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Placental Trophoblast-Derived Factors Diminish Endothelial Barrier Function

Departments of Obstetrics and Gynecology (Y.W., D.F.L., Y.G., Y.Z.), and Molecular and Cellular Physiology (J.S.A., D.N.G.), Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130

Address all correspondence and requests for reprints to: Yuping Wang, M.D., Ph.D., Department of Obstetrics and Gynecology, Louisiana State University Health Sciences Center, P.O. Box 33932, Shreveport, Louisiana 71130. E-mail: ywang1{at}lsuhsc.edu.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Although increased vascular permeability is an important event in the pathogenesis of preeclampsia, the origin of the circulating factor(s) that elicits this endothelial barrier dysfunction is not known. In this study, we use coculture of endothelial cells and placental trophoblast cells to determine whether placental trophoblasts are a potential source of the factor(s) that mediate the increased vascular permeability of preeclampsia. Human umbilical vein endothelial cells grown in Transwell inserts or on coverslips were cocultured with trophoblast cells isolated from normal and preeclamptic placentas or placenta conditioned media. Endothelial cell barrier function was determined by: 1) measurements of electrical resistance and leakage of horseradish peroxidase, and 2) immunofluorescent staining of vascular endothelial-cadherin, pan-cadherin, and occludin. Uterine myometrium endothelial cells were also studied for comparison. We observed the following: 1) electrical resistance was significantly (P < 0.01) decreased (compared with control endothelial cells) in endothelial cell monolayers cocultured with normal trophoblast cells and further reduced in endothelial cells cocultured with preeclamptic trophoblast cells; 2) an increased horseradish peroxidase leakage that was correlated with the decreased electrical resistance in cocultured cells; and 3) disorganized tight junction proteins and an altered distribution of vascular endothelial-cadherin and occludin in monolayers of endothelial cells cocultured with preeclamptic trophoblast cells. Similar responses were noted in uterine myometrium endothelial cells. We conclude that: 1) placental trophoblast cells produce factors that diminish the barrier function of endothelial cells; 2) endothelial tight junctions are more susceptible to factors released from preeclamptic trophoblast cells than from normal trophoblast cells; and 3) these results implicate trophoblast-derived factors in the increased vascular permeability associated with preeclampsia.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
INCREASED VASCULAR PERMEABILITY and enhanced vasoconstriction are major underlying pathophysiological events in preeclampsia, a multisystem disorder with systemic endothelial dysfunction (1, 2) during human pregnancy. Clinically, proteinuria and interstitial edema are manifestations of the diminished endothelial barrier function that occur in the maternal circulation during preeclampsia. Electron microscopic studies of myometrial vessels and sc arteries in women with preeclampsia (3, 4) have revealed disrupted and enlarged intercellular junctions between endothelial cells. These structural changes in the endothelial cell lining are consistent with in vivo studies that demonstrate a higher rate of disappearance of intravascular albumin-bound Evans blue dye in preeclamptic women compared with women with normal pregnancies (5, 6). Furthermore, we have recently reported that even in the earliest passage of cultured endothelial cells derived from women with preeclampsia there is an increased endothelial monolayer permeability that is associated with alterations in the junctional adhesion molecule vascular endothelial (VE)-cadherin and tight junctional protein occludin (7). Interestingly, these functional and morphological changes that are observed in endothelial cells derived from preeclamptic women disappear when cells are continually cultured in vitro (7), which suggests that the extracellular environment in preeclampsia contains factors that diminish endothelial barrier function in a reversible manner.

Although the cause of the altered endothelial barrier integrity and function in preeclampsia is not known, there is evidence that implicates abnormal placental trophoblast function, i.e. increased trophoblast deportation into maternal circulation (8, 9) and soluble factors released from placental trophoblasts, as a major causative agent. Placental trophoblast-derived factors may alter endothelial barrier function via direct receptor activation and/or indirectly via activating circulating cells such as leukocytes and platelets. We previously demonstrated that factors released from the placenta can promote neutrophil-endothelial cell adhesion (10), increase the expression of CD11b on neutrophils (11), and proteinase-activating receptor on endothelial cells (12). We and others have also studied the interaction of placental trophoblast cells and vascular endothelial cells. Using our unique cell coculture model, coculture of endothelial cells with placental trophoblasts from normal and preeclamptic pregnancies, we found a significant increase in adhesion molecule P-selectin and E-selectin expression in endothelial cells cocultured with trophoblast cells derived from preeclamptic placentas (13). Gallery et al. (14) and Campbell et al. (15) found that coculture of decidual endothelial cells with trophoblasts decreased trophoblast secretion of matrix metalloproteinase (MMP)-9 and reduced cytotrophoblast migration through collagen-coated cell culture insert. These findings demonstrate that components produced by placental tissue or trophoblast cells exert vascular effects on endothelial cells or vice versa. von Dadelszen et al. (16) also reported that supernatants from cocultured endothelial cells and syncytiotrophoblast microvillous membranes activate peripheral blood leukocytes in vitro. Taken together, these findings indicate the importance of cross-talk between placental cells and maternal cells in situ at the maternal-fetal interface and distal effects in the maternal systemic circulation.

In the present study, using our unique cell coculture model (coculture of endothelial cells with placental trophoblasts derived from normal and preeclamptic pregnancies), we determined whether trophoblast-derived factors can elicit an increase in endothelial monolayer permeability and induce the changes in endothelial junction integrity and junctional protein distribution previously described in preeclampsia (7). Both human umbilical vein endothelial cells (HUVECs) and uterine myometrium endothelial cells (UtMECs) were used in this study. We found that barrier function is diminished when endothelial cells are cocultured with trophoblasts or placental conditioned medium from either normal or preeclamptic placentas. However, the altered barrier function was more dramatic when endothelial cells were cocultured with trophoblasts or conditioned medium from preeclamptic placentas. These results support a role for placenta-derived factors as mediators of the increased vascular permeability that accompanies preeclampsia.

	Materials and Methods

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Human placentas were collected from normal and preeclamptic pregnancies in nonsmoking women immediately after delivery at the Main Hospital, Louisiana State University Health Sciences Center, Shreveport, Louisiana. Normal pregnancy was defined as a pregnancy in which the mother had normal blood pressure (140/90 mm Hg), absence of proteinuria, and no medical complications. Mild preeclampsia was defined as a maternal blood pressure of 140/90 mm Hg or higher with proteinuria (300 mg/24 h) or more than 1+ dipstick on two separate readings. Severe preeclampsia was defined as a maternal blood pressure of at least 160/110 mm Hg with proteinuria more than 2+ dipstick on two separate readings. In this study, a total of 22 placentas (11 from normal and 11 from preeclamptic pregnancies) were used. Table 1 shows the demographics for the women from whom placentas were used either to isolate trophoblast cells or to prepare placental conditioned medium or villous culture. In the preeclampsia group, three patients were diagnosed with mild preeclampsia, and eight patients were diagnosed with severe preeclampsia. The proteinuria was not included because all patients in the preeclamptic group had more than 2+ dipstick protein in urine tests. This study was approved by the Institutional Review Board for Human Research at the Louisiana State University Health Sciences Center.

HUVECs were isolated from normal term pregnancies as previously described (17, 18). HUVECs were incubated with endothelial cell growth medium (BioWhittaker, Walkersville, MD). Placental trophoblast cells were isolated as previously described (13, 19). Isolated trophoblast cells were incubated with serum free DMEM (Sigma Chemical, Inc., St. Louis, MO).

UtMECs were purchased from BioWhittaker. UtMECs were also incubated in endothelial cell growth medium (BioWhittaker).

Coculture of endothelial cells with trophoblasts

For the endothelial monolayer permeability assay, freshly isolated trophoblasts were placed in a 24-well plate with a density of 1 x 10⁵ cells per well. Endothelial cells (HUVECs) were placed in polycarbonate Transwell inserts (8.0-µm pore size)/24-well plate. Trophoblast cells were incubated with serum free DMEM overnight, and then the endothelial cell inserts were transferred onto the trophoblast cultures. After 48 h of coculture, endothelial monolayer permeability assays were conducted, i.e. measurements of endothelial resistance and horseradish peroxidase (HRP) leakage through endothelial inserts. For the study of endothelial junctional structure, trophoblasts were placed into Transwell inserts, and endothelial cells were grown on glass coverslips in a 24-well plate. After 48 h of coculture, trophoblast filters were removed, and endothelial coverslips were fixed with 95% ethanol and then stained with antibodies against specific endothelial junctional proteins. A schematic drawing of an endothelial/trophoblast cell coculture system is shown in Fig. 1. All cocultures were performed in duplicate or triplicate. Endothelial cells were also cocultured with conditioned medium derived from normal and preeclamptic placental villous cultures, followed by an assessment of junction protein distribution.

View larger version (19K): [in this window] [in a new window]   FIG. 1. A schematic drawing of an in vitro model of endothelial/trophoblast coculture system. Transwell system consists of endothelial cell monolayers grown on microporous membranes with trophoblast cells placed in a compartment below the endothelial monolayer (A) or vice versa (B). This coculture system mimics the intimate relationships between trophoblasts in the placental intervillous space and endothelial cells in the maternal circulation and allows for study of bidirectional cellular regulation between the two cell types.

Immunofluorescent photomicrography

Immunofluorescent staining of junctional protein VE-cadherin, occludin, and pan-cadherin was performed as previously described (7). Briefly, endothelial cells were fixed with 95% ethanol at 4 C for at least 30 min and permeabilized with 80% acetone for 1 min at room temperature. Cells were then washed twice with PBS containing 0.1% milk and incubated with specific monoclonal or polyclonal antibodies. The antibodies used in our experiments were monoclonal mouse antihuman VE-cadherin (Immunotech, Marseilles, France), polyclonal rabbit antihuman occludin (Zymed, San Francisco, CA), and monoclonal mouse anti-pan cadherin (Sigma). Cy3 donkey antimouse IgG (H + L) or antirabbit IgG (H + L) was used as a secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Coverslips were then mounted on glass slides. Stained cell coverslips were examined by fluorescent microscopy (Olympus Optical Co., Tokyo, Japan), and fluorescence images at x100 with a 12-sec exposure time were recorded on Kodak T400 films (Eastman Kodak, Rochester, NY).

Measurement of endothelial monolayer electrical resistance

Endothelial cells were grown on polycarbonate cell Transwell inserts with 8.0-µm pore size (Becton Dickinson Labware, Franklin Lakes, NJ.). The resistance across the monolayer was measured by using an Endohm EVOM endothelial ohmmeter (World Precision Instruments, Sarasota, FL) as previously described (20). Briefly, the resistance was measured at 48 h of coculture. The mean reading from duplicated measurements per filter was used for data calculation. The value obtained from a blank insert was subtracted to give the net resistance, which was multiplied by the membrane area to give the resistance in area corrected units. The value for endothelial resistance was expressed as ohms times square centimeters, taking into account the surface area of the filter (0.30 cm²).

HRP leakage assay

Endothelial monolayer permeability was also measured by HRP leakage through endothelial inserts (7, 20). Briefly, 1 ml of medium was equilibrated to all lower chambers, and then HRP (VI-A type, 44,000 molecular weight; Sigma) at a concentration of 0.5 µg/ml was added to the upper endothelial chamber. The cells were incubated for 8 h in a CO₂ incubator at 37 C. An aliquot of 25 µl of medium in the lower chamber was collected in duplicate at 2, 4, and 8 h after HRP was added. To measure HRP leakage, 25 µl of the medium collected from the lower compartment was combined and mixed well with 860 µl of a reaction buffer of 50 mM NaH₂PO₄ with 5 mM guaiacol (Sigma). The reaction was started by adding 100 µl H₂O₂ (0.6 mM in double distilled water) to the mixture and was incubated for 30 min at room temperature. The samples were then read by spectrometer at 470 nM (Ultraspect 3000, Pharmacia Biotech, Cambridge, UK). The data were expressed as OD 470 nM for permeation of HRP across Transwell filters.

Statistical analysis

Data are presented as mean ± SE (unless specifically indicated). Statistical analysis was performed by the computer software program StatView (SAS Institute, Inc., Cary, NC) with the Mann-Whitney U test or one-way ANOVA. Fisher’s protected least significant difference and the Student-Newman-Keuls were used as the post hoc tests. A probability level of P < 0.05 was considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
VE-cadherin expression and distribution

Figure 2 shows representative micrographs of VE-cadherin expression and distribution in endothelial cells cocultured with placental trophoblast cells isolated from two normal or two preeclamptic pregnancies. In this experiment, endothelial cells (HUVECs) were grown on glass coverslips, and trophoblasts were loaded into Transwell inserts. After 48 h of coculture, trophoblast filters were removed, and endothelial cells were either fixed or continually cultured for 24 h (recovery). Uncocultured endothelial cells served as controls. VE-cadherin staining was achieved with a fluorescently labeled binding antibody. In control cells, VE-cadherin was localized around cell borders with spike-like structures projecting into the cell cytoplasm. Endothelial cells cocultured with trophoblasts from normal placentas showed an intact cell membrane around cell borders but were elongated in shape and lacked the spike-like projections. However, cells cocultured with trophoblasts from preeclamptic placentas exhibited multiple morphological changes: 1) cell shape was elongated and irregular; 2) cell fibers were retracted; and 3) gaps or pores were present at cell contact regions. These morphological changes were reversible, i.e. after removal of trophoblasts from the coculture system, cell shape returned to normal, and the expression and distribution of VE-cadherin were restored to cell contact regions with spike-like projects reappearing in the cytoplasm. Consistency of disturbed morphological changes was observed in endothelial cells cocultured with trophoblasts from all preeclamptic placentas.

View larger version (99K): [in this window] [in a new window]   FIG. 2. Representative distribution and expression for VE-cadherin in ECs (HUVECs) cocultured with TCs from two normal and two PE placentas. Nine independent coculture experiments were conducted, with TCs isolated from four normal and five from PE placentas. Uncocultured ECs served as control in each experiment. All cocultures were performed in duplicate for 48 h. VE-cadherin is continuously expressed at regions of cell contact in control ECs (A and F) and in ECs cocultured with normal TCs (B and G). Compared with control ECs, cocultured cells are elongated (B, D, G, and I). Discontinuous expression of VE-cadherin is seen at regions of cell contact in ECs cocultured with PE-TCs (D and I). The disturbed cell morphology was recovered after TCs (both normal and PE) were removed from the coculture system (C, E, H, and J). EC, Endothelial cell; TC, trophoblast cell; Nor, normal; PE, preeclamptic.

Endothelial monolayer permeability was determined by two assays, measurements of endothelial electrical resistance and HRP leakage through confluent monolayers on a Transwell membrane. Figure 3 shows the resistance offered by the endothelial cell monolayer after 48 h of coculture with trophoblast cells from seven normal and five preeclamptic placentas. Uncocultured endothelial cells served as control. Monolayer resistance was significantly reduced in cells cocultured with trophoblasts from normal placentas and further reduced in cells cocultured with trophoblasts derived from preeclamptic placentas compared with control cells (34.91 ± 0.94 and 29.37 ± 2.25 vs. 45.96 ± 1.14 ·cm²; P < 0.01, respectively).

View larger version (26K): [in this window] [in a new window]   FIG. 3. Electrical resistance across endothelial monolayers measured at 48 h of coculture. Monolayer resistance was significantly decreased in ECs cocultured with normal TCs (EC/Nor-TCs) and further decreased in ECs cocultured with PE-TCs (EC/PE-TCs) compared with those of control ECs. **, P < 0.01, cocultured cells compared with control ECs. #, P < 0.05, EC/PE-TCs vs. EC/Nor-TCs, respectively. EC, Endothelial cell; TC, trophoblast cell; Nor, normal; PE, preeclamptic.

View larger version (24K): [in this window] [in a new window]   FIG. 4. Leakage of HRP across endothelial monolayers cocultured with placental trophoblasts. After 48 h of coculture, HRP was added to the endothelial compartment (upper chamber), and HRP leakage to the lower chamber was measured at 2, 4, and 8 h. Uncocultured ECs served as control. HRP leakage was increased in ECs cocultured with normal TCs (EC/Nor-TCs) and further increased in ECs cocultured with PE-TCs (EC/PE-TCs). *, P < 0.05; **, P < 0.01; EC/Nor-TCs (n = 7) or EC/PE-TCs (n = 5) vs. control ECs. #, P < 0.05; ##, P < 0.01; EC/PE-TCs compared with EC/Nor-TCs, respectively. EC, Endothelial cell; TC, trophoblast cell; Nor, normal; PE, preeclamptic.

Morphological changes of endothelial junctions were further studied by fluorescent staining of VE-cadherin, pan-cadherin, and occludin on endothelial monolayers cultured with conditioned media derived from either normal or preeclamptic placental cultures. Figure 5 shows the results from endothelial cells cocultured with one normal conditioned medium and one preeclamptic conditioned medium for 40 h in duplicate. Eight independent coculture experiments were conducted with four normal conditioned media and four preeclamptic conditioned media. Uncocultured endothelial cells served as control. Consistency of disturbed junctional changes was observed in endothelial cells cocultured with preeclamptic placental conditioned medium. Compared with control cells (Fig. 5, A, D, and G), endothelial cells cultured with conditioned media were elongated. VE-cadherin distribution around cell borders was intact in cells cultured with normal placental conditioned media (Fig. 5B), but markedly reduced and discontinuously expressed in cells cultured with preeclamptic placental conditioned media (Fig. 5C). Intercellular gaps were present in cells cultured with conditioned media from normal placentas as evident from the reduced staining of pan-cadherin and occludin at cell junctions (Fig. 5, E and H). However, severe junctional disruption was observed in cells cultured with conditioned media from preeclamptic placentas, with loss of contact between cells as shown for pan-cadherin (Fig. 5F) and occludin (Fig. 5I), respectively. These morphological changes indicate that components released from preeclamptic placentas are more likely to disrupt endothelial junctions than those from normal placentas.

View larger version (86K): [in this window] [in a new window]   FIG. 5. Representative distribution and expression of VE-cadherin, pan-cadherin, and occludin in ECs cocultured with CM derived from normal and PE placental cultures. ECs were cocultured with CM for 40 h. Uncocultured ECs were used as control. Compared with control ECs (A, VE-cadherin; D, pan-cadherin; and G, occludin), the distribution and expression for VE-cadherin was intact (B), whereas pan-cadherin (E) and occludin (H) expression were reduced in ECs cocultured with normal placental CM. However, ECs cocultured with PE placental CM showed a discontinuous distribution and lower expression for VE-cadherin (C) at cell contact areas. Pan-cadherin (F) and occludin (I) expression were completely lost in ECs cocultured with PE placental CM. Eight independent experiments were conducted with ECs cocultured with four normal CM and four PE-CM. EC, Endothelial cell; CM, conditioned medium; TC, trophoblast cell; Nor, normal; PE, preeclamptic.

View larger version (60K): [in this window] [in a new window]   FIG. 6. Representative distribution and expression of VE-cadherin and occludin in UtMECs cocultured with placental CM derived from PE placental cultures. The disturbed VE-cadherin (B) and occludin (E) distributions in UtMECs cocultured with PE-CM showed a similar pattern as those of HUVECs cocultured with PE-CM. Similar to HUVECs, the disturbed VE-cadherin and occludin distribution and expression were recovered after CM was removed (A, VE-cadherin control; D, occludin control; C, VE-cadherin recovery; and F: occludin recovery, respectively). Two repeat experiments were conducted. EC, Endothelial cell; CM, conditioned medium; TC, trophoblast cell; Nor, normal; PE, preeclamptic.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

One of the most important functions of endothelial cells in the vascular system is to create and maintain a restrictive barrier for solute movement between the intravascular and interstitial compartments. The maintenance of this function is largely dependent on the intactness of adhesion and tight junctions between adjacent endothelial cells. VE-cadherin, an endothelial cell-specific adhesion protein, is known to play a major role in the support of endothelial monolayer integrity (21). The establishment of tight junctions also appears to depend on VE-cadherin and its role in establishing normal intercellular adhesion. We previously reported that the expression and distribution of VE-cadherin and occludin in regions of endothelial cell contact are altered when HUVECs are harvested during preeclamptic deliveries. This altered protein expression/distribution was associated with an increase in endothelial monolayer permeability (7). These responses are consistent with electron microscopic studies that demonstrate enlarged intercellular gaps between endothelial cells of myometrial vessels and sc arteries from women with preeclampsia (3, 4). It is known that cell-cell junctions are targets for a variety of signaling processes that have been implicated in the vascular permeability responses to different physiological and pathological stimuli. Our finding of disturbed VE-cadherin, pan-cadherin, and occludin distribution at regions of cell contact further supports the critical role of endothelial junctions as targets for the permeability-inducing substances produced by placental trophoblasts during pregnancy.

In this study, we observed an increased endothelial monolayer permeability not only when monolayers were cocultured with trophoblasts from preeclamptic placentas, but also when they were cocultured with trophoblasts from normal placentas. However, the changes in monolayer permeable function measured by endothelial resistance and HRP leakage and the loss of junctional protein pan-cadherin and occludin were more remarkable in cells cocultured with trophoblasts derived from preeclamptic placentas. Furthermore, we noticed that the normal VE-cadherin distribution was accompanied with partial loss of occludin at cell contact regions in monolayers cocultured with conditioned media from normal placentas. A complete loss of occludin expression was noted in endothelial monolayers cocultured with conditioned media from preeclamptic placentas. These observations suggest that tight junction structure is more susceptible than adhesion components to the permeability-inducing effects of trophoblast-derived factors, which may explain the results of reduced endothelial resistance and disturbed pan-cadherin and occludin distribution in endothelial cells cocultured with trophoblasts from normal placentas. Another explanation is the polarity changes in cocultured cells. It is known that tight junction and polarity are steady-state situations to maintain cell integrity (22). Elongated cell shape is a phenomenon of cocultured endothelial cells. The polarity changes in cocultured cells are accompanied by disturbed tight junction protein distribution, which reverts after removal of trophoblasts or conditioned media from the coculture. These observations demonstrate that assembling and disassembling of tight junction protein with polarity changes represent a highly dynamic arrangement of endothelial plasma membranes in the microenvironment that is created by permeability-inducing substances released from the placenta.

Directional release of factors from trophoblast cells may also contribute to the increased endothelial permeability and altered endothelial junctional structures observed in our coculture system, because the vectorial secretion or polarized release of substances has been demonstrated in both trophoblast cells (23, 24) and endothelial cells (25, 26). For example, studies have shown that more than 90% of MMP-2 and MMP-9 were released from the basolateral surface of syncytiotrophoblast cells when they were cultured on cell inserts (23). While using the same culture system to test polarized release of human cytomegalovirus from trophoblast cells in vitro, less than 1% of virus was detectable in the basal culture chamber compared with 20% of virus detected in the apical culture compartment (24). Directional changes in von Willebrand factor release were also reported in endothelial cells upon stimulation (25). These results suggest that the directional release by cells with polarized phenotype depends on the microenvironment stability under physiological conditions and the compensate response during pathophysiological and mechanical challenges. In our study, we did not test the polarity of trophoblast secretion. It is possible that factors released from apical and/or basolateral surface by trophoblasts may affect functional and morphological responses in endothelial cells in our coculture system (Fig. 1). Nevertheless, our results showed that factors released from trophoblast cells have the ability to disturb endothelial barrier function and induce endothelial permeability.

The possibility that endothelial cells exhibit a unique sensitivity to trophoblast-derived factors was also addressed in this study. Using a coculture system of placental conditioned medium and UtMECs, we found a very similar response to that seen with HUVECs, i.e. reversible changes in VE-cadherin and occludin expression/distribution. These findings suggest that the factors released from placental trophoblasts can activate endothelial cells and exert a permeability-increasing effect in vascular beds possibly throughout the body.

The trophoblast-derived substances that enable the placenta to alter junctional proteins and increase vascular permeability remain undefined. However, some insights into the possible identity of these factors are provided by previously published work. For example, it has been shown that the increased neutrophil-endothelial adhesion induced by placental factors is mediated by platelet-activating factor (10). Endothelial protease-activated receptors, protease thrombin, and trypsin have also been implicated in the endothelial dysfunction induced by placental factors (12). Most recently, we have demonstrated that chymotrypsin released from preeclamptic placentas is responsible for increased expression of P-selectin and E-selectin on endothelial cells (13). Whether the same factors also mediate the increased endothelial monolayer permeability that is induced by placental trophoblasts remains to be determined.

In summary, we employed a unique endothelial/trophoblast coculture model to assess the role of placental trophoblast cells in the regulation of endothelial cell barrier function. We found that endothelial junctions are sensitive targets for TC-derived products and that the tight junction protein occludin is more susceptible to these products than the cadherins. The resulting disturbance in cell-cell contact at the intercellular junctions was associated with a significant increase in endothelial monolayer permeability. Although trophoblasts are not of endothelial cell origin, they are directly exposed to maternal blood in the intervillous space of the placenta. Hence, it is highly likely that placental products released into the intervillous space will enter the maternal circulation during pregnancy. Once in the systemic circulation, these placental factors can mediate an increased vascular permeability at multiple distant sites, leading to the excessive accumulation of plasma proteins and water in the interstitial compartment (edema). Additional work is needed to determine the identity of these placental trophoblast-derived factors that diminish endothelial barrier function during preeclampsia.

	Footnotes

 
This work was supported in part by Grant HD36822 from the National Institutes of Health, National Institute of Child Health Development, and by Grants HL 67997 and HL26441 from the National Heart, Lung, and Blood Institute.

Results of this study were presented at the 50th Annual Meeting of the Society for Gynecologic Investigation, Washington, D.C., March 27–31, 2003.

Abbreviations: HRP, Horseradish peroxidase; HUVEC, human umbilical vein endothelial cell; MMP, matrix metalloproteinase; UtMEC, uterine myometrium endothelial cell; VE, vascular endothelial.

Received October 1, 2003.

Accepted February 1, 2004.

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