Coronary computed tomographic (CT) angiography is a noninvasive test that enables direct visualization of coronary artery disease (CAD) and correlates favorably with invasive coronary angiography (ICA) for measures of stenosis severity.¹ However, CT cannot determine the hemodynamic significance of CAD, and even among CT-identified obstructive stenoses confirmed by ICA, fewer than half are ischemia-causing.^2,3 These findings underscore an unreliable relationship of stenosis severity to ischemia and have raised concerns that use of CT may precipitate unnecessary ICA and coronary revascularization for patients who do not have ischemia.^4,5

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 ( R ² =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.

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— 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.

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 [ ¹¹¹ In]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.

Over the past 2 decades, chronic total occlusion (CTO) percutaneous coronary intervention has developed into its own subspecialty of interventional cardiology. Dedicated terminology, techniques, devices, courses, and training programs have enabled progressive advancements. However, only a few randomized trials have been performed to evaluate the safety and efficacy of CTO percutaneous coronary intervention. Moreover, several published observational studies have shown conflicting data. Part of the paucity of clinical data stems from the fact that prior studies have been suboptimally designed and performed. The absence of standardized end points and the discrepancy in definitions also prevent consistency and uniform interpretability of reported results in CTO intervention. To standardize the field, we therefore assembled a broad consortium comprising academicians, practicing physicians, researchers, medical society representatives, and regulators (US Food and Drug Administration) to develop methods, end points, biomarkers, parameters, data, materials, processes, procedures, evaluations, tools, and techniques for CTO interventions. This article summarizes the effort and is organized into 3 sections: key elements and procedural definitions, end point definitions, and clinical trial design principles. The Chronic Total Occlusion Academic Research Consortium is a first step toward improved comparability and interpretability of study results, supplying an increasingly growing body of CTO percutaneous coronary intervention evidence.