University of Groningen

Role of fetoplacental exosomes in fetoplacental endothelial dysfunction in gestationaldiabetes mellitusSáez Gutiérrez, Tamara Andrea

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Gestational diabetes mellitus (GDM) is a pregnancy complication, which is first recognized during the second trimester of pregnancy (American Diabetes Association, 2017). It is characterized by maternal hyperglycemia and fetal hyperglycemia and hyperinsulinemia. Patients with GDM are treated with diet or insulin to reduce complications, such as macrosomia (Kgosidialwa et al, 2015; Sobrevia et al, 2015). However, despite this treatment, children from mothers who were diagnosed with GDM, have a higher risk to develop metabolic complications in adult life, such as obesity or cardiovascular diseases (Hiersch & Yogev, 2014). Due to the high worldwide prevalence of this complication, around 5-20% of total pregnancies (American Diabetes Association, 2017), GDM has been studied for many decades to try and understand the cellular mechanisms behind metabolic complications in offspring later in life. Such studies have shown that GDM is associated with endothelial dysfunction of the feto-placental vasculature. Studies performed in human umbilical vein endothelial cells (HUVECs) have shown an increased L-arginine/nitric oxide(NO) signaling pathway, characterized by increased expression of the human cationic amino acid transporter type 1 (hCAT-1) and activity of endothelial nitric oxide synthase (eNOS) as well as decreased NO bioavailability (Leiva et al, 2016). Furthermore, an altered angiogenesis as well as a pro-inflammatory state have been shown in feto-placental tissues from GDM patients (Mordwinkin et al, 2013). All these changes indicate that feto-placental endothelial function is impaired. This may suggest that also fetal endothelial function is impaired and if this endothelial dysfunction is permanent, this may be involved in the cardiovascular and metabolic diseases seen in offspring of mothers with GDM. Studies into the mechanisms of this oeto-placental endothelial dysfunction are therefore important and may result in therapeutic opportunities to treat the long-term complications of the offspring of GDM mothers.Exosomes are extracellular vesicles, which are released from various cell types, including HUVECs and monocytes (de Jong et al, 2012; Tang et al, 2016). Acting as a communication mechanism, they can modulate different cellular functions, such as angiogenesis, cell activation and endothelial cell function in general (Müller, 2012). This may suggest that they also could have a role in the development or progression of endothelial dysfunction, for instance in the feto-placental vasculature. Indeed, exosomes have been associated with the progression of different diseases associated with endothelial dysfunction, for instance

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in pregnancy complications, such as GDM and preeclampsia (Salomon et al, 2016; Pillay et al, 2016). Therefore, in the search for understanding the mechanisms associated with feto-placental endothelial dysfunction in GDM, exosomes may be a potential mechanism. In this thesis, therefore, we propose altered exosomes or exosome production from the feto-placental vasculature in GDM, which may modulate endothelial function in the feto-placental vasculature in GDM. In this thesis, we used HUVECs as a model for endothelial cells in the fetal-placental vasculature and.we used high D-glucose treated HUVECs as a model for GDM HUVECs. We determined the role of hyperglycemia and tumor necrosis factor alpha (TNF-α) in the vascular dysfunction, and we also isolated exosomes from HUVECs and monocytes, which were exposed to high D-glucose and/or TNF-α. We evaluated the effect of these exosomes on L-arginine/NO signaling pathway, endothelial cell migration and endothelial cell activation. Since our experiments showed a potential role for exosomes in feto-placental endothelial dysfunction in GDM, in the final chapter we isolated endothelial exosomes derived from HUVECs from GDM and normal pregnancies to evaluate their role in the cellular changes associated with foeto-placental endothelial dysfunction in HUVECs. Our approach would help to understand the mechanism of induction and progression of feto-placental endothelial dysfunction in GDM.

Isolation and characterization of exosomesIn this study, we isolated exosomes based on the study of Théry et al (Théry et al, 2006). Briefly, exosomes were isolated after 24 h incubation of HUVECs (or monocytes) with exosome-free medium culture. The culture medium was filtered and several steps of centrifugation and ultracentrifugation were used. The resulting pellet was used to isolate and purify exosomes from other extracellular vesicles by sucrose gradient, where the floating density was measured. This protocol was used since we were specifically interested in exosomes rather then other extracellular vesicles. In this case, it is important to use a sucrose gradient to isolate exosomes from the other extracellular vesicles (Théry et al, 2006). Exosomes are extracellular vesicles formed by a regulated cellular process, while microvesicles are released by outward budding of the plasma membrane (Huang-Doran et al, 2017), suggesting different roles in cell communication. All fractions obtained after sucrose gradient isolation were washed with PBS. We showed that only fraction 11 contained exosomes, since only this fraction was positive for exosomal markers in westernblotting

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(TSG-101 or CD63 or CD81). Also nanoparticle tracking analysis (NTA) showed that this fraction contained exosomes, since the size of the particles in this fraction was between 40 to no more than 200 nm, depending on the origin cell (Chapter 3 and 5).

Role of exosomes in high glucose-induced feto-placental endothelial dysfunction Fetal and maternal hyperglycemia is one of the main characteristics of GDM (American Diabetes Association, 2017). We and others have shown that hyperglycemia can be involved in inducing endothelial dysfunction in GDM, since it has been shown that HUVECs from healthy pregnant women, exposed to high levels of D-glucose (HG, 25 mmol/L) showed impaired endothelial function similar to HUVECs from patients with GDM. This was characterized by increased L-arginine transport, eNOS activity, hCAT-1 and vascular endothelial growth factor (VEGF) expression (Chapter 3; Vásquez et al 2007). Moreover, HG also induces the expression of intracellular adhesion molecule type 1 (ICAM-1) in HUVECs (Chapter 3; Piconi et al, 2004). The question arose whether these effects of high glucose on endothelial function in HUVECs were mediated by exosomes. To study the role of exosomes in the high glucose-induced endothelial cell dysfunction/activation, in chapter 3 we isolated exosomes from HUVECs incubated with high glucose or basal glucose (5 mmol/L). These exosomes were then incubated with HUVECs incubated with high or basal glucose and effects on eNOS, hCAT-1, VEGF, endothelial cell migration and activation were studied.We first characterized the exosomes. HG increased the concentration of exosomes released from HUVECs. This is in line with the results from Rice et al (Rice et al 2015), who showed that the numbers of exosomes derived from trophoblast cells exposed to HG are also increased as compared with incubation of trophoblast with basal glucose conditions. Also studies using glomerular endothelial cells under HG conditions showed increased production of exosomes (Wu et al, 2016). Cellular stress, such as HG, is known to increase the release and change the content of exosomes (de Jong et al, 2012). The exact mechanism by which high levels of D-glucose induced increased exosome release is unknown. However, high glucose may induce the activation of mitogen-activated protein kinase (MAPK), especially the MEK/ERK signaling pathway (Shang et al, 2017)..This may be involved in increased exosomes release, since exosome release can be modulated by increased MAPK activity, especially MEK signaling (Agarwal et al, 2015).

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Then, we demonstrated that endothelial exosomes derived from HUVECs exposed to HG can induce a similar increased endothelial cell migration, increased activation of eNOS, and increased endothelial cell activation as compared with incubation with high glucose. However, these exosomes could not mimic the increased VEGF or hCAT-1 expression induced by high glucose. Furthermore, exosomes derived from HUVECs incubated under basal glucose levels seem to restore the endothelial cell migration and activated eNOS in HUVECs from HG, towards values seen in HUVECs incubated under basal glucose. Interestingly, exosomes increased the expression of ICAM-1 in HUVECs, suggesting that exosomes could also be involved in endothelial cell activation. Also other studies have shown effects of extracellular vesicles on endothelial ICAM-1 expression (Jansen et al, 2015). This study, however, used microvesicles, rather then exosomes, which were shown to have an anti-inflammatory effect.Our results suggest that exosomes can mediate part of the endothelial dysfunction induced by high glucose in HUVECs. However, the mechanisms by which the exosomes induced these effects are not exactly known. Since our data indicate that exosomes isolated from HUVECs under high or low glucose have different effects on endothelial function, it seems that HG modifies the exosomal cargo. This suggestion is in line with data from de Jong et al, who showed that HG indeed modified, although not massively, exosomal protein cargo from endothelial cells (de Jong et al 2012). Exosomes also contain miRNAs (Huang-Doran et al, 2017) and HG may also change the miRNA content of exosomes, although no studies are available to substantiate this hypothesis. One of these differential miRNAs may be miR-15a, since, miR-15a is increased in plasma of diabetic patients. This miRNA has also been found in exosomes derived from pancreatic β-cells exposed to HG (Kamalden et al, 2017). These pancreatic exosomes may be involved in induction of diabetic complications by inducing oxidative stress in various tissues (Kamalden et al, 2017). Another miRNA, which may be differently expressed in exosomes from high and basal glucose HUVECs, may be miR-137. Down-regulation of this miRNA in HUVECs, suppressed the changes induced by HG in these cells (Li et al, 2016). Our data suggest that it is important in future research to study the cargo of the exosomes induced by HG and BG in HUVECs in search of proteins and miRNAs as potential therapeutic targets to interfere with the effects of GDM in the offspring. It is also not exactly known, how exosomes can be taken up or internalized into the cells.

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The interaction between proteins in exosome membrane and cellular receptors on cellular membrane of the target cells cause a fusion of plasma membranes and release of the exosome cargo into the cells (Colombo et al, 2013; Huang-Doran et al, 2017). Whether the interaction of exosomes with target cells differs between exosomes derived from HUVECs HG or BG is unknown and should be subject of future studies Endothelial dysfunction in HUVECs induced by high glucose thus seems to be mediated by exosomes. However, exosomes isolated from HUVECs incubated with HG cannot mimic the complete high glucose phenotype of HUVECs. Therefore, also other factors seem to play a role in the high glucose induced endothelial dysfunction. Next to exosomes also the larger microvesicles could play a role. However, pro-inflammatory factors, which are present in the fetal placental circulation (Lim et al, 2017; Marseille-Tremblay et al 2008) and which are known to induce endothelial dysfunction, may also be a likely candidate. Therefore, in chapter 4 we focussed on the role of the proinflammatory TNF-α in inducing endothelial dysfunction in HUVECs and tried to answer the following question:

Can TNF-α, with or without high glucose, induce endothelial dysfunction similar to endothelial dysfunction in GDM? And do exosomes play a role? TNF-α is one of the main pro-inflammatory factors (Bradley, 2008). Since TNF-α can modulate the L-arginine/NO signaling pathway by modulation of the expression of eNOS (Zhang et al 2008) and hCAT-1 (Visigalli et al 2004) and can also modulate VEGF and ICAM-1 expression (Yoshida et al 1997; Morzycki et al, 1990), we hypothesized that TNF-α can be another candidate inducing feto-placental endothelial dysfunction in GDM. Therefore in chapter 4 we first evaluated the role of TNF-α alone or together with HG on endothelial cell dysfunction and exosome release. In contrast to HG, TNF-α did not modify the concentration of exosomes from supernantants of HUVECs. However, the coincubation of TNF-α with HG, largely decreased the concentration of exosomes in the supernatant of HUVECs as compared with incubation with HG alone. Since TNF-α induces MAPK signaling (Miller et al, 2005), whether the decreased production of exosomes after coincubation of HG with TNF-α is induced via MAPK signaling remains to be established. Although incubation of HUVECs with HG alone increased endothelial cell migration and incubation of HUVECs with TNF-α did not affect

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endothelial cell migration, the co-incubation of HUVECs with HG and TNF-α decreased endothelial cell migration (Chapter 4). In other studies, TNF-α was able to induce angiogenesis (Yoshida et al 1997, Kim et al, 2017). Differences between our study and the Yoshida and Kim study may be the concentration of TNF-α and different methods used to measure angiogenesis. Our data are in line with the study of Lim et al, who showed that the coincubation of TNF-α and HG impaired angiogenesis (Lim et al, 2017). Although TNF-α affected eNOS, VEGF and ICAM-1 expression in HUVECs, exosomes were not involved in inducing these effects of TNF-α on HUVECs. Although in our study the effects of TNF-α seemed not to depend on exosomes, other studies have shown that TNF-α can induce the production of exosomes which are able to affect other cells, since a study by Lu et al showed that exosomes derived from mesenchymal stem cells pre-incubated with TNF-α, promoted cell proliferation and pro-inflammatory changes in human primary osteoblastic cells (Lu et al, 2017). The effect of TNF-α on exosomes may therefore be cell dependent. Exosomes derived from HUVECs incubated with TNF-α and HG increased ICAM-1 expression in basal glucose cells. It may be speculated that TNF-α induced TNF-α production in HUVECs, which may be packaged into the exosomes. Packaging of TNF-α into exosomes has been shown by other studies: exosomes derived for dendritic cells cause inflammation in HUVECs by carrying TNF-α in exosomal membrane (Gao et al, 2016); also exosomes derived from cardiomyocytes under hypoxic condition, contain TNF-α (Yu et al, 2012). Most effects of TNF-α in our study were observed on endothelial cell activation. This effect of TNF-α is not surprising, since TNF-α is a well-known activator of endothelial cells and immune cells, involving the activation of p38, ERK and NF-kappaB signaling pathways (Nizamutdinova et al, 2007). Interestingly, although de Jong et al showed that TNF-α changed the exosomal cargo (de Jong et al 2012), in our experiments, exosomes were not involved in TNF-α induced increased ICAM-1 expression in HUVECs. The present data show that high glucose and proinflammatory factors, like TNF-α, may play a role in the feto-placental endothelial dysfunction and activation in GDM. Together they may be involved in impaired angiogenesis. Further, high glucose may induce endothelial dysfunction, via dysregulation of the NO signaling pathway, while TNF-α may induce endothelial cell activation in GDM. Exosomes may play an important role in inducing the effects of high glucose,however, the present chapter does not suggest an important role for exosomes

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in the effects of TNF-α on endothelial cells.

Do HUVECs and monocytes communicate with exosomes under high glucose conditions?. As we described above, GDM is associated with hyperglycemia and a pro-inflammatory state in foeto-placental tissue. In chapter 4, we showed that TNF-α may play a role in the endothelial activation in the feto-placental vasculature in this condition. As TNF-α is mainly produced by monocytes (Gane et al, 2016), in this chapter (Chapter 5), we studied the potential role of monocytes in endothelial cell activation under high levels of glucose. Therefore, we first studied whether monocytes were also activated by high D-glucose. We used the monomac-6 cell line (MM6) in this study. This is a monocyte cell line, showing phenotypic and functional features of mature monocytes (Ziegler-Heitbrock, et al 1988). In line with the findings in HUVECs (chapter 3), high glucose also increased ICAM-1 expression in MM6. As HG induces the secretion of TNF-α by monocytes (Moroshi et al 1995), and TNF- is one of the main factors inducing ICAM-1 expression in monocytes (Neuner et al, 1997), it may be suggested that HG induces TNF-α production, which in it’s turn induced ICAM-1 expression in monocytes. In the present chapter, the interaction between MM6 cells and endothelial cells was very obvious in cocultures of MM6 and HUVECs: cocultured cells incubated under high glucose showed increased ICAM-1 expression as compared with monocultured cells under high glucose conditions. This was not the case when cells were incubated under basal glucose conditions. We, and others, have shown an interaction between endothelial cells and monocytes before (Faas et al, 2010; Takahashi et al, 1996). Similar as above, this interaction may be due to high glucose induced TNF-α production. This suggestion needs to be confirmed by measuring cytokines in the supernatants of cocultured and monoculture HUVECs and MM6 cells. However, based on our previous chapter showing a role for exosomes in the high glucose induced endothelial dysfunction and activation, we speculated that in the coculture, exosomes from MM6 and HUVECs collaborate to induce the increased effects of high glucose.To test this hypothesis, we first evaluated whether the effect of glucose on MM6 cells was induced by exosomes, then we studied the effect of exosomes derived from MM6 cells on HUVECs and vice versa and whether this putative effect was dependent on high glucose.In contrast to what was shown for HUVECs in chapter 3, in this chapter we showed that high glucose did not alter the concentration

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of exosomes derived from MM6. However, high glucose increased the size of the exosomes. It has been sown before that the exosome characteristics, including exosomal size, may be affected, depending on environment or cellular stimuli (Bobrie et al, 2012).Then we evaluated whether monocytic exosomes derived from MM6 under HG or BG could affect ICAM-1 expression in MM6 cells. Our results indicate that exosomes are able to induce ICAM-1 expression, however, exosomes from both conditions (BG and HG) increased ICAM-1 in MM6 similarly. These results are similar to our results found in HUVECs (Chapter 3). As exosomes have an important role in cellular communication between different cells (Simons & Raposo, 2009) (Müller, et al 2012), we next determined the effect of exosomes derived from HUVECs under HG or BG condition on MM6 cells. Also in this experiment HUVECs exosomes from BG and HG increased the expression of ICAM-1 in MM6 cells, suggesting communication of HUVECs with monocytes via exosomes. Then, the effect of exosomes derived from MM6 cells under HG or BG was evaluated in HUVECs under the same conditions. Again, exosomes from MM6 under both HG and BG, increased ICAM-1 expression in HUVECs (Chapter 5), suggesting communication of monocytes with HUVECs via exosomes. These results are in line with a recent study: exosomes derived from LPS-activated monocytes induced ICAM-1 expression in HUVECs (Tang et al, 2016) by activation of TLR4 and NF-κB signaling pathways. Whether a similar TLR4 and NF-kB activation is inducing ICAM-1 expression in our experiments remains to be established. These data show that in line with our hypothesis, communication between MM6 and HUVECs via exosomes produced by MM6 and HUVECs in cocultures under HG conditions may be involved in inducing increased ICAM-1 expression seen in coculture vs monoculture.We next studied whether these MM6 and HUVECs exosomes collaborate in inducing their effects on ICAM-1 expression. Our experiments showed that this indeed appeared to be the case, since we found that HUVECs or MM6 cells coincubated with an exosomal mix derived from both cell types incubated under high glucose could mimic the increased ICAM-1 expression seen in cocultures vs monocultures after incubation with HG. Such an effect could not be derived with exosomes from a single cell type. Moreover, the exoMix derived from MM6 and HUVECs under basal glucose was able to decrease ICAM-1 expression in both MM6 and HUVECs cultured with HG (Chapter 5). How these exosomes collaborate remains to be established. It seems likely that both types of exosomes release their cargo into HUVECs

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and MM6 cells and that the cargo of both exosomal types together mediates the final effects.

Role of exosomes derived from HUVECs in fetoplacental endothelial dysfunction associated to GDMPrevious studies from our group showed that HUVECs from GDM pregnancies express an L-Arginine/NO signaling pathway (Westermeier et al, 2015, Leiva et al, 2016). These results were also observed in this thesis (Chapter 6). The maternal plasma concentration is increased in women diagnosed with GDM as compared to healthy women (Salomon et al, 2016), this suggests that probably, fetoplacental exosomes may be altered in GDM. Since, different cellular stress conditions can modulate the endothelial exosome cargo (de Jong et al, 2012), the bioactivity of fetoplacental endothelial exosomes could be altered in GDM. In the previous chapters, we showed that exosomes seem to play a role in endothelial dysfunction in GDM, using a model for GDM, i.e. high glucose treated HUVECs from healthy pregnant women. As shown in chapter 3, HG can mimic the endothelial dysfunction seen in GDM. Based on our previous studies, we speculate that in GDM, endothelial exosomes are released from endothelial cells in increased amounts or with a different cargo. These exosomes then induce endothelial dysfunction as characterized by an impaired L-arginine/NO signaling pathway (Sobrevia et al, 2011). To test this hypothesis, in chapter 6 we isolated exosomes from HUVEC derived from GDM patients and incubated these exosomes with HUVECs from healthy pregnant individuals. We will supply this with chapter 6. Evaluating the role of exosomes in these parameters, we found that exosomes derived from GDM increased the L-Arginine/NO signaling pathway in HUVECs from normal pregnancy. This was observed in HUVECs from normal pregnancies exposed to exosomes derived from GDM condition, which increased the transport of L-arginine via an increased hCAT-1 expression. The eNOS expression and NO synthesis were also increased. Despite of the recent evidence that endothelial exosomes could modulate eNOS (Lu et al, 2016), until nowadays no evidence about the role of fetoplacental endothelial exosomes in fetoplacental endothelial dysfunction have been published. Our results are in line with the first results in this thesis, where exosomes from cells exposed to high glucose modulated the endothelial cell migration and eNOS activity, suggesting that the effect found in this chapter can bi associated to high levels of glucose. Moreover, exosomes from GDM

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could be participate in the increased NO found in HUVECs in GDM, but more studies are needed to confirm this hypothesis. On another hand, the effect of GMD on HUVECs is mediated by MAPK activity. Interestingly, despite of the inhibition of this signaling pathway, exosomes can increased the L-Arginine uptake in cells from GDM, suggesting that increased L-arginine transported can be mediated by another different pathway than MAPK. More studies are needed to evaluate the role of these exosomes on cellular mechanisms associated to L-Arginine transport. We hypothesized that exosomes may carry adenosine and by adenosine increasing the L-Arginine transport. This, due to adenosine can modulate the L-Arginine/NO signaling pathway in HUVECs (Guzmán-Gutiérrez et al, 2012) and because exosomes may contain or promote the production of adenosine in another cellular types and different cellular environment (Chapter 1; Clayton et al, 2011; Schuler et al. 2014). Interestingly, exosomes derived from HUVECs of normal pregnancies reversed the effect of GDM in HUVECs (Chapter 6). This study could help to approach exosomes as a potential candidate for futures therapeutic strategy in pregnancy complications.

Future perspectives

Based on the results from this thesis, it is tempting to speculate that exosomes produced by feto-placental endothelial cells and monocytes are important in modulating feto-placental endothelial (dys)function (figure 1). Although this thesis showed that the exosomes are affected by high glucose, while TNF-α may not affect HUVECs exosomes,. Various questions remain to be answered. For instance, it remains to be established whether TNF-α affects monocyte exosomes. Moreover, the ratio between monocytic and endothelial exosomes in the circulation also is unknown at this time and a predominance of one of the exosomal type can make a great difference in the final role of these exosomes in feto-placental endothelial dysfunction in GDM. The communication between endothelial cells and monocytes by exosomes seems to be important in the changes associated to endothelial cell activation, further studies are needed to determinethe role of exosomes from monocytes in endothelial dysfunction the feto-placental vasculature in GDM. Futures studies are also needed to understand the mechanism by which exosomes modulate the endothelial and monocytic changes observed in the present thesis. It is necessary to evaluate the exosomal cargo from exosomes derived from endothelial cells and monocytes,

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to determine whether which part of the cargo, i.e. miRNA, proteins or mRNAs, is responsible for the effects observed. Such studies may lead to new therapeutic opportunities, especially since the cargo of exosomes derived from cells under basal glucose conditions may revert the effects of high glucose on endothelial cells or monocytes. Additionally, it is also needed to evaluate how exosomes modulate the changes observed in this thesis. For instance, whether the effect of exosomes is mediated by the release of exosomal cargo into the cells or by the interaction of the exosomes with the cell membrane of the target cells. Finally, it is important to evaluate the downstream signaling pathways inducing the effects of the exosomes. And to evaluate the pathways leading to exosomal release and packaging and whether these are different in HUVECs from GDM patients compared with HUVECs from healthy pregnant women.

Figure 1: Proposed model for the role of exosomes in foeto-placental endothelial dysfunction in GDM. GDM may alter feto-placental exosomes (number and size) and these exosomes could affect the L-Arginine/NO signaling pathway and endothelial cell activation, probably by a MAPK-AKT signaling pathway mechanism. The interaction between endothelial cells and monocytes could help to promote changes associated to cellular activation by increasing ICAM-1 expression and participating in feto-placental endothelial dysfunction in GDM.

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