David E. Sosnovik

BACKGROUND: Noninvasive imaging of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1) may identify early stages of inflammation in atherosclerosis. We hypothesized that a novel, second-generation VCAM-1-targeted agent with enhanced affinity had sufficient sensitivity to enable real-time detection of VCAM-1 expression in experimental atherosclerosis in vivo, to quantify pharmacotherapy-induced reductions in VCAM-1 expression, and to identify activated cells in human plaques. METHODS AND RESULTS: In vivo phage display in apolipoprotein E-deficient mice identified a linear peptide affinity ligand, VHPKQHR, homologous to very late antigen-4, a known ligand for VCAM-1. This peptide was developed into a multivalent agent detectable by MRI and optical imaging (denoted VINP-28 for VCAM-1 internalizing nanoparticle 28, with 20 times higher affinity than previously reported for VNP). In vitro, VINP-28 targeted all cell types expressing VCAM-1. In vivo, MRI and optical imaging in apolipoprotein E-deficient mice (n=28) after injection with VINP-28 or saline revealed signal enhancement in the aortic root of mice receiving VINP-28 (P<0.05). VINP-28 colocalized with endothelial cells and other VCAM-1-expressing cells, eg, macrophages, and was spatially distinct compared with untargeted control nanoparticles. Atheromata of atorvastatin-treated mice showed reduced VINP-28 deposition and VCAM-1 expression. VINP-28 enhanced early lesions in juvenile mice and resected human carotid artery plaques. CONCLUSIONS: VINP-28 allows noninvasive imaging of VCAM-1-expressing endothelial cells and macrophages in atherosclerosis and spatial monitoring of anti-VCAM-1 pharmacotherapy in vivo and identifies inflammatory cells in human atheromata. This clinically translatable agent could noninvasively detect inflammation in early, subclinical 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.

Macrophages populate the healthy myocardium and, depending on their phenotype, may contribute to tissue homeostasis or disease. Their origin and role in diastolic dysfunction, a hallmark of cardiac aging and heart failure with preserved ejection fraction, remain unclear. Here we show that cardiac macrophages expand in humans and mice with diastolic dysfunction, which in mice was induced by either hypertension or advanced age. A higher murine myocardial macrophage density results from monocyte recruitment and increased hematopoiesis in bone marrow and spleen. In humans, we observed a parallel constellation of hematopoietic activation: circulating myeloid cells are more frequent, and splenic 18F-FDG PET/CT imaging signal correlates with echocardiographic indices of diastolic dysfunction. While diastolic dysfunction develops, cardiac macrophages produce IL-10, activate fibroblasts, and stimulate collagen deposition, leading to impaired myocardial relaxation and increased myocardial stiffness. Deletion of IL-10 in macrophages improves diastolic function. These data imply expansion and phenotypic changes of cardiac macrophages as therapeutic targets for cardiac fibrosis leading to diastolic dysfunction.

BACKGROUND: Visualizing early changes in valvular cell functions in vivo may predict the future risk and identify therapeutic targets for prevention of aortic valve stenosis. METHODS AND RESULTS: To test the hypotheses that (1) aortic stenosis shares a similar pathogenesis to atherosclerosis and (2) molecular imaging can detect early changes in aortic valve disease, we used in vivo a panel of near-infrared fluorescence imaging agents to map endothelial cells, macrophages, proteolysis, and osteogenesis in aortic valves of hypercholesterolemic apolipoprotein E-deficient mice (30 weeks old, n=30). Apolipoprotein E-deficient mice with no probe injection (n=10) and wild-type mice (n=10) served as controls. Valves of apolipoprotein E-deficient mice contained macrophages, were thicker than wild-type mice (P<0.001), and showed early dysfunction detected by MRI in vivo. Fluorescence imaging detected uptake of macrophage-targeted magnetofluorescent nanoparticles (24 hours after injection) in apolipoprotein E-deficient valves, which was negligible in controls (P<0.01). Valvular macrophages showed proteolytic activity visualized by protease-activatable near-infrared fluorescence probes. Ex vivo magnetic resonance imaging enhanced with vascular cell adhesion molecule-1-targeted nanoparticles detected endothelial activation in valve commissures, the regions of highest mechanical stress. Osteogenic near-infrared fluorescence signals colocalized with alkaline phosphatase activity and expression of osteopontin, osteocalcin, Runx2/Cbfa1, Osterix, and Notch1 despite no evidence of calcium deposits, which suggests ongoing active processes of osteogenesis in inflamed valves. Notably, the aortic wall contained advanced calcification. Quantitative image analysis correlated near-infrared fluorescence signals with immunoreactive vascular cell adhesion molecule-1, macrophages, and cathepsin-B (P<0.001). CONCLUSIONS: Molecular imaging can detect in vivo the key cellular events in early aortic valve disease, including endothelial cell and macrophage activation, proteolytic activity, and osteogenesis.