Farouc A. Jaffer

Noninvasive Vascular Cell Adhesion Molecule-1 Imaging Identifies Inflammatory Activation of Cells in Atherosclerosis

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.

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.

Multimodality Molecular Imaging Identifies Proteolytic and Osteogenic Activities in Early Aortic Valve Disease

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.

Monocyte accumulation in mouse atherogenesis is progressive and proportional to extent of disease

Monocytes participate importantly in the pathogenesis of atherosclerosis, but their spatial and temporal recruitment from circulation remains uncertain. This study tests the hypothesis that monocyte accumulation in atheroma correlates with the extent of disease by using a sensitive and simple quantitative assay that allows tracking of highly enriched populations of blood monocytes. A two-step isolation method yielded viable and functionally intact highly enriched peripheral blood monocyte populations (>90%). Recipient mice received syngeneic monocytes labeled in two ways: by transgenically expressing EGFP or with a radioactive tracer [111In]oxine. After 5 days, more labeled cells accumulated in the aorta, principally the aortic root and ascending aorta, of 10-wk-old ApoE−/− compared with 10-wk-old C57BL/6 mice (223 ± 3 vs. 87 ± 22 cells per aorta). Considerably more monocytes accumulated in 20-wk-old ApoE−/− mice on either chow (314 ± 41 cells) or high-cholesterol diet (395 ± 53 cells). Fifty-week-old ApoE−/− mice accumulated even more monocytes in the aortic root, ascending aorta, and thoracic aorta after both chow (503 ± 67 cells) or high-cholesterol diet (648 ± 81 cells). Labeled monocyte content in the aorta consistently correlated with lesion surface area. These data indicate that monocytes accumulate continuously during atheroma formation, accumulation increases in proportion to lesion size, and recruitment is augmented with hypercholesterolemia. These results provide insights into mechanisms of atherogenesis and have implications for the duration of therapies directed at leukocyte recruitment.

Arterial and Aortic Valve Calcification Abolished by Elastolytic Cathepsin S Deficiency in Chronic Renal Disease

Background— Clinical studies have demonstrated that 50% of individuals with chronic renal disease (CRD) die of cardiovascular causes, including advanced calcific arterial and valvular disease; however, the mechanisms of accelerated calcification in CRD remain obscure, and no therapies can prevent disease progression. We recently demonstrated in vivo that inflammation triggers cardiovascular calcification. In vitro evidence also indicates that elastin degradation products may promote osteogenesis. Here, we used genetically modified mice and molecular imaging to test the hypothesis in vivo that cathepsin S (catS), a potent elastolytic proteinase, accelerates calcification in atherosclerotic mice with CRD induced by 5/6 nephrectomy. Methods and Results— Apolipoprotein-deficient (apoE−/−)/catS+/+ (n=24) and apoE−/−/catS−/− (n=24) mice were assigned to CRD and control groups. CRD mice had significantly higher serum phosphate, creatinine, and cystatin C levels than those without CRD. To visualize catS activity and osteogenesis in vivo, we coadministered catS-activatable and calcification-targeted molecular imaging agents 10 weeks after nephrectomy. Imaging coregistered increased catS and osteogenic activities in the CRD apoE−/−/catS+/+ cohort, whereas CRD apoE−/−/catS−/− mice exhibited less calcification. Quantitative histology demonstrated greater catS-associated elastin fragmentation and calcification in CRD apoE−/−/catS+/+ than CRD apoE−/−/catS−/− aortas and aortic valves. Notably, catS deletion did not cause compensatory increases in RNA levels of other elastolytic cathepsins or matrix metalloproteinases. Elastin peptide and recombinant catS significantly increased calcification in smooth muscle cells in vitro, a process further amplified in phosphate-enriched culture medium. Conclusions— The present study provides direct in vivo evidence that catS-induced elastolysis accelerates arterial and aortic valve calcification in CRD, providing new insight into the pathophysiology of cardiovascular calcification.

Optical Visualization of Cathepsin K Activity in Atherosclerosis With a Novel, Protease-Activatable Fluorescence Sensor

Background— Cathepsin K (CatK), a potent elastinolytic and collagenolytic cysteine protease, likely participates in the evolution and destabilization of atherosclerotic plaques. To assess better the biology of CatK activity in vivo, we developed a novel near-infrared fluorescence (NIRF) probe for imaging of CatK and evaluated it in mouse and human atherosclerosis. Methods and Results— The NIRF imaging agent consists of the CatK peptide substrate GHPGGPQGKC-NH2 linked to an activatable fluorogenic polymer. In vitro, CatK produced a 2- to 14-fold activation of the agent over other cysteine and matrix metalloproteinases (P<0.0001), as well as a >8-fold activation over a control imaging agent (P<0.001). Optical imaging of atheroma revealed >100% NIRF signal increases in apolipoprotein E−/− mice in vivo (n=13; P<0.05, CatK imaging agent versus control agent) and in human carotid endarterectomy specimens ex vivo (n=14; P<0.05). Fluorescence microscopy of plaque sections demonstrated that enzymatically active CatK (positive NIRF signal) localized primarily in the vicinity of CatK-positive macrophages. Augmented NIRF signal (reflecting CatK activity) colocalized with disrupted elastin fibers within the media underlying plaques. Conclusions— Use of this novel protease-activatable NIRF agent for optical imaging in vivo demonstrated preferential localization of enzymatically active CatK to macrophages, consistent with their known greater elastinolytic capabilities compared with smooth muscle cells. Augmented CatK proteolysis in atheromata further links CatK to vascular remodeling and plaque vulnerability.

Intra-arterial catheter for simultaneous microstructural and molecular imaging in vivo

Advancing understanding of human coronary artery disease requires new methods that can be used in patients for studying atherosclerotic plaque microstructure in relation to the molecular mechanisms that underlie its initiation, progression and clinical complications, including myocardial infarction and sudden cardiac death. Here we report a dual-modality intra-arterial catheter for simultaneous microstructural and molecular imaging in vivo using a combination of optical frequency domain imaging (OFDI) and near-infrared fluorescence (NIRF) imaging. By providing simultaneous molecular information in the context of the surrounding tissue microstructure, this new catheter could provide new opportunities for investigating coronary atherosclerosis and stent healing and for identifying high-risk biological and structural coronary arterial plaques in vivo.

Age and Sex Distribution of Subclinical Aortic Atherosclerosis A Magnetic Resonance Imaging Examination of the Framingham Heart Study

Autopsy data demonstrate a correlation between subclinical aortic atherosclerosis and cardiovascular disease. Therefore, noninvasive cardiovascular magnetic resonance (CMR) of subclinical atherosclerosis may provide a novel measure of cardiovascular risk, but it has not been applied to an asymptomatic population-based cohort to establish age- and sex-specific normative data. Participants in the Framingham Heart Study offspring cohort who were free of clinically apparent coronary disease were randomly sampled from strata of sex, quartiles of age, and quintiles of Framingham Coronary Risk Score. Subjects (n=318, aged 60±9 years, range 36 to 78 years, 51% women) underwent ECG-gated T2-weighted black-blood thoracoabdominal aortic CMR scanning. CMR evidence of aortic atherosclerosis was noted in 38% of the women and 41% of the men. Plaque prevalence and all measures of plaque burden increased with age group and were greater in the abdomen than in the thorax for both sexes and across all age groups. In addition, the Framingham Coronary Risk Score was significantly correlated with all plaque prevalence and burden measures for women but only for men after age adjustment. These noninvasive CMR data extend the prior autopsy-based prevalence estimates of subclinical atherosclerosis and may help to lay the foundation for future studies of risk stratification and treatment of affected individuals.

Molecular Imaging of Cardiovascular Disease

Molecular imaging aims at sensing specific molecular targets, fundamental biological processes, and certain cell types in living subjects. An integrative discipline rooted in the biological, chemical, and imaging sciences, molecular imaging has broad applications in biology and drug discovery1–5 and increasingly within cardiovascular disease.6–12 Before discussing key factors spurring the growth of this field, we first briefly review 2 essential components of this technology: imaging agents and imaging hardware. Molecular imaging requires highly sensitive and specific imaging agents. Such agents incorporate 2 key factors: (1) a signal detection compound and the corresponding imaging hardware platform and (2) an affinity ligand that recognizes the intended molecular or cellular target. Favorable targets include those with established biological and clinical importance in a disease of interest, as well as targets with inherent signal amplification potential such as internalizing receptors or enzymes. Inaccessible and low-abundance targets (DNA, RNA, sparsely expressed proteins) present greater challenges, particularly in a noninvasive, clinical setting. Signal detection compounds include radioisotopes for positron emission tomography (PET) and single-photon-emission computed tomography (SPECT) imaging, paramagnetic (gadolinium)/superparamagnetic (iron oxide) agents for magnetic resonance imaging (MRI), fluorochromes for near-infrared fluorescence imaging, and microbubbles for ultrasound imaging. Certain agents can exhibit unique physical changes favorable for signal amplification when spaced close together (eg, quenching of fluorochromes13–18 or augmented relaxivity of magnetic substrates19–21). These tags can form the basis of imaging agents with inherent chemical amplification capabilities. Amplification strategies generally enable higher target-to-background ratios, a key strategy for developing sufficiently sensitive agents for clinical use. Affinity ligands confer molecular or cellular specificity for the target of interest. The application of novel ligand screening methods, emerging new chemistries for conjugation and signal amplification, and nanotechnology have fostered substantial growth in ligand development. From a clinical perspective, qualities of an ideal ligand include (1) …

Seeing Within: Molecular Imaging of the Cardiovascular System

Abstract— Molecular imaging is a rapidly evolving discipline with the goal of developing tools to display and quantify molecular and cellular targets in vivo. The heart of this field is based on the rational design and screening of targeted and activatable imaging reporter agents to sense fundamental processes of biology. Parallel advances in small animal imaging systems and in agent synthesis have allowed molecular imaging applications to extend into the in vivo arena. These advances have permitted, for example, in vivo sensing of inflammation, apoptosis, cell trafficking, and gene expression. In this review, we first review core principles of molecular imaging with an emphasis on smart, activatable agent technology. We then discuss applications of state-of-the-art molecular probes to interrogate important aspects of cardiovascular biology, with a focus on atherosclerosis, thrombosis, and heart failure. In the ensuing years, we anticipate that fundamental aspects of cardiovascular biology will be detectable in vivo, and that promising molecular imaging agents will be translated into the clinical arena to guide diagnosis and therapy of human cardiovascular illness.