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Evidence for basic fibroblast growth factor as a crucial angiogenic growth factor, released from human trophoblasts during early gestation
Placenta
(1998),19,
149-155
Evidence Growth
for
Basic
Factor,
Fibroblast
Released
Growth
from
Human
Factor
as a Crucial
Trophoblasts
Angiogenic
During
Early
Gestation
Y. Hamai,
T. Fujiia, T. Yamashita,
Department of Obstetrics and Gynecology, 113 Japan Paper accepted 23 September 1997
S. Kozuma, Faculty
T. Okai and Y. Taketani
of Medicine,
University
of Tokyo, 7-3-l
Hongo,
Bunkyo-ku,
Tokyo,
The objective of this study was to clarify the possible angiogenesis-promoting factors from human trophoblasts in early stage gestation. The existence of angiogenic growth factors such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) in the condition medium from human villous trophoblasts was determined. Biological activity of angiogenic growth factors released by trophoblasts was examined using vascular endothelial cell lines. The condition medium from trophoblasts enhanced the growth of endothelial cells. Although cultured trophoblasts exhibited immunoreactive products for both bFGF and VEGF in the cytoplasm, only bFGF was detected in the condition medium by ELISA. The growth-enhancing activity of the condition medium was eliminated completely by the addition of anti-bFGF antibody but not with anti-VEGF antibody. Thus, trophoblastic cells seem to play an important role in extensive angiogenesis occurring in early gestation, mainly by releasing bFGF but not VEGF. 0 1998 W. B. Saunders Company Ltd Placenta (1998), 19, 149-155
INTRODUCTION
MATERIALS
Successful pregnancy depends on placental growth and development. Growth of the placenta requires extensive angiogenesis to develop its vascular structure. For instance, fetal villous angiogenesis is dependent upon the proliferation of the pre-existing endothelium and pericytes in early stage of pregnancy. On the other hand, when trophoblasts are penetrating the decidua, the maternal arteries also continue to elongate into the decidua. Thus, extensive angiogenesis occurs both in the fetal villous tissue and in the maternal decidua (Blankenship, Enders and King, 1993). The presence of known angiogenic growth factors such as basic fibroblast growth factor (bFGF) (Carney et al., 1992; Ferriani et al., 1994; Shams and Ahmed, 1994) and vascular endothelial growth factor (VEGF) (Jakeman et al., 1993; Barleon et al., 1994; Jackson et al., 1994; Ahmed et al., 1995) in human trophoblasts has been reported using immunohistochemistry and in situ hybridization. However, thus far, it remains to be clarified whether these angiogenic growth factors are actually released by trophoblasts, and possess angiogenic property. In this study, we attempted to specify angiogenic factors derived from trophoblasts in physiological paradigm. To address this, we examined whether human trophoblastic cells obtained from placental villi at early gestation release bFGF and VEGF, and verified their angiogenic property in vitro.
Culture of human cells (HMvECs)
a To
whom
correspondence
0143-4004/98/020149+07
should $18.00/O
be addressed.
AND
METHODS microvascular
endothelial
HMvECs from microvascula of adult nasal mucosa (Morinaga, Tokyo, Japan) were cultured in the medium MCDB 104 (Morinaga, Tokyo, Japan) supplemented with 10 per cent fetal calf serum (FCS) (Gibco, NY, USA) and growth factor solution@ (Morinaga) containing 0.5 mg/ml endothelial cell growth supplement@ (Morinaga). They were subcultured after being confluent.
Preparation
and culture
of human
trophoblasts
Placental tissue samples were obtained from consenting healthy pregnant women undergoing legal abortion of normal pregnancy at 6-11 weeks of gestation. The obtained placental villi were torn apart from connective tissue in phosphatebuffered saline (PBS) and washed thoroughly with PBS. Chorionic villi were then minced into small pieces and stirred for 30 min at 37°C in medium 199 (Gibco) supplemented with 100 IU/ml collagenase (Wako, Osaka, Japan) and 20 IU/ml deoxyribonuclease (Sigma, St Louis, USA). The resultant cells were passed through a lOO+m nylon mesh (Tokyo Screen, Tokyo, Japan) and centrifuged at 1OOOgfor 5 min. The cells were then resuspended in medium 199 and layered on 40 per 8 1998 W. B. Saunders
Company
Ltd
150
Placenta 600
j :: j
lo2
Fluorescence
cent of percoll (Pharmacia, Uppsala, Sweden) in medium 199 followed by centrifugation at 2200 g for 15 min. The cells at a density of 1.048-1.062 g/ml were collected and resuspended in the medium 199 containing 5 per cent FCS, 0.7 mM L-glutamine and 50 IU/ml penicillin-50 yg/ml streptomycin (Gibco). One millilitre of the cell suspension (1 X lo4 cells/ml) was added evenly onto each well of 24-well culture plates (Corning, NY, USA) which had been precoated with 3 mg/ml collagen (Celtrix, NY, USA) for 30 min at room temperature (Figure 1). The plates were incubated in a continuous flow of 9.5 per cent air, 5 per cent CO, at 37°C. About 90 per cent of cultured cells were proved to be trophoblasts as judged by immunostaining with anti-cytokeratin antibody, CAM 5.2 (Dako, Copenhagen, Denmark) (Sasagawa et al., 1986) (Figure 2). No apparent fibroblasts were found to grow in this culture. During the 72 h of culture, no discernible morphological changes of trophoblasts were observed. After 72 h of culture, almost all the trophoblasts were observed to be viable by staining with trypan blue, and the medium was collected and stored at - 20°C for 3-4 weeks until used.
Vol. 19
j :: j
lo1 Figure 1. Cultured trophoblasts ( x 40). Trophoblasts were prepared and cultured as described in ‘Materials and Methods’. The large oval-shaped cells mainly dispersed in the lower half of the picture were trophoblasts. Fibroblasts were not observed.
(1998),
103 intensity
Figure 2. Flowcytometric analysis of cultured cells isolated from chorionic villi using an anti-cytokeratin antibody, CAM5.2. Cultured cells isolated from chorionic villi were stained with anti-cytokeratin antibody, CAM5.2 (-) or with IgG2a-negative control ( ) and analysed on a FACScan instrument (Becton Dickinson, San Jose, CA, USA).
MlT
proliferation
assay for HMvECs
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasolium bromide; Chemicon International, CA, USA] is a pale yellow substrate that is cleaved by living cells to yield a dark blue formazan product. This process requires mitochondrial function, and even freshly dead cells cleave small amounts of MTT, if any. MTT was dissolved in PBS (pH 7.4) at the concentration of 5 mg/ml. One hundred microlitres of MTT solution were added into each culture well and incubated at 37°C for 4 h. After adding 100 ~1 of isopropanol with 0.04 N of HCl into each well to dissolve the cleaved products of MTT, the light absorbance was measured by an ELISA plate reader (Toyosoda, Tokyo, Japan) with a test wavelength of 570 nm and a reference wavelength of 620 nm. The light absorbance (AOD 570-620 nm ) was proportional to the number of living cells from 100 to 50 000 cells/well (Mosmann, 1983).
Measurement of bFGF and VEGF in the culture media of human trophoblasts Culture medium
of endothelial cells in the condition from trophoblasts
One hundred microlitres of HMvECs suspension (8 x lo3 cells/ml) in MCDB 104 medium supplemented with 10 per cent FCS and growth factor solution@ (Morinaga) were added onto each well of 96-well flat-bottomed culture plates (Corning) which had been precoated with 3 mg/ml collagen for 30 min at room temperature. HMvECs were cultured for overnight to allow to adhere to the well bottom at 37°C in 5 per cent CO, and then the medium was replenished with either medium 199 with 5 per cent FCS or the above-stated condition medium of trophoblasts. After 48 h in culture when cultured HMvECs occupied about 80 per cent of the area of a culture disk, their number was determined by MTT proliferation assay (Mosmann, 1983) as described below.
Human bFGF and VEGF were measured by using sandwich enzyme-linked immunoassay (ELISA) kits (R&D Systems, MN, USA).
Absorption the medium
of angiogenic and culture
growth factors supernatant
from
Basic FGF. A total of 0.6 ml trophoblast condition medium and 0.6 ml medium 199 with 5 per cent FCS was incubated with protein G-sepharose (Sigma) beads which could bind 10 mg of IgG and either 1 pg/ml anti-human bFGF antibody (Austral Biologicals, CA, USA) or 10 pg/ml mouse IgGl negative control (the IgG subclass similar to the antihuman bFGF antibody; Chemicon International), at 4°C for overnight.
Hamai
et al.: Trophoblasts
and Placental
151
Angiogenesis
VEGF. For VEGF, 0.6 ml trophoblast condition medium and 0.6 ml medium 199 with 5 per cent FCS were incubated with protein A-sepharose (Sigma) beads which could bind 10 mg of IgG and either 5 pg/ml anti-human VEGF neutralizing antibody (R&D Systems, MN, USA) or 10 pg/ml mouse IgG2b negative control (the IgG subclass similar to the anti-human VEGF neutralizing antibody; Chemicon International) at 4°C for overnight.
Addition of angiogenic growth factor antibody-treated condition medium
to
Human bFGF (Progen Biotechnik, Heidelberg, Germany) at different concentrations (0.3, 3 and 30 pg/ml) was added into 72-h condition medium of trophoblasts pretreated with anti-bFGF antibody and protein G-sepharose beads. The resultant medium was used for experiment of HMvECs-growth augmentation.
lmmunohistochemical trophoblasts with antibody
3,3’-diaminobenzidine tetrahydrochloride containing 0.02 per cent H,O, for 10 min to visualize reaction products. Finally, the sections were soaked in excess water.
Statistical
analysis
Differences between the two groups were analysed for significance (PcO.05) by Wilcoxon’s test.
RESULTS Growth factors trophoblasts
released
from
cultured
The concentration of bFGF in the 72-h condition medium of trophoblasts was 2.16 f 1.63 pg/ml (mean f s.d.). The concentration of VEGF in the 72-h condition medium was below the sensitivity of ELISA. Both bFGF and VEGF were not detected in the 5 per cent FCS-supplemented medium 199 by ELISA.
staining of cultured anti-bFGF and anti-VEGF
Trophoblasts were stained by using a labelled streptavidin biotin method. Cultured trophoblasts were fixed in acetone at 4°C for 10 min. After being washed with cold tris-buffered saline (TBS) (50 mM Tris-HCl, 150 mM NaCl, pH 7.6), the cells were incubated in 0.03 per cent H,O,, 0.03 per cent NaN, (Peroxidase blocking reagen@; Dako) for 10 min at room temperature to remove the endogenous peroxidase activity. Subsequently, the sections were washed with cold TBS, incubated in 5 per cent rabbit serum-TBS for 10 min at room temperature. After being incubated in 0.1 per cent avidin50 mM tris-HCl with 15 mM NaN3 (Biotin blocking systemE’; Dako) for 15 min at room temperature the cells were washed with cold TBS and then incubated in 0.01 per cent biotin50 mM Tris-HCl with 15 mM NaN, (Biotin blocking systems) for 15 min. Following these procedures, the cells were washed with cold TBS and incubated in 0.025 pg/ml anti-human bFGF antibody or 0.025 pg/ml anti-human VEGF neutralizing antibody diluted in TBS containing carrier protein and 15 mM NaN, (Dako Antibody Diluent@) overnight. For control staining, mouse IgGl, the IgG subclass similar to the anti-human bFGF antibody, and mouse IgG2b, the IgG subclass similar to the anti-human VEGF neutralizing antibody, were used at 0.025 pg/ml. After being washed with cold TBS, the cells were then incubated with 1.2 pg/ml biotinylated F(ab’)2 fragment of rabbit anti-mouse immunoglobulins (Dako), diluted in tris-HCl buffer containing carrier protein and 15 mM NaN, (Dako antibody diluent’m), for 10 min at room temperature. They were further washed with cold TBS, followed by the incubation with peroxidase-labelled streptavidin solution (Dako LSAB kit@) for 10 min. They were washed again with cold TBS and incubated with 1 mg/ml
Elimination of HMvECs growth-promoting activity of bFGF or VEGF by neutralizing antibodies The MTT assay showed that viable HMvECs after 48 h culture in medium 199 with 30 pg/ml of bFGF or 30 pg/ml of VEGF increased significantly in number compared to those cultured in medium 199 without FCS. Neutralization of these growth factors with respective antibody completely eliminated the HMvECs growth-promoting activities of the growth factors (Figures 3 and 4). These results demonstrate that this neutralization method can be used to eliminate the activities of both bFGF and VEGF, which are higher than those present in condition medium of trophoblasts.
Growth of HMvECs in the condition treated with or without the antibody bFGF or VEGF
medium against
Figure 5 compares the number of cells of HMvECs cultured with either medium 199 with 5 per cent FCS or condition medium of trophoblasts. The number of cells increased by 60 per cent when cultured in trophoblast condition medium. The pretreatment of the condition medium with anti-bFGF antibody and protein G-sepharose beads resulted in a decrease in cell numbers by 50 per cent. Incidentally, the pretreatment of the control medium (medium 199 with 5 per cent FCS) with anti-bFGF antibody and protein G-sepharose beads also produced a 20 per cent decrease in the number of cells. The number of cells cultured with control medium pretreated with anti-bFGF antibody and protein G-sepharose beads equalled that cultured with the condition medium pretreated in the
152
Placenta
(1998),
Vol. 19
0.140 0.120 0.100
.
0.080
0.060
-
0.060
0.050
-
i
I . .
i
0.040 0.020 0.000
I
NS Medium
199
A
199 + 30 pg/ml
C bFGF
Figure 3. Effect of bFGF and its antibody and protein G-sepharose beads on the proliferation of HMvECs. HMvECs were cultured for 48 h in medium 199 supplemented with bFGF at concentration of 30 pg/ml. Medium 199 was supplemented with the same concentration of bFGF which was then treated with either anti-bFGF antibody and protein G-sepharose beads (B), or IgGl negative control and protein G-sepharose beads (C). The cell culture was run in parallel using these culture media. Cell numbers were determined by MTT assay. * Statistically significant, P
. 8 ii I . .i
0.140 0.120 0.100
I
0.080
i 0.040
0 i 8
’ :
i !.
0.020 0.000
NS Medium
199
A Medium
I NS
B
Medium
0.060
8
3 : 0
0.000 ’
A Medium
B
B
C VEGF
Figure 4. Effect of VEGF and its antibody and protein A-sepharose beads on the proliferation of HMvECs. HMvECs were cultured for 48 h in medium 199 supplemented with VEGF at concentration of 30 pg/ml. Medium 199 was supplemented with the same concentration of VEGF which was then treated with either anti-VEGF antibody and protein A-sepharose beads (B), or IgG2b negative control and protein A-sepharose beads (C). The cell culture was run in parallel using these culture media. Cell numbers were determined by MTT assay. *Statistically significant, P
same way. The pretreatment with IgGl and protein G-sepharose beads had no effect on cell numbers in both groups.
A
B
C
Condition medium of trophoblasts
Figure 5. Effects of condition medium from trophoblasts pretreated with anti-bFGF antibody and protein G-sepharose beads on the proliferation of HMvECs. HMvECs were cultured with Medium 199 and 5 per cent FCS or condition medium from trophoblasts. Each culture group was randomized to either IgGl and protein G-sepharose beads pretreatment (B), anti-bFGF antibody and protein G-sepharose beads pretreatment (C) or without pretreatment (A). The cell cultures were conducted for another 48 h. Cell numbers were determined by MTT assay. + Statistically significant, P
The growth-inhibiting effect of anti-bFGF antibody and protein G-sepharose beads, when added to the trophoblast condition medium, was obviated by the addition of bFGF at 3 pg/ml, the concentration of which was comparable with that attainable in the condition medium of trophoblasts (Figure 6). Figure 7 illustrates that the addition of anti-VEGF antibody and protein A-sepharose beads into trophoblast condition medium resulted in a 15 per cent reduction in the number of cells (statistically significant, P
199 + 30 pg/ml
C
199 + 5 per cent FCS
staining
The presence of immunoreactive human bFGF and VEGF molecules was demonstrated in cultured trophoblasts. The reaction products for both, which were identified as small granules, were present in the cytoplasm of trophoblasts (Figures 8 and 9). When IgGl or IgG2b was used instead of anti-human bFGF antibody or anti-human VEGF antibody respectively, no apparent staining was observed in the trophoblasts (Figures 10 and 11). DISCUSSION Growth of the placenta requires extensive angiogenesis to develop its vascular structure in both fetal chorionic villi and
Hamai
et al.: Trophoblasts
and Placental
Angiogenesis
153 7
0.090 0.080 0.070
NS
I
I
. : I a .
0.080
: :
Ii . .
0.060
1 i .
*
I
.-~, NS
0.070 1
; fi
0.050
0.060
7,
f
t 0.050 0.040
$
;
*
8 ;
i
1 0.040
*
’
*i
8
0.030
i
l
*I
0.030
0.020
0.000
0.000
L -r
I * ’ A Medium
Condition
medium
of trophoblasts
Figure 6. Effects of anti-bFGF antibody (B) and different concentrations of bFGF on the proliferation of HMvECs cultured with condition medium from trophoblasts. Human bFGF at different concentrations (C, 0.3; D, 3; and E, 30 pg/ml) was added to 72-h condition medium of trophoblasts which had been pretreated with anti-bFGF antibody and protein G-sepharose beads. The resultant medium was used for experiment of HMvECs growth augmentation. Cell numbers were determined by MTT assay. NS, Not significant; A, IgGl and protein G
maternal decidua (Blankenship, Enders and King, 1993). According to the current opinion, failure of establishing the adequate placental vascular structure may cause ischaemia of the placenta and therefore vasoactive substances in the placenta might be released into the maternal blood, which in turn could lead to complications such as pre-eclampsia, placental dysfunction, intrauterine growth restriction or intrauterine fetal death (Sharkey et al., 1993). To date, several angiogenic factors such as bFGF, VEGF and platelet-derived growth factor (PDGF) have been shown to exist in trophoblasts. These growth factors are considered to play a role in the development of fetomaternal vascular system. However, published studies have simply demonstrated the presence of the angiogenic factors, without providing a support for the role of trophoblasts in the extensive neovascularization occurring during pregnancy. In this study, we demonstrated that cultured human trophoblastic cells released angiogenic substances as judged by growth-promoting activity of vascular endothelial cells. In addition, the angiogenic activity was eliminated almost completely by the concomitant addition of anti-bFGF antibody and protein G-sepharose beads, suggesting that an angiogenic factor released by trophoblasts may be bFGF. Unexpectedly, the addition of anti-bFGF antibody and protein G-sepharose beads to control medium resulted in a marginal decrease in cell numbers. This can be explained by the presence of a trace
B
C
199 + 5 per cent FCS
A
B
C
Condition medium of trophoblasts
Figure 7. Effects of condition medium from trophoblasts pretreated with anti-VEGF antibody and protein A-sepharose beads on the proliferation of HMvECs. HMvECs were cultured with medium 199 and 5 per cent FCS or condition medium from trophoblasts. Each culture group was randomized to either IgG2b and protein A-sepharose beads pretreatment (B), anti-VEGF antibody and protein A-sepharose beads pretreatment (C) or without pretreatment (A). The cell cultures were conducted for another 48 h. Cell numbers were determined by MTT assay. * Statistically significant, P
Figure 8. Immunohistochemical staining of bFGF molecule in the cultured trophoblasts (200 x ). Acetone-fixed cultured trophoblasts were reacted with anti-human FGF antibody, and then stained by the labelled streptavidin biotin method (see Materials and Methods).
amount of bFGF, the level of which was too low to be detected. However, the present study demonstrated the presence of VEGF in cultured trophoblastic cells as well, suggesting a role of VEGF as one of trophoblast-derived angiogenic factors. The expression of bFGF has been shown in endothelial cells and smooth muscle cells of placental vessels other than trophoblastic cells (Ferriani et al., 1994; Shams and Ahmed, 1994). Ferriani et al. (1994) demonstrated that bFGF immunoreactivity is present in or around the cytotrophoblast cells. Thus, bFGF produced in these cells may be responsible
Placenta
Figure 9. Immunohistochemical trophoblasts (200 x ). Cultured VEGF antibody.
staining of VEGF molecule in the cultured trophoblasts were reacted with anti-human
Figure 10. Immunohistochemical staining of the cultured trophoblasts with IgGl negative control (200 X ). Acetone-fixed cultured trophoblasts were reacted with IgGl negative control. No reaction product was noticed against the IgGl negative control.
for fetal villous angiogenesis. In this study, bFGF in the condition medium was shown to be mitogenic on endothelial cells. Although bFGF is known to be released through a mechanism of basement membrane degradation, a recent study showed that bFGF may be secreted directly by FGFproducing cells via a pathway independent of the endoplasmic reticulum (Mignatti, Morimoto and Rifkin, 1992). In addition, almost all the cultured trophoblasts were viable after 72 h in culture as judged by trypan blue exclusion, it is probable that bFGF detected in the culture media is secreted by trophoblasts. Also basic FGF is known to cause morphological changes of capillary endothelial cells, and increase the production of proteinases such as plasminogen activator and collagenase in a cell culture system of these cells (Tsuboi, Sato and R&in, 1990), thus enhancing migration of the cells, a prerequisite for angiogenesis. Basic FGF has been shown to be localized in the porcine uterus comprising glandular and luminal epithelia and stromal cells (Katsahambas and Hearn, 1996). In human, FGF mRNA has been detected in the endometrium; its level being higher in the proliferative phase compared with secretory phase
(1998),
Vol. 19
Figure 11. Immunohistochemical staining of the cultured trophoblasts with IgG2b negative control (200 X ). Acetone-fixed cultured trophoblast were reacted with IgG2b negative control. No reaction product was noticed against the IgG2b negative control.
(Fujimoto et al., 1996). The mRNA level does not increase in the early stage of pregnancy (Katsahambas and Hearn, 1996). Assuming that FGF is a key factor responsible for marked neovascularization in the endometrium after implantation, an unchanged level of FGF mRNA during early pregnancy does not explain the prominent progression of vasculature associated with pregnancy. It is reasonable to presume that FGF derived from trophoblasts together with FGF from decidual tissues may enhance decidual vascularization. The finding that FGF is located predominantly in extravillous trophoblasts (Ferriani et al., 1994), the fetal tissue interface, may support this hypothesis. VEGF has been reported also to exist in trophoblasts, decidual macrophages, maternal decidual cells, Hofbauer cells within the villous mesenchyme and embryo, and the smooth muscle cells surrounding vein and arteries of the umbilical cord (Jakeman et al., 1993; Barleon et al., 1994; Jackson et al., 1994; Ahmed et al., 1995). VEGF receptor, flt-1 protein, has been reported to be expressed in trophoblasts, decidual macrophages, maternal decidual cells and Hofbauer cells within the villous mesenchyme (Jakeman et al., 1993; Barleon et al., 1994; Ahmed et al., 1995). These findings strongly support a role of VEGF as one of stimulators of angiogenesis in both fetal and maternal compartments during gestation. However, in this study, the addition of anti-VEGF antibody and protein A-sepharose beads into the condition medium resulted in a slight but significant reduction (15 per cent) in cell numbers, while it decreased control cell number by 30 per cent, suggesting that growth inhibition by anti-VEGF antibody was ascribable to the neutralization of an undetectable amount of VEGF present in control medium and its level, if any, in the condition medium was the same as that in control medium. On the basis of these findings, one can envision that VEGF may act in an autocrine manner. If it can work in a paracrine way at all, it may act only on adjacent cells. Pregnancy seems to be an immunological paradox. The fetus, which is a semi-allograft, can survive for 9 months despite the maternal immune system. It is reasonable to
Hamai
et al.: Trophoblasts
and Placental
Angiogenesis
155
speculate that some special immunological mechanisms are involved in securing fetal growth (Rocklin et al., 1976; Kovithavongs and Dossetor, 1978; McIntyre and Faulk, 1979; Suciu-Foca et al., 1983; Wegmann et al., 1989). Disruption of this immune regulation during pregnancy is suggested to be one of the etiological factors for pre-eclampsia. For instance, serum concentrations of both IL-2 and TNF-a in the first trimester of pregnancy were significantly higher in the women who developed pre-eclampsia after 28 weeks of pregnancy compared with those in the women who completed pregnancy uneventfully (Hamai et al., 1997). Also IL-2 has been proved to be present in the decidual tissue of pre-eclamptic patients (Hara et al., 1995). These immunological abnormalities may cause an injury of the trophoblasts and the angiogenic growth
factor-secretion from trophoblasts may be decreased. Failure to secrete the angiogenic growth factor may lead to the failure in establishing the adequate placental vascular system and may cause pre-eclampsia. In summary, we demonstrated that cultured human trophoblastic cells obtained from early human placental villi release biologically active bFGF. In addition, the majority of angiogenie activity in condition medium from trophoblasts was suggested to be attributable to bFGF released from trophoblasts. Thus, it appears that bFGF is a crucial angiogenic factor derived from human trophoblastic cells and thereby may play an important role in angiogenesis occurring in early gestation.
ACKNOWLEDGEMENTS We sincerely
thank
Dr Takeshi
Sugimoto
for providing
placental
tissue.
REFERENCES Ahmed A, Li XF, Dunk C, Whittle MJ, Rushton DI & Rollason T (1995) Colocalisation of vascular endothelial growth factor and its flt-1 receptor in human placenta. Growth Factors, 12, 235-243. Barleon B, Hauser S, Schollmann C, Weindel K, Marme D, Yayon A & Weich HA (1994) Differential expression of the two VEGF receptors flt and KDR in placenta and vascular endothelial cells. 3 Cell Biochem, 54, 56-66. Blankenship TN, Enders AC & King BF (1993) Trophoblastic invasion and the development of uteroplacental arteries in the macaque: immunohistochemical localization of cytokeratins, desmin, type IV collagen, laminin, and fibronectin. Cell Tissue Res, 272, 227-236. Carney EW, Lye SJ, Paek W, Zander D & Ritchie JW (1992) Cellular localization of basic fibroblast growth factor within human placenta throughout gestation. 39th Annual Meeting of the Society for Gynecological Investigation, 533 (abstract). Ferriani RA, Ahmed A, Sharkey A & Smith SK (1994) Colocalization of acidic and basic fibroblast growth factor (FGF) in human placenta and the cellular effects of bFGF in trophoblast cell line JEG-3. Growth Factors, 10, 259-268. Fujimoto J, Hori M, Ichigo S & Tamaya T (1996) Expression of basic fibroblast growth factor and its mRNA in uterine endometrium during the menstrual cycle. Gynecol Endocrinol, 10, 193-197. Hamai Y, Fujii T, Yamashita T, Nishina H, Kozuma S, Mikami Y & Taketani Y (1997) Evidence for an elevation in serum interleukin-2 and tumour necrosis factor-a levels before the clinical manifestations of preeclampsia. Am 3 Reprod Zmmunol, 38, 89-93. Hara N, Fujii T, Okai T & Taketani Y (1995) Histochemical demonstration of interleukin-2 in decidua cells of patients with preeclampsia. Am 3 Reprod Zmmunol, 34, 44-5 1. Jackson MR, Carney EW, Lye SJ & Ritchie JW (1994) Localization of two angiogenic growth factors (PDECGF and VEGF) in human placentae throughout gestation. Placenta, 15, 341-353. Jakeman LB, Armanini M, Phillips HS & Ferrara N (1993) Developmental expression of binding sites and messenger ribonucleic acid for vascular endothelial growth factor suggests a role for this protein in vasculogenesis and angiogenesis. Endocrinology, 133, 848-859.
Katsahambas S & Hearn MT (1996) Localization of basic tibroblast growth factor mRNA (FGF-2 mRNA) in the uterus of mated and unmated gilts. 3 Histochem Cytochem, 44, 1289-1301. Kovithavongs T & Dossetor JB (1978) Suppressor cells in human pregnancy. Transplant Proc, 10, 911-913. McIntyre JA & Faulk WP (1979) Maternal blocking factors in human pregnancy are found in plasma not serum. Lancer, 2, 821-823. Mignatti P, Morimoto T & Rifkin DB (1992) Basic fibroblast growth factor, a protein devoid of secretory signal sequence, is released by cells via a pathway independent of the endoplasmic reticulum-Golgi complex. 3 Cell Physiol, 151, 81-93. Mosmann T (1983) Rapid calorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. 3 Zmmunol Methods, 65, 55-63. Rocklin R, K&miller JL, Carpenter CB, Garvoy MR & David JR (1976) Maternal-fetal relation: absence of immunological blocking factor from the serum of women with chronic abortions. New Eng 3 Med, 295, 1209-1213. Sasagawa M, Watanabe S, Ohmomo Y, Honma S, Kanazawa K & Takeuchi S (1986) Reactivity of two monoclonal antibodies (Troma 1 and CAM 5.2) on Human Tissue sections: Analysis of their usefulness as a histological trophoblast marker in normal pregnancy and trophoblastic disease. Znt 3 Gynecol Pathol, 5, 345-356. Suciu-Foca N, Reed E, Rohowsky C, Kung P & King DW (1983) Anti-idiotypic antibodies to anti-HLA receptors induced by pregnancy. Proc Nat
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Shams M & Ahmed A (1994) Localization of mRNA for basic fibroblast growth factor in human placenta. Growth Factors, 11, 105-l 11. Sharkey AM, Charnock-Jones DS, Boocock CA, Brown KD & Smith SK (1993) Expression of mRNA for vascular endothelial growth factor in human placenta. 3 Reprod Fertil, 99, 609-615. Tsuboi R, Sato Y & Rifkin DB (1990) Correlation of cell migration, cell invasion, receptor number, proteinase production, and basic fibroblast growth factor levels in endothelial cells. 3 Cell Biol, 110, 51 l-517. Wegmann TG, Athanassakis I, Guilbert L, Branch D, Dy M, Menu E & Chaouat G (1989) The role of M-CSF and GM-CSF in fostering placental growth, fetal growth, and fetal survival. Transplant Proc, 21, 566568.
(1998),19,
149-155
Evidence Growth
for
Basic
Factor,
Fibroblast
Released
Growth
from
Human
Factor
as a Crucial
Trophoblasts
Angiogenic
During
Early
Gestation
Y. Hamai,
T. Fujiia, T. Yamashita,
Department of Obstetrics and Gynecology, 113 Japan Paper accepted 23 September 1997
S. Kozuma, Faculty
T. Okai and Y. Taketani
of Medicine,
University
of Tokyo, 7-3-l
Hongo,
Bunkyo-ku,
Tokyo,
The objective of this study was to clarify the possible angiogenesis-promoting factors from human trophoblasts in early stage gestation. The existence of angiogenic growth factors such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) in the condition medium from human villous trophoblasts was determined. Biological activity of angiogenic growth factors released by trophoblasts was examined using vascular endothelial cell lines. The condition medium from trophoblasts enhanced the growth of endothelial cells. Although cultured trophoblasts exhibited immunoreactive products for both bFGF and VEGF in the cytoplasm, only bFGF was detected in the condition medium by ELISA. The growth-enhancing activity of the condition medium was eliminated completely by the addition of anti-bFGF antibody but not with anti-VEGF antibody. Thus, trophoblastic cells seem to play an important role in extensive angiogenesis occurring in early gestation, mainly by releasing bFGF but not VEGF. 0 1998 W. B. Saunders Company Ltd Placenta (1998), 19, 149-155
INTRODUCTION
MATERIALS
Successful pregnancy depends on placental growth and development. Growth of the placenta requires extensive angiogenesis to develop its vascular structure. For instance, fetal villous angiogenesis is dependent upon the proliferation of the pre-existing endothelium and pericytes in early stage of pregnancy. On the other hand, when trophoblasts are penetrating the decidua, the maternal arteries also continue to elongate into the decidua. Thus, extensive angiogenesis occurs both in the fetal villous tissue and in the maternal decidua (Blankenship, Enders and King, 1993). The presence of known angiogenic growth factors such as basic fibroblast growth factor (bFGF) (Carney et al., 1992; Ferriani et al., 1994; Shams and Ahmed, 1994) and vascular endothelial growth factor (VEGF) (Jakeman et al., 1993; Barleon et al., 1994; Jackson et al., 1994; Ahmed et al., 1995) in human trophoblasts has been reported using immunohistochemistry and in situ hybridization. However, thus far, it remains to be clarified whether these angiogenic growth factors are actually released by trophoblasts, and possess angiogenic property. In this study, we attempted to specify angiogenic factors derived from trophoblasts in physiological paradigm. To address this, we examined whether human trophoblastic cells obtained from placental villi at early gestation release bFGF and VEGF, and verified their angiogenic property in vitro.
Culture of human cells (HMvECs)
a To
whom
correspondence
0143-4004/98/020149+07
should $18.00/O
be addressed.
AND
METHODS microvascular
endothelial
HMvECs from microvascula of adult nasal mucosa (Morinaga, Tokyo, Japan) were cultured in the medium MCDB 104 (Morinaga, Tokyo, Japan) supplemented with 10 per cent fetal calf serum (FCS) (Gibco, NY, USA) and growth factor solution@ (Morinaga) containing 0.5 mg/ml endothelial cell growth supplement@ (Morinaga). They were subcultured after being confluent.
Preparation
and culture
of human
trophoblasts
Placental tissue samples were obtained from consenting healthy pregnant women undergoing legal abortion of normal pregnancy at 6-11 weeks of gestation. The obtained placental villi were torn apart from connective tissue in phosphatebuffered saline (PBS) and washed thoroughly with PBS. Chorionic villi were then minced into small pieces and stirred for 30 min at 37°C in medium 199 (Gibco) supplemented with 100 IU/ml collagenase (Wako, Osaka, Japan) and 20 IU/ml deoxyribonuclease (Sigma, St Louis, USA). The resultant cells were passed through a lOO+m nylon mesh (Tokyo Screen, Tokyo, Japan) and centrifuged at 1OOOgfor 5 min. The cells were then resuspended in medium 199 and layered on 40 per 8 1998 W. B. Saunders
Company
Ltd
150
Placenta 600
j :: j
lo2
Fluorescence
cent of percoll (Pharmacia, Uppsala, Sweden) in medium 199 followed by centrifugation at 2200 g for 15 min. The cells at a density of 1.048-1.062 g/ml were collected and resuspended in the medium 199 containing 5 per cent FCS, 0.7 mM L-glutamine and 50 IU/ml penicillin-50 yg/ml streptomycin (Gibco). One millilitre of the cell suspension (1 X lo4 cells/ml) was added evenly onto each well of 24-well culture plates (Corning, NY, USA) which had been precoated with 3 mg/ml collagen (Celtrix, NY, USA) for 30 min at room temperature (Figure 1). The plates were incubated in a continuous flow of 9.5 per cent air, 5 per cent CO, at 37°C. About 90 per cent of cultured cells were proved to be trophoblasts as judged by immunostaining with anti-cytokeratin antibody, CAM 5.2 (Dako, Copenhagen, Denmark) (Sasagawa et al., 1986) (Figure 2). No apparent fibroblasts were found to grow in this culture. During the 72 h of culture, no discernible morphological changes of trophoblasts were observed. After 72 h of culture, almost all the trophoblasts were observed to be viable by staining with trypan blue, and the medium was collected and stored at - 20°C for 3-4 weeks until used.
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lo1 Figure 1. Cultured trophoblasts ( x 40). Trophoblasts were prepared and cultured as described in ‘Materials and Methods’. The large oval-shaped cells mainly dispersed in the lower half of the picture were trophoblasts. Fibroblasts were not observed.
(1998),
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Figure 2. Flowcytometric analysis of cultured cells isolated from chorionic villi using an anti-cytokeratin antibody, CAM5.2. Cultured cells isolated from chorionic villi were stained with anti-cytokeratin antibody, CAM5.2 (-) or with IgG2a-negative control ( ) and analysed on a FACScan instrument (Becton Dickinson, San Jose, CA, USA).
MlT
proliferation
assay for HMvECs
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasolium bromide; Chemicon International, CA, USA] is a pale yellow substrate that is cleaved by living cells to yield a dark blue formazan product. This process requires mitochondrial function, and even freshly dead cells cleave small amounts of MTT, if any. MTT was dissolved in PBS (pH 7.4) at the concentration of 5 mg/ml. One hundred microlitres of MTT solution were added into each culture well and incubated at 37°C for 4 h. After adding 100 ~1 of isopropanol with 0.04 N of HCl into each well to dissolve the cleaved products of MTT, the light absorbance was measured by an ELISA plate reader (Toyosoda, Tokyo, Japan) with a test wavelength of 570 nm and a reference wavelength of 620 nm. The light absorbance (AOD 570-620 nm ) was proportional to the number of living cells from 100 to 50 000 cells/well (Mosmann, 1983).
Measurement of bFGF and VEGF in the culture media of human trophoblasts Culture medium
of endothelial cells in the condition from trophoblasts
One hundred microlitres of HMvECs suspension (8 x lo3 cells/ml) in MCDB 104 medium supplemented with 10 per cent FCS and growth factor solution@ (Morinaga) were added onto each well of 96-well flat-bottomed culture plates (Corning) which had been precoated with 3 mg/ml collagen for 30 min at room temperature. HMvECs were cultured for overnight to allow to adhere to the well bottom at 37°C in 5 per cent CO, and then the medium was replenished with either medium 199 with 5 per cent FCS or the above-stated condition medium of trophoblasts. After 48 h in culture when cultured HMvECs occupied about 80 per cent of the area of a culture disk, their number was determined by MTT proliferation assay (Mosmann, 1983) as described below.
Human bFGF and VEGF were measured by using sandwich enzyme-linked immunoassay (ELISA) kits (R&D Systems, MN, USA).
Absorption the medium
of angiogenic and culture
growth factors supernatant
from
Basic FGF. A total of 0.6 ml trophoblast condition medium and 0.6 ml medium 199 with 5 per cent FCS was incubated with protein G-sepharose (Sigma) beads which could bind 10 mg of IgG and either 1 pg/ml anti-human bFGF antibody (Austral Biologicals, CA, USA) or 10 pg/ml mouse IgGl negative control (the IgG subclass similar to the antihuman bFGF antibody; Chemicon International), at 4°C for overnight.
Hamai
et al.: Trophoblasts
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Angiogenesis
VEGF. For VEGF, 0.6 ml trophoblast condition medium and 0.6 ml medium 199 with 5 per cent FCS were incubated with protein A-sepharose (Sigma) beads which could bind 10 mg of IgG and either 5 pg/ml anti-human VEGF neutralizing antibody (R&D Systems, MN, USA) or 10 pg/ml mouse IgG2b negative control (the IgG subclass similar to the anti-human VEGF neutralizing antibody; Chemicon International) at 4°C for overnight.
Addition of angiogenic growth factor antibody-treated condition medium
to
Human bFGF (Progen Biotechnik, Heidelberg, Germany) at different concentrations (0.3, 3 and 30 pg/ml) was added into 72-h condition medium of trophoblasts pretreated with anti-bFGF antibody and protein G-sepharose beads. The resultant medium was used for experiment of HMvECs-growth augmentation.
lmmunohistochemical trophoblasts with antibody
3,3’-diaminobenzidine tetrahydrochloride containing 0.02 per cent H,O, for 10 min to visualize reaction products. Finally, the sections were soaked in excess water.
Statistical
analysis
Differences between the two groups were analysed for significance (PcO.05) by Wilcoxon’s test.
RESULTS Growth factors trophoblasts
released
from
cultured
The concentration of bFGF in the 72-h condition medium of trophoblasts was 2.16 f 1.63 pg/ml (mean f s.d.). The concentration of VEGF in the 72-h condition medium was below the sensitivity of ELISA. Both bFGF and VEGF were not detected in the 5 per cent FCS-supplemented medium 199 by ELISA.
staining of cultured anti-bFGF and anti-VEGF
Trophoblasts were stained by using a labelled streptavidin biotin method. Cultured trophoblasts were fixed in acetone at 4°C for 10 min. After being washed with cold tris-buffered saline (TBS) (50 mM Tris-HCl, 150 mM NaCl, pH 7.6), the cells were incubated in 0.03 per cent H,O,, 0.03 per cent NaN, (Peroxidase blocking reagen@; Dako) for 10 min at room temperature to remove the endogenous peroxidase activity. Subsequently, the sections were washed with cold TBS, incubated in 5 per cent rabbit serum-TBS for 10 min at room temperature. After being incubated in 0.1 per cent avidin50 mM tris-HCl with 15 mM NaN3 (Biotin blocking systemE’; Dako) for 15 min at room temperature the cells were washed with cold TBS and then incubated in 0.01 per cent biotin50 mM Tris-HCl with 15 mM NaN, (Biotin blocking systems) for 15 min. Following these procedures, the cells were washed with cold TBS and incubated in 0.025 pg/ml anti-human bFGF antibody or 0.025 pg/ml anti-human VEGF neutralizing antibody diluted in TBS containing carrier protein and 15 mM NaN, (Dako Antibody Diluent@) overnight. For control staining, mouse IgGl, the IgG subclass similar to the anti-human bFGF antibody, and mouse IgG2b, the IgG subclass similar to the anti-human VEGF neutralizing antibody, were used at 0.025 pg/ml. After being washed with cold TBS, the cells were then incubated with 1.2 pg/ml biotinylated F(ab’)2 fragment of rabbit anti-mouse immunoglobulins (Dako), diluted in tris-HCl buffer containing carrier protein and 15 mM NaN, (Dako antibody diluent’m), for 10 min at room temperature. They were further washed with cold TBS, followed by the incubation with peroxidase-labelled streptavidin solution (Dako LSAB kit@) for 10 min. They were washed again with cold TBS and incubated with 1 mg/ml
Elimination of HMvECs growth-promoting activity of bFGF or VEGF by neutralizing antibodies The MTT assay showed that viable HMvECs after 48 h culture in medium 199 with 30 pg/ml of bFGF or 30 pg/ml of VEGF increased significantly in number compared to those cultured in medium 199 without FCS. Neutralization of these growth factors with respective antibody completely eliminated the HMvECs growth-promoting activities of the growth factors (Figures 3 and 4). These results demonstrate that this neutralization method can be used to eliminate the activities of both bFGF and VEGF, which are higher than those present in condition medium of trophoblasts.
Growth of HMvECs in the condition treated with or without the antibody bFGF or VEGF
medium against
Figure 5 compares the number of cells of HMvECs cultured with either medium 199 with 5 per cent FCS or condition medium of trophoblasts. The number of cells increased by 60 per cent when cultured in trophoblast condition medium. The pretreatment of the condition medium with anti-bFGF antibody and protein G-sepharose beads resulted in a decrease in cell numbers by 50 per cent. Incidentally, the pretreatment of the control medium (medium 199 with 5 per cent FCS) with anti-bFGF antibody and protein G-sepharose beads also produced a 20 per cent decrease in the number of cells. The number of cells cultured with control medium pretreated with anti-bFGF antibody and protein G-sepharose beads equalled that cultured with the condition medium pretreated in the
152
Placenta
(1998),
Vol. 19
0.140 0.120 0.100
.
0.080
0.060
-
0.060
0.050
-
i
I . .
i
0.040 0.020 0.000
I
NS Medium
199
A
199 + 30 pg/ml
C bFGF
Figure 3. Effect of bFGF and its antibody and protein G-sepharose beads on the proliferation of HMvECs. HMvECs were cultured for 48 h in medium 199 supplemented with bFGF at concentration of 30 pg/ml. Medium 199 was supplemented with the same concentration of bFGF which was then treated with either anti-bFGF antibody and protein G-sepharose beads (B), or IgGl negative control and protein G-sepharose beads (C). The cell culture was run in parallel using these culture media. Cell numbers were determined by MTT assay. * Statistically significant, P
. 8 ii I . .i
0.140 0.120 0.100
I
0.080
i 0.040
0 i 8
’ :
i !.
0.020 0.000
NS Medium
199
A Medium
I NS
B
Medium
0.060
8
3 : 0
0.000 ’
A Medium
B
B
C VEGF
Figure 4. Effect of VEGF and its antibody and protein A-sepharose beads on the proliferation of HMvECs. HMvECs were cultured for 48 h in medium 199 supplemented with VEGF at concentration of 30 pg/ml. Medium 199 was supplemented with the same concentration of VEGF which was then treated with either anti-VEGF antibody and protein A-sepharose beads (B), or IgG2b negative control and protein A-sepharose beads (C). The cell culture was run in parallel using these culture media. Cell numbers were determined by MTT assay. *Statistically significant, P
same way. The pretreatment with IgGl and protein G-sepharose beads had no effect on cell numbers in both groups.
A
B
C
Condition medium of trophoblasts
Figure 5. Effects of condition medium from trophoblasts pretreated with anti-bFGF antibody and protein G-sepharose beads on the proliferation of HMvECs. HMvECs were cultured with Medium 199 and 5 per cent FCS or condition medium from trophoblasts. Each culture group was randomized to either IgGl and protein G-sepharose beads pretreatment (B), anti-bFGF antibody and protein G-sepharose beads pretreatment (C) or without pretreatment (A). The cell cultures were conducted for another 48 h. Cell numbers were determined by MTT assay. + Statistically significant, P
The growth-inhibiting effect of anti-bFGF antibody and protein G-sepharose beads, when added to the trophoblast condition medium, was obviated by the addition of bFGF at 3 pg/ml, the concentration of which was comparable with that attainable in the condition medium of trophoblasts (Figure 6). Figure 7 illustrates that the addition of anti-VEGF antibody and protein A-sepharose beads into trophoblast condition medium resulted in a 15 per cent reduction in the number of cells (statistically significant, P
199 + 30 pg/ml
C
199 + 5 per cent FCS
staining
The presence of immunoreactive human bFGF and VEGF molecules was demonstrated in cultured trophoblasts. The reaction products for both, which were identified as small granules, were present in the cytoplasm of trophoblasts (Figures 8 and 9). When IgGl or IgG2b was used instead of anti-human bFGF antibody or anti-human VEGF antibody respectively, no apparent staining was observed in the trophoblasts (Figures 10 and 11). DISCUSSION Growth of the placenta requires extensive angiogenesis to develop its vascular structure in both fetal chorionic villi and
Hamai
et al.: Trophoblasts
and Placental
Angiogenesis
153 7
0.090 0.080 0.070
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*
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; fi
0.050
0.060
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f
t 0.050 0.040
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;
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8
0.030
i
l
*I
0.030
0.020
0.000
0.000
L -r
I * ’ A Medium
Condition
medium
of trophoblasts
Figure 6. Effects of anti-bFGF antibody (B) and different concentrations of bFGF on the proliferation of HMvECs cultured with condition medium from trophoblasts. Human bFGF at different concentrations (C, 0.3; D, 3; and E, 30 pg/ml) was added to 72-h condition medium of trophoblasts which had been pretreated with anti-bFGF antibody and protein G-sepharose beads. The resultant medium was used for experiment of HMvECs growth augmentation. Cell numbers were determined by MTT assay. NS, Not significant; A, IgGl and protein G
maternal decidua (Blankenship, Enders and King, 1993). According to the current opinion, failure of establishing the adequate placental vascular structure may cause ischaemia of the placenta and therefore vasoactive substances in the placenta might be released into the maternal blood, which in turn could lead to complications such as pre-eclampsia, placental dysfunction, intrauterine growth restriction or intrauterine fetal death (Sharkey et al., 1993). To date, several angiogenic factors such as bFGF, VEGF and platelet-derived growth factor (PDGF) have been shown to exist in trophoblasts. These growth factors are considered to play a role in the development of fetomaternal vascular system. However, published studies have simply demonstrated the presence of the angiogenic factors, without providing a support for the role of trophoblasts in the extensive neovascularization occurring during pregnancy. In this study, we demonstrated that cultured human trophoblastic cells released angiogenic substances as judged by growth-promoting activity of vascular endothelial cells. In addition, the angiogenic activity was eliminated almost completely by the concomitant addition of anti-bFGF antibody and protein G-sepharose beads, suggesting that an angiogenic factor released by trophoblasts may be bFGF. Unexpectedly, the addition of anti-bFGF antibody and protein G-sepharose beads to control medium resulted in a marginal decrease in cell numbers. This can be explained by the presence of a trace
B
C
199 + 5 per cent FCS
A
B
C
Condition medium of trophoblasts
Figure 7. Effects of condition medium from trophoblasts pretreated with anti-VEGF antibody and protein A-sepharose beads on the proliferation of HMvECs. HMvECs were cultured with medium 199 and 5 per cent FCS or condition medium from trophoblasts. Each culture group was randomized to either IgG2b and protein A-sepharose beads pretreatment (B), anti-VEGF antibody and protein A-sepharose beads pretreatment (C) or without pretreatment (A). The cell cultures were conducted for another 48 h. Cell numbers were determined by MTT assay. * Statistically significant, P
Figure 8. Immunohistochemical staining of bFGF molecule in the cultured trophoblasts (200 x ). Acetone-fixed cultured trophoblasts were reacted with anti-human FGF antibody, and then stained by the labelled streptavidin biotin method (see Materials and Methods).
amount of bFGF, the level of which was too low to be detected. However, the present study demonstrated the presence of VEGF in cultured trophoblastic cells as well, suggesting a role of VEGF as one of trophoblast-derived angiogenic factors. The expression of bFGF has been shown in endothelial cells and smooth muscle cells of placental vessels other than trophoblastic cells (Ferriani et al., 1994; Shams and Ahmed, 1994). Ferriani et al. (1994) demonstrated that bFGF immunoreactivity is present in or around the cytotrophoblast cells. Thus, bFGF produced in these cells may be responsible
Placenta
Figure 9. Immunohistochemical trophoblasts (200 x ). Cultured VEGF antibody.
staining of VEGF molecule in the cultured trophoblasts were reacted with anti-human
Figure 10. Immunohistochemical staining of the cultured trophoblasts with IgGl negative control (200 X ). Acetone-fixed cultured trophoblasts were reacted with IgGl negative control. No reaction product was noticed against the IgGl negative control.
for fetal villous angiogenesis. In this study, bFGF in the condition medium was shown to be mitogenic on endothelial cells. Although bFGF is known to be released through a mechanism of basement membrane degradation, a recent study showed that bFGF may be secreted directly by FGFproducing cells via a pathway independent of the endoplasmic reticulum (Mignatti, Morimoto and Rifkin, 1992). In addition, almost all the cultured trophoblasts were viable after 72 h in culture as judged by trypan blue exclusion, it is probable that bFGF detected in the culture media is secreted by trophoblasts. Also basic FGF is known to cause morphological changes of capillary endothelial cells, and increase the production of proteinases such as plasminogen activator and collagenase in a cell culture system of these cells (Tsuboi, Sato and R&in, 1990), thus enhancing migration of the cells, a prerequisite for angiogenesis. Basic FGF has been shown to be localized in the porcine uterus comprising glandular and luminal epithelia and stromal cells (Katsahambas and Hearn, 1996). In human, FGF mRNA has been detected in the endometrium; its level being higher in the proliferative phase compared with secretory phase
(1998),
Vol. 19
Figure 11. Immunohistochemical staining of the cultured trophoblasts with IgG2b negative control (200 X ). Acetone-fixed cultured trophoblast were reacted with IgG2b negative control. No reaction product was noticed against the IgG2b negative control.
(Fujimoto et al., 1996). The mRNA level does not increase in the early stage of pregnancy (Katsahambas and Hearn, 1996). Assuming that FGF is a key factor responsible for marked neovascularization in the endometrium after implantation, an unchanged level of FGF mRNA during early pregnancy does not explain the prominent progression of vasculature associated with pregnancy. It is reasonable to presume that FGF derived from trophoblasts together with FGF from decidual tissues may enhance decidual vascularization. The finding that FGF is located predominantly in extravillous trophoblasts (Ferriani et al., 1994), the fetal tissue interface, may support this hypothesis. VEGF has been reported also to exist in trophoblasts, decidual macrophages, maternal decidual cells, Hofbauer cells within the villous mesenchyme and embryo, and the smooth muscle cells surrounding vein and arteries of the umbilical cord (Jakeman et al., 1993; Barleon et al., 1994; Jackson et al., 1994; Ahmed et al., 1995). VEGF receptor, flt-1 protein, has been reported to be expressed in trophoblasts, decidual macrophages, maternal decidual cells and Hofbauer cells within the villous mesenchyme (Jakeman et al., 1993; Barleon et al., 1994; Ahmed et al., 1995). These findings strongly support a role of VEGF as one of stimulators of angiogenesis in both fetal and maternal compartments during gestation. However, in this study, the addition of anti-VEGF antibody and protein A-sepharose beads into the condition medium resulted in a slight but significant reduction (15 per cent) in cell numbers, while it decreased control cell number by 30 per cent, suggesting that growth inhibition by anti-VEGF antibody was ascribable to the neutralization of an undetectable amount of VEGF present in control medium and its level, if any, in the condition medium was the same as that in control medium. On the basis of these findings, one can envision that VEGF may act in an autocrine manner. If it can work in a paracrine way at all, it may act only on adjacent cells. Pregnancy seems to be an immunological paradox. The fetus, which is a semi-allograft, can survive for 9 months despite the maternal immune system. It is reasonable to
Hamai
et al.: Trophoblasts
and Placental
Angiogenesis
155
speculate that some special immunological mechanisms are involved in securing fetal growth (Rocklin et al., 1976; Kovithavongs and Dossetor, 1978; McIntyre and Faulk, 1979; Suciu-Foca et al., 1983; Wegmann et al., 1989). Disruption of this immune regulation during pregnancy is suggested to be one of the etiological factors for pre-eclampsia. For instance, serum concentrations of both IL-2 and TNF-a in the first trimester of pregnancy were significantly higher in the women who developed pre-eclampsia after 28 weeks of pregnancy compared with those in the women who completed pregnancy uneventfully (Hamai et al., 1997). Also IL-2 has been proved to be present in the decidual tissue of pre-eclamptic patients (Hara et al., 1995). These immunological abnormalities may cause an injury of the trophoblasts and the angiogenic growth
factor-secretion from trophoblasts may be decreased. Failure to secrete the angiogenic growth factor may lead to the failure in establishing the adequate placental vascular system and may cause pre-eclampsia. In summary, we demonstrated that cultured human trophoblastic cells obtained from early human placental villi release biologically active bFGF. In addition, the majority of angiogenie activity in condition medium from trophoblasts was suggested to be attributable to bFGF released from trophoblasts. Thus, it appears that bFGF is a crucial angiogenic factor derived from human trophoblastic cells and thereby may play an important role in angiogenesis occurring in early gestation.
ACKNOWLEDGEMENTS We sincerely
thank
Dr Takeshi
Sugimoto
for providing
placental
tissue.
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