functionality of the targeted biological component. Objective: To develop a biochemically robust, highly reproducible, and site-specific coupling method using the Staphylococcus aureus sortase A enzyme for the conjugation of a single-chain antibody (scFv) to nanoparticles and cells for molecular imaging and cell homing in cardiovascular diseases. This scFv specifically binds to activated platelets, which play a pivotal role in thrombosis, atherosclerosis, and inflammation. Methods and Results: The conjugation procedure involves chemical and enzyme-mediated coupling steps. The scFv was successfully conjugated to iron oxide particles (contrast agents for magnetic resonance imaging) and to model cells. Conjugation efficiency ranged between 50% and 70%, and bioactivity of the scFv after coupling was preserved. The targeting of scFv-coupled cells and nanoparticles to activated platelets was strong and specific as demonstrated in in vitro static adhesion assays, in a flow chamber system, in mouse intravital microscopy, and in in vivo magnetic resonance imaging of mouse carotid arteries. Conclusions: This unique biotechnological approach provides a versatile and broadly applicable tool for procuring targeted regenerative cell therapy and targeted molecular imaging in cardiovascular and inflammatory diseases and beyond.

Following myocardial infarction (MI), myeloid cells derived from the hematopoietic system drive a sharp increase in systemic leukocyte levels that correlates closely with mortality. The origin of these myeloid cells, and the response of hematopoietic stem and progenitor cells (HSPCs) to MI, however, is unclear. Here, we identify a CCR2(+)CD150(+)CD48(-) LSK hematopoietic subset as the most upstream contributor to emergency myelopoiesis after ischemic organ injury. This subset has 4-fold higher proliferation rates than CCR2(-)CD150(+)CD48(-) LSK cells, displays a myeloid differentiation bias, and dominates the migratory HSPC population. We further demonstrate that the myeloid translocation gene 16 (Mtg16) regulates CCR2(+) HSPC emergence. Mtg16(-/-) mice have decreased levels of systemic monocytes and infarct-associated macrophages and display compromised tissue healing and post-MI heart failure. Together, these data provide insights into regulation of emergency hematopoiesis after ischemic injury and identify potential therapeutic targets to modulate leukocyte output after MI.

Background— Inflammation and myocardial necrosis play important roles in ischemia/reperfusion injury after coronary artery occlusion and recanalization. The detection of inflammatory activity and the extent of myocardial necrosis itself are of great clinical and prognostic interest. We developed a dual, noninvasive imaging approach using molecular magnetic resonance imaging in an in vivo mouse model of myocardial ischemia and reperfusion. Methods and Results— Ischemia/reperfusion injury was induced in 10-week-old C57BL/6N mice by temporary ligation of the left anterior descending coronary artery. Activated platelets were targeted with a contrast agent consisting of microparticles of iron oxide (MPIOs) conjugated to a single-chain antibody directed against a ligand-induced binding site (LIBS) on activated glycoprotein IIb/IIIa (LIBS-MPIOs). After injection and imaging of LIBS-MPIOs, late gadolinium enhancement was used to depict myocardial necrosis; these imaging experiments were also performed in P2Y 12 −/− mice. All imaging results were correlated to immunohistochemistry findings. Activated platelets were detectable by magnetic resonance imaging via a significant signal effect caused by LIBS-MPIOs in the area of left anterior descending coronary artery occlusion 2 hours after reperfusion. In parallel, late gadolinium enhancement identified the extent of myocardial necrosis. Immunohistochemistry confirmed that LIBS-MPIOs bound significantly to microthrombi in reperfused myocardium. Only background binding was found in P2Y 12 −/− mice. Conclusions— Dual molecular imaging of myocardial ischemia/reperfusion injury allows characterization of platelet-driven inflammation by LIBS-MPIOs and myocardial necrosis by late gadolinium enhancement. This noninvasive imaging strategy is of clinical interest for both diagnostic and prognostic purposes and highlights the potential of molecular magnetic resonance imaging for characterizing ischemia/reperfusion injury.

Exposure to psychosocial stress is a risk factor for many diseases, including atherosclerosis. Although incompletely understood, interaction between the psyche and the immune system provides one potential mechanism linking stress and disease inception and progression. Known cross-talk between the brain and immune system includes the hypothalamic-pituitary-adrenal axis, which centrally drives glucocorticoid production in the adrenal cortex, and the sympathetic-adrenal-medullary axis, which controls stress-induced catecholamine release in support of the fight-or-flight reflex. It remains unknown, however, whether chronic stress changes hematopoietic stem cell activity. Here we show that stress increases proliferation of these most primitive hematopoietic progenitors, giving rise to higher levels of disease-promoting inflammatory leukocytes. We found that chronic stress induced monocytosis and neutrophilia in humans. While investigating the source of leukocytosis in mice, we discovered that stress activates upstream hematopoietic stem cells. Under conditions of chronic variable stress in mice, sympathetic nerve fibers released surplus noradrenaline, which signaled bone marrow niche cells to decrease CXCL12 levels through the β3-adrenergic receptor. Consequently, hematopoietic stem cell proliferation was elevated, leading to an increased output of neutrophils and inflammatory monocytes. When atherosclerosis-prone Apoe(-/-) mice were subjected to chronic stress, accelerated hematopoiesis promoted plaque features associated with vulnerable lesions that cause myocardial infarction and stroke in humans.

Background—Exaggerated and prolonged inflammation after myocardial infarction (MI) accelerates left ventricular remodeling. Inflammatory pathways may present a therapeutic target to prevent post-MI heart failure. However, the appropriate magnitude and timing of interventions are largely unknown, in part because noninvasive monitoring tools are lacking. Here, we used nanoparticle-facilitated silencing of CCR2, the chemokine receptor that governs inflammatory Ly-6Chigh monocyte subset traffic, to reduce infarct inflammation in apolipoprotein E–deficient (apoE−/−) mice after MI. We used dual-target positron emission tomography/magnetic resonance imaging of transglutaminase factor XIII (FXIII) and myeloperoxidase (MPO) activity to monitor how monocyte subset–targeted RNAi altered infarct inflammation and healing. Methods and Results—Flow cytometry, gene expression analysis, and histology revealed reduced monocyte numbers and enhanced resolution of inflammation in infarcted hearts of apoE−/− mice that were treated with nanoparticle-encapsulated siRNA. To follow extracellular matrix cross-linking noninvasively, we developed a fluorine-18–labeled positron emission tomography agent (18F-FXIII). Recruitment of MPO-rich inflammatory leukocytes was imaged with a molecular magnetic resonance imaging sensor of MPO activity (MPO-Gd). Positron emission tomography/magnetic resonance imaging detected anti-inflammatory effects of intravenous nanoparticle-facilitated siRNA therapy (75% decrease of MPO-Gd signal; P, National Heart, Lung, and Blood Institute, United States. Dept. of Health and Human Services (contract No. HHSN268201000044C), National Institutes of Health (U.S.) (grant R01-HL096576), National Institutes of Health (U.S.) (grant R01-HL095629), National Institutes of Health (U.S.) (grant T32-HL094301), Deutsche Forschungsgemeinschaft (HE-6382/1-1)

Iron oxide core nanoparticles are attractive imaging agents because their material properties allow the tuning of pharmacokinetics as well as the attachment of multiple moieties to their surface. In addition to affinity ligands, these include fluorochromes and radioisotopes for detection with optical and nuclear imaging. As the iron oxide core can be detected by MRI, options for combining imaging modalities are manifold. Already, preclinical imaging strategies have combined noninvasive imaging with higher resolution techniques, such as intravital microscopy, to gain unprecedented insight into steady-state biology and disease. Going forward, hybrid iron oxide nanoparticles will help to merge modalities, creating a synergy that will enable imaging in basic research and, potentially, also in the clinic.

During progression of atherosclerosis, myeloid cells destabilize lipid-rich plaques in the arterial wall and cause their rupture, thus triggering myocardial infarction and stroke. Survivors of acute coronary syndromes have a high risk of recurrent events for unknown reasons. Here we show that the systemic response to ischaemic injury aggravates chronic atherosclerosis. After myocardial infarction or stroke, Apoe-/- mice developed larger atherosclerotic lesions with a more advanced morphology. This disease acceleration persisted over many weeks and was associated with markedly increased monocyte recruitment. Seeking the source of surplus monocytes in plaques, we found that myocardial infarction liberated haematopoietic stem and progenitor cells from bone marrow niches via sympathetic nervous system signalling. The progenitors then seeded the spleen, yielding a sustained boost in monocyte production. These observations provide new mechanistic insight into atherogenesis and provide a novel therapeutic opportunity to mitigate disease progression.

BACKGROUND: Activated platelets can be found on the surface of inflamed, rupture-prone and ruptured plaques as well as in intravascular thrombosis. They are key players in thrombosis and atherosclerosis. In this study we describe the construction of a radiolabeled single-chain antibody targeting the LIBS-epitope of activated platelets to selectively depict platelet activation and wall-adherent non-occlusive thrombosis in a mouse model with nuclear imaging using in vitro and ex vivo autoradiography as well as small animal SPECT-CT for in vivo analysis. METHODOLOGY/PRINCIPAL FINDINGS: LIBS as well as an unspecific control single-chain antibody were labeled with (111)Indium ((111)In) via bifunctional DTPA ( = (111)In-LIBS/(111)In-control). Autoradiography after incubation with (111)In-LIBS on activated platelets in vitro (mean 3866 ± 28 DLU/mm(2), 4010 ± 630 DLU/mm(2) and 4520 ± 293 DLU/mm(2)) produced a significantly higher ligand uptake compared to (111)In-control (2101 ± 76 DLU/mm(2), 1181 ± 96 DLU/mm(2) and 1866 ± 246 DLU/mm(2)) indicating a specific binding to activated platelets; P

Objective: The accumulation of inflammatory leukocytes is a prerequisite of adipose tissue inflammation during cardiometabolic disease. We previously reported that a genetic deficiency of the intracellular signaling adaptor TRAF5 (TNF [tumor necrosis factor] receptor–associated factor 5) accelerates atherosclerosis in mice by increasing inflammatory cell recruitment. Here, we tested the hypothesis that an impairment of TRAF5 signaling modulates adipose tissue inflammation and its metabolic complications in a model of diet-induced obesity in mice. Approach and Results: To induce diet-induced obesity and adipose tissue inflammation, wild-type or Traf5 −/− mice consumed a high-fat diet for 18 weeks. Traf5 −/− mice showed an increased weight gain, impaired insulin tolerance, and increased fasting blood glucose. Weight of livers and peripheral fat pads was increased in Traf5 −/− mice, whereas lean tissue weight and growth were not affected. Flow cytometry of the stromal vascular fraction of visceral adipose tissue from Traf5 −/− mice revealed an increase in cytotoxic T cells, CD11c + macrophages, and increased gene expression of proinflammatory cytokines and chemokines. At the level of cell types, expression of TNFα, MIP (macrophage inflammatory protein)-1α, MCP (monocyte chemoattractant protein)-1, and RANTES (regulated on activation, normal T-cell expressed and secreted) was significantly upregulated in Traf5 -deficient adipocytes but not in Traf5 -deficient leukocytes from visceral adipose tissue. Finally, Traf5 expression was lower in adipocytes from obese patients and mice and recovered in adipose tissue of obese patients one year after bariatric surgery. Conclusions: We show that a genetic deficiency of TRAF5 in mice aggravates diet-induced obesity and its metabolic derangements by a proinflammatory response in adipocytes. Our data indicate that TRAF5 may promote anti-inflammatory and obesity-preventing signaling events in adipose tissue.