Placenta
Volume 28, Supplement , Pages S23-S32, April 2007

Dual In Vitro Perfusion of an Isolated Cotyledon as a Model to Study the Implication of Changes in the Third Trimester Placenta on Preeclampsia

  • S. Di Santo

      Affiliations

    • Department of Obstetrics and Gynecology, Universitäts-Frauenklinik, Inselspital, Effinger Strasse 102, CH 3010 Berne, Switzerland
    • ,
  • R. Sager

      Affiliations

    • Department of Obstetrics and Gynecology, Universitäts-Frauenklinik, Inselspital, Effinger Strasse 102, CH 3010 Berne, Switzerland
    • ,
  • A.-C. Andres

      Affiliations

    • Department of Clinical Research, University Berne, Berne, Switzerland
    • ,
  • S. Guller

      Affiliations

    • Department of Obstetrics and Gynecology and Reproductive Sciences, Yale University School of Medicine, New Haven, CT, USA
    • ,
  • H. Schneider

      Affiliations

    • Department of Obstetrics and Gynecology, Universitäts-Frauenklinik, Inselspital, Effinger Strasse 102, CH 3010 Berne, Switzerland
    • Corresponding Author InformationCorresponding author. Tel.: +41 (0)31 961 7430.

Accepted 15 January 2007. published online 09 March 2007.

Article Outline

Abstract 

In the current study perfusions of an isolated cotyledon of term placenta using standard medium were compared to medium containing xanthine plus xanthine oxidase (X+XO), which generates reactive oxygen species (ROS). A time-dependant increase in the levels of different cytokines (TNF-α, IL-1ß, IL-6, IL-8 and IL-10) was observed between 1 and 7h with more than 90% of the total recovered from the maternal compartment with no significant difference between the 2 groups. For 8-iso-PGF2α 90% of the total was found in the fetal compartment and a significantly higher total release was seen in the X+XO group.

Microparticles (MPs) isolated from the maternal circuit were identified by flow cytometry as trophoblastic sheddings, whereas MPs from the fetal circuit were predominantly derived from endothelial cells. More than 90% of the total of MPs was found in the maternal circuit. The absolute amount of the total as well as the maternal fraction were significantly higher in the X+XO group.

Immunohistochemistry (IHC) of the perfused tissue revealed staining for IL-1β of villous stroma cells, which became clearly more pronounced in experiments with X+XO. Western blot of tissue homogenate revealed 2 isoforms of IL-1β at 17 and 31kD. In X+XO experiments there was a tendency for increased expression of antioxidant enzymes in the tissue.

Western blot of MPs from the maternal circuit showed increased expression of antioxidant enzymes in the X+XO group and for IL-1β only the 17kD band was detected.

In vitro reperfusion of human placental tissue results in mild tissue injury suggestive of oxidative stress. In view of the increased generation of ROS in perfused tissue with further increase under the influence of X+XO, the overall manifestation of oxidative stress remained rather mild. Preservation of antioxidant capacity of human placental tissue could be a sign of integrity of structure and function being maintained in vitro by dual perfusion of an isolated cotyledon. The observed changes resemble findings seen in placentae from preeclampsia.

Keywords: Placental perfusion, Oxidative stress, Microparticles, Preeclampsia

 

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1. Introduction 

The delivery of the baby normally is followed by the expulsion of the placenta, which undergoes an abrupt change from a state of high flow perfusion of two separate vascular spaces with maternal and fetal blood, respectively, to complete ischemia. Villous explant cultures subjected to hypoxia followed by reoxygenation showed typical signs of oxidative stress including apoptosis of syncytiotrophoblast [1], [2]. We have shown that resumption of oxygenation of placental tissue in vitro leads to tissue damage resembling reperfusion injury. Furthermore, in a direct comparison of villous explant culture and dual perfusion of tissue from the same placenta and using similar experimental conditions, in our hands basic trophoblast viability and functionality for at least 7h is better preserved by dual perfusion [3], which is consistent with the finding of other groups [4], [5].

In this study we asked the question of whether the changes in placental tissue developing during in vitro perfusion are similar to the findings reported for placentae from certain pregnancy pathologies like preeclampsia. The present study adds new insights into the response of placental tissue to conditions of in vitro perfusion and extends the analysis to the effect of pro-oxidant influences. Standard experimental conditions were compared to perfusions with medium containing X+XO as a system generating reactive oxygen species (ROS). It should be noted that data from the standard perfusions were already presented in 2 previous studies from our group [3], [6].

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2. Materials and methods 

2.1. Human subjects 

Human term placentas (n=9) were obtained from uncomplicated pregnancies with a single, normally grown fetus delivered by elective caesarean section. Indications were previous caesarean or patient's request. Informed consent for the experimental use of the placenta was given by each patient according to the regulations of the review board at Inselspital Berne, Switzerland.

2.2. Dual in vitro perfusion 

A suitable cotyledon was set up for dual perfusion as we have previously described [7]. When both maternal and fetal circuits were stable with mean flow rates of 12 and 6ml per min on the maternal and fetal side respectively, dual perfusion was continued with closed circuits holding 150ml perfusion medium in each compartment. Further experimental details including oxygenation and composition of the medium were described before [3], [6]. After a prerun of one hour, medium was exchanged in both circuits and the actual experiment consisted of 2 phases lasting 2 and 4h. After the initial 2h, another exchange of medium was performed. Control perfusions under standard conditions (n=5) were compared to an experimental series with induced oxidative stress by addition of a mixture of xanthine (X, 2.3mM) and xanthine oxidase (XO, 15mU/ml) to the medium in both circuits (n=4).

2.3. Sample collection 

Medium samples were taken at regular intervals from the maternal and the fetal circulation, and after centrifugation at 1500×g for 10min, the supernatant was stored at −20°C. Tissue was randomly sampled from an unperfused area next to the chosen cotyledon before start of the experiment and from the perfused cotyledon at the end and immediately frozen in liquid nitrogen. For tissue extraction 1g was homogenized in 3ml of Ringer solution, centrifuged (1500×g for 10min) and aliquots of the supernatant were frozen. Metabolic parameters like consumption of glucose and oxygen as well as production of lactate were assessed by standard procedures. Placental proteohormones like hCG, hPL and leptin were measured by ELISA and synthesis was calculated as described before [3]. For the determination of various cytokines and 8-iso-PGF2α, commercial ELISA kits were used (R&D).

2.4. Histochemistry 

For immunohistochemistry (IHC) 4μm paraffin sections were treated with the following primary antibodies: rabbit anti-p22phox oxidase. (Santa Cruz 20781), rabbit anti-catalase (Calbiochem 219010), sheep anti-Cu/Zn SOD (Calbiochem 574597), sheep anti-Mn SOD (Calbiochem 574596), rabbit anti-IL-1β (Santa Cruz SC 7884), rabbit anti-four-HNE (Calbiochem 393207) using a standard protocol. Alkaline phosphatase conjugated anti-mouse and anti-rabbit antibodies were purchased from Dako, anti-sheep antibody was from Sigma. Color development was carried out with the Neufuchsin method.

2.5. Western Blot analysis 

For Western blot analysis approximately 100mg placental tissue were homogenized in 1ml lysis buffer (50mM Tris pH 7.5, 150mM NaCl, 1% NP40 (Sigma), 0.5% sodium deoxycholate, 0.1% SDS) containing a Complete Protease Inhibitor (Roche Diagnostics). Homogenates were centrifuged for 20min at 10,000×g to remove insoluble material. Microparticles (MP) isolated from the medium as described below were solubilized in the same buffer. The protein concentration in the supernatant of tissue homogenate and in the MP suspension was determined using Bicinchonic Assay (Sigma).

Equal amounts of protein (50–100μg) were separated on a 12% SDS polyacrylamide gel (Serva) and transferred onto nitrocellulose membrane (Schleicher and Schuell). The membranes were blocked with 5% non-fat dry milk in PBS containing 0.1% Tween 20 (PBS-T), washed in PBS-T and incubated overnight at 4°C with the same primary antibodies used for immunohistochemistry. Subsequently bound antibodies were detected by incubation for 1h with appropriate horseradish peroxidase-conjugated secondary antibodies. Equal loading was verified by stripping and reprobing the blots with mouse anti β-actin (Amersham). Negative controls were performed by omitting the primary antibody. Immunodetection was carried out using the ECL system (Life Technologies).

2.6. Isolation of microparticles (MP) 

MPs were prepared from medium samples recovered at medium exchange after one hour prerun, between experimental phase I and II and at the end of the perfusion. An amended protocol of centrifugation was used [8]. Samples were centrifuged twice at 1500×g for 10min at 4°C. Ten ml of the supernatant of the second spin was centrifuged at 10,000×g for 30min. The pellet was resuspended in 1.5ml of Ringers solution (B. Braun Medical AG, Emmenbrücke, Switzerland) and centrifuged for a second time at 10,000×g for 30min and the final pellet was taken up in 100μl water and stored at −80°C until further processing. The protein content of the MP suspension was quantified with the Bicinchonic Assay Kit (BCA-1, Sigma).

2.7. Flow cytometric analysis of MPs 

One μl of the suspension of MPs collected from the maternal circuit was incubated for 30min at room temperature with mouse monoclonal anti-Placental Alkaline Phosphatase (PLAP, Serotec MCA 2091) antibody or isotype control (Serotec MCA928) diluted 1:100 in TBS/PBS/BSA (25mM, 0.25% BSA, pH 7.4). Subsequently, F(ab′)2 rabbit anti-mouse IgG1-FITC conjugate (Serotec Star9B) diluted 1:100 in TBS/PBS/BSA was added followed by incubation in the dark for another 30min at room temperature. Ten μl of MP suspension collected from the fetal circuit were incubated 30min at room temperature with mouse monoclonal anti-CD62E-FITC labeled antibody (Dako) diluted 1:100. After incubation, MPs were centrifuged for 30min at 10,000×g, the supernatant was removed and the pellet was resuspended in 400μl of TBS/PBS. Stained MPs were analysed by a LSR II flow cytometer (Becton Dickinson). Both forward and sideward scatter were set at a logarithmic gain. Data were collected and subsequently analysed by a computer equipped with FlowJo Software (TreeStar Corp).

2.8. Statistical analysis 

All data are presented as mean±SD. The statistical significance of the difference between experimental groups was calculated using the test of Mann–Whitney. GraphPad PRISM Software (GraphPad Software, San Diego, USA) was used and a p value <0.05 was considered significant.

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3. Results 

Consumption of glucose and oxygen, production of lactate as well as synthesis of hCG, hPL and leptin found under conditions of standard perfusions have been published before and no difference was seen in experiments with X+XO [3]. The transcript level of the two stress proteins (BiP and RTP), which had been found to be increased in tissue perfused under standard conditions compared to unperfused tissue, showed no further increase under the prooxidant influence of X+XO. The low incidence of apoptotic cell death also was unaffected by X+XO. The comparative analysis between the two perfusion protocols showed that under both experimental conditions trophoblast viability and functionality was preserved over the 7h of the experiment. The increased transcript level of the two stress proteins (BiP and RTP) in perfused tissue could be a sign of oxidative stress.

3.1. Signs of oxidative stress in the tissue 

The results from the analysis of perfused tissue were related to unperfused control tissue taken from the same placenta immediately after arrival in the laboratory.

Immunohistochemistry with antibodies against the p22phox oxidase subunit of the superoxide producing enzyme NAD(P)H, for unperfused tissue showed some staining predominantly in Hofbauer cells (HC) inside the villous stroma (Fig. 1A, C). Perfusion under standard conditions resulted in an augmented expression, which was further increased in the X+XO experiments (Fig. 1B, D).

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  • Fig. 1 

    Immunohistochemical detection of p22phox expression in control tissue at the beginning of the experiment (A, C) and after perfusion under standard (B), and prooxidant (X+XO) conditions (D). Arrows and arrowheads indicate trophoblast and Hofbauer cells respectively. Bar=100μm.

Immunohistochemical staining of 4-HNE, a marker of lipidperoxidation, failed to show a significant response, neither in unperfused nor in perfused tissue (data not shown).

Immunostaining of IL-1ß, another marker of oxidative stress, showed clear signals in villous stroma of perfused tissue, whereas control tissue was basically negative (Fig. 2). This reaction was even more intense under prooxidant conditions. While immunostaining of antioxidant enzymes, which will be shown below, was predominantly localized to the trophoblast, the IL-1ß specific stain was seen in HC in the villous stroma. The Western blot of homogenates from perfused tissue interestingly showed 2 bands for Il-1ß at 17 and 31kD.

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  • Fig. 2 

    Immunohistochemical detection (i) and Western blot analysis (ii) of IL-1β expression in control tissue at the beginning of the experiment (A, C) and after perfusion under standard (B), and prooxidant (X+XO) conditions (D). Arrowheads indicate Hofbauer cells. Bar=100μm.

3.2. Release of stress markers into the medium 

A time-dependant rise in accumulation of 8-iso-PGF2α in the medium was found in both groups and under X+XO total release during the whole duration of the perfusion was significantly higher (Fig. 3). Interestingly the majority (standard perfusion=95%; X+XO=89%) of 8-iso-PGF2α was found in the fetal circuit indicating a specific targeting of this molecule. A similar pattern of accumulation in the medium was noticed for IL-1β as well as other cytokines like IL-6, IL-8, IL-10 and TNF-α. However, for these cytokines the major fraction appeared in the maternal circuit (83–98%), with no significant difference between the two groups for any of the cytokines.

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  • Fig. 3 

    Total accumulation of 8-iso-PGF2α over 7h in the fetal (F), the maternal (M) and both circuits (M+F) under standard conditions (n=5) and in the presence of X+XO (n=4). Accumulation values calculated as mean±SD. *Standard vs. X+XO: P<0.04.

3.3. Release of microparticles 

The total of MPs isolated from the pooled medium from the maternal and fetal circuit was quantified by analysis of protein. Ninety% of MPs were found in the maternal circuit. Total accumulation of MPs was significantly higher in pro-oxidant compared to standard perfusions and the maternal fraction increased to 98% (Table 1).

Table 1. Rate of MPs accumulation during perfusion
Mean accumulation rate (μg/ml/min)SDM (%)F (%)
Control6.741.199010
X+XO10.35*1.69982

Mean accumulation rate of protein in the perfusion medium pooled from maternal (M) and fetal circuit (F). *Statistical significant difference (p<0.03).

The percentage of trophoblast-derived MPs as indicated by PLAP increased throughout the perfusion in the maternal compartment reaching a peak of 94% in phase II. Eighty five % of MPs in the fetal compartment were of endothelial origin as indicated by CD62E. There were no significant differences between the two perfusion groups (Fig. 4).

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  • Fig. 4 

    Flow cytometric analysis of the sediment of perfusion medium collected from the maternal circuit and stained for PLAP (i and iii) and from the fetal circuit and stained for CD62E (ii and iv). (i and ii): percentage of positive events in the three perfusate fractions; (iii and iv): representative analysis of the microparticles from the last perfusate fraction. Values are expressed as mean±SD of the percentage of the positive events.

3.4. Antioxidant defences 

The expression of the principle antioxidant enzymes, copper/zinc (Cu/ZnSOD) as well as manganese superoxide dismutase (MnSOD) and catalase was analysed by Western blot and IHC. Cu/ZnSOD was immunolocalized in the trophoblast and after perfusion immunostaining remained unchanged in both groups. This was confirmed by Western blot analysis of tissue homogenates (data not shown). Catalase immunoreactivity was found in trophoblast and few HCs (Fig. 5). In trophoblast staining apparently decreased as a result of perfusion in both groups, whereas HCs showed increased staining in perfusions with X+XO. Western blot in standard perfusions revealed a decline in catalase expression. In contrast, in prooxidant perfusions with X+XO, the overall catalase expression remained unchanged compared to unperfused tissue. For MnSOD a slight increase in staining was observed in HC in perfused tissue, with no clear difference in the two groups. Western blot also revealed an increased intensity of the bands as a result of perfusion in both groups (Fig. 6).

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  • Fig. 5 

    (i) Immunohistochemical detection and (ii) Western blot analysis of catalase expression in control tissue at the beginning of the experiment (A, C) and after perfusion under standard (B), and prooxidant (X+XO) conditions (D). Bar=100μm. Arrows and arrowheads indicate trophoblast and Hofbauer cells respectively.

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  • Fig. 6 

    (i) Immunohistochemical detection and (ii) Western blot analysis of MnSOD expression in control tissue at the beginning of the experiment (A, C) and in placental tissue perfused for 7h under standard (B), and prooxidant (X+XO) conditions (D). Bar=100μm. Arrows indicate trophoblast cells and arrowheads indicate Hofbauer cells.

The tissue expression of the three antioxidant enzymes was compared with trophoblastic MPs. Only material recovered from the maternal circuit was sufficient in quantity to do Western blots. MPs recovered at the end of the experiment, i.e. after 7h of standard perfusion or 1h of control followed by 6h under prooxidant conditions showed clear bands for the antioxidant enzymes Cu/ZnSOD and catalase (Fig. 7). The intensity of the bands for the two enzymes shown for the prooxidant perfusions compared to standard perfusions clearly was higher than the comparison shown for immunoreactivity or Western of tissue homogenates. Western blots from MPs recovered at earlier time points suggested, that there was a time-dependant increase for the two antioxidant enzymes and IL-1β in both groups (data not shown). The increase in intensity of the catalase band in MPs of the X+XO group (Fig. 7) differs from the lack of change in expression in the perfused tissue (Fig. 5D).

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  • Fig. 7 

    Western blot analysis of protein extracts of MPs collected from the maternal circuit at the end of perfusion under standard (Crtl) and pro-oxidant conditions (X+XO).

For the third antioxidant enzyme, MnSOD, no reaction was seen neither in MPs from standard nor from prooxidant perfusions. In view of the expression in IHC and Western blot of tissue samples, this could be explained by the predominant localisation of this isoform of the SOD inside mitochondria of HC.

Particularly remarkable was, that the Western of MPs for the proinflammatory cytokine IL-1ß showed only a single strong band at 17kD, with no difference in intensity between perfusions under standard or prooxidant conditions (Fig. 7). In contrast the Western blot for homogenate of perfused tissue had shown 2 bands at 17 and 31kD (Fig. 2). This suggests that the larger isoform shown at 31kD is tissue bound, whereas only the biologically active 17kD variant is exported in MPs.

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4. Discussion 

Variations in blood flow are normal features of placental hemodynamics. In preeclampsia pronounced regional shifts in perfusion of placental tissue may lead to hypoxia, as well as reperfusion inducing oxidative stress together with an inflammatory response of the tissue [9], [10], [11]. In different in vitro systems, be it villous explant culture or the dual perfusion of an isolated cotyledon, placental tissue, when reexposed to oxygen following an episode of complete ischemia shows signs of reperfusion injury [3]. This is combined with hypoxia [12]. In spite of a very high oxygen consumption rate in vivo, placental tissue in vitro is particularly resistant to reduced oxygen supply [13].

With addition of X+XO superoxide anions (SO), the most common ROS, are generated during the conversion of xanthine (X) to uric acid by xanthine oxidase (XO) [14], [15]. Under prooxidant conditions there is increased expression of NAD(P)H oxidase, which is localised in placental tissue in trophoblastic cells in addition to the phagocytic cells in the villous stroma [16]. In the present study, we found increased expression of p22phox, a subunit of NAD(P)H oxidase, in the HC of perfused tissue with a noticeable augmentation in the X-XO experimental group. This is consistent with our previous finding of increased intracellular levels of ROS, predominantly located in the syncytium in tissue perfused under standard conditions with further increase in intensity after addition of X+XO [6]. These findings are relevant in view of recent reports showing higher expression of NAD(P)H subunits, as well as activity leading to increased ROS generation in placentae from preeclamptic women [17], [18]. SO combines with nitric oxide to produce peroxynitrite anions, which damage proteins [14]. Increased nitrosylation of proteins and other signs of oxidative stress have been shown in placental tissue in preeclampsia [19], [20]. In addition, SO can lead to the production of hydrogen peroxide and formation of the highly reactive hydroxyl radical, which causes lipid peroxidation [21], [22].

The X+XO perfusion was associated with a significant increase of total production of 8-iso-PGF2α a major isoprostane, which as a stable product of lipid peroxidation is a marker of oxidative stress. Eight-iso-PGF2α being a potent vasoconstrictor, platelet activator and mitogen, is involved in the pathogenesis of PE [22]. In this study the mechanism of increased production of 8-iso-PGF2α in the X+XO perfusions was not further investigated, but it is assumed that free radical-induced peroxidation of arachidonic acid is the predominant pathway. It is of special note that the majority of 8-iso-PGF2α during the perfusion was released into the fetal compartment as opposed to most placental proteins, peptides and cytokines, which appear predominantly in the maternal circuit. This may explain the in vivo observation of increased levels of lipid peroxide not only in placental tissue homogenate but also in umbilical venous blood in cases with preeclampsia [23].

Four-HNE staining, another product of lipid peroxidation and indicator of oxidative damage, failed to show a significant difference between tissue perfused under standard as compared to prooxidant conditions. In placental villi exposed to ethanol in vitro there was no difference seen for 4-HNE, whereas other markers of oxidative stress like nitrotyrosine compared to controls showed increased staining [24].

The inflammatory response shown by immunohistochemical staining of IL-1β in HCs in villous stroma is related to oxidative stress. This response clearly appeared in the dually perfused placental tissue and increased under prooxidant conditions. These tissue changes are reflected by a time-dependant increase in accumulation of IL-1β and other cytokines like, IL-6, IL-8, IL-10 and TNF-α in the perfusate.

As we have previously shown in standard perfusion, there is a time-dependant increase of PAI-1 (plasminogen activator inhibitor) release into the maternal compartment, which was associated with a concomitant decrease in PAI-2 levels, resulting in an approximate 3-fold increase in the ratio of PAI-1/PAI-2 [6]. These observations are significant in light of the increase in PAI-1/PAI-2 ratio in maternal blood [25], [26] in preeclampsia and demonstrate another parallel between in vivo changes in this condition and the response of placental tissue to in vitro dual perfusion.

In view of the substantial generation of SO in the tissue perfused with X+XO, signs of oxidative stress except for the release of 8-iso-PGF2α into the medium and the shift in PAI-1/PAI-2 ratio remain rather mild. The placenta is richly endowed with antioxidants like thioredoxin, glutathione and its peroxidase, catalase, Cu/Zn- and MnSOD as well as vitamins. With the exposure of placental tissue to maternal arterial blood antioxidant enzymes like catalase, MnSOD and Cu/ZnSOD appear around 12weeks [27] and antioxidant protection of placental tissue remains intact throughout pregnancy [28]. The presence and localization of catalase, MnSOD and Cu/ZnSOD described in this study confirm the findings of a previous report [14]. Although the difference in expression of the various SODs in tissue from perfusions under standard vs prooxidant conditions is not always convincing, this does not exclude a difference in enzyme activity. Stimulation of antioxidant enzyme activity may be an early compensatory mechanism in situations of acute oxidative stress, and 7h of perfusion may not be long enough to show an increase in enzyme levels by IHC. Chronic exposure to oxidative stress may lead to a decrease in activity of antioxidant enzymes due to oxidation and inactivation by free radicals. In fact, the Cu/ZnSOD expression in placental tissue in preeclampsia is similar to that found in normal pregnancy, however the enzyme activity is reduced [29].

Efficient neutralization of radicals minimizing oxidative damage seems to be linked to trophoblast viability, functionality and metabolic activity. The addition of X+XO to the medium in the explant villous culture, which as a model preserves tissue integrity substantially less well [3], produced signs of considerably more severe damage, albeit at a considerably longer exposure of 24h [30].

In normal pregnancy physiological turnover of syncytiotrophoblast leads to apoptosis with the release of corpuscular syncytial knots into the maternal circulation. In preeclampsia, a substantial increase in circulating MPs derived from trophoblast in the form of STBMs, T cells, granulocytes and endothelial cells, together with changes in their pathogenic potential, have been discussed as a possible placental factor responsible for an increased inflammatory response with endothelial dysfunction in the maternal system [31], [32]. In vivo it is assumed that the production of MPs is initiated by cell activation by different agents or by apoptosis [33]. A number of studies have focused on the interaction between STBMs and endothelial cells as well as leukocytes. The role of a direct effect of STBMs for the impairment of endothelial function [34], [35], [36] vs mediation via stimulation of an inflammatory state [35], [37], [38], [39] or the generation of SO radicals [40] is still controversial. More recently, the comparison of the level of STBMs in peripheral venous blood from normal term pregnancies with early vs late onset preeclampsia and normotensive intrauterine growth restriction showed significantly increased levels only in the early onset preeclampsia group, underlining their role in the pathogenesis of the maternal syndrome of preeclampsia [41].

In vitro studies using villous explants revealed that under low oxygen (2%) there was an increased proliferation of the cytotrophoblast and lack of fusion with syncytiotrophoblast. Shedding of trophoblast predominantly was a result of necrosis [42]. The increase of aponecrotic subcellular material in the periphery of the maternal circulation in preeclampsia, could explain the generation of dysfunction of the endothelial system of the maternal vasculature [43].

Different in vitro techniques for the production of MPs from term placentae varying in size and biological function have been described [44]. Variations in the ratio of MPs generated by apoptosis vs. necrosis seem to be related to differences in the content of nucleic acids [45]. Details of the technique of isolation of MPs must be considered, when interpreting their effect. In this study we have used an amended version of the protocol first described by vanWijk et al. [8].

STBMs may function as a special export system for tissue products. Homogenate of perfused tissue showed both the 31kD proform as well as the biologically active 17kD variant of IL-1β, whereas MPs contained only the 17kD form. This may suggest that the cytokine is produced by the villous stroma and the larger proform is processed in the syncytiotrophoblast to the smaller biologically active variant. It is interesting that the difference in expression of Cu/ZnSOD and catalase, as shown by Western blot in MPs from prooxidant perfusions compared to standard conditions, was more pronounced than in perfused tissue samples. This could reflect a cumulative uptake into MPs. Furthermore, we have documented for the first time, that, MPs carrying markers of endothelial cells are also released into the fetal circulation. Their potential role in producing endothelial dysfunction in the fetus, which has been reported in cases of preeclampsia, requires further investigation [46], [47].

In summary, oxidative stress with increased secretion of pro-inflammatory cytokines and release of STBMs seems to be an in vivo response of the placenta to changes in its environment. This pathological response may be linked to events like endothelial dysfunction in the maternal vasculature in preeclampsia. As was recently emphasised, there is a need for in vitro models to study variables like oxidative stress and the related inflammatory response of the tissue for a better understanding of the underlying molecular dysregulation in conditions like preeclampsia [48]. The dual in vitro perfusion could be a suitable experimental model.

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Acknowledgments 

This work was supported in part by grant 32-061771.00 from the Swiss National Research Foundation (HS) and by HD33909 from the NIH (SG).

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PII: S0143-4004(07)00012-4

doi:10.1016/j.placenta.2007.01.009

Placenta
Volume 28, Supplement , Pages S23-S32, April 2007

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