Filip K. Swirski

The healing myocardium sequentially mobilizes two monocyte subsets with divergent and complementary functions

Healing of myocardial infarction (MI) requires monocytes/macrophages. These mononuclear phagocytes likely degrade released macromolecules and aid in scavenging of dead cardiomyocytes, while mediating aspects of granulation tissue formation and remodeling. The mechanisms that orchestrate such divergent functions remain unknown. In view of the heightened appreciation of the heterogeneity of circulating monocytes, we investigated whether distinct monocyte subsets contribute in specific ways to myocardial ischemic injury in mouse MI. We identify two distinct phases of monocyte participation after MI and propose a model that reconciles the divergent properties of these cells in healing. Infarcted hearts modulate their chemokine expression profile over time, and they sequentially and actively recruit Ly-6Chi and -6Clo monocytes via CCR2 and CX3CR1, respectively. Ly-6Chi monocytes dominate early (phase I) and exhibit phagocytic, proteolytic, and inflammatory functions. Ly-6Clo monocytes dominate later (phase II), have attenuated inflammatory properties, and express vascular–endothelial growth factor. Consequently, Ly-6Chi monocytes digest damaged tissue, whereas Ly-6Clo monocytes promote healing via myofibroblast accumulation, angiogenesis, and deposition of collagen. MI in atherosclerotic mice with chronic Ly-6Chi monocytosis results in impaired healing, underscoring the need for a balanced and coordinated response. These observations provide novel mechanistic insights into the cellular and molecular events that regulate the response to ischemic injury and identify new therapeutic targets that can influence healing and ventricular remodeling after MI.

Identification of Splenic Reservoir Monocytes and Their Deployment to Inflammatory Sites

Monitoring Monocyte Reservoirs Monocytes are cells of the immune system that are recruited to sites of tissue injury and inflammation where they help to resolve the infection and are important for tissue repair. The bone marrow and blood are believed to be the primary reservoirs from which monocytes are mobilized after injury. Swirski et al. (p. 612; see the Perspective by Jia and Pamer) now demonstrate that the spleen also serves as a critical reservoir of monocytes that are recruited during ischemic myocardial injury. Monocytes in the spleen are very similar in phenotype to blood-derived monocytes and are mobilized to the injured heart, where they represent a large fraction of the total monocytes that are recruited. The chemoattractant, angiotensin II, is required for optimal monocyte mobilization from the spleen and emigration into injured tissue. A rapid deployment force of immune cells is identified in the spleen that is important for resolving 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.

Ly-6Chi monocytes dominate hypercholesterolemia-associated monocytosis and give rise to macrophages in atheromata

Macrophage accumulation participates decisively in the development and exacerbation of atherosclerosis. Circulating monocytes, the precursors of macrophages, display heterogeneity in mice and humans, but their relative contribution to atherogenesis remains unknown. We report here that the Ly-6C(hi) monocyte subset increased dramatically in hypercholesterolemic apoE-deficient mice consuming a high-fat diet, with the number of Ly-6C(hi) cells doubling in the blood every month. Ly-6C(hi) monocytes adhered to activated endothelium, infiltrated lesions, and became lesional macrophages. Hypercholesterolemia-associated monocytosis (HAM) developed from increased survival, continued cell proliferation, and impaired Ly-6C(hi) to Ly-6C(lo) conversion and subsided upon statin-induced cholesterol reduction. Conversely, the number of Ly-6C(lo) cells remained unaffected. Thus, we believe that Ly-6C(hi) monocytes represent a newly recognized component of the inflammatory response in experimental atherosclerosis.

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.

Local proliferation dominates lesional macrophage accumulation in atherosclerosis

During the inflammatory response that drives atherogenesis, macrophages accumulate progressively in the expanding arterial wall. The observation that circulating monocytes give rise to lesional macrophages has reinforced the concept that monocyte infiltration dictates macrophage buildup. Recent work has indicated, however, that macrophage accumulation does not depend on monocyte recruitment in some inflammatory contexts. We therefore revisited the mechanism underlying macrophage accumulation in atherosclerosis. In murine atherosclerotic lesions, we found that macrophages turn over rapidly, after 4 weeks. Replenishment of macrophages in these experimental atheromata depends predominantly on local macrophage proliferation rather than monocyte influx. The microenvironment orchestrates macrophage proliferation through the involvement of scavenger receptor A (SR-A). Our study reveals macrophage proliferation as a key event in atherosclerosis and identifies macrophage self-renewal as a therapeutic target for cardiovascular disease.

Therapeutic siRNA silencing in inflammatory monocytes in mice

Excessive and prolonged activity of inflammatory monocytes is a hallmark of many diseases with an inflammatory component. In such conditions, precise targeting of these cells could be therapeutically beneficial while sparing many essential functions of the innate immune system, thus limiting unwanted effects. Inflammatory monocytes—but not the noninflammatory subset—depend on the chemokine receptor CCR2 for localization to injured tissue. Here we present an optimized lipid nanoparticle and a CCR2-silencing short interfering RNA that, when administered systemically in mice, show rapid blood clearance, accumulate in spleen and bone marrow, and localize to monocytes. Efficient degradation of CCR2 mRNA in monocytes prevents their accumulation in sites of inflammation. Specifically, the treatment attenuates their number in atherosclerotic plaques, reduces infarct size after coronary artery occlusion, prolongs normoglycemia in diabetic mice after pancreatic islet transplantation, and results in reduced tumor volumes and lower numbers of tumor-associated macrophages.

Nanoparticle PET-CT Imaging of Macrophages in Inflammatory Atherosclerosis

Background— Macrophages participate centrally in atherosclerosis, and macrophage markers (eg, CD68, MAC-3) correlate well with lesion severity and therapeutic modulation. On the basis of the avidity of lesional macrophages for polysaccharide-containing supramolecular structures such as nanoparticles, we have developed a new positron emission tomography (PET) agent with optimized pharmacokinetics to allow in vivo imaging at tracer concentrations. Methods and Results— A dextranated and DTPA-modified magnetofluorescent 20-nm nanoparticle was labeled with the PET tracer 64Cu (1 mCi/0.1 mg nanoparticles) to yield a PET, magnetic resonance, and optically detectable imaging agent. Peak PET activity 24 hours after intravenous injection into mice deficient in apolipoprotein E with experimental atherosclerosis mapped to areas of high plaque load identified by computed tomography such as the aortic root and arch and correlated with magnetic resonance and optical imaging. Accumulated dose in apolipoprotein E-deficient aortas determined by gamma counting was 260% and in carotids 392% of respective wild-type organs (P<0.05 both). Autoradiography of aortas demonstrated uptake of the agent into macrophage-rich atheromata identified by Oil Red O staining of lipid deposits. The novel nanoagent accumulated predominantly in macrophages as determined by fluorescence microscopy and flow cytometry of cells dissociated from aortas. Conclusions— This report establishes the capability of a novel trimodality nanoparticle to directly detect macrophages in atherosclerotic plaques. Advantages include improved sensitivity; direct correlation of PET signal with an established biomarker (CD68); ability to readily quantify the PET signal, perform whole-body vascular surveys, and spatially localize and follow the trireporter by microscopy; and clinical translatability of the agent given similarities to magnetic resonance imaging probes in clinical trials.

Monocytes: Protagonists of Infarct Inflammation and Repair After Myocardial Infarction

Myocardial infarction (MI) is the most frequent cause of heart failure, which is an incapacitating disease with high prevalence and broad socioeconomic impact. In 2008 in the United States, 5.7 million people suffered from heart failure, and more than 287 000 people died.1 Timely revascularization of ischemic myocardium reduces acute infarct mortality, and current standard therapy with β blockers and angiotensin-converting enzyme (ACE) inhibitors curbs development of post-MI heart failure. For example, ACE inhibitor treatment reduced mortality from 25% to 20% in the Survival and Ventricular Enlargement (SAVE) trial.2 Although this is a major advance, long-term mortality remains high. The combination of reduced acute infarct mortality due to efficient acute care and insufficient options to treat infarct survivors chronically has contributed to an increased heart failure prevalence (Figure 1).3 Figure 1. Disease statistics. A, Deaths per 100 000 population for coronary heart disease in men, ages 35 to 74 years, United States, 1970 to 2005. B, Deaths due to heart failure, United States, 1970 to 2005. C, Hospitalizations per 100 000 population for heart failure, age ≥65 years, United States, 1971 to 2006. Adapted from National Heart, Lung, and Blood Institute Fact Book 2008.3 The need to understand and treat heart failure better has motivated clinicians and basic scientists to explore new therapeutic strategies to repair the failing heart, for instance, with stem cells.4,5 Augmentation of intrinsic wound healing that occurs during the first 1 to 2 weeks after MI is a prospective approach with the potential to prevent heart failure. During this period, the infarct is highly active biologically.6–8 Delicate granulation tissue undergoes rapid turnover of cells and of structural components such as the extracellular matrix. Preexisting collagen is digested and new matrix is laid down. During these extensive changes of tissue …

Leukocyte Behavior in Atherosclerosis, Myocardial Infarction, and Heart Failure

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, MI, and heart failure.

Osteogenesis Associates With Inflammation in Early-Stage Atherosclerosis Evaluated by Molecular Imaging In Vivo

Background— Arterial calcification is associated with cardiovascular events; however, mechanisms of calcification in atherosclerosis remain obscure. Methods and Results— We tested the hypothesis that inflammation promotes osteogenesis in atherosclerotic plaques using in vivo molecular imaging in apolipoprotein E−/− mice (20 to 30 weeks old, n=35). A bisphosphonate-derivatized near-infrared fluorescent imaging agent (excitation 750 nm) visualized osteogenic activity that was otherwise undetectable by x-ray computed tomography. Flow cytometry validated the target specifically in osteoblast-like cells. A spectrally distinct near-infrared fluorescent nanoparticle (excitation 680 nm) was coinjected to simultaneously image macrophages. Fluorescence reflectance mapping demonstrated an association between osteogenic activity and macrophages in aortas of apolipoprotein E−/− mice (R2=0.93). Intravital dual-channel fluorescence microscopy was used to further monitor osteogenic changes in inflamed carotid arteries at 20 and 30 weeks of age and revealed that macrophage burden and osteogenesis concomitantly increased during plaque progression (P<0.01 and P<0.001, respectively) and decreased after statin treatment (P<0.0001 and P<0.05, respectively). Fluorescence microscopy on cryosections colocalized near-infrared fluorescent osteogenic signals with alkaline phosphatase activity, bone-regulating protein expression, and hydroxyapatite nanocrystals as detected by electron microscopy, whereas von Kossa and alizarin red stains showed no evidence of calcification. Real-time reverse-transcription polymerase chain reaction revealed that macrophage-conditioned media increased alkaline phosphatase mRNA expression in vascular smooth muscle cells. Conclusions— This serial in vivo study demonstrates the real-time association of macrophage burden with osteogenic activity in early-stage atherosclerosis and offers a cellular-resolution tool to identify preclinical microcalcifications.