- Email: [email protected]
Activated Endothelial Cells Resist Displacement by Trophoblast In Vitro
Placenta 28 (2007) 743e747
Activated Endothelial Cells Resist Displacement by Trophoblast In Vitro Q. Chen*, P.R. Stone, L.M.E. McCowan, L.W. Chamley Department of Obstetrics and Gynaecology, Faculty of Medical and Health Sciences, The University of Auckland, 85 Park Road, Grafton, Auckland 1001, New Zealand Accepted 4 October 2006
Abstract Background: Transformation of the spiral arteries by invading trophoblasts is an essential prerequisite to the development of a healthy fully grown fetus. Reduced transformation of the spiral arteries is a characteristic feature of pregnancies complicated by several diseases of pregnancy including preeclampsia. The aim of this study was to investigate further the hypothesis that spiral artery endothelial cells can contribute to the mechanism of shallow trophoblast invasion. Method: Fluorescently labeled Jar cells were added to monolayers of fluorescently-labeled endothelial cells that had been activated by treatment with TNFa, INFg or necrotic cell bodies. The ability of the Jars to displace endothelial cells from the monolayers was quantified by measuring the area of Jar cells ‘‘islands’’ formed in the endothelial cell monolayers by confocal microscopy and digital image. Results: The area of Jar cell islands formed in monolayers of activated endothelial cells was significantly smaller that the area of islands formed in control resting/non-activated endothelial cell monolayers regardless of the activator. Discussion: This work demonstrates that activated endothelial cells are more resistant to trophoblast displacement than resting endothelial cells and adds weight to the suggestion that endothelial cells could contribute to shallow invasion of the spiral arteries by trophoblasts in diseases such as preeclampsia. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Trophoblast; Invasion; Endothelial cell; Activation; Implantation
1. Introduction Growth of the human fetus is dependant upon an adequate supply of maternal blood reaching the placenta. However, prior to pregnancy the uterine spiral arteries that would supply blood to the placenta are small muscular arterioles that could not conduct enough maternal blood to support fetal growth in the later stages of pregnancy. Consequently, the human fetus has evolved the ability to transform the spiral arteries into high-flow, low-resistance vessels [1,2]. This transformation of the spiral arteries is effected by extravillous trophoblasts that migrate out of the placenta and invade the lumen and wall of the spiral arteries replacing the endothelial cells that * Corresponding author. Tel.: þ64 9 373 7599x89519; fax: þ64 9 303 5969. E-mail address: [email protected] (Q. Chen). 0143-4004/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.placenta.2006.10.004
line the non-pregnant spiral arteries and digesting the musculo-elastic wall of these vessels. Thus, a fully transformed spiral artery is lined by endovascular trophoblast, is dilated and lacks the ability to respond to vasoactive stimuli. The exact pathway by which trophoblast initially enter the spiral arteries is unclear but once the vessels have been breached, endovascular trophoblasts appear to migrate antidromically within the lumen of the vessels towards the myometrial segments of the spiral arteries [3]. These changes, termed the physiological changes of pregnancy [1] may be deficient in pregnancies complicated by preeclampsia (PE) and small for gestational age babies (SGA) [1,3,4]. In these diseases of pregnancy either the depth of transformation of the spiral arteries is limited or the number of vessels undergoing the transformation is reduced [1,5]. The mechanisms that lead to the invasion and transformation of the spiral arteries by
Q. Chen et al. / Placenta 28 (2007) 743e747
trophoblast and the factors that restrict trophoblast invasion in diseases of pregnancy are still poorly understood. Many studies have focused on factors that may promote/control trophoblast invasion such as vascular endothelial growth factor (VEGF) [6], Nitric Oxide (NO) [7] and adhesion molecule expression [8,9]. In all of these studies the underlying assumption is that trophoblasts control the extent of endovascular invasion. However, recently we and others [10] have suggested that the control of endovascular invasion may not be so simple and that spiral artery endothelial cells may be able to resist invasion by trophoblasts. In the current study we have further investigated that hypothesis by examining whether the activation state of endothelial cells alters their susceptibility to invading trophoblasts and have also investigated whether different activators of endothelial cells have similar effects on susceptibility of endothelial cells to trophoblast invasion. 2. Materials and methods 2.1. Materials All the culture medium and Fetal Bovine Serum (FBS) were purchased from Invitrogen. Cell tracker fluorescent stains CMFDA (5-chloromethyl-7hydroxycoumarin) and SNARF-1 (5-chloromethy carboxyseminapthorhodofluor-1) were purchased from Molecular Probes. Cytokines were purchased from R & D Systems.
2.2. Cell culture The human microvascular endothelial cell line (HMEC-1) was obtained from the National Centre for Infectious Diseases (USA) and grown in MCDB 131 as previously described [11]. The trophoblast-derived choriocarcinoma cell line, Jar was used to model deported trophoblasts and was grown in DMEM/F12 as previously described [11].
2.3. Endothelial cell activation assay Endothelial cells (HMEC-1) were directly exposed to necrotic trophoblasts as described previously [12]. Briefly, Jar cells were induced to either apoptosis by exposing to UV light (30 W) for 1 h or necrosis by freeze-thaw cycle for 1.5 h. Also the HMEC-1 were exposed to PMA (10 ng/ml), Tumor necrosis factor-alpha (TNF-a) (20 ng/ml, 2 107 units/mg) and Interferon gamma (INF g) (10 ng/ml, 1 107 units/mg) for 24 h. To determine whether HMEC-1 became activated following treatment with the above factor a cell based ELISA was employed to quantify the expression of intercellular adhesion molecule-1 (ICAM-1), E -selectin or vascular cell adhesion molecule (VCAM), as described previously [13].
2.4. Coculture of activated endothelial cells with trophoblasts HMEC-1 cells were seeded onto plastic microscope slide coverslips in 6-well plates as described previously [11], and then labeled with fluorescent cell tracker CMFDA (1 mM) for 2 h. HMEC-1 cells were then exposed to necrotic Jar cells or TNFa (20 ng/ml), INFg (10 ng/ml), or the positive controls, for activation, PMA (10 ng/ml). After 24 h, cell tracker SNARF-1 (1 mM) labeled Jar cells (2.5 104/ml) were added to these confluent monolayers of activated endothelial cells for a further 24 h at 37 C, in a humidified 5% CO2 atmosphere. Control cultures consisted of non-treated HMEC-1 monolayers cultured with trophoblasts Jar cells. All the cocultures were then washed, fixed with 4% paraformaldehyde (PFA) and coverslips were mounted onto glass microscope slides using
Citiflour fluorescent mounting medium, and then examined by confocal microscopy using a Leica model TCS SP2 confocal microscopy. The area of Jar cell islands within the endothelial cell monolayers was then quantified in 12 high power fields/treatment using Image J software.
2.5. Statistical analysis All experiments were repeated at least three times. Data are presented as mean SEM. The statistical significance of the results was assessed by ANOVA followed by t-test using Microsoft Excel.
3. Result 3.1. Factors that activate endothelial cells It is well known that various cytokines including TNFa and INFg activate endothelial cells but in order to confirm that these factors were activating the endothelial cells in our system we monitored the expression of ICAM-1 by cell-based ELISA. Treating endothelial cell monolayers with TNFa or INFg for 24 h induced a significant ( p < 0.05) increase in the endothelial cell surface expression ICAM-1 (Fig. 1), while E-selectin, and VCAM expression was not affected at that time point (data not shown). We have previously shown that endothelial cells phagocytose dead trophoblasts [12] but when the trophoblasts have died by necrosis this induces activation of the endothelial cells. We confirmed in the current experiments that necrotic cell bodies but not apoptotic cell bodies produced a significant ( p < 0.05) increase in the cell surface expression of ICAM-1 by endothelial cell monolayers whereas apoptotic cell bodies did not activate the endothelial cells as we have shown previously [12]. 3.2. Comparison of the invasion of activated versus resting endothelial cell monolayers by Jar cells To examine whether activated endothelial cells could resist invasion by Jar cells we activated (green) fluorescently-labeled endothelial cell monolayers with TNF-a, INF
ICAM-1 expression (OD 490nm)
744
2.5 2
*
*
* *
1.5 1 0.5 0 TNF-α
INF-
PMA
Necrotic bodies
Apoptotic bodies
control *p<0.05
Fig. 1. Levels of ICAM-1 expression were measured by cell-based ELISA 24 h after treatment of confluent HMEC-1 monolayers with TNFa, INFg, PMA, necrotic or apoptotic cell bodies. Levels of ICAM-1 expression were compared to untreated control cultures by t-test. All treatments were quantified in triplicate and the assays were repeated on three separate occasions.
Q. Chen et al. / Placenta 28 (2007) 743e747
g, or necrotic cell bodies prior to adding red fluorescently-labeled Jar cells to the endothelial cell monolayers. Positive control monolayers of endothelial cells were activated with PMA. Examination of the cocultures by confocal microscopy suggested that less endothelial cells were displaced from the monolayers by Jar cells when the endothelial cells were activated than non-activated/resting control monolayers (Fig. 2A). Quantification of this effect demonstrated that the area of endothelial cells displaced by Jar cells was more than halved when the endothelial cells were activated regardless of the activator including necrotic cell bodies ( p < 0.05, Fig. 2B). In contrast, addition of apoptotic cell bodies to the endothelial cell monolayers did not significantly reduce the displacement of endothelial cells by Jar cells (Fig. 2B).
745
4. Discussion Transformation of the spiral arteries including the displacement of vascular endothelial cells by extravillous trophoblasts is an essential prerequisite to normal placentation. We have shown in this study that activated endothelial cells are able to resist displacement by trophoblasts using Jar choriocarcinoma cells as a model of invasive trophoblasts. One of the criticisms of this work could be the use of Jar cells which are malignant and unlike extravillous trophoblasts exhibit aggressive and uncontrolled invasion. While that criticism is to some extent valid this study clearly demonstrates the principle that activated endothelial cells can resist invasion and suggests that the extent of trophoblast invasion of the spiral arteries may not be solely dependent upon the invasive capacity of
A a
b
c
d
e
f
Area of HMEC-1 displaced (µm2)
B
30000 25000 20000 15000 10000 5000
*
*
*
*
INF-
PMA
Necrotic bodies
0 TNF-α
Apoptotic Untreated bodies *p <0.05
Fig. 2. A) Representative confocal photomicrographs showing cell ‘‘islands’’ of red fluorescent-labeled Jar cells which have displaced endothelial cells (Green) from monolayers. Prior to the addition of Jar cells the endothelial cells were treated with a) TNFa, b) INFg, c) PMA, d) Necrotic cell bodies, e) Apoptotic cell bodies or f) Untreated controls. B) The area of Jar cell islands which had displaced endothelial cells from monolayers was quantified by digital image analysis following confocal microscopy. Twelve micrographic fields were examined for each treatment and experiments were repeated three times. (*p < 0.05 t-test).
746
Q. Chen et al. / Placenta 28 (2007) 743e747
the invading trophoblasts. Furthermore, if activated endothelial cells can resist displacement by these aggressively invasive trophoblast-derived cells then it would seem intuitively likely that activated endothelial cells might resist displacement by normal trophoblast to an even greater extent. It was interesting to note that activated endothelial cells resisted displacement by Jar cells regardless of their activator. Thus, both TNFa and INFg which are well known physiologic/pathologic activators of endothelial cells had a similar ability to render endothelial cell monolayers resistant to displacement by Jar cells. Both of these cytokines are known to be produced at the materno-fetal interface and production may be increased in pregnancies complicated by preeclampsia, a disease characterized by shallow trophoblast invasion [14e16]. It has previously been shown that elevated levels of TNFa can inhibit trophoblast invasion via effects on trophoblast-derived plasminogen activator inhibitor -1 (PAI-1) suggesting that TNFa may affect trophoblasts directly to reduce their invasive capacity [17]. In a series of elegant experiments Crocker et al. [18] demonstrated that TNFa, at a similar dose to that which we have employed, reduced endovascular trophoblast invasion in vitro and it is possible that TNFa acts both directly on trophoblasts to reduce their invasive potential and also on spiral artery endothelial cells making them resistant to trophoblast invasion. Our data also showed that necrotic trophoblast cell carcasses/debris activated endothelial cells and that the endothelial cells activated in this manner also resisted displacement by trophoblast. This result further confirms that activated endothelial cells resist displacement regardless of the nature of the activator and raises the possibility that increased necrotic cell cellular debris in the spiral arteries could also limit trophoblast invasion. It is widely accepted that one of the hallmark features of preeclampsia is systemic maternal endothelial dysfunction characterized by activation of the endothelium which is thought to be secondary to shallow invasion of the spiral arteries by trophoblast [19]. Our results suggest that endothelial cell activation could contribute not only to the maternal symptoms of preeclampsia seen in later gestation but also to the hallmark shallow invasion of the spiral arteries that is believed to be one of the underlying pathogenic triggers of severe preeclampsia. We do not know the mechanism by which activated endothelial cells resist displacement by Jar cells but activation of endothelial cells is known to induce a wide variety of changes in the endothelial cells including secretion of soluble factors that might have induced apoptosis in the Jar cells. However, our own experience and that of others [20] is that Jar cells are particularly resistant to factors that readily induce apoptosis in other cells and it seems unlikely that the effect we observed was due to increased apoptotic death of the Jar cells. In summary, we have shown that activated endothelial cells resist displacement by trophoblast-derived Jar cells regardless of the mechanism by which the endothelial cells were activated. If this phenomenon also occurs in vivo these results suggest that any blood borne factors that activate endothelial cells, including those we have studied and others such as interleukin-1 [21] or syncytiotrophoblast microparticles [22], could
contribute to the shallow invasion that is characteristic of preeclampsia and other disorders of pregnancy. Acknowledgements This study was supported by grants from the University of Auckland Staff Research Fund, and Auckland Medical Research Foundation (AMRF). References [1] Brosens I, Robertson WB, Dixon HG. The physiological response of the vessels of the placental bed to normal pregnancy. J Pathol Bacteriol 1967;93:569e79. [2] Hamilton WJ, Boyd JD. Trophoblast in human utero-placental arteries. Nature 1966;212:906e8. [3] Pijnenborg R, Dixon G, Robertson WB, Brosens I. Trophoblastic invasion of human decidua from 8 to 18 weeks of pregnancy. Placenta 1980;1:3e19. [4] Khong TY, De Wolf F, Robertson WB, Brosens I. Inadequate maternal vascular response to placentation in pregnancies complicated by preeclampsia and by small-for-gestational age infants. Br J Obstet Gynaecol 1986;93:1049e59. [5] Benirschke K, Kaufmann P. Pathology of the human placenta. 4th ed. New York: Springer-Verlag; 2000. [6] Zhou Y, Bellingard V, Feng KT, McMaster M, Fisher SJ. Human cytotrophoblasts promote endothelial survival and vascular remodeling through secretion of Ang2, PlGF, and VEGF-C. Dev Biol 2003;263:114e25. [7] Lyall F, Bulmer JN, Kelly H, Duffie E, Robson SC. Human trophoblast invasion and spiral artery transformation: the role of nitric oxide. Am J Pathol 1999;154:1105e14. [8] Lyall F, Bulmer JN, Duffie E, Cousins F, Theriault A, Robson SC. Human trophoblast invasion and spiral artery transformation: the role of PECAM-1 in normal pregnancy, preeclampsia, and fetal growth restriction. Am J Pathol 2001;158:1713e21. [9] Kaufmann P, Black S, Huppertz B. Endovascular trophoblast invasion: implications for the pathogenesis of intrauterine growth retardation and preeclampsia. Biol Reprod 2003;69:1e7. [10] Campbell S, Rowe J, Jackson EJ, Gallery ED. In vitro migration of cytotrophoblasts through a decidual endothelial cell monolayer: the role of matrix metalloproteinases. Placenta 2003;24:306e15. [11] Chen Q, Stone PR, McCowan LM, Chamley LW. Interaction of Jar choriocarcinoma cells with endothelial cell monolayers. Placenta 2005;26:617e25. [12] Chen Q, Stone PR, McCowan LM, Chamley LW. Phagocytosis of necrotic but not apoptotic trophoblasts induces endothelial cell activation. Hypertension 2006;47:116e21. [13] Chen Q, Stone PR, Woon ST, Ching LM, Hung S, McCowan LM, et al. Antiphospholipid antibodies bind to activated but not resting endothelial cells: is an independent triggering event required to induce antiphospholipid antibody-mediated disease? Thromb Res 2004;114:101e11. [14] Banerjee S, Smallwood A, Moorhead J, Chambers AE, Papageorghiou A, Campbell S, et al. Placental expression of interferon-gamma (IFNgamma) and its receptor IFN-gamma R2 fail to switch from early hypoxic to late normotensive development in preeclampsia. J Clin Endocrinol Metab 2005;90:944e52. [15] Vince GS, Starkey PM, Austgulen R, Kwiatkowski D, Redman CW. Interleukin-6, tumour necrosis factor and soluble tumour necrosis factor receptors in women with pre-eclampsia. Br J Obstet Gynaecol 1995;102:20e5. [16] Hung TH, Charnock-Jones DS, Skepper JN, Burton GJ. Secretion of tumor necrosis factor-alpha from human placental tissues induced by hypoxia-reoxygenation causes endothelial cell activation in vitro: a potential mediator of the inflammatory response in preeclampsia. Am J Pathol 2004;164:1049e61.
Q. Chen et al. / Placenta 28 (2007) 743e747 [17] Bauer S, Pollheimer J, Hartmann J, Husslein P, Aplin JD, Knofler M. Tumor necrosis factor-alpha inhibits trophoblast migration through elevation of plasminogen activator inhibitor-1 in firsttrimester villous explant cultures. J Clin Endocrinol Metab 2004; 89:812e22. [18] Crocker IP, Wareing M, Ferris GR, Jones CJ, Cartwright JE, Baker PN, et al. The effect of vascular origin, oxygen, and tumour necrosis factor alpha on trophoblast invasion of maternal arteries in vitro. J Pathol 2005;206(4):476e85. [19] Broughton Pipkin F, Roberts JM. Hypertension in pregnancy. J Hum Hypertens 2000;14:705e24.
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[20] Sun QH, Peng JP, Xia HF. IFN gamma pretreatment sensitizes human choriocarcinoma cells to etoposide-induced apoptosis. Mol Hum Reprod 2006;12:99e105. [21] Rusterholz C, Gupta AK, Huppertz B, Holzgreve W, Hahn S. Soluble factors released by placental villous tissue: Interleukin-1 is a potential mediator of endothelial dysfunction. Am J Obstet Gynecol 2005;192: 618e24. [22] Goswami D, Tannetta DS, Magee LA, Fuchisawa A, Redman CW, Sargent IL, et al. Excess syncytiotrophoblast microparticle shedding is a feature of early-onset pre-eclampsia, but not normotensive intrauterine growth restriction. Placenta 2006;27:56e61.
Activated Endothelial Cells Resist Displacement by Trophoblast In Vitro Q. Chen*, P.R. Stone, L.M.E. McCowan, L.W. Chamley Department of Obstetrics and Gynaecology, Faculty of Medical and Health Sciences, The University of Auckland, 85 Park Road, Grafton, Auckland 1001, New Zealand Accepted 4 October 2006
Abstract Background: Transformation of the spiral arteries by invading trophoblasts is an essential prerequisite to the development of a healthy fully grown fetus. Reduced transformation of the spiral arteries is a characteristic feature of pregnancies complicated by several diseases of pregnancy including preeclampsia. The aim of this study was to investigate further the hypothesis that spiral artery endothelial cells can contribute to the mechanism of shallow trophoblast invasion. Method: Fluorescently labeled Jar cells were added to monolayers of fluorescently-labeled endothelial cells that had been activated by treatment with TNFa, INFg or necrotic cell bodies. The ability of the Jars to displace endothelial cells from the monolayers was quantified by measuring the area of Jar cells ‘‘islands’’ formed in the endothelial cell monolayers by confocal microscopy and digital image. Results: The area of Jar cell islands formed in monolayers of activated endothelial cells was significantly smaller that the area of islands formed in control resting/non-activated endothelial cell monolayers regardless of the activator. Discussion: This work demonstrates that activated endothelial cells are more resistant to trophoblast displacement than resting endothelial cells and adds weight to the suggestion that endothelial cells could contribute to shallow invasion of the spiral arteries by trophoblasts in diseases such as preeclampsia. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Trophoblast; Invasion; Endothelial cell; Activation; Implantation
1. Introduction Growth of the human fetus is dependant upon an adequate supply of maternal blood reaching the placenta. However, prior to pregnancy the uterine spiral arteries that would supply blood to the placenta are small muscular arterioles that could not conduct enough maternal blood to support fetal growth in the later stages of pregnancy. Consequently, the human fetus has evolved the ability to transform the spiral arteries into high-flow, low-resistance vessels [1,2]. This transformation of the spiral arteries is effected by extravillous trophoblasts that migrate out of the placenta and invade the lumen and wall of the spiral arteries replacing the endothelial cells that * Corresponding author. Tel.: þ64 9 373 7599x89519; fax: þ64 9 303 5969. E-mail address: [email protected] (Q. Chen). 0143-4004/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.placenta.2006.10.004
line the non-pregnant spiral arteries and digesting the musculo-elastic wall of these vessels. Thus, a fully transformed spiral artery is lined by endovascular trophoblast, is dilated and lacks the ability to respond to vasoactive stimuli. The exact pathway by which trophoblast initially enter the spiral arteries is unclear but once the vessels have been breached, endovascular trophoblasts appear to migrate antidromically within the lumen of the vessels towards the myometrial segments of the spiral arteries [3]. These changes, termed the physiological changes of pregnancy [1] may be deficient in pregnancies complicated by preeclampsia (PE) and small for gestational age babies (SGA) [1,3,4]. In these diseases of pregnancy either the depth of transformation of the spiral arteries is limited or the number of vessels undergoing the transformation is reduced [1,5]. The mechanisms that lead to the invasion and transformation of the spiral arteries by
Q. Chen et al. / Placenta 28 (2007) 743e747
trophoblast and the factors that restrict trophoblast invasion in diseases of pregnancy are still poorly understood. Many studies have focused on factors that may promote/control trophoblast invasion such as vascular endothelial growth factor (VEGF) [6], Nitric Oxide (NO) [7] and adhesion molecule expression [8,9]. In all of these studies the underlying assumption is that trophoblasts control the extent of endovascular invasion. However, recently we and others [10] have suggested that the control of endovascular invasion may not be so simple and that spiral artery endothelial cells may be able to resist invasion by trophoblasts. In the current study we have further investigated that hypothesis by examining whether the activation state of endothelial cells alters their susceptibility to invading trophoblasts and have also investigated whether different activators of endothelial cells have similar effects on susceptibility of endothelial cells to trophoblast invasion. 2. Materials and methods 2.1. Materials All the culture medium and Fetal Bovine Serum (FBS) were purchased from Invitrogen. Cell tracker fluorescent stains CMFDA (5-chloromethyl-7hydroxycoumarin) and SNARF-1 (5-chloromethy carboxyseminapthorhodofluor-1) were purchased from Molecular Probes. Cytokines were purchased from R & D Systems.
2.2. Cell culture The human microvascular endothelial cell line (HMEC-1) was obtained from the National Centre for Infectious Diseases (USA) and grown in MCDB 131 as previously described [11]. The trophoblast-derived choriocarcinoma cell line, Jar was used to model deported trophoblasts and was grown in DMEM/F12 as previously described [11].
2.3. Endothelial cell activation assay Endothelial cells (HMEC-1) were directly exposed to necrotic trophoblasts as described previously [12]. Briefly, Jar cells were induced to either apoptosis by exposing to UV light (30 W) for 1 h or necrosis by freeze-thaw cycle for 1.5 h. Also the HMEC-1 were exposed to PMA (10 ng/ml), Tumor necrosis factor-alpha (TNF-a) (20 ng/ml, 2 107 units/mg) and Interferon gamma (INF g) (10 ng/ml, 1 107 units/mg) for 24 h. To determine whether HMEC-1 became activated following treatment with the above factor a cell based ELISA was employed to quantify the expression of intercellular adhesion molecule-1 (ICAM-1), E -selectin or vascular cell adhesion molecule (VCAM), as described previously [13].
2.4. Coculture of activated endothelial cells with trophoblasts HMEC-1 cells were seeded onto plastic microscope slide coverslips in 6-well plates as described previously [11], and then labeled with fluorescent cell tracker CMFDA (1 mM) for 2 h. HMEC-1 cells were then exposed to necrotic Jar cells or TNFa (20 ng/ml), INFg (10 ng/ml), or the positive controls, for activation, PMA (10 ng/ml). After 24 h, cell tracker SNARF-1 (1 mM) labeled Jar cells (2.5 104/ml) were added to these confluent monolayers of activated endothelial cells for a further 24 h at 37 C, in a humidified 5% CO2 atmosphere. Control cultures consisted of non-treated HMEC-1 monolayers cultured with trophoblasts Jar cells. All the cocultures were then washed, fixed with 4% paraformaldehyde (PFA) and coverslips were mounted onto glass microscope slides using
Citiflour fluorescent mounting medium, and then examined by confocal microscopy using a Leica model TCS SP2 confocal microscopy. The area of Jar cell islands within the endothelial cell monolayers was then quantified in 12 high power fields/treatment using Image J software.
2.5. Statistical analysis All experiments were repeated at least three times. Data are presented as mean SEM. The statistical significance of the results was assessed by ANOVA followed by t-test using Microsoft Excel.
3. Result 3.1. Factors that activate endothelial cells It is well known that various cytokines including TNFa and INFg activate endothelial cells but in order to confirm that these factors were activating the endothelial cells in our system we monitored the expression of ICAM-1 by cell-based ELISA. Treating endothelial cell monolayers with TNFa or INFg for 24 h induced a significant ( p < 0.05) increase in the endothelial cell surface expression ICAM-1 (Fig. 1), while E-selectin, and VCAM expression was not affected at that time point (data not shown). We have previously shown that endothelial cells phagocytose dead trophoblasts [12] but when the trophoblasts have died by necrosis this induces activation of the endothelial cells. We confirmed in the current experiments that necrotic cell bodies but not apoptotic cell bodies produced a significant ( p < 0.05) increase in the cell surface expression of ICAM-1 by endothelial cell monolayers whereas apoptotic cell bodies did not activate the endothelial cells as we have shown previously [12]. 3.2. Comparison of the invasion of activated versus resting endothelial cell monolayers by Jar cells To examine whether activated endothelial cells could resist invasion by Jar cells we activated (green) fluorescently-labeled endothelial cell monolayers with TNF-a, INF
ICAM-1 expression (OD 490nm)
744
2.5 2
*
*
* *
1.5 1 0.5 0 TNF-α
INF-
PMA
Necrotic bodies
Apoptotic bodies
control *p<0.05
Fig. 1. Levels of ICAM-1 expression were measured by cell-based ELISA 24 h after treatment of confluent HMEC-1 monolayers with TNFa, INFg, PMA, necrotic or apoptotic cell bodies. Levels of ICAM-1 expression were compared to untreated control cultures by t-test. All treatments were quantified in triplicate and the assays were repeated on three separate occasions.
Q. Chen et al. / Placenta 28 (2007) 743e747
g, or necrotic cell bodies prior to adding red fluorescently-labeled Jar cells to the endothelial cell monolayers. Positive control monolayers of endothelial cells were activated with PMA. Examination of the cocultures by confocal microscopy suggested that less endothelial cells were displaced from the monolayers by Jar cells when the endothelial cells were activated than non-activated/resting control monolayers (Fig. 2A). Quantification of this effect demonstrated that the area of endothelial cells displaced by Jar cells was more than halved when the endothelial cells were activated regardless of the activator including necrotic cell bodies ( p < 0.05, Fig. 2B). In contrast, addition of apoptotic cell bodies to the endothelial cell monolayers did not significantly reduce the displacement of endothelial cells by Jar cells (Fig. 2B).
745
4. Discussion Transformation of the spiral arteries including the displacement of vascular endothelial cells by extravillous trophoblasts is an essential prerequisite to normal placentation. We have shown in this study that activated endothelial cells are able to resist displacement by trophoblasts using Jar choriocarcinoma cells as a model of invasive trophoblasts. One of the criticisms of this work could be the use of Jar cells which are malignant and unlike extravillous trophoblasts exhibit aggressive and uncontrolled invasion. While that criticism is to some extent valid this study clearly demonstrates the principle that activated endothelial cells can resist invasion and suggests that the extent of trophoblast invasion of the spiral arteries may not be solely dependent upon the invasive capacity of
A a
b
c
d
e
f
Area of HMEC-1 displaced (µm2)
B
30000 25000 20000 15000 10000 5000
*
*
*
*
INF-
PMA
Necrotic bodies
0 TNF-α
Apoptotic Untreated bodies *p <0.05
Fig. 2. A) Representative confocal photomicrographs showing cell ‘‘islands’’ of red fluorescent-labeled Jar cells which have displaced endothelial cells (Green) from monolayers. Prior to the addition of Jar cells the endothelial cells were treated with a) TNFa, b) INFg, c) PMA, d) Necrotic cell bodies, e) Apoptotic cell bodies or f) Untreated controls. B) The area of Jar cell islands which had displaced endothelial cells from monolayers was quantified by digital image analysis following confocal microscopy. Twelve micrographic fields were examined for each treatment and experiments were repeated three times. (*p < 0.05 t-test).
746
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the invading trophoblasts. Furthermore, if activated endothelial cells can resist displacement by these aggressively invasive trophoblast-derived cells then it would seem intuitively likely that activated endothelial cells might resist displacement by normal trophoblast to an even greater extent. It was interesting to note that activated endothelial cells resisted displacement by Jar cells regardless of their activator. Thus, both TNFa and INFg which are well known physiologic/pathologic activators of endothelial cells had a similar ability to render endothelial cell monolayers resistant to displacement by Jar cells. Both of these cytokines are known to be produced at the materno-fetal interface and production may be increased in pregnancies complicated by preeclampsia, a disease characterized by shallow trophoblast invasion [14e16]. It has previously been shown that elevated levels of TNFa can inhibit trophoblast invasion via effects on trophoblast-derived plasminogen activator inhibitor -1 (PAI-1) suggesting that TNFa may affect trophoblasts directly to reduce their invasive capacity [17]. In a series of elegant experiments Crocker et al. [18] demonstrated that TNFa, at a similar dose to that which we have employed, reduced endovascular trophoblast invasion in vitro and it is possible that TNFa acts both directly on trophoblasts to reduce their invasive potential and also on spiral artery endothelial cells making them resistant to trophoblast invasion. Our data also showed that necrotic trophoblast cell carcasses/debris activated endothelial cells and that the endothelial cells activated in this manner also resisted displacement by trophoblast. This result further confirms that activated endothelial cells resist displacement regardless of the nature of the activator and raises the possibility that increased necrotic cell cellular debris in the spiral arteries could also limit trophoblast invasion. It is widely accepted that one of the hallmark features of preeclampsia is systemic maternal endothelial dysfunction characterized by activation of the endothelium which is thought to be secondary to shallow invasion of the spiral arteries by trophoblast [19]. Our results suggest that endothelial cell activation could contribute not only to the maternal symptoms of preeclampsia seen in later gestation but also to the hallmark shallow invasion of the spiral arteries that is believed to be one of the underlying pathogenic triggers of severe preeclampsia. We do not know the mechanism by which activated endothelial cells resist displacement by Jar cells but activation of endothelial cells is known to induce a wide variety of changes in the endothelial cells including secretion of soluble factors that might have induced apoptosis in the Jar cells. However, our own experience and that of others [20] is that Jar cells are particularly resistant to factors that readily induce apoptosis in other cells and it seems unlikely that the effect we observed was due to increased apoptotic death of the Jar cells. In summary, we have shown that activated endothelial cells resist displacement by trophoblast-derived Jar cells regardless of the mechanism by which the endothelial cells were activated. If this phenomenon also occurs in vivo these results suggest that any blood borne factors that activate endothelial cells, including those we have studied and others such as interleukin-1 [21] or syncytiotrophoblast microparticles [22], could
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