help button home button Endocrine Society JCEM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2004-2242
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Belkacemi, L.
Right arrow Articles by Graham, C. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Belkacemi, L.
Right arrow Articles by Graham, C. H.
The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 8 4846-4851
Copyright © 2005 by The Endocrine Society

Inhibition of Human Trophoblast Invasiveness by High Glucose Concentrations

Louiza Belkacemi1, Gendie E. Lash1, Shannyn K. Macdonald-Goodfellow, Jason D. Caldwell and Charles H. Graham

Department of Anatomy and Cell Biology, Queen’s University, Kingston, Ontario, Canada K7L 3N6

Address all correspondence and requests for reprints to: Dr. Charles H. Graham, Department of Anatomy and Cell Biology, Botterell Hall, 9th Floor, Queen’s University, Kingston, Ontario, Canada K7L 3N6. E-mail: grahamc{at}post.queensu.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Context: Trophoblast invasion of the uterus is regulated by local microenvironmental factors.

Objective: Because certain conditions may affect uterine glucose levels during placentation, the aim of this study was to determine the effect of glucose concentration on trophoblast invasion.

Results: Compared with incubation in 0.2 and 2.5 mM glucose, a 24-h incubation in increasing glucose concentrations (5 and 10 mM) resulted in up to a 62% inhibition (P < 0.01) of the in vitro invasiveness of immortalized HTR-8/SVneo trophoblasts. This decreased invasiveness in 5 and 10 mM glucose was paralleled by inhibition of a plasminogen activator (PA) activity corresponding to active urokinase-type PA (uPA). Inhibition of pro-uPA binding to the uPA receptor decreased the invasiveness of cells incubated in 0.2 and 2.5 mM glucose to levels observed in cells incubated in higher glucose concentrations (P < 0.05). Gelatin zymography and Western blot analysis revealed that the levels of matrix metalloproteinase-2 and -9, PA inhibitor-1, and uPA receptor were unaffected by glucose. Glucose transporter-1 levels were 26 and 34% higher in cells cultured in 2.5 and 0.2 mM glucose, respectively, vs. 5 or 10 mM glucose (P < 0.05). In contrast, glucose transporter-3 levels were not affected by incubation in various glucose concentrations.

Conclusions: These findings indicate that high glucose concentrations inhibit the invasiveness of HTR-8/SVneo cells by preventing uPA activation. Therefore, through its effects on uPA activity, glucose may be an important regulator of trophoblast invasiveness during implantation and placentation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TROPHOBLAST INVASION OF the uterus during blastocyst implantation and subsequent placental development is a process that is strictly regulated by local microenvironmental factors. Some of these endometrial factors, such as TGF-ß and TNF-{alpha}, inhibit trophoblast invasiveness (1, 2), whereas other molecules, such as hepatocyte growth factor, have been shown to stimulate invasiveness (3, 4). Studies in our laboratory revealed that local oxygen concentrations may also play a role in regulating trophoblast invasiveness (5, 6).

It is also known that alterations in glucose levels at the fetal-maternal interface are associated with abnormal fetal development (7, 8). Type I diabetes mellitus during pregnancy is associated with dysregulation of glucose metabolism, which affects placental villous growth and function (8). In addition, the incidence of preeclampsia, a disease of pregnancy characterized by shallow placental trophoblast invasion of the uterine spiral arterioles, is increased in women with either nongestational type I diabetes or gestational diabetes (9, 10, 11, 12). However, the effects of local glucose concentrations on placental development and trophoblast invasion are not fully understood. Furthermore, although the physiological blood glucose levels are approximately 5 mM, the precise glucose concentration at the fetal-maternal interface is not known. Nevertheless, it was recently reported that glucose levels are considerably lower in the intervillous fluid of first trimester placentas than in the maternal serum (13). Thus, it is likely that normal placental development takes place under glucose concentrations lower than those present in maternal blood.

Studies revealed that the invasive and metastatic potentials of various mouse carcinoma cell lines were increased after a period of glucose starvation (14, 15). Invasive trophoblast cells share several phenotypic properties with malignant cells, including some of the molecular mechanisms that regulate invasion through the extracellular matrix (16). Therefore, it is possible that the low glucose environment of the first trimester fetal-maternal interface provides the optimal conditions for trophoblast invasion.

Cellular invasion requires proteolytic degradation of extracellular matrix molecules. Both the matrix metalloproteinase (MMP) and the urokinase plasminogen activator (uPA) systems play major roles in the breakdown of extracellular matrix and cellular invasion (17). MMPs are a family of more than 23 zinc-binding enzymes that include the collagenases (MMP-1 and -4), the stromelysins (MMP-3 and -10), and the gelatinases (MMP-2 and -9) (18, 19). They are secreted as latent proenzymes that are activated outside the cell or when bound to the cell membrane. The activity of the MMPs is primarily regulated by the tissue inhibitors of metalloproteinases-1, -2, and -3.

The main components of the uPA system include the serine proteinase uPA, the uPA receptor (uPAR), and the plasminogen activator inhibitor-1 and -2 (PAI-1 and -2) (20). uPA is secreted as a single-chain proenzyme (pro-uPA) that has little enzymatic activity (21). However, upon binding to uPAR, pro-uPA is converted into a two-chain, high molecular mass (52 kDa), active enzyme (22). Binding of pro-uPA to uPAR occurs via the amino-terminal domain of pro-uPA. Free active uPA and uPA bound to uPAR can convert plasminogen into plasmin, which, in turn, degrades extracellular matrix proteins, such as fibronectin, laminin, and collagen, or activates other invasion-associated enzymes, including the MMPs (23). As in the case of MMP inhibition by tissue inhibitors of metalloproteinases, inhibition of free uPA and uPAR-bound uPA is mostly mediated by PAI-1 through the formation of irreversible complexes (24, 25, 26, 27).

In the present study, an immortal line of invasive trophoblast cells was used to model the effects of glucose on placental trophoblast invasion of the uterus. Specifically, we investigated the effects of glucose concentration on the ability of HTR-8/SVneo cells to invade extracellular matrix in vitro as well as on the expression of certain components of the uPA and MMP systems by these cells.


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

The HTR-8/SVneo trophoblast cells were obtained from explant cultures of human first trimester placenta (8–10 wk gestation) and were immortalized by transfection with a cDNA construct that encodes the simian virus 40 large T antigen (28). These cells, although nontumorigenic and metastatic, are highly invasive in vitro and exhibit phenotypic properties of extravillous cytotrophoblasts (29), including the expression of cytokeratins 7, 8, and 18; placental alkaline phosphatase; uPAR; human leukocyte antigen (HLA) framework antigen W6/32; IGF-II mRNA and protein; as well as an integrin profile characteristic of invasive cytotrophoblasts (30). When plated on Matrigel (BD Biosciences, Bedford, MA), these cells express HLA-G (29), a nonpolymorphic HLA molecule expressed by extravillous cytotrophoblasts in situ.

Culture medium containing 1, 2.5, 5, or 10 mM glucose (final concentrations) was prepared by adding corresponding amounts of glucose to glucose-free RPMI 1640 medium (Invitrogen Life Technologies, Inc., Burlington, Canada) supplemented with 5% fetal bovine serum (FBS; Invitrogen Life Technologies, Inc.). Using a glucose-lactate-electrolyte analyzer (EML-105, Radiometer, London Scientific, London, Canada), it was determined that glucose levels in the FBS were 4 mM. Therefore, medium with 0.2 mM glucose consisted of glucose-free RPMI 1640 and 5% FBS without glucose supplementation. The fasting physiological blood glucose concentration in humans is approximately 5 mM. Cells were incubated in a standard Sanyo CO2 incubator (5% CO2 in air, 37 C; Esbe Scientific, Markham, Canada).

In vitro invasion assay

To determine the effect of glucose on the invasiveness of HTR-8/SVneo cells, we used a previously described assay that employs reconstituted extracellular matrix (Matrigel) as the substrate for invasion (31). The relative invasiveness was calculated after counting under a microscope the total number of cells that invaded through the Matrigel after a 24-h incubation under various glucose concentrations in the presence or absence of 10 or 100 ng/ml of the amino-terminal fragment (ATF) of uPA (Molecular Innovation, Inc., Southfield, MI). Each experiment was performed in triplicate and was repeated three times. In a pilot study we determined that the rate of HTR-8/SVneo cell proliferation is identical in low (0.2 mM) vs. high (10 mM) glucose concentrations for at least 48 h, thereby indicating that differences in cell numbers that invaded the Matrigel at the end of the assay are reflective of altered invasive capacity alone.

Substrate gel zymography

Zymographic analysis was performed as described previously (32) using medium samples (2 µg protein) and cell lysates (10 µg protein) resolved in 12% sodium dodecyl sulfate-polyacrylamide gels containing 2 mg/ml casein and 0.025 U/ml plasminogen (American Diagnostica, Inc., Greenwich, CT). To specifically assess the presence of uPA in the samples (conditioned media and cell extracts), gels were incubated overnight with 100 mM amiloride (selectively blocks uPA activity) in 50 mM Tris and 5 mM CaCl2. Alternatively, samples were resolved in gels containing casein, but no plasminogen. To determine secretion of gelatinases by zymography, gelatin at a final concentration of 2 mg/ml was used instead of casein and plasminogen. After incubation, the gels were stained with Coomassie Brilliant Blue R250, destained, preserved, and dried.

Evaluation of uPA activity in culture media

uPA activity was determined by measuring plasminogen activation using a kit (Chemicon International, Temecula, CA) according to the manufacturer’s instructions. Briefly, 50 µl of 10x concentrated supernatants from cultures of cells incubated under various glucose concentrations for 24 h were added to a sample buffer (100 mM Tris and 0.5% Triton X-100, pH 8.8) containing a chromogenic tripeptide uPA substrate (supplied by the manufacturer) in a final volume of 100 µl. Human urine substrate (also provided by the manufacturer) was used as a positive control. After a 20-min incubation at 37 C, triplicate samples were analyzed spectrophotometrically at 405 nm in a SpectraMax Plus plate reader (Molecular Devices, Sunnyvale, CA). Three independent determinations of uPA activity were performed using this assay.

Western blot analysis

After culture, cells were lysed in sample buffer (2% sodium dodecyl sulfate, 10 mM Tris-Cl, and 0.15 mM NaCl, pH 7.5), and protein samples (10–25 µg) were subjected to SDS-PAGE and transferred to Immobilon-P membranes (Millipore Corp., Bedford, MA). The membranes were blocked overnight at 4 C in 0.1% Tween 20 and 5% dry milk powder and were subsequently probed with primary goat anti-uPAR antibody (Chemicon International, catalog no. AB8903; 1:500 dilution), goat anti-PAI-1 antibody (American Diagnostica, Inc., catalog no. 395G; 1:200 dilution), rabbit anti-glucose transporter-1 (GLUT-1) antibody (Chemicon International, catalog no. AB1341; 1:3000 dilution), or rabbit anti-GLUT-3 antibody (Chemicon International, catalog no. AB1345; 1:8000 dilution) for at least 1 h. Membranes were incubated with horseradish peroxidase-conjugated secondary antibodies, developed using a chemiluminescence Western blotting plus kit (PerkinElmer, Woodbridge, Canada), and subsequently exposed on X-OMAT film (Eastman Kodak Co., Rochester, NY).

To determine the uniformity of loading, blots were probed with a monoclonal anti-ß-actin antibody (Sigma-Aldrich Corp., St. Louis, MO; clone AC-15; 1:8000 dilution). Band intensities were measured using a SigmaGel densitometry software package (Jandel Scientific, San Rafael, CA), and protein bands were normalized to their respective ß-actin bands. Western blot analyses were performed in duplicate from three to six separate experiments.

Statistical analysis

Statistical analysis of uPA activity and Western blots was performed using PRISM software (GraphPad, Inc., San Diego, CA). Statistical significance was determined using one-way ANOVA, followed by a post hoc Tukey-Kramer test. All data were presented as means ± SE and were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of glucose on trophoblast invasion

Compared with cells incubated in 0.2 or 2.5 mM glucose, HTR-8/SVneo trophoblasts incubated for 24 h in physiological blood glucose levels (5 mM) or standard cell culture glucose levels (10 mM) exhibited a 49–62% decrease (P < 0.01) in their in vitro invasive capacity (Fig. 1Go). Addition of 10 or 100 ng/ml of the ATF of uPA to cells incubated in 0.2 or 2.5 mM glucose, to block the binding of pro-uPA to uPAR, decreased their invasiveness to levels exhibited by cells incubated in 5 or 10 mM glucose (P < 0.05; Fig. 1Go).



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 1. Invasion of first trimester HTR-8/SVneo extravillous trophoblast cells through Matrigel in response to incubation under various glucose concentrations in the presence or absence of the noncatalytically active ATF of uPA. Bars represent the invasion index (mean ± SE) normalized to the invasiveness of cells incubated in 2.5 mM glucose. Three independent experiments were performed. Statistical analysis was performed by ANOVA, followed by the Tukey-Kramer test. *, Significant difference (P < 0.01) in the invasiveness in 10 or 5 mM glucose vs. 2.5 or 0.2 mM glucose; **, significant difference (P < 0.05) in the invasiveness in 2.5 or 0.2 mM glucose in the absence vs. the presence of ATF; ***, significant difference (P < 0.001) in the invasiveness in 2.5 or 0.2 mM glucose in the absence vs. the presence of 100 ng/ml ATF.

 
Effects of various glucose concentrations on uPA and MMP secretion

Casein zymography revealed the presence of two prominent bands in samples of 24-h conditioned culture media and cell fractions (Fig. 2Go, A and B). These bands probably represent uPA, because the absence of plasminogen in the gels or incubation of the gels with amiloride resulted in the abolition of the bands (data not shown). Based on their electrophoretic mobility of 55 and 52 kDa, it is likely that these two prominent bands represent the single chain inactive and the two-chain active forms of uPA, respectively. The 52-kDa band was detectable only in the conditioned media and extracts of cells incubated in 0.2, 1, and 2.5 mM glucose (Fig. 2Go, A and B). In addition to the 52- and 55-kDa caseinolytic bands, the results of some experiments revealed the presence of a faint band of approximately 80 kDa in the culture medium (not shown). The levels of this caseinolytic band were not affected by incubation under various glucose concentrations.



View larger version (42K):
[in this window]
[in a new window]
  		FIG. 2. Zymographic analysis of cell-associated and secreted uPA (A and B) and secreted MMPs (C) in cultures of HTR-8/SVneo cells incubated under various glucose concentrations. Clear areas within the gels represent caseinolytic or gelatinolytic activity, the former resulting from activation of plasminogen. Caseinolytic bands at 55 and 52 kDa probably represent single-chain (inactive; Pro-uPA) and two-chain (active; tcuPA) uPA, respectively. Note the absence of the 52-kDa caseinolytic band in cell extracts and conditioned media from cultures incubated with concentrations of glucose greater than 2.5 mM (*). The results presented in this figure are representative of four independent experiments with similar outcomes.

 
To assess the effect of low glucose concentrations on uPA activation, uPA activity was measured in conditioned medium of HTR-8/SVneo cells cultured in 0.2, 2.5, 5, and 10 mM glucose for 24 h. Secreted uPA enzymatic activity was detected in all samples of conditioned medium (Table 1Go). Moreover, uPA activity was more than 2-fold higher in the medium of cultures incubated with 0.2 mM glucose vs. 5 or 10 mM glucose (P < 0.05; Table 1Go).


View this table:
[in this window]
[in a new window]
  		TABLE 1. Secreted uPA activity in cultures of HTR-8/SV neo cells

 
The results of gelatin-based zymograms revealed a prominent band of 72 kDa (MMP-2) and a less intense band of 92 kDa (MMP-9) in the culture medium of cells incubated with various concentrations of glucose (Fig. 2CGo). An additional faint band of approximately 85 kDa was observed in some experiments (not shown). Densitometric analysis of the bands revealed that glucose concentrations did not significantly affect the levels of gelatinase in the culture supernatants.

Effect of glucose on uPAR and PAI-1 levels

The effect of glucose concentration on uPAR and PAI-1 protein levels was determined by Western immunoblot analysis. Results revealed no significant differences in the uPAR and PAI-1 protein levels in lysates of cells incubated under various glucose concentrations for 24 h (Fig. 3Go).



View larger version (41K):
[in this window]
[in a new window]
  		FIG. 3. Representative Western blot analysis of uPAR in cell lysates (A) and PAI-1 in cell lysates and culture-conditioned media (B and C) of HTR-8/SVneo cultures exposed to various concentrations of glucose for 24 h. This experiment was repeated three times with similar results.

 
Effect of glucose concentrations on GLUT-1 and GLUT-3 protein levels

Immunoblot analysis of proteins extracted from HTR-8/SVneo cells revealed two bands of 55 and 48 kDa, corresponding to GLUT-1 and GLUT-3, respectively (Fig. 4Go). The levels of GLUT-1 protein in cells cultured in 0.2 and 2.5 mM glucose were 34 and 26% higher, respectively, than that in cells cultured in 5 mM glucose (P < 0.05). In contrast, the levels of GLUT-3 protein were not affected by incubation under various glucose concentrations.



View larger version (64K):
[in this window]
[in a new window]
  		FIG. 4. Representative Western blot analysis of GLUT-1 (A) and GLUT-3 (B) in lysates of HTR-8/SVneo cells incubated in various concentrations of glucose for 24 h. Compared with cells incubated in 5 or 10 mM glucose, densitometric analysis revealed that the levels of GLUT-1 protein were increased by 34 and 26%, respectively, in cells incubated in 0.2 and 2.5 mM glucose (P < 0.05). This experiment was repeated four times with similar results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The main finding of this study is that the in vitro invasiveness of immortalized placental trophoblast cells with properties of extravillous cytotrophoblasts was inversely correlated with the glucose concentrations to which these cells were exposed. Specifically, the invasion of reconstituted extracellular matrix by trophoblast cells decreased by approximately 50% when glucose concentrations in the 24-h invasion assay were increased from 2.5 to 5 mM and by up to 62% when glucose concentrations were increased from 0.2 to 5 mM. Normal maternal blood glucose levels are approximately 4–5 mM, whereas glucose levels in the intervillous space of the placenta in the first trimester of pregnancy are believed to be substantially lower (13). Thus, the present findings provide evidence that physiologically relevant glucose concentrations play an important role in regulating trophoblast invasiveness. Furthermore, because the first trimester of pregnancy is a time of maximal placental growth and development, the low glucose levels present in the first trimester placenta (13) may be important for adequate trophoblast invasion.

Trophoblast invasion and remodeling of the uterine spiral arterioles are important aspects of normal placentation. A hallmark of this form of vascular adaptation is the replacement of the maternal endothelial lining of the spiral arterioles with fetal trophoblast cells and a complete loss of the musculoelastic tissue of the vessels up to about the inner third of the myometrium. Consequently, the spiral arterioles become vessels with large diameters and low resistance, and also lose their responsiveness to maternal vasoactive molecules (33). This physiologically normal vascular remodeling process is necessary to accommodate the high placental blood flow demanded by the fetus later in gestation (34). In contrast to normal pregnancy, trophoblast remodeling of the spiral arterioles is markedly reduced in preeclampsia, a common pathological condition of pregnancy associated with maternal hypertension and proteinuria. Thus, in preeclampsia, the spiral arterioles maintain their responsiveness to vasoactive molecules and have much reduced internal diameters (35). At present, it is not known whether uterine glucose levels affect trophoblast remodeling of the spiral arterioles or whether placental glucose concentrations are elevated in the first trimester of pregnancies that later develop into preeclampsia. However, there is evidence that the incidence of preeclampsia is increased in women with type I diabetes mellitus (9, 10) as well as in women with gestational diabetes (11, 12). In light of the present results showing that relatively small changes in glucose concentrations can lead to significant alterations in the invasive capacity of trophoblast cells, it is possible that abnormally high blood glucose levels in the first trimester of pregnancy, as would be the case in women with nongestational type I diabetes, lead to uterine glucose concentrations that restrict trophoblast invasiveness and remodeling of the spiral arterioles.

In our study, we observed that the high invasive potential of cells incubated in low glucose concentrations was dependent on the ability of pro-uPA to bind to uPAR. This conclusion is based on the observation that addition of the catalytically inactive ATF inhibited invasion. Upon binding to uPAR, pro-uPA is converted into the active, two-chain form (22). As indicated by the results of the chromogenic uPA activity assay, the reduced invasiveness of trophoblast cells incubated in high glucose concentrations was associated with decreased uPA activity. Similarly, we found that the decreased invasiveness of immortalized HTR-8/SVneo cells incubated in 5 or 10 mM glucose was paralleled by the absence of a caseinolytic activity of approximately 52 kDa present in cultures of cells incubated in 0.2 and 2.5 mM glucose. Based on the observation that all caseinolytic activity was absent in control zymograms using gels lacking plasminogen or in plasminogen-containing gels incubated with amiloride, we conclude that all bands present in the casein zymograms belong to members of the uPA family. Amiloride has been shown to inhibit all forms of uPA, but not tissue PA, activity (36). Because of its close association with the 55-kDa pro-uPA, it is likely that the 52-kDa band present in cultures incubated in low glucose levels represents the active, two-chain form of uPA. It is also likely that the 52-kDa caseinolytic activity in the cell extracts represents cell membrane, uPAR-bound uPA. The precise identity of an 80-kDa caseinolytic activity revealed in some of the casein zymograms is presently unknown. However, because of its electrophoretic mobility, it is possible that it represents a complex made up of the A or B chain of uPA bound to uPAR, PAI-1, or even maspin.

The final mechanism by which high glucose concentrations inhibit uPA activation requires additional investigation. Inhibition of uPA activity by high glucose concentrations may not be a phenomenon limited to invasive trophoblast cells, as it has been reported that cultured human mesangial cells have a decreased capacity to generate plasmin in a high glucose environment (37). Furthermore, Chang et al. (38) demonstrated decreased gingival plasminogen activator activity in diabetic rats. Based on the observations of the present study, we can conclude that trophoblast invasion in low glucose concentrations requires uPA activity and the interaction of uPA with uPAR. Although incubation under low glucose did not affect the levels of PAI-1, uPAR, and gelatinases, it is possible that other members of the MMP family and/or the cathepsins are also regulated by glucose.

An additional objective of the present study was to determine whether changes in glucose concentrations affect the levels of expression of GLUT-1 and GLUT-3 as part of a compensatory mechanism to maintain homeostatic intracellular glucose concentrations. These transporters have been shown to be expressed in human invasive extravillous cytotrophoblasts (39, 40). Although the levels of expression of GLUT-1 were slightly higher in low vs. high glucose concentrations, this increase in GLUT-1 expression was not sufficient to prevent the cellular adaptive response and inhibit invasiveness. Additional research is required to elucidate the intracellular signaling pathway(s) activated by low glucose levels that eventually leads to uPA activation and invasion.

In summary, the results of the present study suggest a role for glucose in the regulation of trophoblast invasion of the uterus. Additional elucidation of the mechanisms involved in this regulation of invasiveness may reveal potential links between abnormalities of glucose metabolism and certain diseases of pregnancy.


   Acknowledgments
 
We are thankful to Dr. Anne Croy for the critical review of the manuscript.


   Footnotes
 
This work was supported by a grant from the Heart and Stroke Foundation of Ontario (T-5722; to C.H.G.). G.E.L. was a postdoctoral fellow of the Canadian Hypertension Society.

Current address of G.E.L.: School of Surgical and Reproductive Sciences, 3rd Floor, William Leech Building, University of Newcastle, Newcastle, United Kingdom NE2 4HH.

First Published Online May 10, 2005

1 L.B. and G.E.L. contributed equally to the current study. Back

Abbreviations: ATF, Amino-terminal fragment; FBS, fetal bovine serum; GLUT, glucose transporter; HLA, human leukocyte antigen; MMP, matrix metalloproteinase; PA, plasminogen activator; uPA, urokinase PA; uPAR, uPA receptor.

Received November 15, 2004.

Accepted May 2, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Graham CH, Lala PK 1991 Mechanism of control of trophoblast invasion in situ. J Cell Physiol 148:228–234[CrossRef][Medline]
  2. Bauer S, Pollheimer J, Hartmann J, Husslein P, Aplin JD, Knofler M 2004 Tumor necrosis factor-{alpha} inhibits trophoblast migration through elevation of plasminogen activator inhibitor-1 in first-trimester villous explant cultures. J Clin Endocrinol Metab 89:812–822[Abstract/Free Full Text]
  3. Cartwright JE, Holden DP, Whitley GS 1999 Hepatocyte growth factor regulates human trophoblast motility and invasion: a role for nitric oxide. Br J Pharmacol 128:181–189[CrossRef][Medline]
  4. Kauma SW, Bae-Jump V, Walsh SW 1999 Hepatocyte growth factor stimulates trophoblast invasion: a potential mechanism for abnormal placentation in preeclampsia. J Clin Endocrinol Metab 84:4092–4096[Abstract/Free Full Text]
  5. Graham CH, Fitzpatrick TE, McCrae KR 1998 Hypoxia stimulates urokinase receptor expression through a heme protein-dependent pathway. Blood 91:3300–3307[Abstract/Free Full Text]
  6. Postovit LM, Adams MA, Graham CH 2001 Does nitric oxide play a role in the aetiology of pre-eclampsia? Placenta 22(Suppl A):S51–S55
  7. Singh SD 1986 Gestational diabetes and its effect on the umbilical cord. Early Hum Dev 14:89–98[CrossRef][Medline]
  8. Burleigh DW, Stewart K, Grindle KM, Kay HH, Golos TG 2004 Influence of maternal diabetes on placental fibroblast growth factor-2 expression, proliferation, and apoptosis. J Soc Gynecol Invest 11:36–41[CrossRef][Medline]
  9. Garner PR, D’Alton ME, Dudley DK, Huard P, Hardie M 1990 Preeclampsia in diabetic pregnancies. Am J Obstet Gynecol 163:505–508[Medline]
  10. Ang C, Lumsden MA 2001 Diabetes and the maternal resistance vasculature. Clin Sci 101:719–729[Medline]
  11. Ostlund I, Haglund B, Hanson U 2004 Gestational diabetes and preeclampsia. Eur J Obstet Gynecol Reprod Biol 113:12–16[CrossRef][Medline]
  12. Yogev Y, Langer O, Brustman L, Rosenn B 2004 Pre-eclampsia and gestational diabetes mellitus: does a correlation exist early in pregnancy? J Matern Fetal Neonatal Med 15:39–43[CrossRef][Medline]
  13. Jauniaux E, Hempstock J, Teng C, Battaglia FC, Burton GJ 2005 Polyol concentrations in the fluid compartments of the human conceptus during the first trimester of pregnancy: maintenance of redox potential in a low oxygen environment. J Clin Endocrinol Metab 90:1171–1175[Abstract/Free Full Text]
  14. Jang A, Hill RP 1997 An examination of the effects of hypoxia, acidosis, and glucose starvation on the expression of metastasis-associated genes in murine tumor cells. Clin Exp Metastasis 15:469–483[CrossRef][Medline]
  15. Cuvier C, Jang A, Hill RP 1997 Exposure to hypoxia, glucose starvation and acidosis: effect on invasive capacity of murine tumor cells and correlation with cathepsin (L + B) secretion. Clin Exp Metastasis 15:19–25[CrossRef][Medline]
  16. Yagel S, Parhar RS, Jeffrey JJ, Lala PK 1988 Normal nonmetastatic human trophoblast cells share in-vitro invasive properties of malignant cells. J Cell Physiol 136:455–462[CrossRef][Medline]
  17. Behrendt N, Ronne E, Dano K 1995 The structure and function of the urokinase receptor, a membrane protein governing plasminogen activation on the cell surface. Biol Chem Hoppe-Seyler 376:269–279[Medline]
  18. Kim HS, Shang T, Chen Z, Pflugfelder SC, Li DQ 2004 TGF-ß1 stimulates production of gelatinase (MMP-9), collagenases (MMP-1, -13) and stromelysins (MMP-3, -10, -11) by human corneal epithelial cells. Exp Eye Res 79:263–274[CrossRef][Medline]
  19. Van Themsche C, Alain T, Kossakowska AE, Urbanski S, Potworowski EF, St Pierre Y 2004 Stromelysin-2 (matrix metalloproteinase 10) is inducible in lymphoma cells and accelerates the growth of lymphoid tumors in vivo. J Immunol 173:3605–3611[Abstract/Free Full Text]
  20. Vassalli JD, Sappino AP, Belin D 1991 The plasminogen activator/plasmin system. J Clin Invest 88:1067–1072
  21. Petersen LC, Lund LR, Nielsen LS, Dano K, Skriver L 1988 One-chain urokinase-type plasminogen activator from human sarcoma cells is a proenzyme with little or no intrinsic activity. J Biol Chem 263:11189–11195[Abstract/Free Full Text]
  22. Dano K, Behrendt N, Brunner N, Ellis V, Ploug M, Pyke C 1994 The urokinase receptor. Protein structure and role in plasminogen activation and cancer invasion. Fibrinolysis 8:189–203[CrossRef]
  23. Moser TL, Enghild JJ, Pizzo SV, Stack MS 1993 The extracellular matrix proteins laminin and fibronectin contain binding domains for human plasminogen and tissue plasminogen activator. J Biol Chem 268:18917–18923[Abstract/Free Full Text]
  24. Shetty S, Idell S 2000 Posttranscriptional regulation of plasminogen activator inhibitor-1 in human lung carcinoma cells in vitro. Am J Physiol 278:L148–L156
  25. Kwaan HC 1992 The plasminogen-plasmin system in malignancy. Cancer Metastasis Rev 11:291–311[CrossRef][Medline]
  26. Ellis V, Behrendt N, Dano K 1991 Plasminogen activation by receptor-bound urokinase. A kinetic study with cell associated and isolated receptor. J Biol Chem 266:12752–12758[Abstract/Free Full Text]
  27. Nykjaer A, Petersen CM, Moller B, Jensen PH, Moestrup SK, Holtet TI, Etzerodt M, Thogersen HC, Munch M, Andreasen PA, Gliemann J 1992 Purified {alpha}2-macroglobulin receptor/LDL receptor-related protein binds urokinase plasminogen activator inhibitor type-1 complex. Evidence that the {alpha}2-macroglobulin receptor mediates cellular degradation of urokinase receptor-bound complexes. J Biol Chem 268:14543–14546
  28. Graham CH, Hawley TS, Hawley RG, MacDougall JR, Kerbel RS, Khoo N, Lala PK 1993 Establishment and characterization of first trimester human trophoblast cells with extended lifespan. Exp Cell Res 206:204–211[CrossRef][Medline]
  29. Kilburn BA, Wang J, Duniec-Dmuchowski ZM, Leach RE, Romero R, Armant DR, Duniec-Dmuchkowski ZM 2000 Extracellular matrix composition and hypoxia regulate the expression of HLA-G and integrins in a human trophoblast cell line. Biol Reprod 62:739–747[Abstract/Free Full Text]
  30. Irving JA, Lala PK 1995 Functional role of cell surface integrins on human trophoblast cell migration: regulation by TGF-ß, IGF-II, and IGFBP-1. Exp Cell Res 217:419–427[CrossRef][Medline]
  31. Graham CH, Forsdike J, Fitzgerald CF, Macdonald-Goodfellow S 1999 Hypoxia-mediated stimulation of carcinoma cell invasiveness via upregulation of urokinase receptor expression. Int J Cancer 80:617–623[CrossRef][Medline]
  32. Graham CH, McCrae KR 1996 Altered expression of gelatinase and surface-associated plasminogen activator activity by trophoblast cells isolated from placentae of preeclamptic patients. Am J Obstet Gynecol 175:555–562[CrossRef][Medline]
  33. Robertson WB, Brosens I, Dixon HG 1967 The pathological response of the vessels of the placental bed to hypertensive pregnancy. J Pathol Bacteriol 93:581–592[CrossRef][Medline]
  34. Gant NF, Madden JD, Chand S, Worley RJ, Sineri PK, MacDonald PC 1976 Metabolic clearance of dehydroisoandrosterone sulfate. VI. Studies of eclampsia. Obstet Gynecol 47:327–330[Medline]
  35. Khong TY, De Wolf F, Robertson WB, Brosens I 1986 Inadequate maternal vascular response to placentation in pregnancies complicated by pre-eclampsia and by small-for-gestational age infants. Br J Obstet Gynaecol 93:1049–1059[Medline]
  36. Vassalli JD, Belin D 1987 Amiloride selectively inhibits the urokinase-type plasminogen activator. FEBS Lett 214:187–191[CrossRef][Medline]
  37. Fisher EJ, McLennan SV, Yue DK, Turtle JR 1997 High glucose reduces generation of plasmin activity by mesangial cells. Microvasc Res 53:173–178[CrossRef][Medline]
  38. Chang K, Rani AS, Chang K, Kumar S 1996 Plasminogen activator activity is decreased in rat gingiva during diabetes. J Periodontol 67:743–747[Medline]
  39. Sakata M, Kurachi H, Imai T, Tadokoro C, Yamaguchi M, Yoshimoto Y, Oka Y, Miyake A 1995 Increase in human placental glucose transporter-1 during pregnancy. Eur J Endocrinol 132:206–212[Abstract/Free Full Text]
  40. Hahn D, Blaschitz A, Korgun ET, Lang I, Desoye G, Skofitsch G, Dohr G 2001 From maternal glucose to fetal glycogen: expression of key regulators in the human placenta. Mol Hum Reprod 7:1173–1178[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Biol. Reprod.Home page
S. J. Renaud, S. K. Macdonald-Goodfellow, and C. H. Graham
Coordinated Regulation of Human Trophoblast Invasiveness by Macrophages and Interleukin 10
Biol Reprod, March 1, 2007; 76(3): 448 - 454.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a related Letter to the Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Belkacemi, L.
Right arrow Articles by Graham, C. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Belkacemi, L.
Right arrow Articles by Graham, C. H.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals