Sialic acid-binding immunoglobulin-like lectin-6 (SIGLEC6) is increased by hypoxia and inflammation; relevance to preeclampsia

Sialic acid-binding immunoglobulin-like lectin-6 (SIGLEC6) is increased by hypoxia and inflammation; relevance to preeclampsia | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Sialic acid-binding immunoglobulin-like lectin-6 (SIGLEC6) is increased by hypoxia and inflammation; relevance to preeclampsia Manju Kandel, Lucy Bartho, Natalie J Hannan, Ping Cannon, Tuong-Vi Nguyen, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7164266/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract SIGLEC6, a human-specific transmembrane receptor, is highly expressed in placenta. Our team have identified elevated SIGLEC6 in maternal circulation preceding preeclampsia and in women with preeclampsia, correlating with disease severity. This study aimed to characterise SIGLEC6 relevant to preeclampsia using human (cyto)trophoblast stem cells (hTSCs) and primary Human Umbilical Vein Endothelial Cells (HUVECs). SIGLEC6 was measured across three placental cell types by differentiating hTSCs: (cyto)trophoblasts, syncytiotrophoblasts, and extravillous trophoblasts. SIGLEC6 was expressed in all cell types and upregulated during differentiation into syncytiotrophoblasts and extravillous trophoblasts. Exposure of syncytialised hTSCs to hypoxia (1% vs 8% O 2 ) elevated SIGLEC6 expression (p = 0.0079), and protein secretion (p = 0.0079). Similarly, inflammatory cytokines (IL-6 or TNFα) increased SIGLEC6 expression (IL-6: p = 0.0016, and TNFα: p = 0.0015) and protein secretion (IL-6: p = 0.002, and TNFα: p = 0.01) from syncytialised hTSCs. Treatment with Brefeldin A (impairs protein trafficking) reduced SIGLEC6 secretion in cell lysates (p = 0.001) and conditioned media (p = 0.02). To evaluate functional effects, HUVECs were treated with recombinant SIGLEC6. No significant changes were observed in endothelial dysfunction markers (VCAM1, ICAM1, ET-1), pro-angiogenic factors (PlGF, VEGF) or anti-angiogenic splice variants (sFlt-1 e15a, sFlt-1 i13). SIGLEC6 expression is induced by hypoxia and inflammation in syncytialised hTSCs, but recombinant SIGLEC6 does not induce features of endothelial dysfunction observed in preeclampsia. Biological sciences/Cell biology Health sciences/Diseases Biological sciences/Immunology Health sciences/Medical research SIGLEC6 Placenta Preeclamspia Pregnancy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Preeclampsia is serious pregnancy complication affecting 2–8% of pregnancies worldwide 1 . It is a leading cause of maternal and neonatal morbidity and mortality 1 . It is characterised by placental hypoxia, oxidative stress, and production of excessive anti-angiogenic and pro-inflammatory factors leading to widespread systemic endothelial dysfunction 2 . Given the significant burden of preeclampsia, there is a need to better understand its molecular pathophysiology to improve diagnostics, management and therapeutic options. Preeclampsia is often associated with abnormal trophoblast differentiation 3 . Recent single-cell sequencing and spatial multi-omics studies have revealed cell-type-specific dysregulation in preeclamptic placentas. Syncytiotrophoblast, which forms the maternal-fetal interface, exhibit premature differentiation and a senescence-associated secretory phenotype in preeclampsia 4 . These changes contribute to systemic maternal effects, highlighting the placenta’s central role in driving the disease 4 . SIGLEC6 is a member of the sialic acid-binding immunoglobulin-like lectin (SIGLEC) family, a group of negatively charged carbohydrate-binding proteins that bind to sialyated glycans in the pericellular matrix of various cell types 5 . It is a transmembrane receptor highly expressed in the placenta in a human-specific manner 6 . While SIGLEC6 is expressed in B cells across all studied primates, its unique expression in human placenta highlights its potential significance in preeclampsia, a complication predominantly observed in human pregnancies. Although its precise function remains unclear, SIGLEC6 is known to bind sialyl-Tn glycans and leptin 7 . Leptin is a hormone that plays diverse roles in reproduction, including regulating gonadotrophins, facilitating blastocyst development, implantation, and placentation and communication between the fetus and the placenta 8 . SIGLEC6 has previously been identified as highly expressed in preeclamptic placentas 9 , and its mRNA and protein are measurable in syncytiotrophoblast derived extracellular vesicles 7 , 10 , 11 . A recent study suggests SIGLEC6 may be a signalling molecule in human trophoblasts, potentially influencing placental development and immune interactions 12 . However, more studies need to be done to understand its biological function in placenta. Recent work 13 from our team identified that plasma SIGLEC6 levels are significantly elevated prior to preeclampsia onset and among those with established disease, correlating with disease severity. It was also upregulated in several large proteomic screens 14 – 16 . SIGLEC6 may therefore be an important molecule in preeclampsia pathogenesis as well as a potential biomarker. Therefore, this study aimed to investigate the regulatory mechanisms of placental SIGLEC6 and its involvement in known pathways associated with preeclampsia pathogenesis. First, we assessed SIGLEC6 mRNA expression and protein secretion across all trophoblast subpopulations and examined whether SIGLEC6 is regulated by hypoxia or inflammation using primary trophoblasts and human trophoblast stem cells (hTSCs). We also examined whether elevated levels of circulating SIGLEC6 contributes to endothelial dysfunction, given vascular dysfunction is a hallmark of preeclampsia. Materials and Methods Ethics approval for placenta/umbilical cord collection Ethics approval for this study was granted by the Mercy Health Human Research Ethics Committee (R11/34). Participants presenting at Mercy Hospital for Women (Heidelberg, Victoria) provided informed, written consent for the collection of term placentas and umbilical cord following caesarean delivery. All methods were performed in accordance with the National Health and Medical Research Council of Australia (NHMRC) National Statement on Ethical Conduct in Human Research. Culture and differentiation of first trimester human trophoblast stem cells (hTSCs) First trimester human trophoblast stem cell lines (hTSCs) were imported from the RIKEN BRC via the National BioResource Project of the MEXT/AMED, Japan. Cells were cultured and differentiated into either syncytiotrophoblasts or extravillous trophoblasts according to the publication from Okae and colleagues 17 . Treatment of syncytialised hTSCs with hypoxia, interleukin 6 (IL-6), and tumor necrosis factor α (TNFα) Cells were plated at 60,000 cells/well in a 24-well cell culture plate in syncytial (ST(2D)) media and incubated at 37 0 C, 8% O 2 , and 5% CO 2 for 72 hours to allow for syncytialisation. Cells undergoing hypoxic exposure were placed at 1% O 2 whilst normoxic cells were maintained at 8% O 2 for 48 hours. For inflammatory stimuli, cells were treated with increasing doses of IL-6 or TNFα at 0 (vehicle control), 0.1, and 1ng/ml for 24 hours. Conditioned media, cell lysates and mRNA were collected for analysis using ELISA and qRT-PCR respectively. Each treatment was performed in triplicate and repeated five times (n = 5). Treatment of syncytialised hTSCs with recombinant human SIGLEC6 Cells were plated at 60,000 cells/well in a 24-well cell culture plate in syncytial (ST(2D)) media and incubated at 37 0 C, 8% O 2 , and 5% CO 2 for 72 hours to allow syncytialisation. Cells were treated with increasing doses of recombinant human (rh)SIGLEC6 (In Vitro Technologies) at 10, 20, and 40ng/ml or control for 48 hours. Conditioned media, cell lysates and mRNA were collected for analysis using ELISA and qRT-PCR respectively. Each treatment was performed in triplicate and repeated five times (n = 5). Treatment of syncytialised hTSCs with Brefeldin A Cells were plated at 60,000 cells/well in a 24-well cell culture plate in syncytial (ST(2D)) media and incubated at 37 0 C, 8% O 2 , and 5% CO 2 for 72 hours to allow syncytialisation. Cells were treated with increasing doses of Brefeldin A at 0, 5, and 10 ng/ml or vehicle control for 24 hours. Conditioned media, and cell lysates were collected for analysis using ELISA. Each treatment was performed in triplicate and repeated five times (n = 5). Collection of umbilical cord and isolation of primary human umbilical vein endothelial cells (HUVECs) Human umbilical vein endothelial cells (HUVECs) were isolated as previously described 18 . The cells were cultured using M199 media (Life Technologies) supplemented with 20% fetal bovine serum (FBS), 1mg/ml heparin, 2mg/ml of endothelial cell growth factor (ECGS) (Sigma, Missouri, USA), and 1% antibiotic-antimycotic (AA) (Life Technologies). They were then incubated at 37 0 C, 20% O 2 , and 5% CO 2 . HUVECs were used between the first 1 to 4 passages. Treatment of primary HUVECs with recombinant human SIGLEC6 Cells were seeded at 40,000/well in a 24-well cell culture plate and incubated overnight at 37 0 C, 20% O 2 , and 5% CO 2 . Cells were then treated with increasing doses of recombinant (rh)SIGLEC6 (In Vitro Technologies) at 10, 20, and 40ng/ml or vehicle control for 48 hours. Conditioned media, cell lysates and mRNA were collected for analysis using ELISA and qRT-PCR respectively. Each treatment was performed in triplicate and repeated five times (n = 5). Enzyme Linked Immunosorbent Assay (ELISA) Concentrations of SIGLEC6 in conditioned cell culture media were measured using human SIGLEC6 DuoSet ELISA kit (RnD systems, Minnesota, USA) according to the manufacturer’s instructions. This kit has an inter-assay and intra-assay precision of < 15%. RNA Isolation RNA was isolated from primary trophoblasts, hTSCs, and HUVECs using the GenElute™ Mammalian Total RNA Miniprep Kit (Sigma-Aldrich, Missouri, USA) according to manufacturer’s instructions. mRNA concentration was quantified using a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies Inc.), and equivalent amounts were converted to cDNA as described below. Quantitative Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) mRNA was reverse transcribed into cDNA using the Applied Biosystems High-Capacity cDNA Reverse Transcriptase kit (Life Technologies) as per manufacturer’s instructions. Reverse transcription was carried out using the iCycler iQ5 (Bio-Rad) under the following conditions: 25°C for 10 minutes, 37°C for 60 minutes, and 85°C for 5 minutes. Gene expression of SIGLEC6 (Sialic Acid-Binding Immunoglobulin-like Lectin-6, Assay ID: Hs00609663_m1), TEAD4 (TEA domain transcription factor 4, Assay ID: Hs01125032_m1), SDC1 (Syndecan 1, Assay ID: Hs00896423_m1), HLAG (Human Leukocyte Antigen G, Assay ID: Hs03045108_m1), VCAM-1 (Vascular Cell Adhesion Molecule-1, Assay ID: Hs01003372_m1), ICAM-1 (Intracellular Adhesion Molecule-1, Assay ID: Hs00164932_m1), ET1 (Endothelin 1, Assay ID: Hs00174961_m1), CYC1 (Cytochrome C1, Assay ID: Hs00357717_m1), TOP1 (DNA Topoisomerase I, Assay ID: Hs00243257_m1), YHWAZ (Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein Zeta, Assay ID: Hs01122454_m1), and GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase, Assay ID: Hs99999905_m1) were quantified by quantitative real-time PCR (qRT-PCR) on the CFX384 (Bio-Rad, Hercules, CA) using FAM-labelled Taqman™ Fast Advanced Master Mix (Applied Biosystems) and their specific Taqman™ Gene expression Assays (Life Technologies). The run conditions were 95°C for 20 seconds, followed by 40 cycles of: 95°C for 3 seconds, and 60°C for 30 seconds. SYBR qRT-PCR was carried out to assess gene expression of sFLT-1 e15a , sFLT-1 i13 and YWHAZ on the CFX384 (Bio-Rad, Hercules, CA) using Fast SYBR™ Green Master Mix (Applied Biosystems) with specific forward and reverse primers. The run conditions were 95°C for 20 seconds, followed by 40 cycles of 95°C for 1 second and 60°C for 20 seconds, with a melt curve from 65°C to 95°C in 0.5°C increments for 0.05 seconds each. No product was detected in the non-template control. Gene expression was normalised to the geometric mean of CYC1 and TOP1 for hypoxia experiment, GAPDH for syncytial hTSCs, and YHWAZ for HUVECs samples. All samples were run in duplicate, and the average threshold (Ct) value was used. Results were calibrated against the average Ct of controls and expressed as fold change relative to controls. Statistical analysis All in vitro experiments were performed in technical triplicates and repeated five times. Normality and lognormality were assessed using the Anderson-Darling, D’Agostino & Pearson, Shapiro-Wilk, and Kolmogorov-Smirnov tests, and the appropriate statistical methods were selected based on data distribution. Parametric tests were used for normally distributed data, while non-parametric tests were applied to data that were not normally distributed. For comparisons between two unpaired groups, an unpaired t-test (parametric) or Mann-Whitney test (non-parametric) was used. For comparisons with three or more groups, either one-way ANOVA (parametric) or Kruskal-Wallis test (non-parametric) was used. All data are expressed as mean ± SEM or median (interquartile range, IQR), as appropriate. A p-value of < 0.05 was considered statistically significant. All analyses were performed using GraphPad Prism version 10.3.0 (GraphPad Software Inc., San Diego, California). Results SIGLEC6 expression and secretion increases with trophoblast differentiation To characterise SIGLEC6 expression in the placental cells, hTSCs were differentiated into either syncytiotrophoblasts or extravillous trophoblasts over 96 hours, and mRNA levels and protein secretion were measured at 0, 48, 72 and 96 hours. Syncytialisation was confirmed by a significant reduction in the progenitor marker, TEAD4 mRNA expression (Fig. 1 A, p = 0.0018) and increased mRNA expression of an established syncytiotrophoblast marker, SDC1 (Fig. 1 B, p = 0.001) at 72 and 96 hours post-differentiation. We have previously shown reduced TEAD4 protein expression and increased SDC1 protein expression with hTSC syncytialisation 19 . SIGLEC6 mRNA expression (Fig. 1 C, p = 0.004) and protein secretion (Fig. 1 D, p = 0.0009) were both significantly elevated following differentiation into syncytiotrophoblasts over 96 hours. Differentiation of the hTSCs into EVTs was confirmed by a reduction in progenitor marker, TEAD4 mRNA expression (Fig. 1 E, p = 0.002) and an increase in the EVT marker, HLAG mRNA expression (Fig. 1 F, p = 0.0005) at 72 and 96 hours. Similarly, SIGLEC6 mRNA expression (Fig. 1 G, p = 0.002) and protein secretion (Fig. 1 H, p = 0.001) were significantly increased following EVT differentiation over 96 hours. These data suggets that SIGLEC6 is expressed in all trophoblast subpopulations and its expression is upregulated during both syncytiotrophoblast and extravillous trophoblast differentiation. Effect of hypoxia and inflammatory stimuli on SIGLEC6 expression and secretion in syncytialised first trimester placental stem cells Preeclampsia can be accompanied by impaired placental perfusion, leading to intermittent hypoxia and inflammation 20 . To investigate the role of SIGLEC6 in a preeclampsia in vitro model, we exposed syncytialised hTSCs to either hypoxia (1% O 2 ) or normoxia (8% O 2 ), or inflammatory stimuli: interleukin-6 (IL-6) and tumour necrosis factor α (TNFα). Given syncytiotrophoblasts are likely the primary source of SIGLEC6 in preeclampsia (Figure: 1E, 1G), they were utilised for further investigation. SIGLEC6 mRNA expression was significantly increased following hypoxic exposure of syncytialised hTSCs (Fig. 2 A, p = 0.008). Similarly, protein secretion in cell culture media was also significantly elevated in syncytialised hTSCs (Fig. 2 B, p = 0.008). In preeclampsia, pro-inflammatory cytokines IL-6 and TNFα are known to be elevated in the circulation and contribute to placental and systemic inflammation 21 . To investigate whether these cytokines regulate placental SIGLEC6 expression or secretion, syncytialised hTSCs were treated with increasing doses of IL-6 (0, 0.1, and 1ng/ml) and TNFα (0, 0.1, and 1ng/ml). Both IL-6 and TNFα significantly upregulated SIGLEC6 mRNA expression (Fig. 2 C, p = 0.002 and Fig. 2 E, p = 0.009) and protein secretion (Fig. 2 D, p = 0.001 and Fig. 2 F, p = 0.01) in a dose-dependent manner, compared to controls. Overall, hypoxia and pro-inflammatory cytokines may contribute to driving modest increases in SIGLEC6 expression and secretion from syncytiotrophoblast in the placenta. Effect of recombinant SIGLEC6 on anti-angiogenic markers in syncytialised first trimester placental stem cells. Recent studies have shown SIGLEC6 expression is significantly elevated in placental tissue complicated by preeclampsia 9 , 13 , 22 . To further understand the function of SIGLEC6 in the placenta complicated by preeclampsia, we examined the effect of high levels of recombinant SIGLEC6 on syncytialised hTSCs. We measured the expression of anti-angiogenic molecule sFlt-1 and its variants, e15a and i13, both of which are known to be altered in preeclampsia. Our results showed no significant changes in mRNA expression of sFlt -1 variant s ( e15a – Fig. 3 A, and i13 – Fig. 3 B) or in sFlt-1 protein secretion (Fig. 3 C) following SIGLEC6 treatment of syncytiotrophoblast cells. Mechanisms of SIGLEC6 secretion in syncytialised syncytialised first trimester placental stem cells. To investigate the mechanisms contributing to SIGLEC6 secretion, syncytialised hTSCs were treated with increasing doses of Brefeldin A (0, 5, and 10ng/ml). Brefeldin A inhibits protein secretion by blocking protein transport from the endoplasmic reticulum to the Golgi apparatus 23 . A significant dose-dependent reduction in SIGLEC6 was observed, both in the cell culture media (Fig. 4 A, p = 0.02 at 5 ng/ml) and in cell lysates (Fig. 4 B, p = 0.03 at 5 ng/ml, p = 0.005 at 10 µM). MTS assay results suggest no impact on cell viability at these concentrations (data not shown). Thus, this data suggests that SIGLEC6 may be actively secreted via the Golgi apparatus from placenta, rather than being passively released into the circulation through syncytial shedding or turnover. Administering recombinant SIGLEC6 to endothelial cells does not alter the expression of markers of endothelial dysfunction, or angiogenic molecules. Preeclampsia features widespread maternal endothelial cell dysfunction, largely attributed to the increased release of anti-angiogenic factors 24 . Given that SIGLEC6 protein levels are significantly elevated in the circulation of women with preeclampsia 13 , 22 , we examined the effect of recombinant SIGLEC6 at levels comparable to those found in maternal circulation in preeclampsia, on primary HUVECs. Treatment of HUVECs with recombinant SIGLEC6 had no significant effect on the mRNA expression of the endothelial dysfunction markers VCAM1 (Fig. 5 A), ICAM1 (Fig. 5 B), or the vasoconstrictor ET-1 (Fig. 5 C). Similarly, recombinant SIGLEC6 did not alter mRNA levels of pro-angiogenic molecules VEGF (Fig. 5 D) or PlGF (Fig. 5 E). Furthermore, recombinant SIGLEC6 had no significant effect on the mRNA expression of sFlt-1 variants e15a (Fig. 5 F) and i13 (Fig. 5 G), nor did it affect sFlt-1 protein secretion (Fig. 5 H). These findings suggests that high circulating levels of SIGLEC6 may not modulate endothelial dysfunction or angiogenic imbalance in preeclampsia. Discussion This study showed that SIGLEC6 is expressed in hTSCs as well as syncytialised hTSCs and those differentiated into EVTs. Further, in vitro studies showed hypoxia and inflammation increased SIGLEC6 expression in human (cyto)trophoblast stem cells. Additionally, Brefeldin A was found to reduce the secretion of SIGLEC6 from syncytialised hTSCs. However, treating primary endothelial cells with levels of recombinant SIGLEC6 equivalent to those found in the circulation of women with preeclampsia did not induce markers of endothelial dysfunction. SIGLEC6, a molecule known to facilitate cell-cell interactions, is highly expressed in the placenta 7 , 25 . Our findings demonstrate that SIGLEC6 is expressed in all trophoblast subpopulations, with its mRNA expression and protein secretion significantly upregulated during the differentiation of first trimester human (cyto)trophoblast stem cells into syncytiotrophoblasts and extravillous trophoblasts over a 96-hour period. These findings suggest that SIGLEC6 is dynamically regulated throughout trophoblast development. Previous studies have also shown SIGLEC6 expression in cytotrophoblasts and syncytiotrophoblasts within chorionic villi, as well as in extravillous trophoblast of the decidua basalis of human placenta 7 , 26 . Some studies have also reported SIGLEC6 expression decreases with advancing gestation (after 8 weeks) but remains detectable in proliferative cytotrophoblasts and syncytiotrophoblasts 7 , 27 . SIGLEC6 is a known regulator of immune interactions and has been implicated in trophoblast signalling through Src kinase tyrosine phosphorlation and SHP-2 recruitment 12 . SHP-2 and Src kinase phosphorylation regulate various cellular processes, including proliferation, differentiation, motility, and adhesion 28 , 29 . This is particularly significant given that preeclampsia is associated with impaired trophoblast invasion and differentiation. However, further research is still needed to elucidate the mechanisms by which SIGLEC6 influences trophoblast function. Given placental hypoxia and inflammation are hallmarks of preeclampsia, we examined the effects of hypoxia (1% O 2 vs 8% O 2 ) and pro-inflammatory cytokines (IL-6 and TNFα) on SIGLEC6 mRNA expression and protein secretion in syncytialised hTSCs. Exposure to hypoxia (1% O 2 ) significantly increased SIGLEC6 mRNA expression and protein secretion compared to normoxia (8% O 2 ). This aligns with previous findings: Guan et al demonstrated that hypoxia-inducible factor-1α (HIF-1α) accumulation promoted SIGLEC6 expression and secretion in HTR8/SVeno and BeWo cells 26 . Furthermore, a 2018 study reported a significant upregulation of SIGLEC6 mRNA expression in primary human mast cells following 24 hours of hypoxia exposure 30 . Similarly, exposure to inflammatory stimuli (IL-6 and TNFα), demonstrated a significant dose-dependent upregulation of SIGLEC6 mRNA expression and protein secretion from syncytialised hTSCs. While studies examining the effects of IL-6 and TNFα on SIGLEC6 expression in placental cells are limited, a 2018 study reported increased SIGLEC6 expression in human mast cells co-cultured with colon cancer cells lines. The authors suggests cancer cell-derived factors, possibly inflammatory mediators, may modulate SIGLEC6 levels 30 . As our study only measured IL6 and TNFα, future work should assess additional imflammatory mediators to better understand their contribution to SIGLEC6 upregulation in preeclamspia. Collectively, these findings suggest hypoxia and inflammation contribute to elevated SIGLEC6 levels in maternal blood from patients diagnosed with preeclampsia 22 . In our recent publication, we demonstrated a strong association between elevated SIGLEC6 and preeclampsia 13 . Our team identified significantly elevated SIGLEC6 levels in the maternal circulation preceding preeclampsia (as early as 15–20 weeks’ gestation), in women diagnosed with preeclampsia, and elevated levels strongly correlate with disease severity 13 . SIGLEC6 was highly abundant in maternal circulation, which led us to hypothesise that despite being a transmembrane receptor, it may be secreted from the placental surface. To investigate the secretory mechanism of SIGLEC6, syncytialised hTSCs were treated with Brefeldin A, a macrolide antibiotic that inhibits protein secretion by blocking protein transport from the endoplasmic reticulum to the Golgi apparatus. While Brefeldin A treatment resulted in a modest reduction in SIGLEC6 secretion in the conditioned media, this incomplete inhibition suggests other mechanisms, beyond canonical Golgi-dependent secretion, may be involved in SIGLEC6 secretion. While this study enhances our understanding of SIGLEC6 expression in trophoblasts, its precise role in placental function and pathogenesis of preeclampsia remains unclear. Previous studies on the role of SIGLEC6 in trophoblast function have yielded inconsistent findings. Jia et al., reported that SIGLEC6 inhibits trophoblast migration and invasion by impairing mitochondrial function in HTR-8/SVneo and JAR cells 31 . In contrast, Guan et al. found that SIGLEC6 overexpression had no significant impact on proliferation, migration, or invasion in HTR-8/SVneo cells 26 . Additionally, a 2012 study suggested a complex interplay between SIGLEC6 and leptin, where SIGLEC6 promoted proliferation in a leptin-dependent manner, while also enhancing invasion and reducing apoptosis independently of leptin 27 . Moreover, SIGLEC6 has been shown to bind glycodelin-A (a glycoprotein), suppressing trophoblast invasion by down-regulating the ERK/c-Jun signalling pathway, critical for trophoblast invasion and vascular remodelling 32 . Given these inconsistencies, further investigation into the interaction with SIGLEC6, leptin and other signalling molecules, may offer deeper insights into mechanisms underlying trophoblast differentiation and invasion and the development of preeclampsia. Preeclampsia is characterized by widespread systemic endothelial dysfunction, driven primarily by an imbalance between pro-angiogenic and anti-angiogenic factors 24 . In the current study, SIGLEC6 had no effect on endothelial dysfunction markers, pro-angiogenic markers, and anti-angiogenic markers. These findings suggest the elevated levels of circulating SIGLEC6 levels in preeclampsia are unlikely to directly contribute to endothelial dysfunction, indicating its role may be more confined to the placenta rather than vascular dysfunction. A significant strength of this study is the use of differentiated trophoblast suntypes and primary HUVECs to assess the role of SIGLEC6 in preeclampsia. However, a limitation of this work was that we could not mimic the over-expression of SIGLEC6 that is apparent in placentas from pregnancies complicated by preeclampsia 9 . While this was attempted via plasmid transfection of syncytialised hTSCs, we could not obtain elevated protein expression or secretion. Future studies to over-express SIGLEC6 in primary trophoblast may assist in furthering our understanding of its biology. In our interrogation of the impacts of SIGLEC6 on endothelial dysfunction, we only examined a subset of markers. Future studies could expand to explore alternative aspects of endothelial dysfunction, such as nitric oxide signalling pathway (vasodilation/constriction) and vascular reactivity, to further elucidate whether the high circulating levels of SIGLEC6 apparent in preeclampsia 13 , have any effect on vascular dysfunction. In conclusion, this study provides evidence to suggest the expression of SIGLEC6 across all trophoblast subpopulation, with its highest expression observed in syncytiotrophoblast and extravillous trophoblasts. We showed that SIGLEC6 may be regulated by placental hypoxia and inflammation. While SIGLEC6 is unlikely to directly contribute to endothelial dysfunction or angiogenic imbalance, its upregulation in trophoblast differentiation and in hypoxic and proinflammatory environments warrants further investigation into its role in placental function in preeclampsia. Declarations Author Contributions Statement Conceptualisation: M.K. and T.J.K.-L.; Methodology, M.K., L.B., N.J.H, P.C., T.-V.N., A.N., C.N.M., G.P.W., S.T., and T.J.K.-L; Formal analysis: M.K. and T.J.K.-L; Investigation: M.K. and T.J.K.-L; Resources: T.J.K.-L., S.T., N.H., and L.B.; Writing—original draft preparation, M.K. and T.J.K.-L.; Writing—review and editing, all co-authors; Funding Acquisition, T.J.K.-L, S.T., and L.B. All authors have revised and approved the final version of the manuscript. Additional Information The authors have no conflicts of interest to declare. Funding Funding for this work was provided by: National Health and Medical Research Council (#1065854, 2000732). Salary support was received from the National Health and Medical Research Council Fellowship for S.T (#1136418), and from Australian Research Council Future Fellowships for T.J.K.-L (FT230100125) and N.H (FT210100193). The funders played no role in study design or analysis. Author Contribution Conceptualisation: M.K. and T.J.K.-L.; Methodology, M.K., L.B., N.J.H, P.C., T.-V.N., A.N., C.N.M., G.P.W., S.T., and T.J.K.-L; Formal analysis: M.K. and T.J.K.-L; Investigation: M.K. and T.J.K.-L; Resources: T.J.K.-L., S.T., N.H., and L.B.; Writing—original draft preparation, M.K. and T.J.K.-L.; Writing—review and editing, all co-authors; Funding Acquisition, T.J.K.-L, S.T., and L.B. All authors have revised and approved the final version of the manuscript. Acknowledgement We thank Kaitlin Constable, Melissa Sutton, Gabrielle Fleming, Rachel Murdoch, Genevieve Christophers, Anna Middleton, Kirsty Dane, Alex Roddy-Mitchell, Brooke Henshall, Katelyn Dark and Danica Idzes for their assistance in recruiting and characterising participants. We also wish to thank the pathology, health information services, and prenatal clinic staff at the Mercy Hospital for Women for their assistance in conducting this research and patients for agreeing to participate. 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PloS one . 14 , e0217273 (2019). Okae, H. et al. Derivation of human trophoblast stem cells. Cell stem cell 22, 50–63. e56 (2018). Brownfoot, F., Hannan, N., Onda, K. & Tong, S. Kaitu'u-Lino, T. Soluble endoglin production is upregulated by oxysterols but not quenched by pravastatin in primary placental and endothelial cells. Placenta 35 , 724–731 (2014). Wong, G. P. et al. Stem Cell Markers LGR5, LGR4 and Their Immediate Signalling Partners are Dysregulated in Preeclampsia. Stem Cell. Reviews Reports , 1–25 (2024). Redman, C. W. & Sargent, I. L. Latest advances in understanding preeclampsia. Science 308 , 1592–1594 (2005). Vitoratos, N., Economou, E., Iavazzo, C., Panoulis, K. & Creatsas, G. Maternal serum levels of TNF-alpha and IL‐6 long after delivery in preeclamptic and normotensive pregnant women. Mediators of inflammation 908649 (2010). (2010). Kaitu'u-Lino, T. J. et al. in REPRODUCTIVE SCIENCES. 75A-75A (SPRINGER HEIDELBERG TIERGARTENSTRASSE 17, D-69121 HEIDELBERG, GERMANY). Chardin, P., McCormick, F. & Brefeldin, A. the advantage of being uncompetitive. Cell 97 , 153–155 (1999). Boeldt, D. & Bird, I. Vascular adaptation in pregnancy and endothelial dysfunction in preeclampsia. J. Endocrinol. 232 , R27 (2016). Crocker, P. R., Paulson, J. C. & Varki, A. Siglecs and their roles in the immune system. Nat. Rev. Immunol. 7 , 255–266 (2007). Guan, X. et al. Elevated trophoblastic Siglec6 contributes to the impairment of vascular endothelial cell functions by downregulating Wnt6/β-catenin signaling in preeclampsia. Arch. Biochem. Biophys. 730 , 109396 (2022). Rumer, K. K. et al. Siglec-6 is expressed in gestational trophoblastic disease and affects proliferation, apoptosis and invasion. Endocr. Relat. Cancer . 19 , 827–840 (2012). Bjorge, J. D., Jakymiw, A. & Fujita, D. J. Selected glimpses into the activation and function of Src kinase. Oncogene 19 , 5620–5635 (2000). Zhang, S. Q. et al. Shp2 regulates SRC family kinase activity and Ras/Erk activation by controlling Csk recruitment. Mol. Cell . 13 , 341–355 (2004). Yu, Y. et al. Functional inhibitory siglec-6 is upregulated in human colorectal cancer-associated mast cells. Front. Immunol. 9 , 2138 (2018). Jia, Y. et al. Upregulation of Siglec-6 induces mitochondrial dysfunction by promoting GPR20 expression in early-onset preeclampsia. J. Translational Med. 22 , 674 (2024). Lam, K. K. et al. Glycodelin-A protein interacts with Siglec-6 protein to suppress trophoblast invasiveness by down-regulating extracellular signal-regulated kinase (ERK)/c-Jun signaling pathway. J. Biol. Chem. 286 , 37118–37127 (2011). Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7164266","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":500174007,"identity":"07a3353b-6c2c-4529-bf2d-d74dca511838","order_by":0,"name":"Manju Kandel","email":"data:image/png;base64,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","orcid":"","institution":"University of Melbourne","correspondingAuthor":true,"prefix":"","firstName":"Manju","middleName":"","lastName":"Kandel","suffix":""},{"id":500174008,"identity":"01201a43-e9b6-436e-b854-801b9c5de736","order_by":1,"name":"Lucy Bartho","email":"","orcid":"","institution":"University of Melbourne","correspondingAuthor":false,"prefix":"","firstName":"Lucy","middleName":"","lastName":"Bartho","suffix":""},{"id":500174009,"identity":"c07ba29d-2395-4f78-962c-b91696f72c18","order_by":2,"name":"Natalie J Hannan","email":"","orcid":"","institution":"University of Melbourne","correspondingAuthor":false,"prefix":"","firstName":"Natalie","middleName":"J","lastName":"Hannan","suffix":""},{"id":500174010,"identity":"f0ae5001-a917-4499-816a-15efc7fe4bcf","order_by":3,"name":"Ping Cannon","email":"","orcid":"","institution":"University of Melbourne","correspondingAuthor":false,"prefix":"","firstName":"Ping","middleName":"","lastName":"Cannon","suffix":""},{"id":500174011,"identity":"f28583eb-7b3e-4176-966e-00284f5309f5","order_by":4,"name":"Tuong-Vi Nguyen","email":"","orcid":"","institution":"University of Melbourne","correspondingAuthor":false,"prefix":"","firstName":"Tuong-Vi","middleName":"","lastName":"Nguyen","suffix":""},{"id":500174012,"identity":"cd642dca-198f-4044-80cf-db4a0e5f0ca4","order_by":5,"name":"Anna Nguyen","email":"","orcid":"","institution":"University of Melbourne","correspondingAuthor":false,"prefix":"","firstName":"Anna","middleName":"","lastName":"Nguyen","suffix":""},{"id":500174013,"identity":"e57b32b7-88d5-439a-9326-06ac01886d74","order_by":6,"name":"Ciara N Murphy","email":"","orcid":"","institution":"University of Melbourne","correspondingAuthor":false,"prefix":"","firstName":"Ciara","middleName":"N","lastName":"Murphy","suffix":""},{"id":500174014,"identity":"5fdd4abf-1524-4aad-9380-bb56c47ce0c7","order_by":7,"name":"Georgia P Wong","email":"","orcid":"","institution":"University of Melbourne","correspondingAuthor":false,"prefix":"","firstName":"Georgia","middleName":"P","lastName":"Wong","suffix":""},{"id":500174015,"identity":"99787aa7-d217-4acb-a71f-4dcf49b88d43","order_by":8,"name":"Stephen Tong","email":"","orcid":"","institution":"University of Melbourne","correspondingAuthor":false,"prefix":"","firstName":"Stephen","middleName":"","lastName":"Tong","suffix":""},{"id":500174016,"identity":"622098d2-cab6-40ee-8aee-353984bcc977","order_by":9,"name":"Tu’uhevaha J Kaitu’u-Lino","email":"","orcid":"","institution":"University of Melbourne","correspondingAuthor":false,"prefix":"","firstName":"Tu’uhevaha","middleName":"J","lastName":"Kaitu’u-Lino","suffix":""}],"badges":[],"createdAt":"2025-07-19 11:53:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7164266/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7164266/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89051212,"identity":"8d19d41d-a961-473b-b0a7-32559a253642","added_by":"auto","created_at":"2025-08-14 07:41:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":52679,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSIGLEC6 is expressed in all trophoblast subpopulation\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eFirst trimester placental cytotrophoblast cells were differentiated into either syncytiotrophoblast (ST) or extravillous trophoblast (EVT) cells over 96 hours. Syncytiotrophoblast differentiation was confirmed by reduced in \u003cem\u003eTEAD4\u003c/em\u003eexpression (cytrotrophoblast marker) (A) and increase in \u003cem\u003eSDC1\u003c/em\u003e(syncytiotrophoblast marker) (B) expression across time. \u003cem\u003eSIGLEC6\u003c/em\u003e mRNA expression (C) and protein secretion (D) increases with syncytiotrophoblast differentiation across 96 hours. EVT differentiation was confirmed by reduced expression of \u003cem\u003eTEAD4\u003c/em\u003e (cytotrophoblast marker) (E) and increased expression of \u003cem\u003eHLAG\u003c/em\u003e (EVT marker) (F) across time. \u003cem\u003eSIGLEC6\u003c/em\u003e mRNA expression (G) and protein secretion (H) increases with EVT differentiation across 96 hours. All experiments were repeated n = 5 times in triplicate. Data is expressed as mean ± SEM; *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p\u0026lt;0.001\u003c/p\u003e","description":"","filename":"Slide1.png","url":"https://assets-eu.researchsquare.com/files/rs-7164266/v1/0df54733f0b00f94b3432aef.png"},{"id":89051210,"identity":"5a9662fb-8da3-43e4-90a2-de76eccf0506","added_by":"auto","created_at":"2025-08-14 07:41:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":48412,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of hypoxia and inflammation on SIGLEC6 in syncytialised human (cyto)trophoblast stem cells (hTSCs)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eHuman (cyto)trophoblast stem cells were first syncytialised and then cultured under hypoxic conditions (1%O\u003csub\u003e2 \u003c/sub\u003eor 8%O\u003csub\u003e2\u003c/sub\u003e) or exposed to inflammatory stimuli (IL-6 or TNFα) at 0, 0.1, and 1ng/ml. \u003cem\u003eSIGLEC6\u003c/em\u003e expression \u003cstrong\u003e(A)\u003c/strong\u003e and protein secretion \u003cstrong\u003e(B) \u003c/strong\u003ewere significantly elevated in hypoxia (1%O\u003csub\u003e2\u003c/sub\u003e) compared to normoxic conditions. SIGLEC6 expression and secretion were also significantly elevated following IL-6 \u003cstrong\u003e(C and D\u003c/strong\u003e) and TNFα (\u003cstrong\u003eE and F\u003c/strong\u003e) treatments, respectively. All experiments were repeated n = 5 times with triplicate repeats. Data is expressed as mean ± SEM; *p \u0026lt; 0.05, **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Slide2.png","url":"https://assets-eu.researchsquare.com/files/rs-7164266/v1/6c96c870691c5f0e60890c0b.png"},{"id":89051209,"identity":"65068fc0-41ef-4723-9f98-3da7a072289d","added_by":"auto","created_at":"2025-08-14 07:41:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":17479,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of increasing doses of SIGLEC6 in syncytialised hTSCs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFirst trimester human trophoblast stem cells were first differentiated into syncytiotrophoblasts and treated with increasing doses of SIGLEC6 (10, 20, and 40ng/ml). Increasing doses of recombinant SIGLEC6 had no effect on mRNA expression of \u003cem\u003esFlt-1-e15a \u003c/em\u003e(A), \u003cem\u003esFlt-1-i13\u003c/em\u003e (B), and protein secretion of sFlt-1 (C). All experiments were repeated n = 5 times with triplicate repeats. Data is expressed as mean ± SEM; *p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Slide3.png","url":"https://assets-eu.researchsquare.com/files/rs-7164266/v1/fc027c65b7536ba89833fbe0.png"},{"id":89051211,"identity":"9b077bf1-a595-410e-a271-32c3ee464718","added_by":"auto","created_at":"2025-08-14 07:41:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":18359,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTreatment of syncytialised human (cyto)trophoblast stem cells (hTSCs)with brefeldin A.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFirst trimester human (cyto)trophoblast stem cells were first differentiated into syncytiotrophoblasts and treated with increasing doses (0, 5, and 10 ng/ml) of Brefeldin A. SIGLEC6 protein secretion significantly decreased with higher doses of Brefeldin A in both cell culture media (A) and cell lysates (B). All experiments were repeated n = 5 times in triplicate. Data is expressed as mean ± SEM; *p \u0026lt; 0.05, **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Slide4.png","url":"https://assets-eu.researchsquare.com/files/rs-7164266/v1/38796c116d6d9fbc2a6f9f7c.png"},{"id":89051213,"identity":"bb9a6581-d747-4d18-9bd0-aa3767267e70","added_by":"auto","created_at":"2025-08-14 07:41:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":39210,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of circulating SIGLEC6 on endothelial dysfunction, pro-angiogenic and anti-angiogenic markers.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIncreasing doses of recombinant SIGLEC6 had no effect on mRNA expression of endothelial dysfunction markers: \u003cem\u003eVCAM1\u003c/em\u003e (A), \u003cem\u003eICAM1\u003c/em\u003e (B), and \u003cem\u003eET-1 \u003c/em\u003e(C), pro-angiogenic markers: \u003cem\u003eVEGF\u003c/em\u003e (D), and \u003cem\u003ePlGF\u003c/em\u003e (E), or anti-angiogenic markers: \u003cem\u003esFlt-1-i13\u003c/em\u003e (F), and \u003cem\u003esFlt-1-e15a\u003c/em\u003e (G). Recombinant SIGLEC6 also had no effect on sFlt-1 protein secretion (H). All experiments were repeated n = 5 times with triplicate repeats. Data is expressed as mean ± SEM.\u003c/p\u003e","description":"","filename":"Slide5.png","url":"https://assets-eu.researchsquare.com/files/rs-7164266/v1/196d0ff99e20f29eeec9816d.png"},{"id":90485537,"identity":"f7109dd5-2043-4a6b-9cae-259a85b3431e","added_by":"auto","created_at":"2025-09-03 08:47:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1349642,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7164266/v1/ef95ecde-edc3-4c9b-b7ff-54896316c4c3.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Sialic acid-binding immunoglobulin-like lectin-6 (SIGLEC6) is increased by hypoxia and inflammation; relevance to preeclampsia","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePreeclampsia is serious pregnancy complication affecting 2\u0026ndash;8% of pregnancies worldwide \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. It is a leading cause of maternal and neonatal morbidity and mortality \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. It is characterised by placental hypoxia, oxidative stress, and production of excessive anti-angiogenic and pro-inflammatory factors leading to widespread systemic endothelial dysfunction \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Given the significant burden of preeclampsia, there is a need to better understand its molecular pathophysiology to improve diagnostics, management and therapeutic options.\u003c/p\u003e\u003cp\u003ePreeclampsia is often associated with abnormal trophoblast differentiation \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Recent single-cell sequencing and spatial multi-omics studies have revealed cell-type-specific dysregulation in preeclamptic placentas. Syncytiotrophoblast, which forms the maternal-fetal interface, exhibit premature differentiation and a senescence-associated secretory phenotype in preeclampsia \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. These changes contribute to systemic maternal effects, highlighting the placenta\u0026rsquo;s central role in driving the disease \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eSIGLEC6 is a member of the sialic acid-binding immunoglobulin-like lectin (SIGLEC) family, a group of negatively charged carbohydrate-binding proteins that bind to sialyated glycans in the pericellular matrix of various cell types \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. It is a transmembrane receptor highly expressed in the placenta in a human-specific manner \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. While SIGLEC6 is expressed in B cells across all studied primates, its unique expression in human placenta highlights its potential significance in preeclampsia, a complication predominantly observed in human pregnancies. Although its precise function remains unclear, SIGLEC6 is known to bind sialyl-Tn glycans and leptin \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Leptin is a hormone that plays diverse roles in reproduction, including regulating gonadotrophins, facilitating blastocyst development, implantation, and placentation and communication between the fetus and the placenta \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eSIGLEC6 has previously been identified as highly expressed in preeclamptic placentas \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, and its mRNA and protein are measurable in syncytiotrophoblast derived extracellular vesicles \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. A recent study suggests SIGLEC6 may be a signalling molecule in human trophoblasts, potentially influencing placental development and immune interactions \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. However, more studies need to be done to understand its biological function in placenta.\u003c/p\u003e\u003cp\u003eRecent work \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e from our team identified that plasma SIGLEC6 levels are significantly elevated prior to preeclampsia onset and among those with established disease, correlating with disease severity. It was also upregulated in several large proteomic screens \u003csup\u003e\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. SIGLEC6 may therefore be an important molecule in preeclampsia pathogenesis as well as a potential biomarker.\u003c/p\u003e\u003cp\u003eTherefore, this study aimed to investigate the regulatory mechanisms of placental SIGLEC6 and its involvement in known pathways associated with preeclampsia pathogenesis. First, we assessed \u003cem\u003eSIGLEC6\u003c/em\u003e mRNA expression and protein secretion across all trophoblast subpopulations and examined whether SIGLEC6 is regulated by hypoxia or inflammation using primary trophoblasts and human trophoblast stem cells (hTSCs). We also examined whether elevated levels of circulating SIGLEC6 contributes to endothelial dysfunction, given vascular dysfunction is a hallmark of preeclampsia.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003efor placenta/umbilical cord collection\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthics approval\u0026nbsp;for this study was granted by the Mercy Health Human Research Ethics Committee (R11/34). Participants presenting at Mercy Hospital for Women (Heidelberg, Victoria) provided informed, written consent for the collection of term placentas and umbilical cord following caesarean delivery.\u003c/p\u003e\n\u003cp\u003eAll methods were performed in accordance with the National Health and Medical Research Council of Australia (NHMRC) National Statement on Ethical Conduct in Human Research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCulture and differentiation of first trimester human trophoblast stem cells (hTSCs)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFirst trimester human trophoblast stem cell lines (hTSCs) were imported from the RIKEN BRC via the National BioResource Project of the MEXT/AMED, Japan. Cells were cultured and differentiated into either syncytiotrophoblasts or extravillous trophoblasts according to the publication from Okae and colleagues \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTreatment of syncytialised hTSCs with hypoxia, interleukin 6 (IL-6), and tumor necrosis factor \u0026alpha; (TNF\u0026alpha;)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were plated at 60,000 cells/well in a 24-well cell culture plate in syncytial (ST(2D)) media and incubated at 37\u003csup\u003e0\u003c/sup\u003eC, 8% O\u003csub\u003e2\u003c/sub\u003e, and 5% CO\u003csub\u003e2\u003c/sub\u003e for 72 hours to allow for syncytialisation.\u003c/p\u003e\n\u003cp\u003eCells undergoing hypoxic exposure were placed at 1% O\u003csub\u003e2\u003c/sub\u003e whilst normoxic cells were maintained at 8% O\u003csub\u003e2\u003c/sub\u003e for 48 hours. For inflammatory stimuli, cells were treated with increasing doses of IL-6 or TNF\u0026alpha; at 0 (vehicle control), 0.1, and 1ng/ml for 24 hours. Conditioned media, cell lysates and mRNA were collected for analysis using ELISA and qRT-PCR respectively. Each treatment was performed in triplicate and repeated five times (n\u0026thinsp;=\u0026thinsp;5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTreatment of syncytialised hTSCs with recombinant human SIGLEC6\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were plated at 60,000 cells/well in a 24-well cell culture plate in syncytial (ST(2D)) media and incubated at 37\u003csup\u003e0\u003c/sup\u003eC, 8% O\u003csub\u003e2\u003c/sub\u003e, and 5% CO\u003csub\u003e2\u003c/sub\u003e for 72 hours to allow syncytialisation. Cells were treated with increasing doses of recombinant human (rh)SIGLEC6 (In Vitro Technologies) at 10, 20, and 40ng/ml or control for 48 hours. Conditioned media, cell lysates and mRNA were collected for analysis using ELISA and qRT-PCR respectively. Each treatment was performed in triplicate and repeated five times (n\u0026thinsp;=\u0026thinsp;5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTreatment of syncytialised hTSCs with Brefeldin A\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were plated at 60,000 cells/well in a 24-well cell culture plate in syncytial (ST(2D)) media and incubated at 37\u003csup\u003e0\u003c/sup\u003eC, 8% O\u003csub\u003e2\u003c/sub\u003e, and 5% CO\u003csub\u003e2\u003c/sub\u003e for 72 hours to allow syncytialisation. Cells were treated with increasing doses of Brefeldin A at 0, 5, and 10 ng/ml or vehicle control for 24 hours. Conditioned media, and cell lysates were collected for analysis using ELISA. Each treatment was performed in triplicate and repeated five times (n\u0026thinsp;=\u0026thinsp;5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCollection of umbilical cord and isolation of primary human umbilical vein endothelial cells (HUVECs)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman umbilical vein endothelial cells (HUVECs) were isolated as previously described \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. The cells were cultured using M199 media (Life Technologies) supplemented with 20% fetal bovine serum (FBS), 1mg/ml heparin, 2mg/ml of endothelial cell growth factor (ECGS) (Sigma, Missouri, USA), and 1% antibiotic-antimycotic (AA) (Life Technologies). They were then incubated at 37\u003csup\u003e0\u003c/sup\u003eC, 20% O\u003csub\u003e2\u003c/sub\u003e, and 5% CO\u003csub\u003e2\u003c/sub\u003e. HUVECs were used between the first 1 to 4 passages.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTreatment of primary HUVECs with recombinant human SIGLEC6\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were seeded at 40,000/well in a 24-well cell culture plate and incubated overnight at 37\u003csup\u003e0\u003c/sup\u003eC, 20% O\u003csub\u003e2\u003c/sub\u003e, and 5% CO\u003csub\u003e2\u003c/sub\u003e. Cells were then treated with increasing doses of recombinant (rh)SIGLEC6 (In Vitro Technologies) at 10, 20, and 40ng/ml or vehicle control for 48 hours. Conditioned media, cell lysates and mRNA were collected for analysis using ELISA and qRT-PCR respectively. Each treatment was performed in triplicate and repeated five times (n\u0026thinsp;=\u0026thinsp;5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnzyme Linked Immunosorbent Assay (ELISA)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConcentrations of SIGLEC6 in conditioned cell culture media were measured using human SIGLEC6 DuoSet ELISA kit (RnD systems, Minnesota, USA) according to the manufacturer\u0026rsquo;s instructions. This kit has an inter-assay and intra-assay precision of \u0026lt;\u0026thinsp;15%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA Isolation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA was isolated from primary trophoblasts, hTSCs, and HUVECs using the GenElute\u0026trade; Mammalian Total RNA Miniprep Kit (Sigma-Aldrich, Missouri, USA) according to manufacturer\u0026rsquo;s instructions. mRNA concentration was quantified using a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies Inc.), and equivalent amounts were converted to cDNA as described below.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003emRNA was reverse transcribed into cDNA using the Applied Biosystems High-Capacity cDNA Reverse Transcriptase kit (Life Technologies) as per manufacturer\u0026rsquo;s instructions. Reverse transcription was carried out using the iCycler iQ5 (Bio-Rad) under the following conditions: 25\u0026deg;C for 10 minutes, 37\u0026deg;C for 60 minutes, and 85\u0026deg;C for 5 minutes.\u003c/p\u003e\n\u003cp\u003eGene expression of \u003cem\u003eSIGLEC6\u003c/em\u003e (Sialic Acid-Binding Immunoglobulin-like Lectin-6, Assay ID: Hs00609663_m1), \u003cem\u003eTEAD4\u003c/em\u003e (TEA domain transcription factor 4, Assay ID: Hs01125032_m1), \u003cem\u003eSDC1\u003c/em\u003e (Syndecan 1, Assay ID: Hs00896423_m1), \u003cem\u003eHLAG\u003c/em\u003e (Human Leukocyte Antigen G, Assay ID: Hs03045108_m1), \u003cem\u003eVCAM-1\u003c/em\u003e (Vascular Cell Adhesion Molecule-1, Assay ID: Hs01003372_m1), \u003cem\u003eICAM-1\u003c/em\u003e (Intracellular Adhesion Molecule-1, Assay ID: Hs00164932_m1), \u003cem\u003eET1\u003c/em\u003e (Endothelin 1, Assay ID: Hs00174961_m1), \u003cem\u003eCYC1\u003c/em\u003e (Cytochrome C1, Assay ID: Hs00357717_m1), \u003cem\u003eTOP1\u003c/em\u003e (DNA Topoisomerase I, Assay ID: Hs00243257_m1), \u003cem\u003eYHWAZ\u003c/em\u003e (Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein Zeta, Assay ID: Hs01122454_m1), and \u003cem\u003eGAPDH\u003c/em\u003e (Glyceraldehyde-3-Phosphate Dehydrogenase, Assay ID: Hs99999905_m1) were quantified by quantitative real-time PCR (qRT-PCR) on the CFX384 (Bio-Rad, Hercules, CA) using FAM-labelled Taqman\u0026trade; Fast Advanced Master Mix (Applied Biosystems) and their specific Taqman\u0026trade; Gene expression Assays (Life Technologies). The run conditions were 95\u0026deg;C for 20 seconds, followed by 40 cycles of: 95\u0026deg;C for 3 seconds, and 60\u0026deg;C for 30 seconds.\u003c/p\u003e\n\u003cp\u003eSYBR qRT-PCR was carried out to assess gene expression of \u003cem\u003esFLT-1 e15a\u003c/em\u003e, \u003cem\u003esFLT-1 i13 and YWHAZ\u003c/em\u003e on the CFX384 (Bio-Rad, Hercules, CA) using Fast SYBR\u0026trade; Green Master Mix (Applied Biosystems) with specific forward and reverse primers. The run conditions were 95\u0026deg;C for 20 seconds, followed by 40 cycles of 95\u0026deg;C for 1 second and 60\u0026deg;C for 20 seconds, with a melt curve from 65\u0026deg;C to 95\u0026deg;C in 0.5\u0026deg;C increments for 0.05 seconds each. No product was detected in the non-template control.\u003c/p\u003e\n\u003cp\u003eGene expression was normalised to the geometric mean of \u003cem\u003eCYC1\u003c/em\u003e and \u003cem\u003eTOP1\u003c/em\u003e for hypoxia experiment, \u003cem\u003eGAPDH\u003c/em\u003e for syncytial hTSCs, and \u003cem\u003eYHWAZ\u003c/em\u003e for HUVECs samples. All samples were run in duplicate, and the average threshold (Ct) value was used. Results were calibrated against the average Ct of controls and expressed as fold change relative to controls.\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eStatistical analysis\u003c/h2\u003e\n\u003cp\u003eAll \u003cem\u003ein vitro\u003c/em\u003e experiments were performed in technical triplicates and repeated five times. Normality and lognormality were assessed using the Anderson-Darling, D\u0026rsquo;Agostino \u0026amp; Pearson, Shapiro-Wilk, and Kolmogorov-Smirnov tests, and the appropriate statistical methods were selected based on data distribution. Parametric tests were used for normally distributed data, while non-parametric tests were applied to data that were not normally distributed. For comparisons between two unpaired groups, an unpaired t-test (parametric) or Mann-Whitney test (non-parametric) was used. For comparisons with three or more groups, either one-way ANOVA (parametric) or Kruskal-Wallis test (non-parametric) was used. All data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM or median (interquartile range, IQR), as appropriate. A p-value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant. All analyses were performed using GraphPad Prism version 10.3.0 (GraphPad Software Inc., San Diego, California).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eSIGLEC6 expression and secretion increases with trophoblast differentiation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo characterise SIGLEC6 expression in the placental cells, hTSCs were differentiated into either syncytiotrophoblasts or extravillous trophoblasts over 96 hours, and mRNA levels and protein secretion were measured at 0, 48, 72 and 96 hours. Syncytialisation was confirmed by a significant reduction in the progenitor marker, \u003cem\u003eTEAD4\u003c/em\u003e mRNA expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, p\u0026thinsp;=\u0026thinsp;0.0018) and increased mRNA expression of an established syncytiotrophoblast marker, \u003cem\u003eSDC1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, p\u0026thinsp;=\u0026thinsp;0.001) at 72 and 96 hours post-differentiation. We have previously shown reduced TEAD4 protein expression and increased SDC1 protein expression with hTSC syncytialisation \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eSIGLEC6\u003c/em\u003e mRNA expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, p\u0026thinsp;=\u0026thinsp;0.004) and protein secretion (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, p\u0026thinsp;=\u0026thinsp;0.0009) were both significantly elevated following differentiation into syncytiotrophoblasts over 96 hours.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eDifferentiation of the hTSCs into EVTs was confirmed by a reduction in progenitor marker, \u003cem\u003eTEAD4\u003c/em\u003e mRNA expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, p\u0026thinsp;=\u0026thinsp;0.002) and an increase in the EVT marker, \u003cem\u003eHLAG\u003c/em\u003e mRNA expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, p\u0026thinsp;=\u0026thinsp;0.0005) at 72 and 96 hours. Similarly, \u003cem\u003eSIGLEC6\u003c/em\u003e mRNA expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG, p\u0026thinsp;=\u0026thinsp;0.002) and protein secretion (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH, p\u0026thinsp;=\u0026thinsp;0.001) were significantly increased following EVT differentiation over 96 hours. These data suggets that SIGLEC6 is expressed in all trophoblast subpopulations and its expression is upregulated during both syncytiotrophoblast and extravillous trophoblast differentiation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffect of hypoxia and inflammatory stimuli on SIGLEC6 expression and secretion in syncytialised first trimester placental stem cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePreeclampsia can be accompanied by impaired placental perfusion, leading to intermittent hypoxia and inflammation \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. To investigate the role of SIGLEC6 in a preeclampsia \u003cem\u003ein vitro\u003c/em\u003e model, we exposed syncytialised hTSCs to either hypoxia (1% O\u003csub\u003e2\u003c/sub\u003e) or normoxia (8% O\u003csub\u003e2\u003c/sub\u003e), or inflammatory stimuli: interleukin-6 (IL-6) and tumour necrosis factor α (TNFα). Given syncytiotrophoblasts are likely the primary source of SIGLEC6 in preeclampsia (Figure: 1E, 1G), they were utilised for further investigation.\u003c/p\u003e\u003cp\u003e\u003cem\u003eSIGLEC6\u003c/em\u003e mRNA expression was significantly increased following hypoxic exposure of syncytialised hTSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, p\u0026thinsp;=\u0026thinsp;0.008). Similarly, protein secretion in cell culture media was also significantly elevated in syncytialised hTSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, p\u0026thinsp;=\u0026thinsp;0.008).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn preeclampsia, pro-inflammatory cytokines IL-6 and TNFα are known to be elevated in the circulation and contribute to placental and systemic inflammation \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. To investigate whether these cytokines regulate placental SIGLEC6 expression or secretion, syncytialised hTSCs were treated with increasing doses of IL-6 (0, 0.1, and 1ng/ml) and TNFα (0, 0.1, and 1ng/ml). Both IL-6 and TNFα significantly upregulated \u003cem\u003eSIGLEC6\u003c/em\u003e mRNA expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, p\u0026thinsp;=\u0026thinsp;0.002 and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, p\u0026thinsp;=\u0026thinsp;0.009) and protein secretion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, p\u0026thinsp;=\u0026thinsp;0.001 and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, p\u0026thinsp;=\u0026thinsp;0.01) in a dose-dependent manner, compared to controls.\u003c/p\u003e\u003cp\u003eOverall, hypoxia and pro-inflammatory cytokines may contribute to driving modest increases in SIGLEC6 expression and secretion from syncytiotrophoblast in the placenta.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffect of recombinant SIGLEC6 on anti-angiogenic markers in syncytialised first trimester placental stem cells.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRecent studies have shown SIGLEC6 expression is significantly elevated in placental tissue complicated by preeclampsia \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. To further understand the function of SIGLEC6 in the placenta complicated by preeclampsia, we examined the effect of high levels of recombinant SIGLEC6 on syncytialised hTSCs. We measured the expression of anti-angiogenic molecule sFlt-1 and its variants, e15a and i13, both of which are known to be altered in preeclampsia. Our results showed no significant changes in mRNA expression of sFlt\u003cem\u003e-1\u003c/em\u003e variant\u003cem\u003es\u003c/em\u003e (\u003cem\u003ee15a\u003c/em\u003e \u0026ndash; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, and i13 \u0026ndash; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) or in sFlt-1 protein secretion (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) following SIGLEC6 treatment of syncytiotrophoblast cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eMechanisms of SIGLEC6 secretion in syncytialised syncytialised first trimester placental stem cells.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the mechanisms contributing to SIGLEC6 secretion, syncytialised hTSCs were treated with increasing doses of Brefeldin A (0, 5, and 10ng/ml). Brefeldin A inhibits protein secretion by blocking protein transport from the endoplasmic reticulum to the Golgi apparatus \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. A significant dose-dependent reduction in SIGLEC6 was observed, both in the cell culture media (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, p\u0026thinsp;=\u0026thinsp;0.02 at 5 ng/ml) and in cell lysates (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, p\u0026thinsp;=\u0026thinsp;0.03 at 5 ng/ml, p\u0026thinsp;=\u0026thinsp;0.005 at 10 \u0026micro;M). MTS assay results suggest no impact on cell viability at these concentrations (data not shown). Thus, this data suggests that SIGLEC6 may be actively secreted via the Golgi apparatus from placenta, rather than being passively released into the circulation through syncytial shedding or turnover.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eAdministering recombinant SIGLEC6 to endothelial cells does not alter the expression of markers of endothelial dysfunction, or angiogenic molecules.\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePreeclampsia features widespread maternal endothelial cell dysfunction, largely attributed to the increased release of anti-angiogenic factors \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Given that SIGLEC6 protein levels are significantly elevated in the circulation of women with preeclampsia \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, we examined the effect of recombinant SIGLEC6 at levels comparable to those found in maternal circulation in preeclampsia, on primary HUVECs.\u003c/p\u003e\u003cp\u003eTreatment of HUVECs with recombinant SIGLEC6 had no significant effect on the mRNA expression of the endothelial dysfunction markers VCAM1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), ICAM1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), or the vasoconstrictor ET-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Similarly, recombinant SIGLEC6 did not alter mRNA levels of pro-angiogenic molecules VEGF (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD) or PlGF (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Furthermore, recombinant SIGLEC6 had no significant effect on the mRNA expression of sFlt-1 variants e15a (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF) and i13 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG), nor did it affect sFlt-1 protein secretion (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). These findings suggests that high circulating levels of SIGLEC6 may not modulate endothelial dysfunction or angiogenic imbalance in preeclampsia.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study showed that SIGLEC6 is expressed in hTSCs as well as syncytialised hTSCs and those differentiated into EVTs. Further, \u003cem\u003ein vitro\u003c/em\u003e studies showed hypoxia and inflammation increased SIGLEC6 expression in human (cyto)trophoblast stem cells. Additionally, Brefeldin A was found to reduce the secretion of SIGLEC6 from syncytialised hTSCs. However, treating primary endothelial cells with levels of recombinant SIGLEC6 equivalent to those found in the circulation of women with preeclampsia did not induce markers of endothelial dysfunction.\u003c/p\u003e\u003cp\u003eSIGLEC6, a molecule known to facilitate cell-cell interactions, is highly expressed in the placenta \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Our findings demonstrate that SIGLEC6 is expressed in all trophoblast subpopulations, with its mRNA expression and protein secretion significantly upregulated during the differentiation of first trimester human (cyto)trophoblast stem cells into syncytiotrophoblasts and extravillous trophoblasts over a 96-hour period. These findings suggest that SIGLEC6 is dynamically regulated throughout trophoblast development. Previous studies have also shown SIGLEC6 expression in cytotrophoblasts and syncytiotrophoblasts within chorionic villi, as well as in extravillous trophoblast of the decidua basalis of human placenta \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Some studies have also reported SIGLEC6 expression decreases with advancing gestation (after 8 weeks) but remains detectable in proliferative cytotrophoblasts and syncytiotrophoblasts \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. SIGLEC6 is a known regulator of immune interactions and has been implicated in trophoblast signalling through Src kinase tyrosine phosphorlation and SHP-2 recruitment \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. SHP-2 and Src kinase phosphorylation regulate various cellular processes, including proliferation, differentiation, motility, and adhesion \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. This is particularly significant given that preeclampsia is associated with impaired trophoblast invasion and differentiation. However, further research is still needed to elucidate the mechanisms by which SIGLEC6 influences trophoblast function.\u003c/p\u003e\u003cp\u003eGiven placental hypoxia and inflammation are hallmarks of preeclampsia, we examined the effects of hypoxia (1% O\u003csub\u003e2\u003c/sub\u003e vs 8% O\u003csub\u003e2\u003c/sub\u003e) and pro-inflammatory cytokines (IL-6 and TNFα) on \u003cem\u003eSIGLEC6\u003c/em\u003e mRNA expression and protein secretion in syncytialised hTSCs. Exposure to hypoxia (1% O\u003csub\u003e2\u003c/sub\u003e) significantly increased \u003cem\u003eSIGLEC6\u003c/em\u003e mRNA expression and protein secretion compared to normoxia (8% O\u003csub\u003e2\u003c/sub\u003e). This aligns with previous findings: Guan et al demonstrated that hypoxia-inducible factor-1α (HIF-1α) accumulation promoted SIGLEC6 expression and secretion in HTR8/SVeno and BeWo cells \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Furthermore, a 2018 study reported a significant upregulation of \u003cem\u003eSIGLEC6\u003c/em\u003e mRNA expression in primary human mast cells following 24 hours of hypoxia exposure \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eSimilarly, exposure to inflammatory stimuli (IL-6 and TNFα), demonstrated a significant dose-dependent upregulation of \u003cem\u003eSIGLEC6\u003c/em\u003e mRNA expression and protein secretion from syncytialised hTSCs. While studies examining the effects of IL-6 and TNFα on SIGLEC6 expression in placental cells are limited, a 2018 study reported increased \u003cem\u003eSIGLEC6\u003c/em\u003e expression in human mast cells co-cultured with colon cancer cells lines. The authors suggests cancer cell-derived factors, possibly inflammatory mediators, may modulate SIGLEC6 levels \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. As our study only measured IL6 and TNFα, future work should assess additional imflammatory mediators to better understand their contribution to SIGLEC6 upregulation in preeclamspia. Collectively, these findings suggest hypoxia and inflammation contribute to elevated SIGLEC6 levels in maternal blood from patients diagnosed with preeclampsia \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn our recent publication, we demonstrated a strong association between elevated SIGLEC6 and preeclampsia \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Our team identified significantly elevated SIGLEC6 levels in the maternal circulation preceding preeclampsia (as early as 15\u0026ndash;20 weeks\u0026rsquo; gestation), in women diagnosed with preeclampsia, and elevated levels strongly correlate with disease severity \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. SIGLEC6 was highly abundant in maternal circulation, which led us to hypothesise that despite being a transmembrane receptor, it may be secreted from the placental surface. To investigate the secretory mechanism of SIGLEC6, syncytialised hTSCs were treated with Brefeldin A, a macrolide antibiotic that inhibits protein secretion by blocking protein transport from the endoplasmic reticulum to the Golgi apparatus. While Brefeldin A treatment resulted in a modest reduction in SIGLEC6 secretion in the conditioned media, this incomplete inhibition suggests other mechanisms, beyond canonical Golgi-dependent secretion, may be involved in SIGLEC6 secretion.\u003c/p\u003e\u003cp\u003eWhile this study enhances our understanding of SIGLEC6 expression in trophoblasts, its precise role in placental function and pathogenesis of preeclampsia remains unclear. Previous studies on the role of SIGLEC6 in trophoblast function have yielded inconsistent findings. Jia et al., reported that SIGLEC6 inhibits trophoblast migration and invasion by impairing mitochondrial function in HTR-8/SVneo and JAR cells \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. In contrast, Guan et al. found that SIGLEC6 overexpression had no significant impact on proliferation, migration, or invasion in HTR-8/SVneo cells \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Additionally, a 2012 study suggested a complex interplay between SIGLEC6 and leptin, where SIGLEC6 promoted proliferation in a leptin-dependent manner, while also enhancing invasion and reducing apoptosis independently of leptin \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Moreover, SIGLEC6 has been shown to bind glycodelin-A (a glycoprotein), suppressing trophoblast invasion by down-regulating the ERK/c-Jun signalling pathway, critical for trophoblast invasion and vascular remodelling \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Given these inconsistencies, further investigation into the interaction with SIGLEC6, leptin and other signalling molecules, may offer deeper insights into mechanisms underlying trophoblast differentiation and invasion and the development of preeclampsia.\u003c/p\u003e\u003cp\u003ePreeclampsia is characterized by widespread systemic endothelial dysfunction, driven primarily by an imbalance between pro-angiogenic and anti-angiogenic factors \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. In the current study, SIGLEC6 had no effect on endothelial dysfunction markers, pro-angiogenic markers, and anti-angiogenic markers. These findings suggest the elevated levels of circulating SIGLEC6 levels in preeclampsia are unlikely to directly contribute to endothelial dysfunction, indicating its role may be more confined to the placenta rather than vascular dysfunction.\u003c/p\u003e\u003cp\u003eA significant strength of this study is the use of differentiated trophoblast suntypes and primary HUVECs to assess the role of SIGLEC6 in preeclampsia. However, a limitation of this work was that we could not mimic the over-expression of SIGLEC6 that is apparent in placentas from pregnancies complicated by preeclampsia \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. While this was attempted via plasmid transfection of syncytialised hTSCs, we could not obtain elevated protein expression or secretion. Future studies to over-express SIGLEC6 in primary trophoblast may assist in furthering our understanding of its biology. In our interrogation of the impacts of SIGLEC6 on endothelial dysfunction, we only examined a subset of markers. Future studies could expand to explore alternative aspects of endothelial dysfunction, such as nitric oxide signalling pathway (vasodilation/constriction) and vascular reactivity, to further elucidate whether the high circulating levels of SIGLEC6 apparent in preeclampsia\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, have any effect on vascular dysfunction.\u003c/p\u003e\u003cp\u003eIn conclusion, this study provides evidence to suggest the expression of SIGLEC6 across all trophoblast subpopulation, with its highest expression observed in syncytiotrophoblast and extravillous trophoblasts. We showed that SIGLEC6 may be regulated by placental hypoxia and inflammation. While SIGLEC6 is unlikely to directly contribute to endothelial dysfunction or angiogenic imbalance, its upregulation in trophoblast differentiation and in hypoxic and proinflammatory environments warrants further investigation into its role in placental function in preeclampsia.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eAuthor Contributions Statement\u003c/h2\u003e\u003cp\u003eConceptualisation: M.K. and T.J.K.-L.; Methodology, M.K., L.B., N.J.H, P.C., T.-V.N., A.N., C.N.M., G.P.W., S.T., and T.J.K.-L; Formal analysis: M.K. and T.J.K.-L; Investigation: M.K. and T.J.K.-L; Resources: T.J.K.-L., S.T., N.H., and L.B.; Writing\u0026mdash;original draft preparation, M.K. and T.J.K.-L.; Writing\u0026mdash;review and editing, all co-authors; Funding Acquisition, T.J.K.-L, S.T., and L.B. All authors have revised and approved the final version of the manuscript.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eAdditional Information\u003c/h2\u003e\u003cp\u003eThe authors have no conflicts of interest to declare.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003cp\u003eFunding for this work was provided by: National Health and Medical Research Council (#1065854, 2000732). Salary support was received from the National Health and Medical Research Council Fellowship for S.T (#1136418), and from Australian Research Council Future Fellowships for T.J.K.-L (FT230100125) and N.H (FT210100193). The funders played no role in study design or analysis.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualisation: M.K. and T.J.K.-L.; Methodology, M.K., L.B., N.J.H, P.C., T.-V.N., A.N., C.N.M., G.P.W., S.T., and T.J.K.-L; Formal analysis: M.K. and T.J.K.-L; Investigation: M.K. and T.J.K.-L; Resources: T.J.K.-L., S.T., N.H., and L.B.; Writing\u0026mdash;original draft preparation, M.K. and T.J.K.-L.; Writing\u0026mdash;review and editing, all co-authors; Funding Acquisition, T.J.K.-L, S.T., and L.B. All authors have revised and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank Kaitlin Constable, Melissa Sutton, Gabrielle Fleming, Rachel Murdoch, Genevieve Christophers, Anna Middleton, Kirsty Dane, Alex Roddy-Mitchell, Brooke Henshall, Katelyn Dark and Danica Idzes for their assistance in recruiting and characterising participants. We also wish to thank the pathology, health information services, and prenatal clinic staff at the Mercy Hospital for Women for their assistance in conducting this research and patients for agreeing to participate. First trimester cytotrophoblast stem cell lines were obtained from the RIKEN BRC through the National BioResource Project of the MEXT/AMED, Japan.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data from this study are available within the publication. Other resources used in this study are available from the corresponding author upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDuley, L. in \u003cem\u003eSeminars in perinatology\u003c/em\u003e. 130\u0026ndash;137 (Elsevier).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRoberts, J. M. \u0026amp; Gammill, H. S. 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Glycodelin-A protein interacts with Siglec-6 protein to suppress trophoblast invasiveness by down-regulating extracellular signal-regulated kinase (ERK)/c-Jun signaling pathway. \u003cem\u003eJ. Biol. Chem.\u003c/em\u003e \u003cb\u003e286\u003c/b\u003e, 37118\u0026ndash;37127 (2011).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"SIGLEC6, Placenta, Preeclamspia, Pregnancy","lastPublishedDoi":"10.21203/rs.3.rs-7164266/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7164266/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSIGLEC6, a human-specific transmembrane receptor, is highly expressed in placenta. Our team have identified elevated SIGLEC6 in maternal circulation preceding preeclampsia and in women with preeclampsia, correlating with disease severity. This study aimed to characterise SIGLEC6 relevant to preeclampsia using human (cyto)trophoblast stem cells (hTSCs) and primary Human Umbilical Vein Endothelial Cells (HUVECs).\u003c/p\u003e\n\u003cp\u003eSIGLEC6 was measured across three placental cell types by differentiating hTSCs: (cyto)trophoblasts, syncytiotrophoblasts, and extravillous trophoblasts. SIGLEC6 was expressed in all cell types and upregulated during differentiation into syncytiotrophoblasts and extravillous trophoblasts. Exposure of syncytialised hTSCs to hypoxia (1% vs 8% O\u003csub\u003e2\u003c/sub\u003e) elevated SIGLEC6 expression (p = 0.0079), and protein secretion (p = 0.0079). Similarly, inflammatory cytokines (IL-6 or TNFα) increased \u003cem\u003eSIGLEC6\u003c/em\u003e expression (IL-6: p = 0.0016, and TNFα: p = 0.0015) and protein secretion (IL-6: p = 0.002, and TNFα: p = 0.01) from syncytialised hTSCs. Treatment with Brefeldin A (impairs protein trafficking) reduced SIGLEC6 secretion in cell lysates (p = 0.001) and conditioned media (p = 0.02).\u003c/p\u003e\n\u003cp\u003eTo evaluate functional effects, HUVECs were treated with recombinant SIGLEC6. No significant changes were observed in endothelial dysfunction markers (VCAM1, ICAM1, ET-1), pro-angiogenic factors (PlGF, VEGF) or anti-angiogenic splice variants (sFlt-1 e15a, sFlt-1 i13).\u003c/p\u003e\n\u003cp\u003eSIGLEC6 expression is induced by hypoxia and inflammation in syncytialised hTSCs, but recombinant SIGLEC6 does not induce features of endothelial dysfunction observed in preeclampsia.\u003c/p\u003e","manuscriptTitle":"Sialic acid-binding immunoglobulin-like lectin-6 (SIGLEC6) is increased by hypoxia and inflammation; relevance to preeclampsia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-14 07:40:57","doi":"10.21203/rs.3.rs-7164266/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7a4e27a1-53a2-4991-849a-5d145e87e4a2","owner":[],"postedDate":"August 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":53134731,"name":"Biological sciences/Cell biology"},{"id":53134732,"name":"Health sciences/Diseases"},{"id":53134733,"name":"Biological sciences/Immunology"},{"id":53134734,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2025-09-03T08:39:08+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-14 07:40:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7164266","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7164266","identity":"rs-7164266","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}