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Female Reproductive Tract |
a INSERM U 361, Université René Descartes, Pavillon Baudelocque, 75014 Paris, France
b Service of Microcinema (Department of Scientific Information and Communications), INSERM, 78110 Le Vésinet, France
c Maternité Port-Royal, Hopital Cochin, AP-HP, Université René Descartes, 75014 Paris, France
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-actin immunostaining. Taken together, these observations show that the uterine smooth muscle cells are contractile and respond appropriately to a potent uterotonic agent. Based on these findings, a cultured uterine smooth muscle cell model, which could be used to elucidate the mechanisms controlling uterine activity, is proposed.
mechanisms of hormone action, parturition, signal transduction, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The primary function of mature uterine smooth muscle is reflected by its inherent ability to contract. In vitro contractile studies have demonstrated that intact human myometrial strips maintain their normal in vivo contractile properties [2]. However, whether cultured human myometrial cells maintain any of these features in vitro is not certain. Previous studies [3] have indicated that uterine smooth muscle cells undergo a transformation from a "contractile" to a "synthetic" phenotype during primary culture. The phenotypic modulation in culture was first reported by Chamley-Campbell et al. [4], who demonstrated that vascular smooth muscle cells (VSMC) lose the ability to contract when subcultured. This process of VSMC phenotypic modulation involves not only a decrease in the expression of contractile and cytoskeletal proteins but also the reorganization of these proteins [5]. However, this whole concept is now being challenged. As early as 1990, it was demonstrated that cultured VSMC do not necessarily undergo phenotypic modulation with a loss of contractility after prolonged culture [6]. Cavaillé et al. [7] have defined culture conditions that allow preservation of the expression of smooth muscle markers of differentiation in cultured myometrial cells. This is an important finding, but it is not sufficient to conclude whether myometrial cells do or do not preserve their contractile properties in culture.
The present study was therefore designed to characterize the contractile response of cultured uterine smooth muscle cells to a potent uterotonic agent, endothelin-1 (ET-1). This 21-amino acid peptide is one of three distinct peptide isoforms known as ET-1, ET-2, and ET-3. The ET-1 stimulates the force and frequency of human myometrial contractions in vitro [8]. Two distinct endothelin receptors—denoted endothelin A (ETA), which is ET-1 selective, and endothelin B (ETB), which is equally sensitive to all three endothelins and to sarafotoxin 6c (S6c)—have been identified in human myometrium, but we and others have demonstrated that only the ETA receptors mediate the contractile effect of ET-1 in this tissue [9, 10]. An increase in the density of myometrial ETA receptors [11, 12] accompanies the increase of uterine contractile responsiveness to ET-1 at the end of pregnancy [13]. Because only ETA receptors functionally coupled to the phosphoinositide-specific phospholipase C/Ca2+ pathway are found in human myometrial cells [14], we hypothesized that the "contractile phenotype" was preserved in our culture conditions. This hypothesis, which has yet to be confirmed, is compatible with the fact that in fresh tissue, the ETA receptors are strongly related to the contractile state of the uterus.
Because myometrial ETA-receptor activation is a good biological indicator of uterine activity, we investigated the contractility of cultured myometrial cells in response to ET-1. For this purpose, we used two in vitro systems developed previously to assess the contractility of cultured VSMC. These experiments yielded direct evidence for the contractility of individual myometrial cells cultured on a silicone rubber substratum [15]. This qualitative method is based on the ability of contractile cells to distort a silicone rubber sheet. To confirm that the changes were representative of the contractile state of these cells, we quantified the ability of ET-1 to modify myometrial cell tension in a conventional collagen gel retraction assay [16]. The demonstration that the contractile responses were maintained in cultured myometrial cells indicates their potential usefulness as a convenient model system for studying the physiology and/or pathophysiology of uterine contractility in humans.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Dulbecco modified Eagle medium (DMEM), trypsin-EDTA, penicillin-streptomycin mixture, PBS with and without calcium, and fetal calf serum (FCS) were supplied by In Vitrogen Life Technologies (Cergy-Pontoise, France). Silicone fluid (Dow Corning) was obtained from BDH Laboratories (Poole, U.K.). Collagen was purchased from Becton Dickinson Biosciences (Bedford, MA). The ET-1, S6c, and ET-receptor antagonists BQ 123 and BQ 788 were obtained from Neosystem Laboratoire (Strasbourg, France). The antibody against smooth muscle -actin was from Novocastra (Newcastle, U.K.), and the fluorescein isothiocyanate (FITC)-labeled conjugated antibody against mouse immunoglobulin (Ig) G was from Southern Biotechnology Associates, Inc. (Birmingham, U.K.). The fluorescent mounting medium was obtained from DAKO (Carpinteria, CA). Other drugs and chemicals used were of the highest quality available from Sigma Chemical Co. (St. Louis, MO).
Culture of Human Myometrial Cells
Myometrial biopsies were collected from 10 women undergoing hysterectomies for benign gynecological indications. Tissue samples were excised from normal muscle in the uterine corpus (myometrial outer layer) in areas free of macroscopically visible anomalies. This study was approved by the (Comité Consultatif de Protection des Personnes Pour la Recherche Biomédicale; Paris-Cochin, France) hospital Ethics Committee.
After collection, the biopsies were placed in DMEM supplemented with 100 U/ml of penicillin and 100 µg/ml of streptomycin. Human myometrial cells were obtained by the explant method as previously described [7]. Cells were cultured in DMEM supplemented with antibiotic solution and 10% (v/v) FCS and routinely passaged when 90–95% of the cells were confluent. The experiments presented in this report were performed with cells between their third and sixth passages, with no noticeable difference between results obtained with cells from individual passages and with cells obtained from different uteri. Each population of myometrial cells studied had been taken from a different patient. Confluent myometrial cells were identified by their typical "hill and valley" microscopic appearance and by their positive reaction to a monoclonal antibody against smooth muscle -actin.
Silicone Assay
The contractile responses of cultured myometrial cells to ET-1 stimulation were evaluated using the method of Harris et al. [15] with slight modifications. A thin layer of silicone fluid 200 (viscosity, 30 000 centistokes) was spread on one surface of a microscope coverslip. This surface was then turned face down and exposed to a low flame from a Bunsen burner so that a film of tiny wrinkles formed on the fluid surface. The silicone took 2 sec to polymerize. The plates were placed in 35-mm dishes, and cells were added to the dishes in DMEM supplemented with 10% FCS and antibiotic solution. Next, they were incubated at 37°C for 6 days. All experiments with ET-1 administration were then performed using coated coverslips in a "Rose chamber" culture [17]. The behavior of the cells was recorded at different times using a Zeiss inverted microscope (ICM 405; Le Pecq, France) equipped with a 16x Ph N.A. 0.40 Neofluar objective and a 16-mm standard Arriflex cine camera (Stouen, France). The microscope was placed in a box maintained at 37°C by a DIP temperature controller device (Multitop; Chauvin Arnoux, Paris, France). Pictures were taken every 30 sec before and after adding the test agents, Paris France. The light was turned off between two successive frames to avoid cell damage. AGFA Copex Rapid AHU film (Paris, France) was used. Myometrial cells isolated from five separate uteri were studied from the fourth to the sixth passage.
Preparation of Three-Dimensional Hydrated Collagen Lattices
Human myometrial cells were included in collagen gels as described by Kelley et al. [16]. Briefly, confluent cells harvested with 0.25% trypsin-0.02% EDTA were centrifuged at 1000 x g for 5 min and resuspended in 10% FCS-DMEM at the required cell density. A type-I collagen solution (4.1 mg/ml in 0.1 N HCl) was adjusted to pH 7.2 with 0.1 N NaOH. The final concentration of collagen was 1.5 mg/ml. The appropriate concentration of myometrial cells (150 000 cells/well) was then added to the neutralized collagen solution. Collagen gel-cell suspensions were incubated in untreated culture dishes (diameter, 35 mm) for 2 h at 37°C to allow gelling, and then 2 ml of fresh DMEM supplemented with 10% FCS was added over the cell-collagen lattice. Three days later, the culture medium was replaced. The lattices were then gently detached from the sides and lifted off the bottom of the well containing 2 ml of serum-free medium and the agents to be tested. When ETA-receptor antagonist (BQ 123) or ETB-receptor antagonist (BQ 788) was used, the myometrial cell preparations were exposed for 30 min to one of these antagonists at the concentration of 100 µM and then to ET-1 (50 nM). The areas of the lattices were measured daily up to 3 days. Maximum attenuation of gel contraction was obtained after 24 h of culture. Cells cultured on plastic dishes or into collagen gels were removed with 1 ml of 0.25% trypsin-0.02% EDTA in PBS. The number of cells liberated was counted in triplicate wells with a hemocytometer, and the viability of cells was checked by trypan blue exclusion. We have verified that the growth rate of myometrial cells into collagen gels was similar to that of cells on plastic dishes (data not shown). Images of the floating gels were captured and digitized using a scanner (Studio Scan IISI; AGFA) before adding the test agents and after incubating for 24 h. The lattice was assimilated to an ellipse, and the area was calculated after measuring the major and minor diameters of the gel. Collagen contraction was expressed as percentage contraction ± SEM of triplicate determinations from 5 to 10 separate experiments, where percentage contraction is the percentage decrease in terms of the original surface area.
Immunocytochemistry
Immunostaining comparisons were made with cells grown on glass coverslips (control) as well as within collagen lattices. Cells were fixed with 4% paraformaldehyde in PBS for 10 min as previously described [18]. The cells were then rendered permeable by incubating for 15 min with 0.1% Triton X-100 in PBS containing 10% FCS and incubated overnight at 4°C with the first monoclonal antibody against smooth-muscle -actin and for 1 h at room temperature with the second FITC antibody against mouse IgG. Coverslips were mounted on slides using fluorescent mounting medium. A Nikon E-600 inverted microscope (Champigny, France) was used for conventional fluorescence microscopy, and photographs were taken using Coolsnap Software (RS Photometrics, Evry, France).
Statistical Analysis
Results are expressed as the mean ± SEM. Groups of data were evaluated by ANOVA. A Bonferroni correction was performed to adjust for multiple comparisons of gel areas. Values of P < 0.05 were considered to be significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To explore whether ET-1 induces contractility in myometrial cells, we used a modified version of the flexible rubber substrate assay of Harris et al. [15]. Myometrial cells were grown on the surface of distortable silicone rubber substrata for 6 days and then cultured for 72 h in serum-free media to make them quiescent. Under these conditions, confluent cells generate tension in the underlying substrate that is revealed by wrinkles in the silicone rubber (Fig. 1A). Stimulating myometrial cells with ET-1 (500 nM) led to a gradual decrease in wrinkling of the silicone rubber underneath the cells, indicating that contractile responses had occurred (Fig. 1B). This effect was detected after 20 min of treatment and peaked after incubating with ET-1 for 1 h.
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Effects of ET-1 on Ability of Myometrial Cells to Contract Floating Collagen Gels
No contraction of the lattices was observed when collagen lattices without myometrial cells were incubated with serum-free DMEM for 24 h (Fig. 2, A and B).
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When myometrial cells were cultured in collagen lattices and incubated under the same conditions, a slight reduction was observed in the area of the floating gel, demonstrating their basal contractile tone (Fig. 2, C and D). When 10 nM ET-1 was added to the medium, a subsequent contraction of the lattice was observed (Fig. 2, E and F). As shown in Figure 3, ET-1 (50 nM) induced a time-dependent decrease in the area of the lattices. After exposing the floating collagen gels to ET-1, the contractile response rose during the first 6 h. Most of the reduction in lattice diameter occurred within the first 24 h after release. Incubating for a further 2 days did not result in more intense contraction. When the floating collagen gels were maintained in serum-free DMEM alone, the area decrease shared the same profile, with minimal values around 24 h. When added to "collagen gel-incorporated cells," ET-1 induced a dose-dependent decrease in the diameter of the area, as shown in Figure 4. The reduction in the area of the lattices was significant at concentrations as low as 1 nM, and maximum stimulation of myometrial cell contraction occurred at 50 nM ET-1.
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Characterization of the ET-Receptor Subtype Mediating Myometrial Cell Contraction
The effects of ET-receptor antagonists were evaluated to identify the subtype of ET-receptor involved in the contractile response mediated by ET-1. The selective ETA-receptor antagonist BQ123 produced a substantial inhibition of the contraction of collagen gels achieved by incubating for 24 h with 50 nM ET-1 (Fig. 5). On the other hand, treatment with 100 µM BQ788, an ETB-receptor antagonist, failed to inhibit the contractile effects induced by ET-1 in myometrial cells on collagen lattices. Similarly, S6c, a selective ETB-receptor agonist, had no significant effect on the original area of the lattice at concentrations up to 100 µM. No ETA- or ETB-receptor antagonist alone had any effect on the size of the gels at concentrations up to 100 µM.
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Effect of Cytochalasin B on Collagen Lattice Contraction
Cytochalasin B, an intracellular microfilament-disrupting agent, was used to evaluate the role of actin filaments in the contraction of collagen lattices by myometrial cells. A dose-response curve of myometrial cell contraction in the presence of 0.001–1 µg/ml of cytochalasin B is shown in Figure 6. Incubation with low concentrations of cytochalasin B (0.001 and 0.01 µg/ml) resulted in a decrease of the basal myometrial cell contraction (7%–5%) compared to the control (20%), whereas higher concentrations (0.1 and 1 µg/ml) had a slight dilatory effect.
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Morphology and Immunocytochemistry
When cultured in hydrated collagen gels for 3 days, myometrial cells entrapped in the collagen meshwork presented an elongated morphology similar to that of myometrial cells grown on glass coverslips. The expression of the smooth muscle phenotypic marker (-actin) was consistently present in myometrial cells grown within collagen lattices (Fig. 7).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To obtain direct evidence for myometrial cell contraction, we used two in vitro systems developed previously to assess the contractility of VSMC. The use of silicone rubber substrata is a common method first described by Harris et al. [15] for assessing changes in cell tension and for detecting small changes in the shear forces exerted by smooth muscle cells. The presence of wrinkling on the silicone rubber surface suggests that the unstimulated cells exerted some basal tension. When observed by phase-contrast microscopy, myometrial cells respond to exposure to ET-1 by producing major distortions in the silicone surface, as revealed by reduced wrinkling of the substrate. This phenomenon is apparent from the first hour. Nevertheless, our findings conflict with those of earlier reports, in which contraction of subcultured VSMC in response to contractile agonists led to increased wrinkling [6, 15, 16, 19]. The reason for distortion differences between the two cellular models (i.e., decreased vs. increased wrinkling) is not clear and remains to be investigated. When contraction occurs, some of the cell attachment sites that need to be broken in myometrial cells cultured on artificial elastic substrates could be quite different from those of VSMC. Little is known about integrins, the transmembrane adhesion and signaling receptors that physically link the extracellular matrix to the cytoskeleton in the human myometrium. However, only one report demonstrated that the smooth muscle cells of human myometrium exhibit a pattern of cell adhesion molecules (integrins and cadherins) differing from the thick-walled vessels of myometrium [20].
Because this qualitative analysis on a silicone rubber sheet does not readily lend itself to measuring the amount of force generated by the stimulated cells, we developed a quantitative method for measuring the contraction of myometrial cells cultured within a collagen lattice. This technique can be used to measure the response of a whole population of myometrial cells rather than that of individual cells as measured by the silicone rubber method. When myometrial cells are cultured in a three-dimensional collagen gel and detached from the underlying surface, the myometrial cells contract the gels over 24 h in serum-free media. The ET-1 has been shown to affect the myometrial cell-mediated contraction of collagen gels. Interestingly, the concentration of ET-1 (50 nM) required to induce the maximum reduction in collagen gel size was similar to that which produced a maximum contraction in a human uterine bath experiment [9]. The lack of contractile effects of the ETB-selective agonist, S6c, further confirms that myometrial contraction was not mediated by the ETB-receptor subtype. Additional evidence for this was provided by the fact that adding a selective ETA-receptor antagonist, BQ 123, significantly inhibited the contraction of free-floating cell-collagen lattices induced by ET-1, whereas BQ 788, a selective ETB antagonist, was ineffective. This agrees well with numerous observations linking ETA receptors to uterine contractions [9, 10, 12].
Our culture system preserves the myometrial cell response to ET-1 in terms of the contractility and receptors involved in this effect. These findings imply that cultured myometrial cells retain the normal physiological properties that occur in vivo in fully differentiated smooth muscle cells in a normal environment. One reason for our success in observing contractile responses may lie in the tissue source itself. In adult women, myometrium is a tissue primarily composed of finished, differentiated smooth muscle cells. A second possible reason lies in the stimuli used to elicit contraction. We showed previously that from the 3rd to the 10th passage, the response to ET-1 in terms of inducing inositol phosphate accumulation and subsequent Ca2+ mobilization were equivalent regardless of the culture passage [14]. However, contractile responses to some other agents may have been lost during prolonged subculturing. This was definitely not the case for oxytocin (OT), a well-known contractile agonist of uterine smooth muscle, because functional OT receptors are maintained over several passages in cultured human myometrial cells [21–23]. In addition, Carrasco et al. [24] demonstrated that subcultured human myometrial cells retained their physiological properties and, particularly, their sensitivity to prostaglandin F2 (PGF2), another potent uterine stimulant. Any increase of the inositol-1,4,5-phosphate/Ca2+ pathway, reflecting ETA, OT, and PGF2 receptor activation, would lead to enhancement of the contractile activity of the uterus, which is of crucial importance at the end of pregnancy.
Intact actin filaments, or stress fibers, appear to be part of the mechanism of human myometrial cell contraction [25], and when myometrial cells were incorporated into collagen lattices, cytochalasin B, at a concentration known to destroy actin filaments, inhibited basal contraction. Using collagen type I as a substrate for the culture of many cell types may improve differentiation, so we checked whether the expression of smooth muscle -actin was modified by this culture system. When myometrial cells are cultured within three-dimensional collagen gels, they exhibit similar staining patterns at a comparable passage level to those of cells cultured on a classical solid support, such as glass coverslips. Similarly, Ehrlich et al. [26] have shown that the phenotype of VSMC grown on collagen type I gel is quite similar to that of cells grown on plastic alone and that the cells are extremely elongated. Profound modification of expression of smooth muscle cell markers of differentiation has been described in our present model of cultured human uterine smooth muscle cells [7]. Indeed, it was shown by Western blot analysis that the expression of smooth muscle -actin and desmin was down-regulated by subculturing but enhanced by suppression of serum in the culture medium after the cells had reached confluence [7]. Similarly, in spite of a very low level of SM1 and SM2 myosin heavy chain (MHC) in cultured myometrial cells, MHC was detected in confluent cells until the 12th passage, when they were deprived of serum during culture. However, this apparent loss of phenotypic markers did not appear to influence in vitro myometrial cell contractility. These findings are in line with those of Kropp et al. [27], who showed that a low pattern of expression of -actin is not necessarily correlated with an absence of contraction of bladder smooth muscle cells, but we cannot exclude that smooth muscle cell phenotypic modulation involves not only quantitative changes in contractile and cytoskeletal proteins but also a reorganization of the cytoskeletal network [5]. We have no data about the possible reorganization of the cytoskeleton, which sometimes acts as a spatial regulator of signaling molecules for mediating the changes in function associated with human uterine smooth muscle cell phenotypic modulation.
In summary, we found that passaged cells were representative of the tissue of origin—that is, that human myometrial cells exhibited the smooth muscle cell phenotype and responded appropriately to the potent uterotonic agent, ET-1, via selective activation of the ETA receptor. We propose that the "contractile phenotype" was preserved under our culture conditions, because in fresh tissue, ETA receptors are closely related to the contractile state of the uterus. Therefore, the culture of human myometrial cells provides a convenient myometrial tissue model for identifying potential regulatory pathways and for studying the action of a variety of signaling factors that modulate both normal and pathological uterine activity.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received: 13 June 2002.
First decision: 5 July 2002.
Accepted: 24 September 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. J Clin Endocrinol Metab 1992 75:1027-1032[Abstract] is required for endothelin-1-induced proliferation of human myometrial cells. Biol Reprod 2002 66:44-49
tal lung (IMR-90) cells: myofibroblasts with smooth muscle-like contractile properties. Cell Motil Cytoskelet 1996 34:288-298[CrossRef][Medline]This article has been cited by other articles:
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  I. Garcia-Verdugo, Z. Tanfin, E. Dallot, M.-J. Leroy, and M. Breuiller-Fouche Surfactant Protein A Signaling Pathways in Human Uterine Smooth Muscle Cells Biol Reprod, August 1, 2008; 79(2): 348 - 355. [Abstract] [Full Text] [PDF]
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 M. O'Brien, J. J. Morrison, and T. J. Smith Upregulation of PSCDBP, TLR2, TWIST1, FLJ35382, EDNRB, and RGS12 Gene Expression in Human Myometrium at Labor Reproductive Sciences, April 1, 2008; 15(4): 382 - 393. [Abstract] [PDF]
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 M. Breuiller-Fouche, C. Moriniere, E. Dallot, S. Oger, R. Rebourcet, D. Cabrol, and M.-J. Leroy Regulation of the Endothelin/Endothelin Receptor System by Interleukin-1{beta} in Human Myometrial Cells Endocrinology, November 1, 2005; 146(11): 4878 - 4886. [Abstract] [Full Text] [PDF]
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 G. D. Liberto, E. Dallot, I. E.-L. Parco, D. Cabrol, F. Ferre, and M. Breuiller-Fouche A critical role for PKC{zeta} in endothelin-1-induced uterine contractions at the end of pregnancy Am J Physiol Cell Physiol, September 1, 2003; 285(3): C599 - C607. [Abstract] [Full Text] [PDF]
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