Ganga Karunamuni

VESGEN 2D: automated, user-interactive software for quantification and mapping of angiogenic and lymphangiogenic trees and networks.

Quantification of microvascular remodeling as a meaningful discovery tool requires mapping and measurement of site‐specific changes within vascular trees and networks. Vessel density and other critical vascular parameters are often modulated by molecular regulators as determined by local vascular architecture. For example, enlargement of vessel diameter by vascular endothelial growth factor (VEGF) is restricted to specific generations of vessel branching (Parsons‐Wingerter et al., Microvascular Research72: 91, 2006). The averaging of vessel diameter over many successively smaller generations is therefore not particularly useful. The newly automated, user‐interactive software VESsel GENeration Analysis (VESGEN) quantifies major vessel parameters within two‐dimensional (2D) vascular trees, networks, and tree‐network composites. This report reviews application of VESGEN 2D to angiogenic and lymphangiogenic tissues that includes the human and murine retina, embryonic coronary vessels, and avian chorioallantoic membrane. Software output includes colorized image maps with quantification of local vessel diameter, fractal dimension, tortuosity, and avascular spacing. The density of parameters such as vessel area, length, number, and branch point are quantified according to site‐specific generational branching within vascular trees. The sole user input requirement is a binary (black/white) vascular image. Future applications of VESGEN will include analysis of 3D vascular architecture and bioinformatic dimensions such as blood flow and receptor localization. Branching analysis by VESGEN has demonstrated that numerous regulators including VEGF165, basic fibroblast growth factor, transforming growth factor β‐1, angiostatin and the clinical steroid triamcinolone acetonide induce ‘fingerprint’ or ‘signature’ changes in vascular patterning that provide unique readouts of dominant molecular signaling. Anat Rec, 292:320–332, 2009.

Ethanol exposure alters early cardiac function in the looping heart: a mechanism for congenital heart defects?

Alcohol-induced congenital heart defects are frequently among the most life threatening and require surgical correction in newborns. The etiology of these defects, collectively known as fetal alcohol syndrome, has been the focus of much study, particularly involving cellular and molecular mechanisms. Few studies have addressed the influential role of altered cardiac function in early embryogenesis because of a lack of tools with the capability to assay tiny beating hearts. To overcome this gap in our understanding, we used optical coherence tomography (OCT), a nondestructive imaging modality capable of micrometer-scale resolution imaging, to rapidly and accurately map cardiovascular structure and hemodynamics in real time under physiological conditions. In this study, we exposed avian embryos to a single dose of alcohol/ethanol at gastrulation when the embryo is sensitive to the induction of birth defects. Late-stage hearts were analyzed using standard histological analysis with a focus on the atrio-ventricular valves. Early cardiac function was assayed using Doppler OCT, and structural analysis of the cardiac cushions was performed using OCT imaging. Our results indicated that ethanol-exposed embryos developed late-stage valvuloseptal defects. At early stages, they exhibited increased regurgitant flow and developed smaller atrio-ventricular cardiac cushions, compared with controls (uninjected and saline-injected embryos). The embryos also exhibited abnormal flexion/torsion of the body. Our evidence suggests that ethanol-induced alterations in early cardiac function have the potential to contribute to late-stage valve and septal defects, thus demonstrating that functional parameters may serve as early and sensitive gauges of cardiac normalcy and abnormalities.

Apoptosis in the developing mouse heart.

Apoptosis occurs at high frequency in the myocardium of the developing avian cardiac outflow tract (OFT). Up‐ or down‐regulating apoptosis results in defects resembling human conotruncal heart anomalies. This finding suggested that regulated levels of apoptosis are critical for normal morphogenesis of the four‐chambered heart. Recent evidence supports an important role for hypoxia of the OFT myocardium in regulating cell death and vasculogenesis. The purpose of this study was to determine whether apoptosis in the outflow tract myocardium occurs in the mouse heart during developmental stages comparable to the avian heart and to determine whether differential hypoxia is also present at this site in the murine heart. Apoptosis was detected using a fluorescent vital dye, Lysotracker Red (LTR), in the OFT myocardium of the mouse starting at embryonic day (E) 12.5, peaking at E13.5–14.5, and declining thereafter to low or background levels by E18.5. In addition, high levels of apoptosis were detected in other cardiac regions, including the apices of the ventricles and along the interventricular sulcus. Apoptosis in the myocardium was detected by double‐labeling with LTR and cardiomyocyte markers. Terminal deoxynucleotidyl transferase–mediated deoxyuridinetriphosphate nick end‐labeling (TUNEL) and immunostaining for cleaved Caspase‐3 were used to confirm the LTR results. At the peak of OFT apoptosis in the mouse, the OFT myocardium was relatively hypoxic, as indicated by specific and intense EF5 staining and HIF1α nuclear localization, and was surrounded by the developing vasculature as in the chicken embryo. These findings suggest that cardiomyocyte apoptosis is an evolutionarily conserved mechanism for normal morphogenesis of the outflow tract myocardium in avian and mammalian species. Developmental Dynamics 235:2592–2602, 2006.

Altered hypoxia-inducible factor-1 alpha expression levels correlate with coronary vessel anomalies.

The outflow tract myocardium and other regions corresponding to the location of the major coronary vessels of the developing chicken heart, display a high level of hypoxia as assessed by the hypoxia indicator EF5. The EF5‐positive tissues were also specifically positive for nuclear‐localized hypoxia inducible factor‐1 alpha (HIF‐1α), the oxygen‐sensitive component of the hypoxia inducible factor‐1 (HIF‐1) heterodimer. This led to our hypothesis that there is a “template” of hypoxic tissue that determines the stereotyped pattern of the major coronary vessels. In this study, we disturbed this template by altering ambient oxygen levels (hypoxia 15%; hyperoxia 75–40%) during the early phases of avian coronary vessel development, in order to alter tissue hypoxia, HIF‐1α protein expression, and its downstream target genes without high mortality. We also altered HIF‐1α gene expression in the embryonic outflow tract cardiomyocytes by injecting an adenovirus containing a constitutively active form of HIF‐1α (AdCA5). We assayed for coronary anomalies using anti‐alpha‐smooth muscle actin immunohistology. When incubated under abnormal oxygen levels or injected with a low titer of the AdCA5, coronary arteries displayed deviations from their normal proximal connections to the aorta. These deviations were similar to known clinical anomalies of coronary arteries. These findings indicated that developing coronary vessels may be subject to a level of regulation that is dependent on differential oxygen levels within cardiac tissues and subsequent HIF‐1 regulation of gene expression. Developmental Dynamics 238:2688–2700, 2009.

Expression of Active Notch1 in Avian Coronary Development

Notch1 is an important regulator of intercellular interactions in cardiovascular development. We show that the nuclear‐localized, cleaved and active form of Notch1, the Notch1 intracellular domain (N1ICD), appeared in mesothelial cells of the pro‐epicardium during epicardial formation at looped heart stages. N1ICD was also present in mesothelial cells and mesenchymal cells specifically within the epicardium at sulcus regions. N1ICD‐positive endothelial cells were detected within the nascent vessel plexus at the atrioventricular junction and within the compact myocardium (Hamburger and Hamilton stage [HH] 25–HH30). The endothelial cells expressing N1ICD were surrounded by N1ICD‐positive smooth muscle cells after coronary orifice formation (HH32–HH35), while N1ICD expression was absent in the mesenchymal and mesothelial cells surrounding mature coronary vessels. We propose that differential activation of the hypoxia/HIF1‐VEGF‐Notch pathway may play a role in epicardial cell interactions that promote epicardial epithelial/mesenchymal transition and coronary progenitor cell differentiation during epicardial development and coronary vasculogenesis in particularly hypoxic sulcus regions. Developmental Dynamics 238:162–170, 2009.

Using optical coherence tomography to rapidly phenotype and quantify congenital heart defects associated with prenatal alcohol exposure

Background: The most commonly used method to analyze congenital heart defects involves serial sectioning and histology. However, this is often a time‐consuming process where the quantification of cardiac defects can be difficult due to problems with accurate section registration. Here we demonstrate the advantages of using optical coherence tomography, a comparatively new and rising technology, to phenotype avian embryo hearts in a model of fetal alcohol syndrome where a binge‐like quantity of alcohol/ethanol was introduced at gastrulation. Results: The rapid, consistent imaging protocols allowed for the immediate identification of cardiac anomalies, including ventricular septal defects and misaligned/missing vessels. Interventricular septum thicknesses and vessel diameters for three of the five outflow arteries were also significantly reduced. Outflow and atrioventricular valves were segmented using image processing software and had significantly reduced volumes compared to controls. This is the first study to our knowledge that has 3D reconstructed the late‐stage cardiac valves in precise detail to examine their morphology and dimensions. Conclusions: We believe, therefore, that optical coherence tomography, with its ability to rapidly image and quantify tiny embryonic structures in high resolution, will serve as an excellent and cost‐effective preliminary screening tool for developmental biologists working with a variety of experimental/disease models. Developmental Dynamics 244:607–618, 2015.

Expression of Lymphatic Markers During Avian and Mouse Cardiogenesis

The adult heart has been reported to have an extensive lymphatic system, yet the development of this important system during cardiogenesis is still largely unexplored. The nuclear‐localized transcription factor Prox‐1 identified a sheet of Prox‐1‐positive cells on the developing aorta and pulmonary trunk in avian and murine embryos just before septation of the four heart chambers. The cells coalesced into a branching lymphatic network that spread within the epicardium to cover the heart. These vessels eventually expressed the lymphatic markers LYVE‐1, VEGFR‐3, and podoplanin. Before the Prox‐1‐positive cells were detected in the mouse epicardium, LYVE‐1, a homologue of the CD44 glycoprotein, was primarily expressed in individual epicardial cells. Similar staining patterns were observed for CD44 in avian embryos. The proximity of these LYVE‐1/CD44‐positive mesenchymal cells to Prox‐1‐positive vessels suggests that they may become incorporated into the lymphatics. Unexpectedly, we detected LYVE‐1/PECAM/VEGFR‐3‐positive vessels within the embryonic and adult myocardium, which remained Prox‐1/podoplanin‐negative. Lymphatic markers were surprisingly found in adult rat and embryonic mouse epicardial cell lines, with Prox‐1 also exhibiting nuclear‐localized expression in primary cultures of embryonic avian epicardial cells. Our data identified three types of cells in the embryonic heart expressing lymphatic markers: (1) Prox‐1‐positive cells from an extracardiac source that migrate within the serosa of the outflow tract into the epicardium of the developing heart, (2) individual LYVE‐1‐positive cells in the epicardium that may be incorporated into the Prox‐1‐positive lymphatic vasculature, and (3) LYVE‐1‐positive cells/vessels in the myocardium that do not become Prox‐1‐positive even in the adult heart. Anat Rec, 2010.

Capturing structure and function in an embryonic heart with biophotonic tools

Disturbed cardiac function at an early stage of development has been shown to correlate with cellular/molecular, structural as well as functional cardiac anomalies at later stages culminating in the congenital heart defects (CHDs) that present at birth. While our knowledge of cellular and molecular steps in cardiac development is growing rapidly, our understanding of the role of cardiovascular function in the embryo is still in an early phase. One reason for the scanty information in this area is that the tools to study early cardiac function are limited. Recently developed and adapted biophotonic tools may overcome some of the challenges of studying the tiny fragile beating heart. In this chapter, we describe and discuss our experience in developing and implementing biophotonic tools to study the role of function in heart development with emphasis on optical coherence tomography (OCT). OCT can be used for detailed structural and functional studies of the tubular and looping embryo heart under physiological conditions. The same heart can be rapidly and quantitatively phenotyped at early and again at later stages using OCT. When combined with other tools such as optical mapping (OM) and optical pacing (OP), OCT has the potential to reveal in spatial and temporal detail the biophysical changes that can impact mechanotransduction pathways. This information may provide better explanations for the etiology of the CHDs when interwoven with our understanding of morphogenesis and the molecular pathways that have been described to be involved. Future directions for advances in the creation and use of biophotonic tools are discussed.

Cardiac neural crest ablation results in early endocardial cushion and hemodynamic flow abnormalities

Cardiac neural crest cell (CNCC) ablation creates congenital heart defects (CHDs) that resemble those observed in many syndromes with craniofacial and cardiac consequences. The loss of CNCCs causes a variety of great vessel defects, including persistent truncus arteriosus and double-outlet right ventricle. However, because of the lack of quantitative volumetric measurements, less severe defects, such as great vessel size changes and valve defects, have not been assessed. Also poorly understood is the role of abnormal cardiac function in the progression of CNCC-related CHDs. CNCC ablation was previously reported to cause abnormal cardiac function in early cardiogenesis, before the CNCCs arrive in the outflow region of the heart. However, the affected functional parameters and how they correlate with the structural abnormalities were not fully characterized. In this study, using a CNCC-ablated quail model, we contribute quantitative phenotyping of CNCC ablation-related CHDs and investigate abnormal early cardiac function, which potentially contributes to late-stage CHDs. Optical coherence tomography was used to assay early- and late-stage embryos and hearts. In CNCC-ablated embryos at four-chambered heart stages, great vessel diameter and left atrioventricular valve leaflet volumes are reduced. Earlier, at cardiac looping stages, CNCC-ablated embryos exhibit abnormally twisted bodies, abnormal blood flow waveforms, increased retrograde flow percentage, and abnormal cardiac cushions. The phenotypes observed in this CNCC-ablation model were also strikingly similar to those found in an established avian fetal alcohol syndrome model, supporting the contribution of CNCC dysfunction to the development of alcohol-induced CHDs.

Connecting teratogen-induced congenital heart defects to neural crest cells and their effect on cardiac function

Neural crest cells play many key roles in embryonic development, as demonstrated by the abnormalities that result from their specific absence or dysfunction. Unfortunately, these key cells are particularly sensitive to abnormalities in various intrinsic and extrinsic factors, such as genetic deletions or ethanol-exposure that lead to morbidity and mortality for organisms. This review discusses the role identified for a segment of neural crest in regulating the morphogenesis of the heart and associated great vessels. The paradox is that their derivatives constitute a small proportion of cells to the cardiovascular system. Findings supporting that these cells impact early cardiac function raises the interesting possibility that they indirectly control cardiovascular development at least partially through regulating function. Making connections between insults to the neural crest, cardiac function, and morphogenesis is more approachable with technological advances. Expanding our understanding of early functional consequences could be useful in improving diagnosis and testing therapies.