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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2004-0803
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 2 1083-1090
Copyright © 2005 by The Endocrine Society

Hypoxia and Transforming Growth Factor-ß1 Act Independently to Increase Extracellular Matrix Production by Placental Fibroblasts

Chie-Pein Chen, Yuh-Cheng Yang, Tsung-Hsien Su, Chia-Yu Chen and John D. Aplin

Division of High Risk Pregnancy (C.-P.C.), and Departments of Obstetrics and Gynecology (C.P.-C., Y.-C.Y., T.-H.S.) and Medical Research (C.-P.C., Y.-C.Y., C.-Y.C.), Mackay Memorial Hospital, Taipei 104, Taiwan; Mackay Medicine, Nursing and Management College (C.-P.C., Y.-C.Y., T.-H.S.), Taipei 112, Taiwan; and Academic Unit of Obstetrics and Gynaecology, Medical School (J.D.A.), University of Manchester, Manchester M13 0JH, United Kingdom

Address all correspondence and requests for reprints to: John D. Aplin, Ph.D., Research Floor, St. Mary’s Hospital, Manchester M13 0JH, United Kingdom. E-mail: John.Aplin{at}man.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Villous fibrosis is associated with oxygen deprivation in placental pathology, but the signaling networks and growth factors involved in activating the relevant cellular repair mechanisms are largely unknown. TGF is a powerful enhancer of extracellular matrix (ECM) production and an important immune suppressor that has been linked with fibrosis in several tissues. Here, cell culture methods were used to investigate possible links between hypoxia, elevated TGFß1, and altered ECM production in placenta. Term placental fibroblasts were isolated and cultured under hypoxia (3% O2) or in the presence of TGFß1, and the expression of fibronectin, collagen I, and collagen IV was examined using immunohistochemistry, ELISA of cell monolayers with associated ECM, and real-time RT-PCR. The effect of hypoxia on endogenous production of TGFß1–3 was also examined. Both TGFß1 and hypoxia increased fibronectin, collagen I, and collagen IV protein and mRNA in placental fibroblasts. However, TGFß1–3 production was not increased by culturing the cells under hypoxic conditions for 5 d. Thus, increased ECM expression under hypoxia was not mediated directly by increased TGFß. We conclude that ECM production can be stimulated independently by hypoxia and TGFß1.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PLACENTAL FIBROSIS IS associated with increased production of extracellular matrix (ECM) and is a dynamic process resulting from the chronic activation of tissue repair mechanisms after insult or injury. Placental fibrosis has been found to be associated with pathologies including intrauterine growth restriction, fetal death in utero, and placental vascular occlusion (1). It is thought that trophoblasts, villous stroma, and vessels all respond to variations in the intrauterine oxygen supply. Chronic hypoxia results in minimal fibrosis of the villi, whereas a marked fibrosis with increase in stromal connective tissue and villous avascularity results from thrombosis in main stem and surface vessels (1). In a related degenerative pathway, McDermott and Gillan (2) have shown that a chronic reduction of fetal blood flow is intimately associated with villous infarction.

ECM and the resident fibroblasts of the villous stroma play a key role in villous architecture. ECM production is one of the major functions of placental fibroblasts. ECM molecules such as collagen types I, III, V, VI, laminin, fibronectin, fibrillin, thrombospondin, and tenascin have been described in the stroma of chorionic villi (3, 4, 5, 6, 7, 8). These macromolecules support morphogenetic processes including growth, angiogenesis, and tissue remodeling (9). Collagens I and IV appear to be essential to villous architecture, whereas fibronectin has been suggested to play an important role in placental anchorage (8, 10).

Hypoxia can increase the production of ECM by placental fibroblasts (8). Similarly, human dermal fibroblast cultures exposed to hypoxia, a condition known to occur during tissue repair (11) and in fibrotic skin (12) shows increased {alpha}1 (I) procollagen mRNA levels (13). Type I collagen and fibronectin mRNA expression increase, whereas proteoglycans decrease in response to elevated concentrations of O2 in rat fetal lung fibroblasts, suggesting oxygen tension selectively up- or down-regulates gene expression of different matrix molecules (14). Furthermore, it has been reported that oxygen deprivation induces both ECM and TGFß1 production in peritoneal (15), renal (16), and dermal fibroblasts (17).

The stimulatory effect of TGFß on ECM formation has been well established in prior reports (18, 19). Principal effects of TGFß on cells include inhibition of the growth of hematopoietic, epithelial, and endothelial cells, stimulation of chemotaxis of cells including lymphocytes, macrophages, and fibroblasts, and stimulation of matrix protein production by mesenchymal cells (20). TGFß has been associated with causative pathways in fibrosis; for example, TGFß plays a pivotal role in the human lung fibrosis (21). In mammals, the cytokine has three isoforms, TGFß1–3, whose biologic properties are similar but distinct (19). The TGFß1 gene is up-regulated in response to tissue injury, and TGFß1 is the isoform most clearly implicated in fibrosis (20). TGFß1 and TGFß3 have the effect of increasing ECM deposition and decreasing matrix metalloproteinase-1 (interstitial collagenase) secretion by human lung fibroblasts (21).

TGFß has been shown to be present at the human fetal-maternal interface (19, 22, 23); in light of the above findings, we hypothesized that exposure of placental fibroblasts to low oxygen might enhance TGFß synthesis leading to autocrine or paracrine stimulation of ECM expression. To test this hypothesis, the effect of varying oxygen partial pressure on production in vitro of three key ECM components and TGFß1–3 by term placental fibroblasts was examined, as well as the effect of exogenous TGFß1 on their ECM production.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Fibroblast culture

Placental fibroblasts were obtained as described by Haigh et al. (24). Briefly, term placental tissue was minced and subjected to four to five 10-min sequential digestions with 0.25% trypsin-EDTA solution (Invitrogen, Carlsbad, CA) and 10 U/ml DNAse I (Sigma-Aldrich, St. Louis, MO) in DMEM (Invitrogen). In each fraction, the tissue was pipetted vigorously up and down for 5 min. Then, the tube was allowed to sit for 5 min undisturbed to let the pieces of tissue settle. The supernatant was transferred to a fresh tube. Supernatants were pooled and spun at 1000 x g for 10 min. The pellet was resuspended in 5 ml culture medium and seeded in a 25-cm2 flask. After a 1-h incubation to allow for cell adherence, the flask was washed with DMEM to remove loosely adherent (mainly trophoblastic) cells. Cultures maintained in DMEM/10% fetal calf serum were characterized using a panel of markers. They express vimentin, smooth muscle {alpha}-actin and fibroblast surface protein, but not cytokeratin 7, pregnancy-specific ß-1 glycoprotein (SP1), CD34, endoglin, or smooth muscle myosin and so are not endothelial, trophoblast, or vascular smooth muscle cells. The cells grow rapidly in vitro and exhibit tightly aligned bipolar morphology at confluence and an absence of multinucleated cells.

The effect of TGFß1 on the production of ECM was examined using term placental fibroblasts cultured in the presence of recombinant human TGFß1 (R&D Systems, Minneapolis, MN). Initially, cells at different days of culture were incubated with TGFß1 (1, 3, or 5 d), and different doses (1, 5, 10 ng/ml) of TGFß1 were used to establish optimal assay conditions. The use of 10 ng/ml TGFß1 for 5 d, added 2 d after initial plating, caused a maximal change in ECM production, and this concentration was chosen for subsequent experiments.

For the study of hypoxic effects, fibroblasts at passage 5–7 were incubated at 37 C, 5% CO2 in air for 2 d, then transferred at approximately 70% confluence to incubators with oxygen tension set at 3 or 20% oxygen with 5% CO2 for 5 further d. The hypoxic conditions in the incubator and culture medium were confirmed using oxygen sensor electrode measurements as in Khaliq et al. (25). In blocking experiments, chicken-neutralizing antibody against TGFß1 (10 µg/ml; R&D) was added to the fresh culture medium 24 h after the medium was changed. According to the manufacturer, this antibody is specific to recombinant human TGFß1 and shows less than 2% cross-reactivity with recombinant human TGFß3 on ELISA and Western blot. Nonspecific rabbit immunoglobulin (Vector, Burlingame, CA) was added at the same concentration as a control. The fibroblasts were cultured for 5 further d at different oxygen concentrations without further changing the medium.

In total, 10 term placental fibroblasts strains were isolated from 10 different placentas in this study. Each experiment was performed in duplicate with at least three independent fibroblast cultures. Approval for this study was obtained from the institutional review board at Mackay Memorial Hospital (Taipei, Taiwan; NSC-91-2314-B-195-029).

Immunofluorescence of cells in culture

Fibroblasts were seeded into multichamber culture slides (Lab-Tek II Chamber slide system, Lab-Tek, Naperville, IL) at a density of 5 x 104 cells/chamber. The medium was removed 24 h after plating, and fibroblasts were incubated for 5 d in culture medium containing 50 µg/ml L-ascorbic acid (Sigma-Aldrich) with or without 10 ng/ml TGFß1 (R&D Systems). The cells were washed twice with PBS and fixed with methanol at room temperature for 30 min. Immunofluorescence was then performed as described previously (8). Briefly, they were rehydrated with PBS for 5 min, treated with protein block (Dako, High Wycombe, United Kingdom) for 20 min, and incubated in primary antibody against fibronectin (1:200, Chemicon International, Temecula, CA), collagen I (clone COL-1, Sigma-Aldrich; recognizes the native helical form of collagen type I; 1:500), or collagen IV (clone COL-94, Sigma-Aldrich; recognizes {alpha}1 and/or {alpha}2 chains of human collagen IV; 1:250) for 1 h at room temperature. After three washes in PBS (5 min each), cells were incubated in fluorescein isothiocyanate-conjugated secondary antibody (Dako) for 1 h, washed quickly in PBS three times, and mounted in nonfade aqueous mountant (Immumount, Life Sciences International, Basingstoke, United Kingdom). Control experiments were carried out in which the primary antibody or secondary antibody was omitted or in which irrelevant primary antibodies (to keratan sulfate or MUC-4) were included. The results were negative.

Cell ELISA (CELISA) for ECM

CELISA of ECM in the cell layer is a direct, biologically relevant measurement that can be used to compare deposition under different culture conditions. Fibroblasts at passages 7–10 were seeded into 96-well flat-bottom microplates and incubated at 37C, 5% CO2 for 7 d. Initially, different cell densities including 1 x 104/well and 2 x 104/well were tested after 7 d of culture. ECM production was reduced at the lower cell density. Therefore, the higher plating density of 2 x 104/well was used for further experiments. Cells were at confluence for the final 3–4 d in culture. The culture medium was aspirated, and wells were washed twice with PBS, then fixed by adding 200 µl methanol and incubated at room temperature for 30 min. Each well was washed twice with PBS, treated for 1 h with 150 µl protein block [5% (wt/vol) dried milk solids and 0.05% (vol/vol) Tween 20 in PBS], then washed with PBS-Tween 20. After adding primary antibody (50 µl/well, diluted in 0.05% PBS-Tween 20) against fibronectin (1:400, Chemicon International), collagen I (clone COL-1 1:500), or collagen IV (clone COL-94, 1:200) and incubating for 3 h at room temperature, the plates were washed three times for 5 min for each by PBS-Tween 20. Secondary antibodies (goat antimouse horseradish peroxidase-conjugated antibody, Dako, 1:500 in PBS-Tween 20, 50 µl/well) were added for 1 h at room temperature. Plates were washed three times for 5 min each using PBS-Tween 20 and then once for 5 min with citrate/phosphate buffer (0.1 M citric acid and 0.1 M Na2HPO4). Finally, the substrate 2,2'-azino-di-[3-ethylbenzthiazoline]-sulfonate (1 mM in citrate/phosphate buffer with 0.005% H2O2 was added to each well (100 µl), and the reaction was allowed to develop for 30 min. Absorbance measurements were performed at 405 nm on an automated plate reader (Bio-Tek Instruments). Primary and secondary antibody concentrations were optimized by titration. These dilutions produced absorbances in the range 0.9–2.0 after 30 min. Absolute absorbance values detected in CELISA depend on antibody characteristics and are not a comparative measure of the amounts of the different ECM components present. Data from four wells in each of three independent experiments were grouped and analyzed under experimental and control conditions. Grouped absorbance values were examined for statistical significance.

ELISA for TGFß

For the study of hypoxic effects on TGFß production, fibroblasts were incubated at 37 C, 5% CO2 in air for 2 d, then transferred to incubators with oxygen tension set at 3 or 20% and 5% CO2 for 5 further d. Initially, different days of culture incubated in hypoxia (1, 3, or 5 d) and cells cultured in serum-free medium containing 1% BSA (Sigma-Aldrich) were used to examine differences in production of TGFßs. However, no significant differences were observed after the different times except that the values observed in serum-free medium were lower.

Cell lysates and cell culture medium were used to analyze production of TGFß1–3 by fibroblasts. Fibroblasts were harvested by cell scraper (Nunc, Nalge Nunc Int., Roskilde, Denmark) and collected in PBS buffer with proteinase inhibitors (2 g/ml aprotinin, leupeptin, pepstatin A, and 120 g/ml phenylmethylsulfonylfluoride), followed by homogenization (Polytron RT MR3100, Kinematica AG, Littau-Luzem, Switzerland). The sample containers were cooled in ice and cells homogenized for three times at 10-sec intervals. The homogenates were spun at 5000 x g for 10 min at 4 C to remove debris. Supernatants were aliquoted and stored at –80 C until required. Protein was assayed in 96-well plates (Dynatech Labs, Chantilly, VA); to 3 µl of standard (BSA) and samples was added 150 µl of protein assay dye reagent (Bio-Rad, Hercules, CA) diluted 1:5 with distilled water. Absorbance at 630 nm was measured immediately on an automated plate reader (Bio-Tek Instruments, Winooski, VT).

The samples (cell lysates or cell culture supernatant) were either assayed directly for bioactive TGFß or, for the detection of total TGFß (bioactive and latent forms), activated before immunoassay. To 0.5 ml sample (cell lysate or cell culture supernatant), 0.1 ml 1M HCl was added, mixed, and incubated for 10 min at room temperature. Then, the acidified sample was neutralized by adding 0.1 ml of 1.2 M NaOH/0.5 M HEPES. This assay detects total TGFß (bioactive and latent forms) expressed by the cells.

Plates (Nunc, Nalge Nunc Int.) were coated with 100 µl of 2 µg/ml capture antibody in PBS overnight at room temperature. After three washes [0.05% Tween 20 in PBS (pH 7.4)], plates were blocked for 1 h with PBS containing 1% BSA and 5% sucrose. Plates were washed again; then, 100 µl of standards or samples was added. Samples were diluted in TBS (pH 7.3) containing 0.05% Tween 20 and 0.1% BSA. After a 3-h incubation and three washes, 100 µl biotinylated detection anti-TGF antibody (100 ng/well) diluted 1:250 in sample diluent buffer was added for 2 h. After three further washes, 100 µl of 1:200 streptavidin-horseradish peroxidase solution diluted in PBS containing 0.1% BSA was added for 20 min. Three more washes were performed; then, 100 µl 2,2'-azino-di-[3-ethylbenzthiazoline]-sulfonate substrate (Roche, Mannheim, Germany) was added for 45 min. The optical density of each well was determined immediately by using a microplate reader set to 450 nm and followed by correcting at 540 nm. The range of the assay was 32–2000 pg/ml. Complete culture medium was used as one of the controls. The specificity of the assay was <5% reactivity between TGFß1–3 at 10 ng/ml. All of the samples were measured within the linear range of the standards. The final concentration of TGFß1–3 in the sample was expressed as picograms of TGFß per milligram of sample protein.

RNA isolation and first strand cDNA synthesis by RT

RNA was isolated from fibroblasts cultured under different oxygen tensions. Cells were homogenized in TRIZOL reagent (Life Technologies, Inc.-BRL, Gaithersburg, MD), and extraction of RNA was performed according to the instructions of the manufacturer. At the end of the procedure, the RNA pellet was briefly dried in air and dissolved in RNase-free water. Total RNA content was evaluated by A260 measurement, and its integrity was checked by 1% agarose gel electrophoresis.

First strand cDNA was prepared by RT using 2 µg total RNA in a reaction mixture containing 0.5 µg random hexamer primers (Promega, Madison, WI), 0.5 mM dNTPs, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, and 4 U Maloney murine leukemia virus reverse transcriptase (Promega). The reaction mixture was incubated for 1 h at 42 C and stopped by incubation at 70 C for 10 min.

Real-time PCR

Real-time PCR was performed as described previously (26). In brief, PCR was performed in an ABI PRISM 7700 sequence detector in a 25-µL final volume. Amplification reactions were performed using a SYBR Green PCR Master Mix reagent kit (Applied Biosystems, Foster City, CA). 18S rRNA was used as an internal control because ribosomal RNA constitutes the majority of cellular RNA, and its level was less likely to vary in amount under different physiological conditions (27). The primer sequences for gene amplification, designed using Primer Express (Applied Biosystems), are shown in Table 1Go.


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  		TABLE 1. The sequences of primers used for gene amplification

 
PCR was initiated after activation of the Amplitaq Gold enzyme (Applied Biosystems) in the reaction mixture by heating for 10 min at 95 C. All genes were amplified by a first step of 15 sec at 95 C, followed by 1 min at 60 C for 40 cycles. Cross-reactivity of the primers to genomic DNA was excluded by demonstration of a lack of amplification with human DNA. Additionally, target gene amplification was not detected in the absence of reverse transcriptase. Each amplicon was observed at the expected size after 2% agarose gel electrophoresis, then eluted and sequenced using an ABI 310 automated laser fluorescent sequencer (Applied Biosystems). Relative quantitation of target gene expression was calculated by the comparative threshold cycle (CT) method, which normalizes the copy number of the target gene to that of an endogenous reference gene. A validation experiment was performed to demonstrate that efficiencies of amplification of the various target genes examined and the 18S rRNA reference gene were approximately equal. All the absolute values of the slope of log input amount vs. {Delta}{Delta}CT were less than 0.1. Based on exponential amplification of the target and reference genes, the amount of amplified molecules at the CT value was given. Normalized target gene expression relative to 18S rRNA is given by calculating the difference in CT values ({Delta}CT) (26). Then, the {Delta}{Delta}CT value of the target gene is calculated by subtracting the mean {Delta}CT of the reference gene from the value of {Delta}CT for each target gene. The relative change in target transcript expression, normalized to an 18S rRNA and relative to the reference gene, is computed as 2–{Delta}{Delta}CT.

Statistical analysis

The distributions of data were determined to be parametric or nonparametric by using the Kolmogorov-Smirnov one-sample test. The differences were assessed by using a {chi}2 test, independent-samples Student’s t test, paired-samples Student’s t test, Mann-Whitney U test, or Wilcoxon Signed Ranks test, when appropriate. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Term placental fibroblasts were cultured in the presence of recombinant human TGFß1 (10 ng/ml, R&D). Fibronectin, collagen I, and collagen IV were distributed as both intracellular and extracellular deposits. TGFß1 significantly increased the ECM associated with term placental fibroblasts as assayed by indirect immunofluorescence (Fig. 1Go).



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  		FIG. 1. Immunofluorescence staining of fibronectin (A and B), collagen I (C and D), and collagen IV (E and F) in term placental fibroblasts. TGFß1 significantly increased the expression of these ECM molecules by placental fibroblasts. A, C, and E, Control. B, D, and F, Fibroblasts were treated by TGFß1 10 ng/ml for 5 d; representative features are shown from three independent experiments. Magnification, x500. Scale bar, 10 µm.

 
Placental fibroblasts were cultured with TGFß1 (10 ng/ml) for 5 d, then CELISA was used to quantify ECM production. Transforming growth factor-ß1 significantly increased the production of fibronectin and collagen I (Fig. 2Go, A and B). The production of collagen IV was also higher, although not statistically significant (Fig. 2CGo; P = 0.109).



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  		FIG. 2. TGFß1 effects on ECM production by term placental fibroblasts. Cells were treated with TGFß1 (10 ng/ml) for 5 d, increasing prominently expression of fibronectin and collagen I. A, Fibronectin, P < 0.05. B, Collagen I, P < 0.05. C, Collagen IV, P = 0.109. The results show the average of three independent experiments. Error bars = SD.

 
To study the effects of hypoxia on ECM production by term placental fibroblasts, cells were cultured at different oxygen tensions. At least three independent experiments were performed using five term fibroblast cell strains. As we have described (8), no morphologic abnormalities or significant changes in cell proliferation rates were noted in confluent fibroblast cultures exposed to low oxygen. Hypoxia significantly increased fibronectin, collagen I, and collagen IV production by term placental fibroblasts detected by CELISA (Fig. 3Go).



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  		FIG. 3. Effects of hypoxia on ECM production by term placental fibroblasts. The fibroblasts were cultured at two different oxygen tensions (3 and 20%) for 5 d. Hypoxia significantly increased ECM production by fibroblasts compared with the control value observed at 20% pO2. A, Fibronectin, P < 0.05. B, Collagen I, P < 0.05. C, Collagen IV, P < 0.05. Each value was obtained from an average of three experiments. Error bars = SD.

 
These alterations were further verified by real-time PCR. For the comparative CT method to be valid, the amplification efficiencies of the target and reference (18S rRNA) genes must be approximately equal. A validation method to assess whether amplification of target and reference genes has the same efficiency is to look at how {Delta}CT varies with different template dilutions. If the amplification efficiencies of target and referenced genes are approximately equal, the slope derived from the plot of log input amount of total RNA vs. {Delta}CT should be less than 0.1. All the levels of the input RNA/cDNA fell in the range of 0.4 to 1.602 (after log transformation) of the validation curves. The absolute values of the slope of log input amount vs. {Delta}CT was shown in Fig. 4Go for each target gene, respectively. Therefore, relative to 18S rRNA, the amplification efficiency of real-time PCR for each target gene is approximately equal.



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  		FIG. 4. Amplification efficiencies of target and 18S rRNA genes in real-time PCR. Plots of log input amount vs. {Delta}CT demonstrated that the amplification efficiencies of target and reference genes are approximately equal (slopes approximately zero). Data were obtained from three independent experiments. TGFß1, y = 0.0342x + 15.994, r2 = 0.2042; TGFß2, y = 0.0339x + 19.876, r2 = 0.228; TGFß3, y = 0.006x + 19.462, r2 = 0.0594; fibronectin, y = 0.0772x + 17.809, r2 = 0.8579; collagen I, y = 0.0187x + 15.456, r2 = 0.6587; collagen IV, y = 0.0161x + 16.977, r2 = 0.0789.

 
Low oxygen tension significantly increased the fibronectin, collagen I {alpha}1 (COL1A1), and collagen IV {alpha}1 (COL4A1) chain mRNA production by term placental fibroblasts (Fig. 5Go).



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  		FIG. 5. Effects of hypoxia on ECM production by term placental fibroblasts quantified by real-time PCR. The fibroblasts were cultured at two different oxygen tensions (3% pO2 and 20% pO2) for 5 d. Hypoxia significantly increased the mRNA levels in fibroblasts compared with the control value observed at 20% pO2 (P < 0.05). A, Fibronectin, P < 0.05. B, Collagen I, P < 0.05. C, Collagen IV, P < 0.01. Each value was obtained from an average of three experiments. Error bars = SD.

 
Data obtained so far suggested the possibility that the elevated production of ECM under hypoxic conditions might be mediated by increased endogenous TGFß. Therefore, levels of TGFßs were measured in culture medium (data not shown) and lysates of placental fibroblasts (Fig. 6Go) cultured under different oxygen tension. TGFß1–3 could all be detected in the cells. However, there were no significant differences in production between hypoxic cells cultured for 1, 3, or 5 d and controls (Fig. 6Go; 1 and 3 d; data not shown). Similarly, mRNA levels for the three TGFß isoforms were detectable but not significantly altered under hypoxic conditions (Fig. 7Go). Neutralizing antibody to TGFß1 added to culture medium failed to inhibit the increase of ECM caused by hypoxia (data not shown).



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  		FIG. 6. Effects of hypoxia on TGFß1–3 production by term placental fibroblasts. The fibroblasts were cultured at two different oxygen tensions (3% pO2 and 20% pO2) for 5 d. Cell lysates were analyzed by ELISA. There was no significant difference in TGFß1–3 in fibroblasts cultured in 3% pO2 compared with controls (20% pO2) (P > 0.05). A, TGFß1. B, TGFß2. C, TGFß3. Each value was obtained from an average of three experiments. Error bars = SD.

 


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  		FIG. 7. Effects of hypoxia on TGFß1–3 production by term placental fibroblasts quantified by real-time PCR. The fibroblasts were cultured at two different oxygen tensions (3% pO2 and 20% pO2) for 5 d. mRNAs were analyzed by real-time PCR. There were no significant differences in TGFß1–3 mRNA expression in fibroblasts cultured in 3% O2 compared with controls (20% O2) (P > 0.05). A, TGFß1; B, TGFß2; C, TGFß3. Each value was obtained from an average of three experiments. Error bars = SD.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In previous work, we demonstrated the presence of fibronectin, collagen I, and collagen IV in term and first trimester placental villi (8). Fibronectin is abundant throughout the villous stroma, including vessel walls and areas beneath the trophoblast basement membrane. Collagen IV is not confined to the basement membranes beneath the villous trophoblast and in vessel walls; it is also notably abundant in areas of villous stroma lacking detectable collagen I. In other villous profiles, collagen I dominates the stroma, with collagen IV confined to basement membranes (8). Consistent with these observations, villous fibroblasts in culture produce an abundant ECM containing collagens I and IV and fibronectin, suggesting the importance of these cells in elaboration of the villous ECM in vivo (8).

TGFß stimulates increased expression of collagen I, collagen IV, and fibronectin by term fibroblasts. Increases were observed in deposition of mature protein into ECM, as monitored by immunofluorescence and CELISA. Similar effects have been observed in other connective tissue cells (18, 29, 30). Collagen assembly into heterotrimer is required for secretion; the immunofluorescence evidence of ECM deposition in monolayers indicates that levels of the mature trimeric collagens I and IV are increased by TGFß stimulation.

Placental fibrosis has not been widely studied, but it is apparent from classical pathological examination that it is associated with conditions of later pregnancy in which oxygen deprivation is a feature of the villous tissue environment (1). For example, collagen IV up-regulation has been observed in certain types of IUGR (31, 32). However, the mechanisms underlying the initiation and progression of placental fibrosis have not been identified. The present study set out to challenge cells with an oxygen environment representing the lower extreme of the physiological range. Levels around 3% have been reported in early pregnancy, rising to 11–14% O2 during the third trimester (33, 34, 35, 36). Fibroblasts cultured at 20% are hyperoxic, but this level served as a reference to relate the present study to others that have used standard culture conditions.

It was found that ECM production by placental fibroblasts is higher under hypoxic than hyperoxic conditions. Consistent effects have been seen in other cell systems (13, 37, 38). Increases were observed both at the mRNA and protein level under hypoxia and were seen in fibronectin, collagen I and collagen IV. An increase in production by placental fibroblasts of the oncofetal fibronectin isoform was recently reported in response to either TGFß or hypoxia (39).

There is some evidence to suggest a link between hypoxia and the TGFß pathway. Thus, it has been reported that hypoxia results in an increase in collagen I, fibronectin, tissue inhibitor of metalloproteinase-1, TGFß1, and TGFß2 levels in peritoneal fibroblasts (15). Similarly, hypoxia stimulates TGFß1 and collagen I production in kidney proximal tubular epithelial cells (40). This increase could arise by up-regulation of matrix production or decreased turnover or both (16). Furthermore, the response of different ECM molecules to hypoxia may be not consistent across cell types; thus Orphanides et al. (40) showed that hypoxia stimulated collagen I production but suppressed collagen IV in kidney cells. Neutralizing anti-TGFß1 antibody did not abolish the hypoxia-induced changes in gelatinase activity, tissue inhibitor of metalloproteinase-1, collagen IV, or collagen I mRNA expression, implying that TGFß1 was not the mediator (40). Similarly, in our study, neutralizing antibody to TGFß1 failed to prevent the effect of hypoxia on ECM expression by placental fibroblasts. This indicates that alterations in active TGFß1 are not responsible for the effect and additionally argues against a change in receptor sensitivity occasioned by hypoxic challenge. Likewise, plasminogen activator inhibitor-1 expression in first trimester trophoblast is stimulated under hypoxia by a pathway that does not require TGFß (41).

At about 11 weeks of pregnancy, maternal blood gains access to the intervillous space and the local oxygen tension increases (42, 43). TGFß levels in placenta (44, 45, 46) are low and invariant between 7 and 19 weeks’ gestation (44, 45, 46), suggesting no oxygen effect on TGF production in vivo. Our observation that placental fibroblasts do not increase TGFß1–3 under hypoxia in vitro is consistent.

Because the effect of hypoxia in vitro on placental fibroblast ECM production is not mediated directly through increased endogenous TGFß1 production, other signaling mechanisms must be sought. Hypoxia activates a variety of signal transduction pathways, including protein kinase C, protein kinase A, and tyrosine kinases (47, 48, 49). Hypoxia may induce other cytokines like platelet-derived growth factor, basic fibroblast growth factor, TNF-{alpha}, and IL-1ß that in turn could affect ECM production (20, 50, 51). Other possible mechanisms might include paracrine stimuli from trophoblasts or endothelium in the villus. The TGFß family of growth factors has a critical role in modulation of vascular inflammatory responses and remodeling, and up-regulation of TGFß production by endothelium is likely to be a critical factor affecting the course of vascular inflammation (50, 51).

We conclude that TGFß1 and hypoxia independently increase ECM expression in placental fibroblasts. TGFß expression is low in the normal villus but may increase in situations where immune suppression is required; placental TGF may also derive from maternal sources (52, 53). Hypoxia and fibrosis are both associated with related pregnancy pathologies, but TGFß is not the primary mediator of increased ECM production under hypoxic conditions. Oxygen deprivation may induce placental villous injury and activate repair processes involving fibroblast ECM production. However, further work will be needed to understand the role of interactions between fibroblasts and other villous cell types including endothelium, macrophages, and trophoblasts, as well as other diffusible factors, in the response to injury and control of villous ECM production.


   Footnotes
 
This work was supported by the Mackay Memorial Hospital and Nation Science Council, Taipei, Taiwan (Research Grants MMH-9129 and NSC-91-2314-B-195-029 to C.-P.C.).

First Published Online November 2, 2004

Abbreviations: CELISA, Cell ELISA; CT, threshold cycle; ECM, extracellular matrix.

Received April 29, 2004.

Accepted October 22, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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