Mapping Molecular Agents Distributions in Whole Mice Hearts Using Born-Normalized Optical Projection Tomography

To date there is a lack of tools to map the spatio-temporal dynamics of diverse cells in experimental heart models. Conventional histology is labor intensive with limited coverage, whereas many imaging techniques do not have sufficiently high enough spatial resolution to map cell distributions. We have designed and built a high resolution, dual channel Born-normalized near-infrared fluorescence optical projection tomography system to quantitatively and spatially resolve molecular agents distribution within whole murine heart. We validated the use of the system in a mouse model of monocytes/macrophages recruitment during myocardial infarction. While acquired, data were processed and reconstructed in real time. Tomographic analysis and visualization of the key inflammatory components were obtained via a mathematical formalism based on left ventricular modeling. We observed extensive monocyte recruitment within and around the infarcted areas and discovered that monocytes were also extensively recruited into non-ischemic myocardium, beyond that of injured tissue, such as the septum

Rapid monocyte kinetics in acute myocardial infarction are sustained by extramedullary monocytopoiesis

IL-1b signaling augments continued splenic monocyte supply during acute inflammation

Monocyte-Directed RNAi Targeting CCR2 Improves Infarct Healing in Atherosclerosis-Prone Mice

Background—Exaggerated and prolonged inflammation after myocardial infarction (MI) accelerates left ventricular remodeling. Inflammatory pathways may present a therapeutic target to prevent post-MI heart failure. However, the appropriate magnitude and timing of interventions are largely unknown, in part because noninvasive monitoring tools are lacking. Here, we used nanoparticle-facilitated silencing of CCR2, the chemokine receptor that governs inflammatory Ly-6Chigh monocyte subset traffic, to reduce infarct inflammation in apolipoprotein E–deficient (apoE−/−) mice after MI. We used dual-target positron emission tomography/magnetic resonance imaging of transglutaminase factor XIII (FXIII) and myeloperoxidase (MPO) activity to monitor how monocyte subset–targeted RNAi altered infarct inflammation and healing. Methods and Results—Flow cytometry, gene expression analysis, and histology revealed reduced monocyte numbers and enhanced resolution of inflammation in infarcted hearts of apoE−/− mice that were treated with nanoparticle-encapsulated siRNA. To follow extracellular matrix cross-linking noninvasively, we developed a fluorine-18–labeled positron emission tomography agent (18F-FXIII). Recruitment of MPO-rich inflammatory leukocytes was imaged with a molecular magnetic resonance imaging sensor of MPO activity (MPO-Gd). Positron emission tomography/magnetic resonance imaging detected anti-inflammatory effects of intravenous nanoparticle-facilitated siRNA therapy (75% decrease of MPO-Gd signal; P<0.05), whereas 18F-FXIII positron emission tomography reflected unimpeded matrix cross-linking in the infarct. Silencing of CCR2 during the first week after MI improved ejection fraction on day 21 after MI from 29% to 35% (P<0.05). Conclusion—CCR2-targeted RNAi reduced recruitment of Ly-6Chigh monocytes, attenuated infarct inflammation, and curbed post-MI left ventricular remodeling.National Heart, Lung, and Blood InstituteUnited States. Dept. of Health and Human Services (contract No. HHSN268201000044C)National Institutes of Health (U.S.) (grant R01-HL096576)National Institutes of Health (U.S.) (grant R01-HL095629)National Institutes of Health (U.S.) (grant T32-HL094301)Deutsche Forschungsgemeinschaft (HE-6382/1-1

Local proliferation dominates lesional macrophage accumulation in atherosclerosis

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

Myocardial Infarction Accelerates Atherosclerosis

During progression of atherosclerosis, myeloid cells destabilize lipid-rich plaque in the arterial wall and cause its 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 ischemic 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. When seeking the source of surplus monocytes in plaque, we found that myocardial infarction liberated hematopoietic stem and progenitor cells from bone marrow niches via sympathetic nervous system signaling. 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

Sensitive and Direct Detection of Circulating Tumor Cells by Multimarker µ-Nuclear Magnetic Resonance12

Identifying circulating tumor cells (CTCs) with greater sensitivity could facilitate early detection of cancer and rapid assessment of treatment response. Most current technologies use EpCAM expression as a CTC identifier. However, given that a significant fraction of cancer patients have low or even absent EpCAM levels, there is a need for better detection methods. Here, we hypothesize that a multimarker strategy combined with direct sensing of CTC in whole blood would increase the detection of CTC in patients. Accordingly, molecular profiling of biopsies from a patient cohort revealed a four-marker set (EpCAM, HER-2, EGFR, and MUC-1) capable of effectively differentiating cancer cells from normal host cells. Using a point-of-care micro-nuclear magnetic resonance (µNMR) system, we consequently show that this multimarker combination readily detects individual CTC directly in whole blood without the need for primary purification. We also confirm these results in a comparative trial of patients with ovarian cancer. This platform could potentially benefit a broad range of applications in clinical oncology

Validation of tomographic reconstructions.

<p>Comparison between OPT reconstructions and histological sections of molecular probe activity distribution (Prosense-680) in the inflamed heart after MI. Comparisons between reconstructions obtained with fluorescence OPT (A), Bornnormalized OPT (B), and the corresponding histological section (C). Born normalization preserves the molecular distributions in the reconstructed fluorescence channels. This is particularly evident for the papillary muscles located deep within the left ventricle, which appear less fluorescent without normalization, and the epicardium, which always shows bright fluorescent signal in absence of normalization. Born-normalized OPT reconstructions were obtained on the whole heart. The histological section (500 µm) belongs to the same specimen.</p

Born-normalized molecular optical projection tomography of inflamed hearts.

<p>(A) Dual channel imaging. A cocktail containing two different molecular imaging agents is administered intravenously. (B) Dual channel Born-normalized near-infrared transillumination fluorescence image on day 5 after myocardial infarction (MI), 24 hours after probe injection. High signal in both the 680 nm (left) and 750 nm (right) fluorescence channels can be observed in the heart region, indicating elevated protease and phagocytic activity. (C) A narrow bandwidth light source excites the fluorophores attached to a molecular probe located within the sample, and fluorescent light is then emitted. Both excitation and emission light are, in part, absorbed by the sample on the way to and from the fluorophore, respectively. Using a Born-normalized approach, fluorescence tomographic reconstructions can be obtained, after correcting for the sample's absorption map. (D) Image processing algorithms are applied to tomographically obtain signal distribution reconstructions in whole hearts and to remove noise and artifacts contributions.</p

Quantification of probe activity in infarct area.

<p>(A) Cylindrical (left) and Bull's Eye (right) maps of molecular probe and bead distribution of an excised heart at day 5 after myocardial infarction. High cathepsin B and phagocytic activity, are evident in the proximity of the injured area with a high degree of colocalization. Fluorescently labeled (FITC) microbeads, injected in vivo into the left ventricle before harvesting, are homogeneously distributed across the whole heart, with the exception of the non-perfused infarct area. The white dotted line indicates the border of the necrotic infarct scar, localized in apical and lateral segments. Continuous white line indicates the border of the interventricular septum. (B) Axial reconstructions of probe activities at the indicated plane. (C) Cylindrical (left) and Bull's Eye (right) representation of reparative (Ly6C^lo, green) and inflammatory (Ly6C^hi, red) monocyte subsets together with the an axial (D) and tomographic (E) reconstruction.</p

Probe activity distribution maps in inflamed left ventricle.

<p>(A) Bull's Eye (top) and cylindrical (right) maps of molecular probe activity at day 5 post myocardial infarction. Inflammation is accentuated along the border of the injured tissue, while the core of the infarct displays low signal. Continuous white line indicates the border of the interventricular septum. (B) One-dimensional plots showing the intensities of the probes activity, along the directions indicated in the maps (white faded bands). Signal plots indicate a high correlation in probe activities. (C) Axial reconstructions of cell-associated probe activity along the indicated plane, with their corresponding co-localization maps. (D) Overlapping cross correlation functions (CCF) are calculated for the two channels, taking into account both translational and rotational shifts. Overall, a high degree of correlation is present, however there are distinct differences in each channel reflecting the different spatial distribution of the molecular target.</p