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Department of Obstetrics and Gynecology, Catholic University of the Sacred Heart, Largo Gemelli 8, 00168 Rome, Italy
1 Department of Microbiology, Catholic University of the Sacred Heart, Largo Gemelli 8, 00168 Rome, Italy
(Requests for offprints should be addressed to N Di Simone; Email: nicoletta.disimone{at}virgilio.it)
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Abstract |
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In human choriocarcinoma cells (BeWo), resistin (10–100 ng/ml) enhanced both MMP-2 protein and mRNA expression, significantly reduced TIMP-1 and TIMP-2 and increased trophoblast-like cell invasiveness.
We analyzed the effect of resistin on an in vitro angiogenesis system for endothelial cells (HUVEC) and we evaluated its ability to modulate the secretion of an angiogenic factor, vascular endothelial growth factor (VEGF). Our data showed that resistin induced VEGF production and we observed that the addition of resistin stimulated endothelial cell tube formation.
These findings suggest that resistin might be able to induce BeWo cell invasiveness and to contribute to the control of placental vascular development.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, resistin has been postulated to play a role in pregnancy (Kaaja et al. 1999, Innes et al. 2001) and resistin gene expression has been observed in human placenta (Yura et al. 2003). Resistin mRNA expression was detected in a trophoblastic cell line, and similarly, resistin immunostaining was detected in placental villi and in extravillous cytotrophoblast spread in the decidual tissue (Yura et al. 2003). The invasive ability of extravillous cytotrophoblastic cells is regulated by a variety of proteinases (matrix metalloproteinases, MMPs), their activators and inhibitors (Huppertz et al. 1998, Liu et al. 1998, Hu et al. 1999, Feng et al. 2000). As the most important features of the placenta are the trophoblast invasive behavior and the angiogenic processes, and as resistin has been identified in some of the main components of this organ (Yura et al. 2003), we postulated that resistin could influence and modulate the invasive behavior of the trophoblast and the proliferation of the fetoplacental vascular system by endothelial cells. In this study, we used a choriocarcinoma cell line (BeWo), a widely used model for first trimester trophoblast, to test the influence of human resistin on MMPs and the tissue inhibitors of metalloproteinases (TIMPs) and we analyzed the effect of resistin on an in vitro angiogenesis system for endothelial cells. Our data indicate that resistin is able to modify the proteolytic phenotype of BeWo cells in culture and to increase capillary tube formation related to significant vascular endothelial growth factor (VEGF) production.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BeWo choriocarcinoma cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were cultured in F12-K medium (ATCC) containing 10% FBS (HyClone Laboratories, Logan, UT, USA) and 2% penicillin/streptomycin (Sigma-Aldrich).
The cells were plated on 6-well plates (Falcon multiwell cell culture plates; BD Biosciences, Palo Alto, CA, USA) at 4 x 104 cells/well and cultured for 24 h at 37 °C in 5% CO2/95% air. Cell cultures were performed for 24 h in medium containing resistin (human recombinant, 10–100 ng/ml; Phoenix Pharmaceutical Inc, Belmont, CA, USA).
Human umbilical vein endothelial cells (HUVEC, CRL-1730) were obtained from the ATCC. The cells were cultured in F12-K medium (ATCC) supplemented with 10% FCS (Sigma-Aldrich), 100 IU/ml penicillin (Sigma-Aldrich), 100 µg/ml streptomycin (Sigma-Aldrich), 30 µg/ml endothelial cell growth supplement (ECGS; BD Bioscience, Bedford, MA, USA) and 10 µg/ml heparin (Aventis Pharma, Milan, Italy) at 37 °C in 5% CO2/95% air.
MMPs activity assay
The determination of active MMP-2 and MMP-9 in cell culture supernatants after treatment with resistin (0, 10, 50 and 100 ng/ml) in serum-free medium for 24 h was performed using the Biotrack MMPs activity assay (Amersham Biosciences; sensitivity: 0.5 ng/ml; range: 0.75–12 ng/ml) according to the manufacturer’s instructions. The assay uses the pro form of a detection enzyme that can be activated by captured active MMP into an active detection enzyme, through a single proteolytic event. MMP activated detection enzyme can then be measured using a specific chromogenic peptide substrate. Standard and samples were incubated in microplate wells precoated with anti-MMP-2 or anti-MMP-9 antibody. Any MMP present will be bound to the wells, other components of the sample being removed by washing and aspiration. Either the endogenous levels of free active MMP or the total levels of free MMP in a sample can be detected following incubation with a substrate solution. The resultant color is read at 405 nm in a microplate spectrophotometer.
Western blot analysis
At 24 h culture MMP-2, MMP-9 and two tissue inhibitors of metalloproteinases, TIMP-1 and TIMP-2 were investigated by Western blot analysis as previously described (Di Simone et al. 2003). Eighty micrograms of each sample were separated on a 12% SDS-polyacrylamide gel, and after electroblotting onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA) they were incubated with 5% non-fat dry milk in 1 mol/l Trizma/base, 1.54 mol/l NaCl, 0.05% Tween 20 (Tris buffered solution plus Tween 20, TBST, pH 7.4) and then exposed overnight at 4 °C to TBST containing primary antibodies (MMP-2 (mouse monoclonal IgG, 2C1, sc-13594, dilution 1:500), MMP-9 (mouse monoclonal IgG, 2C3, sc-21733, dilution 1:500), TIMP-1 (mouse monoclonal IgG, 2A5, sc-21734, dilution 1:500), or TIMP-2 (mouse monoclonal IgG, 3A4, sc-21735, dilution 1:500) obtained from Santa Cruz Biotechnology, Santa Cruz, CA, USA). After incubation with secondary antibody (anti-mouse horseradish peroxidase (HPR)-conjugated, sc-2031, dilution 1:10 000, Santa Cruz Bio-technology) the immunocomplexes were visualized using the ECL-Plus detection system (Amersham Biosciences) according to the instructions of the manufacturer. Bands were analyzed on the image analysis system, Gel Doc 200 system (Bio-Rad Laboratories), using the Quantity One Quantitation software (Bio-Rad Laboratories). The levels of MMP-2 and TIMPs were estimated compared with the constant level of a 42 kDa protein present in the cytosolic extract (ß-actin; mouse monoclonal, AC-15, A5441, Sigma-Aldrich).
Total RNA extraction
Total cellular RNA was extracted using the ‘QuickPrep’ Total RNA Extraction Kit (Amersham Biosciences) according to the manufacturer’s protocol. Briefly, cell pellets, obtained from BeWo cells grown in F12-K medium with resistin (10, 50 and 100 ng/ml) were suspended in lithium chloride solution, ß-mercaptoethanol, and extraction buffer. Then, samples were homogenized and incubated for 10 min on ice with caesium trifluoro-acetate solution. After centrifugation at 14 000 r.p.m. for 15 min, RNA pellets were washed and dissolved in 50 µl diethylpyrocarbonate (DEPC)-treated water. RNA concentration was evaluated by monitoring absorbance at 260/280 nm.
Quantitative real-time RT-PCR
Quantitative expression of the TIMP-1, TIMP-2 and MMP-2 genes was performed by real-time PCR using the i-Cycler iQTM system (Bio-Rad Laboratories). For the target genes and the endogenous housekeeping gene encoding for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a primer pair and Taqman probe, which hybridizes to the region between primers, were designed using Beacon Designer 2 v. 3.00 software (Premier Biosoft International, Palo Alto, CA, USA) and synthesized by MWG Biotech (Florence, Italy) (Table 1
). Quantitative PCR was performed in a 50-µl volume containing the following reagents: 25 µl Platinum Quantitative RT-PCR ThermoScript reaction mix (Invitrogen Inc), 1.5 U ThermoScript Plus/Platinum Taq mix (Invitrogen), each primer pair and the Taqman probe at a concentration of 0.5 µM, 5 µl total RNA sample and distilled water up to the final volume. Samples were subjected to an initial step at 52 °C for 45 min for RT, 94 °C for 5 min to inactivate the ThermoScript Plus reverse transcriptase and to activate the Platinum Taq polymerase, and 50 cycles, each consisting of 15 s at 94 °C and 1 min at 59 °C. Fluorescent data were collected during the 59 °C annealing/extension step and analyzed with the iCycler iQTM software. Each reaction was run in quadruplicate. Mean threshold cycle (Ct) was determined for each transcript and was plotted versus RNA concentration input to calculate the slope. Amplification efficiency for all genes was then determined (Pfaffl 2001, Peirson et al. 2003). For relative quantification of the target genes, each set of primer pairs and Taqman probe were used in combination with that of the GAPDH gene in separate reactions. The relative mRNA expression levels of the target genes in each sample were calculated using the comparative cycle time (Ct) method (Meijerink et al. 2001). Briefly, the target PCR Ct value (i.e. the cycle number at which emitted fluorescence exceeds 10 times the standard deviation (S.D.) of baseline emissions as measured from cycles 3 to 15) is normalized to the GAPDH PCR Ct value by subtracting the GAPDH Ct value from the target PCR Ct value, which gives the 
Ct value. From this Ct value, the relative mRNA expression level to GAPDH for each target PCR can be calculated using the following equation: relative mRNA expression = 2(CT target-CT GADPH). To assess the validity of GAPDH as a reference gene for comparative studies of gene expression with and without resistin treatment, an absolute quantification of GAPDH transcripts was performed. To this end, a standard curve was constructed by plotting serial dilutions of a cloned GAPDH gene fragment (range 1012 to 106 copies/reaction) and this was used to quantify GAPDH mRNA in samples of RNA extracted from cells treated or not with resistin (50 ng total RNA for each sample). The results were expressed as GAPDH copies per µg total RNA. Similar amounts of GAPDH mRNA were found in cells grown in the absence or presence of resistin (1.2 x 1011 versus 0.9 x 1011 copies/µg RNA). This finding demonstrates that resistin does not affect GAPDH expression in BeWo choriocarcinoma cells.
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To investigate the effects of resistin on choriocarcinoma cell invasiveness we used a Matrigel invasion assay (Biocoat Matrigel Chamber; BD Biosciences).
BeWo cells were labeled for 36 h with 10 µCi/ml tritium-labeled thymidine ([3H]TdR, Amersham Biosciences) in F12-K complete medium. The cells were then trypsinized, washed and resuspended in complete medium at a concentration of 106 cells/ml (Di Simone et al. 1999). A 500 µl aliquot of the cell suspension with or without different concentrations of resistin (10, 50 100 ng/ml) was added in duplicate to the upper well of transwell chambers; 700 µl media containing fibronectin (Sigma-Aldrich; 5 µg/ml) were added to the lower wells. After 48 h incubation, the media from the upper and lower wells were removed and placed in separate tubes. The upper wells were washed once with phosphate-buffered saline (PBS) and the washings pooled with the media from the upper wells.
The membranes were carefully removed with a small scalpel and placed in separate tubes for the determination of radioactivity. A 500 µl portion of a 0.01% trypsin solution in PBS was added to each lower well. The trypsin solutions of the lower wells were pooled with the incubation media from the lower wells. The fractions from each compartment of the invasion chambers as well as the Matrigel-coated filters were counted in a Beckman scintillation counter to determine associated radioactivity (Beckman, Fullerton, CA, USA). The invasion index was calculated from the amount of radioactivity in the lower wells expressed as a percentage of the sum of the radioactivity in all compartments.
Angiogenesis assay
Endothelial cell differentiation into capillary-like tube structures was monitored by the BD Biocoat angiogenesis system (BD Biosciences). HUVEC were seeded on Matrigel-coated plates (2 x 104 cells/well) in endothelial cell culture medium (EBM-2) MV SingleQuots (CAM-BREX, Baltimore, MD, USA) containing resistin (10, 50 and 100 ng/ml) or suramin (Calbiochem, San Diego, CA, USA) (40 µM) as a negative control, and incubated for 16–18 h at 37 °C, 5% CO2 atmosphere.
Following incubation, the plates were washed twice with Hank’s balanced salt solution (HBSS) and the cells were labeled by adding 50 µl/well calcein AM (8 µg/ml) for 30 min at 37 °C. Tube formation was observed using an inverted phase fluorescent microscope (Carl Zeiss S.p.A, Milan, Italy). Images were acquired with a digital camera (Nikon, Tokyo, Japan) and quantified by Photoshop software (San Jose, CA, USA) measuring the number and the total length of the tubules in each well.
Measurement of VEGF
VEGF165 secretion was detected by a human VEGF colorimetric ELISA kit (Pierce Endogen, Rockford, IL, USA; sensitivity 5.0 pg/ml; range 31.3–2000 pg/ml) according to the manufacturer’s instructions. Briefly, HUVEC were plated in 24-well plates at 50 000 cells/well in endothelial cell culture medium with 5% FBS containing various concentrations of resistin (from 0 to 100 ng/ ml) and incubated for 24 h at 37 °C and 5% CO2 atmosphere.
Sample or standard (50 µl) was added to each well, previously coated with human monoclonal anti-VEGF antibody. After 2 h incubation, wells were washed and incubated with an enzyme-linked polyclonal anti-VEGF antibody. 3,3',5,5'-Tetramethyl benzidine substrate solution (TMB) was added to each well and the color developed in proportion to the amount of VEGF bound in the initial step. The plate was read on a Titertek Multiscan Plus Mk II plate reader (Flow Laboratories, GmbH, Meckenheim, Germany) measuring the absorbance at a wavelength of 450 nm minus 550 nm.
Statistical analyses
The results are presented as means ± S.E. The data were analyzed using ANOVA followed by a post hoc test (Bonferroni test). Statistical significance was determined at P < 0.05.
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Results |
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In human choriocarcinoma cells (BeWo cells), MMP-2 was dominant, whereas the amount of MMP-9 was negligible (data not shown). To examine the effects of resistin on trophoblast pro-MMP-2 and TIMPs, we have treated BeWo cells with resistin and analyzed the MMP-2 and TIMPs protein levels on cell lysates by Western blot analysis. The protein levels of active MMP-2 have been evaluated in the conditioned media of BeWo cell cultures by ELISA. Resistin, at concentrations that are reached in vivo (10 ng/ml), greatly enhanced both pro-MMP2 and its active form (Fig. 1). To examine whether the effects of resistin on MMP-2 were also indirect, we measured the level of TIMPs. We observed basal expression of TIMP-1 and TIMP-2 in BeWo cells with Western blot analysis. After 24 h culture, resistin (from 10 ng/ml) induced a significant decrease in TIMPs protein expression (Fig. 2).
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The mRNA for MMP-2 and TIMPs was quantitated by real-time RT-PCR. We observed constitutive expression of MMP-2 and TIMPs mRNA in BeWo cells. The intensity of the MMP-2 and TIMPs mRNAs were normalized with the internal control, the GAPDH gene. As shown in Fig. 3, resistin induced MMP-2 mRNA threefold and significantly reduced by four- to fivefold the expression of TIMP-1 and TIMP-2 in this trophoblast-like cell line.
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In vitro invasion assay
Trophoblast differentiation is characterized by the development of extravillous trophoblast that migrates into maternal myometrium. Matrigel cultures have been reported to be useful in in vitro assays to evaluate trophoblast invasiveness. We found that incubation with resistin significantly increased BeWo cells invasiveness. Figure 4 shows the results of a 48 h in vitro Matrigel invasion assay. Resistin at 10 ng/ml did not show a significant effect on choriocarcinoma cell invasiveness, whereas this was significantly induced with doses of resistin of 50 ng/ml and greater (P<0.01).
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To investigate whether resistin affects the ability of endothelial cells to form capillary-like tubes, HUVEC, exposed to resistin, were seeded in extracellular matrix gels and examined for tube formation microscopically (Fig. 5). Quantitative analysis showed that the number and total length of the tubules following 22 h of culture were significantly greater in the presence of resistin (10–100 ng/ml; Table 2). To better understand the effect of resistin on angiogenesis, we tested the ability of resistin to modulate the secretion of an angiogenic factor, VEGF. Our data showed that resistin induced VEGF production (Table 2). The increase in VEGF was significant starting at 10 ng/ml, but no further increase was observed when higher concentrations of resistin were tested.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although expression of resistin mRNA and protein was detected also in the first trimester chorionic villous tissues and in extravillous cytotrophoblast spread in the decidual tissue, the functional roles of resistin have not yet been clarified. In the present study we were able to show that resistin, in addition to its invasion-stimulating action on the BeWo cells, has a direct angiogenic effect. Trophoblast invasion requires degradation of extracellular matrix molecules by the activity of specific proteases (Bischof et al. 1991, Graham & Lala 1991). It has previously been demonstrated that MMP-2 is expressed in the extravillous cytotrophoblast cells (Castellucci et al. 1993) where Yura et al.(2003) showed mRNA expression of resistin. These observations led us to consider resistin as a potential specific regulator of the production of these molecules. Our in vitro studies support this hypothesis because resistin regulates MMP-2 and TIMPs. We showed that resistin increased the secretion of MMP-2 protein in a dose-dependent manner and enhanced the activity of MMP-2 in BeWo cells. Basal levels of MMP-2 mRNA expression were also stimulated by resistin. The observed discordance between mRNA and protein increase suggest that BeWo cells must have a mechanism for post-transcriptional regulation of MMP-2 expression.
The activity of MMPs is physiologically regulated by the TIMPs (Stetler-Stevenson et al. 1989, Goldberg et al. 1992), and so the balance between the two seems to be important. We have demonstrated that resistin regulated the invasive capacity of BeWo cells by acting on MMP-2 at three levels: transcription, conversion of the proenzyme into the active form and synthesis of the specific inhibitors, TIMPs. Although it is assumed that the majority of metalloproteinase inhibitors that function in the placental bed are probably of maternal origin, TIMPs produced by the trophoblast cells themselves may have a role to play in local regulation of extracellular matrix degradation.
The BeWo cell line produces large quantities of latent and active MMP-2 but negligible amounts of MMP-9. These results are consistent with other studies (Morgan et al. 1998, Staun-Ram et al. 2004). Staun-Ram and colleagues found that MMP-2 production was dominant between 6 and 8 weeks of gestation and then declined, whereas MMP-9 production significantly increased from 8 to 11 weeks, with a shift of dominant gelatinase from MMP-2 to MMP-9 from week 9 of gestation. In order to detect the contribution of each gelatinase to this invasive process, inhibitory antibodies to MMP-2 and MMP-9 were added to cell culture and the cell invasive ability was examined (Staun-Ram et al. 2004). Only MMP-2 inhibitory antibody caused a significant decrease in BeWo cell invasion. This indicated that most probably MMP-2 and not MMP-9 is the key enzyme in the invasion process of BeWo cells and in early first trimester trophoblast (Puistola et al. 1989, Turpeenniemi-Hujanen et al. 1992), whereas MMP-9 plays a role in late first trimester trophoblast (Shimonovitz et al. 1994). Furthermore, a recent paper confirmed that MMP-2 seems be the key regulator of invasiveness (Vegh et al. 1999).
New observations suggest that MMPs, involved in degradation of the extracellular matrix (ECM), also play critical roles in endothelial cell migration and matrix remodeling during angiogenesis (Wu et al.2005). Different results indicated that MMPs contribute more to angiogenesis than just degrading ECM components (Rundhaug 2005). Specific MMPs have been shown to enhance angiogenesis by helping to detach pericytes from vessels undergoing angiogenesis, by releasing ECM-bound angiogenic factors, by exposing cryptic proangiogenic integrin binding sites in the ECM and by cleaving endothelial cell–cell adhesions. MMP-2 is constitutively secreted by endothelial cells (Nguyen et al. 2001), as is VEGF, which is able to induce expression of MMP in endothelial cells. On the other hand, exogenous MMP has been shown to enhance endothelial cell growth in vitro (Pozzi et al. 2002). In the present research we observed an increase in BeWo cell MMP-2 secretion and in endothelial VEGF secretion when cells were treated with resistin. These observations indicate that resistin might be able to induce MMP expression in endothelial cells directly, or via VEGF. Furthermore, increased trophoblast MMP-2 secretion might contribute to the control of placental vascularization in vivo.
Angiogenesis plays an important role in embryo implantation and placentation. Maternal blood flow in the intervillous space increases during pregnancy due to the vasomotor changes of the distal intramyometrial portions of uteroplacental arteries and the transformation and dilatation of decidual segments. This results essentially from the invasive behaviour of cytotrophoblast cells that invade and colonize the endometrial spiral arteries allowing for a loss of their elasticity (Fisher 2000). The increased blood requirement is also met by intense angiogenic processes taking place in the placental villi and the maternal endometrium. Although several angiogenic factors have been identified at the feto-maternal interface, the regulation of this process remains obscure (Folkman & Klagsbrun 1987, Smith 2000). In the present study, we used a tridimensional system to explore angiogenesis, and we observed that the addition of resistin stimulated HUVEC tube formation. This observation is consistent with the increase in endothelial VEGF secretion, identified as an endothelium-specific mitogen and inducer of angiogenesis and of endothelial cell survival.
In conclusion, the present data reflect a complex interaction of regulatory factors on placental growth. Caution is needed when extrapolating results obtained in choriocarcinoma cells to normal trophoblast cells. However, we speculate that these models can help identify the roles of resistin in trophoblast invasion and fetoplacental vascular development.
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Funding |
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References |
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Received in final form 21 March 2006
Accepted 24 March 2006
Made available online as an Accepted Preprint 24 March 2006
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