Scott E. Klewer

Disruption of hyaluronan synthase-2 abrogates normal cardiac morphogenesis and hyaluronan-mediated transformation of epithelium to mesenchyme

We identified hyaluronan synthase-2 (Has2) as a likely source of hyaluronan (HA) during embryonic development, and we used gene targeting to study its function in vivo. Has2(-/-) embryos lack HA, exhibit severe cardiac and vascular abnormalities, and die during midgestation (E9.5-10). Heart explants from Has2(-/-) embryos lack the characteristic transformation of cardiac endothelial cells into mesenchyme, an essential developmental event that depends on receptor-mediated intracellular signaling. This defect is reproduced by expression of a dominant-negative Ras in wild-type heart explants, and is reversed in Has2(-/-) explants by gene rescue, by administering exogenous HA, or by expressing activated Ras. Conversely, transformation in Has2(-/-) explants mediated by exogenous HA is inhibited by dominant-negative Ras. Collectively, our results demonstrate the importance of HA in mammalian embryogenesis and the pivotal role of Has2 during mammalian development. They also reveal a previously unrecognized pathway for cell migration and invasion that is HA-dependent and involves Ras activation.

Cell Biology of Cardiac Cushion Development

The valves of the heart develop in the embryo from precursor structures called endocardial cushions. After cardiac looping, endocardial cushion swellings form and become populated by valve precursor cells formed by an epithelial-mesenchymal transition (EMT). Endocardial cushions subsequently undergo directed growth and remodeling to form the valvular structures and the membranous septa of the mature heart. The developmental processes that mediate cushion formation include many prototypic cellular actions including adhesion, signaling, migration, secretion, replication, differentiation, and apoptosis. Cushion morphogenesis is unique in that these cellular possesses occur in a functioning organ where the cushions act as valves even while developing into definitive valvular structures. Cardiovascular defects are the most common congenital defects, and one of the most common causes of death during infancy. Thus, there is significant interest in understanding the mechanisms that underlie this complex developmental process. In this regard, substantial progress has been made by incorporating an understanding of cardiac morphology and cell biology with the rapidly expanding repertoire of molecular mechanisms gained through human genetics and research using animal models. This article reviews cardiac morphogenesis as it relates to heart valve formation and highlights selected growth factors, intracellular signaling mediators, and extracellular matrix components involved in the creation and remodeling of endocardial cushions into mature cardiac structures.

Heart-valve mesenchyme formation is dependent on hyaluronan-augmented activation of ErbB2-ErbB3 receptors

Heart septation and valve malformations constitute the most common anatomical birth defects. These structures arise from the endocardial cushions within the atrioventricular canal (AVC) through dynamic interactions between cushion cells and the extracellular matrix (termed cardiac jelly). Transformation of endothelial cells to mesenchymal cells is essential for the proper development of the AVC and subsequent septation and valve formation. Atrioventricular septal defects can result from incomplete endocardial cushion morphogenesis. We show that hyaluronan-deficient AVC explants from Has2−/− embryos, which normally lack mesenchyme formation, are rescued by heregulin treatment, which restores phosphorylation of ErbB2 and ErbB3. These events were blocked using a soluble ErbB3 molecule, as well as with an inhibitor of ErbB2, herstatin. We show further that ErbB3 is activated during hyaluronan treatment of Has2−/− explants. These data provide a link between extracellular matrix-hyaluronan and ErbB receptor activation during development of early heart-valve and septal mesenchyme.

Dobutamine stress echocardiography: a sensitive indicator of diminished myocardial function in asymptomatic doxorubicin-treated long-term survivors of childhood cancer.

Doxorubicin is an effective anticancer chemotherapeutic agent known to cause acute and chronic cardiomyopathy. To develop a more sensitive echocardiographic screening test for cardiac damage due to doxorubicin, a cohort study was performed using dobutamine infusion to differentiate asymptomatic long-term survivors of childhood cancer treated with doxorubicin from healthy control subjects. Echocardiographic data from the experimental group of 21 patients (mean age 16 +/- 5 years) treated from 1.6 to 14.3 years (median 5.3) before this study with 27 to 532 mg/m2 of doxorubicin (mean 196) were compared with echocardiographic data from 12 normal age-matched control subjects. Graded dobutamine infusions of 0.5, 2.5, 5 and 10 micrograms/kg per min were administered. Echocardiographic Doppler studies were performed before infusion and after 15 min of infusion at each rate. Dobutamine infusion at 10 micrograms/kg per min was discontinued after six studies secondary to a 50% incidence rate of adverse symptoms. The most important findings were that compared with values in control subjects, end-systolic left ventricular posterior wall dimension and percent of left ventricular posterior wall thickening in doxorubicin-treated patients were decreased at baseline study and these findings were more clearly delineated with dobutamine stimulation. End-systolic left ventricular posterior wall dimension at baseline for the doxorubicin-treated group was 11 +/- 1.9 mm versus 13.1 +/- 1.5 mm for control subjects (p less than 0.01). End-systolic left ventricular posterior wall dimension at the 5-micrograms/kg per min dobutamine infusion for the doxorubicin-treated group was 14.1 +/- 2.4 mm versus 19.3 +/- 2.6 mm for control subjects (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of type VI collagen in the developing mouse heart

During development, the embryonic atrioventricular (AV) endocardial cushions undergo a morphogenic process to form mature valve leaflets and the membranous septa in the heart. Several extracellular matrix (ECM) proteins are expressed in the developing AV endocardial cushions, but it remains to be established if any specific ECM proteins are necessary for normal cushion morphogenesis. Abnormal development of the cardiac AV valves is a frequent cause of congenital heart defects, particularly in infants with trisomy 21 (Down syndrome). The genes encoding the α1 and α2 chains of type VI collagen are located on human chromosome 21 within the region thought to be critical for congenital heart defects in trisomy 21 infants. This suggests that the type VI collagen α1(VI) and α2(VI) chains may be important in normal AV valve morphogenesis. As a first step in understanding the role of type VI collagen in valve development, the authors examined the normal spatial and temporal expression patterns of mRNA and protein for type VI collagen in the embryonic mouse heart. Ribonuclease protection assay analysis demonstrates cardiac expression of the type VI collagen for α1(VI), α2(VI), and α3(VI) transcripts beginning at embryonic days 11–11.5 of mouse development. In situ hybridization studies demonstrate a coordinated pattern of cardiac expression within the AV valves for each type VI collagen chain from embryonic day 11.5 through the neonatal period. Immunohistochemical studies confirm a concentrated type VI collagen localization pattern in the endocardial cushions from the earliest stages of valve development through the neonatal period. These data indicate that type VI collagen is expressed in the developing AV canal in a pattern consistent with cushion tissue mesenchymal cell migration and proliferation, and suggest that type VI collagen plays a role in the morphogenesis of the developing cardiac AV endocardial cushions into the valve leaflets and membranous septa of the heart. Dev. Dyn. 1998;211:248–255.

Dysregulation of the PDGFRA gene causes inflow tract anomalies including TAPVR: integrating evidence from human genetics and model organisms

Total anomalous pulmonary venous return (TAPVR) is a congenital heart defect inherited via complex genetic and/or environmental factors. We report detailed mapping in extended TAPVR kindreds and mutation analysis in TAPVR patients that implicate the PDGFRA gene in the development of TAPVR. Gene expression studies in mouse and chick embryos for both the Pdgfra receptor and its ligand Pdgf-a show temporal and spatial patterns consistent with a role in pulmonary vein (PV) development. We used an in ovo function blocking assay in chick and a conditional knockout approach in mouse to knock down Pdgfra expression in the developing venous pole during the period of PV formation. We observed that loss of PDGFRA function in both organisms causes TAPVR with low penetrance (approximately 7%) reminiscent of that observed in our human TAPVR kindreds. Intermediate inflow tract anomalies occurred in a higher percentage of embryos (approximately 30%), suggesting that TAPVR occurs at one end of a spectrum of defects. We show that the anomalous pulmonary venous connection seen in chick and mouse is highly similar to TAPVR discovered in an abnormal early stage embryo from the Kyoto human embryo collection. Whereas the embryology of the normal venous pole and PV is becoming understood, little is known about the embryogenesis or molecular pathogenesis of TAPVR. These models of TAPVR provide important insight into the pathogenesis of PV defects. Taken together, these data from human genetics and animal models support a role for PDGF-signaling in normal PV development, and in the pathogenesis of TAPVR.

Latrophilin-2 is a novel component of the epithelial-mesenchymal transition within the atrioventricular canal of the embryonic chicken heart

Endothelial cells in the atrioventricular canal of the heart undergo an epithelial‐mesenchymal transition (EMT) to form heart valves. We surveyed an on‐line database (http://www.geisha.arizona.edu/) for clones expressed during gastrulation to identify novel EMT components. One gene, latrophilin‐2, was identified as expressed in the heart and appeared to be functional in EMT. This molecule was chosen for further examination. In situ localization showed it to be expressed in both the myocardium and endothelium. Several antisense DNA probes and an siRNA for latrophilin‐2 produced a loss of EMT in collagen gel cultures. Latrophilin‐2 is a putative G‐protein‐coupled receptor and we previously identified a pertussis toxin‐sensitive G‐protein signal transduction pathway. Microarray experiments were performed to examine whether these molecules were related. After treatment with antisense DNA against latrophilin‐2, expression of 1,385 genes and ESTs was altered. This represented approximately 12.5% of the microarray elements. In contrast, pertussis toxin altered only 103 (0.9%) elements of the array. There appears to be little overlap between the two signal transduction pathways. Latrophilin‐2 is thus a novel component of EMT and provides a new avenue for investigation of this cellular process. Developmental Dynamics 235:3213–3221, 2006.

Arsenic Exposure Perturbs Epithelial-Mesenchymal Cell Transition and Gene Expression In a Collagen Gel Assay

Arsenic is a naturally occurring metalloid and environmental contaminant. Arsenic exposure in drinking water is reported to cause cancer of the liver, kidneys, lung, bladder, and skin as well as birth defects, including neural tube, facial, and vasculogenic defects. The early embryonic period most sensitive to arsenic includes a variety of cellular processes. One key cellular process is epithelial-mesenchymal transition (EMT) where epithelial sheets develop into three-dimensional structures. An embryonic prototype of EMT is found in the atrioventricular (AV) canal of the developing heart, where endothelia differentiate to form heart valves. Effects of arsenic on this cellular process were examined by collagen gel invasion assay (EMT assay) using explanted AV canals from chicken embryo hearts. AV canals treated with 12.5-500 ppb arsenic showed a loss of mesenchyme at 12.5 ppb, and mesenchyme formation was completely inhibited at 500 ppb. Altered gene expression in arsenic-treated explants was investigated by microarray analysis. Genes whose expression was altered consistently at exposure levels of 10, 25, and 100 ppb were identified, and results showed that 25 ppb in vitro was particularly effective. Three hundred and eighty two genes were significantly altered at this exposure level. Cytoscape analysis of the microarray data using the chicken interactome identified four clusters of altered genes based on published relationships and pathways. This analysis identified cytoskeleton and cell adhesion-related genes whose disruption is consistent with an altered ability to undergo EMT. These studies show that EMT is sensitive to arsenic and that an interactome-based approach can be useful in identifying targets.

Still's-like innocent murmur can be produced by increasing aortic velocity to a threshold value

Abstract Stills murmur is a musical or vibratory systolic ejection murmur found in many normal school-aged children and adolescents. Its origin has been attributed to vibration of a cardiac structure during ventricular contraction, turbulent blood flow, or pressure changes across normal valves. 1 We previously related Stills murmur to a small ascending aorta with concomitant high aortic velocity. 2 Functional murmurs are accentuated in high cardiac output states such as fever, exercise and anemia. Dobutamine, a β 1 agonist with primarily inotropic action at low doses, allows study of high cardiac output states. We investigated whether dobutamine infusion could produce a Stills-like murmur in subjects without murmurs at rest and evaluated those factors correlating best with murmur presence.

Comparison of accuracy of diagnosis of congenital heart disease by history and physical examination versus echocardiography

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