Monocytes are critical mediators of healing following acute myocardial infarction (AMI), making them an interesting target to improve myocardial repair. The purpose of this study was a gain of insight into the source and recruitment of monocytes following AMI in humans.
Methods and results
Post-mortem tissue specimens of myocardium, spleen and bone marrow were collected from 28 patients who died at different time points after AMI. Twelve patients who died from other causes served as controls. The presence and localization of monocytes (CD14+ cells), and their CD14+CD16– and CD14+CD16+ subsets, were evaluated by immunohistochemical and immunofluorescence analyses. CD14+ cells localized at distinct regions of the infarcted myocardium in different phases of healing following AMI. In the inflammatory phase after AMI, CD14+ cells were predominantly located in the infarct border zone, adjacent to cardiomyocytes, and consisted for 85% (78–92%) of CD14+CD16– cells. In contrast, in the subsequent post-AMI proliferative phase, massive accumulation of CD14+ cells was observed in the infarct core, containing comparable proportions of both the CD14+CD16– [60% (31–67%)] and CD14+CD16+ subsets [40% (33–69%)]. Importantly, in AMI patients, of the number of CD14+ cells was decreased by 39% in the bone marrow and by 58% in the spleen, in comparison with control patients (P = 0.02 and <0.001, respectively).
Overall, this study showed a unique spatiotemporal pattern of monocyte accumulation in the human myocardium following AMI that coincides with a marked depletion of monocytes from the spleen, suggesting that the human spleen contains an important reservoir function for monocytes.
Acute myocardial infarction; Inflammation; Monocytes; Spleen; Bone marrow
Dysfunctional endothelium contributes to more disease than any other tissue in the body. Small interfering RNAs (siRNAs) have the potential to help study and treat endothelial cells in vivo by durably silencing multiple genes simultaneously, but efficient siRNA delivery has so far remained challenging. Here we show that polymeric nanoparticles made of low molecular weight polyamines and lipids can deliver siRNA to endothelial cells with high efficiency, thereby facilitating the simultaneous silencing of multiple endothelial genes in vivo. Unlike lipid or lipid-like nanoparticles, this formulation does not significantly reduce gene expression in hepatocytes or immune cells even at the dosage necessary for endothelial gene silencing. It mediates the most durable non-liver silencing reported to date, and facilitates the delivery of siRNAs that modify endothelial function in mouse models of vascular permeability, emphysema, primary tumour growth, and metastasis. We believe these nanoparticles improve the ability to study endothelial gene function in vivo, and may be used to treat diseases caused by vascular dysfunction.
Exposure to psychosocial stress is a risk factor for many diseases, including atherosclerosis1,2. While incompletely understood, interaction between the psyche and the immune system provides one potential mechanism linking stress and disease inception and progression. Known crosstalk 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 reflex3,4. It remains unknown however if chronic stress changes hematopoietic stem cell activity. Here we show that stress increases proliferation of these most primitive 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. Sympathetic nerve fibers release surplus noradrenaline, which uses the β3 adrenergic receptor to signal bone marrow niche cells to decrease CXCL12 levels. Consequently, elevated hematopoietic stem cell proliferation increases output of neutrophils and inflammatory monocytes. When atherosclerosis–prone ApoE−/− mice encounter chronic stress, accelerated hematopoiesis promotes plaque features associated with vulnerable lesions that cause myocardial infarction and stroke in humans.
In response to lung infection, pleural innate response activator B cells produce GM-CSF–dependent IgM and ensure a frontline defense against bacterial invasion.
Pneumonia is a major cause of mortality worldwide and a serious problem in critical care medicine, but the immunophysiological processes that confer either protection or morbidity are not completely understood. We show that in response to lung infection, B1a B cells migrate from the pleural space to the lung parenchyma to secrete polyreactive emergency immunoglobulin M (IgM). The process requires innate response activator (IRA) B cells, a transitional B1a-derived inflammatory subset which controls IgM production via autocrine granulocyte/macrophage colony-stimulating factor (GM-CSF) signaling. The strategic location of these cells, coupled with the capacity to produce GM-CSF–dependent IgM, ensures effective early frontline defense against bacteria invading the lungs. The study describes a previously unrecognized GM-CSF-IgM axis and positions IRA B cells as orchestrators of protective IgM immunity.
Only a few decades ago, students of the pathophysiology of cardiovascular disease paid little heed to the involvement of inflammation and immunity. Multiple lines of evidence now point to the participation of innate and adaptive immunity and inflammatory signaling in a variety of cardiovascular conditions. Hence, interest has burgeoned in this intersection. This review will focus on the contribution of innate immunity to both acute injury to the heart muscle itself, notably myocardial infarction, and to chronic inflammation in the artery wall, namely atherosclerosis, the cause of most myocardial infarctions. Our discussion of the operation of innate immunity in cardiovascular diseases will focus on functions of the mononuclear phagocytes with special attention to emerging data regarding the participation of different functional subsets of these cells in cardiovascular pathophysiology.
myocardial infarction; atherosclerosis; acute coronary syndromes; inflammation; mononuclear phagocytes; innate immunity
The important role of monocytes and macrophages in diseases like cancer and atherosclerosis has started to get uncovered in the last decade. In addition, subsets of these cell types are believed to participate in the initiation and aggravation of several diseases including cancer and cardiovascular disease. For this reason, monocytes and macrophages have recently been identified as interesting targets for both diagnosis and treatment of the aforementioned pathologies. Compared with free therapeutic or imaging agents, nanoparticle formulations provide several advantages that improve the pharmacokinetics and bioavailability of these agents. In addition, the possibility of surface functionalization creates numerous ways to optimize nanoparticle delivery. Recent advances in nanomedicine have led to the development of multifunctional nanoparticles that allow simultaneous diagnosis and treatment of monocytes and macrophages with high specificity. Relying on the inherent ability of monocytes and macrophages to easily take up foreign particles, the use of nanoparticles provides a precious opportunity for the management of several inflammatory diseases.
anti-inflammatory; diagnosis; macrophage; monocyte; nanomedicine; nanoparticles; treatment
Iron oxide core nanoparticles are attractive imaging agents because their material properties allow the tuning of pharmacokinetics as well as attachment of multiple moieties to their surface. In addition to affinity ligands, these include fluorochromes and radioisotopes for detection with optical and nuclear imaging. As the iron oxide core can be detected by MRI, options for combining imaging modalities are manifold. Already, preclinical imaging strategies combine non-invasive imaging with higher resolution techniques such as intravital microscopy to gain unprecedented insight into steady state biology and disease. Going forward, hybrid iron oxide nanoparticles will likely help to merge modalities, creating a synergy that enables imaging in basic research and, potentially, also in the clinic.
nanoparticle; iron oxide; multimodality; multiscale; molecular imaging; MRI; PET; optical imaging
Inflammation is a key feature of atherosclerosis and a target for therapy. Statins have potent anti-inflammatory properties but these cannot be fully exploited with oral statin therapy due to low systemic bioavailability. Here we present an injectable reconstituted high-density lipoprotein (rHDL) nanoparticle carrier vehicle that delivers statins to atherosclerotic plaques. We demonstrate the anti-inflammatory effect of statin-rHDL in vitro and show this effect is mediated through inhibition of the mevalonate pathway. We also apply statin-rHDL nanoparticles in vivo in an apolipoprotein E-knockout mouse model of atherosclerosis and show they accumulate in atherosclerotic lesions where they directly affect plaque macrophages. Finally we demonstrate that a three-month low-dose statin-rHDL treatment regimen inhibits plaque inflammation progression, while a one-week high-dose regimen markedly decreases inflammation in advanced atherosclerotic plaques. Statin-rHDL represents a novel potent atherosclerosis nanotherapy that directly affects plaque inflammation.
Identification of key molecular players in myocardial healing could lead to improved therapies, reduction of scar formation, and heart failure after myocardial infarction (MI). We hypothesized that clotting factor XIII (FXIII), a transglutaminase involved in wound healing, may play an important role in MI given prior clinical and mouse model data.
Methods and Results
To determine whether a truly causative relationship existed between FXIII activity and myocardial healing, we prospectively studied myocardial repair in FXIII-deficient mice. All FXIII−/− and FXIII−/+ (FXIII activity <5% and 70%) mice died within 5 days after MI from left ventricular rupture. In contradistinction, FXIII−/− mice that received 5 days of intravenous FXIII replacement therapy had normal survival rates; however, cardiac MRI demonstrated worse left ventricular remodeling in these reconstituted FXIII−/− mice. Using a FXIII-sensitive molecular imaging agent, we found significantly greater FXIII activity in wild-type mice and FXIII−/− mice receiving supplemental FXIII than in FXIII−/− mice (P<0.05). In FXIII−/− but not in reconstituted FXIII−/− mice, histology revealed diminished neutrophil migration into the MI. Reverse transcriptase–polymerase chain reaction studies suggested that the impaired inflammatory response in FXIII−/− mice was independent of intercellular adhesion molecule and lipopolysaccharide-induced CXC chemokine, both important for cell migration. After MI, expression of matrix metalloproteinase-9 was 650% higher and collagen-1 was 53% lower in FXIII−/− mice, establishing an imbalance in extracellular matrix turnover and providing a possible mechanism for the observed cardiac rupture in the FXIII−/− mice.
These data suggest that FXIII has an important role in murine myocardial healing after infarction.
factor XIII; healing; heart failure; myocardial infarction; remodeling
In a cell-free approach to regenerative therapeutics, transient application of paracrine factors in vivo could be used to alter the behavior and fate of progenitor cells to achieve sustained clinical benefits. Here we show that intramyocardial injection of synthetic modified RNA (modRNA) encoding human vascular endothelial growth factor-A (VEGF-A) resulted in the expansion and directed differentiation of endogenous heart progenitors in a murine myocardial infarction model. VEGF-A modRNA markedly improved heart function and enhanced long-term survival of recipients. This improvement was in part due to mobilization of epicardial progenitor cells and redirection of their differentiation toward cardiovascular cell types. Direct in vivo comparison with DNA vectors, and temporal control with VEGF inhibitors, documented the markedly increased efficacy of pulse-like delivery of VEGF-A. Our results suggest that modRNA is a versatile approach for expressing paracrine factors as cell fate switches to control progenitor cell fate and thereby enhance long term organ repair.
Monocytes and macrophages are innate immune cells that reside and accumulate in the healthy and injured heart. The cells and their subsets pursue distinct functions in steady state and disease, and their tenure may range between hours to months. Some subsets are highly inflammatory, while others support tissue repair. This review discusses current concepts of lineage relationships and systems’ cross talk, highlights open questions, and describes tools for studying monocyte and macrophage subsets in the murine and human heart.
heart failure; myocardial infarction; healing; monocyte; macrophage
Autophagy is a biological process during which cells digest organelles in their cytoplasm and recycle the constituents. The impact of autophagy in the heart, however, remains unclear in part due to the inability to noninvasively image this process in living animals.
Methods and Results
Here, we report the use of fluorescence molecular tomography (FMT) and a cathepsin activatable fluorochrome to image autophagy in the heart in vivo following ischemia-reperfusion and rapamycin therapy. We show that cathepsin-B activity in the lysosome is upregulated by rapamycin and that this allows the expanded lysosomal compartment in autophagy to be imaged in vivo with FMT. We further demonstrate that the delivery of diagnostic nanoparticles to the lysosome by endocytosis is enhanced during autophagy. The upregulation of autophagy by rapamycin was associated with a 23% reduction (p<0.05) of apoptosis in the area-at-risk (AAR), and a 45% reduction in final infarct size (19.6 +/− 5.6% of AAR with rapamycin versus 35.9 +/− 9.1% of AAR without rapamycin, p<0.05).
The ability to perform noninvasive tomographic imaging of autophagy in the heart has the potential to provide valuable insights into the pathophysiology of autophagy, particularly its role in cardiomyocyte salvage. While additional data are needed, our study supports the investigation of rapamycin therapy in patients with acute coronary syndromes.
autophagy; apoptosis; molecular imaging; rapamycin; myocardium
High-resolution imaging of the heart in vivo is challenging due to the difficulty in accessing the heart and the tissue motion caused by the heartbeat.
Here, we describe a suction-assisted endoscope for visualizing fluorescently labeled cells and vessels in the beating heart tissue through a small incision made in the intercostal space.
Methods and Results
A suction tube with a diameter of 2-3 mm stabilizes the local tissue motion safely and effectively at a suction pressure of 50 mmHg. Using a minimally invasive endoscope integrated into a confocal microscope, we performed fluorescence cellular imaging in both normal and diseased hearts in live mice for an hour per session repeatedly over a few weeks. Real-time imaging revealed the surprisingly rapid infiltration of CX3CR1+ monocytes into the injured site within several minutes after acute myocardial infarction (MI).
The time-lapse analysis of flowing and rolling (patrolling) monocytes in the heart and the peripheral circulation provide evidence that the massively recruited monocytes come first from the vascular reservoir and later from the spleen. The imaging method requires minimal surgical preparation and can be implemented into standard intravital microscopes. Our results demonstrate the applicability of our imaging method for a wide range of cardiovascular research.
Myocardial infarction; leukocytes; imaging; circulation; monocyte
Innate immune cells are critically involved in ischemic complications of atherosclerosis. While new insight emerged on the origin and role of leukocytes in steady state, the knowledge about myeloid cell's sources, functions and fate after stroke is limited. In our review, we highlight open questions in this important area while examining potential parallels in the immune response after stroke and myocardial infarction. We stress the need to better understand systemic interactions between ischemic tissue, immunity and hematopoiesis, as turn over of leukocytes in inflammatory sites can be rapid, and cell production and supply may serve as future therapeutic targets to modulate inflammation in the vessel wall, the brain and heart.
During the inflammatory response that drives atherogenesis, macrophages accumulate progressively in the expanding arterial wall1,2. The observation that circulating monocytes give rise to lesional macrophages3–9 has reinforced the concept that monocyte infiltration dictates macrophage build-up. Recent work indicates, however, that macrophages do not depend on monocytes in some inflammatory contexts10. We therefore revisited the mechanism of macrophage accumulation in atherosclerosis. We show that murine atherosclerotic lesions experience a surprisingly rapid, 4-week, cell turnover. Replenishment of macrophages in these experimental atheromata depends predominantly on local macrophage proliferation rather than monocyte influx. The microenvironment orchestrates macrophage proliferation via the involvement of scavenger receptor (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.
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.
Nanoparticles are employed for delivering therapeutics into cells1,2. However, size, shape, surface chemistry and the presentation of targeting ligands on the surface of nanoparticles can affect circulation half-life and biodistribution, cell specific internalization, excretion, toxicity, and efficacy3-7. A variety of materials have been explored for delivering small interfering RNAs (siRNAs) - a therapeutic agent that suppresses the expression of targeted genes8,9. However, conventional delivery nanoparticles such as liposomes and polymeric systems are heterogeneous in size, composition and surface chemistry, and this can lead to suboptimal performance, lack of tissue specificity and potential toxicity10-12. Here, we show that self-assembled DNA tetrahedral nanoparticles with a well-defined size can deliver siRNAs into cells and silence target genes in tumours. Monodisperse nanoparticles are prepared through the self-assembly of complementary DNA strands. Because the DNA strands are easily programmable, the size of the nanoparticles and the spatial orientation and density of cancer targeting ligands (such as peptides and folate) on the nanoparticle surface can be precisely controlled. We show that at least three folate molecules per nanoparticle is required for optimal delivery of the siRNAs into cells and, gene silencing occurs only when the ligands are in the appropriate spatial orientation. In vivo, these nanoparticles showed a longer blood circulation time (t1/2 ∼ 24.2 min) than the parent siRNA (t1/2 ∼ 6 min).
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.
To use molecular imaging targeting coagulation pathway and inflammation to 1) better understand the pathophysiology of silent brain ischemia (SBI) and 2) monitor the effects of factor XIIa inhibition.
Silent brain ischemia can be observed in patients who undergo invasive vascular procedures. Unlike acute stroke, the diffuse nature of SBI and its less tangible clinical symptoms make this disease difficult to diagnose and treat.
We induced SBI in mice by intra-arterial injection of fluorescently-labeled microbeads or fractionated clot into the carotid artery. After SBI induction, diffusion-weighted (DWI) magnetic resonance imaging (MRI) was performed to confirm the presence of microinfarcts in asymptomatic mice. Molecular imaging targeting the downstream factor XIII activity (SPECT-CT) at three hours and myeloperoxidase activity (MRI) on day three after SBI induction were performed, without and with the intravenous administration of a recombinant selective factor XIIa inhibitor derived from the hematophagous insect Triatoma infestans (rHA-Infestin-4). Statistical comparisons between two groups were evaluated by the Student’s t-test or the Mann-Whitney U test.
In SBI-induced mice, we found abnormal activation of the coagulation cascade (factor XIII activity) and increased inflammation (myeloperoxidase activity) close to where emboli lodge in the brain. rHA-Infestin-4 administration significantly reduced ischemic damage (53–85% reduction of infarct volume, p < 0.05) and pathological coagulation (35–39% reduction of factor XIII activity, p < 0.05) without increasing hemorrhagic frequency. Myeloperoxidase activity, when normalized to the infarct volume, did not significantly change with rHA-Infestin-4 treatment, suggesting that this treatment does not further decrease inflammation other than that resulted from the reduction in infarct volume.
Focal intracerebral clotting and inflammatory activity are part of the pathophysiology underlying SBI. Inhibiting factor XIIa with rHA-Infestin-4 may present a safe and effective treatment to decrease the morbidity from SBI.
Silent brain ischemia; coagulation; inflammation; imaging; infestin
In vivo imaging is often severely compromised by cardiovascular and respiratory motion. Highly successful motion compensation techniques have been developed for clinical imaging (e.g. magnetic resonance imaging) but the use of more advanced techniques for intravital microscopy is largely unexplored. Here, we implement a sequential cardiorespiratory gating scheme (SCG) for averaged microscopy. We show that SCG is very efficient in eliminating motion artifacts, is highly practical, enables high signal-to-noise ratio (SNR) in vivo imaging, and yields large field of views. The technique is particularly useful for high-speed data acquisition or for imaging scenarios where the fluorescence signal is not significantly above noise or background levels.
(180.0180) Microscopy; (170.2520) Fluorescence microscopy; (170.5810) Scanning microscopy; (170.3880) Medical and biological imaging
MRI; molecular imaging; myocardial infarction; inflammation; iron oxide nanoparticles
Despite significant advancements in medical and device-based therapies, cardiovascular disease remains the number one cause of death in the United States. Early detection of atherosclerosis, prevention of myocardial infarction and sudden cardiac death, and modulation of adverse ventricular remodeling still remain elusive goals. Molecular imaging focuses on identifying critical cellular and molecular targets and therefore plays an integral role in understanding these biological processes in vivo. Since many imaging targets are upregulated before irreversible tissue damage occurs, early detection could ultimately lead to development of novel, preventive therapeutic strategies. This review addresses recent work on radionuclide imaging of cardiovascular inflammation, infection, and infarct healing. We further discuss opportunities provided by multimodality approaches such as PET/MRI and PET/optical imaging.
molecular imaging; cardiovascular; nuclear medicine; multi-modality; MRI