Department of Cell Biology and Anatomy, University of Arizona, Tucson, Arizona 85724
Received January 15, 1999; accepted May 11, 1999
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ABSTRACT |
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Key Words: cardiogenesis; epithelial-mesenchymal cell transformation; cardiac valve formation; TCE; Mox-1.
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INTRODUCTION |
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Cardiac teratogenicity of TCE was subsequently studied in both chicken and rat (Dawson et al., 1990, 1993
; Loeber et al., 1988
). In chicken, TCE (doses calculated to be 15150 ppm) were injected in ovo at various stages of development and 7.3% of TCE-treated hearts had defects (Loeber et al., 1988
). Cardiac defects observed in the chick study included both inflow and outflow anomalies. Rats were exposed to TCE by either intrauterine osmotic minipumps (Dawson et al., 1990
) or through maternal drinking water (Dawson et al., 1993
). These studies demonstrated TCE cardiac teratogenicity in rats down to a dose of 1.5 ppm (Dawson et al., 1993
; Johnson et al., 1998
).
Other metabolites of TCE, including dichloroethylene, trichloroacetic acid and dichloroacetic acid, were also shown to produce heart defects (Epstein et al., 1992; Johnson et al., 1998
). Interestingly, exposure to dichloroacetic acid produced a specific cardiac defect in the rat, a high ventricular (membranous) septal defect (Epstein et al., 1992
). In both species, the effective level of TCE or metabolites in these experiments remained at least 2 orders of magnitude higher than seen in the Tucson groundwater.
Our understanding of valve and septal formation in the heart provides an avenue for further cellular and molecular analysis of the teratologic effects of TCE. The basic events of cardiac valve formation can be summarized as follows. Early in development, the heart is a hollow tube-like structure with 2 cell layers. The outer surface is a myocardial cell layer and the inner luminal surface is an endothelium. Between the two cell layers lies an expanse of extracellular matrix (ECM). At a specific time in development, a subpopulation of endothelial cells lining the atrioventricular (AV) canal detaches from adjacent cells and invades the underlying ECM (Markwald et al., 1984). This event is termed an epithelial-mesenchymal cell transformation (Fig. 1A
). These endothelial-derived mesenchymal cells migrate towards the surrounding myocardium and begin to proliferate in order to populate the entire AV canal ECM. Cardiac mesenchyme provides the cellular constituents of the septum intermedium and the valvular leaflets of the mitral and tricuspid valves. The septum intermedium subsequently contributes to the lower portion of the atrial septum and the membranous portion of the ventricular septum (Markwald et al., 1984
; Wessels et al., 1996
) (Fig. 1B
).
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Progress in understanding epithelial-mesenchymal cell transformation in the AV canal of the heart is mainly due to the development of an in vitro culture system (Bernanke and Markwald, 1982; Runyan and Markwald, 1983
). The in vitro AV canal culture mimics the in situ temporal and regional specificity of cardiac epithelial-mesenchymal cell transformation. Components of cardiac epithelial-mesenchymal cell transformation, including activities of cardiac cushion cells, endothelial cell activation, and mesenchymal cell transformation and migration have been extensively studied in the chick system (Boyer et al., 1999a
,b
; Brown et al., 1996
, 1999
; Krug et al., 1987
, 1985
; Loeber and Runyan, 1990
; Mjaatvedt et al., 1987
; Potts et al., 1991
, 1992
; Potts and Runyan, 1989
; Ramsdell and Markwald, 1997
; Runyan et al., 1992
).
In order to examine the molecular mechanisms of TCE effects on cardiac development, we used the in vitro chick AV canal explants model to study the process of epithelial-mesenchymal cell transformation in the presence of TCE. Although other metabolites may be more potent, application of this toxicant to the target tissue permitted a direct evaluation of TCE as a potential cardiac teratogen. In this study, we show that TCE perturbs endothelial cell separation and mesenchymal cell formation processes during epithelial-mesenchymal cell transformation.
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MATERIALS AND METHODS |
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Measurement of epithelial-mesenchymal cell transformation.
Epithelial-mesenchymal cell transformation was measured by counting the number of mesenchymal cells inside the collagen gel matrix (Fig. 1D) for each explant on an Olympus IMT-2 inverted microscope equipped with Hoffman Modulation Optics (Hoffman Optics, Brooklyn, NY). A total of 15 explants were counted for each dose of TCE or control.
Endothelial cell density measurement.
Endothelial cells from 250 ppm TCE-treated and control AV explant cultures were visualized with Hoffman Modulation Optics. Micrographs of endothelial cells were taken with a Dage CCD camera and a Scion frame grabber on a Macintosh 7500 computer using NIH Image software. A frame of 220 µm x 220 µm was placed over each micrograph, with the center in between the edge of the endothelial cells and the edge of myocardium. The number of endothelial cells within the frame was counted. Micrographs from 7 explants were counted for each treatment. Statistical analysis was performed using the Student's t-test.
Cell migration assay.
In order to produce a population of mesenchymal cells, AV-canal explants from Stage 17 chick embryos were placed onto collagen gels and incubated for 18 h. Cultures then were treated with medium 199+ or TCE (250 ppm). Thirty min after the addition of medium 199+ or TCE, the lateral migration of a population of mesenchymal cells in each culture was videotaped for 2 h. Videomicrography was performed, using an inverted Olympus IMT microscope equipped with an incubator stage, maintained at 37°C and infused with CO2 to maintain the pH of the medium. Cellular migration distance was obtained by measuring the change in location of the centroid of each cell at 5-min intervals using a computer equipped with a Bioquant Image Analysis System (R & M Biometrics, Nashville, TN).
Immunohistochemistry.
AV canal explants from Stage 16 chick embryos were placed onto medium 199+ (control for TCE) or TCE-treated (250 ppm) collagen gels as described above. The explants were treated with medium 199+ or TCE after 6 h of incubation. Explants then were incubated for an additional 48-h and fixed in 1% PFA for 30 min. Collagen gels were rinsed extensively in phosphate-buffered saline (PBS) before immunostaining. Primary antibodies used were anti--smooth muscle actin (mouse monoclonal, 1:400 dilution, Sigma, St. Louis, MO), Mox-1 (mouse monoclonal, 1:200 dilution, gift of Dr. C. Wright), and fibrillin 2 (mouse monoclonal, 1:200 dilution, gift of Dr. C. Little). Primary antibody incubation was carried out overnight at 4°C. Secondary antibody (Cy-5 conjugated anti-mouse antibody at 1:200 dilution) incubation was overnight at 4°C. Immunostained collagen gels were placed on slides, covered with glycerol, and sealed with nail polish. Immunostained AV explants then were viewed on a Leica confocal microscope. For each experimental group (control vs. TCE), the immunostaining procedures, and the settings on the confocal microscope were identical.
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RESULTS |
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In order to test the effects of TCE on epithelial-mesenchymal cell transformation, AV-canal explants from Stage 16 chick embryos were placed on the surface of collagen gels containing 0250 ppm TCE, and cultured as described in Materials and Methods. After 6 h of culture, additional medium containing TCE at the desired concentration was added, and the explants were cultured for an additional 48 h before fixation in 4% PFA. The effect of TCE on epithelial-mesenchymal cell transformation is shown in Figure 2. Endothelial cells form a monolayer on the surface of untreated control cultures, displaying a polygonal organization and the cellcell separation indicative of activation (Fig. 2A
) (Bolender et al., 1980
; Crossin and Hoffman, 1991
; Krug et al., 1985
). In TCE-treated cultures, endothelial cells formed a monolayer with a lesser degree of cellcell separation (Fig. 2B
). Figure 2C
shows mesenchymal cells from control explant invading inside the collagen gel. TCE-treated cultures (250 ppm) had fewer mesenchymal cells compared to control (compare Figs. 2C and 2D
). Therefore, TCE treatment inhibited both endothelial cell activation and normal mesenchymal cell formation in AV cushion cultures.
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Immunofluorescent staining revealed that expression of Mox-1 and fibrillin 2 was affected by TCE (Fig. 5). Mox-1 and fibrillin-2 immunostaining intensity in mesenchymal cells was strongly inhibited by TCE (compare Figs. 5A and 5B, 5C and 5D
). In contrast,
-smooth muscle actin expression was not altered by TCE treatment (Figs. 5
E and 5F). These data suggest that TCE has no effect on early stages of endothelial cell activation prior to cellcell separation. However, TCE does have a specific effect on a subset of mesenchymal cell markers expressed during endothelial cellcell separation and mesenchymal cell formation.
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DISCUSSION |
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Based on these studies, we hypothesize that TCE mediates cardiac valve and septa malformation by perturbing one or more specific events of epithelial-mesenchymal cell transformation. The objectives of our studies were to investigate the cellular and molecular mechanism of the cardiac teratogenicity of TCE. First, we sought to determine whether TCE perturbs epithelial-mesenchymal cell transformation. Second, using specific proteins as molecular markers of transformation, the effects of TCE on cardiac epithelial-mesenchymal cell transformation were confirmed at the molecular level. We exposed Stage 16 chicken AV canal cultures to 0250 ppm TCE in vitro and determined that the epithelial-mesenchymal cell transformation is sensitive to TCE exposure. In particular, we observed that mesenchymal cell numbers, as a measurement of epithelial-mesenchymal cell transformation, are inhibited by TCE at all doses tested. In order to identify TCE-sensitive events during valve morphogenesis, we treated Stage 14 AV canal cultures with TCE and found that there is no difference in the effect of TCE on epithelial-mesenchymal cell transformation between younger and older stages. This observation suggests that TCE is perturbing processes immediately proximal to the actual change in endothelial cell phenotype and that it has little effect on the preparatory and inductive events during epithelial-mesenchymal cell transformation that took place at earlier stages (Boyer et al. 1999a; Ramsdell and Markwald, 1997
). The decrease of mesenchymal cell numbers in TCE-treated cultures is probably due to a perturbation of cellcell adhesion that occurs within the endothelial layer.
An inhibition of endothelial cellcell separation similar to that seen with TCE was also observed in cultures treated with pertussis toxin (an inhibitor of Gi proteins), or blocking antibodies toward both TGFß-2 and the TGFß Type III receptor (Boyer et al, 1999a,b
). Since all of these reagents also inhibited mesenchymal cell migration, the observation that TCE has no effect on cell migration suggests that TCE may perturb epithelial-mesenchymal cell transformation through a separate mechanism from TGFß and G protein signal transduction processes.
To determine the TCE effect on epithelial-mesenchymal cell transformation at the molecular level, the expression of molecular markers that are associated with epithelial-mesenchymal cell transformation was examined. Though a variety of proteins is known, the 3 molecules chosen here provide a representative sample of markers. Mox-1 is a mediator of mesenchymal cell formation and Mox-1 protein and mRNA are expressed in both endothelial and mesenchymal cells in the AV canal at the time of transformation (Boyer et al., 1999a; Wendler and Runyan, in preparation). We found that expression of Mox-1 protein was dramatically inhibited by exposure to TCE. The expression of an ECM protein fibrillin 2, as a marker of mesenchymal cell formation, was also greatly inhibited. In comparison, the early endothelial cell activation marker,
-smooth muscle actin, was unaffected by TCE. In addition, a mesenchymal cell migration marker, Type I collagen (Sinning et al., 1988
) was not affected by TCE (data not shown).
The contrasting effects of TCE on fibrillin 2 and -smooth muscle actin and the normal rate of migration seen in the presence of TCE suggest that this toxicant perturbs specific developmental processes. The expression of cell migration marker Type I collagen and the rate of mesenchymal cell migration are unperturbed by the TCE, indicating that transformed mesenchymal cells are insensitive to TCE. TCE-sensitive events during epithelial-mesenchymal cell transformation are restricted to the period of visible cellcell separation and cell shape change. The observation that Mox-1 expression is reduced could account for the loss of mesenchymal cell formation and the reduced expression of fibrillin 2. Antisense oligonucleotide experiments demonstrate that Mox-1 is required for epithelial-mesenchymal cell transformation in cardiac explant cultures (Wendler and Runyan, unpublished). However, since several transcription factors are specifically expressed in the AV canal of the heart, including brachyury (Huang et al., 1995
), slug (Romano and Runyan, submitted), GATA 4/5/6 (Jiang et al., 1998
), Id (Evans and O'Brien, 1993
), and NF-ATc (de la Pompa et al., 1998
; Ranger et al., 1998
), we were unable to distinguish whether the effect of TCE on Mox-1 is direct or indirect. Since our current model of epithelial-mesenchymal cell transformation in the heart suggests that mesenchymal cell formation is the product of multiple, independent signal transduction pathways into and within the target cell (Boyer et al., 1999a
), TCE inhibition of Mox-1 may be sufficient to reduce mesenchymal cell numbers.
Although the present study points to TCE inhibition of elements of epithelial-mesenchymal cell transformation as the basis of cardiac valvular and septal defects, not all cardiac defects seen in treated animals are due to defects in early valve formation. Other potential sources of cardiac defects include a loss of neural crest cells (Kirby and Waldo, 1990), altered blood flow patterns (Hogers et al., 1997
), and myocardial cell deficits (Vikstrom et al., 1996
). It is likely that several of these elements could be affected by TCE. In a concurrent study, TCE-treated rat embryo hearts are being examined for changes in gene expression compared to untreated controls. To date, we have identified more than 40 molecules whose expressions are either up- or down-regulated in response to 110 ppm TCE exposure in maternal drinking water. These molecules include a variety of stress-response genes, housekeeping genes, cytoskeletal components, and developmentally expressed genes (Collier et al., in preparation). Ongoing studies will explore the localization of gene expression in the developing heart and the functional significance of changes in several of these candidates.
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ACKNOWLEDGMENTS |
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NOTES |
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2 To whom correspondence should be addressed at Department of Cell Biology and Anatomy, College of Medicine, University of Arizona, Life Sciences North, Rm. 421, Tucson, AZ 85724. Fax: (520) 626-2097. E-mail: rrunyan{at}u.arizona.edu.
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