A comparison of fibronectin, laminin, and galactosyltransferase adhesion mechanisms during embryonic cardiac mesenchymal cell migration in vitro.

Embryonic hearts contain a homogeneous population of mesenchymal cells which migrate through an extensive extracellular matrix (ECM) to become the earliest progenitors of the cardiac valves. Since these cells normally migrate through an ECM containing several adhesion substrates, this study was undertaken to examine and compare three ECM binding mechanisms for mesenchymal cell migration in an in vitro model. Receptor mechanisms for the ECM glycoproteins fibronectin (FN) and laminin (LM) and the cell surface receptor galactosyltransferase (GalTase), which binds an uncharacterized ECM substrate, were compared. Primary cardiac explants from stage 17 chick embryos were cultured on three-dimensional collagen gels. Mesenchymal cell outgrowth was recorded every 24 hr and is reported as a percentage of control. Migration was perturbed using specific inhibitors for each of the three receptor mechanisms. These included the hexapeptide GRGDSP (300-1000 micrograms/ml), which mimics a cell binding domain of FN, the pentapeptide YIGSR (300-1000 micrograms/ml), which mimics a binding domain of LM, and alpha-lactalbumin (1-10 mg/ml), a protein modifier of GalTase activity. The functional role of these adhesion mechanisms was further tested using antibodies to avian integrin (JG22) and avian GalTase. While the FN-related peptide had no significant effect on cell migration it did produce a rounded cellular morphology. The LN-related peptide inhibited mesenchymal migration 70% and alpha-lactalbumin inhibited cell migration 50%. Antibodies against integrin and GalTase inhibited mesenchymal cell migration by 80 and 50%, respectively. The substrate for GalTase was demonstrated to be a single high molecular weight substrate which was not LM or FN. Control peptides, proteins and antibodies demonstrated the specificity of these effects. These data demonstrate that multiple adhesion mechanisms, including cell surface GalTase, are potentially functional during cardiac mesenchymal cell migration. The sensitivity of cell migration to the various inhibitors suggests that occupancy of specific ECM receptors can modulate the activity of other, unrelated, ECM adhesion mechanisms utilized by these cells.

Signal transduction of a tissue interaction during embryonic heart development.

During early cardiac development, progenitors of the valves and septa of the heart are formed by an epithelial-mesenchymal cell transformation of endothelial cells of the atrioventricular (AV) canal. We have previously shown that this event is due to an interaction between the endothelium and products of the myocardium found within the extracellular matrix. The present study examines signal transduction mechanisms governing this differentiation of AV canal endothelium. Activators of protein kinase C (PKC), phorbol myristate acetate (PMA) and mezerein, both produced an incomplete phenotypic transformation of endothelial cells in an in vitro bioassay for transformation. On the other hand, inhibitors of PKC (H-7 and staurosporine) and tyrosine kinase (genistein) blocked cellular transformation in response to the native myocardium or a myocardially-conditioned medium. Intracellular free calcium concentration ([Ca2+]i) was measured in single endothelial cells by microscopic digital analysis of fura 2 fluorescence. Addition of a myocardial conditioned medium containing the transforming stimulus produced a specific increase in [Ca2+]i in "competent" AV canal, but not ventricular, endothelial cells. Epithelial-mesenchymal cell transformation was inhibited by pertussis toxin but not cholera toxin. These data lead to the hypothesis that signal transduction of this tissue interaction is mediated by a G protein and one or more kinase activities. In response to receptor activation, competent AV canal endothelial cells demonstrate an increase in [Ca2+]i. Together, the data provide direct evidence for a regional and temporal regulation of signal transduction processes which mediate a specific extracellular matrix-mediated tissue interaction in the embryo.

Trichloroethylene: a cardiac teratogen in developing chick embryos.

Prior studies have evaluated the teratogenicity of TCE, a contaminant of drinking water. However, none specifically examined effects of TCE on cardiogenesis. The purpose of this study was to determine if TCE is a cardiac teratogen in early embryogenesis in an avian model. Fertile White Leghorn chicken eggs were incubated under standard conditions. At stage 6, 12, 18, or 23, TCE, in concentrations of 5 to 25 microM (2 to 28 micrograms/g body weight) was injected into the air space of the egg (vol = 0.03 ml). Mineral oil and saline served as control solutions. For this double-blinded study, solutions were coded and remained so until all observations were made and recorded. Embryonic hearts (n = 1055) were examined at stage 29, 34, or 44. Gross examination was performed, followed by microdissection. Cardiac malformations were found in 7.3% of TCE-treated hearts, compared to 2.3% of saline controls (p less than 0.01), and 1.5% of mineral oil controls (p less than 0.001). No significant difference in incidence of malformations was found when comparing saline and mineral oil controls. Cardiac defects include septal defects, cor biloculare, conotruncal abnormalities, atrioventricular canal defects, and abnormal cardiac muscle. These data demonstrate that TCE is a cardiac teratogen in an avian model.

Does transducer selection affect aortic arch velocities?

The hypothesis tested was that transducers of different types and shapes would produce different peak and mean ascending aortic (AAo) velocities. Additionally, we sought to determine if mean and peak velocity recorded from the descending aorta (DAo) were similar to velocities in the AAo. Twenty-eight consecutive individuals who had normal hearts were studied. AAo velocities were measured with four transducers including a nonimaging device that transmitted Doppler at right angles to the transducer handle, a 30-degree angled continuous wave transducer, an imaging transducer that transmitted Doppler in line with the transducer handle, and a second imaging transducer that sectored at 25 degrees to the transducer handle. DAo was studied with a standard in-line imaging transducer. Results showed that mean and peak AAo velocities recorded by transducers that transmitted off the axis of the transducer handle were similar, but the transducer that imaged along the transducer handle axis produced significantly lower peak and mean velocities. The problem that caused lower velocity for the on-axis transducer was inability to image the area immediately posterior to the sternum to permit alignment in the azimuthal dimension. The continuous wave transducer provided a wide spectral dispersion. Mean DAo velocity was similar to mean AAo velocity, but variability was large.

How much does aortic and pulmonary artery area vary during the cardiac cycle?

Cardiac output (CO), as measured by Doppler, is computed by multiplying vessel area by mean velocity. One unresolved problem is the magnitude of vessel area change during the cardiac cycle. Prior estimates have ranged from 2% to 30% area change during the cardiac cycle. The purpose of this study was to measure cyclic dimensional changes in the aorta and pulmonary artery of dogs by a very sensitive technique, mercury strain gauge plethysmography. Gauges were fixed around the pulmonary artery and the aorta and changes in vessel circumference were recorded under the following conditions: increased preload, increased afterload, decreased preload, and during administration of dobutamine, 5 and 10 micrograms/kg/min. Percent change in circumference of the aorta at baseline was small (mean circumference change = 4%, SD = 1%, SEE = 0.5). Percent change in the pulmonary artery at baseline was slightly larger (mean circumference change = 6%, SD = 5%, SEE = 2%). Mean aortic area change was 9% (SD = 2%) and for the pulmonary artery it was 12% (SD = 10%). No experimental manipulation significantly altered the percent cyclic change in vessel size from baseline except for clamping the inferior vena cava, in which mean percent change in aortic circumference decreased to 1% (SD = 1%, SEE = 5%) (p less than 0.05). Percent change in the aorta correlated only weakly with heart rate (r = 0.48), blood pressure (r = 0.4), and CO (r = 0.46). Similar values were obtained for the pulmonary artery. These data demonstrate a small but definite cyclic change in pulmonary artery area, and to a lesser extent in aortic area.

Doppler echocardiographic comparison of flows distal to the four cardiac valves.

Cardiac flows measured by the Doppler technique and invasive methods correlate well, but no prior study has correlated Doppler flows obtained distal to the four cardiac valves in the same individual. The purpose of this investigation was to measure the four flows in normal subjects using the range-gated pulsed Doppler echocardiographic technique. Velocities were obtained from 22 subjects aged 4 to 29 years at a beam-flow intercept angle close to 0 degree in the ascending aorta, distal main pulmonary artery and the tricuspid and mitral valve outflow areas. Vessel and orifice sizes of the ascending aorta, main pulmonary artery and tricuspid valve orifice were measured directly from two-dimensional echocardiographic images. The mitral valve orifice was measured by a previously described method. Results show that flow values for the ascending aorta, main pulmonary artery and tricuspid valve inflow area were similar in absolute magnitude and correlated strongly (r = 0.93 to 0.98). Slopes for these relations were approximated at 1.0. The absolute magnitude of mitral valve flows was highly variable and showed the poorest correlation with flows from the other areas (r = 0.59 to 0.67). The high correlation of ascending aorta, main pulmonary artery and tricuspid valve outflow areas was considerably assisted by recording of velocity at a verified angle near 0 degree and obtaining accurate vessel and valve diameters. Improved angle accuracy was possible in the ascending aorta with the use of a new transducer designed to image anteroinferiorly from the suprasternal notch. A relatively simple method for measuring tricuspid flow was developed.(ABSTRACT TRUNCATED AT 250 WORDS)