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BOR - Papers in Press, published online ahead of print March 2, 2005.
Biol Reprod 2005, 10.1095/biolreprod.104.036509
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BIOLOGY OF REPRODUCTION 73, 14–19 (2005)
DOI: 10.1095/biolreprod.104.036509
© 2005 by the Society for the Study of Reproduction, Inc.

Trophoblast Migration Under Flow Is Regulated by Endothelial Cells1

Arlen Soghomonians 3, Abdul I. Barakat 3, Twanda L. Thirkill 4, and Gordon C. Douglas 2 4

Department of Mechanical and Aeronautical Engineering3 Department of Cell Biology and Human Anatomy,4 School of Medicine, University of California, Davis, Davis, California 95616-8643


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During pregnancy, trophoblasts enter the uterine vasculature and are found in spiral arteries far upstream of uterine capillaries. It is unknown whether trophoblasts reach the spiral arteries by migration within blood vessels against blood flow or by intravasation directly into spiral arteries after interstitial migration. We have developed an in vitro system consisting of early gestation macaque monkey trophoblasts cocultured with uterine endothelial cells and have exposed the cells in a parallel plate flow chamber to physiological levels of shear stress. Videomicroscopy followed by quantitative image analysis revealed that the migratory activity (expressed as average displacement and average migration velocity) of trophoblasts cultured on top of endothelial cells remained unchanged between shear stresses of 1–30 dyne/cm2 whereas activity of trophoblasts alone increased with increasing shear stress. When the direction of migration was assessed at 1 and 7.5 dyne/cm2, the extent of migration against and with flow was roughly equal for both trophoblasts alone and cocultured trophoblasts. At shear stress levels of 15 and 30 dyne/cm2, trophoblasts incubated alone showed a significant decrease in migration against flow and corresponding increased migration in the direction of flow. In contrast, trophoblasts cocultured with uterine endothelial cells maintained the same extent of migration against flow at all shear stress levels. Migration against flow was also maintained when trophoblasts were cultured with endothelial cell-conditioned medium or fixed endothelial cells. The results indicate that factors expressed on the surface of uterine endothelial cells and factors released by endothelial regulate trophoblast migration under flow.

placenta, pregnancy, trophoblast, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Early in pregnancy in the human and in the macaque monkey, a specialized subset(s) of trophoblast cells breach the uterine epithelium and enter superficial venule-like, nonarteriolar vessels [1–3]. Eventually, trophoblasts become locally established within the lumens of maternal spiral arteries. This invasion of the uterine vasculature results in continuity between the maternal blood supply and the trophoblast-lined spaces that form the placenta [3, 4].

Intravascular trophoblasts have certain unusual features. First, they must be able to adhere to endothelium with sufficient strength to resist displacement by blood flow. Second, some trophoblasts are thought to migrate extensively within small endometrial vessels against the direction of blood flow and eventually reside within the spiral arteries of the uterus [1, 3, 5–8]. Over time, some endovascular trophoblasts extravasate by migrating between and displacing arterial endothelial cells [3, 9, 10]. Ultimately, trophoblast cells form the luminal surface of these invaded maternal vessels [8–11]. Other trophoblasts, apparently unable to attach with sufficient strength to the endothelium, are carried by the blood and become lodged in microvessels within maternal lung tissue. It can be speculated that these different behavior characteristics reflect distinct intravascular trophoblast phenotypes.

While migration within vessels against blood flow could account for the presence of intravascular trophoblasts within the spiral arteries, another, and not necessarily mutually exclusive, possibility is that trophoblasts migrate extensively within the uterine stroma and then enter the spiral arteries by intravasation. This may be the case in the human but is less likely in the macaque, where interstitial trophoblasts are seen less frequently [12]. In both species, trophoblasts enter superficial venule-like, nonarteriolar vessels [1–3].

Although trophoblast invasion of the vasculature and adhesion to vascular endothelium appear to be accompanied by changes in the expression of proteases, cadherins, and various integrins[13–16], the factors that might mediate trophoblast migration against the direction of fluid flow have not been identified. Trophoblast migration on extracellular matrix in vitro can be regulated by EGF, TNF, and TGF-ß [17–20].

Recently, we demonstrated that macaque trophoblast migration is also regulated by fluid mechanical shear stress [21]. Migration increased with the magnitude of applied shear stress. However, unlike the in vivo situation, flow-induced migration of trophoblasts alone occurs in the direction of the applied flow. An obvious component that was missing from our previous experiments was endothelium. We have now tested the hypothesis that the presence of endothelium plays an essential role in regulating the character and directionality of trophoblast migration in response to flow.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Trophoblast Isolation and Culture

All procedures involving animals were performed in accordance with the NIH Guide for the Care and Use Laboratory Animals and under the approval of the University of California, Davis, Animal Care and Use Committee. For most experiments, trophoblast cells were isolated from 40–65-day macaque placentas using procedures we have previously detailed [22]. Limited experiments were also performed on term human placental trophoblast cells (HuPla), which were isolated as we have described previously [23]. Yields in all cases were approximately 3 x 106 cells/g tissue. When the cells were cultured for 24 h on LabTek slides and then stained, at least 98% were positive for cytokeratin and negative for HLA-ABC/DR, factor VIII, and vimentin, consistent with trophoblast. The remaining cells were vimentin positive and HLA positive. The cells were subjected to an additional purification step using immunomagnetic microspheres coated with anti-HLA antibodies [23]. As we reported for human trophoblast cells [23], this step removes contaminating HLA-positive cells leaving pure (cytokeratin-positive, HLA-ABC/DR-negative, vimentin-negative) trophoblast cells. FACS analysis of this purified trophoblast population revealed that 75% of the cells were ß1 integrin positive [21], consistent with a predominantly extravillous trophoblast phenotype.

Endothelial Cell Culture

Human uterine microvascular endothelial cells (UtMVEC, passage 3) were purchased from Clonetics Corporation (San Diego, CA) and maintained in endothelial basal medium-2 (Clonetics) supplemented with human recombinant epidermal growth factor, human fibroblast growth factor, vascular endothelial growth factor, ascorbic acid (vitamin C), hydrocortisone, human recombinant insulin-like growth factor, heparin, gentamicin, amphotercin, and 5% fetal bovine serum. Cells in passages 4–7 were used in the experiments. Bovine aortic endothelial cells (BAEC, passage 2) were purchased from Cell Systems, Inc. (Kirkland, WA), and cultured in Dulbecco Modified Eagle Medium (DMEM)-Ham F-12 (50:50 v/v) (Gibco) supplemented with 10% heat-inactivated calf serum and 1% gentamycin. Cells in passages 5–6 were used in the experiments.

Coculture and Flow Experiments

In the macaque trophoblast-UtMVEC experiments, UtMVEC were cultured to confluence on tissue culture plastic slides (Permonox, Fisher Scientific) and were subsequently exposed in a standard parallel plate flow chamber to a flow-preconditioning period of 12 h at a shear stress of 15 dyne/cm2 using protocols that we have previously described [24, 25]. Briefly, cell culture medium (EBM-2 + 10% FCS), gently gassed with CO2, was drawn from a reservoir using a peristaltic flow pump (Cole-Parmer Instruments, Chicago, IL) and passed through two smaller buffer reservoirs inserted between the pump and the flow chamber to dampen pulsatility. All reservoirs were maintained at 37°C by placing them in a temperature-controlled water bath. Flow was recirculated back into the feed reservoir. Following flow preconditioning, flow within the parallel plate flow chamber was halted, and 2.5-3 x 106 trophoblast cells were gently added and allowed to attach to the endothelial cells for 2–3 h at 37°C and 5% CO2. The cocultures were then subjected in the flow chamber to shear stress (1–30 dyne/cm2) for a period of 12 h. Under static conditions, trophoblast viability as determined by Trypan blue staining was 84%. After exposure to shear stress of 15 dyne/cm2 for 24 h, trophoblast viability was 90%.

For some experiments, flow-preconditioned endothelial monolayers were lightly fixed by immersion in 0.05% glutaraldehyde at 4°C for 1 min. The fixed cells were then extensively washed in PBS and incubated in 1% BSA and 1% glycine to block any remaining free aldehyde groups [26]. After further washes with PBS, trophoblasts were then added as described previously. The possibility that trophoblast viability was compromised by residual endothelial-associated aldehydes was negated by the observation (not shown) that trophoblasts responded to the fixed endothelial cells by showing an increase in ß1 integrin expression, similar to the response seen when trophoblasts are added to viable endothelial cells [27].

In addition to the macaque trophoblast-UtMVEC cocultures, control experiments were performed with cocultures of macaque trophoblast-BAEC as well as human term trophoblast-UtMVEC. Furthermore, some studies of the impact of endothelial-conditioned medium on trophoblast migration were conducted. In these studies, two slides of UtMVEC were placed within the parallel plate flow chamber upstream of a slide of trophoblasts, and trophoblast migration under flow was studied. Endothelial cells in the trophoblast-BAEC, human trophoblast-UtMVEC, and UtMVEC-conditioned medium experiments were exposed to a flow preconditioning period as described previously.

Analysis of Trophoblast Cell Migration

The flow chamber was placed on the stage of an inverted phase-contrast microscope (Nikon TE300, Tokyo, Japan) equipped with a digital CCD camera (QImaging, Retiga 1300, Canada) and interfaced with a computer and software for image acquisition. Images of trophoblasts in coculture with endothelial cells were captured every 15 min for 12 h. Similar recordings were performed on no-flow control cells. Following the 12-h recording period, individual trophoblast cell contours were traced, and the data were quantitatively analyzed using image analysis software (Scion Image version beta 3, http://www.scioncorp.com). For each cell, the image analysis involved determination at the end of each hour during the 12-h recording period of cell area, perimeter, major and minor axes of a best-fit ellipse to the cell contour, and x- and y-coordinates of the center of gravity (cog). Trophoblast average migration velocity was determined as the total distance traversed by the cell (based on changes in the x- and y-coordinates of the cog) during the recording period divided by 12. Absolute cell displacement in the flow coordinate direction (x-direction) was determined as the absolute value of the difference between the x-coordinates of the cell cog at the 0- and 12-h time points. To assess the directionality of trophoblast migration, the fraction of the 12-h total monitoring period during which cells moved in the direction opposite that of flow was determined and denoted as the "counterflow migration index." By definition, the value of this index falls between zero and unity.

Statistical Analysis

Experiments were repeated at least three times using trophoblasts from different placentas in each case. Cells from different placentas were not pooled. Statistical analysis was performed by ANOVA followed by Tukey-Kramer multiple comparison and linear trend posttests using the Prism software program (GraphPad Inc., San Diego, CA). Data are means ± SEM. Differences in means were considered significant if P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Macaque Trophoblast-Endothelial Cell Cocultures

We have previously described in detail the kinetics of trophoblast attachment to UtMVEC and the morphological characteristics of the cocultures under static (no-flow) conditions [28]. Trophoblasts are readily distinguished from underlying endothelial cells based on their smaller size and shape and by the manner in which they refract light. To validate the visual method of selecting trophoblasts under these coculture conditions, trophoblasts were live-labeled with the fluorescent tracer calcein-AM and then added to confluent UtMVEC cultures. Figure 1A is a phase-contrast image showing many small, refractile cells (an example is labeled T in Fig. 1A) sitting on top of the flattened endothelial cell monolayer (EC). Figure 1B shows the same field viewed under fluorescence, and it can be seen that the fluorescently labeled trophoblasts colocalize with the rounded refractile cells visualized under phase-contrast conditions. For routine experiments, trophoblasts were therefore identified by their rounded and refractile character under phase contrast. Small colonies of trophoblasts were often observed; however, quantitation of cell migration was based on data derived from single cells.



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  		FIG. 1. Trophoblast-UtMVEC coculture. A) Phase-contrast image showing small, refractile trophoblast cells on top of flattened UtMVEC. B) Fluorescence image of the same field showing calcein-AM labeled trophoblast cells. Bar = 20 µm. T, trophoblast; EC, Endothelial cell

Trophoblast Migration on Endothelial Cell Monolayers

We wanted to determine whether attachment of trophoblasts to endothelial cells affected trophoblast migration and, particularly, the trophoblast migratory response to shear stress. Trophoblasts were added to confluent UtMVEC cultures, and trophoblast migratory behavior was evaluated under a range of shear stresses by videomicroscopy and subsequent image analysis of individual cells. Migratory behavior was expressed as either average cell migration velocity or absolute cell displacement in the flow coordinate direction (see Materials and Methods). Figure 2A shows the average migration velocity of trophoblasts cultured in the presence or absence of UtMVEC at different levels of shear stress. As we previously reported [21], the migratory activity of trophoblasts cultured alone increased in response to increasing levels of shear stress. This response was not seen in trophoblasts cultured on top of UtMVEC, where velocity remained essentially the same at all levels of shear stress. A similar pattern was seen when trophoblast displacement was considered (Fig. 2B). In addition to displacement in the flow coordinate direction, migration for both trophoblasts cultured alone and cocultured with UtMVEC also exhibited a significant component orthogonal to flow. For trophoblasts alone, the ratio of displacement normal to flow to that parallel to flow ranged from ~0.2 to 1. The equivalent range for trophoblasts cocultured with UtMVEC was somewhat narrower (~0.6– 0.8).



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  		FIG. 2. Effect of shear stress on trophoblast cell migration for cells cultured alone or cocultured with UtMVEC. A) Cell average migration velocity. B) Cell absolute displacement in the flow coordinate direction. C) Counterflow migration index (see Materials and Methods for definition). Data for trophoblast cells cultured alone are partially derived from [21]. Data are means ± SEM from three to six experiments. There was a significant (P < 0.05) linear trend in the response of trophoblasts alone to shear stress for all parameters (A, B, and C) measured. All values for trophoblasts alone at 15 and 30 dyne/cm2 were significantly different from cocultured trophoblasts under the same shear stress conditions. There was no significant difference in any of the measured parameters between cocultured trophoblasts incubated at different levels of shear stress

We next considered the directional migration of trophoblasts. From Figure 2C, it can be seen that at 1 and 7.5 dyne/cm2, both trophoblasts alone and cocultured trophoblasts showed similar directional migration characteristics with a counterflow migration index (as defined in Materials and Methods) of approximately 0.5. Interestingly, at higher and more physiological shear stress levels (15 and 30 dyne/ cm2), the extent of migration against flow decreased significantly for trophoblasts alone, whereas migration against flow remained virtually unchanged (counterflow migration index of ~0.5) for cocultured trophoblasts. At these higher shear stress levels, migration against flow was almost twice as great for trophoblasts cultured with endothelial cells than for trophoblasts cultured alone.

Trophoblast Migration Against Flow Is Retained at Physiological Shear Stress Levels by Contact with Uterine Endothelial Cells or Endothelial Cell-Conditioned Medium

The results described previously suggest that trophoblast migration against flow is maintained at physiological shear stresses when trophoblasts are cocultured with UtMVEC. To determine whether this retention of directional migratory activity was the result of direct contact with endothelial cells or the response to a soluble component released by endothelial cells, trophoblasts were incubated under shear stress conditions (15 dyne/cm2) with UtMVEC-conditioned medium. The data in Figure 3, A and B, show that trophoblast absolute x-direction displacement in the presence of conditioned medium was significantly reduced compared to control trophoblasts alone, although the reduction was not quite to the same extent as seen with cocultured trophoblasts. The average migration velocity of trophoblasts cultured in the presence of conditioned medium was lower than that of trophoblasts alone, but the difference was not significant. However, when directionality was considered (Fig. 3C), trophoblasts incubated with conditioned medium retained, as did trophoblasts cocultured with UtMVEC, significant migration against flow. When trophoblasts were cocultured with UtMVECs that had been lightly fixed in glutaraldehyde to render them metabolically inactive, overall migratory behavior was significantly reduced compared to trophoblasts incubated with conditioned medium or with viable UtMVEC (Fig. 3, A and B); however, the extent of migration against flow was similar to trophoblasts cocultured with UtMVECs and significantly greater than trophoblasts alone. Other experiments depicted in Figure 3 show that when macaque trophoblasts were cocultured with BAEC, the extent of migration against flow was not significantly different from that of trophoblasts alone. Also, the extent of human term trophoblast migration against flow when cocultured with UtMVEC was similar to trophoblasts alone. These results show that retention of counterflow migration under shear stress conditions is specific to early gestation macaque trophoblasts cocultured with UtMVE-C or with UtMVEC-conditioned medium.



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  		FIG. 3. Effect of different culture conditions on trophoblast migration at a shear stress of 15 dyne/cm2. A) Cell average migration velocity. B) Cell absolute displacement in the flow coordinate direction. C) Counterflow migration index. Values are means ± SEM for three to six experiments. In A, the value for trophoblast alone was significantly different (P < 0.005) from all other treatment groups with the exception of trophoblasts + conditioned medium. In B, the value for trophoblasts alone was significantly different (P < 0.001) from all other treatment groups. In C, values for trophoblast + UtMVEC, trophoblast + conditioned medium, and trophoblast + fixed UtMVEC were significantly different (P < 0.05) from trophoblast alone


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have previously shown that macaque and human trophoblasts adhere to endothelial cells under static (no-flow) conditions and have characterized some of the adhesion molecules involved [28, 29]. We also showed that, under nonflow conditions and in the absence of endothelial cells, macaque trophoblasts were highly dynamic with continuous nondirectional shifting in their centers of gravity and no net migration [21]. Application of steady shear stress increased cellular average migration velocity and cell displacement. In the present paper, we have described an in vitro coculture system that allows the migration of trophoblast cells on the apical surface of endothelial cells to be studied under shear stress conditions.

Compared to trophoblasts alone, the overall migratory activity of early gestation trophoblasts was significantly reduced in the presence of endothelial cells, most likely because of greater adherence under coculture conditions compared to adherence to collagen-coated culture dishes. Other significant differences were observed when the direction of migration was considered. At lower levels of shear stress, trophoblasts alone and cocultured trophoblasts spent nearly as much time migrating in the direction of flow as they did against flow. At the more physiological shear stress levels of 15 and 30 dyne/cm2, trophoblasts incubated alone showed a significant decrease in migration against flow and a corresponding increased migration in the direction of flow. In contrast, trophoblasts that were cocultured with uterine endothelial cells maintained the same extent of migration against flow at all shear stress levels. Thus, the ability of trophoblasts to migrate against flow at physiological levels of shear stress is dependent on the presence of endothelial cells. These in vitro results support the idea that retrograde migration of endovascular trophoblasts occurs within the uterine vasculature in the macaque. The possibility that trophoblasts reach the spiral arteries after extended migration within uterine stroma is not discounted by these findings but may be less likely, particularly in the macaque, where few interstitial trophoblasts are observed [12].

The mechanism by which trophoblasts are able to maintain directional migration on endothelium under shear stress conditions is clearly of importance. The results presented here indicate that factors expressed on the surface of uterine endothelial cells as well as factors released by endothelial cells play a role in regulating trophoblast migration against flow. The corollary is that endothelium-derived factors also function to prevent excessive "loss" of trophoblasts in the direction of flow (i.e., away from the spiral arteries). Migration of early gestation macaque trophoblasts against flow was specific since migration against flow was not maintained (i.e., it was similar to trophoblasts alone) when human term villous trophoblast cells were cocultured with UtMVEC or when macaque trophoblasts were cultured with bovine aortic endothelial cells. Directional migration likely depends on direct shear stress-mediated effects on both trophoblast cells and endothelium. Unfortunately, very little is known about the response of trophoblasts to shear stress. Trophoblast migration in the absence of endothelium and trophoblast attachment to endothelium involves trophoblast ß1 integrin [28]. We have previously shown that macaque trophoblasts incubated in the absence of endothelium respond to shear stress by increasing their expression of ß1 integrin [21]. Also, we have recently shown that trophoblast ß1 integrin expression is increased by contact with endothelial cells and by contact with PECAM-1 and {alpha}vß3 integrin [27] independently of shear stress. These trophoblast responses may be important in enhancing their attachment to endothelium and ensuring that cells are able to withstand the shear stresses encountered in vivo. Further studies will be required to better characterize the trophoblast response to shear stress and to identify potential molecules that could be involved in regulating directional migration.

Large-vessel endothelial cells exhibit a variety of responses to shear stress, and these have been well documented [30–37]. In addition to quantitative up-regulation or down-regulation of specific endothelial cell surface molecules, trophoblast migratory behavior could also be modulated by changes in the surface distribution of endothelial cell adhesion molecules or chemokines. Identification of the endothelial component(s) responsible for the change in trophoblast migratory behavior will require further studies and further refinement of the present in vitro system.

The trophoblast response to endothelial cells and/or shear stress was not uniform. Some cells continued to move in the direction of flow or showed little migratory activity. These trophoblast populations could represent either trophoblasts that are not yet committed to differentiate and/or trophoblasts that have already differentiated into a nonmigratory phenotype and are no longer responsive. Indeed, our cell isolation procedure most likely yields a mixed population of trophoblasts at different stages of differentiation. Approximately 75% of the isolated trophoblasts express ß1 integrin, characteristic of migratory extravillous trophoblast [21], and 25% express ß4 integrin, characteristic of villous trophoblast. We previously reported that villous trophoblasts isolated from late gestation macaque placentas (which do not express ß1 integrin) show little or no migratory activity [21].

The phenomenon of retrograde migration of trophoblast cells against blood flow is unusual. While metastasizing tumor cells also attach to and migrate on endothelium [38], the extent to which this occurs against flow is not known. The migration of trophoblasts against flow serves to move the cells to regions of progressively higher oxygen concentrations. This raises the question whether trophoblast migration against flow might be additionally regulated by oxygen, a possibility that could be tested using this system.


   ACKNOWLEDGMENTS
 
Tissue was made available through the cooperation of the veterinary and animal care staff at the California National Primate Research Center, University of California, Davis.


   FOOTNOTES
 
1 Supported by grants from the National Institutes of Health (NIHR01HL068035-01A1) and Philip Morris USA. Back

2 Correspondence: G.C. Douglas, Department of Cell Biology and Human Anatomy, School of Medicine, Tupper Hall, One Shields Ave., University of California, Davis, CA 95616-8643. FAX: 530 752 8520; gcdouglas{at}ucdavis.edu Back

Received: 21 September 2004.

First decision: 14 October 2004.

Accepted: 23 February 2005.


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TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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