Florence Rothenberg

Proinflammatory phenotype of perivascular adipocytes: influence of high-fat feeding.

Adipose tissue depots originate from distinct precursor cells, are functionally diverse, and modulate disease processes in a depot-specific manner. However, the functional properties of perivascular adipocytes, and their influence on disease of the blood vessel wall, remain to be determined. We show that human coronary perivascular adipocytes exhibit a reduced state of adipocytic differentiation as compared with adipocytes derived from subcutaneous and visceral (perirenal) adipose depots. Secretion of antiinflammatory adiponectin is markedly reduced, whereas that of proinflammatory cytokines interleukin-6, interleukin-8, and monocyte chemoattractant protein-1, is markedly increased in perivascular adipocytes. These depot-specific differences in adipocyte function are demonstrable in both freshly isolated adipose tissues and in vitro–differentiated adipocytes. Murine aortic arch perivascular adipose tissues likewise express lower levels of adipocyte-associated genes as compared with subcutaneous and visceral adipose tissues. Moreover, 2 weeks of high-fat feeding caused further reductions in adipocyte-associated gene expression, while upregulating proinflammatory gene expression, in perivascular adipose tissues. These changes were observed in the absence of macrophage recruitment to the perivascular adipose depot. We conclude that perivascular adipocytes exhibit reduced differentiation and a heightened proinflammatory state, properties that are intrinsic to the adipocytes residing in this depot. Dysfunction of perivascular adipose tissue induced by fat feeding suggests that this unique adipose depot is capable of linking metabolic signals to inflammation in the blood vessel wall.

Ultrahigh-speed optical coherence tomography imaging and visualization of the embryonic avian heart using a buffered Fourier Domain Mode Locked laser

The embryonic avian heart is an important model for studying cardiac developmental biology. The mechanisms that govern the development of a four-chambered heart from a peristaltic heart tube are largely unknown due in part to a lack of adequate imaging technology. Due to the small size and rapid motion of the living embryonic avian heart, an imaging system with high spatial and temporal resolution is required to study these models. Here, an optical coherence tomography (OCT) system using a buffered Fourier Domain Mode Locked (FDML) laser is applied for ultrahigh-speed non-invasive imaging of embryonic quail hearts at 100,000 axial scans per second. The high scan rate enables the acquisition of high temporal resolution 2D datasets (195 frames per second or 5.12 ms between frames) and 3D datasets (10 volumes per second). Spatio-temporal details of cardiac motion not resolvable using previous OCT technology are analyzed. Visualization and measurement techniques are developed to non-invasively observe and quantify cardiac motion throughout the brief period of systole (less than 50 msec) and diastole. This marks the first time that the preseptated embryonic avian heart has been imaged in 4D without the aid of gating and the first time it has been viewed in cross section during looping with extremely high temporal resolution, enabling the observation of morphological dynamics of the beating heart during systole.

The pros and cons of apoptosis assays for use in the study of cells, tissues, and organs.

Programmed cell death or apoptosis occurs in many tissues during normal development and in the normal homeostasis of adult tissues. Apoptosis also plays a significant role in abnormal development and disease. Increased interest in apoptosis and cell death in general has resulted in the development of new techniques and the revival of old ones. Each assay has its advantages and disadvantages that can render it appropriate and useful for one application, but inappropriate or difficult to use in another. Understanding the strengths and limitations of the assays would allow investigators to select the best methods for their needs.

4D embryonic cardiography using gated optical coherence tomography

Simultaneous imaging of very early embryonic heart structure and function has technical limitations of spatial and temporal resolution. We have developed a gated technique using optical coherence tomography (OCT) that can rapidly image beating embryonic hearts in four-dimensions (4D), at high spatial resolution (10-15 mum), and with a depth penetration of 1.5 - 2.0 mm that is suitable for the study of early embryonic hearts. We acquired data from paced, excised, embryonic chicken and mouse hearts using gated sampling and employed image processing techniques to visualize the hearts in 4D and measure physiologic parameters such as cardiac volume, ejection fraction, and wall thickness. This technique is being developed to longitudinally investigate the physiology of intact embryonic hearts and events that lead to congenital heart defects.

In vivo spectral domain optical coherence tomography volumetric imaging and spectral Doppler velocimetry of early stage embryonic chicken heart development

Progress toward understanding embryonic heart development has been hampered by the inability to image embryonic heart structure and simultaneously measure blood flow dynamics in vivo. We have developed a spectral domain optical coherence tomography system for in vivo volumetric imaging of the chicken embryo heart. We have also developed a technique called spectral Doppler velocimetry (SDV) for quantitative measurement of blood flow dynamics. We present in vivo volume images of the embryonic heart from initial tube formation to development of endocardial cushions of the same embryo over several stages of development. SDV measurements reveal the influence of heart tube structure on blood flow dynamics.

Enhanced transmural fiber rotation and connexin 43 heterogeneity are associated with an increased upper limit of vulnerability in a transgenic rabbit model of human hypertrophic cardiomyopathy

Human hypertrophic cardiomyopathy, characterized by cardiac hypertrophy and myocyte disarray, is the most common cause of sudden cardiac death in the young. Hypertrophic cardiomyopathy is often caused by mutations in sarcomeric genes. We sought to determine arrhythmia propensity and underlying mechanisms contributing to arrhythmia in a transgenic (TG) rabbit model (&bgr;-myosin heavy chain–Q403) of human hypertrophic cardiomyopathy. Langendorff-perfused hearts from TG (n=6) and wild-type (WT) rabbits (n=6) were optically mapped. The upper and lower limits of vulnerability, action potential duration (APD) restitution, and conduction velocity were measured. The transmural fiber angle shift was determined using diffusion tensor MRI. The transmural distribution of connexin 43 was quantified with immunohistochemistry. The upper limit of vulnerability was significantly increased in TG versus WT hearts (13.3±2.1 versus 7.4±2.3 V/cm; P=3.2e−5), whereas the lower limits of vulnerability were similar. APD restitution, conduction velocities, and anisotropy were also similar. Left ventricular transmural fiber rotation was significantly higher in TG versus WT hearts (95.6±10.9° versus 79.2±7.8°; P=0.039). The connexin 43 density was significantly increased in the mid-myocardium of TG hearts compared with WT (5.46±2.44% versus 2.68±0.77%; P=0.024), and similar densities were observed in the endo- and epicardium. Because a nearly 2-fold increase in upper limit of vulnerability was observed in the TG hearts without significant changes in APD restitution, conduction velocity, or the anisotropy ratio, we conclude that structural remodeling may underlie the elevated upper limit of vulnerability in human hypertrophic cardiomyopathy.

Quantitative Measurement of Blood Flow Dynamics in Embryonic Vasculature Using Spectral Doppler Velocimetry

The biophysical effects of blood flow are known to influence the structure and function of adult cardiovascular systems. Similar effects on the maturation of the cardiovascular system have been difficult to directly and non‐invasively measure due to the small size of the embryo. Optical coherence tomography (OCT) has been shown to provide high spatial and temporal structural imaging of the early embryonic chicken heart. We have developed an extension of Doppler OCT, called spectral Doppler velocimetry (SDV), that will enable direct, non‐invasive quantification of blood flow and shear rate from the early embryonic cardiovascular system. Using this technique, we calculated volumetric flow rate and shear rate from chicken embryo vitelline vessels. We present blood flow dynamics and spatial velocity profiles from three different vessels in the embryo as well as measurements from the outflow tract of the embryonic heart tube. This technology can potentially provide spatial mapping of blood flowand shear rate in embryonic cardiovascular structures, producing quantitative measurements that can be correlated with gene expression and normal and abnormal morphology. Anat Rec, 2009.

Initiation of apoptosis in the developing avian outflow tract myocardium.

Apoptosis occurs within the cardiac outflow tract (OFT) myocardium during normal development of chick hearts. This peak of apoptosis occurs at stage 30–31 and coincides with dramatic remodeling of the OFT, suggesting that apoptosis occurs to allow proper alignment of the great vessels over their respective ventricles. The signals that initiate apoptosis in this setting are unknown. The aim of this study was to characterize the cells undergoing apoptosis in the cardiac OFT myocardium and the cells that may influence this process. Two cell populations that may initiate apoptosis of the cardiomyocytes are the cardiac neural crest (CNC) cells and epicardial cells. We examined stage 30–31 chick embryos that had undergone removal of the CNC cells or had delayed epicardial growth for alterations of apoptosis. Removal of the CNC cells did not reduce the levels or pattern of apoptosis in the OFT myocardium. In contrast, impeding the growth of the epicardium over the OFT resulted in a 57% reduction in apoptotic cells in the OFT myocardium. Analysis of the apoptotic cells within the OFT myocardium showed that as many as 92% of them expressed cardiomyocyte markers. In the quail, the endothelial marker QH1 identified a component from the epicardium, endothelial cells, in regions where apoptosis is elevated in the OFT myocardium. These results suggest that a component from the epicardium, possibly endothelial cells, is required for the initiation of apoptosis in OFT cardiomyocytes.

The influence of long-term nifedipine or indomethacin therapy on neurologic recovery from experimental spinal cord injury.

Inhibition of prostaglandin pathways and calcium channel conduction has been shown to improve neurological outcome after spinal cord injury. Functional recovery from such intervention has been routinely evaluated by a simple motor examination or somatosensory evoked potentials (SSEPs) after short-term drug administration. We comprehensively evaluated the influence of continuously administered indomethacin and nifedipine on functional outcome after impact spinal cord injury. SSEP and cortico-motor evoked potential records and neurologic examinations were obtained over 6 weeks after injury. Terminal histopathologic changes within the spinal cord were also examined. Only indomethacin significantly improved neurological function and reduced the severity of histopathologic change. Evoked potential analysis was not found to be of prognostic value. Modulation of prostaglandin pathways may enhance neurological recovery after spinal cord injury.

Emerging patterns of cardiac conduction in the chick embryo: Waveform analysis with photodiode array-based optical imaging

Major difficulties investigating the developing cardiac conduction system stem from that the embryonic heart is extremely small (< 2 mm) and cardiac activation is relatively rapid (< 8 msec). The objective of this study was to investigate the electrophysiology of the embryonic chick cardiac conduction system at periseptation stages with a photodiode array‐based detection method of optical mapping capable of high spatial and temporal resolution. Previous work indicated that, in chicken embryos, a switch occurs in ventricular activation pattern from immature base‐to‐apex to mature apex‐to‐base pattern at the time of ventricular septation. It was our aim to map activation in more detail to identify the active pathway or pathways of atrioventricular conduction at these particular stages. Analysis of preseptated hearts (n = 10) showed that the latest atrial activation took place just above the site of the earliest ventricular activation at the ventral left ventricular base. Analysis of postseptated hearts (n = 11) showed apex‐to‐base conduction consistent with activation through the maturing His–Purkinje system. Evaluation of hearts during septation revealed a gradual transition of ventricular activation patterns rather than an abrupt “switch.” External pacing of preseptated hearts revealed significant slowing of interventricular conduction compared with spontaneous beats (spontaneous, 61.7 cm/sec ± 9 cm/sec vs. paced, 36.5 cm/sec ± 10 cm/sec). The more detailed mapping revealed that, before septation, the pattern of activation of the ventricular myocardium is consistent with direct atrial–ventricular myocardial connections at the left lateral atrioventricular junction; however, functional evidence for a preferential conduction pathway within the ventricles was present before septation. Developmental Dynamics 233:456–465, 2005.