Faculty Bibliography

Angiogenesis in breast cancer helps fulfill the metabolic demands of the progressing tumor and plays a critical role in tumor metastasis. Therefore, various imaging modalities have been used to characterize tumor angiogenesis. While micro-CT (μCT) is a powerful tool for analyzing the tumor microvascular architecture at micron-scale resolution, magnetic resonance imaging (MRI) with its sub-millimeter resolution is useful for obtaining in vivo vascular data (e.g. tumor blood volume and vessel size index). However, integration of these microscopic and macroscopic angiogenesis data across spatial resolutions remains challenging. Here we demonstrate the feasibility of 'multiscale' angiogenesis imaging in a human breast cancer model, wherein we bridge the resolution gap between ex vivo μCT and in vivo MRI using intermediate resolution ex vivo MR microscopy (μMRI). To achieve this integration, we developed suitable vessel segmentation techniques for the ex vivo imaging data and co-registered the vascular data from all three imaging modalities. We showcase two applications of this multiscale, multi-modality imaging approach: (1) creation of co-registered maps of vascular volume from three independent imaging modalities, and (2) visualization of differences in tumor vasculature between viable and necrotic tumor regions by integrating μCT vascular data with tumor cellularity data obtained using diffusion-weighted MRI. Collectively, these results demonstrate the utility of 'mesoscopic' resolution μMRI for integrating macroscopic in vivo MRI data and microscopic μCT data. Although focused on the breast tumor xenograft vasculature, our imaging platform could be extended to include additional data types for a detailed characterization of the tumor microenvironment and computational systems biology applications.

Atrial fibrillation (AF), the most common cardiac arrhythmia, is a significant cause of embolic stroke. Although systemic anticoagulation is the primary strategy for preventing the thromboembolic complications of AF, anticoagulants carry major bleeding risks, and many patients have contraindications to their use. Because thromboembolism typically arises from a clot in the left atrial appendage (LAA), local therapeutic alternatives to systemic anticoagulation involving surgical or percutaneous exclusion of the LAA have been developed. Surgical exclusion of the LAA is typically performed only as an adjunct to other cardiac surgeries, thus limiting the number of eligible patients. Furthermore, surgical exclusion of the LAA is frequently incomplete, and thromboembolism may still occur. Percutaneous LAA exclusion includes two approaches: transseptal delivery of an occlusion device to the LAA and epicardial suture ligation of the LAA, the LARIAT procedure. In the LARIAT procedure, a pretied snare is placed around the epicardial surface of the LAA orifice via pericardial access. Proper snare placement is achieved with epicardial and endocardial magnet-tipped guidewires. The endocardial wire is advanced transvenously to the LAA apex after transseptal puncture. The epicardial wire, introduced into the pericardial space, achieves end-to-end union with the endocardial wire at the LAA apex. The snare is then placed over the LAA, tightened, and sutured. On the basis of early clinical experience, the LARIAT procedure has a high success rate of LAA exclusion with low risk for complications. The authors describe the indispensable role of real-time transesophageal echocardiography in the guidance of LAA epicardial suture ligation with the LARIAT device.

BACKGROUND: Triggered activity in cardiac Purkinje cells (PCs) has been implicated in the genesis of inherited and acquired ventricular arrhythmias. PC arrhythmogenicity is attributed, at least in part, to dysregulation of intracellular calcium homeostasis; however, the molecular basis for differential calcium handling in PCs vs ventricular myocytes (VMs) is incompletely characterized. The goal of this study was to identify and characterize novel molecular mechanisms responsible for altered calcium handling in PCs. METHODS AND RESULTS: Compound transgenic mice harboring both a Cntn2-eGFP BAC reporter and an alpha-MHC-Cre/floxed tdTomato reporter were used to isolate cardiac PCs (eGFP(+)/tomato(+)) from VMs (eGFP(-)/tomato(+)) by FACS. Gene profiling was performed on total RNA extracted from each cell population. The transcript encoding Purkinje cell protein 4 (PCP4), a modulator of calmodulin-dependent signaling, was significantly enriched in PCs. Restricted expression of PCP4 protein in PCs was confirmed by immunohistochemistry. PCP4 knockout mice were obtained and crossed with the Cntn2-eGFP reporter mouse, and isolated VMs and PCs from wild-type (WT) and mutant hearts were distinguished by epifluorescence and intracellular Ca(2+) dynamics recorded by microfluorometry. PCP4(-/-) PCs displayed significantly slower kinetics of relaxation [tau(decay)] than that observed in wild-type PCs (328.9 +/- 24.9 ms vs 235.8 +/- 20.7 ms, P <.01). PCP4(-/-)) PCs were also more likely to develop rate dependent early aftertransients [63.6% (21/33) vs 8.3% (3/36) at 0.5 Hz, P <.0001] and delayed aftertransients [36.4% (12/33) vs 8.3% (3/36) at 0.5 Hz, P <.01). Intraperitoneal injection of caffeine and epinephrine in adult WT mice (n = 5) elicited no arrhythmias, whereas injection into PCP4(-/-)) mice elicited PVCs (4/5, P <.5) and bidirectional VT (1/5). CONCLUSIONS: PCP4 is preferentially expressed in the murine ventricular conduction system, where it modulates intracellular calcium homeostasis. Loss of function of PCP4 results in an increased propensity for the development of early and delayed aftertransients and triggered arrhythmias.

PURPOSE: There is an impending need for noninvasive biomarkers of breast cancer angiogenesis to evaluate the efficacy of new anti-angiogenic therapies in vivo. The purpose of this study was to systematically evaluate the sensitivity of in vivo steady-state susceptibility contrast-MRI biomarkers of angiogenesis in a human breast cancer model. METHODS: Orthotopic MDA-MB-231 human breast cancer xenografts were imaged by steady-state susceptibility contrast-MRI at post-inoculation week 3 and post-inoculation week 5, followed by ex vivo whole tumor 3D micro-CT angiography. "Absolute" (i.e., measures of vascular morphology in appropriate units) and "relative" (i.e., proportional to measures of vascular morphology) MRI biomarkers of tumor blood volume, vessel size, and vessel density were computed and their ability to predict the corresponding micro-CT analogs assessed using cross-validation analysis. RESULTS: All MRI biomarkers significantly correlated with their micro-CT analogs and were sensitive to the micro-CT-measured decreases in tumor blood volume and vessel density from post-inoculation week 3 to post-inoculation week 5. However, cross-validation analysis revealed there was no significant difference between the predictive accuracy of "absolute" and "relative" biomarkers. CONCLUSION: As "relative" biomarkers are more easily computed from steady-state susceptibility contrast-MRI (i.e., without additional MRI measurements) than "absolute" biomarkers, it makes them promising candidates for assessing breast cancer angiogenesis in vivo.

Cardiac gap junctions are specialized membrane structures comprised of arrays of intercellular channels responsible for propagation of the cardiac impulse. These channels are formed by oligomerization of individual protein subunits known as connexins. In response to a broad array of pathologic stressors, gap junction expression is disturbed, resulting in aberrant cardiac conduction and increased propensity for rhythm disturbances. In this article, we review some of the recently identified molecular regulators of connexin assembly, membrane targeting, and degradation, focusing on the role of post-translational phosphorylation of connexin 43, the major gap junctional protein expressed in ventricular myocardium. We also describe efforts to engineer "designer" gap junctions that are resistant to pathologic remodeling.

Abnormal vascular phenotypes have been implicated in neuropathologies ranging from Alzheimer's disease to brain tumors. The development of transgenic mouse models of such diseases has created a crucial need for characterizing the murine neurovasculature. Although histologic techniques are excellent for imaging the microvasculature at submicron resolutions, they offer only limited coverage. It is also challenging to reconstruct the three-dimensional (3D) vasculature and other structures, such as white matter tracts, after tissue sectioning. Here, we describe a novel method for 3D whole-brain mapping of the murine vasculature using magnetic resonance microscopy (μMRI), and its application to a preclinical brain tumor model. The 3D vascular architecture was characterized by six morphologic parameters: vessel length, vessel radius, microvessel density, length per unit volume, fractional blood volume, and tortuosity. Region-of-interest analysis showed significant differences in the vascular phenotype between the tumor and the contralateral brain, as well as between postinoculation day 12 and day 17 tumors. These results unequivocally show the feasibility of using μMRI to characterize the vascular phenotype of brain tumors. Finally, we show that combining these vascular data with coregistered images acquired with diffusion-weighted MRI provides a new tool for investigating the relationship between angiogenesis and concomitant changes in the brain tumor microenvironment.

Knowledge of the three-dimensional (3D) architecture of blood vessels in the brain is crucial because the progression of various neuropathologies ranging from Alzheimer's disease to brain tumors involves anomalous blood vessels. The challenges in obtaining such data from patients, in conjunction with development of mouse models of neuropathology, have made the murine brain indispensable for investigating disease induced neurovascular changes. Here we describe a novel method for "whole brain" 3D mapping of murine neurovasculature using magnetic resonance microscopy (μMRI). This approach preserves the vascular and white matter tract architecture, and can be combined with complementary MRI contrast mechanisms such as diffusion tensor imaging (DTI) to examine the interplay between the vasculature and white matter reorganization that often characterizes neuropathologies. Following validation with micro computed tomography (μCT) and optical microscopy, we demonstrate the utility of this method by: (i) combined 3D imaging of angiogenesis and white matter reorganization in both, invasive and non-invasive brain tumor models; (ii) characterizing the morphological heterogeneity of the vascular phenotype in the murine brain; and (iii) conducting "multi-scale" imaging of brain tumor angiogenesis, wherein we directly compared in vivo MRI blood volume measurements with ex vivo vasculature data.