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Purpose: The study aimed to assess the feasibility and image quality of dual-source photon-counting detector computed tomography (PCD-CT) in evaluating small-sized coronary artery stents with respect to different acquisition modes in a phantom model., Methods: Utilizing a phantom setup mimicking the average patient's water-equivalent diameter, we examined six distinct coronary stents inflated in a silicon tube, with stent sizes ranging from 2.0 to 3.5 mm, applying four different CT acquisition modes of a dual-source PCD-CT scanner: "high-pitch," "sequential," "spiral" (each with collimation of 144 × 0.4 mm and full spectral information), and "ultra-high-resolution (UHR)" (collimation of 120 × 0.2 mm and no spectral information). Image quality and diagnostic confidence were assessed using subjective measures, including a 4-point visual grading scale (4 = excellent; 1 = non-diagnostic) utilized by two independent radiologists, and objective measures, including the full width at half maximum (FWHM)., Results: A total of 24 scans were acquired, and all were included in the analysis. Among all CT acquisition modes, the highest image quality was obtained for the UHR mode [median score: 4 (interquartile range (IQR): 3.67-4.00)] ( P = 0.0015, with 37.5% rated as "excellent"), followed by the sequential mode [median score: 3.5 (IQR: 2.84-4.00)], P = 0.0326 and the spiral mode [median score: 3.0 (IQR: 2.53-3.47), P > 0.05]. The lowest image quality was observed for the high-pitch mode [median score: 2 (IQR: 1- 3), P = 0.028]. Similarly, diagnostic confidence for evaluating stent patency was highest for UHR and lowest for high-pitch ( P < 0.001, respectively). Measurement of stent dimensions was accurate for all acquisition modes, with the UHR mode showing highest robustness (FWHM for sequential: 0.926 ± 0.061 vs. high-pitch: 0.990 ± 0.083 vs. spiral: 0.962 ± 0.085 vs. UHR: 0.941 ± 0.036, P = non-significant, respectively)., Conclusion: Assessing small-sized coronary stents using PCD-CT technology is feasible. The UHR mode offers superior image quality and diagnostic confidence, while all modes show consistent and accurate measurements., Clinical Significance: These findings highlight the potential of PCD-CT technology, particularly the UHR mode, to enhance non-invasive coronary stent evaluation. Confirmatory research is necessary to influence the guidelines, which recommend cardiac CT only for stents of 3 mm or larger., Competing Interests: Fabian Bamberg Siemens Healthineers AG (unrestricted research grant, speaker’s bureau), Christopher L. Schlett Siemens Healthineers AG (speaker’s bureau), Muhammad Taha Hagar Siemens Healthineers AG (speaker‘s bureau). Other authors have nothing to disclose.

To compare the diagnostic value of ultrahigh-resolution CT-angiography (UHR-CTA) compared with high-pitch spiral CTA (HPS-CTA) using a first-generation, dual-source photon-counting CT (PCD-CT) scanner for preprocedural planning of transcatheter aortic valve replacement (TAVR). Clinically referred patients with severe aortic valve stenosis underwent both, retrospective ECG-gated cardiac UHR-CTA (collimation: 120 × 0.2 mm) and prospective ECG-triggered aortoiliac HPS-CTA (collimation: 144 × 0.4 mm, full spectral capabilities) for TAVR planning from August 2022 to March 2023. Radiation dose was extracted from the CT reports, and the effective dose was calculated. Two radiologists analyzed UHR-CTA and HPS-CTA datasets, assessing the image quality of the aortic annulus, with regard to the lumen visibility and margin delineation using a 4-point visual-grading scale (ranges: 4 = "excellent" to 1 = "poor"). Aortic annulus area (AAA) measurements were taken for valve prosthesis sizing, with retrospective UHR-CTA serving as reference standard. A total of 64 patients were included (mean age, 81 years ± 7 SD; 28 women) in this retrospective study. HPS-CTA showed a lower radiation dose, 4.1 mSv vs. 12.6 mSv (p < 0.001). UHR-CTA demonstrated higher image quality to HPS-CTA (median score, 4 [IQR, 3-4] vs. 3 [IQR, 2-3]; p < 0.001). Quantitative assessments of AAA from both CTA datasets were strongly positively correlated (mean 477.4 ± 91.1 mm 2 on UHR-CTA and mean 476.5 ± 90.4 mm 2 on HPS-CTA, Pearson r 2  = 0.857, p < 0.001) with a mean error of 22.3 ± 24.6 mm 2 and resulted in identical valve prosthesis sizing in the majority of patients (91%). Patients with lower image quality on HPS-CTA (score value 1 or 2, n = 28) were more likely to receive different sizing recommendations (82%). Both UHR-CTA and HPS-CTA acquisitions using photon-counting CT technology provided reliable aortic annular assessments for TAVR planning. While UHR-CTA offers superior image quality, HPS-CTA is associated with lower radiation exposure. However, severely impaired image quality on HPS-CTA may impact on prosthesis sizing, suggesting that immediate post-scan image evaluations may require complementary UHR-CTA scanning., (© 2024. The Author(s).)

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 TNF[alpha], MIP (macrophage inflammatory protein)-1[alpha], 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.

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)-α, IL (interleukin)-1β, and TLRs (toll-like receptors)., Methods and Results: Mice deficient 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 β3-adrenergic and inflammatory signaling and required the presence of both TRAF-1-deficient 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 findings identify TRAF-1 as a regulator of dysmetabolism in mice and humans and question the pathogenic role of chronic inflammation in metabolism., (© 2018 American Heart Association, Inc.)

Integrin-based therapeutics have garnered considerable interest in the medical treatment of inflammation. Integrins mediate the fast recruitment of monocytes and neutrophils to the site of inflammation, but are also required for host defense, limiting their therapeutic use. Here, we report a novel monoclonal antibody, anti-M7, that specifically blocks the interaction of the integrin Mac-1 with its pro-inflammatory ligand CD40L, while not interfering with alternative ligands. Anti-M7 selectively reduces leukocyte recruitment in vitro and in vivo. In contrast, conventional anti-Mac-1 therapy is not specific and blocks a broad repertoire of integrin functionality, inhibits phagocytosis, promotes apoptosis, and fuels a cytokine storm in vivo. Whereas conventional anti-integrin therapy potentiates bacterial sepsis, bacteremia, and mortality, a ligand-specific intervention with anti-M7 is protective. These findings deepen our understanding of ligand-specific integrin functions and open a path for a new field of ligand-targeted anti-integrin therapy to prevent inflammatory conditions.

Identification of individuals who are at risk of suffering from acute coronary syndromes (ACS) may allow to introduce preventative measures. We aimed to identify ACS-related urinary peptides, that combined as a pattern can be used as prognostic biomarker. Proteomic data of 252 individuals enrolled in four prospective studies from Australia, Europe and North America were analyzed. 126 of these had suffered from ACS within a period of up to 5 years post urine sampling (cases). Proteomic analysis of 84 cases and 84 matched controls resulted in the discovery of 75 ACS-related urinary peptides. Combining these to a peptide pattern, we established a prognostic biomarker named Acute Coronary Syndrome Predictor 75 (ACSP75). ACSP75 demonstrated reasonable prognostic discrimination (c-statistic = 0.664), which was similar to Framingham risk scoring (c-statistics = 0.644) in a validation cohort of 42 cases and 42 controls. However, generating by a composite algorithm named Acute Coronary Syndrome Composite Predictor (ACSCP), combining the biomarker pattern ACSP75 with the previously established urinary proteomic biomarker CAD238 characterizing coronary artery disease as the underlying aetiology, and age as a risk factor, further improved discrimination (c-statistic = 0.751) resulting in an added prognostic value over Framingham risk scoring expressed by an integrated discrimination improvement of 0.273 ± 0.048 (P < 0.0001) and net reclassification improvement of 0.405 ± 0.113 (P = 0.0007). In conclusion, we demonstrate that urinary peptide biomarkers have the potential to predict future ACS events in asymptomatic patients. Further large scale studies are warranted to determine the role of urinary biomarkers in clinical practice.

Cell accumulation is a prerequisite for adipose tissue inflammation. The leukocyte integrin Mac-1 (CD11b/CD18, α M β 2 ) is a classic adhesion receptor critically regulating inflammatory cell recruitment. Here, we tested the hypothesis that a genetic deficiency and a therapeutic modulation of Mac-1 regulate adipose tissue inflammation in a mouse model of diet-induced obesity (DIO). C57Bl6/J mice genetically deficient (Mac-1 -/- ) or competent for Mac-1 (WT) consumed a high fat diet for 20 weeks. Surprisingly, Mac-1 -/- mice presented with increased diet-induced weight gain, decreased insulin sensitivity in skeletal muscle and in the liver in insulin-clamps, insulin secretion deficiency and elevated glucose levels in fasting animals, and dyslipidaemia. Unexpectedly, accumulation of adipose tissue macrophages (ATMs) was unaffected, while gene expression indicated less inflamed adipose tissue and macrophages in Mac-1 -/- mice. In contrast, inflammatory gene expression at distant locations, such as in skeletal muscle, was not changed. Treatment of ATMs with an agonistic anti-Mac-1 antibody, M1/70, induced pro-inflammatory genes in cell culture. In vivo, treatment with M1/70 induced a hyper-inflammatory phenotype with increased expression of IL-6 and MCP-1, whereas accumulation of ATMs did not change. Finally, inhibition of Mac-1's adhesive interaction to CD40L by the peptide inhibitor cM7 did not affect myeloid cell accumulation in adipose tissue. We present the surprising finding that adhesive properties of the leukocyte integrin Mac-1 are not required for macrophage accumulation in adipose tissue. Instead, Mac-1 modulates inflammatory gene expression in macrophages. These findings question the net effect of integrin blockade in cardio-metabolic disease.

Clinical, but not experimental evidence has suggested that air pollution particulate matter (PM) aggravates myocardial infarction (MI). Here, we aimed to describe mechanisms and consequences of PM exposure in an experimental model of MI. C57BL/6J mice were challenged with a PM surrogate (Residual Oil Fly Ash, ROFA) by intranasal installation before MI was induced by permanent ligation of the left anterior descending coronary artery. Histological analysis of the myocardium 7 days after MI demonstrated an increase in infarct area and enhanced inflammatory cell recruitment in ROFA-exposed mice. Mechanistically, ROFA exposure increased the levels of the circulating pro-inflammatory cytokines TNF-α, IL-6, and MCP-1, activated myeloid and endothelial cells, and enhanced leukocyte recruitment to the peritoneal cavity and the vascular endothelium. Notably, these effects on endothelial cells and circulating leukocytes could be reversed by neutralizing anti-TNF-α treatment. We identified alveolar macrophages as the primary source of elevated cytokine production after PM exposure. Accordingly, in vivo depletion of alveolar macrophages by intranasal clodronate attenuated inflammation and cell recruitment to infarcted tissue of ROFA-exposed mice. Taken together, our data demonstrate that exposure to environmental PM induces the release of inflammatory cytokines from alveolar macrophages which directly worsens the course of MI in mice. These findings uncover a novel link between air pollution PM exposure and inflammatory pathways, highlighting the importance of environmental factors in cardiovascular disease.

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 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.

To detect unstable atherosclerotic plaques early and noninvasively would be of great clinical interest. Activated platelets are an interesting molecular target for detecting early lesions or unstable plaques. We therefore developed an MRI contrast agent consisting of magnetoliposomes (ML) linked to an antibody (anti-LIBS) specifically targeting the ligand-induced binding site of the activated GPIIb/IIIa receptor of platelets. ML were prepared by dual centrifugation (DC). ML pegylation up to a total PEG content of 7.5 mol% positively influenced the stability and amount of entrapped SPIOs, and also reduced SPIO-membrane interactions, while higher PEG contents destabilized PEG-ML. Stable anti-LIBS-ML with high amounts of entrapped SPIOs (∼86%, ∼0.22 mol Fe/mol liposomal lipid) and high MRI sensitivity (relaxivity r2 = 422 s(-1) mM(-1) and r2(∗) = 452 s(-1) mM(-1)) were obtained by coupling anti-LIBS to ML in a two-step post-insertion technique. We confirmed specific binding to the GPIIb/IIIa receptor's activated conformation on activated human platelets and cell lines expressing activated GPIIb/IIIa receptor ex vivo. The immuno-ML obtained in this study constitute an important step towards developing a potentially human-compatible MRI contrast agent for the timely detection of plaque rupture by targeting activated platelets., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Atherosclerosis and its sequelae have a major impact on morbidity and mortality. The rupture of an inflamed atherosclerotic plaque is a crucial event, since it can result in acute thrombotic closure of an arterial vessel, resulting e. g. in myocardial infarction or stroke. Not only detection of early plaque rupture with imminent closure is therefore of clinical interest, but also timely detection of vascular inflammation and atherosclerotic plaque progression. However, plaque inflammation or even plaque rupture without vessel occlusion is not reliably detectable by current imaging techniques. Coronary angiography is the gold standard for evaluation of the coronary vessels, but only allows visualization of the vessel lumen without characterizing the important pathophysiology of the vessel wall. Therefore, highly inflamed and rupture prone plaques can be missed, or appear as a minor vessel narrowing. Although currently available techniques such as intravascular ultrasound or optical coherence tomography allow a further characterization of atherosclerotic plaques, it would be desirable to detect plaque inflammation, early plaque rupture or vascular thrombosis by non-invasive techniques such as magnetic resonance imaging (MRI), since they could allow early identification of patients at risk or triage of symptomatic patients. In this manuscript, different strategies for detection of vascular inflammation, plaque-rupture and thrombosis by MRI will be discussed, with a special focus on molecular imaging contrast agents.

Objective: Nucleotides such as ATP, ADP, UTP, and UDP serve as proinflammatory danger signals via purinergic receptors on their release to the extracellular space by activated or dying cells. UDP binds to the purinergic receptor Y6 (P2Y6) and propagates vascular inflammation by inducing the expression of chemokines such as monocyte chemoattractant protein 1, interleukin-8, or its mouse homologsCCL1 (chemokine [C-C motif] ligand 1)/keratinocyte chemokine, CXCL2 (chemokine [C-X-C motif] ligand 2)/macrophage inflammatory protein 2, and CXCL5 (chemokine [C-X-C motif] ligand 5)/LIX, and adhesion molecules such as vascular cell adhesion molecule 1 and intercellular cell adhesion molecule 1. Thus, P2Y6 contributes to leukocyte recruitment and inflammation in conditions such as allergic asthma or sepsis. Because atherosclerosis is a chronic inflammatory disease driven by leukocyte recruitment to the vessel wall, we hypothesized a role of P2Y6 in atherogenesis., Approach and Results: Intraperitoneal stimulation of wild-type mice with UDP induced rolling and adhesion of leukocytes to the vessel wall as assessed by intravital microscopy. This effect was not present in P2Y6-deficient mice. Atherosclerotic aortas of low-density lipoprotein receptor-deficient mice consuming high-cholesterol diet for 16 weeks expressed significantly more transcripts and protein of P2Y6 than respective controls. Finally, P2Y6 (-/-)/low-density lipoprotein receptor-deficient mice consuming high-cholesterol diet for 16 weeks developed significantly smaller atherosclerotic lesions compared with P2Y6 (+/+)/low-density lipoprotein receptor-deficient mice. Bone marrow transplantation identified a crucial role of P2Y6 on vascular resident cells, most likely endothelial cells, on leukocyte recruitment and atherogenesis. Atherosclerotic lesions of P2Y6-deficient mice contained fewer macrophages and fewer lipids as determined by immunohistochemistry. Mechanistically, RNA expression of vascular cell adhesion molecule 1 and interleukin-6 was decreased in these lesions and P2Y6-deficient macrophages took up less modified low-density lipoprotein cholesterol., Conclusions: We show for the first time that P2Y6 deficiency limits atherosclerosis and plaque inflammation in mice., (© 2014 American Heart Association, Inc.)

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 P2Y12 (-/-) 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 P2Y12 (-/-) 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., (© 2014 American Heart Association, Inc.)

The co-stimulatory immune molecule CD40L figures prominently in a variety of inflammatory conditions including arterial disease. Recently, we made the surprising finding that CD40L mediates atherogenesis independently of its classic receptor CD40 via a novel interaction with the leukocyte integrin Mac-1. Here, we hypothesised that selective blockade of the CD40L-Mac-1 interaction may also retard restenosis. We induced neointima formation in C57/BL6 mice by ligation of the left carotid artery. Mice were randomised to daily intraperitoneal injections of either cM7, a small peptide selectively inhibiting the CD40L-Mac-1 interaction, scM7, a scrambled control peptide, or saline for 28 days. Interestingly, cM7-treated mice developed neointima of similar size compared with mice receiving the control peptide or saline as assessed by computer-assisted analysis of histological cross sections. These data demonstrate that the CD40L-Mac-1 interaction is not required for the development of restenosis. In contrast, CD40-deficient mice subjected to carotid ligation in parallel, developed significantly reduced neointimal lesions compared with respective wild-type controls (2872 ± 843 µm² vs 35469 ± 11870 µm²). Flow cytometry in CD40-deficient mice revealed reduced formation of platelet-granulocyte and platelet-inflammatory monocyte- aggregates. In vitro, supernatants of CD40-deficient platelet-leukocyte aggregates attenuated proliferation and increased apoptosis of smooth muscle cells. Unlike in the setting of atherosclerosis, CD40L mediates neointima formation via its classic receptor CD40 rather than via its recently described novel interaction with Mac-1. Therefore, selective targeting of CD40L-Mac-1 binding does not appear to be a favorable strategy to fight restenosis.

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/objectives: We examined the applicability of contrast-enhanced ultrasound (CEUS) for imaging of murine deep vein thrombosis (DVT) and measured the effects of enoxaparin, ticagrelor and P2Y(12) receptor deficiency in vivo., Methods: Deep vein thrombosis was induced by exposure to ferric chloride or ligation of the infrarenal vena cava of C57BL/6 mice after pretreatment with enoxaparin, ticagrelor or vehicle and in P2Y(12-/-) mice. Initial thrombus growth was visualized by intravital microscopy. Thrombi were weighed and examined by immunohistochemistry. CEUS was performed with a standard ultrasound system (Vivid 7, GE Healthcare) in the open abdominal cavity after injection of stabilized sulphur hexafluoride microbubbles., Results: Incubation with ferric chloride resulted in non-occluding platelet-containing thrombus growth within 15-25 min. Sham-operated mice, enoxaparin- and ticagrelor-pretreated wild-type and P2Y(12-/-) mice developed only small thrombi. After injection of the contrast agent, growing thrombi were delineated clearly as negative contrast on CEUS. Thrombus size on CEUS after 25 min was significantly smaller in enoxaparin- (0.3 ± 0.1 mm(2)) and ticagrelor-treated (0.5 ± 0.1 mm(2)) wild-type and in P2Y(12-/-) mice (0.4 ± 0.1 mm(2)) as compared with vehicle-treated wild-type mice (2.0 ± 0.3 mm(2)) in the maximal sagittal plane (P < 0.001, n = 5-10). CEUS-derived thrombus size correlated linearly with thrombus weight and also reflected the extent of ligation-induced DVT., Conclusions: Contrast-enhanced ultrasound allowed the real-time quantification of DVT in living mice. Genetic and pharmacologic antithrombotic interventions were well reflected by CEUS and suggested an important role of the platelet P2Y(12) receptor in early DVT formation., (© 2013 International Society on Thrombosis and Haemostasis.)

Objective: The endothelial protein C-receptor (EPCR) is an endothelial transmembrane protein that binds protein C and activated protein C (APC) with equal affinity, thereby facilitating APC formation. APC has anticoagulant, antiapoptotic and antiinflammatory properties. Soluble EPCR, released by the endothelium, may bind activated neutrophils, thereby modulating cell adhesion. EPCR is therefore considered as a possible link between the anticoagulant properties of protein C and the inflammatory response of neutrophils. In the present study, we aimed to provide proof of concept for a direct binding of EPCR to the β2-integrin Mac-1 on monocytic cells under static and physiological flow conditions., Measurements and Main Results: Under static conditions, human monocytes bind soluble EPCR in a concentration dependent manner, as demonstrated by flow cytometry. Binding can be inhibited by specific antibodies (anti-EPCR and anti-Mac-1). Specific binding was confirmed by a static adhesion assay, where a transfected Mac-1 expressing CHO cell line (Mac-1+ cells) bound significantly more recombinant EPCR compared to Mac-1+ cells blocked by anti-Mac-1-antibody and native CHO cells. Under physiological flow conditions, monocyte binding to the endothelium could be significantly blocked by both, anti-EPCR and anti-Mac-1 antibodies in a dynamic adhesion assay at physiological flow conditions. Pre-treatment of endothelial cells with APC (drotrecogin alfa) diminished monocyte adhesion significantly in a comparable extent to anti-EPCR., Conclusions: In the present study, we demonstrate a direct binding of Mac-1 on monocytes to the endothelial protein C receptor under static and flow conditions. This binding suggests a link between the protein C anticoagulant pathway and inflammation at the endothelium side, such as in acute vascular inflammation or septicaemia.

Noninvasive diagnosis of atherosclerosis via single biomarkers has been attempted but remains elusive. However, a previous polymarker or pattern approach of urine polypeptides in humans reflected coronary artery disease with high accuracy. The aim of the current study is to use urine proteomics in ApoE(-/-) mice to discover proteins with pathophysiological roles in atherogenesis and to identify urinary polypeptide patterns reflecting early stages of atherosclerosis. Urine of ApoE(-/-) mice either on high fat diet (HFD) or chow diet was collected over 12 weeks; urine of wild type mice on HFD was used to exclude diet-related proteome changes. Capillary electrophoresis coupled to mass spectrometry (CE-MS) of samples identified 16 polypeptides specific for ApoE(-/-) mice on HFD. In a blinded test set, these polypeptides allowed identification of atherosclerosis at a sensitivity of 90% and specificity of 100%, as well as monitoring of disease progression. Sequencing of the discovered polypeptides identified fragments of α(1)-antitrypsin, epidermal growth factor (EGF), kidney androgen-regulated protein, and collagen. Using immunohistochemistry, α(1)-antitrypsin, EGF, and collagen type I were shown to be highly expressed in atherosclerotic plaques of ApoE(-/-) mice on HFD. Urinary excretion levels of collagen and α(1)-antitrypsin fragments also significantly correlated with intraplaque collagen and α(1)-antitrypsin content, mirroring plaque protein expression in the urine proteome. To provide further confirmation that the newly identified proteins are relevant in humans, the presence of collagen type I, α(1)-antitrypsin, and EGF was also confirmed in human atherosclerotic disease. Urine proteome analysis in mice exemplifies the potential of a novel multimarker approach for the noninvasive detection of atherosclerosis and monitoring of disease progression. Furthermore, this approach represents a novel discovery tool for the identification of proteins relevant in murine and human atherosclerosis and thus also defines potential novel therapeutic targets.

The development of magnetic resonance imaging (MRI) contrast agents targeting epitopes in atherosclerosis is of general interest. In particular, early detection of activated platelets as key players in plaque rupture could provide improved triage of patients. However, so far the efficiency of contrast agents targeting human pathologies can only be examined in animal experiments, which do not necessarily reflect human in vivo conditions. We therefore describe application of a contrast agent targeting activated human platelets in an MRI tissue flow chamber, allowing detection and characterization of contrast agent binding. Microparticles of iron oxide (MPIO) were conjugated to an antibody targeting ligand-induced binding sites (LIBS) on the activated platelet glycoprotein IIb/IIIa-receptor or to control antibody, resulting in LIBS-MPIO or control-MPIO contrast agent. Human endarterectomy specimens from patients with acute stroke or transient ischemic attack were imaged ex vivo before and after contrast agent perfusion using a 9.4 T MRI system. Specimens were measured under static (n = 18) or flow conditions (n = 18) in a specially designed flow chamber setup, simulating physiological conditions in a stenosed vessel. A significant MPIO-induced negative contrast was achieved in MRI by LIBS-MPIO in specimens under static and flow conditions (LIBS-MPIO vs control-MPIO: p < 0.01), and the location of LIBS-MPIO binding corresponded well between histology and MRI (p < 0.05). The number of MPIOs per platelet area on endarterectomy specimens in histology was significantly higher with LIBS-MPIO (p < 0.001). Furthermore, the intensity of contrast agent binding and signal change showed the potential to reflect the severity of clinical symptoms. LIBS-MPIO allows the detection of activated platelets on the surface of symptomatic atherosclerotic human plaques using molecular MRI. Furthermore, the MRI tissue flow chamber setup described could help to evaluate binding properties of contrast agents, and might therefore be an interesting tool for contrast agent development from animal experiments towards clinical application., (Copyright © 2012 John Wiley & Sons, Ltd.)

Background: Adipose tissue inflammation fuels the metabolic syndrome. We recently reported that CD40L--an established marker and mediator of cardiovascular disease--induces inflammatory cytokine production in adipose cells in vitro. Here, we tested the hypothesis that CD40L deficiency modulates adipose tissue inflammation in vivo., Methodology/principal Findings: WT or CD40L(-/-) mice consumed a high fat diet (HFD) for 20 weeks. Inflammatory cell recruitment was impaired in mice lacking CD40L as shown by a decrease of adipose tissue macrophages, B-cells, and an increase in protective T-regulatory cells. Mechanistically, CD40L-deficient mice expressed significantly lower levels of the pro-inflammatory chemokine MCP-1 both, locally in adipose tissue and systemically in plasma. Moreover, levels of pro-inflammatory IgG-antibodies against oxidized lipids were reduced in CD40L(-/-) mice. Also, circulating low-density lipoproteins and insulin levels were lower in CD40L(-/-) mice. However, CD40L(-/-) mice consuming HFD were not protected from the onset of diet-induced obesity (DIO), insulin resistance, and hepatic steatosis, suggesting that CD40L selectively limits the inflammatory features of diet-induced obesity rather than its metabolic phenotype. Interestingly, CD40L(-/-) mice consuming a low fat diet (LFD) showed both, a favorable inflammatory and metabolic phenotype characterized by diminished weight gain, improved insulin tolerance, and attenuated plasma adipokine levels., Conclusion: We present the novel finding that CD40L deficiency limits adipose tissue inflammation in vivo. These findings identify CD40L as a potential mediator at the interface of cardiovascular and metabolic disease.

Background: Early and non-invasive detection of platelets on micro atherothrombosis provides a means to identify unstable plaque and thereby allowing prophylactic treatment towards prevention of stroke or myocardial infarction. Molecular magnetic resonance imaging (mMRI) of activated platelets as early markers of plaque rupture using targeted contrast agents is a promising strategy. In this study, we aim to specifically image activated platelets in murine atherothrombosis by in vivo mMRI, using a dedicated animal model of plaque rupture., Methods: An antibody targeting ligand-induced binding sites (LIBS) on the glycoprotein IIb/IIIa-receptor of activated platelets was conjugated to microparticles of iron oxide (MPIO) to form the LIBS-MPIO contrast agent causing a signal-extinction in T2*-weighted MRI. ApoE(-/-) mice (60 weeks-old) were fed a high fat diet for 5 weeks. Using a small needle, the surface of their carotid plaques was scratched under blood flow to induce atherothrombosis. In vivo 9.4 Tesla MRI was performed before and repetitively after intravenous injection of either LIBS-MPIO versus non-targeted-MPIO., Results: LIBS-MPIO injected animals showed a significant signal extinction (p<0.05) in MRI, corresponding to the site of plaque rupture and atherothrombosis in histology. The signal attenuation was effective for atherothrombosis occupying ≥ 2% of the vascular lumen. Histology further confirmed significant binding of LIBS-MPIO compared to control-MPIO on the thrombus developing on the surface of ruptured plaques (p<0.01)., Conclusion: in vivo mMRI detected activated platelets on mechanically ruptured atherosclerotic plaques in ApoE(-/-) mice with a high sensititvity. This imaging technology represents a unique opportunity for noninvasive detection of atherothrombosis and the identification of unstable atherosclerotic plaques with the ultimate promise to prevent strokes and myocardial infarctions.

Rationale: CD40L figures prominently in chronic inflammatory diseases such as atherosclerosis. However, since CD40L potently regulates immune function and hemostasis by interaction with CD40 receptor and the platelet integrin GPIIb/IIIa, its global inhibition compromises host defense and generated thromboembolic complications in clinical trials. We recently reported that CD40L mediates atherogenesis independently of CD40 and proposed Mac-1 as an alternate receptor., Objective: Here, we molecularly characterized the CD40L-Mac-1 interaction and tested whether its selective inhibition by a small peptide modulates inflammation and atherogenesis in vivo., Methods and Results: CD40L concentration-dependently bound to Mac-1 I-domain in solid phase binding assays, and a high-affinity interaction was revealed by surface-plasmon-resonance analysis. We identified the motif EQLKKSKTL, an exposed loop between the α1 helix and the β-sheet B, on Mac-1 as binding site for CD40L. A linear peptide mimicking this sequence, M7, specifically inhibited the interaction of CD40L and Mac-1. A cyclisized version optimized for in vivo use, cM7, decreased peritoneal inflammation and inflammatory cell recruitment in vivo. Finally, LDLr(-/-) mice treated with intraperitoneal injections of cM7 developed smaller, less inflamed atherosclerotic lesions featuring characteristics of stability. However, cM7 did not interfere with CD40L-CD40 binding in vitro and CD40L-GPIIb/IIIa-mediated thrombus formation in vivo., Conclusions: We present the novel finding that CD40L binds to the EQLKKSKTL motif on Mac-1 mediating leukocyte recruitment and atherogenesis. Specific inhibition of CD40L-Mac-1 binding may represent an attractive anti-inflammatory treatment strategy for atherosclerosis and other inflammatory conditions, potentially avoiding the unwanted immunologic and thrombotic effects of global inhibition of CD40L.

Objective: : The final event leading to myocardial infarction is adhesion and activation of platelets after rupture of an atherosclerotic plaque, ending in thrombotic occlusion of the coronary artery. Imaging of imminent vessel occlusion may improve patient care. The feasibility of molecular magnetic resonance imaging (MRI) for the detection of coronary artery thrombosis in mice was examined., Materials and Methods: : The left anterior descending coronary artery was exposed by lateral thoracotomy and incubated with ferric chloride to induce nonocclusive thrombosis in C57Bl/6 mice. A single chain antibody targeting ligand-induced binding sites (LIBS) of the activated glycoprotein IIb/IIIa or control antibody was conjugated to 1 μm-sized microparticles of iron oxide (MPIOs), resulting in LIBS-MPIO or control-MPIO MRI contrast agent, and injected intravenously. Hearts were subjected to histology and ex vivo MRI at 9.4 Tesla., Results: : Thrombus size was comparable in mice injected with control-MPIO and LIBS-MPIO in histology. Significant binding of MPIOs to thrombi was observed in LIBS-MPIO-injected animals while no binding was observed in control animals (P < 0.05). In MRI, LIBS-MPIO binding to thrombi of the left anterior descending coronary artery resulted in significant MPIO-induced signal void compared with controls (P < 0.05). MRI signal void and the amount of bound contrast agent particles in histology showed a significant positive linear correlation (r = 0.939, P < 0.001)., Conclusions: : We established a new mouse model of nonocclusive coronary artery thrombosis. LIBS-MPIO contrast agent binds to activated platelets in this model, allowing molecular MRI of coronary thrombosis. This could have important implications on the timely noninvasive detection of arterial thrombosis, helping to initiate early therapeutic interventions.

Objective: Spleen tyrosine kinase (SYK) has come into focus as a potential therapeutic target in chronic inflammatory diseases, such as rheumatoid arthritis and asthma, as well as in B-cell lymphomas. SYK has also been involved in the signaling of immunoreceptors, cytokine receptors, and integrins. We therefore hypothesized that inhibition of SYK attenuates the inflammatory process underlying atherosclerosis and reduces plaque development., Methods and Results: Low-density lipoprotein receptor-deficient mice consuming a high-cholesterol diet supplemented with 2 doses of the orally available SYK inhibitor fostamatinib for 16 weeks showed a dose-dependent reduction in atherosclerotic lesion size by up to 59±6% compared with the respective controls. Lesions of fostamatinib-treated animals contained fewer macrophages but more smooth muscle cells and collagen-characteristics associated with more stable plaques in humans. Mechanistically, fostamatinib attenuated adhesion and migration of inflammatory cells and limited macrophage survival. Furthermore, fostamatinib normalized high-cholesterol diet -induced monocytosis and inflammatory gene expression., Conclusions: We present the novel finding that the SYK inhibitor fostamatinib attenuates atherogenesis in mice. Our data identify SYK inhibition as a potentially fruitful antiinflammatory therapeutic strategy in atherosclerosis.

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.