Eric A. Osborn

A Mechanistic Model of the Actin Cycle

We have derived a broad, deterministic model of the steady-state actin cycle that includes its major regulatory mechanisms. Ours is the first model to solve the complete nucleotide profile within filaments, a feature that determines the dynamics and geometry of actin networks at the leading edges of motile cells, and one that has challenged investigators developing models to interpret steady-state experiments. We arrived at the nucleotide profile through analytic and numerical approaches that completely agree. Our model reproduces behaviors seen in numerous experiments with purified proteins, but allows a detailed inspection of the concentrations and fluxes that might exist in these experiments. These inspections provide new insight into the mechanisms that determine the rate of actin filament treadmilling. Specifically, we find that mechanisms for enhancing Pi release from the ADP.Pi intermediate on filaments, for increasing the off rate of ADP-bound subunits at pointed ends, and the multiple, simultaneous functions of profilin, make unique and essential contributions to increased treadmilling. In combination, these mechanisms have a theoretical capacity to increase treadmilling to levels limited only by the amount of available actin. This limitation arises because as the cycle becomes more dynamic, it tends toward the unpolymerized state.

The advancing clinical impact of molecular imaging in CVD.

Molecular imaging seeks to unravel critical molecular and cellular events in living subjects by providing complementary biological information to current structural clinical imaging modalities. In recent years, molecular imaging efforts have marched forward into the clinical cardiovascular arena, and are now actively illuminating new biology in a broad range of conditions, including atherosclerosis, myocardial infarction, thrombosis, vasculitis, aneurysm, cardiomyopathy, and valvular disease. Development of novel molecular imaging reporters is occurring for many clinical cardiovascular imaging modalities (positron emission tomography, single-photon emission computed tomography, magnetic resonance imaging), as well as in translational platforms such as intravascular fluorescence imaging. The ability to image, track, and quantify molecular biomarkers in organs not routinely amenable to biopsy (e.g., the heart and vasculature) open new clinical opportunities to tailor therapeutics based on a cardiovascular disease molecular profile. In addition, molecular imaging is playing an increasing role in atherosclerosis drug development in phase II clinical trials. Here, we present state-of-the-art clinical cardiovascular molecular imaging strategies, and explore promising translational approaches positioned for clinical testing in the near term.

The year in molecular imaging.

Molecular imaging aims to enable personalized medicine via imaging-specific molecular and cellular targets that are relevant to the diagnosis and treatment of disease. By providing in vivo readouts of biological detail, molecular imaging complements traditional anatomical imaging modalities to allow: 1) visualization of important disease-modulating molecules and cells in vivo; 2) serial investigations to image evolutionary changes in disease attributes; and 3) evaluation of the in vivo molecular effects of biotherapeutics. The added information garnered by molecular imaging can improve risk assessment and prognosticative studies, this is of particular benefit in the management of cardiovascular disease (CVD).

Advances in Molecular Imaging of Atherosclerotic Vascular Disease

Purpose of review Molecular imaging aims to illuminate vital molecular and cellular aspects of disease in vivo, and is rapidly translating into the clinical arena. Advantages of this field include enabling serial biological studies in living subjects, assessment of pharmaceutical efficacy, and in-vivo characterization of clinical diseases. Here we present recent exciting advances in molecular imaging of atherosclerotic vascular disease. Recent findings Atherosclerosis molecular imaging approaches are now available for magnetic resonance, nuclear, computed tomography, ultrasound, and near-infrared fluorescence imaging. Advances in agent synthesis and detection technology are now enabling in-vivo imaging of endothelial cell activation, macrophages, cellular metabolism, protease activity, apoptosis, and osteogenic activity. Several agents show clinical utility for the detection of high-risk plaques. Summary Molecular imaging is actively unraveling the biological basis of atherosclerosis in living subjects. In the near-term, molecular imaging will play an important role in assessing novel atherosclerosis pharmacotherapies in clinical trials. Longer term, molecular imaging should enable accurate identification of high-risk plaques responsible for myocardial infarction, stroke, and ischemic limbs.

Imaging atherosclerosis and risk of plaque rupture.

Atherosclerosis imaging strategies can delineate characteristics of plaques at risk of rupture and thrombosis. Structural plaque imaging identifies high-risk plaque features, including lipid pools, thin fibrous caps, and intraplaque hemorrhage. New molecular imaging techniques complement structural imaging approaches by illuminating important features of plaque biology, with a prominent focus on detecting inflammation as a high-risk phenotype. As we unravel the molecular and structural characteristics underlying thrombosis-prone plaques, there is significant promise for eventual early identification and prediction of atherosclerotic plaque complications before they occur. Here we focus on recent imaging insights into high-risk arterial plaques, the etiologic agent of acute myocardial infarction, stroke, and sudden cardiac death.

Impact of papillary and trabecular muscles on quantitative analyses of cardiac function in hypertrophic cardiomyopathy.

To examine the impact of cardiovascular magnetic resonance (CMR) partitioning methods on volumetric analysis in hypertrophic cardiomyopathy (HCM) patients. The standard CMR method for partitioning ventricular myocardium from ventricular cavity includes the myocardial papillary and trabecular muscles in the cavity volume. This approach may misrepresent ventricular mass and volume in patients with HCM due to large papillary muscles and extensive trabeculations.

Imaging inflammation and neovascularization in atherosclerosis: clinical and translational molecular and structural imaging targets.

Purpose of review The purpose of this study is to showcase advances in molecular imaging of atheroma biology in living individuals. Recent findings 18F-fluorodeoxyglucose (FDG) PET/computed tomography (CT) continues to be the predominant molecular imaging approach for clinical applications, particularly in the large arterial beds. Recently, there has been significant progress in imaging of neovascularization and inflammation to delineate high-risk atheroma and to evaluate drug efficacy. In addition, new hardware detection technology and imaging agents are enabling in-vivo imaging of new targets on diverse imaging platforms. Summary In this review, we present recent exciting developments in molecular and structural imaging of atherosclerotic plaque inflammation and neovascularization. Building upon prior studies, these advances develop key technology that will play an important role in propelling new diagnostic and therapeutic strategies identifying high-risk plaque phenotypes and assessing new plaque stabilization therapies in clinical trials.

Quantitative intravascular biological fluorescence-ultrasound imaging of coronary and peripheral arteries in vivo.

Aims (i) to evaluate a novel hybrid near-infrared fluorescence-intravascular ultrasound (NIRF-IVUS) system in coronary and peripheral swine arteries in vivo; (ii) to assess simultaneous quantitative biological and morphological aspects of arterial disease. Methods and results Two 9F/15MHz peripheral and 4.5F/40MHz coronary near-infrared fluorescence (NIRF)-IVUS catheters were engineered to enable accurate co-registrtation of biological and morphological readings simultaneously in vivo. A correction algorithm utilizing IVUS information was developed to account for the distance-related fluorescence attenuation due to through-blood imaging. Corrected NIRF (cNIRF)-IVUS was applied for in vivo imaging of angioplasty-induced vascular injury in swine peripheral arteries and experimental fibrin deposition on coronary artery stents, and of atheroma in a rabbit aorta, revealing feasibility to intravascularly assay plaque structure and inflammation. The addition of ICG-enhanced NIRF assessment improved the detection of angioplasty-induced endothelial damage compared to standalone IVUS. In addition, NIRF detection of coronary stent fibrin by in vivo cNIRF-IVUS imaging illuminated stent pathobiology that was concealed on standalone IVUS. Fluorescence reflectance imaging and microscopy of resected tissues corroborated the in vivo findings. Conclusions Integrated cNIRF-IVUS enables simultaneous co-registered through-blood imaging of disease related morphological and biological alterations in coronary and peripheral arteries in vivo. Clinical translation of cNIRF-IVUS may significantly enhance knowledge of arterial pathobiology, leading to improvements in clinical diagnosis and prognosis, and helps to guide the development of new therapeutic approaches for arterial diseases.

Metabolic and Molecular Imaging of Atherosclerosis and Venous Thromboembolism

Metabolic and molecular imaging continues to advance our understanding of vascular disease pathophysiology. At present, 18F-FDG PET imaging is the most widely used clinical tool for metabolic and molecular imaging of atherosclerosis. However, novel nuclear tracers and intravascular optical near-infrared fluorescence imaging catheters are emerging to assess new biologic targets in vivo and in coronary arteries. This review highlights current metabolic and molecular imaging clinical and near-clinical applications within atherosclerosis and venous thromboembolism, and explores the potential for metabolic and molecular imaging to affect patient-level risk prediction and disease treatment.

Simultaneous subacute coronary artery stent thrombosis in a carrier of two CYP2C19 loss–of function polymorphisms (*2/*3)

Clopidogrel, a second generation thienopyridine and a selective inhibitor of the platelet adenosine diphosphate (ADP) P2Y12 receptor, is the historical standard anti-platelet treatment added to aspirin following percutaneous coronary intervention (PCI) in order to reduce the risk of stent thrombotic complications. Clopidogrel is an inactive pro-drug that requires hepatic metabolism by cytochrome P450 2C19 (CYP2C19) into its active form in order to inhibit platelet aggregation. Recently, substantial subpopulations have been recognized that exhibit an inadequate response to clopidogrel leading to insufficient antiplatelet effects [1]. Individuals carrying at least one loss-of function allele (either *2 or *3) of the CYP2C19 gene demonstrate reduced active clopidogrel metabolites and suppressed antiplatelet activity [2]. Such patients have been identified to carry a significantly higher risk of stent thrombosis, a major adverse event that is associated with >60% risk of death or myocardial [3]. Of note, the stent thrombosis risk in patients carrying a CYP2C19 loss-of function allele appears to be similarly elevated across stable and unstable patient populations, regardless of elective or urgent PCI, or treatment with bare metal or drug-eluting stents [4,5]. A 61-year-old dyslipidemic, non-diabetic male with stable angina presented for elective cardiac catheterization. Coronary angiography showed complex multivessel disease consisting of a proximal RCA chronic total occlusion (CTO) with collateral flow from conus branch of RCA and a diagonal branch (Fig. 1a, white arrow), and severe proximal LAD stenosis (Fig. 1d, white arrow). Based on the angiographic results, we first treated the RCA-CTO lesion by deploying three overlapping drug eluting stents (DES) (Promus Premier, Boston Scientific)with proximal to distal sizes of 3.5 × 24 mm, 3.0 × 24 mm and 2.5 × 28 mm. The mid stent and proximal stent were then post-dilated with 3.0 mm and 3.75 mm diameter non-compliant balloons, respectively. Post-PCI intravascular ultrasound (IVUS) imaging confirmed good stent apposition with a sufficient stent/lumen diameter ratio in comparison to the proximal and distal reference vessel segments (Fig. 2, upper left panel). Four days later, staged PCI of the proximal LAD lesion was performed with implantation of a 3.0 × 23 mm DES (Xience Alpine, Abott Vascular) with post-dilatation using a 3.0 mm diameter noncompliant balloon. Similar to the RCA results, good stent apposition was shown by IVUS (Fig. 2, upper right panel). Both procedures were successful with no complications (Fig. 1b and e, white arrows). The patient was continued uninterrupted on treatment with dual-antiplatelet therapy consisting of aspirin 100 mg and clopidogrel 75 mg daily, which had been started 30 days prior to the index PCI without adverse effects. He was discharged to home without angina and in stable condition. Fig. 1 Coronary angiography at initial presentation for elective coronary stent placement, and following representation with ST elevation myocardial infarction due to subacute stent thrombosis. Before (a and d) and after (b and e) initial percutaneous coronary ... Fig. 2 Intravascular ultrasound (IVUS) after initial PCI and intracoronary optical coherence tomography (OCT) imaging of multivessel stent thrombosis. Upper panels: IVUS demonstrated good stent expansion and apposition to the vessel wall. Lower panels: Massive ... The day following discharge, the patient developed sudden-onset severe chest pain and diaphoresis at rest. He presented within 2 hours of symptom onset in hemodynamically stable condition (blood pressure 157/92 mmHg, heart rate 83 beats per minute). The electrocardiogram revealed new ST elevations (>2 mm) with inverted T waves in V1 through V3, and stat echocardiography showed severe hypokinesis of the mid-distal-apical anterior wall. Blood tests returned with leukocytosis (12,500 cells/µl). Emergent coronary angiography for acute anterior ST elevation myocardial infarction (STEMI) was performed after administration of unfractionated heparin 5000 units, supported by an intra-aortic balloon pump (IABP), that showed thrombotic occlusion of both the RCA (Fig. 1c, black arrowhead) and LAD stents (Fig. 1f, black arrowhead). Given the clopidogrel failure, a loading dose of prasugrel (20 mg) was orally administered. GPIIb/IIIa inhibitor was not used. We then proceeded to restore flow in the LAD by passing a coronary guidewire through the stent and performing manual aspiration thrombectomy followed by subsequent percutaneous transluminal angioplasty (PTCA) with a 3.0 mm diameter balloon within the stent. Intracoronary optical coherence tomography demonstrated massive thrombus within the LAD stent despite excellent stent strut apposition throughout (Fig. 2, lower right panel). After achieving TIMI3 flow in the LAD (Fig. 1f, right bottom corner), we moved to treat the RCA with aspiration and PTCA in similar fashion resulting in return of TIMI3 flow in RCA (Fig. 1c, right bottom corner). Intracoronary OCT imaging in the RCA also showed massive thrombus in the distal stent (Fig. 2, lower left panel). The patient was discharged day 16 post-PCI on a medical regimen of an αβ-blocker (carvedilol, 5 mg once daily), an angiotensin converting enzyme inhibitor (Perindopril erbumine, 2 mg once daily) and dual anti-platelet therapy with prasugrel 3.75 mg and aspirin 100 mg once daily. At four months follow-up, the patient has been doing well without any evidence of recurrent myocardial ischemia. Genotyping of CYP2C19 gene polymorphyrisms revealed that the patient carries two reduced-function alleles (*2/*3) of the CYP2C19 gene, and he was thus defined as a poor clopidogrel metabolizer. In this case, the patient carried two non-functioning polymorphisms (*2/*3) of the CYP2C19 gene, a deficit known to result in critically reduced clopidogrel antiplatelet activity, that precipitated simultaneous subacute stent thrombosis of two major epicardial coronary arteries. The frequency for the most common loss-of-function variant CYP2C19*2 is <15% in Caucasians and Africans, but affects up to 35% of those of Asian descent; in comparison, CYP2C19*3 is the second-most common genetic mutation, occurring in fewer than 10% of Asians [1]. Therefore, especially in Asian population, the number of patients defined as a “poor clopdigrel metabolizer” carrying the CYP2C19*2/*2 or *2/*3 polymorphisms may be higher than previously postulated. To date, cardiovascular society guidelines recommend against routine gene testing for CYP2C19 polymorphisms for patients treated with clopidogrel after PCI [6,7], primarily because a priori knowledge of CYP2C19 genetic mutations or the results of platelet function testing have not demonstrated an improvement in outcomes [8]. However, while clinical trials may be equivocal, in certain high-risk individual patients, as presented in this case of an Asian patient undergoing elective complex PCI and suffering simultaneous subacute stent thromobisis, it might be reasonable to perform risk estimation using genetic screening or platelet reactivity testing before coronary stenting. In carriers of *2 and/or *3 alleles of CYP2C19 that have less or no CYP2C19 enzymatic activity (intermediate and poor metabolizers) on standard doses of clopidogrel (75 mg daily), two alternative strategies have been considered: 1) increasing the clopidogrel dose by two- to four-fold [9], which more potently decreases platelet activity but has not been proven to reduce cardiovascular events [10], or 2) the preferred approach of switching to alternative P2Y12 inhibitors, such as the third generation thienopyridines prasugrel and ticagrelor that are less influenced by polymorphisms of the CYP2C19 gene. For prasugrel, carriers of the CYP2C19*2 allele are known to produce equivalent concentrations of active prasugrel metabolite and achieve a similar antiplatelet effect to those that do not carry this allele [5]. In this patient, clopidogrel was replaced by prasugrel with no clinical or adverse events observed in early follow up. Based on the clinical course of this case and related evidence, we suggest that when elective PCI is planned in certain patients at high risk for CYP2C19 polymorphisms, such as those of Asian decent, that are also at high risk for critical stent thrombosis due to complex multivessel or left main coronary artery disease, that physicians consider performing genetic testing for CYP2C19 polymorphisms or platelet function testing to evaluate the potential risk of using clopidogrel, or to simply use a third generation thienopyridine primarily.