Claudio Vinegoni

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.

Chronic variable stress activates hematopoietic stem cells

Exposure to psychosocial stress is a risk factor for many diseases, including atherosclerosis. Although incompletely understood, interaction between the psyche and the immune system provides one potential mechanism linking stress and disease inception and progression. Known cross-talk 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 reflex. It remains unknown, however, whether chronic stress changes hematopoietic stem cell activity. Here we show that stress increases proliferation of these most primitive hematopoietic 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. Under conditions of chronic variable stress in mice, sympathetic nerve fibers released surplus noradrenaline, which signaled bone marrow niche cells to decrease CXCL12 levels through the β3-adrenergic receptor. Consequently, hematopoietic stem cell proliferation was elevated, leading to an increased output of neutrophils and inflammatory monocytes. When atherosclerosis-prone Apoe−/− mice were subjected to chronic stress, accelerated hematopoiesis promoted plaque features associated with vulnerable lesions that cause myocardial infarction and stroke in humans.

PET/MRI of inflammation in myocardial infarction.

OBJECTIVES The aim of this study was to explore post-myocardial infarction (MI) myocardial inflammation. BACKGROUND Innate immune cells are centrally involved in infarct healing and are emerging therapeutic targets in cardiovascular disease; however, clinical tools to assess their presence in tissue are scarce. Furthermore, it is currently not known if the nonischemic remote zone recruits monocytes. METHODS Acute inflammation was followed in mice with coronary ligation by 18-fluorodeoxyglucose ((18)FDG) positron emission tomography/magnetic resonance imaging, fluorescence-activated cell sorting, polymerase chain reaction, and histology. RESULTS Gd-DTPA-enhanced infarcts showed high (18)FDG uptake on day 5 after MI. Cell depletion and isolation data confirmed that this largely reflected inflammation; CD11b(+) cells had 4-fold higher (18)FDG uptake than the infarct tissue from which they were isolated (p < 0.01). Surprisingly, there was considerable monocyte recruitment in the remote myocardium (approximately 10(4)/mg of myocardium, 5.6-fold increase; p < 0.01), a finding mirrored by macrophage infiltration in the remote myocardium of patients with acute MI. Temporal kinetics of cell recruitment were slower than in the infarct, with peak numbers on day 10 after ischemia. Quantitative polymerase chain reaction showed a robust increase of recruiting adhesion molecules and chemokines in the remote myocardium (e.g., 12-fold increase of monocyte chemoattractant protein-1), although levels were always lower than in the infarct. Finally, matrix metalloproteinase activity was significantly increased in noninfarcted myocardium, suggesting that monocyte recruitment to the remote zone may contribute to post-MI dilation. CONCLUSIONS This study shed light on the innate inflammatory response in remote myocardium after MI.

Real-Time Catheter Molecular Sensing of Inflammation in Proteolytically Active Atherosclerosis

Background— To enable intravascular detection of inflammation in atherosclerosis, we developed a near-infrared fluorescence (NIRF) catheter–based strategy to sense cysteine protease activity during vascular catheterization. Methods and Results— The NIRF catheter design was based on a clinical coronary artery guidewire. In phantom studies of NIRF plaques, blood produced only a mild (<30%) attenuation of the fluorescence signal compared with saline, affirming the favorable optical properties of the NIR window. Catheter evaluation in vivo used atherosclerotic rabbits (n=11). Rabbits received an injection of a cysteine protease–activatable NIRF imaging agent (Prosense750; excitation/emission, 750/770 nm) or saline. Catheter pullbacks through the blood-filled iliac artery detected NIRF signals 24 hours after injection of the probe. In the protease agent group, the in vivo peak plaque target-to-background ratio was 558% greater than controls (6.8±1.9 versus 1.3±0.3, mean±SEM; P<0.05). Ex vivo fluorescence reflectance imaging corroborated these results (target-to-background ratio, 10.3±1.8 for agent versus 1.8±0.3 for saline group; P<0.01). In the protease group only, saline flush–modulated NIRF signal profiles further distinguished atheromata from normal segments in vivo (P<0.01). Good correlation between the in vivo and ex vivo plaque target-to-background ratio was present (r=0.82, P<0.01). Histopathological analyses demonstrated strong NIRF signal in plaques only from the protease agent group. NIRF signals colocalized with immunoreactive macrophages and the cysteine protease cathepsin B. Conclusions— An intravascular fluorescence catheter can detect cysteine protease activity in vessels the size of human coronary arteries in real time with an activatable NIRF agent. This strategy could aid in the detection of inflammation and high-risk plaques in small arteries.

Multispectral photoacoustic imaging of fluorochromes in small animals

Fluorochromes have become essential reporter molecules in biological research. We show that the depth-resolved distribution of fluorochromes in small animals can be imaged with 25 fmol sensitivity and 150 microm spatial resolution by means of multispectral photoacoustic imaging. The major advantage of the multispectral approach is the sensitive differentiation of chromophores and fluorochromes of interest based on self-reference measurements, as evidenced in this study by resolving a commonly used fluorochrome (Alexa Fluor 750) in mouse. The suggested method is well suited for enhancing visualization of functional and molecular information in vivo and longitudinally.

Hybrid PET-optical imaging using targeted probes.

Fusion imaging of radionuclide-based molecular (PET) and structural data [x-ray computed tomography (CT)] has been firmly established. Here we show that optical measurements [fluorescence-mediated tomography (FMT)] show exquisite congruence to radionuclide measurements and that information can be seamlessly integrated and visualized. Using biocompatible nanoparticles as a generic platform (containing a 18F isotope and a far red fluorochrome), we show good correlations between FMT and PET in probe concentration (r2 > 0.99) and spatial signal distribution (r2 > 0.85). Using a mouse model of cancer and different imaging probes to measure tumoral proteases, macrophage content and integrin expression simultaneously, we demonstrate the distinct tumoral locations of probes in multiple channels in vivo. The findings also suggest that FMT can serve as a surrogate modality for the screening and development of radionuclide-based imaging agents.

Indocyanine Green Enables Near-Infrared Fluorescence Imaging of Lipid-Rich, Inflamed Atherosclerotic Plaques

Indocyanine green, a clinically approved near-infrared fluorescence imaging agent, rapidly targets atheromas for in vivo detection of lipid-rich, inflammatory plaques. Greenify Your Arteries Many have taken it upon themselves to “go green,” perhaps by turning down the thermostat, swapping out old light bulbs, or even buying a hybrid car. But who would have thought that even your heart doctor can take part in this green initiative? As described by Vinegoni et al., going green may be just what you and your arteries need to detect atherosclerotic plaques residing within them. Indocyanine green (ICG) is a Food and Drug Administration–approved dye for imaging the vascular system at near-infrared (NIR) wavelengths (~800 nm)—wavelengths that boast limited photon absorption by blood and low tissue autofluorescence. ICG is also quickly absorbed by lipid-rich plaques and cells, making it a potentially useful plaque-imaging agent. Vinegoni and colleagues decided to test this hypothesis in rabbit models of atherosclerosis. Lipid-rich, inflamed atheromas were induced in 19 cholesterol-fed rabbits with balloon injury of the aorta. Eight weeks after injury, rabbits received an injection of ICG. Only 45 min later, the animals were killed for fluorescence imaging, which showed strong focal signals in the abdominal aorta and iliac arteries—areas that colocalized with atherosclerotic plaques. Conversely, control animals showed minimal NIR fluorescence signal. The authors then performed in vivo NIR fluorescence imaging of ICG in live animals. Using a clinical-type intravascular guidewire and a previously described “pullback” technique, they were able to sense atheroma in the coronary arteries of five rabbits. The location of these plaques was confirmed by x-ray angiography and intravascular ultrasound. Furthermore, ICG localization to human atheroma was confirmed ex vivo with freshly resected carotid endarterectomy specimens from four patients. Together, these animal and human data suggest direct translation to the clinic and highlight the potential application of ICG as a routine, green screening tool for atherosclerosis. New high-resolution molecular and structural imaging strategies are needed to visualize high-risk plaques that are likely to cause acute myocardial infarction, because current diagnostic methods do not reliably identify at-risk subjects. Although molecular imaging agents are available for low-resolution detection of atherosclerosis in large arteries, a lack of imaging agents coupled to high-resolution modalities has limited molecular imaging of atherosclerosis in the smaller coronary arteries. Here, we have demonstrated that indocyanine green (ICG), a Food and Drug Administration–approved near-infrared fluorescence (NIRF)–emitting compound, targets atheromas within 20 min of injection and provides sufficient signal enhancement for in vivo detection of lipid-rich, inflamed, coronary-sized plaques in atherosclerotic rabbits. In vivo NIRF sensing was achieved with an intravascular wire in the aorta, a vessel of comparable caliber to human coronary arteries. Ex vivo fluorescence reflectance imaging showed high plaque target-to-background ratios in atheroma-bearing rabbits injected with ICG compared to atheroma-bearing rabbits injected with saline. In vitro studies using human macrophages established that ICG preferentially targets lipid-loaded macrophages. In an early clinical study of human atheroma specimens from four patients, we found that ICG colocalized with plaque macrophages and lipids. The atheroma-targeting capability of ICG has the potential to accelerate the clinical development of NIRF molecular imaging of high-risk plaques in humans.

WNT5A/JNK and FGF/MAPK pathways regulate the cellular events shaping the vertebrate limb bud.

BACKGROUND The vertebrate limb is a classical model for understanding patterning of three-dimensional structures during embryonic development. Although decades of research have elucidated the tissue and molecular interactions within the limb bud required for patterning and morphogenesis of the limb, the cellular and molecular events that shape the limb bud itself have remained largely unknown. RESULTS We show that the mesenchymal cells of the early limb bud are not disorganized within the ectoderm as previously thought but are instead highly organized and polarized. Using time-lapse video microscopy, we demonstrate that cells move and divide according to this orientation. The combination of oriented cell divisions and movements drives the proximal-distal elongation of the limb bud necessary to set the stage for subsequent morphogenesis. These cellular events are regulated by the combined activities of the WNT and FGF pathways. We show that WNT5A/JNK is necessary for the proper orientation of cell movements and cell division. In contrast, the FGF/MAPK signaling pathway, emanating from the apical ectodermal ridge, does not regulate cell orientation in the limb bud but instead establishes a gradient of cell velocity enabling continuous rearrangement of the cells at the distal tip of the limb. CONCLUSIONS Together, these data shed light on the cellular basis of vertebrate limb bud morphogenesis and uncover new layers to the sequential signaling pathways acting during vertebrate limb development.

Real-time in vivo imaging of the beating mouse heart at microscopic resolution

Real-time imaging of moving organs and tissues at microscopic resolutions represents a major challenge in studying the complex biology of live animals. Here we present a technique based on a novel stabilizer setup combined with a gating acquisition algorithm for the imaging of a beating murine heart at the single-cell level. The method allows serial in vivo fluorescence imaging of the beating heart in live mice in both confocal and nonlinear modes over the course of several hours. We demonstrate the utility of this technique for in vivo optical sectioning and dual-channel time-lapse fluorescence imaging of cardiac ischaemia. The generic method could be adapted to other moving organs and thus broadly facilitate in vivo microscopic investigations.

In vivo imaging of Drosophila melanogaster pupae with mesoscopic fluorescence tomography

We report a technique for fluorescence tomography that operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The method uses multi-projection illumination and photon transport description in opaque tissues. We demonstrate whole-body three-dimensional visualization of the morphogenesis of GFP-expressing salivary glands and wing imaginal discs in living Drosophila melanogaster pupae in vivo and over time.