editorials; atherosclerosis; folate; folate receptor; immunotoxins; macrophages
Macrophages frequently infiltrate tumors and can enhance cancer growth, yet the origins of the macrophage response are not well understood. Here we address molecular mechanisms of macrophage production in a conditional mouse model of lung adenocarcinoma. We report that over-production of the peptide hormone Angiotensin II (AngII) in tumor-bearing mice amplifies self-renewing hematopoietic stem cells (HSCs) and macrophage progenitors. The process occurred in the spleen but not the bone marrow, and was independent of hemodynamic changes. The effects of AngII required direct hormone ligation on HSCs, depended on S1P1 signaling, and allowed the extramedullary tissue to supply new tumor-associated macrophages throughout cancer progression. Conversely, blocking AngII production prevented cancer-induced HSC and macrophage progenitor amplification and thus restrained the macrophage response at its source. These findings indicate that AngII acts upstream of a potent macrophage amplification program and that tumors can remotely exploit the hormone’s pathway to stimulate cancer-promoting immunity.
Cardiovascular diseases claim more lives worldwide than any other. Etiologically, the dominant trajectory involves atherosclerosis, a chronic inflammatory process of lipid-rich lesion growth in the vascular wall that can cause life-threatening myocardial infarction (MI). Those who survive MI can develop congestive heart failure, a chronic condition of inadequate pump activity that is frequently fatal. Leukocytes – white blood cells – are important participants at the various stages of cardiovascular disease progression and complication. This review will discuss leukocyte function in atherosclerosis, myocardial infarction, and heart failure.
Macrophages (MΦ) predominate among the inflammatory cells in rejecting allografts. These innate immune cells, in addition to allospecific T cells, can damage cardiomyocytes directly.
Methods and Results
We explored if sensitive PET/CT imaging of MΦ-avid nanoparticles detects rejection of heart allografts in mice. In addition, we employed the imaging method to follow the immunomodulatory impact of angiotensin converting enzyme inhibititor therapy (ACEi) on myeloid cells in allografts. Dextran nanoparticles were derivatized with the PET isotope copper-64 and imaged seven days after transplantation. C57/BL6 recipients of BALB/c allografts displayed robust PET signal (standard uptake value allograft, 2.8±0.3; isograft control, 1.7±0.2; p<0.05). Autoradiography and scintillation counting confirmed the in vivo findings. We then imaged the effects of ACEi (5mg/kg Enalapril). ACEi significantly decreased nanoparticle signal (p<0.05). Histology and flow cytometry showed a reduced number of myeloid cells in the graft, blood and lymph nodes, as well as diminished antigen presentation (p<0.05 versus untreated allografts). ACEi also significantly prolonged allograft survival (12 versus 7 days, p<0.0001).
Nanoparticle MΦ PET-CT detects heart transplant rejection and predicts organ survival by reporting on myeloid cells.
heart transplantation; macrophages; imaging; PET/CT
Macrophages are central regulators of disease progression in both atherosclerosis and myocardial infarction. In atherosclerosis, macrophages are the dominant leukocyte population that influences lesional development. In myocardial infarction, which is caused by atherosclerosis, macrophages accumulate readily and play important roles in inflammation and healing. Molecular imaging has grown considerably as a field and can reveal biological process at the molecular, cellular, and tissue levels. Here we explore how various imaging modalities, from intravital microscopy in mice to organ-level imaging in patients, are contributing to our understanding of macrophages and their progenitors in cardiovascular disease.
From many perspectives, cardiovascular diseases and cancers are fundamentally different. On the one hand, atherosclerosis is a disease of lipid accumulation driven by diet and lifestyle, whereas cancer is an attack “from within” driven by mutations. Nevertheless, studies over the last 20 years have forced us to re-evaluate such a view. We are learning that, among other factors, the immune system is indispensable to the development and progression of both diseases. Its components are not only reactive but can also orchestrate both tumor and atherosclerotic lesion growth. In this Viewpoint, we explore how monocytes, which are key constituents of the immune system, forge links between cardiovascular diseases and cancers.
Monocytes are frequently described as bone marrow-derived precursors of macrophages. Although many studies support this view, we now appreciate that monocytes neither develop exclusively in the bone marrow nor give rise to all macrophages and dendritic cells. In addition to differentiating to specific leukocyte populations, monocytes, as monocytes, are functionally and ontogenically heterogeneous. In this review we will focus on the development and activity of monocytes and their subsets in mice (Ly-6Chigh/low) and humans (CD14+/dim/− CD16+/−) in the context of atherosclerosis and its complications.
monocyte subsets; monocyte heterogeneity; Ly-6C; CD14; CD16; macrophage; dendritic cells; atherosclerosis; myocardial infarction; cardiovascular disease; extramedullary hematopoeisis; inflammation
Myeloid cell content in atherosclerotic plaques associates with rupture and thrombosis. Thus, imaging of lesional monocyte and macrophages (Mo/Mϕ) could serve as a biomarker of disease progression and therapeutic intervention.
To noninvasively assess plaque inflammation with dextran nanoparticle-facilitated hybrid PET/MR imaging.
Methods and Results
Using clinically approved building blocks, we systematically developed 13nm polymeric nanoparticles consisting of crosslinked short chain dextrans which were modified with desferoxamine for zirconium-89 radiolabeling (89Zr-DNP) and a near infrared fluorochrome (VT680) for microscopic and cellular validation. Flow cytometry of cells isolated from excised aortas showed DNP uptake predominantly in Mo/Mϕ (76.7%) and lower signal originating from other leukocytes such as neutrophils and lymphocytes (11.8% and 0.7%, p<0.05 versus Mo/Mϕ). DNP colocalized with the myeloid cell marker CD11b on immunohistochemistry. PET/MRI revealed high uptake of 89Zr-DNP in the aortic root of ApoE−/− mice (standard uptake value, ApoE−/− mice versus wild type controls, 1.9±0.28 versus 1.3±0.03, p<0.05), corroborated by ex vivo scintillation counting and autoradiography. Therapeutic silencing of the monocyte-recruiting receptor CCR2 with siRNA decreased 89Zr-DNP plaque signal (p<0.05) and inflammatory gene expression (p<0.05).
Hybrid PET/MR imaging with a 13nm DNP enables noninvasive assessment of inflammation in experimental atherosclerotic plaques and reports on therapeutic efficacy of anti-inflammatory therapy.
PET/MRI; inflammation; atherosclerosis; molecular imaging; nanoparticles
Acute rupture of vulnerable plaques frequently leads to myocardial infarction and stroke. Within the last decades, several cellular and molecular players have been identified that promote atherosclerotic lesion formation, maturation and plaque rupture. It is now widely recognized that inflammation of the vessel wall and distinct leukocyte subsets are involved throughout all phases of atherosclerotic lesion development. The mechanisms that render a stable plaque unstable and prone to rupture, however, remain unknown and the identification of the vulnerable plaque remains a major challenge in cardiovascular medicine. Imaging technologies used in the clinic offer minimal information about the underlying biology and potential risk for rupture. New imaging technologies are therefore being developed, and in the preclinical setting have enabled new and dynamic insights into the vessel wall for a better understanding of this complex disease. Molecular imaging has the potential to track biological processes, such as the activity of cellular and molecular biomarkers in vivo and over time. Similarly, novel imaging technologies specifically detect effects of therapies that aim to stabilize vulnerable plaques and silence vascular inflammation. Here we will review the potential of established and new molecular imaging technologies in the setting of atherosclerosis, and discuss the cumbersome steps required for translating molecular imaging approaches into the clinic.
Molecular imaging; Inflammation; Atherosclerosis
Ralph van Furth and Zanvil A. Cohn proposed, in their classic 1968 paper, that bone marrow-dwelling promonocytes differentiate to monocytes, which then intravasate, circulate and, upon tissue entry, differentiate to sessile macrophages. Since then, understanding of the macrophage family relationship has undergone substantial enhancement and occasional revision. It is currently recognized that, in addition to their role in the bone marrow, hematopoietic progenitors circulate and give rise to their descendants in extramedullary niches. Monocytes, of which there are several subsets, are not merely circulating macrophage precursors, but participate in the immune response in their own right. Macrophages are highly heterogeneous and, recent studies indicate, can arise in the absence of a monocyte intermediate. These spatial and developmental relationships reveal a complex interactive network and underscore the importance of context in evaluating biological systems. The observations have significant implications for how we image, target and treat disease.
hematopoietic progenitors; monocytes; macrophages
Monocytes serve as a central defense system against infection and injury but can also promote pathological inflammatory responses. Considering the evidence that monocytes exist in at least two subsets committed to divergent functions, we investigated whether distinct factors regulate the balance between monocyte subset responses in vivo. We identified a microRNA (miRNA), miR-146a, which is differentially regulated both in mouse (Ly-6Chi/Ly-6Clo) and human (CD14hi/CD14loCD16+) monocyte subsets. The single miRNA controlled the amplitude of the Ly-6Chi monocyte response during inflammatory challenge whereas it did not affect Ly-6Clo cells. miR-146a–mediated regulation was cell-intrinsic and depended on Relb, a member of the non-canonical NF-κB/Rel family, which we identified as a direct miR-146a target. These observations not only provide novel mechanistic insights into the molecular events that regulate responses mediated by committed monocyte precursor populations but also identify novel targets to manipulate Ly-6Chi monocyte responses while sparing Ly-6Clo monocyte activity.
Coagulase-positive Staphylococcus aureus (S. aureus) is the major causal pathogen of acute endocarditis, a rapidly progressing, destructive infection of the heart valves. Bacterial colonization occurs at sites of endothelial damage, where (together with fibrin and platelets) it initiates the formation of abnormal growths known as vegetations. Here we report that an engineered analog of prothrombin detected S. aureus in endocarditic vegetations via noninvasive fluorescence or PET imaging. These prothrombin derivatives bound to staphylocoagulase and intercalated into growing bacterial vegetations. We also present evidence for bacterial quorum sensing in the regulation of staphylocoagulase expression by S. aureus. Staphylocoagulase expression was limited to the growing edge of mature vegetations, where it was exposed to the host and co-localized with the imaging probe. When endocarditis was induced with an S. aureus strain with genetic deletion of coagulases, survival of mice improved, highlighting the role of staphylocoagulase as a virulence factor.
endocarditis; staphylocoagulase; prothrombin; noninvasive imaging; von Willebrand factor binding protein
Recognition and clearance of bacterial infection is a fundamental property of innate immunity. Here we describe an effector B cell population that protects against microbial sepsis. Innate response activator (IRA)-B cells are phenotypically and functionally distinct, develop and diverge from B1a B cells, depend on pattern recognition receptors, and produce GM-CSF. Specific deletion of IRA-B cell activity impairs bacterial clearance, elicits a cytokine storm, and precipitates septic shock. These observations enrich our understanding of innate immunity, position IRA-B cells as gatekeepers of bacterial infection, and identify new treatment avenues for infectious diseases.
To explore the capacity of human CD14+CD16++ and CD14++CD16- monocytes to phagocyte iron-oxide nanoparticles in vitro.
Human monocytes were labeled with four different magnetic nanoparticle preparations (Ferumoxides, SHU 555C, CLIO-680, MION-48) exhibiting distinct properties and cellular uptake was quantitatively assessed by flow cytometry, fluorescence microscopy, atomic absorption spectrometry and Magnetic Resonance Imaging (MRI). Additionally we determined whether cellular uptake of the nanoparticles resulted in phenotypic changes of cell surface markers.
Cellular uptake differed between the four nanoparticle preparations. However for each nanoparticle tested, CD14++CD16- monocytes displayed a significantly higher uptake compared to CD14+CD16++ monocytes, this resulted in significantly lower T1 and T2 relaxation times of these cells. The uptake of iron-oxide nanoparticles further resulted in a remarkable shift of expression of cell surface proteins indicating that the labeling procedure affects the phenotype of CD14+CD16++ and CD14++CD16- monocytes differently.
Human monocyte subsets internalize different magnetic nanoparticle preparations differently, resulting in variable loading capacities, imaging phenotypes and likely biological properties.
Monocyte; atherosclerosis; myocardial infarction; heart failure; wound healing; remodeling
Current management of aortic aneurysms (AAs) primarily relies on size criteria to determine whether invasive repair is indicated to preempt rupture. We hypothesized that emerging molecular imaging tools could be used to more sensitively gauge local inflammation. Since macrophages are key effector cells that destabilize the extracellular matrix in the arterial wall, it seemed likely that they would represent suitable imaging targets. We here aimed to develop and validate macrophage-targeted nanoparticles labeled with fluorine-18 (18F) for PET-CT detection of inflammation in AAs.
Methods and Results
Aneurysms were induced in apoE−/− mice via systemic administration of angiotensin-II. Mice were imaged using PET-CT and a monocyte/macrophage-targeted nanoparticle. AAs were detected by contrast-enhanced micro-CT and had a mean diameter of 1.85 ± 0.08 mm, whereas normal aortas measured 1.07 ± 0.03 (p < 0.05). The in vivo PET signal was significantly higher in aneurysms (SUV 2.46 ± 0.48) when compared to wild type aorta (0.82 ± 0.05, p<0.05). Validation with scintillation counting, autoradiography, fluorescence and immunoreactive histology and flow cytometry demonstrated that nanoparticles predominantly localized to monocytes and macrophages within the aneurysmatic wall.
PET-CT imaging with 18F-CLIO (cross-linked iron oxide) nanoparticles allows quantitation of macrophage content in a mouse model of AAs.
Aortic aneurysm; inflammation; macrophage; PET-CT; nanoparticle
Monocytes recruited to ischemic myocardium originate from a reservoir in the spleen, and the release from their splenic niche relies on angiotensin-II (Ang-II) signaling.
Since monocytes are centrally involved in tissue repair after ischemia, we here hypothesized that early ACE inhibitor therapy impacts healing after myocardial infarction partly via effects on monocyte traffic.
Methods and Results
In a mouse model of permanent coronary ligation, Enalapril arrested the release of monocytes from the splenic reservoir and consequently reduced their recruitment into the healing infarct by 45%, as quantified by flow cytometry of digested infarcts. Time-lapse intravital microscopy revealed that Enalapril reduces monocyte motility in the spleen. In vitro migration assays and Western blotting showed that this was caused by reduced signaling through the Ang-II receptor subtype 1. We then studied the long-term consequences of blocked splenic monocyte release in atherosclerotic apoE-/- mice, in which infarct healing is impaired due to excessive inflammation in the cardiac wound. Enalapril improved histological healing biomarkers and reduced inflammation in infarcts measured by fluorescence molecular tomography (FMT-CT) of proteolytic activity. ACE inhibition improved MRI-derived ejection fraction by 14% on day 21, despite initially comparable infarct size. In apoE-/- mice, ischemia reperfusion injury resulted in larger infarct size, enhanced monocyte recruitment and was reversible by Enalapril treatment. Splenectomy reproduced anti-inflammatory effects of Enalapril.
This study suggests that benefits of early ACE inhibition after MI can partially be attributed to its potent anti-inflammatory impact on the splenic monocyte reservoir.
Monocyte; spleen; ACE inhibitor; myocardial infarction; heart failure; wound healing
Monocytes and macrophages play active roles in atherosclerosis, a chronic inflammatory disease that is a leading cause of death in the developed world. The prevailing paradigm states that, during human atherogenesis, monocytes accumulate in the arterial intima and differentiate into macrophages, which then ingest oxidized lipoproteins, secrete a diverse array of pro-inflammatory mediators, and eventually become foam cells, the key constituents of a vulnerable plaque. Yet monocytes are heterogeneous. In the mouse, one subset (Ly-6Chi) promotes inflammation, expands in hypercholesterolemic conditions, and selectively gives rise to macrophages in atheromata. A different subset (Ly-6Clo) attenuates inflammation and promotes angiogenesis and granulation tissue formation in models of tissue injury, but its role in atherosclerosis is largely unknown. In the human, monocyte heterogeneity is preserved but it is still unresolved how subsets correspond functionally. These cells’ contradistinctive properties suggest commitment for specific function prior to infiltrating tissue. Such commitment argues for discriminate targeting of deleterious subsets while sparing host defense and repair mechanisms. In addition to advancing our understanding of atherosclerosis, the ability to target and image monocyte subsets would allow us to evaluate drugs designed to selectively inhibit monocyte subset recruitment or function, and to stratify patients at risk for developing complications such as myocardial infarction or stroke. In this review we summarize recent advances of our understanding of the behavioral heterogeneity of monocytes during disease progression, and outline emerging molecular imaging approaches to address key questions in the field.
Rates of graft rejection are high among recipients of heart transplants. The onset and progression of clinically significant heart transplant rejection are currently monitored by serial biopsy, but this approach is highly invasive and lacks sensitivity. Here, we have developed what we believe to be a new technique to measure organ rejection noninvasively that involves the exploration of tissue-infiltrating leukocytes as biomarker sources for diagnostic imaging. Specifically, we profiled the myeloid response in a murine model of heart transplantation with the aim of defining and validating an imaging signature of graft rejection. Ly-6Chi monocytes, which promote inflammation, accumulated progressively in allografts but only transiently in isografts. Ly-6Clo monocytes, which help resolve inflammation, did not accumulate, although they composed the majority of the few remaining monocytes in isografts. The persistence of Ly-6Chi monocytes in allografts prompted us to screen for a Ly-6Chi monocyte–associated imaging marker. Low-density array data revealed that Ly-6Chi monocytes express 10-fold higher levels of myeloperoxidase (MPO) than Ly-6Clo monocytes. Noninvasive magnetic resonance imaging of MPO with an MPO-activatable Gd-chelate revealed a spatially defined T1-weighted signal in rejected allografts but not in isografts or MPO-deficient allograft recipients. Flow cytometry, enzymography, and histology validated the approach by mapping MPO activity to Ly-6Chi monocytes and neutrophils. Thus, MPO imaging represents a potential alternative to the current invasive clinical standard by which transplants are monitored.
To test if blood monocytosis in mice with atherosclerosis affects infarct healing.
Monocytes are cellular protagonists of tissue repair and their specific subtypes regulate the healing program after MI. Inflammatory Ly-6Chi monocytes dominate on day 1-4 and digest damaged tissue; reparative Ly-6Clo monocytes dominate on day 5-10 and promote angiogenesis and scar formation. However, the monocyte repertoire is disturbed in atherosclerotic mice: Ly-6Chi monocytes expand selectively, which may disrupt the resolution of inflammation.
Methods and Results
Ex vivo analysis of 5 day-old infarcts showed >10-times more Ly-6Chi monocytes in atherosclerotic (apoE-/-) mice compared to wild type mice. The injured tissue in apoE-/- mice also showed a more pronounced inflammatory gene expression profile (e.g. increased TNF-α and MPO and decreased TGF-β) and a higher abundance of proteases, which are associated with the activity of Ly-6Chi monocytes. To relate inflammatory activity to left ventricular remodeling, we used a combination of noninvasive molecular and physiologic imaging. FMT-CT on day 5 post MI showed higher proteolysis and phagocytosis in infarcts of atherosclerotic mice. Serial MRI showed accelerated deterioration of EF between day 1 and 21 after MI in apoE-/-. Finally, we could recapitulate these features in wild-type mice with artificially–induced Ly-6Chi monocytosis.
Ly-6Chi monocytosis disturbs resolution of inflammation in murine infarcts and consequently enhances left ventricular remodeling. These findings position monocyte subsets as potential therapeutic targets to augment tissue repair after infarction and to prevent post-MI heart failure.
Proteases are emerging biomarkers of inflammatory diseases. In atherosclerosis, these enzymes are often secreted by inflammatory macrophages, digest the extracellular matrix of the fibrous cap and destabilize atheromata. Protease function can be monitored with protease activatable imaging probes and quantitated in vivo by fluorescence molecular tomography (FMT). To address two major constraints currently associated with imaging of murine atherosclerosis (lack of highly sensitive probes and absence of anatomical information), we compared protease sensors (PS) of variable size and pharmacokinetics and co-registered FMT datasets with computed tomography (FMT-CT).
Methods and results
Co-registration of FMT and CT was achieved with a multimodal imaging cartridge containing fiducial markers detectable by both modalities. A high-resolution CT angiography protocol accurately localized fluorescence to the aortic root of atherosclerotic apoE−/− mice. To identify suitable sensors, we first modeled signal kinetics in-silico and then compared three probes with identical oligo-L-lysine cleavage sequences: PS-5, 5nm in diameter containing 2 fluorochromes , PS-25, a 25nm version with an elongated lysine chain and PS-40, a polymeric nanoparticle. Serial FMT-CT showed fastest kinetics for PS-5 but, surprisingly, highest fluorescence in lesions of the aortic root for PS-40. PS-40 robustly reported therapeutic effects of atorvastatin, corroborated by ex vivo imaging and qPCR for the model protease cathepsin B.
FMT-CT is a robust and observer-independent tool for non-invasive assessment of inflammatory murine atherosclerosis. Reporter-containing nanomaterials may have unique advantages over small molecule agents for in vivo imaging.
FMT-CT; molecular imaging; atherosclerosis; protease activity; inflammation
A current paradigm states that monocytes circulate freely and patrol blood vessels but differentiate irreversibly into dendritic cells (DCs) or macrophages upon tissue entry. Here we show that bona fide undifferentiated monocytes reside in the spleen and outnumber their equivalents in circulation. The reservoir monocytes assemble in clusters in the cords of the subcapsular red pulp and are distinct from macrophages and DCs. In response to ischemic myocardial injury, splenic monocytes increase their motility, exit the spleen en masse, accumulate in injured tissue, and participate in wound healing. These observations uncover a role for the spleen as a site for storage and rapid deployment of monocytes and identify splenic monocytes as a resource that the body exploits to regulate inflammation.
molecular imaging; consensus; atherosclerosis; heart failure; myocardial infarction
Monocytes play a key role in atherogenesis, but their participation has been largely discerned via ex vivo analyses of atherosclerotic lesions. We sought to establish a noninvasive technique to determine monocyte trafficking to atherosclerotic lesions in live animals.
Methods and Results
Using a micro-single-photon-emission-computed-tomography (microSPECT/CT) small animal imaging system and an FDA-approved radiotracer ([111Indium] oxyquinoline, 111In-oxine), we demonstrate here that monocyte recruitment to atherosclerotic lesions can be visualized in a noninvasive, dynamic, and three-dimensional fashion in live animals. We demonstrate in vivo that monocytes are recruited avidly to plaques within days of adoptive transfer. Using microSPECT/CT imaging as a screening tool, we were able to investigate modulatory effects on monocyte recruitment in live animals. We found that HMG-CoA-reductase inhibitors rapidly and substantially reduce monocyte recruitment to existing atherosclerotic lesions as imaged here in vivo.
This novel approach to track monocytes to atherosclerotic plaques in vivo should have broad applications and create new insights into the pathogenesis of atherosclerosis and other inflammatory diseases.
Imaging; atherosclerosis; plaque; cells
Monocytes are circulating macrophage and dendritic cell precursors that populate healthy and diseased tissue. In humans, monocytes consist of at least two subsets whose proportions in the blood fluctuate in response to coronary artery disease, sepsis, and viral infection. Animal studies have shown that specific shifts in the monocyte subset repertoire either exacerbate or attenuate disease, suggesting a role for monocyte subsets as biomarkers and therapeutic targets. Assays are therefore needed that can selectively and rapidly enumerate monocytes and their subsets. This study shows that two major human monocyte subsets express similar levels of the receptor for macrophage colony stimulating factor (MCSFR) but differ in their phagocytic capacity. We exploit these properties and custom-engineer magnetic nanoparticles for ex vivo sensing of monocytes and their subsets. We present a two-dimensional enumerative mathematical model that simultaneously reports number and proportion of monocyte subsets in a small volume of human blood. Using a recently described diagnostic magnetic resonance (DMR) chip with 1 µl sample size and high throughput capabilities, we then show that application of the model accurately quantifies subset fluctuations that occur in patients with atherosclerosis.