Department of Anatomy, Saitama Medical School, Moroyama-cho, Iruma-gun, Saitama, 350-04 Japan; and * Department of Biochemistry, The Cancer Institute, Japanese Foundation for Cancer Research, Kami-Ikebukuro, Toshima-ku, Tokyo, 170 Japan
Transforming growth factor- (TGF
) is a
dimeric peptide growth factor which regulates cellular
differentiation and proliferation during development.
Most cells secrete TGF
as a large latent TGF
complex containing mature TGF
, latency associated peptide, and latent TGF
-binding protein (LTBP)-1. The
biological role of LTBP-1 in development remains
unclear. Using a polyclonal antiserum specific for
LTBP-1 (Ab39) and three-dimensional collagen gel culture assay of embryonic heart, we examined the tissue distribution of LTBP-1 and its functional role during the formation of endocardial cushion tissue in the
mouse embryonic heart. Mature TGF
protein was required at the onset of the endothelial-mesenchymal
transformation to initiate endocardial cushion tissue
formation. Double antibody staining showed that
LTBP-1 colocalized with TGF
1 as an extracellular fibrillar structure surrounding the endocardial cushion
mesenchymal cells. Immunogold electronmicroscopy
showed that LTBP-1 localized to 40-100 nm extracellular fibrillar structure and 5-10-nm microfibrils. The
anti-LTBP-1 antiserum (Ab39) inhibited the endothelial-mesenchymal transformation in atrio-ventricular
endocardial cells cocultured with associated myocardium on a three-dimensional collagen gel lattice. This
inhibitory effect was reversed by administration of mature TGF
proteins in culture. These results suggest
that LTBP-1 exists as an extracellular fibrillar structure
and plays a role in the storage of TGF
as a large latent
TGF
complex.
Transforming growth factor- TGF There are at least three forms of LTBP (LTBP-1,
LTBP-2, and LTBP-3) which recently have been cloned
and sequenced (Kanzaki et al., 1990 Epithelial-mesenchymal interaction is one of the most
important embryonic phenomena in the organization of
the body axis, as well as in organogenesis. It is regulated via
genes transcribed in a spatiotemporally restricted manner
during development. Endothelial-mesenchymal transformation during the formation of endocardial cushion tissue is one of the most studied examples of epithelial-mesenchymal interaction in the embryogenesis (Markwald et al.,
1975 Using an antiserum specific for LTBP-1 (Ab39), and an
AV endothelial bioassay employing a three-dimensional
collagen culture system (Bernanke and Markwald, 1982 Materials
Rabbit antiserum (Ab39) was raised against native LTBP-1 purified from
human platelets (Kanzaki et al., 1990
Animal Breeding
ICR (Jcl:ICR) mice were purchased from Japan Clea Co. (Tokyo, Japan).
The mice were kept at 23°C with a 12/12-h light/dark cycle and were allowed free access to water and a standard pellet diet. Females were placed
together with a male for 12 h, and mating was confirmed by the presence
of a vaginal sperm plug (day 0 of gestation). On day 9.5, pregnant mice
were sacrificed by cervical dislocation and a Caesarean section performed.
The heart was removed from each embryo and subjected to the experiments described below.
Three-dimensional Collagen Gel Culture of the
AV Region
Hydrated collagen gel (1 mg/ml type I rat-tail collagen; Collaborative Research, Waltham, MA) was prepared in 4-well dishes (Nunc, Roskilde,
Denmark) as described by Bernanke and Markwald (1982) Gel Electrophoresis and Immunoblotting
Whole embryos (9.5 or 14.5 d) were homogenized in ice-cooled RIPA
buffer (50 mM Tris-HCl at pH 7.2, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate). The deoxycholate-insoluble pellet was collected at
8,000 g for 10 min. The pellet was washed in PBS, and then digested with
plasmin (0.1 U/ml) in PBS for 1 h at 37°C. The resulting supernatant, containing ~50 µg of protein, was concentrated by ultrafiltration (10-kD cutoff; Millipore, Tokyo), diluted into nonreducing SDS-PAGE solubilization buffer (63 mM Tris-HCl at pH 6.8, containing 2% SDS, and 6 M
urea), boiled for 5 min, and subjected to 6% SDS-PAGE (Laemmli, 1970 cDNA Transfection, Metabolic Labeling,
Immunoprecipitation, and Gel Electrophoresis
COS-1 cells were transfected with pSV7d-LTBP-1 (Kanzaki et al., 1990 Indirect Immunofluorescence Microscopy
Embryos (9.5-10.5 d) were equilibrated in a graded series of sucrose solutions (10-20%, wt/vol) in PBS at 4°C for 12 h, embedded in OCT compound (Miles, IN), and frozen in 2-methylbutane cooled over liquid nitrogen. Frozen sections (6-8 µm) were cut on a cryostat, mounted on slides
coated with 3-triethoxysilyl propylamine (Merck, Germany), and air
dried. After rinsing in PBS for 15 min, sections were blocked with 1% bovine serum albumin (BSA) in PBS for 1 h, and incubated with primary antibody (Ab39 was diluted 1:500-1,000 in blocking solution) in a moist
chamber for 2 h at room temperature. They were then rinsed in PBS, incubated with a tetramethylrhodamine-5-(and-6)-isothiocyanate (TRITC)-
conjugated goat antibody against rabbit IgG (Cappel, Malvern, PA) for
1 h, rinsed with PBS and coverslipped with the mounting medium (0.2 M
N-propylgallate in 10% PBS/90% glycerol). Similarly prepared fresh frozen sections were incubated with plasmin (0.1 U/ml in PBS) at 37°C for 1 h.
The resulting sections were rinsed in PBS for 30 min and stained with
Ab39, as described above.
The fresh frozen sections were processed for double antibody staining
for anti-TGF AV explant cultures grown on collagen gels were processed for wholemount double immunostaining. Cultures were drained of medium, rinsed
with PBS, fixed with 4% paraformaldehyde in PBS for 1 h, rinsed with
PBS, blocked for 1 h with 1% BSA in PBS, incubated with primary antibody mixture (Ab39 and anti-TGF Immunogold Electron Microscopy
Post-embedding for indirect immunoelectron microscopy was performed
using the method described by Tamaki and Yamashina (1994) The pre-embedding method for AV-regions was as follows. They were
dissected out from 9.5-d embryos and incubated with Ab39 in 0.1% BSA/
PBS for 12 h at 4°C, rinsed in PBS, and incubated in colloidal gold-conjugated secondary antibody in 0.1% BSA/PBS at 4°C for 12 h. After the extensive washing in PBS, they were fixed in 2.5% glutaraldehyde/4%
paraformaldehyde in PBS for 2 h at 4°C and postfixed in 1% OsO4/PBS
for 40 min at 4°C. Samples were dehydrated in a graded ethanol series and
embedded in Epon. Ultra-thin sections were cut, mounted on 150-mesh
grids and stained with uranyl acetate and lead citrate. Specificity for immunostaining was confirmed by preincubating the antiserum with purified
human LTBP-1 or recombinant human LTBP-1. Samples were observed
using a transmission electron microscope (JEM-100C; JEOL, Tokyo) operated at 80 kV.
Ab39 Recognizes Mouse LTBP-1
In this study, we used a polyclonal antiserum (Ab39)
raised against a purified human LTBP-1 (Kanzaki et al.,
1990
TGF Before examining the function of LTBP-1 during endocardial cushion tissue formation, we examined whether TGF Table I.
Anti-TGFs (TGF
s) are 25-kD
dimeric peptide growth factors and are thought to
regulate the signals by which primary and secondary inductions are initiated at different stages of embryogenesis in vertebrates. Several TGF
s and their receptors
are expressed in developing organs, and their tissue distribution pattern has possible significance for signaling roles in epithelial-mesenchymal interaction during embryogenesis (Sporn et al., 1986
; Barnard et al., 1990
; MacLellan et al.,
1993
; Akhurst, 1994
). Although it is clear that TGF
s are
important molecules in the regulation of cellular differentiation and proliferation, it is still unknown how the activity of this growth factor is controlled under the physiological conditions. The biological activity of TGF
is thought
to be carefully controlled in a number of ways, via mRNA
expression and protein synthesis, tissue or cellular distribution of receptors, presence of a latent form of TGF
, activation of the latent form of TGF
, and inactivation of
the active form (Miyazono et al., 1993
, 1994).
is released from cells in a secretory form consisting of the mature growth factor associated with an NH2terminal peptide, a latency associated peptide (LAP)1, and a
latent TGF
-binding protein (LTBP)-1. This complex, the so-called large latent TGF
complex, is unable to bind
TGF
receptors and is physiologically inactive (Pircher et
al., 1986
; Lawrence et al., 1985
; Miyazono et al., 1991
). The
high molecular weight form of the large latent TGF
complex contains LTBP-1, which is a glycoprotein of more
than 190 kD and associated with the LAP via a disulfide
bond (Miyazono et al., 1988
; Wakefield et al., 1988
; Saharinen et al., 1996
). The mature TGF
is noncovalently associated with the LAP; this complex is called the small latent TGF
complex. The dissociation of the LAP renders the
TGF
biologically active. The large latent TGF
complex
is activated by proteolysis of the latent complex by plasmin (Lyons et al., 1988
; Sato and Rifkin, 1989
; Taipale et
al., 1992
). The latent TGF
is also activated by thrombospondin in certain cell types (Schultz-Cherry and MurphyUllrich, 1993; Souchelnitskiy et al., 1995
).
; Tsuji et al., 1990
;
Moren et al., 1994
; Gibson et al., 1995
; Yin et al., 1995
).
LTBP-1 possesses 16-18 EGF-like domains, two of which
contain hydroxyasparagine posttranscriptional modifications, and a novel motif containing eight cysteine residues (Kanzaki et al., 1990
; Tsuji et al., 1990
). These sequence
characteristics of LTBP are also found in microfibrillar
components, fibrillins, which suggests that LTBPs are involved in protein-protein interaction with cell surface
molecules, as well as with the extracellular matrix (Apella
et al., 1988; Rao et al., 1995
). In the case of bovine endothelial cells cocultured with smooth muscle cells, targeting
of the latent TGF
complex to smooth muscle cells is required for the activation of latent TGF
. Since LTBP-1
contains structural motifs involved in protein-protein interaction, LTBP-1 is thought to target the large latent
TGF
complex on the surface of certain cells in the triggering of the biological activity of TGF
. Flaumenhaft et
al. (1993)
reported that LTBP-1 may play a role in the activation of the large latent TGF
complex on the endothelial cell surface by concentrating the latent complex on the
cell surface. In a model based on a human fibroblast culture, secreted TGF
1 associates with the extracellular matrix via the targeting mechanism involving LTBP-1 of the
large latent TGF
1 complex. In this model, the association
between LTBP-1 and the extracellular matrix is mainly covalent, and the release of latent TGF
is due to a cleavage
of LTBP-1 by plasmin (Taipale et al., 1992
, 1994). It remains unclear what the biological functions and tissue distributions of LTBP-1 actually are in those regions where
TGF
-dependent cellular differentiation occurs during
development.
, 1977
; Krug et al., 1985
, 1987; Mjaatvedt and Markwald, 1989
; Mjaatvedt et al., 1991
; Nakajima et al., 1994
,
1996). During its early development, the heart consists of
two concentric epithelial layers, endocardium and myocardium, which are separated by an expanded acellular extracellular matrix (cardiac jelly). As development proceeds,
some of the endothelial cells of both the outflow tract
(OT) and atrioventricular (AV) regions change their phenotype to that of mesenchymal cells and migrate into the
adjacent cardiac jelly. This is known as endothelial-mesenchymal transformation. It leads to the formation of endocardial cushion tissue of the primordia of both the
valves and septa of the adult heart. This endothelial-mesenchymal transformation is regulated, at least in part, by
TGF
3 during endocardial cushion tissue formation in the
chicken embryonic heart (Potts et al., 1989, 1991; Nakajima et al., 1994
). During murine endocardial cushion tissue formation, three TGF
s (TGF
1, TGF
2, and TGF
3)
are preferentially distributed within the cushion tissueforming region in the heart, TGF
1 being expressed in endothelial/mesenchymal cells and TGF
2 and
3 in the
outer myocardium (Akhurst et al., 1990
; Dickson et al.,
1993
; Mahmood et al., 1992
, 1995
). Although the tissue
distribution of TGF
s has been well examined at mRNA
and protein levels, the functional significance of TGF
in
the formation of murine endocardial cushion tissue has yet
to be determined. In addition, the tissue distribution and
functional role of LTBP-1 during cushion tissue formation
remains unknown.
),
we have examined the tissue distribution of LTBP-1 and
its functional role during TGF
-dependent endocardial
cushion tissue formation in the mouse embryonic heart. Double immunohistochemical staining of antibodies specific for LTBP-1 (Ab39) and TGF
1 revealed that an
LTBP-1 molecule was codistributed with TGF
1 as a fibrillar extracellular matrix in the cushion tissue at the onset
of, or during cushion tissue formation. Immunoelectron microscopy showed that LTBP-1 was associated with 40-
100 nm fibrillar structures in the extracellular matrix surrounding mesenchymal cells. Ab39 inhibited mesenchymal
formation in the AV endothelial bioassay and this inhibitory effect was reversed by administration of mature
TGF
proteins. These results suggest that LTBP-1 exists as an extracellular fibrillar structure and plays a role in the storage of the latent TGF
complex during the formation
of the endocardial cushion tissue.
Materials and Methods
; Miyazono et al., 1991
); it does not
cross-react with LTBP-2 (Moren et al., 1994
; Fig. 1). This antiserum did
not cross-react with mouse fibronectin in immunoblot (data not shown).
Rabbit polyclonal antiserum (Ab178) was raised against a synthetic peptide for human LTBP-2 (Moren et al., 1994
). A neutralizing antibody,
anti-human TGF-
1 antibody (anti-TGF
1, King Brewing, Kakogawashi, Japan), was raised against human recombinant TGF
1 in rabbit, and
affinity purified. This antibody reacts with TGF
1 and TGF
2 and inhibits the TGF
1-induced production of collagen by NRK-49F to 75% of
control at 50 µg/ml (manufacturer's description). Anti-TGF
1-IgY (R & D
systems, Minneapolis, MN) was raised against recombinant human
TGF
1 in chicken and this antibody shows<2% cross-reactivity with
TGF
2 and TGF
3 in immunoblot (manufacturer's description). This antibody did not cross-react with LTBP-1 and mouse fibronectin in immunoblot (data not shown). A neutralizing antibody against chicken recombinant TGF
3 (anti-TGF
3) raised in goat was purchased from R & D
Systems. This antibody completely neutralizes the TGF
3-dependent inhibition of IL-4-dependent thymidine incorporation by HT-2 cells at 10 µg/ml, but does not inhibit the activity of TGF
1, TGF
2, or TGF
5 (manufacturer's description). Recombinant human TGF
1, purified human TGF
2, and recombinant chicken TGF
3 molecules were purchased from King Brewing, Nakarai tesque (Tokyo, Japan), and R & D Systems, respectively. Recombinant human LTBP-1 was generously donated by H. Ohashi (Kirin Brewery Co. Ltd., Maebashi, Japan).
Fig. 1.
Specificity of anti-LTBP-1 antiserum (Ab39). COS-1
cells were transfected with human LTBP-1 (BP-1) or LTBP-2
(BP-2) cDNA. The cells were metabolically labeled, and resulting
conditioned medium was immunoprecipitated with Ab39 or anti-
LTBP-2 antiserum (Ab178) and analyzed by SDS-PAGE (5-
20% linear gradient gel) in the presence of dithiothreitol.
[View Larger Version of this Image (32K GIF file)]
. The hearts
from the mouse embryos were collected and placed in Dulbecco's PBS.
The AV region was dissected out and cut longitudinally to expose the lumen, and then placed on the drained collagen gel that had been saturated
with CM199 (CM199; medium 199 containing 5% FBS, 5 µg/ml insulin, 5 µg/
ml transferrin, 5 ng/ml selenium, and streptomycin/penicillin, ITS; Collaborative Research, strept/pen; GIBCO BRL, Gaithersburg, MD, FBS;
SANKO JYUNYAKU, Tokyo). After 12 h incubation, individual cultures
were processed under the various test conditions described below. To examine the involvement of TGF
s in endothelial-mesenchymal transformation,
AV endothelial cells cocultured with associated myocardium (AV explant)
were incubated with antibodies against TGF
(anti-TGF
1, anti-TGF
3, or both antibodies together) in CM199 and the results compared with that
observed with CM199. To determine the biological activity of LTBP-1
during endothelial-mesenchymal transformation, AV explants were treated
with various concentrations of Ab39 in CM199, with CM199 alone, or with
CM199 containing Ab39 and TGF
protein (TGF
1, TGF
2, or TGF
3).
Cultures were examined under a Hoffman modulation microscope for endothelial outgrowth and for the characteristics of epithelial-mesenchymal
transformation (such as cell hypertrophy, loss of cell-cell contacts, formation of migratory processes, and mesenchymal invasion). The number of
invading mesenchymal cells is shown as the mean ±SD. In these bioassays, an "inhibition of transformation" was considered to have occurred
when the number of invading mesenchymal cells was more than two standard deviations below the number seen in a control explant cultured with
CM199 alone. The determination of the endothelial outgrowth was measured with the end of an eyepiece equipped with a grid pattern and classified as follows: "
", no endothelial outgrowth surrounding the myocardium; "+", maximum diameter of endothelial monolayer was less than
twice the maximum diameter of the myocardium; "++", maximum diameter of endothelial monolayer was more than twice the maximum diameter of the myocardium.
).
Separated proteins were transferred to an Immobilon-P membrane (Millipore, Bedford, MA), nonspecific binding sites were blocked with 5% nonfat dried milk in Tris buffered saline, and the membrane was incubated
with Ab39 (diluted 1:500) or Ab178 (diluted 1:200). After extensive washing, immunoreactivity was detected with the aid of an alkaline phosphatase-conjugated goat antibody against rabbit IgG (BioRad, Hercules,
CA) and BCIP/NBT.
),
pcDNA3-LTBP-2, or pcDNA3 using LIPOFECTAMIN reagent (GIBCO
BRL) following the manufacturer's recommendation. 2 d after transfection, cells were rinsed twice with PBS and the culture medium was exchanged to methionine- and cysteine-free DMEM supplemented with 0.1 mCi/ml of [35S]methionine and cysteine mixture (Pro-mix, Amersham,
England). After 12 h of metabolic labeling, conditioned media were collected, concentrated, split equally into two tubes, subjected to immunoprecipitation by Ab39 or anti-LTBP-2 (Ab178) and analyzed by SDSPAGE (reducing condition) using 5-20% linear gradient gel as described
previously (Moren et al., 1994
). The gels were fixed, dried, and analyzed with a Fuji BAS 2000 Bio-Imaging Analyzer (FUJIFILM, Tokyo).
1-IgY and Ab39. Sections were incubated with primary antibody mixture (Ab39, 1:500; anti-TGF
1-IgY, 10 µg/ml in the blocking
solution) for 2 h at room temperature, rinsed in PBS and incubated with
TRITC-conjugated goat antibody against rabbit IgG for 1 h. They were
then rinsed extensively in PBS and stained with fluorescein-5-isothiocyanate (FITC)-conjugated rabbit antibody against chicken IgY (Promega,
Madison, WI), rinsed in PBS, and coverslipped with the mounting medium. The FITC-conjugated rabbit antibody against chicken IgY did not
recognize Ab39-TRITC-conjugated goat antibody against rabbit IgG
complex on tissue sections (data not shown).
1-IgY) at 4°C overnight, rinsed with
PBS, incubated with TRITC-conjugated goat antibody against rabbit IgG
for 2 h, rinsed with PBS, incubated with FITC-conjugated rabbit antibody
against chicken IgY, rinsed with PBS, transferred to slides, and coverslipped with the mounting medium. Specificity of immunostaining for
LTBP-1 or TGF
1 was confirmed by preincubating the antibody for 30 min with antigen (Ab39 vs purified human LTBP-1 or recombinant human LTBP-1; anti-TGF
1-IgY vs recombinant human TGF
1). Samples
were observed under a conventional fluorescence microscope (BX60;
OLYMPUS, Tokyo) and photographed using Tmax 400 (Kodak) or
PROVIA 400 (FUJI FILM). The exposure times for FITC and TRITC
images were 32 s and 40 s, respectively.
. In brief,
the AV region was resected from the heart of each 9.5-d mouse embryo
and fixed with 0.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M
PBS with the aid of microwave irradiation (150 W; 120 s; maximum temperature, 37°C). Samples were then fixed for an additional 1 h at 4°C,
rinsed in 7% sucrose in PBS for 12 h, and postfixed in 1% OsO4 containing 1.5% potassium ferrocyanide for 30 min at 4°C. Samples were dehydrated in a graded ethanol series (20 min at each concentration), embedded in LR White resin (London Resin, Berkshire, UK) after replacement,
and the resin allowed to polymerize for 24 h at 58°C. Ultra-thin sections
were cut, mounted on 200-mesh nickel grids, and pretreated for etching
with a saturated aqueous solution of sodium metaperiodate for 30 s followed by five subsequent washes in distilled water. Nonspecific binding
sites were blocked with 1% BSA in PBS for 30 min. The sections were incubated with Ab39 (diluted 1:500) for 12 h at 4°C, rinsed in PBS, and incubated with a 5-nm colloidal gold-conjugated secondary antibody (Amersham) for 1 h at room temperature. They were rinsed in PBS followed by
distilled water, and stained with uranyl acetate and lead citrate.
Results
; Miyazono et al., 1991
). It was important to confirm
that Ab39 does not cross-react with LTBP-2 (Moren et al.,
1994
) and recognizes mouse LTBP-1 (Nunes et al., 1995
).
Radiolabeled conditioned medium obtained from COS-1
cells transfected with human LTBP-1 or LTBP-2 cDNA was subjected to immunoprecipitation with Ab39 or antiLTBP-2 antiserum (Ab178). Resulting immunoprecipitates
were analyzed by gel electrophoresis/BAS 2000. As shown
in Fig. 1, Ab39 immunoprecipitated 205-kD protein (LTBP-1)
in the conditioned medium from COS-1 cells transfected with human LTBP-1 cDNA, whereas it did not precipitate
any detectable protein in that transfected with human
LTBP-2 cDNA. Ab178 recognized LTBP-2 protein and did
not cross-react with LTBP-1 (Fig. 1). Using these antisera,
we also performed an immunoblot analysis of the plasmin
digest of a deoxycholate insoluble fraction obtained from homogenizing embryos (9.5 or 14.5 d). Both the 9.5- and
14.5-d embryos contained an Ab39 immunoreactive 190kD protein (Fig. 2). Our preliminary immunoblot showed
that Ab178 reacted with a 180-kD band in the extracts of
14.5-d embryos; there was no detectable immunoreactivity
in the extracts of 9.5-d embryos (data not shown). Thus,
Ab39 appears to recognize mouse LTBP-1 and does not
react with mouse LTBP-2.
Fig. 2.
Immunoblot of plasmin
digest of a deoxycholate insoluble
fraction obtained from homogenizing 9.5- or 14.5-d whole embryo probed with anti-LTBP-1
antiserum (Ab39). Deoxycholate
insoluble fraction of whole homogenate of the embryos was digested
with plasmin, and the resulting
supernatant containing 50 µg of
protein was electrophoresed (6%
SDS-PAGE) under nonreducing
conditions. Separated proteins
were transferred to Immobilon-P
and stained with Ab39. Ab39 recognizes a 190-kD band both in
9.5- and in 14.5-d embryo (arrowhead).
[View Larger Version of this Image (18K GIF file)]
Is Required to Initiate the Formation of
Endocardial Cushion Tissue
s
were required for the initiation of this embryonic phenomenon in an AV endothelial coculture with associated myocardium (AV explant) on a three-dimensional collagen gel
lattice. Although there have been studies of the tissue distribution of TGF
s during mouse endocardial cushion formation, there is no evidence of a regulatory effect of
TGF
s on endothelial-mesenchymal transformation in
mouse embryonic heart. AV explants, containing endocardium and myocardium, were prepared from 9.5-d mouse
embryonic hearts and cultured with or without neutralizing antibodies against TGF
s in CM199 (Table I). We administrated either anti-TGF
1, anti-TGF
3, or both antibodies to the explant culture at an effective concentration
of antibody according to manufacturer's description. From
each AV explant cultured in CM199 alone, there was an
invasion of mesenchymal cells into the gel lattice and
many mesenchymal cells were observed after 60 h in culture. An anti-TGF
1 inhibited this mesenchymal invasion:
the number of mesenchymal cells was significantly lower and the percentage of the explant invaded was reduced to
75% of control. Culture with both anti-TGF
1 and antiTGF
3 antibodies completely inhibited mesenchymal formation. However, an anti-TGF
3 antibody alone did not
inhibit mesenchymal formation in the gel lattice. These results indicate that TGF
s are required for the generation
of the endothelial-mesenchymal transformation. However, it is still uncertain which TGF
s are involved in endothelial-mesenchymal transformation in vivo during endocardial cushion tissue formation.
Antibody Blocking Experiment in AtrioVentricular Explant Culture
Large Latent TGF Complex Is Distributed
as an Extracellular Material in Mouse Endocardial
Cushion Tissue
At 9.0-9.5 d of gestation, the heart consists of two concentric epithelial layers, endocardium and myocardium, which
are separated by an expanded cardiac jelly. At 9.5-10 d of
gestation, endothelial cells in the OT and AV regions
change their phenotype to that of mesenchymal cells. They
invade into the adjacent cardiac jelly leading to the formation of endocardial cushion tissue (Markwald et al., 1975,
1977
; Kaufman, 1992
).
At the onset of this endothelial-mesenchymal transformation, a fibrous structure containing LTBP-1-like molecules was observed beneath hypertrophied endothelial
cells, transforming cells, of OT and AV (Fig. 3, a and b). As
development proceeds, endothelially derived mesenchymal cells accumulate within the AV and OT cardiac jelly,
forming endocardial cushion tissue. A fibrillar arrangement of LTBP-1-like molecule was observed surrounding the
migrating mesenchymal cells within the cushion tissue (Fig.
3, a-d). This Ab39-immunoreactive molecule could be removed by plasmin treatment in nonfixed fresh frozen tissue-sections before incubation with the primary antibody
(Fig. 3, e and f). These results suggested that the fibrillar
arrangement of LTBP-1-like molecule was associated with the extracellular matrix via the plasmin-sensitive extracellular matrix binding sequence (Taipale et al., 1994).
The extracellular matrix of mesenchymal tissue surrounding the dorsal aorta, gut epithelium, and neural tube also
contained LTBP-1-like molecule (our unpublished observations). Immunogold electronmicroscopy revealed that a
40-100-nm low electron dense extracellular fibrillar network surrounding the mesenchymal cells was decorated by
Ab39-gold particles (Fig. 4 a). In addition to 40-100-nm
extracellular fibrillar structure, microfibrils having a diameter of 5-10 nm were stained with Ab39 by the pre-embedding method (Fig. 4 b). The immunostaining was blocked
by a preincubation of Ab39 with purified human LTBP-1 (5 µg/ ml; Fig. 3, g and h and 4 c) or recombinant human
LTBP-1 (50 µg/ml; data not shown).
To determine whether extracellular TGF1 (Akhurst et
al., 1990
) colocalizes with LTBP-1 during the endocardial
cushion tissue formation, we examined immunohistochemical colocalization of TGF
1 and LTBP-1 in tissue
sections (9.75 d embryonic heart) and cultured AV explants on collagen gel lattice. There are several anti-TGF
1 antibodies, some antibodies are able to detect a cellular
TGF
1, while the others react with extracellular TGF
1
(Thompson et al., 1989). We used anti-TGF
1 IgY, purchased from R & D systems, because this antibody stained
extracellular TGF
1. Fresh frozen sections from 9.75-d
embryos were processed for double immunostaining for TGF
1 and LTBP-1. Results showed that extracellular
anti-TGF
1 immunoreactive fibers were observed surrounding the mesenchymal cells and the TGF
1-immunoreactive materials coincided with Ab39 immunoreactivity (Fig. 5, a and b). Double antibody staining was also
performed against cultured AV explant on collagen gel
lattice. AV explants were prepared from 9.5-d embryonic
heart and cultured on the gel lattice. After 48 h in culture,
where endothelial-mesenchymal transformation progressively occurred, cultures were fixed and double-stained
with anti-TGF
1 IgY and Ab39. The extracellular fibrillar
codistribution of TGF
1 and LTBP-1 surrounding the invaded mesenchymal cells was observed (Fig. 5, e and f).
The double antibody staining was blocked by preincubation of an antibody-mixture containing purified human
LTBP-1 (5 µg/ml) and recombinant human TGF
1 (1 µg/ml;
Fig. 5, c and d and g and h). Our present observations indicate that the large latent TGF
complex is distributed as an extracellular fibrillar matrix in the developing endocardial cushion tissue.
Ab39 Inhibits Mesenchymal Formation in Culture
To determine whether LTBP-1 was required for the generation of the endothelial-mesenchymal transformation, we cultured AV explants from 9.5-d embryonic hearts with or without the presence of Ab39 in CM199 (Table II). Antiserum against LTBP-1 (AB39) inhibited the mesenchymal cell invasion into the gel lattice in a dose-dependent manner. At a concentration of 1 µl/ml of Ab39 in CM199, not only the endothelial outgrowth on the gel lattice, but also mesenchymal invasion into the gel lattice was inhibited, by comparison with culture in CM199 alone (Fig. 6, a-d) or normal rabbit serum in CM199. At lower concentrations of Ab39, only the mesenchymal invasion (number of invading mesenchymal cells and percentage of explant transformed into mesenchyme) was inhibited. These results indicate that LTBP-1 was required for the generation of the endothelial outgrowth and for endothelial-mesenchymal transformation. They also suggest that a smaller amount of LTBP-1 was required for the formation of the endothelial outgrowth.
Table II. Anti-LTBP-1 Antiserum (Ab39) Blocking Experiment in AV Explant Culture |
To resolve the question as to whether LTBP-1 was required for activation of the latent TGF complex or as an
extracellular scaffold for mesenchymal cell migration, we
performed an explant culture rescue experiment. In this,
we added TGF
proteins to the above-mentioned neutralizing culture experiment with Ab39. The results of this rescue experiment are summarized in Table II. The previous
Ab39 neutralizing experiment had indicated that 1 µl/ml of
Ab39 in CM199 completely blocked the mesenchymal cell invasion into the gel lattice. In the rescue experiment, the
concentration of Ab39 was maintained at 1 µl/ml in
CM199. TGF
1 or TGF
2 each restored the inhibitory effect of Ab39 at a concentration of 2 ng/ml (Fig. 6, e and f).
The rescue effect of TGF
3 was less than that of TGF
1
or TGF
2. A high concentration of exogenously applied TGF
s (20 ng/ml) did not exert any greater rescue effect
than that observed with 2 ng/ml on the endothelial outgrowth and mesenchymal cell invasion (data not shown).
The results of our culture experiments suggested that
LTBP-1 was required to induce the biological activity of
the mature TGF
, rather than for the erection of an extracellular scaffold during the TGF
-dependent endothelialmesenchymal transformation. They also suggested that an
excess amount of TGF
appeared to inhibit both cellular
differentiation and proliferation in this culture model.
Functional Importance of TGF during the
Endothelial-Mesenchymal Transformation
Antibodies against TGFs, which were exogenously administered to the AV endocardium cocultured with associated myocardium, effectively blocked the invasion of mesenchymal cells into the collagen gel lattice. Therefore, TGF
s
are presumably required in the endocardium-to-mesenchymal cell transformation that occurs during cushion tissue
formation. Three different types of TGF
are preferentially expressed in the endocardial cushion tissue-forming region of the mouse embryonic heart at mRNA and protein levels. TGF
1 is expressed in both the premigratory
endocardium and invading mesenchymal cells, whereas
TGF
2 and TGF
3 are expressed in the external myocardium (Akhurst et al., 1990
; Dickson et al., 1993
; Mahmood et
al., 1993 and 1995). In the chicken embryo, TGF
3 is thought to be involved in endocardial cushion tissue formation because administration of either antisense oligonucleotides or an antibody specific to TGF
3 effectively blocked
mesenchymal formation in a three-dimensional collagen
gel culture model (Potts et al., 1991
; Runyan et al., 1993).
The tissue distribution pattern of TGF
3 in the chicken
endocardial cushion tissue-forming region is similar to that
of TGF
1 in the embryonic heart of the mouse (Akhurst
et al., 1990
; Choy et al., 1991
; Nakajima et al., 1994
). In the
development of the mouse heart, it appears that TGF
s do play a role in eliciting endothelial-mesenchymal transformation during the formation of endocardial cushion tissue.
However, it is still unclear which TGF
isoform is predominantly involved in this embryonic phenomenon.
A combined administration of antibodies (anti-TGF1
and anti-TGF
3) to an AV explant culture inhibited mesenchymal formation more effectively than did a single antibody. Furthermore, TGF
1, TGF
2, or TGF
3 all led to
some recovery of the inhibitory effect of Ab39 against endothelial-mesenchymal transformation in an AV explant
culture. It has been reported that there is no obvious cardiovascular abnormality in the early development of
TGF
1 null (TGF
1
/
) embryos (Letterio et al., 1994
;
Diebold et al., 1995
; Dickson et al., 1995
), despite the fact
that TGF
1 is expressed within the developing heart
(Akhurst et al. 1990
; Akhurst, 1994
; Dickson et al., 1993
;
Mahmood et al., 1992
, 1995
). Furthermore, fetuses from a
TGF
1 null mother are affected with secondary cardiac muscle hypertrophy caused by severe vascular defects and
defective hematopoiesis within the extra-embryonic mesoderm of the yolk sac (Letterio et al., 1994
; Dickson et al.,
1995
). These results, together with our observations, strongly
suggest that there is a functional redundancy of TGF
s in
the formation of endocardial cushion tissue, although
TGF
s are required there during mouse cardiogenesis.
Extracellular Distribution of LTBP-1
Immunohistochemical observation of LTBP-1-like proteins within the developing endocardial cushion tissue
showed that LTBP-1-like molecule was distributed as a
fibrillar structure surrounding mesenchymal cells. Immunogold electronmicroscopy revealed the LTBP-1-like molecule localized to a 40-100-nm fibrillar network structure.
The tissue distribution pattern of this 40-100-nm fibrillar structure is similar to that of LTBP-2, a component of elastin-associated microfibrils (Gibson et al., 1995). In addition, LTBP-1 was associated with 5-10-nm microfibrils.
Sequence analysis of LTBPs has revealed a characteristic
of matrix or adhesion molecules (Kanzaki et al., 1990
; Tsuji
et al., 1990
; Moren et al. 1994
; Gibson et al., 1995
; Yin et
al., 1995
) and they are classified as the fibrillin family of
extracellular matrix proteins (Sakai et al., 1986
, 1991
;
Maddox et al., 1989
; Rosenbloom et al., 1993
; Zhang et al.,
1994
). Immunohistochemical examination of mature TGF
1
in developing endocardial cushion tissue revealed not only
a cellular distribution, but also an extracellular fibrillar
deposition of TGF
1 (Fig. 5, a and e; Akhurst et al., 1990
;
Mahmood et al., 1993, 1995). We showed that the extracellular distribution of mature TGF
1 coincided with that of
LTBP-1 (Figs. 5 b and 6 b). The tissue distribution pattern of LTBP-1 is similar to that of both tenascin (Akhurst et
al., 1990
) and fibronectin (Mjaatvedt et al., 1987). Recently, LTBP-2 was shown to associate with the microfibrillar component of elastic fibers in the bovine fetal
nuchal ligament and aorta (Gibson et al., 1995
). During fetal rat calvarial cell culture formation of extracellular
fibrillar, LTBP-1 is observed and this structure precedes
the presence of type I collagen fibers (Dallas et al., 1995
).
In human fibroblast culture, LTBP-1 is associated with cellular fibronectin as a component of extracellular microfibrils (Taipale et al., 1996
). In COS-1 transfected with the
LTBP-1 cDNA, interaction of LTBP-1 with collagen type
I could also be seen (Olofsson et al., 1995
). Immunohistochemical colocalization of the extracellular LTBP-1 and
TGF
1, as well as immunoelectron microscopic observation for LTBP-1 suggest that the large latent TGF
complex is presumably a component of extracellular microfibrils that are associated with 40-100 nm extracellular
fibrillar network. The tissue distribution pattern of LTBP-1
and mature TGF
1 is compatible with the hypothetical
role of LTBP-1, i.e., that LTBP-1 concentrates/stores
TGF
within those tissues in which TGF
is a prerequisite
for the progression of endothelial-mesenchymal transformation.
Role for LTBP-1 during Cushion Tissue Formation
The antibody specific for LTBP-1 (Ab39) inhibited the endothelially derived mesenchymal-cell invasion into the
collagen gel lattice in culture and the inhibition was reversed by the exogenous administration of a mature form
of TGF proteins. Therefore, these results suggested that
LTBP-1 may be required for the activation or concentration of TGF
within certain regions. Most cells secrete TGF
as a high molecular mass of latent TGF
which contains the mature growth factor associated with LAP and
LTBP-1 (Kanzaki et al., 1990
; Tsuji et al., 1990
). Neither
the large latent TGF
complex nor the small latent TGF
complex has any biological activity as a growth factor. Active TGF
s are highly hydrophobic and basic proteins
which are rapidly lost from solution (Brown et al., 1990
); they therefore bind to many proteins with a relatively high
affinity. Matrix components binding active TGF
could
have a role in rendering nascently activated TGF
more
soluble and in delivering it to the cell surface receptors
(Lopez-Casillas et al., 1993
; Taipale et al., 1994
; Dallas et
al., 1995
). On the basis of results obtained using heterotypic culture of bovine endothelial and smooth muscle cells, in which targeting of latent TGF
to smooth muscle
cells is required for the activation of latent TGF
, it has
been proposed that delivery of the latent TGF
complex
to the cell surface occurs via LAP or LTBP-1 (Sato et al.,
1993
; Flaumenhaft et al., 1993
). The mechanism underlying the targeting process such as LAP-mediated activation
remains unknown but mannose 6-phosphate in LAP appears to play an important role in certain cell types (Dennis and Rifkin, 1991
). Flaumenhaft et al. (1993)
have reported that either the antibody specific to LTBP-1 (Ab39)
or an excess of exogenously applied free LTBP-1 can inhibit the activation of latent TGF
. This suggests that
LTBP-1 may participate in the activation of TGF
by concentrating the latent TGF
on the cell surface, where activation occurs. The large latent form of TGF
associates
with the extracellular matrix covalently via LTBP-1 and the
release of the small latent complex from the extracellular
matrix is a consequence of the proteolytic cleavage of
LTBP-1 (Taipale et al., 1993). To date, it remains unknown which sequences confer this putative targeting mechanism on LTBP-1. Recently, LTBP-1 with a longer NH2-terminal portion (LTBP-1L) has been identified, and this
LTBP-1L binds more effectively to the extracellular matrix than does LTBP-1 (Oloffson et al., 1995). Together
with previously reported results, our observation may suggest a two-step process for the activation for latent TGF
.
In the first step, the secreted large latent TGF
complex
would be targeted to the extracellular matrix via LTBP-1
for the storage/concentration of TGF
. In the second, proteinase-released latent TGF
complex from the matrix may possibly retarget the TGF
to cell surface binding
sites by some mechanism such as mannose 6-phosphate receptor.
In conclusion, during the formation of endocardial cushion tissue formation, the large latent TGF complex, containing LTBP-1, LAP, and the mature form of TGF
1, is
distributed as an extracellular fibrillar structure surrounding cells where TGF
-dependent endothelial-mesenchymal transformation is regulated. LTBP-1 may play a role
in either the storage or concentration of TGF
for the activation of TGF
.
Received for publication 4 April 1996 and in revised form 5 September 1996.
K. Miyazono is supported by Japan Heart Foundation & IBM Japan Research Grant Foundation for 1995.AV, atrioventricular; EX, explant; LAP, latency associated peptide; LTBP, latent transforming growth factor-beta; binding protein; M, myocardium; Me, mesenchymal cell; OT, outflow tract; PL, plasmin.