Nonpreferential but Detrimental Accumulation of Macrophages With Clonal Hematopoiesis-Driver Mutations in Cardiovascular Tissues-Brief Report.

BACKGROUND: Clonal hematopoiesis of indeterminate potential (CHIP) is an acquired genetic risk factor for both leukemia and cardiovascular disease. It results in proinflammatory myeloid cells in the bone marrow and blood; however, how these cells behave in the cardiovascular tissue remains unclear. Our study aimed at investigating whether CHIP-mutated macrophages accumulate preferentially in cardiovascular tissues and examining the transcriptome of tissue macrophages from DNMT3A (DNA methyltransferase 3 alpha) or TET2 (Tet methylcytosine dioxygenase 2) mutation carriers. METHODS: We recruited patients undergoing carotid endarterectomy or heart surgeries to screen for CHIP mutation carriers using targeted genomic sequencing. Myeloid and lymphoid cells were isolated from blood and cardiovascular tissue collected during surgeries using flow cytometry. DNA and RNA extracted from these sorted cells were subjected to variant allele frequency measurement using droplet digital polymerase chain reaction and transcriptomic profiling using bulk RNA sequencing, respectively. RESULTS: Using droplet digital polymerase chain reaction, we detected similar variant allele frequency of CHIP in monocytes from blood and macrophages from atheromas and heart tissues, even among heart macrophages with and without CCR2 (C-C motif chemokine receptor 2) expression. Bulk RNA sequencing revealed a proinflammatory gene profile of myeloid cells from DNMT3A or TET2 mutation carriers compared with those from noncarriers. CONCLUSIONS: Quantitatively, CHIP-mutated myeloid cells did not preferentially accumulate in cardiovascular tissues, but qualitatively, they expressed a more disease-prone phenotype.

Scalable and modular 8-channel transmit and 8-channel flexible receive coil array for 19 F MRI of large animals.

PURPOSE: To introduce an RF coil system consisting of an 8-channel transmit (Tx) and 8-channel receive (Rx) coil arrays for 19 F MRI of large animals. METHODS: The Tx efficiency and homogeneity of the 8-element loop coil array (loop size: 6 × 15 cm2 ) were simulated for two different pig models rendered from MR images. An 8-channel Rx coil array consisting of a flexible 6-channel posterior and a 2-channel planar anterior array was designed to fit on the abdomen of an average-sized pig in supine position. Measurements were performed in a grid phantom and ex vivo on a pig model with perfluoroctylbromide (PFOB)-filled tubes inserted in the thorax. RESULTS: Measured and simulated Tx efficiency and homogeneity for the 8-channel and 5-channel arrays were in good agreement: 1.87 ± 0.22µT/√kW versus 1.96 ± 0.29µT/√kW, and 2.29 ± 0.39µT/√kW versus 2.41 ± 0.37µT/√kW. An isolation of 38 ± 8 dB is achieved between the 19 F Tx and Rx elements, and over 30 dB between the 1 H and 19 F elements. The PFOB-filled vials could be clearly identified within the cadaver abdomen with an SNR of 275 ± 51 for a 3D gradient-echo sequence with 2-mm isotropic resolution and 12 averages, acquired in 9:52 min:s. Performance of the Tx array was robust against phase and amplitude mismatches at the input ports. CONCLUSIONS: A modular and scalable Tx array offers improved Tx efficiency in 19 F MRI of large animals with various sizes. Although conventional birdcage coils have superior Tx efficiency within the target region of interest, scalability of the Tx array to animal size is a major benefit. The described 19 F coil provides homogeneous excitation and high sensitivity detection in large pig models.

Impact of the COVID-19 pandemic on aortic valve replacement procedures in Germany.

BACKGROUND: COVID-19 has caused the deferral of millions of elective procedures, likely resulting in a backlog of cases. We estimate the number of postponed surgical aortic valve replacement (sAVR) and transcatheter aortic valve replacement (TAVR) procedures during the first two waves of the COVID-19 pandemic in Germany. METHODS: Using German national records, all isolated TAVR and sAVR procedures between 2007 and 2020 were identified. Using weekly TAVR and sAVR procedures between 2017 and 2019, we created a forecast for 2020 and compared it with the observed number of procedures in 2020. RESULTS: In Germany, a total of 225,398 isolated sAVR and 159,638 isolated TAVR procedures were conducted between 2007 and 2020 that were included in our analysis. The reduction in all AVR procedures (sAVR and TAVR) for the entire year 2020 was 19.07% (95%CI: 15.19-22.95%). During the first wave of the pandemic (week 12-21), the mean weekly reduction was 32.06% (23.44-40.68%) and during the second wave of the pandemic (week 41-52), the mean weekly reduction was 25.58% (14.19-36.97%). The number of sAVR procedures decreased more than the number of TAVR procedures (24.63% vs. 16.42% for the entire year 2020). CONCLUSION: The first year of the COVID-19 pandemic saw a substantial postponing of AVR procedures in Germany. Postponing was higher for sAVR than for TAVR procedures and less pronounced during the second wave of the COVID-19 pandemic.

Elevated platelet-leukocyte complexes are associated with, but dispensable for myocardial ischemia-reperfusion injury.

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.

P2Y12-dependent activation of hematopoietic stem and progenitor cells promotes emergency hematopoiesis after myocardial infarction.

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 P2Y12-mediated signaling on emergency hematopoiesis and cardiac remodeling after MI. Permanent coronary ligation was performed to induce MI in a murine model. BM activation, inflammatory cell composition and cardiac function were assessed using global and platelet-specific gene knockout and pharmacological inhibition models for P2Y12. Complementary in vitro studies allowed for investigation of ADP-dependent effects on LSK cells. We found that ADP acts as a danger signal for the hematopoietic BM and fosters emergency hematopoiesis by promoting Akt phosphorylation and cell cycle progression. We were able to detect P2Y12 in LSK, implicating a direct effect of ADP on LSK via P2Y12 signaling. P2Y12 knockout and P2Y12 inhibitor treatment with prasugrel reduced emergency hematopoiesis and the excessive inflammatory response to MI, translating to lower numbers of downstream progeny and inflammatory cells in the blood and infarct. Ultimately, P2Y12 inhibition preserved cardiac function and reduced chronic adverse cardiac remodeling after MI. P2Y12-dependent signaling is involved in emergency hematopoiesis after MI and fuels post-ischemic inflammation, proposing a novel, non-canonical value for P2Y12 antagonists beyond inhibition of platelet-mediated atherothrombosis.

Genetic Deficiency of TRAF5 Promotes Adipose Tissue Inflammation and Aggravates Diet-Induced Obesity in Mice.

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.

Ventral calcification in the common femoral artery: A risk factor for major transcatheter aortic valve intervention access site complications.

OBJECTIVES: We aimed to identify risk factors for major transcatheter aortic valve intervention (TAVI) access site complications based on detailed analysis of the preprocedural computed tomography angiogram (CTA). BACKGROUND: Transfemoral TAVI has become the treatment of choice for severe aortic stenosis in elderly patients, especially with increased perioperative risk. Frailty, however, favors complications at the vascular access site due to the large bore vascular sheath devices necessary for valve deployment. METHODS: In this monocentric study, we retrospectively analyzed the preprocedural CTA of 417 consecutive patients that received transfemoral TAVI between 2015 and 2019 to quantify vessel diameter, calcification volume and calcified plaque location in detail within 10 cm proximal to the femoral bifurcation. RESULTS: The mean age of the study cohort was 81.4 ± 6.5 years with a STS of 8 ± 5.2 representing a population at increased periprocedural risk. 54.4% of patients were female. Major vascular access site complications occurred in 8.2% of patients. Major vascular complications correlated statistically with a sheath-to-vessel diameter (SFAR) when measured 1 cm proximal to the femoral bifurcation using a line-derived diameter and ventral calcification within the first 5 cm proximal to the bifurcation. In contrast, overall calcification volume had no influence. CONCLUSIONS: Transfemoral TAVI harbors a considerable risk for vascular access site complications especially if vessel diameter is too small to comfortably host the sheath diameter at the area of the femoral bifurcation. For preprocedural TAVI planning and risk assessment, location of calcification, especially if located ventrally, seems to be more relevant than consideration of overall calcification alone.

Impact of Preprocedural Aortic Valve Calcification on Conduction Disturbances after Transfemoral Aortic Valve Replacement.

AIM: The present study analyzes in depth the impact of different calcification patterns on disturbances of the conduction system in transcatheter aortic valve replacement (TAVR) patients. METHODS AND RESULTS: A total of 169 preprocedural TAVR multislice computed tomography scans from consecutive transfemoral (TF) TAVRs performed between 2014 and 2017 using either Edwards SAPIEN or Medtronic Evolut R valves were retrospectively evaluated. The volume, distribution, and orientation of annular and valvular aortic valve calcification were measured and their impact on postoperative conduction disturbances was determined using linear and logistic regression analyses. The total volume of calcification and distribution at the aortic annulus or valve did not influence the conduction system. Oval calcification of the left aortic cusp was independently associated with an elevated risk for an increase in atrioventricular block degree (+0.6, p = 0.03). Moreover, orthogonal calcifications at the level of the aortic annulus were associated with an increased risk for QRS prolongation (+26 ms, p = 0.004) and an increased risk for permanent pacemaker implantation (OR 4.3, p = 0.03) after TF TAVR. This was more pronounced in patients undergoing TF TAVR using a balloon-expandable Edwards SAPIEN 3 valve (QRS +38.195 ms, p < 0.001; OR permanent pacemaker 15.48, p = 0.013). CONCLUSION: Orthogonal annular calcification confers an increased risk for conduction disturbances after TAVR. This is even more pronounced after implantation of balloon-expandable valves.

Inhibition of macrophage proliferation dominates plaque regression in response to cholesterol lowering.

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.

Nationwide outcomes of aortic valve replacement for pure aortic regurgitation in Germany 2008-2015.

BACKGROUND: Transcatheter aortic valve replacement (TAVR) is routinely used in patients with severe aortic stenosis at increased operative risk. Due to potential technical difficulties, TAVR is not recommended for pure aortic regurgitation (AR). Smaller studies reported its use in AR, but data from big registries are lacking. The present study analyzes the nationwide use of surgical aortic valve replacement (SAVR) and TAVR in patients with AR from 2008 until 2015. METHODS: We identified 138,237 cases of aortic valve replacement in Germany based on ICD and OPS codes. RESULTS: Of 13.2% SAVR-cases and 1.3% of TAVR cases were performed in AR. AR patients undergoing SAVR were younger with lower logistic EuroSCORE (stenosis: 6.1 ± 5.6; AR: 4.5 ± 4.9). Nevertheless, stroke rates, bleedings, prolonged mechanical ventilation, and in-hospital mortality were higher (mortality: stenosis 2.6%, AR: 4.7%). In the TAVR group, patients with AR were at higher operative risk (logistic EuroSCORE: transfemoral (TF)-TAVR: stenosis: 14.3 ± 10.4; AR: 17.3 ± 13.3. Transapical (TA)-TAVR: stenosis: 16.1 ± 11.4; AR: 15.7 ± 12.2). Stroke rates were lower, but bleedings and prolonged ventilation occurred more frequently after TF-TAVR in AR compared to stenosis. The mortality varied markedly (TF-TAVR: 15.2% in 2011; 2.8% in 2015; TA-TAVR: 17.7% in 2012 and 0% in 2014). CONCLUSION: TAVR is off-label used in AR in clinical practice. TAVR seems to be a safe option for AR with regard to in-hospital outcomes. However, further research evaluating long-term outcomes is required to establish the feasibility of TAVR in pure AR.

Inflammatory Pathways Regulated by Tumor Necrosis Receptor-Associated Factor 1 Protect From Metabolic Consequences in Diet-Induced Obesity.

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)-1ß, 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 ß3-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.

Magnetic resonance imaging for pathobiological assessment and interventional treatment of the coronary arteries.

X-ray-based fluoroscopy is the standard tool for diagnostics and intervention in coronary artery disease. In recent years, computed tomography has emerged as a non-invasive alternative to coronary angiography offering detection of coronary calcification and imaging of the vessel lumen by the use of iodinated contrast agents. Even though currently available invasive or non-invasive techniques can show the degree of vessel stenosis, they are unable to provide information about biofunctional plaque properties, e.g. plaque inflammation. Furthermore, the use of radiation and the necessity of iodinated contrast agents remain unfavourable prerequisites. Magnetic resonance imaging (MRI) is a radiation-free alternative to X-ray which offers anatomical and functional imaging contrasts fostering the idea of non-invasive biofunctional assessment of the coronary vessel wall. In combination with molecular contrast agents that target-specific epitopes of the vessel wall, MRI might reveal unique plaque properties rendering it, for example, 'vulnerable and prone to rupture'. Early detection of these lesions may allow for early or prophylactic treatment even before an adverse coronary event occurs. Besides diagnostic imaging, advances in real-time image acquisition and motion compensation now provide grounds for MRI-guided coronary interventions. In this article, we summarize our research on MRI-based molecular imaging in cardiovascular disease and feature our advances towards real-time MRI-based coronary interventions in a porcine model.

La fluoroscopia con rayos X es la herramienta estándar para el diagnóstico y la intervención de coronariopatías. En los últimos años, la tomografía computarizada se ha convertido en una alternativa atraumática a la coronariografía, ya que se puede detectar la calcificación coronaria y ver a través de imágenes las luces de los vasos sanguíneos mediante el uso de medios de contraste yodados. Si bien las técnicas traumáticas o atraumáticas disponibles actualmente pueden mostrar el grado de la estenosis vascular, no pueden proporcionar información sobre las propiedades biofuncionales de la placa de ateroma, por ejemplo, inflamación de la placa de ateroma. Por otra parte, el uso de radiación y la necesidad de agentes de contraste yodados siguen siendo requisitos desfavorables. La resonancia magnética (RM) es una alternativa sin radiación a los rayos X que proporciona contrates de imagen con información anatómica y funcional, lo cual refuerza la idea del diagnóstico biofuncional atraumático de las paredes de los vasos coronarios. En combinación con medios de contraste molecular que actúan sobre epítopos específicos de las paredes de los vasos, la RM puede poner de manifiesto propiedades particulares de la placa de ateroma mediante su representación, por ejemplo, «vulnerabilidad y predisposición a rotura¼. La detección precoz de este tipo de lesiones puede facilitar un tratamiento a tiempo o preventivo antes de que tenga lugar una complicación coronaria grave.Además del diagnóstico por imagen, los avances en la adquisición de imágenes en tiempo real y la compensación del movimiento sirven de base para las intervenciones coronarias guiadas por RM. En este artículo, ofrecemos un resumen de nuestra investigación sobre imagen molecular con resonancia magnética en enfermedades cardiovasculares y presentamos nuestros avances hacia las intervenciones coronarias con RM en tiempo real en un modelo porcino.

Purinergic receptor Y2 (P2Y2)- dependent VCAM-1 expression promotes immune cell infiltration in metabolic syndrome.

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 P2Y2. The gene expression of ATP receptor P2Y2 did not change in several tissues in the course of obesity, but was increased within epididymal fat. Adipose tissue from P2Y 2-/- mice consuming high-fat diet (HFD) contained less crown-like structures with a reduced frequency of adipose tissue macrophages (ATMs). This was likely due to decreased leukocyte migration because of missing VCAM-1 exposition on P2Y2 deficient hypertrophic adipose tissue endothelial cells. Accordingly, P2Y 2-/- mice showed blunted traits of the metabolic syndrome: they gained less weight compared to P2Y 2+/+ controls, while intake of food and movement behaviour remained unchanged. Liver and adipose tissue were smaller in P2Y 2-/- animals. Insulin tolerance testing (ITT) performed in obese P2Y 2-/- mice revealed a better insulin sensitivity as well as lower plasma C-peptide and cholesterol levels. We demonstrate that interfering with somatic P2Y2 signalling prevents excessive immune cell deposition in diet-induced obesity (DIO), both attenuating adipose tissue inflammation and ameliorating the metabolic phenotype. Thus, blocking the P2Y2 cascade may be a promising strategy to limit metabolic disease and its sequelae.

Proliferation and Recruitment Contribute to Myocardial Macrophage Expansion in Chronic Heart Failure.

RATIONALE: Macrophages reside in the healthy myocardium, participate in ischemic heart disease, and modulate myocardial infarction (MI) healing. Their origin and roles in post-MI remodeling of nonischemic remote myocardium, however, remain unclear. OBJECTIVE: This study investigated the number, origin, phenotype, and function of remote cardiac macrophages residing in the nonischemic myocardium in mice with chronic heart failure after coronary ligation. METHODS AND RESULTS: Eight weeks post MI, fate mapping and flow cytometry revealed that a 2.9-fold increase in remote macrophages results from both increased local macrophage proliferation and monocyte recruitment. Heart failure produced by extensive MI, through activation of the sympathetic nervous system, expanded medullary and extramedullary hematopoiesis. Circulating Ly6C(high) monocytes rose from 64±5 to 108±9 per microliter of blood (P<0.05). Cardiac monocyte recruitment declined in Ccr2(-/-) mice, reducing macrophage numbers in the failing myocardium. Mechanical strain of primary murine and human macrophage cultures promoted cell cycle entry, suggesting that the increased wall tension in post-MI heart failure stimulates local macrophage proliferation. Strained cells activated the mitogen-activated protein kinase pathway, whereas specific inhibitors of this pathway reduced macrophage proliferation in strained cell cultures and in the failing myocardium (P<0.05). Steady-state cardiac macrophages, monocyte-derived macrophages, and locally sourced macrophages isolated from failing myocardium expressed different genes in a pattern distinct from the M1/M2 macrophage polarization paradigm. In vivo silencing of endothelial cell adhesion molecules curbed post-MI monocyte recruitment to the remote myocardium and preserved ejection fraction (27.4±2.4 versus 19.1±2%; P<0.05). CONCLUSIONS: Myocardial failure is influenced by an altered myeloid cell repertoire.

Myocardial infarction accelerates atherosclerosis.

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.

Ischemic stroke activates hematopoietic bone marrow stem cells.

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<0.05 versus pre tMCAO). Flow cytometry and cell cycle analysis showed activation of the entire hematopoietic tree, including myeloid progenitors. The cycling fraction of the most upstream hematopoietic stem cells increased from 3.34%±0.19% to 7.32%±0.52% after tMCAO (P<0.05). In vivo microscopy corroborated proliferation of adoptively transferred hematopoietic progenitors in the bone marrow of mice with stroke. The hematopoietic system's myeloid bias was reflected by increased expression of myeloid transcription factors, including PU.1 (P<0.05), and by a decline in lymphocyte precursors. In mice after tMCAO, tyrosine hydroxylase levels in sympathetic fibers and bone marrow noradrenaline levels rose (P<0.05, respectively), associated with a decrease of hematopoietic niche factors that promote stem cell quiescence. In mice with genetic deficiency of the ß3 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.

Targeting Interleukin-1ß Reduces Leukocyte Production After Acute Myocardial Infarction.

BACKGROUND: Myocardial infarction (MI) is an ischemic wound that recruits millions of leukocytes. MI-associated blood leukocytosis correlates inversely with patient survival, yet the signals driving heightened leukocyte production after MI remain incompletely understood. METHODS AND RESULTS: With the use of parabiosis surgery, this study shows that soluble danger signals, among them interleukin-1ß, increase bone marrow hematopoietic stem cell proliferation after MI. Data obtained in bone marrow reconstitution experiments reveal that interleukin-1ß enhances hematopoietic stem cell proliferation by both direct actions on hematopoietic cells and through modulation of the bone marrow's hematopoietic microenvironment. An antibody that neutralizes interleukin-1ß suppresses these effects. Anti-interleukin-1ß treatment dampens the post-MI increase in hematopoietic stem cell proliferation. Consequently, decreased leukocyte numbers in the blood and infarct reduce inflammation and diminish post-MI heart failure in ApoE(-/-) mice with atherosclerosis. CONCLUSIONS: The presented insight into post-MI bone marrow activation identifies a mechanistic target for muting inflammation in the ischemically damaged heart.

Atheroprotection through SYK inhibition fails in established disease when local macrophage proliferation dominates lesion progression.

Macrophages in the arterial intima sustain chronic inflammation during atherogenesis. Under hypercholesterolemic conditions murine Ly6C(high) 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 Ly6C(high) monocytes and macrophages. SYK inhibition limited Ly6C(high) 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.

Differential contribution of monocytes to heart macrophages in steady-state and after myocardial infarction.

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<0.01). Macrophages self-renewed locally through proliferation: 2.1±0.3% incorporated bromodeoxyuridine 2 hours after a single injection, and 13.7±1.4% heart macrophages stained positive for the cell cycle marker Ki-67. The cells likely participate in defense against infection, because we found them to ingest fluorescently labeled bacteria. In ischemic myocardium, we observed that tissue-resident macrophages died locally, whereas some also migrated to hematopoietic organs. If the steady state was perturbed by coronary ligation or diphtheria toxin-induced macrophage depletion in CD11b(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.

Dual-contrast molecular imaging allows noninvasive characterization of myocardial ischemia/reperfusion injury after coronary vessel occlusion in mice by magnetic resonance imaging.

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.