Molecular Human Reproduction, Vol. 7, No. 8, 771-777,
August 2001
© 2001 European Society of Human Reproduction and Embryology
Implantation and pregnancy |
In-vitro evidence of autocrine secretion of vascular endothelial growth factor by endothelial cells from human placental blood vessels
1 Division of Pharmacology and Chemotherapy and 2 Division of Pathology, Department of Oncology, Transplants and Advanced Technologies in Medicine, 3 Division of Gynaecology and Obstetrics, Department of Reproductive Medicine and Child Development, University of Pisa, Pisa, Italy
Abstract
Vascular endothelial growth factor (VEGF), a highly specific mitogen for vascular endothelial cells, is involved in placental vascular growth and remodelling. The aim of this study was to investigate whether placental endothelial cells secrete VEGF in an autocrine manner and if this secretion is correlated with endothelial cell growth. Blood vessels, excised from the apical surface of three human placentae, were sectioned into 40 fragments per placenta and cultured in fibrin gel matrix for 27 days. Immunohistochemical detection of placental endothelial cells was performed by positive staining with anti-human factor VIII-associated antigen and negative staining with anti-human 
-actin and desmin. To investigate the production and autocrine action of VEGF, VEGF concentrations in culture media were measured and the effect of an anti-VEGF neutralizing antibody on endothelial cell growth was observed. The results demonstrate that soluble VEGF is secreted by placental endothelial cells reaching a plateau from day 24 (68.74 ± 7.52 pg/ml) to day 27 (67.20 ± 6.28 pg/ml). Furthermore, VEGF concentrations in media collected on days 6, 12, 18, 21 and 27 of culture were found to be directly correlated to the sprouting parameter of endothelial cells, as calculated by image analysis on the same day (
P
< 0.001,
r
2
= 0.95
). The use of 10 and 100 ng/ml of a neutralizing antibody against human VEGF suppressed cell proliferation, compared to that observed in the untreated controls, by 74.8 ± 7.3 and 89.4 ± 3.9% respectively. In conclusion, this study reports the first evidence of autocrine secretion of VEGF by human placental endothelial cells and demonstrates the involvement of VEGF in endothelial cell growth within a fibrin gel culture.
endothelial cells/image analysis/immunohistochemistry/placental blood vessels/VEGF
Introduction
Angiogenesis, the development of new capillaries from pre-existing blood vessels, is a tightly controlled phenomenon in the adult and occurs in a physiological manner almost exclusively in the female reproductive system. The uterus, ovary and placenta exhibit distinct phases of blood vessel growth, maturation and regression accompanied by changes in their patency to blood flow. Thus, these tissues are among the few adult structures in which angiogenesis occurs as a normal process (
Folkman and Shing, 1992
;
Folkman, 1995
;
Norrby, 1997
).
The members of the VEGF family are prominent among the extracellular signalling molecules that guide vascular development (
Ferrara et al., 1992
). VEGF, a 34–46 kDa homodimeric glycoprotein also known as vascular permeability factor (VPF), acts as a highly specific mitogen for vascular endothelial cells (
Achen and Stacker, 1998
), promotes angiogenesis in several in-vivo and in-vitro models (
Ferrara et al., 1992
), markedly induces vascular permeability (
Keck et al., 1989
) and acts as a survival factor for newly formed blood vessels (
Benjamin and Keshet, 1997
).
VEGF has recently been demonstrated to be an important factor in physiological angiogenesis of human endometrium throughout the menstrual cycle (
Shifren et al., 1996
;
Agrawal et al., 1999
), and its secretion into the lumina of endometrial glands has been suggested as a possible endometrial signal for blastocyst implantation and development (
Hornung et al., 1998
).
In the ovary, vascularization is a prerequisite for corpus luteum formation, since a complex vascular network is formed within the thecal cell layer during follicular growth. Furthermore, a rapid neovascularization occurs toward the granulosa cell layer after ovulation (
Otani et al., 1999
). Recently, definitive evidence has been provided showing that the development and endocrine function of the ovarian corpus luteum are dependent on VEGF-mediated growth of new capillary vessels (
Ferrara et al., 1998
). In ovarian diseases, such as endometriomata (
Fasciani et al., 2000
), tumours (
Orre and Rogers, 1999
) and hyperstimulation syndrome (
Artini et al., 1998
), angiogenesis driven by VEGF has also been described.
With respect to normal development and maturation of human placenta, VEGF plays a crucial role in blood vessel growth (
Wheeler et al., 1995
;
Shiraishi et al., 1996
). Indeed, VEGF mRNA has been detected in placental tissue (
Achen et al., 1997
;
Athanassiades et al., 1998
) and an immunohistochemical analysis has demonstrated the presence of VEGF and its receptors within the placental vascular network (
Winther et al., 1999
), thus suggesting that this growth factor is important in the development and maintenance of vascular function during pregnancy (
Cheung, 1997
). Finally, evidence has been provided on the role played by VEGF in embryonic vasculogenesis and angiogenesis (
Carmeliet et al., 1996
;
Ferrara et al., 1996
); inactivation of the
VEGF
gene in knock-out mice resulted in embryonic lethality in heterozygous embryos associated with severe defects in the vasculature of placenta and nervous system (
Ferrara, 1999
). The mRNA encoding VEGF has been located in decidual macrophages, in the villous mesenchyme and decidual glands (
Clark et al., 1998
), as well as in trophoblast (
Winther et al., 1999
). Recently, VEGF has been demonstrated to increase trophoblast motility, although no effect on extravillous trophoblast invasion and proliferation was observed (
Lash et al., 1999
).
The aims of this study were to investigate whether endothelial cells obtained from human placenta after spontaneous delivery are able to secrete VEGF in vitro and to establish a relationship between the amount of secreted VEGF and endothelial cell sprouting.
Materials and methods
Materials
Cell culture Medium 199 without Phenol Red was purchased from Gibco BRL (Paisley, UK); neutralizing monoclonal anti-human vascular endothelial growth factor, clone 26503.11 (anti-hVEGF mAb), cell culture supplements and all other chemicals not listed in this section were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Plastics for cell culture were supplied by Costar (Cambridge, MA, USA). Monoclonal antibodies against human factor VIII-associated antigen and -actin and desmin were purchased from Dako (Glostrup, Denmark); biotin-labelled secondary antibody and avidin–biotin complex were from Vector Laboratories (Burlingame, CA, USA).
The use of explants from human placentae, as described below, was authorized by the Ethics Review Committee of Pisa University Hospital. A written informed consent on the procedures and aims of the study was obtained from each placenta donor in compliance with regulations concerning the use of human tissues.
In-vitro cultures of human placental vessels
A previously described experimental procedure was adopted (
Brown et al., 1996
) with the following modifications. Immediately after spontaneous delivery and section of the umbilical cord, the placenta was collected in sterile conditions and placed on ice; superficial blood vessels, ~1–1.5 mm in diameter and from 1 to 5 cm in length, were quickly excised from the apical surface of three human placentae using a sterile scalpel.
Forty vessel fragments from each placenta were placed in phosphate-buffered saline (PBS) solution containing 2.5 µg/ml of amphotericin B and 50 µg/ml of gentamycin and cut into ~1 mm fragments using fine dissecting forceps and iridectomy scissors with the aid of a magnifier. Vessel explants were then cleared of residual clots and placed in PBS before their use. Cultures were performed in 24-well culture plates; 0.5 ml/well of a solution of fibrinogen 3 mg/ml in Medium 199 without Phenol Red was added to each well and mixed with 15 µl of thrombin (50 NIH IU/ml in 0.15 mol/l NaCl).
Vessel fragments were quickly placed in the centre of the wells after clot formation and covered by an additional 0.5 ml/well of the fibrinogen/thrombin solution mentioned above, to hold them at the same level between the two clots. After gel formation, 1 ml/well of Medium 199 without Phenol Red was added, supplemented with 5% of heat-inactivated fetal bovine serum (FBS), 0.1% 
-aminocaproic acid (to prevent lysis of the fibrin clot),
L
-glutamine (2 mmol/l) and antibiotics (streptomycin 50 µg/ml, penicillin 50 IU/ml and amphotericin B 2.5 µg/ml). In separate experiments, vessel fragments were treated every 2 days with the neutralizing anti-hVEGF mAb at the concentrations of 10 and 100 ng/ml. Vessels were cultured at 37°C in 95% air/5% CO
2
in a humidified environment for 27 days, and the medium was changed every 3 days. Vessel explants were photographed on days 0, 6, 12, 18, 21 and 27 with a phase-contrast Leitz MD IL microscope (Leica, Heerbrugg, Switzerland) and pictures of at least six explants per day were subjected to image analysis as described below.
Histology and immunohistochemistry
In order to document the characteristics of microvascular structures, at the end of experiments, vessel explants with their sprouted vascular cells
en bloc
with fibrin clots were fixed in 10% formalin. Sections 5 µm thick were stained with haematoxylin–eosin for histological examination and additional sections were used for immunohistochemistry. Sections were deparaffinized in xylene and dehydrated in alcohols. Endogenous peroxide activity was blocked by incubating the slides in 1% hydrogen peroxide in methanol for 10 min. After blocking non-specific binding with normal serum, sections were incubated with the following monoclonal antibodies: (i) anti-human factor VIII-associated antigen as an endothelial cell marker, and (ii) anti -actin and desmin as smooth muscle cell markers. Then slides were incubated with biotin-labelled secondary antibody (dilution 1:500) and finally with 3,3'-diaminobenzidine tetrahydrochloride as the chromogen. Sections were counterstained with haematoxylin, dehydrated and mounted. Negative controls were obtained by omitting primary antibodies.
VEGF detection in culture media
The culture media of 40 vessel fragments from each placenta were collected on days 0, 3, 6, 9, 12, 15, 18, 21, 24 and 27 of culture and immediately centrifuged at 228
g
for 5 min at 4°C to separate the debris from the supernatant which was then stored for a maximum of 1 month at –80°C. Each sample was assayed twice for VEGF concentrations by an enzyme-linked immunosorbent assay (ELISA) which measures the secreted forms of VEGF (CYT
Elisa
Human VEGF; Cytimmune Sciences Inc., College Park, MD, USA
), with a sensitivity of 18.6 pg/ml, an intra-assay variability of ±8.9% and an inter-assay variability of ±11.1%.
For best accuracy of the assay, Medium 199 without Phenol Red was used for cultures to avoid possible interferences with VEGF detection by ELISA, since this step consists of absorbance reading of the amount of a deep red-coloured product with maximum absorbance at 492 nm wavelength.
Image analysis
The image analysis was performed as previosly described (
Bocci et al., 1999
). Briefly, pictures of the placental fragments were digitized in a 512x512 pixel matrix, using a colour video camera TK-1280E (JVC, Tokyo, Japan) and a microcomputer. Digitized pictures were visualized on high resolution colour display (Sampo, Tao-Yuan Hsien, Taiwan) and the true colour image analysis software package KS 300 v.1.2 (Kontron Elektronik GmbH, Eching, Germany) was run for interactive manipulation, quantification of images and data collection. Geometric calibrations were set with a sample of known dimensions and a gray-scale analysis was performed to measure the density of the image that was in the range of 0–255, where 0 is black (presence of endothelial sprouts) and 255 is white (absence of endothelial sprouts). The threshold levels for each sample were set interactively by the software in the respective red, green, and blue (RGB) image partitions that form the full-colour image. The area occupied by the sprouts was calculated by the computer in a binary picture that resulted after application of the RGB-discrimination step. In the fibrin culture of placental vessel fragments, the mean grey level of the endothelial sprouting area was measured and the sprouting index was defined as follows:
sprouting index = [(sprouting area/mean grey level of sprouting area)/perimeter of fragments]x100
Furthermore, the average area (mm 2 ) and radius (mm) of endothelial cell sprouts were calculated interactively by measuring the distance between the explant and the farthest edge of cell growth.
Data analysis
Results of the ELISA assay and image analysis are reported as the mean ± SEM of
n
observations as calculated by the GraphPad Prism
©
software package (GraphPad Software Inc., San Diego, CA, USA). Statistical significance of differences was assessed by the Student's
t
-test and
P
< 0.05 was considered to be significant. The correlation between VEGF concentrations in the culture media and the sprouting index was analysed by linear regression analysis using GraphPad prism
©
. The percentage of inhibition of cell growth was expressed as the mean ± SEM of controls.
Results
The explants sprouting within the fibrin matrix were characterized by numerous microvessels around the placental fragments. Vascular cells organized radially to form microvessels that underwent continuous remodelling (Figure 1
)
. The maximal growth of the 3-D microvascular network occurred during the third to fourth week and reached a plateau 27 days after explant.
|
A subtle framework of sprouting cells was observed within the fibrin gel; these cells stained positive for the endothelial marker factor VIII-associated antigen (Figure 2A , B
)
, and negative for -actin and desmin (data not shown). In most cases endothelial cells organized to form microvessels with lumina (Figure 3A
)
; in others the lumen was absent and only endothelial cells were observed. The vessel explants became relatively acellular and with abundant fibrosis (Figure 3B
). The few cells present in the explants were negative for -actin and desmin.
|
|
The sub-macroscopic picture of the placental explants on the first day of culture is shown in Figure 4A
; the appearance of the outgrowth of endothelial cells from the placental vessel fragments was observed on approximately the 6th day of culture (sprouting index = 0.055 ± 0.004 mm/mean grey; Figure 4B
]. On the 18th day of culture, the capillaries sprouted within the fibrin matrix as shown in Figure 4C
(sprouting index = 0.76 ± 0.12 mm/mean grey
) and the growth of the 3-D microvascular network reached its plateau on the 27th day (sprouting index = 2.30 ± 0.4 mm/mean grey; Figure 4D
). The VEGF concentrations measured in the media of placental vessel explants during the days in culture are reported as a concentration versus time plot in
Figure
4E
alongside the sprouting index values measured on the same days. The VEGF concentration measured on day 0 (a concentration close to the limit of detection of the ELISA immunoassay) was most likely derived from the FCS added to the medium or from the damaged cells of the explant.
|
The areas occupied by endothelial cells increased from 0.5 ± 0.01 mm 2 on day 6 to 24.2 ± 2.6 mm 2 on day 27, while the radius of the area covered by cell sprouts increased from 0.4 ± 0.01 mm at day 6 to 2.1 ± 0.3 mm on day 27, as shown in Figure 5
. The VEGF concentrations in the media collected on days 6, 12, 18, 21 and 27 of culture were found to be directly related to the sprouting index calculated on the same days (
P
< 0.001,
r
2
= 0.95; Figure 6
)
.
|
|
The proliferation of microvascular sprouts, calculated by the sprouting index, was markedly inhibited by the addition of the anti-hVEGF mAb to the culture medium; such decrease of the sprouting index, as compared to controls on day 27, was 74.8 ± 7.3 and 89.4 ± 3.9% after having added 10 and 100 ng/ml respectively of the anti-hVEGF mAb ( P < 0.05 versus untreated controls ).
Discussion
This study demonstrates that soluble VEGF is actively secreted by endothelial cells derived from blood vessels of human placenta and that VEGF production is correlated with microvessel growth and sprouting within a fibrin gel culture.
Although the formation of capillary-like tubules on a collagen matrix seems to be not specific for endothelial cells (Berdichevisky
et al
., 1994
), this may not be the case for fibrin gels. Nevertheless one of the features of endothelial cells is tubulogenesis, because nearly all endothelial cells, whether primary or immortalized, show the ability to form a capillary-like network when cultured on basement membrane matrix
in vitro
(
Maru et al., 1998
).
The 3-D fibrin gel culture was adopted in this study because of the evidence that VEGF induces the migration of endothelial cells within the extracellular matrix (
Qu-Hong et al., 1995
), and such a model mimics the VEGF-induced microenviroment in which neo-angiogenesis develops
in vivo
. Indeed, VEGF has been demonstrated to make blood vessels hyperpermeable, leading to extravasation of plasma fibrinogen and its clotting to form fibrin in both ascites and solid tumours (
Nagy et al., 1988
). Furthermore, extravascular fibrin deposits provide a provisional stroma that serves as a substratum for cell migration during angiogenic phenomena and supports the growth of new blood vessels (
Nagy et al., 1995
). In addition, VEGF promotes angiogenesis in 3-D in-vitro models, inducing confluent microvascular endothelial cells to invade collagen gels and form capillary-like structures (
Pepper et al., 1992
).
Although there has been some controversy concerning the ability of endothelial cells to produce VEGF (
Weindel et al., 1992
;
Qu-Hong et al., 1995
), the autocrine VEGF production by endothelial cells has been suggested on the basis of the presence of VEGF mRNA transcripts in cultures of rat brain capillary endothelial cells (
Ladoux and Frelin, 1993
). This finding was then confirmed in cultured endothelial cells derived from microvessels of derma (HMEC) and umbilical vein (HUVEC) under hypoxic conditions (
Namiki et al., 1995
;
Nomura et al., 1995
). Moreover, it has been demonstrated that VEGF stimulates the growth of capillary endothelial cells from bovine retina (BREC) through an autocrine pathway, based on the detection of both VEGF mRNA within cells and VEGF protein in the BREC-conditioned medium (
Simorre-Pinatel et al., 1994
). Recently, it has been shown that the proliferation and tube formation of skin microvascular endothelial cells is promoted by insulin through the induction of autocrine VEGF (
Yamagishi et al., 1999
).
The evidence that the placental endothelial cells are the main source of VEGF measured in the conditioned medium of the present study, is supported by the finding that cell sprouts were composed of endothelial cells as demonstrated by immunohistochemistry for the endothelial marker factor VIII-associated antigen. In addition to this, the presence of VEGF mRNA in outgrowths from placental vessel explants has been demonstrated in the same experimental model (
Brown et al., 1996
). Moreover, the negative staining for the markers of vascular smooth muscle cells and myofibroblasts, such as the -actin and desmin proteins, and the signs of tissue regression of the explants provide additional evidence suggesting that the only reasonable source of the increasing concentrations of VEGF in our cultures were the endothelial sprouting cells.
Our data are consistent with the wide range of endothelial growth pattern observed in different mammalian species. In fact, the endothelial cell proliferation and the VEGF secretion in this human model of placental vessel explant was different from that previously described (
Nicosia et al., 1997
) in a rat model of vessel explants where VEGF was secreted mostly in the first week of a 2 week culture. However, both the human and the rat models provide the evidence that endogenous VEGF plays an important and critical role in microvessel formation in a 3-D fibrin matrix.
In this study, the quantification of cell sprouts was performed by an image analysis parameter that permits a reliable measurement of the microvascular growth; this method, indeed, allows the calculation of vascular cell proliferation in a 3-D extracellular matrix, where cells migrate and produce microvessel-like structures that interact with the fibrin microenviroment (
Bocci et al., 1999
).
The detection of VEGF in the culture media of placental explants and the specific action of such growth factors on endothelial cell proliferation (
Ferrara et al., 1992
), led us to establish a direct correlation between VEGF concentrations and enhancement of vascular endothelial outgrowth in fibrin clots. These data indicate that VEGF is a mitogen for placental endothelial cells and that its release into the extracellular environment is a requisite for establishing an autocrine loop along with the paracrine effect of VEGF in stimulating placental angiogenesis. Indeed, previous reports have provided evidence of the inhibitory effect of neutralizing antibodies against VEGF on the growth of microvessel sprouts of placenta (
Brown et al., 1996
) and the ability of anti-VEGF antibodies to inhibit the proliferation of cultured bovine retinal endothelial cells (
Simorre-Pinatel et al., 1994
). In this study, an additional demonstration that cell proliferation is driven by this specific growth factor is provided by the observation that the neutralizing anti-hVEGF mAb markedly inhibits the endothelial cell outgrowth. Indeed, these results are comparable to those obtained by (
Nicosia et al., 1997
) in the rat model, thus confirming the same biological activity of VEGF in different species.
In conclusion, this study shows that endothelial cells from human placenta are not only the target, as previously envisaged, but could also be a relevant source of VEGF which supports endothelial cell proliferation and microvessel formation in both normal and pathological conditions.
Acknowledgements
The authors wish to thank Ms Anna Fioravanti for her technical assistance and Ms Cristiana Matteucci for providing assistance in the delivery room.
Notes
4 To whom correspondence should be addressed at: Division of Pharmacology and Chemotherapy, Department of Oncology, Transplants and Advanced Technologies in Medicine, University of Pisa, Via Roma, 55, I-56126 Pisa, Italy.
E-mail: m.deltacca{at}do.med.unipi.it 
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Submitted on February 7, 2001; accepted on May 16, 2001.
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