Hypoxia Induces FGF2 Production by Vascular Endothelial Cells And

Journal of Reproduction and Development, Vol. 57, No. 1, 2011, 10-008K —Original Article— Hypoxia Induces FGF2 Production by Vascular Endothelial Cells and Alters MMP9 and TIMP1 Expression in Extravillous Trophoblasts and Their Invasiveness in a Cocultured Model Jianying LUO1), Fuyuan QIAO2) and Xianghua YIN1) 1)Department of Obstetrics and Gynecology, Northern Jiangsu Province Hospital, Clinical Medical College, Yangzhou University, Jiangsu 225001 and 2)Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology, Hubei 430030, China Abstract. The role of fibroblast growth factor 2 (FGF2) secretion by vascular endothelial cells during trophoblast invasion was assessed. The human extravillous trophoblast cell line, TEV-1, and umbilical vein endothelial cell line, HUVE-12, were cocultured under normal and hypoxic conditions. FGF2 expression in HUVE-12 cells and matrix metalloproteinase 9 (MMP9) and tissue inhibitor of metalloproteinase 1 (TIMP1) expression in TEV-1 cells were analyzed using quantitative RT-PCR and Western blot analyses. TEV-1 cell invasion was also examined. FGF2 expression in the HUVE-12 cells cocultured with TEV-1 cells was significantly increased under hypoxic conditions. In the TEV-1 cells cocultured with HUVE-12, hypoxia reduced MMP9 expression and increased TIMP1 expression; it also reduced cell invasion by 43%. However, the expression of MMP9 and TIMP1 and ratio of MMP9/TIMP1 were increased when the TEV-1 cells were cultured alone under hypoxic conditions. These findings suggest that FGF2 release by stressed endothelial cells of uterine spiral arteries play roles in decreasing MMP9 and increasing TIMP1 production in extravillous trophoblasts (EVT) in response to stress, resulting in reduced EVT invasion and possibly shallow implantation of the placenta. Key words: Basic fibroblast growth factor 2, Extravillous trophoblast, Matrix metalloproteinase 9, Placenta invasion, Tissue inhibitor of metalloproteinase 1 (J. Reprod. Dev. 57: 84–91, 2011)

ormal placental development is characterized by uterine spiral ence the MMP9-TIMP1 balance in EVTs is necessary. artery remodeling through extravillous trophoblast (EVT) Many angiogenic factors can regulate the secretion of MMP9 in invasion of the uterine spiral arteries and the rebuilding process [1]. trophoblast cells. Anteby et al. [7] observed that exogenous vascu- Impaired uterine spiral artery remodeling and reduced trophoblast lar endothelial growth factor (VEGF), epidermal growth factor invasion is associated with susceptibility to preeclampsia. This (EGF), fibroblast growth factor 4 (FGF4) and fibroblast growth pathological state is often manifested as shallow implantation of factor 10 (FGF10) promoted trophoblast secretion of MMP9; how- the placenta [2]. ever, basic fibroblast growth factor 2 (FGF2) inhibited it. In Matrix metalloproteinases (MMPs) belong to the neutral pro- patients with preeclampsia, systemic small artery spasm leads to tease family, which degrades extracellular matrix. MMPs are maternal endothelial cell damage and stress response, resulting in regulated by tissue inhibitor of metalloproteinases (TIMPs). the production of a series of angiogenic factors, including VEGF Among the identified MMPs, matrix metalloproteinase 9 (MMP9) [8, 9], EGF [10–12] and FGF2 [13]. plays an important role in a series of physiological and pathological Angiogenic factors have a protective role in repairing damaged processes, including implantation, wound healing, angiogenesis endothelial cells; however, few reports have assessed their effects and tumor metastasis [3]. MMP9 is principally regulated by on EVT invasion. Therefore, in the present study, we hypothesized endogenous TIMP1; therefore, the MMP9-TIMP1 balance plays an that shallow placenta invasion was mediated by reduced MMP9 essential and rate-limiting role in the invasion of trophoblast cells expression and was related to FGF2 production by vascular endot- [4, 5]. Abnormal expression of MMP9 by EVTs can result in shal- helial cells under hypoxic conditions. To test this hypothesis, a low trophoblast invasion and thus impaired uterine spiral artery human umbilical vein endothelial cell line, HUVE-12, was cocul- remodeling, which is associated with several diseases of preg- tured with an EVT cell line, TEV–1, and maintained under two- nancy, including preeclampsia [6]. In order to understand the dimensional hypoxic conditions in vitro, thereby simulating the in pathogenesis of preeclampsia, analysis of factors that may influ- vivo hypoxic environment of uterine spiral artery endothelial cells and trophoblast cells in preeclampsia. The association of FGF2 Received: January 26, 2010 expression by HUVE-12 cells and MMP9 and TIMP1 expression Accepted: September 16, 2010 Published online in J-STAGE: October 28, 2010 by TEV-1 cells as well as TEV-1 invasion was explored. Deter- ©2011 by the Society for Reproduction and Development mining the factors involved in endothelial cell repair and Correspondence: F Qiao (e-mail: [email protected]) trophoblast invasion may provide new targets for the treatment of X Yin (e-mail: [email protected]) preeclampsia. HYPOXIA ALTERS MMP9 AND INVASION IN EVTS 85

Materials and Methods TATACCAGCGTTATGA-3', sense, and 5'-GAGAAACTCCTC- GCTGCGGTT-3', antisense, for TIMP1 (157 bp); and 5'-CAT- Cell conditions TAAGGAGAAGCTGTGCT-3', sense, and 5'-GTTGAAGGT- The human EVT cell line, TEV-1 (a gift from the Chinese Uni- AGTTTCGTGGA-3 ', antisense, for β-actin (208 bp; Invitrogen). versity of Hong Kong, which first set up this cell line, Hong Kong, In the amplification reaction, 1 μl of template, 100 nM (final China), was derived from primary trophoblast cells isolated from concentration) each of the sense and antisense primers, 12.5 μl of human placental villi early in a normal pregnancy, which were sub- SYBR Green PCR Master Mix (ABI, Foster City, CA, USA) and sequently immortalized through transfection with a retroviral 9.5 μl of dd2H2O were combined. The amplification conditions vector expressing human papillomavirus type 16 (HPV16) E6/E7 consisted of the following cycles: 1 cycle of predegeneration at 95 oncoproteins (PLXSN-E6/E7) [14]. The EVT properties and inva- C for 5 min and 45 cycles of denaturation at 94 C for 20 sec, sion potential of these cells have been characterized, but no annealing at 55 C for 20 sec and extension at 72 C for 20 sec. The malignant transformation capability has been observed [14]. TEV- ratio of the target gene to the internal reference (ΔCt) was obtained, 1 cells were cultured in Dulbecco's Modified Eagle Medium: Nutri- and the sample with the lowest content was used as the standard. ent Mixture F-12 (DMEM/F12) medium purchased from HyClone The contents of the remaining samples were relative multiples of (Logan, UT, USA) with 5% fetal bovine serum (FBS) purchased those samples (ΔΔCt), and the target gene mRNA was the relative from Hangzhou Sijiqing Biological Engineering Material (Hang- quantitative value (2-ΔΔCt) [16]. zhou, Zhejiang, China); no antibiotics were added. Human umbilical vein endothelial cells (HUVE-12) were purchased from Western blot analysis Xiangya Central Laboratory (Changsha, Hunan, China), which pur- At the end of the culture period, cells were washed with ice-cold chased the HUVE-12 from ATCC (Manassas, VA , USA). The PBS and lysed by sonication in lysis buffer (RIPA, ProMab Bio- HUVE-12 cells were cultured and maintained in DMEM/F12 technologies, Richmond, CA, USA). Insoluble material was medium with 5% FBS without antibiotics. All cells were cultured removed by centrifugation (12000 × g, 4 C, 10 min), and the pro- at 37 C in a humidified incubator containing 5% CO2 in air. tein content in the supernatant was determined using a BCA Protein For cocultures, a 2-ml sample of TEV-1 cell suspension (5 × 104 Assay Kit purchased from Beyotime Institute of Biotechnology cells/ml in DMEM/F12 medium supplemented with 5% FBS) was (Shanghai, China). Aliquots of protein (10 μg) were resolved by added in the upper chamber of a Transwell (0.4 μm aperture and SDS-PAGE and electrotransferred to nitrocellulose membranes. 24-mm membrane diameter; 6-well plate, Corning Costar, Lowell, The membranes were incubated for 2 h at room temperature in MA, USA), and a 1-ml HUVE-12 cell suspension (1 × 104 cells/ml blocking buffer (Tris-buffered saline, pH 7.6, 0.05% Tween-20 in DMEM/F12 supplemented with 5% FBS) was added to the [TBS-T] and 0.5% dehydrated non-fat milk). After rinsing with lower chamber. The medium was replaced after 24 h. Upon reach- TBS-T, membranes were immunoblotted with primary antibodies ing 70% confluency, the cells were divided into two groups: a specific for FGF2, MMP9, TIMP1 or GAPDH (Beijing Seajet Sci- normal control group and a hypoxia group that received cobalt entific, Beijing, China) for one hour at room temperature. After chloride (150 μM) in the upper and lower chambers. Cobalt chlo- rinsing with TBS-T, the goat anti -mouse IgG secondary antibodies ride interferes with iron (heme) interaction with oxygen [15]. The conjugated with horseradish peroxidase, purchased from Maxim cells in the upper and lower chambers were collected at 24, 48 and Biotechnology (Fuzhou, Fujian, China), were added for binding by 72 h, and quantitative RT-PCR and Western blot analyses were incubation at room temperature for one hour. The bands were visu- used to detect MMP9 and TIMP1 expression in the TEV-1 cells in alized using ECL reagents (Thermo Scientific Pierce, Rockford, IL, the upper chamber and FGF2 expression in the HUVE-12 cells in USA) and quantified with the Gel-Pro Analyzer software (Gel-Pro the lower chamber. In another experiment, the TEV-1 cells were Version 4.0, Media Cybernetics, Bethesda, MD, USA) for the rela- cultured alone under normal and hypoxic conditions for various tive protein band optical density (OD) reading. lengths of time (24, 48 and 72 h). At the end of each culture period, the cells were collected to analyze MMP9 and TIMP1 expression Invasion analysis by quantitative RT-PCR and Western blot analyses. Cell invasion assays were performed according to the method established by Sato et al. [17]; the filter membrane surface of the Quantitative RT-PCR Transwell plate (8-μm aperture and 24-mm membrane diameter, 6- After the cells were collected, 1 ml of Trizol purchased from well plate, Corning Costar) was coated with 50 mg/l Matrigel Invitrogen (Carlsbad, CA, USA) was added, and the total RNA was (Sigma, St. Louis, MO, USA) diluted at a ratio of 1:8 (100 μl/aper- extracted using the phenol-chloroform extraction method. After ture), which was placed at 37 C for 30 min. A 2-ml sample of removing the genomic DNA, cDNA was synthesized using an RT- TEV-1 cells (5 × 104 cells/ml in DMEM/F12 medium containing cDNA Kit (Fermentas, Burlington, ON, Canada) following the 0.2% BSA) was added to the upper chamber of the Transwell, and manufacturer’s instructions. FGF2, MMP9, TIMP1 and β-actin in the coculture model, 1 ml of HUVE-12 cells (1 × 104 cells/ml in mRNA was amplified using the following primers: 5'-CTAAC- DMEM/F12 containing 5% FBS) was added to the lower chamber. CGTTACCTGGCTATG-3', sense, and 5'-TTATACTGCCCA- After 24 h, the TEV-1 cells from coculture with the HUVE-12 cell GTTCGTTT-3', antisense, for FGF2 (166 bp); 5'-GCAGAG- model or culture alone model were divided into two groups: the GAATACCTGTACCGC-3', sense, and 5'-AGGTTTGGAATC- normal control group and the hypoxia group that received cobalt TGCCCAGGT-3', antisense, for MMP9 (196 bp); 5'-CACCT- chloride (150 μM) in the upper and lower chambers. The cells 86 LUO et al.

Fig. 1. Effects of hypoxia on FGF2 mRNA expression in cocultured HUVE-12 cells. Expression of mRNA was determined by quantitative RT-PCR analysis. FGF2 expression was normalized to β-actin expression in all samples, and relative FGF2 mRNA expression was determined using the ΔΔCt method. *P<0.05 compared with the control group at each time point. †P<0.05 compared with the 24-h group. ‡P<0.05 compared with the 48-h group.

were cultured in air containing 5% CO2 at 37 C for 72 h. A cotton swab was used to wipe the cells and Matrigel in the upper chamber; only those cells that penetrated through the membrane were retained. The cells were fixed with 95% alcohol for 15–20 min, and they were then stained with hematoxylin for 10 min. Immuno- cytochemistry analysis of the lower chamber was undertaken to confirm the absence of TEV-1 cell migration into the lower cham- ber (data not shown). Five visual fields were randomly selected from each aperture using an inverted microscope (300× magnifica- tion), and the cells were counted; an average cell count was calculated. Fig. 2. Effects of hypoxia on FGF2 protein expression in cocultured HUVE-12 cells. A: FGF2 protein expression was determined by Western blot analysis and normalized to GAPDH expression. B: Statistical analyses Band intensities were quantified using the Gel-Pro Analyzer Data were described as means and standard deviations of three software. *P<0.05 compared with the control group at each time separate experiments. The comparisons were performed using t- point. †P<0.05 compared with the 24-h group. ‡P<0.05 compared with the 48-h group. tests and ANOVA with Bonferroni adjustment. Data were ana- lyzed using the SAS 9.0 statistical software (SAS Institute, Cary, NC, USA). All P-values were two-sided, and differences were tion, Western blot analysis revealed that FGF2 protein expression considered significant if the P-value was less than 0.05. was significantly higher in the hypoxia group at each time point as compared with the control group (Fig. 2, all, P<0.05). These Results results suggest that hypoxia conditions stimulate FGF2 expression in both mRNA and protein in HUVE-12 cells cocultured with Effects of hypoxia on FGF2 expression in HUVE-12 cells TEV-1 cells. cocultured with TEV-1 cells We first detected FGF2 expression in HUVE-12 cells cocultured Effects of hypoxia on MMP9 and TIMP1 expression in TEV-1 with TEV-1 cells under hypoxia conditions. As determined by cells cocultured with HUVE-12 cells quantitative RT-PCR analysis, relative FGF2 mRNA expression Next, we wanted to know whether MMP9 and TIMP1 expres- was significantly elevated in cells maintained under hypoxic condi- sion by extravillous trophoblast cells cocultured with endothelial tions for 24, 48 and 72 h as compared with those cultured under cells is also affected under hypoxic conditions. As shown in Figs. control conditions (Fig. 1, all P<0.05). The highest expression of 3 and 4, significantly lower MMP9 mRNA and protein expression, FGF2 mRNA was observed in the hypoxia group at 72 h. In addi- respectively, was observed in cells cultured under hypoxic condi- HYPOXIA ALTERS MMP9 AND INVASION IN EVTS 87

Fig. 3. Effects of hypoxia on MMP9 and TIMP1 mRNA expression in TEV-1 cells cocultured with HUVE-12 cells. Expression of mRNA was determined by quantitative RT-PCR analysis. MMP9 and TIMP1 expression was normalized to β-actin in all samples, and relative MMP9 and TIMP1 mRNA expression was determined using the ΔΔCt method. *P<0.05 compared with the control group at each time point; †P<0.05 compared with the 24 - h group. ‡P<0.05 compared with the 48-h group.

tions for 24, 48 and 72 h as compared with those maintained under control conditions (all, P<0.05). At the same time, TIMP1 mRNA and protein expression was significantly higher in all hypoxia groups as compared with the control group (Figs. 3 and 4, all, P<0.05). In addition, the ratio of MMP-9/TIMP-1 protein was negatively correlated with FGF-2 protein expression in HUVE-12 cells (r=–0.978, P<0.05). To determine whether the hypoxic conditions could directly affect MMP9 and TIMP-1 expression of TEV-1 cells, TEV-1 cells were cultured alone under hypoxic conditions, and then the MMP9 and TIMP-1 mRNA levels were determined (Table 1). The mRNA levels of both MMP9 and TIMP1 were upregulated in TEV-1 cells cultured alone under hypoxic conditions, and an increased MMP9/ TIMP1 ratio was also observed, indicating that downregulation of MMP9 in TEV-1 cell cocultures maintained under hypoxic condi- tions was not caused by hypoxia itself (Table 1). These results indicate that hypoxia increases TIMP1 expression while reducing MMP9 expression in TEV-1 cells cocultured with HUVE-12 cells. Fig. 4. Effects of hypoxia on MMP9 and TIMP1 protein expression in TEV-1 cells cocultured with HUVE-12 cells. A: MMP9 and TIMP1 protein expression was determined by Western blot Effects of hypoxia on invasion of TEV-1 cells cocultured with analysis and normalized to GAPDH expression. B: Band HUVE-12 cells intensities were quantified using the Gel-Pro Analyzer software. *P<0.05 compared with the control group at each time point Because cytotrophoblasts acquire an invasive phenotype upon †P<0.05 compared with the 24-h group. ‡P<0.05 compared with culture on Matrigel [18], TEV-1 cell invasion in response to the 48-h group. HUVE-12 cell coculture and hypoxia was determined using Matri- gel invasion assays. Reduced cell invasion was observed in TEV-1 cells grown under hypoxic coculture conditions as compared with was due to the secretion of a soluble factor from HUVE-12 cells or those maintained under control conditions (Fig. 5A). Specifically, in response to hypoxia, analysis of TEV-1 cell invasion in TEV-1 the average number of invasive cells per visual field was 50.8 ± 4.5 cells cultured under hypoxic conditions only was carried out. In in the control group and 29.6 ± 3.9 in the hypoxia group, represent- response to hypoxia, increased TEV-1 cell invasion was observed ing a 43% reduction in cell invasion (Fig. 5B, P<0.05). (Fig. 5A). Specifically, the average number of invasive TEV-1 Furthermore, to determine whether altered TEV-1 cell invasion cells increased by about 120% under the hypoxia only culture con- 88 LUO et al.

Table 1. The relative mRNA expression of MMP9 and TIMP1 in TEV-1 cells cultured alone under hypoxic conditions MMP9 mRNA TIMP1 mRNA MMP9/TIMP1 ratio 24 h 48 h 72 h 24 h 48 h 72 h 24 h 48 h 72 h Normoxia 1.00 ± 0.09 1.48 ± 0.03 1.79 ± 0.04 1.00 ± 0.26 1.13 ± 0.04 1.15 ± 0.35 1.00 ± 0.01 1.31 ± 0.01 1.55 ± 0.01 Hypoxia 2.40 ± 0.16* 3.89 ± 0.14* 4.57 ± 0.14* 1.60 ± 0.37* 1.86 ± 0.69* 2.04 ± 0.19* 0.67 ± 0.01* 2.09 ± 0.01* 2.24±0.01* * P<0.05 in comparison with normoxia.

Fig. 5. Effects of hypoxia on invasion of TEV-1 cells cocultured with HUVE-12 cells or cultured alone. TEV-1 cell invasion was determined using Matrigel invasion assays. A: The lower surface of an 8-μm pore size membrane was stained with hematoxylin after 72 h. Visualization of the cells maintained under hypoxic (right panel) or normal (left panel) conditions was performed using an inverted microscope (300× magnification). B: Cell invasion was quantified by counting the number of cells in five randomly selected visual fields. *P<0.05 compared with the control group.

ditions (117.0 ± 14.4 ) compared with those maintained under peak takes place within 10 weeks in early pregnancy. At that time, control conditions (53.0 ± 1.4; Fig. 5B). The cultured alone data EVTs invade the vascular wall and extend in a retrograde manner suggested that the hypoxia condition itself might increase TEV-1 to the decidual-myometrial junction. Simultaneously, muscle and cell invasive ability. The reduced invasive ability of the TEV-1 elastic fiber damage in the vascular media and deposition of a cells cocultured with HUVE-12 cells in response to hypoxia possi- fibrin-like substance occur, and these changes are the physiological bly occurs through release of soluble factors such as FGF2 by changes of the uterine spiral arteries during pregnancy [1]. The HUVE-2 cells. second invasion occurs within 14 to 20 weeks of gestation, at which time vascular physiological changes of the myometrial segment of Discussion the spiral artery have been observed, and sometimes the end of the radiate artery is involved [19], inducing progressive dilation of the Using cocultures of HUVE-12 and TEV-1 cells maintained spiral artery and increasing placental and fetal blood supply. The under normal or hypoxic conditions, we observed elevated FGF2 second peak of EVT invasion is absent in patients with preeclamp- expression in HUVE-12 cells in response to hypoxia. In TEV-1 sia [20]. Because the myometrial segment of the spiral artery cells, reduced MMP9 and elevated TIMP1 levels were detected in remains unchanged, the lumen of the spiral arteries remains small, cells subjected to hypoxia; decreased cell invasion was also and the arteries are rich in elastic fibers. This pathological state is observed. often manifested as shallow implantation of the placenta. The cytotrophoblast cell columns and cytotrophoblastic shell The mechanism of shallow implantation is not fully understood. around the entire embryo at early stage of gestation consist of In part, limited information has been obtained from animal models, EVTs [19]. EVT invasion into the uterine spiral arteries and the as spiral artery remodeling is unique to human pregnancy [21, 22]. rebuilding process is known as uterine spiral artery remodeling, Studies using human spiral arteries generally rely on placental which is crucial for normal placental development [1]. In normal biopsy, histochemical or immunohistochemical analysis on pregnancy, EVT invasion occurs during two peak periods; the first resected uterus specimens [23–31]; however, histological analyses HYPOXIA ALTERS MMP9 AND INVASION IN EVTS 89 only reflect the condition of specimens at a certain time and loca- MMP2 and TIMP1 levels in preeclamptic pregnant women were tion, thereby providing very limited information concerning the higher than those in normotensive pregnant women while the mechanism of the remodeling process. In addition, use of single plasma MMP9 level was lower in pregnant women with preec- cells does not fully reflect the in vivo microenvironment. To better lampsia [37–39]. The patients with preeclampsia might secret less simulate the in vivo hypoxic environment of the spiral artery endot- MMP9 from the placenta. For example, reduced MMP9 levels helial cells and trophoblasts in preeclampsia, we cocultured the were observed in placenta extracts from preeclamptic women as human umbilical vein endothelial cell line, HUVE-12, and EVT compared with normal pregnant women [40]. At the same time, the cell line, TEV–1, in two-dimensional hypoxic conditions in vitro. vascular endothelial cells secreted more MMP2 and TIMP1, which This experimental model may provide a basis to determine the in turn damaged vascular endothelial cells [37, 41]. mechanism of shallow implantation in preeclampsia. The MMP9-TIMP1 balance was negatively correlated with the Preeclampsia generally occurs after 20 weeks of gestation and FGF2 protein levels in HUVE-12 cells, suggesting that vascular causes systemic small artery spasms that lead to maternal endothe- endothelial cell production of FGF2 may participate in MMP9/ lial damage and stress response, resulting in the production of a TIMP1 regulation. The influence of FGF2 on MMP9 in various series of angiogenic factors, including VEGF [8, 9], EGF [10–12] cell-types has been well-documented; in some cells, it synergisti- and FGF2 [13]. Those angiogenic factors have a role in repairing cally promotes MMP9 secretion [42–44], while in others, it has no damaged endothelial cells, but their effects on the invasiveness of effects [45–48]. Specific cellular characteristics as well as differ- extravillous trophoblast cells have yet to be determined. Compared ent extracellular environments may determine the FGF2 influence with other angiogenic cytokines such as VEGF, FGF2 induces on MMP9. In addition, the exact regulatory mechanism by which endothelial cell proliferation to a greater extent [32]. Many embry- FGF2 influences trophoblast MMP9 expression remains unclear. onic germ layer-derived cells and endothelial cells themselves In breast cancer MCF-7 cells, FGF2-mediated MMP9 secretion secrete FGF2 [33], which may maintain endothelial cell growth via through Ras/ERK signaling was observed [43]. Further studies are autocrine and paracrine signaling. FGF2 binds heparan sulfate required to determine the signaling mechanism by which FGF2 within the extracellular matrix of basement membrane cells and is may influence MMP9 expression in EVTs. stored within the tissue. During tissue ischemia and hypoxia, extra- There are some limitations regarding the present study. Firstly, cellular matrix damage induces release of stored FGF2, which may we did not examine the direct effect of FGF2 on MMP9 and TIMP1 promote proliferation and differentiation of endothelial cells and expression. It remains possible that other paracrine factors may tissue repair [34, 35]. This is the stress response of endothelial also mediate MMP9 and TIMP1 expression as well as EVT inva- cells in response to hypoxia. In the present study, HUVE-12 cells sion. Anteby et al. reported that addition of FGF2 to trophoblast cultured under hypoxic conditions produced increased FGF2 cell medium resulted in reduced MMP9 expression [7]. In that mRNA and protein levels as compared with cells maintained under study, four other factors, VEGF, EGF, FGF10 and FGF4, also normal conditions. However, increased FGF2 expression was increased MMP9 expression in trophoblasts [7]. Studies using obviously associated with duration of hypoxia (Figs. 1 and 2). FGF2 siRNA in HUVE-12 cells or FGF2 neutralizing antibodies These results are consistent with those reported by Lygnos et al. will be undertaken in the future to determine if FGF2 directly influ- [13], who reported observation of elevated serum FGF2 levels in ences MMP9 and TIMP1. In addition, the use of cobalt chloride to patients with preeclampsia. mimic hypoxia may be viewed as a study limitation. However, pre- MMP9 plays an important role in trophoblast invasion [36]. vious studies comparing the effects of cobalt chloride-induced MMP9 is a proteolytic enzyme that mainly degrades the extracellu- hypoxia with environmental hypoxia found no obvious differences lar matrix. TIMP1 is a specific inhibitor of MMP9; it binds MMP9 [15, 49–51]. However, high levels of both MMP9 and TIMP1 in through noncovalent bonds in a 1:1 ratio. Therefore, the balance mRNA (Table 1) and protein (Luo et al., in press, Acta Medicinae between MMP9 and TIMP1 influences cell infiltration and its rate- Universitatis Science of Technologiae Huazhong, April 2010, Vol- limiting steps [4, 5]. In this study, EVTs expressed lower levels of ume 2) were observed in TEV-1 cells cultured alone under hypoxic MMP9 mRNA and protein and increased levels of TIMP1 mRNA conditions. Thus, altered MMP9 and TIMP1 expression was not and protein in response to coculture hypoxic conditions. There was due to hypoxia but rather signaling factors associated with cocul- a correlation between the duration of hypoxia and the observed lev- ture, which is consistent with a previous study that observed els of MMP9 and TIMP1 mRNA and protein (Figs. 3 and 4). decreased MMP9 expression by cytotrophoblasts in response to Under the same conditions, the invasiveness of EVTs was signifi- coculture with endothelial cells [5]. The use of endothelial and cantly decreased, indicating that cocultures maintained under cytotrophoblasts cell lines rather than primary cultures represents hypoxic conditions reduced MMP9 and increased TIMP1 expres- another study limitation. However, the TEV-1 cell line displays sion, resulting in decreased EVT invasiveness. In addition, similar functions as compared with normal extravillous tropho- analysis of TEV-1 cells alone revealed increased invasion in blasts, and they do not have the malignant invasive capability of the response to hypoxia (data not shown), indicating that hypoxia villous tumor cell lines, BeWo, JEG and JAR [14]. In addition, induces release of soluble factors such as FGF2 by HUVE-2 cells, HUVE-12 cells, which are frequently used to analyze angiogenesis, resulting in reduced MMP9 expression and TEV-1 cell invasion. also respond to hypoxia [52]. Previous studies comparing levels of plasma MMPs and their In summary, the coculture model better simulates the in vivo inhibitors in normal pregnant and preeclamptic women have environment of preeclampsia. Thus, it may help further our under- reported conflicting results. 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