Soluble syndecan-1 and glycosaminoglycans in preeclamptic and normotensive pregnancies

Preeclampsia, an important cause of maternal and fetal morbidity and mortality, is associated with increased sFLT1 levels and with structural and functional damage to the glycocalyx contributing to endothelial dysfunction. We investigated glycocalyx components in relation to preeclampsia in human samples. While soluble syndecan-1 and heparan sulphate were similar in plasma of preeclamptic and normotensive pregnant women, dermatan sulphate was increased and keratan sulphate decreased in preeclamptic women. Dermatan sulphate was correlated with soluble syndecan-1, and inversely correlated with blood pressure and activated partial thromboplastin time. To determine if syndecan-1 was a prerequisite for the sFlt1 induced increase in blood pressure in mice we studied the effect of sFlt1 on blood pressure and vascular contractile responses in syndecan-1 deficient and wild type male mice. The classical sFlt1 induced rise in blood pressure was absent in syndecan-1 deficient mice indicating that syndecan-1 is a prerequisite for sFlt1 induced increase in blood pressure central to preeclampsia. The results show that an interplay between syndecan-1 and dermatan sulphate contributes to sFlt1 induced blood pressure elevation in pre-eclampsia.

The endothelial glycocalyx that covers the vascular wall is in direct contact with the blood flow. It consists of a complex network of membrane-bound proteoglycans and attached negatively-charged glycosaminoglycans (GAGs) that regulate various vascular functions such as permeability, leucocyte adhesion, coagulation and vascular tone by mediating shear-dependent nitric oxide (NO) release 1 and facilitates VEGF signalling 2 .
Scavenging of vascular endothelial growth factor (VEGF) in the maternal circulation by its soluble receptor, vascular endothelial growth factor receptor 1 (sFLT1), contributes to endothelial dysfunction in preeclampsia 3,4 . Preeclampsia is defined by de novo hypertension and proteinuria during pregnancy and is an important cause of maternal and fetal morbidity and mortality 5 . Preeclampsia may trigger complex disorders in the endogenous coagulative pathways resulting in increased activated Partial Thromboplastin Time (aPTT) 6 .
In preeclampsia, excess sFLT1 is produced by the placenta, possibly in response to hypoxia that results from inadequate placentation 7,8 .
Syndecan-1 is the most abundant member of the syndecan family of proteoglycans that is also abundantly expressed in the syncytiotrophoblast layer in the chorionic villi of the human placenta 9,10 . The syndecan-1 signalling function is in part determined by the associated GAGs including heparan sulphate and dermatan sulphate 10 . Syndecan-1 regulates VEGF signalling by formation of a complex with the type 2 vascular endothelial growth factor receptor (VEGFR-2), thereby modulating VEGF-induced motility and migration of endothelial cells 11 . The binding of VEGF to VEGFR-2 is mediated by heparan sulphate that acts as a co-receptor 12,13 and depletion of endothelial cell surface heparan sulphate results in reduced phosphorylation of VEGFR-2 14  www.nature.com/scientificreports/ also possess a binding domain for sFLT1 serving as reservoir and limiting excess placental sFLT1 release into the circulation 15 . Proteolytic shedding of transmembrane syndecan-1 yields (plasma) soluble syndecan-1, which retains its ability to interact with GAGs and growth factors in the circulation 16,17 . Placental SDC1 expression has been shown to be diminished in preeclampsia 14,15,18,19 , and reduced soluble syndecan-1 at mid-pregnancy before the clinical onset of preeclampsia 20 . We hypothesize that the interaction between soluble syndecan-1 and its associated GAGs synergistically contributes to sFLT1-induced BP elevation characteristic of preeclampsia. In the present study, we assess whether circulating amounts of soluble syndecan-1 and associated GAGs are altered in preeclampsia compared to normotensive pregnancies and whether Sdc1 deficient mice have a differential BP response to sFlt1 compared to wild type controls.

Results
Clinical characteristics of participants. A total of 125 pregnant women, 65 preeclamptic and 60 normotensive, were included. Clinical characteristics of pregnant women with and without preeclampsia are summarized in Table 1. HELLP syndrome (Hemolysis Elevated Liver enzymes and Low Platelets) was present in 18 (28%) women with preeclampsia. Magnesiumsulphate (MgSO 4 ) was administered to 26 (40%) and antihypertensive treatment to 47 (72%) preeclamptic women. Of all women treated with antihypertensive medication, 40 (85%) were treated with a calcium-antagonist (nifedipine retard), 15 (32%) received a combined α1 and β-blocking agent (labetalol), 36 (77%) received a central α1 agonist (methyldopa) and 2 (4%) were treated with a selective serotonine 5-HT2-antagonist (ketanserin). Eleven (17%) women with preeclampsia were not treated with any type of antihypertensive medication, of these women, one received MgSO 4 prior to delivery. There was no difference in gestational age at delivery between normotensive and preeclamptic women (239 ± 27 days vs 236 ± 27, p = 0.47). Part of the normotensive women were admitted for imminent spontaneous preterm labor. A calcium antagonist (nifedipine retard) was administered to 9 (15%) normotensive women as a tocolytic agent to delay imminent preterm labor.
Glycosaminoglycans in preeclamptic and normotensive pregnancy and at 3 months postpartum. Plasma heparan sulphate concentrations in women with preeclampsia were similar to normotensive women prior to delivery (N = 14-11, p = 0.36) and significantly decreased at 3 months postpartum, (Fig. 1A). Plasma dermatan sulphate concentrations were significantly higher in preeclamptic women compared to nor-  Fig. 1B). In both groups, dermatan sulphate levels decreased to a similar extent 3 months postpartum (N = 3-2). In contrast, plasma keratan sulphate was decreased in preeclamptic women compared to normotensive controls (N = 14-11, p = 0.01) prior to delivery with comparable levels postpartum (N = 3-2, Fig. 1C).
Soluble syndecan-1 is inversely correlated with blood pressure in preeclamptic women. In preeclamptic women, soluble syndecan-1 was inversely correlated with BP showing higher BP values with decreasing plasma concentrations of soluble syndecan-1 ( Fig. 2A). This association was also evident from the number of required antihypertensive medication, which tends to be lower in women with higher soluble syndecan-1 values (Fig. 2B). This trend however, was not statistically significant. In normotensive pregnant women, soluble syndecan-1 was not correlated with systolic BP (r = 0.05, p = 0.75).  www.nature.com/scientificreports/ Dermatan sulphate is strongly correlated with soluble Syndecan-1 and blood pressure in preeclamptic women. Plasma heparan sulphate and dermatan sulphate were strongly correlated with soluble syndecan-1 in women with preeclampsia ( Fig. 3A,B). In contrast, dermatan sulphate was inversely correlated with systolic blood pressure in women with preeclampsia compared to normotensive women (Fig. 3C), but not in normotensive pregnant women (r = 0.46, p = 0.25). Interestingly, the relatively low dermatan sulphate levels associated with increased systolic blood pressure also correlated with high activated Partial Thromboplastin Time (aPTT) (Fig. 3D).

Discussion
In the present study, we show that in contrast to normotensive pregnant controls, circulating dermatan sulphate is significantly increased in pre-eclampsia and strongly correlates with soluble syndecan-1 in preeclamptic women. In preeclampsia, both soluble syndecan-1 and dermatan sulphate are inversely correlated with blood pressure www.nature.com/scientificreports/ (BP). In addition, sFlt1 induces BP elevation in wild type mice, but not in syndecan-1 deficient mice, indicating that syndecan-1 may be an important mediator in sFLT1 induced blood pressure elevation in preeclampsia and anti-angiogenic hypertension. The circulating soluble syndecan-1 levels we measure are similar to those reported previously 19,21 , but in contrast to previous studies, we dit not observe any difference compared to gestationaly aged matched normotensive controls 22 . This discrepancy may be explained by increased gestational age of controls in the previous studies compared to ours 19,21 or by the high incidence of low birthweight (under 10th percentile) in our control group. Kaitu'u-Lino et al., has shown that in pregnancies resulting in the birth of a baby small for gestational age, syndecan-1 levels are significantly lower compared to controls 23 . The gestational age of normotensive controls in the current study was matched to that of the preeclamptic group by including women with preterm labor of unknown cause. Gandley et al. showed that soluble syndecan-1 increases with gestational age, possibly explaining the lower soluble syndecan-1 among normotensive controls in the present study.
Our pilot data confirms the massive decrease of soluble syndecan-1 after delivery in both normotensive and preeclamptic pregnancies reported previously 21 . Due to the small sample size this outcome needs to be confirmed in a larger patient cohort. This finding suggests that in pregnant women soluble syndecan-1 mainly originates from the placenta. SDC1 is expressed on syncytiotrophoblast cells, which are in direct contact with maternal blood 21 . Shedding of syndecan-1 from the syncytiotrophoblast would likely increase soluble syndecan-1 in maternal plasma during pregnancy. Placental expression of SDC1 has been shown to be reduced in preeclampsia by multiple independent studies 14,15,18,19 , possibly explaining the reduced plasma soluble syndecan-1 at midpregnancy reported previously 21 . In the present study, we corroborate the observation by Gandley et al. that soluble syndecan-1 is correlated with BP in women with pre-eclampsia 21 . Soluble syndecan-1 was not significantly different in preeclamptic and normotensive pregnancies.
In our experiments male mice were used. The main focus of the mice experiments was to gain more insight in the underlying mechanism by which soluble syndecan-1, GAGs and sFLT-1 alter blood pressure irrespective of pregnancy. The use of male mice could be a limitation in our study. However, a randomized controlled trial comparing placebo to monoclonal antibodies to VEGF in (mostly male) patients with metastatic clear-cell renal carcinoma resulted in a significant increase in the occurrence of hypertension and proteinuria in the treatment group 24 . Therapies that target VEGF and its receptors are known to induce " preeclampsia like syndrome" (hypertension and proteinuria) 25 . These side effects occur in both females and males. The same phenomenon can be observed in mice 26,27 . This suggest a comparable underlying mechanism for anti-VEGF induced hypertension and proteinuria in both males and females.
In a Sdc1 knockout mouse model, infusion of sFlt1 did not induce BP elevation. We previously showed that increased endothelin-1-mediated vasoconstriction and decreased methacholine-induced vasodilatation contributed to sFlt1-induced BP elevation in wild type mice 26 . These ex vivo effects on contractility were absent in syndecan-1 deficient mice in our study, suggesting that syndecan-1 is essential for sFLT1-induced hypertension. The potential effect of soluble syndecan-1 on BP may be mediated through its attached glycosaminoglycans, as the structure of these attached glycosaminoglycans are generally not affected in syndecan-1 deficient mice 28 . Administration of sulodexide, a mixture of GAGs containing 20% dermatan sulphate, significantly lowers BP in hypertensive and normotensive individuals 29 , possibly by mediating binding of circulating VEGF to the endothelium 30 . This hypothesis is supported by the positive correlation of soluble syndecan-1 with heparan sulphate and dermatan sulphate and the strong inverse correlation of dermatan sulphate with BP in preeclamptic women in this study. The blood pressure used in our analysis was the highest measured blood pressure during admission. The blood sample was taken at an independent time point during admission. The effect of this difference in sampling on the correlation is difficult to predict. Most of our patients with preeclampsia used one or more types of blood pressure medication. We must therefore take into account the potential role of blood pressure medication on syndecan-1 and blood pressure levels.
Like soluble syndecan-1, heparan sulphate is drastically increased in both preeclamptic and normotensive pregnancy and much lower postpartum, suggesting that heparan sulphate predominantly originates from the placenta. However, the syncytiotrophoblast, which is in direct contact with maternal blood has been shown to be devoid of dermatan sulphate 31 , providing support to a maternal endothelium origin of excess dermatan sulphate. In addition, endocan, an endothelial-cell specific soluble dermatan sulphate proteoglycan, is elevated in women with preeclampsia and positively correlates with circulating sFLT1 32 . One could hypothesize a potential protective effect by the increase of dermatan sulphate originating from maternal endothelium resulting in counteracting the increase in blood pressure seen in preeclampsia. In addition to interaction with sFLT1, dermatan sulphate is able to bind Transforming Growth Factor-β (TGF-β) 33 . Disruption of TGF-β signaling has been linked to endothelial dysfunction and decreased nitric-oxide availability in preeclampsia 3 . TGF-β signaling is modulated by soluble endoglin, which is also positively correlated with the soluble dermatan sulphate proteoglycan endocan in preeclampsia. Although merely speculative, elevated circulating dermatan sulphate levels may also be related to BP via interaction with TGF-β. Further support for a functional role of elevated dermatan sulphate in the circulation of preeclamptic women is provided by our observation that dermatan sulphate is strongly correlated with activated Partial Thromboplastin Time (aPTT) that reflects the integrity of the intrinsic clotting cascade known to be disrupted in preeclampsia 6 . The increase in aPTT has been associated with severe preeclampsia and the inverse correlation with dermatan sulphate may suggest a protective effect of dermatan sulphate in preeclampsia 6 . Accordingly, dermatan sulphate has previously been identified as a major determinant of coagulation in the placenta via activation of the thrombin inhibitor heparin cofactor II within fetal vessel walls 31 .
In conclusion, dermatan sulphate is significantly elevated in preeclampsia and inversely correlated with soluble syndecan-1 and BP, suggesting that the association of soluble syndecan-1 with BP in preeclampsia might be mediated by dermatan sulphate. Prospective studies are needed to assess whether administration of dermatan sulphate reduces the risk of preeclampsia or limits its severity.

Materials and methods
Study population. Clinical data and plasma samples were obtained from participants admitted to the department of obstetrics of the Amsterdam Medical Centre, The Netherlands after obtaining informed consent. Blood samples were drawn from participating pregnant women admitted to our obstetric ward. The participating women were not in active labor at the time of sample collection. Samples that had sufficient residual plasma left were selected for analysis of circulating GAGs. The study has been performed in accordance with the Decleration of Helsinki and was approved by the Medical Review Ethics Committee of the Amsterdam University Medical Centre, The Netherlands. Plasma was immediately stored at − 80 °C. Blood was also drawn at 3 months postpartum from the same study population. Preeclampsia was defined by systolic BP ≥ 140 mmHg or diastolic BP ≥ 90 mmHg recorded on two occasions at least 4 h apart, after 20 weeks gestation in a previously normotensive woman combined with new-onset proteinuria with urinary protein excretion ≥ 300 mg/24-h. BP was measured manually in the sitting position at the right upper arm using an aneroid sphygmomanometer. Diastolic BP was determined at Korotkoff sound V. For this study, the highest measured blood pressure during admission was included. Birth weight percentiles were assessed according to the local Dutch birth weight percentiles. The appropriate chart was chosen based on parity and gender of the baby (http://www.perin atreg .nl/). Small for gestational age was defined as birth weight below 10th percentile. HELLP syndrome was defined by lactate dehydrogenase ≥ 600 U/L or haptoglobin < 0.2 g/L, aspartate or alanine aminotransferase ≥ 70 U/L, and platelet count < 100 * 10 9 /L. Experiments were carried out in accordance with the declaration of Helsinki after informed consent from the participants was obtained. Experiments were approved by an independent ethics committee 4,34,35 . Effect of sFlt1 on blood pressure in wild type and syndecan-1 deficient mice. To investigate the role of syndecan-1 signalling in sFlt1-induced BP elevation, we compared in vivo BP response and isometric tension measurements ex vivo in isolated arteries of syndecan-1 deficient mice and wild type controls. All experimental procedures including housing conditions, BP measurement and isometric tension measurements have been described in detail previously 26 . Briefly, adult (12-14 weeks old) male C57/BL6N mice (Charles River, Maastricht, The Netherlands) and Sdc1 -/-mice on a C57/BL6N background were individually housed in a temperature controlled room with a 12:12 light-dark cycle with food and water ad libitum. After acclimatisation, mice were anesthetized for implantation of osmotic minipumps (Alzet, California USA). Pumps were filled with either vehicle (PBS) or sFlt1 (Creative Biomart, New York, USA, Catalog no: Flt1-1785 M) for continuous 0.5 µl/h compound release (equals 500 ng/h sFlt1) during 2 weeks. During treatment, BP was recorded at fixed time points using the non-invasive tail cuff BP measurement system, according to the previously described protocol (CODA system Kent Scientific Corporation, CT, USA). To reduce the adverse stress response to BP measurements, animals were trained during one week prior to the experiment. After 2 weeks, mice were euthanized and carotid arteries were isolated for isometric analysis of vasomotor tone. All experimental procedures were performed in compliance with the ARRIVE guidelines and approved by the Animal Ethics Committee of the Amsterdam University Medical Centre, The Netherlands 34 .