In higher vertebrates, coronary arteries supply blood to the thick muscular component of the heart where metabolic demand cannot be met by diffusion of oxygen from blood flowing through the interior of the heart. Although it was long believed that coronary vessels formed by angiogenesis as an outgrowth of the proximal aorta, it is now recognized that the coronary vessels initially arise by vasculogenesis (Mikawa and Fischman, 1992; Olivey et al., 2004). Thus, vessels are well-formed before contacting and eventually penetrating the proximal aorta. Precursor cells that form the coronary vascular system are contained within the proepicardium (PE), a cluster of somatopleural cells that arise anterior of the liver primordium adjacent to the sinus venosus (Ho and Shimada, 1978; Viragh and Challice, 1981).
In the chick, the PE forms at stage 14 (Hamburger and Hamilton, 1951) and contacts the heart at stage 16 in the region of the atrioventricular (AV) sulcus. Cells derived from the PE migrate over the myocardium as an epithelial sheet, covering the AV groove first and the truncus arteriosus last to form the epicardium (Viragh et al., 1993). Although migration is not complete until stage 24, epicardial cells initiate epithelial–mesenchymal transformation (EMT) at stage 17, immediately after migration over the heart has begun (Mikawa and Fischman, 1992). EMT can be divided into three steps: activation, invasion, and migration (Ramsdell and Markwald, 1997; and references therein). During activation, cells lose contact with adjacent cells and elongate. Cells enter the underlying matrix in a step termed invasion. Finally, cells migrate through the extracellular matrix. Epicardially derived mesenchymal cells invade the myocardium and give rise to cardiac fibroblasts and vascular smooth muscle cells (Poelmann et al., 1993).
Reports from our laboratory and others have demonstrated a role for transforming growth factor-β (TGFβ) signaling during EMT in the developing heart (Potts and Runyan, 1989; Potts et al., 1992; Brown et al., 1996, 1999; Desgrosellier et al., 2005). We chose to examine, therefore, the role of TGFβ signaling during EMT in the PE. TGFβ ligands bind to the type II TGFβ receptor (TBRII), allowing TBRII to phosphorylate the type I receptor activin receptor-like kinase (ALK) 5. ALK5 then phosphorylates intracellular signaling molecules, including members of the Smad family of transcription factors (Attisano and Wrana, 1998; Mulder, 2000). ALK5 activation signals TGFβ-dependent cell cycle arrest and induction of plasminogen activator inhibitor 1 (Bassing et al., 1994a, b). TBRII can also interact with another type I receptor, ALK2 (Ebner et al., 1993). ALK2 has been implicated in mediating TGFβ-stimulated EMT in AV cushion endocardial cells (Lai et al., 2000; Desgrosellier et al., 2005) and cultured NMuMG breast cancer epithelial cells (Miettinen et al., 1994). These data suggest that TGFβ may signal by the activation of ALK5 or ALK2.
Here, we demonstrate that addition of either TGFβ1 or TGFβ2 to PE explants increases the number of cells that enter a collagen matrix, a direct measure of EMT. Incubation of PE explants with TGFβ1 or TGFβ2 is also associated with decreased cytokeratin expression and a redistribution of the adherens junction protein ZO1, consistent with a loss of epithelial character. Both ALK5 and ALK2 are expressed in the PE before and after contact with the myocardium. caALK2 increases epithelial cell activation, whereas expression of caALK5 is without effect. Furthermore, we show that Smad6, an inhibitor of ALK2 signaling, is expressed in the PE, and overexpression of Smad6 decreases activation of PE explant epithelial cells. Neither fibroblast growth factor (FGF) 1, FGF7, nor bone morphogenetic protein (BMP) 7 affected transformation in PE explants, suggesting that the effects seen with TGFβ were specific. These data demonstrate that TGFβ stimulates EMT in the PE and suggest that this effect may be partially mediated through ALK2 in a Smad-dependent manner.
To assess EMT in response to TGFβ, we directly measured invasion of cells into a three-dimensional collagen matrix (Hay, 1995; Markwald et al., 1996). This culture system has been most widely used to study endocardial cell EMT and was originally shown to mimic closely the morphology and behavior of transformed mesenchymal cells in situ during endocardial cushion development (Bernanke and Markwald, 1982). Other investigators successfully have adapted this collagen culture system to examine EMT in PE explants (Mikawa and Gourdie, 1996; Dettman et al., 1998). Although the collagen matrix does not mimic the complex extracellular matrix environments found in either the endocardial cushions or the subepicardial matrix, this system does facilitate the study of the cellular behaviors that occur during EMT and has been used effectively to identify important signals that regulate EMT (Barnett and Desgrosellier, 2003; Olivey et al., 2004).
PE explants incubated on three-dimensional collagen gels with 200 pM TGFβ1 or TGFβ2 display a distinct phenotype by 48 hr in culture when compared with explants incubated with vehicle (Fig. 1A). At 48 hr, explants incubated with vehicle have an expansive epithelial sheet with elongate cells visible at the edges. In contrast, explants incubated with TGFβ1 or TGFβ2 had relatively small epithelial sheets, with an abundance of elongate, widely separated cells radiating from the explant. This difference between TGFβ1- or TGFβ2-incubated explants vs. vehicle-incubated explants was still evident at 72 hr. The number of transformed cells, that is, cells found in the collagen gel, was determined for both TGFβ- and vehicle-incubated explants at 72 hr. Both TGFβ1- and TGFβ2-incubated explants exhibited a significant increase in transformed cells when compared with explants incubated with vehicle (TGFβ1: 188.8 ± 10.1% of vehicle response, mean ± SEM; TGFβ2: 195.5 ± 12.3%; n = 21 TGFβ1-incubated explants, average 124 transformed cells per explant; n = 19 TGFβ2-incubated explants, average 129 transformed cells per explant; n = 22 vehicle-incubated explants, average 66 transformed cells per explant; P < 0.05, Student's t-test, Fig. 1B).
Because FGF has been demonstrated to stimulate EMT in PE-derived epicardium (Morabito et al., 2001), we tested FGF1 and FGF7 and observed that neither stimulates cell transformation in PE explants, even when cultured for 96 hr in the presence of growth factor (FGF1: 94.0 ± 6.2% of vehicle response, mean ± SEM; n = 39 FGF1-incubated explants, n = 38 vehicle-incubated explants; FGF7: 103 ± 7.2% of vehicle response, n = 37 FGF7-incubated explants, n = 41 vehicle-incubated explants; P > 0.05, Student's t-test; Fig. 1C). The number of transformed cells in TGFβ2-incubated explants remained elevated at 96 hr when compared with vehicle-incubated explants (data not shown). Taken together, these data demonstrate a difference in growth factor-stimulated cell transformation between TGFβ and FGF.
To support our morphological observations, we examined the expression and subcellular distribution of the epithelial markers cytokeratin and ZO1 in PE cells incubated on collagen-coated chamber slides. Others have reported that PE explants undergo the initial steps of EMT in two-dimensional culture systems (Lu et al., 2001). Consistent with these results, we observed that PE explants incubated with vehicle on collagen-coated slides form an expansive epithelial sheet composed of cells that express cytokeratin abundantly. These cells also express ZO1 at their periphery, demarcating cell–cell contact points between adjacent epithelial cells (Fig. 2A,D). In PEs incubated on collagen-coated chamber slides with either TGFβ1 or TGFβ2 we observed a decrease in cytokeratin staining (Fig. 2B,C) consistent with these cells undergoing the initial steps of EMT. The addition of either TGFβ1 or TGFβ2 altered the distribution of ZO1 in cells such that ZO1 immunoreactivity was less prominent at the cell periphery, consistent with a loss of cell–cell contacts and the transition from an epithelial to a mesenchymal cell phenotype (Fig. 2E,F).
Having observed that TGFβ increases the number of transformed cells in PE explants, we hypothesized that this effect is mediated by activation of a type I receptor. We performed in situ hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR) to examine expression of ALK5 and ALK2 in the PE. As TGFβ2 expression has been documented in the mouse PE and epicardium (Molin et al., 2003), we used a TGFβ2 probe as a positive control for in situ hybridization experiments. Consistent with observations in the mouse, we observed abundant TGFβ2 expression in the chick PE from stage 14 until stage 18, demonstrating expression both before and after contact with the myocardium (Fig. 3A,B). ALK2 (Fig. 3C,D) and ALK5 (Fig. 3E,F) are uniformly expressed at low levels throughout the PE. We also noted that Smad6, a known regulator of ALK2 signaling, is expressed in the PE (Fig. 3G,H). RT-PCR analysis confirmed the presence of each of these transcripts in the PE (Fig. 3I). Whereas Molin and colleagues did not observe TGFβ1 mRNA in the mouse PE using in situ hybridization (Molin et al., 2003), we did detect TGFβ1 in the chick PE by means of RT-PCR. This finding may reflect species differences in the expression pattern of this ligand or may be due to the ability of RT-PCR to detect smaller quantities of transcript.
To determine whether ALK2 or ALK5 could mimic the effects of TGFβ, we infected PEs with an adenovirus encoding green fluorescent protein (GFP) and caALK5, GFP and caALK2 (Wieser et al., 1995; Ward et al., 2002), or GFP alone. After 72 hr in culture, GFP-expressing cells were classified by morphology and position in or on the collagen gel as epithelial, activated, or transformed (Fig. 4C). We defined epithelial cells as rounded cells having more than one direct contact with the epithelial sheet. Activated cells were defined as elongate cells on the surface of the collagen gel having no more than one contact with the epithelial sheet. Transformed cells were defined as elongate cells in the collagen gel that lack contact with the epithelial sheet. The number of GFP-positive cells in each group—epithelial, activated, and transformed—was divided by the total number of GFP-positive cells to determine the percentage of each.
Cells infected with caALK2 adenovirus were significantly more likely to be activated (41.7 ± 2.4% vs. 34.0 ± 2.6%, mean ± SEM; n = 1,097 caALK2/GFP-infected cells in 34 explants examined, n = 1,039 GFP-infected cells in 31 explants examined; P < 0.05, Student's t-test; Fig. 4A) and less likely to be epithelial (46.3 ± 2.6% vs. 54.0 ± 1.7%; n = 1,341 caALK2/GFP-infected cells in 34 explants examined, n = 1,112 GFP-infected cells in 31 explants examined; P < 0.05, Student's t-test) than cells infected with adenovirus encoding GFP alone. We did not observe a difference in the percentage of transformed cells between explants infected with caALK2 adenovirus vs. GFP adenovirus (12.1 ± 0.7% vs. 11.9 ± 1.4%; n = 334 caALK2/GFP-infected cells in 34 explants examined, n = 233 GFP-infected cells in 31 explants examined; P > 0.05, Student's t-test). Infecting PE explants with caALK5 adenovirus did not alter the distribution of GFP-expressing epithelial, activated, and transformed cells when compared with cells infected with adenovirus expressing only GFP (epithelial: 53.9 ± 1.3% vs. 49.6 ± 3.3%, mean ± SEM; n = 1,560 caALK5/GFP-infected cells, n = 3,585 GFP-infected cells; activated: 33.0 ± 1.8% vs. 36.1 ± 4.1%; n = 939 caALK5/GFP- infected cells, n = 2,879 GFP-infected cells; transformed: 13.0 ± 0.7% vs. 14.4 ± 2.6%, n = 378 caALK5/GFP-infected cells, n = 1,005 GFP-infected cells; 29 GFP/ALK5-infected explants examined, 37 GFP-infected explants examined; P > 0.05 for all groups; Fig. 4B). These experiments demonstrate that ALK2 can mediate epithelial cell activation, the initial step in transformation, in PE explants, whereas ALK5 does not effect transformation in these cells.
As a second approach to support our implication of ALK2 in mediating epithelial cell activation, we overexpressed Smad6, a molecule demonstrated to inhibit signaling downstream of ALK2, and scored for cell activation in PE explants. Smad6 inhibits ALK2 signaling through Smad1 but has no effect on signaling through Smads 2 and 3 that are downstream of ALK5 (Macias-Silva et al., 1996, 1998; Imamura et al., 1997; Liu et al., 1997; Hata et al., 1998). Explants were infected with adenovirus coding for chicken Smad6 (Yamada et al., 1999) and GFP or adenovirus coding for GFP alone. PE explant cells overexpressing Smad6 were more likely to be epithelial (61.7 ± 2.6% vs. 51.0 ± 1.7%, mean ± SEM; n = 2,069 Smad6/GFP-infected cells in 30 explants examined, n = 5,163 GFP-infected cells in 32 explants examined; P < 0.05, Student's t-test; Fig. 5) and less likely to be activated (26.3 ± 2.9% vs. 37.8 ± 1.3%; n = 832 Smad6/GFP-infected cells in 30 explants examined, n = 3,905 GFP-infected cells in 32 explants examined; P < 0.05, Student's t-test) than were cells expressing only GFP, indicating that Smad6 inhibited epithelial cell activation. However, the percentage of transformed cells in explants infected with Smad6 adenovirus was similar to explants infected with GFP adenovirus (12.0 ± 0.4% vs. 11.2 ± 0.9%, n = 403 Smad6/GFP-infected cells in 30 explants examined, n = 1,171 GFP-infected cells in 32 explants examined; P > 0.05, Student's t-test). Inhibition of epithelial cell activation by Smad6 is confirmatory of our data that ALK2 increases epithelial cell activation.
ALK2 has been reported to signal downstream of BMP7 (ten Dijke et al., 1994; Macias-Silva et al., 1998). We thus asked if addition of BMP7 to PE explants could stimulate epithelial cell activation or transformation. PE explants were first infected with adenovirus encoding GFP to facilitate the analysis of cell phenotype as epithelial, activated, or transformed. Explants were incubated with BMP7 or vehicle for 72 hr. Unlike results obtained in explants infected with virus encoding caALK2, the distribution of epithelial, activated, and transformed cells in explants incubated with BMP7 was not significantly different than the distribution in explants incubated with vehicle (epithelial: 49.6 ± 6.2% vs. 52.7 ± 4.9%, n = 477 BMP7-incubated cells, n = 847 vehicle-incubated cells; activated: 29.6 ± 5.6% vs. 26.8 ± 4.8%, n = 365 BMP7-incubated cells, n = 490 vehicle-incubated cells; transformed: 20.8 ± 1.7% vs. 20.5 ± 2.9%, n = 227 BMP7-incubated cells, n = 358 vehicle-incubated cells; 22 BMP7-incubated explants and 24 vehicle-incubated explants examined; P > 0.05, Student's t-test; Fig. 6). These data suggest that ALK2-mediated epithelial cell activation is not downstream of BMP7 in the PE.
We report that both TGFβ1 and TGFβ2 but neither FGF1 nor FGF7 stimulate EMT in PE explants. PEs incubated with either TGFβ1 or TGFβ2 display altered expression or distribution of both cytokeratin and ZO1, consistent with cells undergoing EMT. caALK2 promotes epithelial cell activation, the initial step in EMT, whereas caALK5 has no effect on either activation or transformation. Furthermore, epithelial cell activation in explants is decreased by overexpressing Smad6, an inhibitor of Smad signaling downstream of ALK2. These data demonstrate that TGFβ stimulates PE cell EMT and that ALK2 mediates epithelial cell activation. BMP7, a known ligand for ALK2, does not affect EMT. Taken together, these data suggest that ALK2 signaling downstream of TGFβ may initiate EMT (Fig. 7).
Our finding that TGFβ stimulates EMT in the PE is consistent with the well-described role of TGFβ in stimulating EMT during embryonic development and tumorigenesis. Surprisingly, we found that caALK5, the canonical type I TGFβ receptor, did not mimic the effects of TGFβ in PE cells. However, caALK2, a type I receptor that can interact with the type II TGFβ receptor, initiates cell activation, the first step in EMT. ALK2 is reported to play a similar role in the TGFβ-stimulated EMT of endothelial cells in the heart during early valvulogenesis. Experiments using explants of a valve-forming region of the heart, the AV cushion, demonstrated that neutralizing antisera to ALK2 but not ALK5 blocked EMT (Lai et al., 2000). Furthermore, caALK2 introduced into normally nontransforming ventricular endocardial cells stimulated these cells to undergo EMT, whereas caALK5 did not (Desgrosellier et al., 2005). Therefore, our experiments using PE explants are a second example of a TGFβ-stimulated EMT in the developing heart that is not mimicked by ALK5 signaling alone.
Additionally, we have implicated Smad6, an inhibitor of ALK2 signaling, in regulating EMT in PE explants. The finding that Smad6 specifically inhibits cells from undergoing activation complements and supports our data that caALK2 causes PE cell activation. Our observations that Smad6 is expressed in the PE and inhibits epithelial cell activation in PE explants is especially significant, given the report that Smad6 null mice display abnormal coronary vessels (Galvin et al., 2000). In these animals, subepicardial vessels lack sufficient smooth muscle cells to maintain proper vascular wall integrity. It is unclear if this defect is caused by improperly regulated EMT or by deficient recruitment or differentiation of coronary vascular smooth muscle cell precursors. Our observation that Smad6 is expressed at the earliest stages of PE development suggests that loss of Smad6 may affect coronary vessel development at any stage, including formation of the PE, PE migration over the heart, EMT, or vessel assembly. Together, ALK2 and Smad6 may represent components of an important regulatory system that controls the number of PE-derived cells that can undergo activation and, ultimately, transformation, to supply the precursors for proper coronary vessel assembly.
Our data suggest that TGFβ and not BMP7 may activate ALK2 in PE cells. In 14-day-old in ovo chick atrial myocytes, TGFβ signals by means of ALK2 to decrease Gαi2 expression (Ward et al., 2002), whereas TGFβ signals primarily by means of ALK5 and decreases Gαi2 in 5 days in ovo cardiac myocytes. This dual regulation of cell responsiveness by a single growth factor, TGFβ, may be relevant to the report by Morabito and colleagues that TGFβ inhibits EMT in the PE-derived epicardium (Morabito et al., 2001), an effect completely opposite to what we observed in the cells of the PE itself. Our experiments were performed on PEs cultured before contact with the myocardium and formation of the epicardium, whereas Morabito and colleagues examined the effects of TGFβ in cultured epicardium. As was observed in the regulation of Gαi2 expression in 5 days in ovo vs. 14 days in ovo cardiac myocytes, it is possible that TGFβ elicits different cellular effects in the PE vs. the epicardium by activating different ALKs.
The failure of caALK2 to mimic fully the effects of TGFβ in mediating EMT may reflect a requirement for additional downstream signaling components. In endothelial cells, TGFβ requires both ALK5 and ALK1 signaling to regulate endothelial cell proliferation and migration. At lower concentrations, TGFβ stimulates endothelial cell proliferation and migration by means of ALK1 in an ALK5-dependent manner. As the TGFβ concentration is increased, TGFβ activates only ALK5-mediated pathways to inhibit endothelial cell proliferation and migration. Therefore, by activating ALK1 and ALK5, or ALK5 alone, TGFβ can both stimulate and inhibit endothelial cell proliferation and migration to balance angiogenesis (Goumans et al., 2002, 2003).
In our studies, not all epithelial cells undergo activation in response to caALK2, nor are all cells inhibited from undergoing activation by Smad6 overexpression. This heterogeneous cell response could be explained by several different mechanisms. First, the PE is composed of precursors for at least three different cell types—epicardial cells, vascular smooth muscle cells, and cardiac fibroblasts. Because most proepicardially derived cells contribute to the epicardium and remain epithelial, it may be that only cells not yet committed to an epicardial fate are competent to initiate EMT. Thus, cells in PE explants committed to become epicardium may be refractory to the manipulations described in our study. Furthermore, although both vascular cell precursors and cardiac fibroblast precursors undergo EMT, they may undergo EMT in response to different signals. Thus, misexpression of caALK2 or Smad6 would reasonably only affect a subset of PE cells.
In summary, we demonstrate a role for TGFβ in promoting EMT of PE cells. We have implicated ALK2 and Smad6 but not ALK5 in cell activation, the first step in EMT. These data suggest that ALK2 may be a component of TGFβ signaling pathways that regulate EMT during organogenesis and tumorigenesis.
Immunohistochemistry, In Situ Hybridization, and RT-PCR
For immunofluorescence experiments, PEs were cultured in BioCoat Collagen I-coated four-well chamber slides (BD Biosciences, Bedford, MA). For cytokeratin (Dako, Carpinteria, CA) staining, explants were fixed with 2% paraformaldehyde for 30 min at room temperature and permeabilized with phosphate buffered saline (PBS) plus 0.1% Triton X-100 for 5 min. For Zona Occludens-1 (ZO-1; Zymed, San Francisco, CA) staining, explants were fixed with ice-cold 70% methanol for 10 min. All explants were blocked in 2% bovine serum albumin (BSA), PBS for 1 hr and incubated with dilute primary antibody (cytokeratin-1:100; ZO-1–2 μg/ml) overnight at 4°C. Primary antibody was detected with goat anti-rabbit cy3 secondary antibody (1:800, Jackson ImmunoResearch, West Grove, PA). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma, St. Louis, MO).
In situ hybridization was performed as described previously, with the substitution of [α-33P]UTP for [α-35S]UTP (Barnett et al., 1994). Riboprobes against chicken TGFβ2 were made as described (Barnett et al., 1994). ALK5 nucleotides (nt) 184 to 468, ALK2 nt 247 to 467 (Lai et al., 2000), and Smad6 nt 886 to 1233 (Yamada et al., 1999) were subcloned into pGEM-3Z (Invitrogen, Carlsbad, CA) such that generation of both antisense and sense riboprobes was under control of the T7 RNA polymerase promoter. After hybridization, slides were coated with emulsion (Kodak NTB-2, Eastman Kodak Co., Rochester, NY) and exposed for 2 weeks. Sections were developed using Kodak Developer and Fixer and counterstained with hematoxylin. Light micrographs were captured on an Olympus Provis AX microscope using an Optronics digital camera.
RT-PCR reactions were carried out on RNA isolated from PEs harvested from chicks between Hamburger and Hamilton stages 15 and 18. Tissue was flash-frozen before total RNA was harvested using the NucleoSpin RNA II kit (Clontech, Palo Alto, CA). Primers for the RT-PCR reaction were designed to amplify the following genes and regions: TGFβ1, nt 214-526 (5′-ATGGACCCGATGAGTATTGG-3′ and 5′-GACGATGAGTGGCTCTCCTT-3′; Jakowlew et al., 1988); TGFβ2, nt 1151-1286 (5′-TCCAGTGGTGATGTGAAAGC-3′ and 5′-CTAGAGCACGCTTCTTCCGC-3′; Jakowlew et al., 1990); ALK5, nt 601-1057 (5′-TACAATTTCCATGTGAAGAT-3′ and 5′-TCTGAAGGAACTACTTTGAA-3′; Lai et al., 2000); ALK2, nt 181-635 (5′-AATGAATGTGTGTGTGAAGG-3′ and 5′-CTCGAGAATTGAGTCTCTCCATGT-3′; Lai et al., 2000); Smad6, nt 886-1383 (5′-TGCTGCAATCCGTACCACTTCAGC and 5′-AAAGTCCTACAGTTGGGGAT-3′; Yamada et al., 1999); and glyceraldehyde-3-phosphate dehydrogenase, nt 234-579 (5′-GGGCACGCCATCACTATCTTCC-3′ and 5′-GAGGGGCCATCCACCGTCTT-3′; Dugaiczyk et al., 1983). RT-PCR amplifications were performed using the Titan One Tube RT-PCR System (Roche, Indianapolis, IN). The identity of each amplification product was verified by mobility on a 4% agarose gel and by restriction endonuclease digestion.
PE Explants Incubated With TGFβ or FGF
Proepicardia were harvested from 3-day-old in ovo white Leghorn chicken embryos (Charles River/SPAFAS, N. Franklin, CT) and placed onto 0.12% collagen gels as previously described (Brown et al., 1996). Explants were incubated with M199 (Cambrex Bio Science, Walkersville, MD) containing 1:400 antibiotic/antimycotic (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum (Sigma) and either 200 pM recombinant human (rh) TGFβ1, 200 pM rhTGFβ2, 10 ng/ml rhFGF1, 10 ng/ml rhFGF7 (R&D Systems, Minneapolis, MN) or vehicle (0.1% BSA in 4 mM HCl for TGFβ, 0.1% BSA in PBS for FGF). Explants were cultured at 37°C in a 5% CO2 atmosphere for 72–96 hr and examined daily. At the end of the culture period, explants were fixed for 5 min with 2% paraformaldehyde in PBS at room temperature and washed extensively with PBS. Transformation was assayed directly by quantitating the number of cells that invaded the collagen matrix beneath each explant as previously described (Dettman et al., 1998). Briefly, each individual explant was optically sectioned using Hoffman optics by a naive observer to determine unequivocally whether individual cells were on the surface of the collagen gel or beneath it. All elongate cells clearly beneath the surface of the collagen gel under each explant were scored as transformed. A subset of explants were re-read by a second naive observer to ensure consistency. The mean number of invasive cells per explant was determined and compared between TGFβ1- and vehicle-, TGFβ2- and vehicle-, FGF1- and vehicle-, or FGF7- and vehicle-incubated groups using Student's t-test. Results from either three or four consecutive, independent experiments are shown for each treatment group.
PE Explants Infected With Adenovirus
Adenoviral constructs coding for GFP alone or GFP and caALK2, caALK5 (Ward et al., 2002), or Smad6 (Yamada et al., 1999) were made according to the method of He et al. (1998). Constitutive activity of proteins produced by each virus was tested using luciferase reporter plasmids that are activated by either ALK5 (3TP-lux; Wrana et al., 1992) or ALK2 (pVent-luc; Onichtchouk et al., 1999). Activity of Smad6 protein produced by viral infection was determined by its ability to decrease Smad1-dependent alkaline phosphatase activity in C3H10T1/2 cells (McDonnell et al., 2001). Data regarding activity of viral constructs have been reported (Desgrosellier et al., 2005).
Proepicardia were harvested as described above. After harvest, explants were incubated in 50 μl of culture medium (M199 containing 10% fetal calf serum and 1:400 antibiotic/antimycotic) and infected with 2 × 106 to 2 × 107 pfu adenovirus coding for either GFP alone or both GFP and caALK2, caALK5, or Smad6. After infection, explants were cultured as described above for 72 hr. At the end of the culture period, explants were fixed in PBS containing 0.05% glutaraldehyde and 0.8% formaldehyde for 5 min at room temperature and washed extensively in PBS.
Infected cells, identified by GFP expression, were counted using Hoffman optics on a Nikon Eclipse TE2000-E inverted fluorescent microscope and photographed with a QImaging digital camera. Cells were identified as epithelial, activated, or transformed based on morphology and position in the gel. Epithelial cells were defined as rounded cells having more than one direct contact with the epithelial sheet. Activated cells were defined as elongate cells on the surface with one or zero contacts with the epithelial sheet. Transformed cells were defined as elongate cells in the collagen gel that lack contact with the epithelial sheet. The percentage of GFP-expressing cells that were epithelial, activated, or transformed was determined for each group (GFP alone, GFP–caALK2, GFP–caALK5, GFP–Smad6). Mean percentages from four consecutive, independent experiments were determined for each group and compared with GFP alone using Student's t-test.
PE Explants Infected With GFP Adenovirus and Incubated With BMP7
Explants were infected with GFP adenovirus and subsequently incubated with 15 nM rhBMP7 (R&D Systems) or vehicle (0.1% BSA in 4 mM HCl) for 72 hr. Explants were fixed and GFP-positive cells were scored as epithelial, activated, or transformed as described. Mean percentages from four consecutive, independent experiments were determined for each group and compared with GFP alone using Student's t-test.
We thank Dr. Charles Lin, Dr. Chris Brown, and the members of the Barnett laboratory for their critical reading of the manuscript. We thank Jay Desgrosellier, Leigh Compton, Tiffany Davis, Dru Potash, Brian Culbreath, and Todd Townsend for reagents and assistance. Histological sections were generated by the Vanderbilt Vascular Histology Imaging Core facility. J.V.B. acknowledges the support of the Vanderbilt-Ingram Cancer Center.