University of Groningen

Placental particles in pregnancy and preeclampsiaGöhner, Claudia

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Citation for published version (APA):Göhner, C. (2016). Placental particles in pregnancy and preeclampsia: A comparative investigation of thefunction of syncytiotrophoblast microvesicles versus exosomes during pregnancy and preeclampsia.University of Groningen.

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Chapter 7

General Discussion

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During pregnancy, diverse types of extracellular vesicles (EV) can be found in the maternal circulation originating from, amongst others, blood cells and en-dothelial cells, but interestingly also from the outer fetal layer of the placenta, the syncytiotrophoblast (STB).[1] The placenta produces a plethora of factors, espe-cially syncytiotrophoblast extracellular vesicles (STB EV), which enter the maternal circulation and can perform physiologic systemic functions.[2–4] In general, it has been described that EV show diverse functions related to angiogenesis, cell surviv-al, coagulation, waste management, cell communication and immune adaptation.[3,5–7] Also the placental fraction, the STB EV, is believed to be involved especially in the immunologic and hemostatic adaptation of the maternal body during preg-nancy.[8–10] The plasma concentration of STB EV increases during pregnancy, prob-ably due to the increasing size of the placenta.[1] However, STB EV are also believed to be involved in the pathophysiology of the pregnancy-complication preeclampsia (PE), and it has been shown that the STB EV concentration is increased in PE com-pared to normal pregnancy.[11] The purpose of this thesis was a comparative investigation of the function of two groups of STB EV, syncytiotrophoblast microvesicles (STB MV) and exosomes in normal and preeclamptic pregnancies. Though secreted from the same cell, the STB, STB MV and exosomes are formed by different mechanisms and may have a (at least partially) different molecular load.[3,5,7,12] Therefore, it is believed that they mediate different functions, although direct comparisons of the functionality of STB MV and exosomes have, to our knowledge, never been made. In theory, STB MV are thought to be immune activating [3,13] e.g. by inducing the expression of inflamma-tory cytokines in peripheral blood mononuclear cells [14]. They are also thought to be anti-angiogenic and pro-coagulant.[15] STB MV also have been associated with the pathophysiology of PE, partially because of the increased plasma concentration of STB MV during PE.[11,14,16] In contrast, placental exosomes are thought to be immune suppressive/tolerance-inducing e.g. by reducing cytotoxicity of NK cells, CD8+ T cells and gamma delta T cells.[17] However, especially most studies on STB MV function and concentration worked either with total plasma samples or EV from pellets after ultracentrifugation at 150,000 g, and thus experiments and measure-ments were most likely performed with a mixture of STB MV and exosomes. There-fore it cannot clearly be stated which vesicle fraction was causing the observed effects. As especially the immune system and the hemostasis are of extraordinary importance for a healthy pregnancy [14,18–22] and shown to be impaired during PE [23–28], we aimed to systematically compare the immune-modulatory and co-agulant functions of STB MV and exosomes. And indeed, the results of this thesis suggest that the general classification of the function of STB MV and exosomes into activating (bad) and tolerance-inducing (good), respectively, may be too simplistic. One of the problems in research on EV is the fact that it is very difficult to quantify the vesicles. Three methods are often used - flow cytometry [29], na-noparticle tracking analysis [30] and protein quantification [2]. Flow cytometry

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is a common method to perform multiparametric analysis of cells and EV.[29,31,32] However, flow cytometry relies on the forward scatter and side scatter of particles crossing a laser beam and these parameters are strongly dependent of the size and granularity of the particles themselves. It has been reviewed that MV constitute the lower detectable size boarder for flow cytometric analyses and that exosomes are actually too small to be analyzed via flow cytometry. [29,31,32]. As with flow cytometry, also nanoparticle tracking analysis relies on light scattering of particles crossing a laser beam. However, in this method, the scattered light is tracked to follow the Brownian motion of the particles in a liquid sample, which allows calcu-lation of the particle size and concentration. Technical improvements during the last years even allow the application of fluorescence-/quantum dot-labeled antibodies to label EV, e.g. based on origin-specific markers, and to track them by nanoparticle tracking analysis.[30] However, the method requires the dye to have a very high in-tensity and half-life, restricting the applicability of fluorescence-labeled antibodies. In several studies, the total protein concentration of EV has been used as a relative measurement of the EV concentration.[2,33] However, it has been described that the molecular load of EV differs during health and disease and may also influence the total protein concentration and thus impedes a comparable quantification of EV. For STB EV, an enzyme-linked immunosorbent assay (ELISA) has been described by the group of Redman and Sargent in Oxford, based on human placental alkaline phosphatase.[11] This method immobilizes STB EV from fluid samples, based on their expression of human placental alkaline phosphatase, but the results may be affected by free human placental alkaline phosphatase in the sample if the vesicles are not carefully separated from the sample by centrifugation on beforehand. Due to the difficulties in quantifying EV, we developed a reliable but simple and quick method to quantify STB EV in fluid samples such as plasma or suspension from ex vivo placenta perfusion. As described in chapter 2, we managed to de-velop an enzyme-linked sorbent assay based on the well-known ELISA-technique, but working without an actual immune component. In this enzyme-linked sorbent assay, STB EV are captured from fluid samples by binding of immobilized annexin V in a microtiter plate to phosphatidylserines on the surface of STB EV. A colorimetric detection reaction is catalyzed by human placental alkaline phosphatase exposed on STB EV. Since our method is using annexin V to immobilize EV based on the phosphatidylserines exposure, it is most likely less sensitive to disturbances in the detection reaction by e.g. soluble human placental alkaline phosphatase in the fluid sample. However, it has been described that not only MV but also a certain amount of exosomes exposes phosphatidylserines on their surface.[34] Thus, we cannot completely rule out that results from our ELSA might be influenced by exosomes and we also recommend a strict centrifugation protocol to separate MV from ex-osomes. To reach a better understanding of the influences of STB EV in healthy and preeclamptic pregnancy, we next aimed to quantify the peripheral STB EV plasma

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concentration in normal pregnancy and PE. Equipped with our enzyme-linked sorb-ent assay, we performed the MORE PrePARd study (Microparticle Orientated Risk Evaluation for Preeclampsia Prediction Among Risk graviDas -the MORE PrePARd Study) as described in chapter 3. This multi-center, prospective, blinded, prognostic marker study explored the usability of the peripheral plasma STB EV concentration as an accessory marker to mid-gestational uterine artery Doppler velocimetry in the prediction of the development of PE in a high risk population. We compared the peripheral plasma STB EV concentration of women who developed PE to that of women (within the high risk group) who did not develop PE. We observed an increase of the STB EV concentration over the time of preg-nancy in the PE group and the control group, but did not detect an significant dif-ference between the two groups. This is in contrast to other studies, that found a significant increase of STB EV in PE [11,16]. The primary outcome of our study was a comparison of the peripheral plasma STB EV concentration at the 20th week of gestation as an accessory tool for PE prediction before PE symptoms occur. There-fore, we included only STB EV concentrations of specimens to our study before PE was diagnosed, while other groups measured the STB EV concentration after PE symptoms had occurred. Although STB EV are secreted from the placenta during nearly the whole pregnancy [2], it may be possible that the PE-related increase of the STB EV concentration only accompanies PE symptoms, but does not precede them. Therefore, we might have not been able to find a significant difference in the STB EV concentration before appearance of PE symptoms. Furthermore, more than half of the patients in our PE group (8 out of 15) had to be excluded from the analysis between the 20th and 36th week of gestation because of the appearance of PE-symptoms and associated complications. Therefore, we potentially lost statis-tical power at our 36 weeks samples and it may be possible that we did not see an increased concentration. Since we did not find increased STB EV levels during PE in our study, while STB EV are suggested to be involved in the pathophysiology of PE [3,4], we hypoth-esized that the pathophysiological effects of STB EV in PE might be due to their differences in molecular load in preeclamptic STB EV versus normal STB EV [35] and therefore a different functionality.

The immunologic function of STB MV and exosomes in normal pregnancy and preeclampsia

In chapters 4 and 5, we stimulated whole blood samples from non-preg-nant women with STB EV from normal and preeclamptic placentae to analyze the potential immunologic function of STB EV. We stimulated the blood samples with physiological late pregnancy concentrations of STB EV as measured in the MORE PrePARd study (36th week of gestation, chapter 3). We used STB EV from preec-lamptic women at similar concentrations, which allowed us to focus on potential

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differences in functionality between STB EV from healthy placentae and preeclamp-tic placentae. In chapter 4, we focused on the effects of STB EV on two of the most abun-dant innate immune cell types, monocytes and granulocytes. Both STB MV and ex-osomes from normal placentae induced a shift from CD16— classical monocytes towards CD16+ intermediate monocytes. They also activated classical monocytes, intermediate monocytes and granulocytes. Although STB MV and exosomes from normal placentae induced similar effects on monocytes and granulocytes, the ex-osomal effect was more pronounced. Also STB EV from preeclamptic placentae in-duced a shift from classical to intermediate monocytes and an activation of classical and intermediate monocytes and granulocytes. The effect of STB EV from normal placentae and STB EV from preeclamptic placentae appeared to be similar. Chapter 5 describes the effects of STB EV on lymphocyte subpopulations, namely CD8- and CD8+ T cells, natural killer (NK) cells and natural killer T (NKT) cells. STB EV from normal placentae induced an activated phenotype in T cells and mem-ory T cells, especially regulatory T (Treg) cells, as well as the production of perforin and granzyme B by NK and NKT cells. STB EV from normal placentae did not signifi-cantly alter the percentage of T helper (Th) cell subsets 1, 2 and 17. In contrast, STB EV from preeclamptic placentae neither induced an activated phenotype in T cells, memory T cells and Treg cells, nor the expression of perforin and granzyme B by NK and NKT cells. Like STB EV from normal placentae, also STB EV from preeclamptic placentae had no significant effect on Th cell subsets. In our experiments, STB EV from normal placentae induced monocyte mat-uration and an activation of monocytes, granulocytes, CD8- and CD8+ T cells, NK cells and NK T cells. Especially Treg cells and regulatory CD16+CD56++ NK cells were shown to be activated. Our study is in line with the conclusions from other studies, showing that both normal STB MV and exosomes affected the phenotype and ac-tivity of monocytes [9,36,37], and showing that cell culture supernatants from pla-cental explants increased the number and suppressive phenotype of Treg cells [38]. Normal pregnancy has been associated with a systemic inflammatory state [14,18], characterized by an increased number of peripheral monocytes, granulocytes and an increased activation of these cells.[39] Also, the number of Treg cells are in-creased in normal pregnancy, next to a predominant type-2 immunity based on a decreased Th1/Th2 ratio and type-2 cytokine production by NK cells and NKT cells.[23–26,40–43] However, also Th1 and especially Th17 cells are active, but strongly regulated during normal pregnancy.[26,44] Our data thus suggest that this pheno-type (i.e., monocyte and granulocyte activation and increased numbers of Treg) is partly induced by STB EV from normal placentae. Interestingly, the STB EV did not affect the distribution of Th1, Th2 and Th17 cells in lymphocytes. Therefore, other factors may be involved in the pregnancy-induced Th2 skewing and decreased Th17 response. Our data suggest that STB EV from normal placentae support the estab-lishment of a tolerant environment by boosting the abilities of Treg, NK cells and

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NKT cells to regulate effector cells by an inflammatory-like phenotype. This sugges-tion is in line with the general idea of a physiological secretion of EV into the blood stream as a mode of communication with target cells.[3–5] Already the endometrium and early placenta are enriched with maternal leucocytes, especially uterine NK cells and macrophages, but their origin is not fully revealed yet – they might be recruited from the periphery or differentiate locally.[39,45] While invading the maternal endometrium, extravillous trophoblasts are getting into contact with the maternal endometrial leucocytes and are protected from an immune attack by the expression of MHC class I molecules.[46–48] Since the villous trophoblast does not have this protection through MHC class I mole-cules [47], it has been discussed that the secretion of factors such as STB EV might mediate protection of the villous trophoblasts from the maternal immune system. However, in our experiments monocytes did not only get activated by STB EV but also showed signs of differentiation into macrophages (increased granularity and CD11b expression, chapter 4). This indicates that STB EV might recruit monocytes to migrate into the decidua and to refresh the pool of placental macrophages. Ad-ditionally, especially CD16+CD56++ NK cells became activated by STB EV and those cells are potential precursors of CD56++ uterine NK cells.[49] That suggests that STB EV also have a potential role for the local immune reactions in the placental bed and support local tolerance towards the semi-allogenic fetus. However, to our knowledge, there are no studies investigating a direct local function of STB EV in the decidua so far. STB EV from preeclamptic placentae induced activation of monocytes and granulocytes and monocyte maturation nearly to the same extend than STB EV from normal placentae did. However, STB EV from preeclamptic placentae failed to activate T cells, NK cells and NKT cells. Especially the STB MV from preeclamptic placentae seemed to reveal a reduced function compared to STB EV from normal placentae, but there was no significant difference between preeclamptic and nor-mal STB MV. In earlier studies, STB EV have been associated with the disturbed immune reactions [14] and endothelial dysfunction [16] during PE. PE features an exaggeration of the pregnancy-related inflammatory state, apparent by a strong-er activation of monocytes and granulocytes, downregulation of Treg cells, and an increase of the Th1/Th2 ratio combined with a stronger activity of Th17 cells and type-1 cytokine production by NK cells and NKT cells.[23–26] Although PE features a pathologic increase of inflammatory signs, STB EV from preeclamptic placentae did not have significantly different effects on inflammatory cells compared with STB EV from normal placentae in our studies. However, the STB EV from preeclamp-tic placentae also did not induce the regulatory, protective phenotype in lympho-cytes which was induced by STB EV from normal placentae. They were not able to induce immunoregulation, especially by Treg and regulatory CD16+CD56++ NK cells, to the same level than STB EV from normal placentae did. Also, despite the fact that the inflammatory cells are more activated during PE, the STB EV from

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preeclamptic placentae did not induce more activation in monocytes and granulo-cytes. We stimulated whole blood of non-pregnant women with similar amounts of STB EV from normal and preeclamptic placentae, since we were interested in functional differences, although some studies showed an increased number of STB EV during PE [11].Therefore, from the present study, we cannot rule out an effect of the increased number of STB EV in PE. However, we speculate that the exaggerated inflammatory state in PE might not be induced by a direct effect of STB EV on mono-cytes and granulocytes. Rather it results from an indirect effect of the function loss of STB EV from preeclamptic placentae, especially STB MV, which are not able to induce the protective immunoregulation (especially on Treg and CD16+CD56++ NK cells) anymore. This may then result in activation of monocytes and granulocytes. The direct comparison of the immunologic effects of STB MV and exosomes from normal placentae in chapters 4 and 5 revealed that both STB EV subtypes have similar immunologic functions, and are not necessarily opponents. In our experi-ments, STB MV and exosomes were isolated from placental perfusates. The ratio of the concentrations of the STB MV and exosomes that we used for stimulation was similar to the STB MV/exosome ratio produced by the placenta. Our own pre-liminary, unpublished data suggest that the STB MV/exosome ratio is nearly 1 in suspensions from ex vivo placenta perfusion. We assume that this ratio also reflects the physiologic STB MV/exosome ratio in plasma during pregnancy. In contrast to what has been thought about the function of STB MV and exosomes, we found that STB MV and exosomes did not perform different functions. Both STB MV and exosomes from normal placentae activated monocytes, granulocytes, T cells, NK cells and NK cells, and induced an activated phenotype in T cells, memory T cells and Treg cells. Based on the phenotypes of the immune cell subsets observed in this thesis, we suggest that both STB MV and exosomes from normal placentae have similar immunologic functions, engage in the systemic inflammatory state during normal pregnancy, and support the establishment of a regulatory, tolerance-induc-ing phenotype in immune cells. Although mediating similar effects, the effect of exosomes was more prominent than the effect of STB MV. This might be a feature of a (partially) different molecular load of STB MV and exosomes. To our knowledge, no study has been published so far which directly compares the molecular load of STB MV and exosomes. However, it has been shown that specific markers exist for MV (exposure of phosphatidylserines, selectins, integrins) and for exosomes (e.g. CD9, CD63, CD81).[3,5,7,50] Therefore, it is reasonable to assume that the molecu-lar load of both vesicle types differs also in further parts. As the STB MV/exosomes ratio in our perfusion suspensions was about 1, a potential superior number of STB exosomes over STB MV cannot be the cause of the stronger exosomal effect. Also for the vesicles of preeclamptic placentae, STB MV and exosomes appeared to have similar effects on the various immune cells, although the effects of the ex-osomes appeared more pronounced. Potentially, STB MV from preeclamptic pla-centae are more effected by the described loss of function than STB exosomes from

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preeclamptic placentae. The mode in which STB EV mediate their effects on immune cells has not been completely clarified yet. It has been shown earlier that monocytes phagocyt-ize STB MV, while this has been barley shown for the other immune cell subsets.[7,27] In our studies, only whole blood stimulations have been performed to mimic physiologic conditions as close as possible. We got an impression of how immune cells may respond to STB EV. However, we cannot judge whether all identified cell subsets actually directly interact with STB EV or some effects are merely down-stream consequences of the influences on other cells. It has been shown that STB EV induce cytokine production in monocytes [9], and these could be potentially involved in a further activation of lymphocytes. Especially dendritic cells are a major player in the adaptive immune system and very important for the regulation of T cell responses.[51] In pregnancy, dendritic cell function seems to be suppressed to avoid an activation of the T cell response against the fetus.[51] The exact mode of the suppression of dendritic cells during pregnancy is not fully clarified yet. How-ever, since we observed activation of T cells in response to STB EV stimulation of whole blood, an involvement of dendritic cells cannot be ruled out and may be an interesting subject for future studies.

The pro-coagulant function of STB MV and exosomes in normal pregnancy and preeclampsia

In chapter 6, we assessed the pro-coagulant function of STB EV by analyz-ing the effects of suspension from ex vivo placenta perfusion on thrombin forma-tion and platelet aggregation. We showed that perfusion suspension from normal placentae induced a low thrombin formation and a low platelet aggregation at a variable, individual aggregation rate. As an artificial model for PE [52], we perfused normal placentae with low oxygen contents. Suspension from these low-oxygen perfusions induced a high thrombin formation and a strongly dysregulated platelet aggregation at a low aggregation rate. By stepwise exclusion of cell debris/macrove-sicles, STB MV and exosomes from the low-oxygen perfusion suspension, we could ascribe the described effects to STB MV and not to exosomes. In this study, we used a different approach to identify the placental factor that induced thrombin formation and platelet aggregation. (1) To study the pro-co-agulant activity from healthy and preeclamptic placentae we used a placental mod-el for PE: i.e. the hypoxic placenta. As described by Jain et al., the perfusion of normal placentae with low oxygen contents induces a PE-like reaction of the placen-tal tissue with increased levels of inflammatory cytokines.[52] Since preeclamptic placentae are rare (PE occurs only in 2-8 % of pregnancies worldwide, even less in developed countries [53]), we thus decided to use this model for the placenta of PE patients. Normal placentae were ex vivo perfused with low oxygen contents to investigate the coagulant impact of preeclamptic placental factors. (2) We first

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analyzed the coagulant impact of total suspension after normoxic and low oxygen ex vivo placenta perfusion. After we saw that the suspension from low oxygen per-fusion features a very different coagulant impact than the suspension from normox-ic perfusion, we wanted to figure out which factor in the solution is actually causing the observed effects. The placenta is known to produce a plethora of factors, not only STB EV, but also soluble molecules like the pro-coagulant proteins plasmino-gen activator inhibitor type 2 or tissue factor [21]. In contrast to our immunologi-cal studies, we decided not to test the coagulant effects of isolated STB MV or ex-osomes, but rather to stepwise exclude the single STB EV fractions (STB cell debris/macrovesicles, STB MV and STB exosomes) from the perfusion suspension and to test the effect of the remaining suspension. Since we already knew the effect of the whole mixture of placental factors, we wanted to see whether the stepwise ex-clusion of the single factors could partially or completely reverse the effects of the complete perfusion suspension. This way, we assumed to get a better impression of the effect of the single STB EV fractions and of a potentially complementary effect of the single factors. Samples of suspension from ex vivo perfusion of normal placentae induced low thrombin formation and high platelet aggregation. Normal pregnancy is char-acterized by hypercoagulability represented by an increase of pro-coagulant factors (e.g. thrombin, fibrin, von Willebrand factor) without compensation by anti-coag-ulant factors (e.g. anti-thrombin III).[19–22] This hypercoagulability is most likely induced to avoid premature bleeding.[19,20] Our data suggest that the placenta supports the controlled hypercoagulability of normal pregnancy. STB EV might en-gage in this hypercoagulable state by inducing coagulation based on the exposure of phosphatidylserines or tissue factor on their surface to regulate coagulation on a controlled level during pregnancy.[7,54,55] Due to the relative low effects of STB EV from healthy placentae we did not perform stepwise exclusion of cell debris/mac-rovesicle, STB MV and exosomes in the samples of the healthy placentae. Therefore we cannot affiliate the observed effects to any subtype of STB EV. In contrast to normal placenta perfusion suspension, the low-oxygen pla-cental perfusion suspension induced a strong thrombin formation and deregula-tion of platelet aggregation. With stepwise exclusion of cell debris/macrovesicles, STB MV and exosomes from this low-oxygen suspension, the strong pro-coagulant effects disappeared after exclusion of STB MV. Since the perfusion suspension de-pleted from STB MV still included exosomes and soluble placental factors, we as-cribed the pro-coagulant function of PE-like STB EV mainly to the STB MV fraction, and not to the exosome fraction. Potentially, STB MV also may have induced the pro-coagulant effect in the normoxic perfusion suspension, but the lack of oxygen during the ex vivo placenta perfusion altered the functionality of the STB MV low oxygen perfusion suspension compared to normal perfusion suspension. PE is as-sociated with a deregulation of hemostasis, represented for example by coagulopa-thies or thrombocytopenia.[53] Our data suggest that preeclamptic STB MV might

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contribute to the impaired hemostasis during PE, although we used an artificial model of PE to obtain our data. In PE, the placenta suffers from oxidative stress [56] which may activate the STB and influence the secretion of STB MV. The different effects of STB MV and exosomes on coagulation may be ex-plained by a different molecular load of STB MV and exosomes (e.g. tissue factor or phosphatidylserine). Since the release of phosphatidylserine-exposing MV is a feature of activated or apoptotic cells [5], the release of MV from the oxidative-ly stressed preeclamptic STB may lead to an increased exposure of phosphatidyl-serines due to a stronger turnover of the membranes during STB MV formation. Furthermore, predominantly STB MV expose phosphatidylserines on their surface, while only a little fraction of exosomes exposes phosphatidylserines.[7,54] The ex-posure of phosphatidylserines encourages coagulation by binding to coagulation factors, and supports activation of coagulation factors (e.g. thrombin as shown in our experiments) and platelet aggregation.[15,57] Also tissue factor, which may play a role in coagulation, was discovered on STB EV.[55] However, this study was performed on a 150,000 g fraction of EV after centrifugation of suspension from ex vivo placenta perfusion.[55] At 150,000 g both MV and exosomes are pelleted together and thus it is not clear whether the tissue factor was exposed on both STB MV and exosomes or only STB MV. Additionally, our data are in line with earlier studies that described an inhibition of platelet aggregation by normal chorioepi-thelial brush border membrane vesicles and basal plasma membrane vesicles, but these studies also did not identify which EV type caused these effects.[58,59] The described hemostatic effects of STB MV but not exosomes also support the hypoth-esis of a different, maybe even opposite function of STB MV and exosomes.

How can the functions of syncytiotrophoblast microvesicles and exosomes in nor-mal pregnancy and preeclampsia be aligned?

As discussed before, the overall impression of the function of STB MV and exosomes in normal pregnancy and PE appears to be very static. Often, STB MV are believed to be rather activating and strongly related to the pathogenesis of PE, while exosomes are believed to be rather tolerance-inducing. The results of this thesis suggest that this view is too simplistic. The functional similarities and differences of STB MV and exosomes seem to be strongly dependent on the physiologic compart-ment in which they are assessed, e.g. on immune cells or hemostasis. While STB MV and exosomes seem to have a similar effect on the immune system, only STB MV seem to have a strong regulatory effect on the coagulation compared to exosomes. Furthermore, the functionality of STB EV from normal and preeclamptic placentae indeed seems to differ from each other. STB EV from normal placentae seem to in-duce a regulated, tolerance-inducing immune reaction and the inflammatory state of normal pregnancy. They also appear to support the regulated hypercoagulable state of pregnancy, which prevents premature bleeding or excessive post-partum

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bleeding. In contrast, STB EV from preeclamptic placentae seem to have lost some of the functions of STB EV from normal placentae. In our studies, STB EV from preeclamptic placentae failed to induce a regulated, potentially protective, immune reaction and even caused a strong deregulation of coagulant functions. The results of this thesis may offer new perspectives on the function of STB MV and exosomes in normal pregnancy and PE and initiate novel approaches to the topic.

Future perspectives

We neither completely understand yet how the maternal immune system tolerizes fetal antigens nor how disturbances in the maternal immune system are triggered in PE. However, it is generally agreed that STB EV are associated with the pathophysiology of PE and evidence is rising that they are also more important in normal pregnancy than expected earlier. This is also apparent by the results achieved in this thesis, i.e. the STB EV from the normal placenta induce immuno-logical changes in whole blood of non-pregnant women which are also seen during pregnancy. To further the understanding of the differential functions of STB MV and exosomes and the effects of STB EV from normal and PE placentae, a deeper understanding of the molecular mechanisms underlying the interaction of STB EV with their target cells is needed. This thesis and other studies indicate an interaction of STB EV with certain immune cell subsets and the hemostatic system. However, especially in the case of immune cells, it is not clear whether STB EV directly attach to or are phagocytized by the affected cells or if the activation of the influenced cells is just a consequence of the signaling of other immune cell subsets. Thus, an identification of cell subsets which actually take-up STB EV and the way of the uptake (e.g. clathrin-mediated or caveolin-dependent endocytosis, micropinocytosis, phagocytosis, lipid rafts or cell surface membrane fusion [60]) may improve our understanding of the mecha-nisms by which STB EV affect other cells. In this thesis, cell stimulations have been performed in whole blood to align as close as possible to physiologic conditions. Based on these results, cell types which were affected by STB EV might be isolated and their direct interaction with STB EV may be investigated in more depth in future studies. Furthermore, it may be advisable to also analyze the effects of STB MV and exosomes together and to compare them to the separate investigations of this the-sis to assess a potentially synergistic effect of STB MV and exosomes. Additionally, a systematic comparison of the molecular load of STB MV and exosomes and of STB EV from normal and preeclamptic placentae may explain differences in their func-tionalities. When comparing the effects of STB MV and exosomes, it is necessary to reflect on difficulties in the separation of these vesicle types from each other. The isolation of MV and exosomes from fluid samples via a protocol based on sequen-tial centrifugation and ultra-centrifugation is a widely accepted method.[3,5,54]

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However, contamination of either vesicle fraction with bigger or smaller vesicles cannot completely be ruled out. Also unpublished nanoparticle tracking analysis data from our group indicate a certain low contamination of STB MV with exosomes and vice versa. As described by Théry et al., exosome purity can be improved by a further ultra-centrifugation on a sucrose gradient or by isolation with immuno-la-beled magnetic beads.[54] A comprehensive comparison of the molecular load of STB MV and exosomes is currently lacking, but may support the search for specific markers for either STB EV fraction and thus also a more specific isolation e.g. via immuno-magnetic separation as suggested by Théry et al. Till date, no effective treatment, other than pregnancy termination, has been found to cure PE. A better understanding of the role of STB EV in normal preg-nancy and PE may open new avenues to successful treatment of PE. A comparison of the molecular load of STB EV from normal and preeclamptic placentae may yield in potential targets for an interaction with the signaling pathways of STB EV and targets for PE therapy. Based on the results of this thesis, we suggested that STB EV potentially suffer from a partial loss of immunologic function and deregulation of hemostatic function in PE compared to normal pregnancy. PE treatment may profit from an induction or inhibition of STB MV or exosomes or both STB EV subgroups as a compensation for the impaired function in PE. To explore the potential benefits of either of these options, an improved understanding of the molecular mechanisms of the STB MV and exosome function is inevitable. A better understanding of the mechanism of the STB EV signaling will not only support obstetrical research but may also be very beneficial for the wider fields of immunology and extracellular vesicles research.

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References

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