Effects of sirtuin 1 deficiency on trophoblasts and its implications in the pathogenesis of pre-eclampsia

Abstract Background Sirtuin 1 (SIRT1) is mainly localised in syncytiotrophoblasts and cytotrophoblasts, and is involved in pregnancy regulation. However, data on the association between SIRT1 and pre-eclampsia (PE) remains limited. This study aimed to investigate the role of SIRT1 in PE pathophysiology. Methods Placental SIRT1 expression, as well as serum SIRT1, placental growth factor (PlGF), and soluble FMS-like tyrosine kinase 1 (sFlt-1) levels, were measured using quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, and enzyme-linked immunosorbent assays in 40 healthy pregnant women (NP group) and 40 women with severe PE (PE group). Additionally, the effects of SIRT1 on the migration, invasion, PlGF, and sFlt-1 secretion of HTR-8/SVneo cells were analysed. Results SIRT1 expression was significantly reduced in the placenta of patients with severe PE compared with that in healthy pregnant women. Compared with the NP group, serum SIRT1 and PlGF expression was significantly lower in the PE group; however, the expression of serum sFlt-1 was significantly higher in the PE group. Correlation analysis showed that in the PE group, placental SIRT1 protein levels positively correlated with serum PlGF levels (r = 0.468, P = .002) and negatively correlated with serum sFlt-1 levels (r = −0.542, P < .001). Cells with downregulated SIRT1 had a significantly shorter migration distance and a prominently reduced number of invasive cells compared with the corresponding negative control group, suggesting that SIRT1 deficiency may inhibit the migration and invasive ability of HTR-8/SVneo cells. The opposite results were observed after transfection with lentivirus overexpressing SIRT1. Compared with the corresponding controls, cells with downregulated SIRT1 had significantly reduced PlGF levels and significantly increased sFlt-1 levels in the cell culture supernatants, whereas SIRT1 overexpression produced the opposite results. Conclusions SIRT1 deficiency may contribute to the pathogenesis of pre-eclampsia by reducing trophoblastic migration, invasion, and PlGF secretion and increasing sFlt-1 secretion. PLAIN LANGUAGE SUMMARY Pre-eclampsia is a serious obstetric disorder that begins in the placenta and can occur midway through pregnancy. However, its exact disease process remains unknown. During early pregnancy, trophoblasts (cells that differentiate from fertilised eggs) evolve into new blood vessels that supply oxygen and nutrients to the placenta and maintain placental formation. In people with pre-eclampsia, problematic trophoblasts lead to abnormal placental formation and release of sFlt-1 and PlGF into the mother’s blood, damaging blood vessels. Experts reported that the intracellular enzyme SIRT1 might be associated with developing pre-eclampsia. SIRT1 expression in the placenta of pregnant women with pre-eclampsia was lower than normal, and the decrease in SIRT1 levels in HTR-8/Svneo trophoblasts prevented their ability to form blood vessels and altered sFlt-1 and PlGF secretion. Hence, our findings suggest that reduced SIRT1 in trophoblasts may lead to pre-eclampsia by affecting their ability to form placental blood vessels and altering enzyme secretion.


Introduction
Preeclampsia (PE) is a severe obstetric multisystem syndrome clinically characterised by new-onset hypertension, with or without proteinuria, in middle and late pregnancy (Brown et al. 2018).As a complex disease, PE affects 2%-8% of all pregnancies and is a leading contributor to maternal and foetal/newborn morbidity and death worldwide (Brown et al. 2018;Yang et al. 2023).Elucidating the intricate pathophysiological mechanisms underlying PE will contribute to its prevention, management, and treatment.
Although the pathogenesis of PE remains unclear, placental ischaemia is considered the central cause of the disease (Sones and Davisson 2016).It is widely known that the initial pathological event in PE is diminished placental perfusion due to insufficient trophoblast invasion and defective recasting of maternal spiral arteries (Granger et al. 2002).Placental ischaemia due to inadequate recasting induces increased production and release of the anti-angiogenic marker soluble FMS-like tyrosine kinase-1 (sFlt-1) by trophoblasts.sFlt-1 binds to the pro-angiogenic markers placental growth factor (PlGF) and vascular endothelial growth factor (VEGF), leading to maternal vascular endothelial cell dysfunction and PE (El-Sayed 2017).However, the current knowledge of the pathological mechanisms underlying placental dysfunction in PE is limited.
Recently, sirtuin 1 (SIRT1), a histone deacetylase, has been suggested to be involved in various biological events in humans, including immune responses, metabolism, antiinflammation, and ageing (Pei et al. 2022).According to previous studies, SIRT1 is ubiquitously expressed in the human placenta, localised mainly in syncytiotrophoblasts and cytotrophoblasts, and involved in pregnancy regulation (Broady et al. 2017;Lappas et al. 2011).However, data on SIRT1 and PE are limited.Several studies have reported decreased SIRT 1 expression in preeclamptic placentas compared to normal placentas, that SIRT1 is a protective factor against PE, and that activation of SIRT1 may reduce sFlt-1 secretion from cytotrophoblast cells (Broady et al. 2017;Hastie et al. 2019;Poidatz et al. 2015;Yin et al. 2017).However, in another study, SIRT 1 protein expression was markedly higher in preeclamptic placentas than in normal placentas (Zhang et al. 2022).Therefore, the relationship between SIRT1 and PE warrants further investigation.
In the present study, we aimed to (1) assess the serum and placental levels of SIRT1 in patients with severe PE and normal pregnancies and (2) investigate whether SIRT1 is involved in developing PE.

Clinical samples
Forty healthy pregnant women (NP group) and 40 patients with severe PE (PE group) were enrolled at Nanchong Central Hospital from February 2022 to January 2023.The Ethics Committee of Nanchong Central Hospital reviewed and approved this study (approval number: NCZXYY-2022-066).Written informed consent was obtained from all pregnant women and their relatives.Severe PE was defined according to the American College of Obstetricians and Gynaecologists (American College of Obstetricians and Gynecologists 2013).All participants underwent caesarean delivery.Women with a history of smoking, diabetes, autoimmune, heart, and infectious diseases, chronic nephritis, multiple pregnancies, or foetal developmental abnormalities were excluded.
Peripheral venous blood samples were obtained immediately upon diagnosis for a follow-up analysis in patients with severe PE.In contrast, peripheral venous blood samples were collected in healthy pregnant women before delivery.Fresh blood samples were clotted at room temperature for 30-60 min to allow a clot formation in the blood collection tube, followed by centrifugation at 3000 rpm at 4 � C for 15 min.The separated serum supernatant was collected and stored immediately in Eppendorf tubes at −80 � C until subsequent testing.Serum levels of SIRT1, sFlt-1, and PlGF were analysed using commercial enzyme-linked immunosorbent assay (ELISA) kits.Placental samples were collected from both groups.After the placentas were separated from the uterus by surgeons, multiple placenta tissue fragments were extracted from the maternal side and stored at −80 � C for follow-up analysis.

Cell culture and treatment
The human placental trophoblast HTR-8/SVneo cell line was purchased from ATCC (Rockville, USA) and maintained at 37 � C in a humidified atmosphere containing 20% O 2 and 5% CO 2 .The cell line was cultured in RPMI-1640 medium, supplemented with 10% foetal bovine serum (Gibco, USA) and 1% penicillin-streptomycin, and the medium was refreshed every 48 hours.

Western blotting
Total proteins from placental tissues or cultured cells were isolated and quantified.After denaturation, proteins were separated.The primary antibodies used included anti-SIRT1 (ab110304, Abcam, USA) and anti-b-actin (ab110304, Abcam, USA).Horseradish peroxidase-conjugated goat anti-rabbit IgG (Proteintech, USA) was used as a secondary antibody to detect protein bands.Band densities were quantified using Image-Pro Plus software (Bio-Rad).Densitometry was used to normalise the protein levels to those of b-actin.

Cell transfection
To determine the effect of SIRT1 on the biological function of trophoblasts, we inhibited and overexpressed the expression levels of SIRT1 in HTR-8/SVneo cells using lentiviral vectors.
The SIRT1 inhibitory lentiviral vector (Si-SIRT1), the corresponding negative control vector (Si-Control) and the SIRT1 overexpressing lentiviral vector (Ov-SIRT1) and the corresponding negative control vector (Ov-Control) were designed and constructed by GenePharma Co. Ltd. (Shanghai, China).Cell transfections were undertaken with the Lipofectamine 3000 kit (Invitrogen Corporation, USA) at an appropriate multiplicity of infection following the supplied protocol.Fluorescence microscopy assessed transduction efficiency, and qRT-PCR and western blotting analysis assessed SIRT1 expression after 48 hours of transfection.Stable clones were screened after three passages and collected for subsequent experiments.

Enzyme-linked immunosorbent assay
SIRT1, PlGF, and sFlt-1 levels were measured using SIRT1 (Abcam, UK), PlGF, and sFlt-1 (R&D Systems, USA) ELISA kits.All kits were used according to the manufacturer's instructions.The concentrations of the substances to be measured in the samples were calculated by optical density interpolation of the standard curve.

Wound-healing assay
Cell migration was evaluated using a wound-healing assay.Cells were incubated in a six-well plate (10 5 cells per well) for 48 hours.Then, a sterile 1 mL pipette tip was used to draw a visible wound on the cell layer.Images of cells at 0 and 48 hours after scratching were obtained using an inverted microscope.Cell migration rate was analysed based on the difference between the width of the scratch after 48 hours and the width of the scratch at 0 hours.The assay was repeated thrice.

Matrigel invasion assay
Cell invasive ability was evaluated using a Matrigel invasion assay in a 24-well Transwell plate with a BD Matrigel matrix (Corning, USA).The suspended cells were seeded evenly into the upper chambers after counting, and then RPMI-1640 medium containing 10% foetal bovine serum was added into the lower chambers and incubated at 37 � C, 5% CO 2 for 24 hours.The non-invasive cells in the upper chamber were removed, and the invasive cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 30 min, followed by staining with 0.1% crystal violet (Sigma-Aldrich) for 15 min.The number of penetrating cells was counted under an optical microscope.The assay was repeated thrice.

Reporting statement declaration
The STREGA checklist was used to write our report (Little et al. 2009).

Statistical analysis
SPSS software (version 16.0; GraphPad Software, Inc., La GraphPad Prism 7.0) was used to construct statistical charts.
Normally distributed data were expressed as the mean ± standard deviation (SD), and a Student's t test was used to compare groups.Non-normally distributed data were expressed as medians and interquartile ranges, and the Mann-Whitney U-test was used for comparisons.Spearman's rho was used to calculate the correlation coefficients.P values < .05indicated statistical significance.

Baseline demographic characteristics of the study population
The baseline demographic characteristics of the study participants in the two groups are shown in Table 1.The gestational age at delivery, body mass index, systolic blood pressure, diastolic blood pressure, and birth weight varied significantly between the PE and NP groups (all P < .05).

SIRT1 was decreased in the placentas and serum samples of PE patients
As revealed by qRT-PCR and western blot analyses, SIRT1 expression was markedly reduced in the placentas of patients with severe PE (PE group) compared with that in normal pregnant women (NP group) (Figure 1A and 1B, P < .001).
As shown by the ELISA data, compared with the NP group, serum SIRT1 and PlGF expression decreased significantly in the PE group (Figure 1C ,D, all P < .001),but serum sFlt-1 expression increased appreciably in the PE group (Figure 1E, P < .001).
To assess whether SIRT1 affects the migration and invasive ability of HTR-8/SVneo cells, we used wound-healing and matrigel invasion assays, respectively, as the corresponding assessment methods.The results showed that cells with downregulated SIRT1 (Si-SIRT1 group) had a significantly shorter migration distance and significantly reduced number of invasive cells compared with the corresponding negative control group (Si-control group), suggesting that SIRT1 deficiency may inhibit the migration and invasive ability of HTR-8/SVneo cells (Supplementary Table 1) (Figures 3A, B, all P < .05).The opposite results were detected following transfection with lentivirus overexpressing SIRT1 (Supplementary Table 1) (Figures 3A,B, all P < .05).
Enzyme-linked immunosorbent assay (ELISA) was used to detect the effects of SIRT1 on PlGF and sFlt-1 secretion in HTR-8/SVneo cells.The data showed that compared with the corresponding controls (Si-control group), cells with downregulated SIRT1 (Si-SIRT1 group) had significantly reduced PlGF levels and significantly increased sFlt-1 levels in the cell culture supernatants, whereas SIRT1 overexpression produced the opposite results (Figure 3D, all P < .05).

Discussion
PE is a multifactorial pregnancy-related disorder involving various pathological mechanisms (El-Sayed 2017).The main pathological features of this disorder include insufficient trophoblast infiltration and subsequent shallow uterine spiral artery recasting, followed by the release of various placentaderived signals and/or inflammatory mediators into the maternal circulation due to abnormal placenta formation.This in turn triggers generalised inflammation and systemic endothelial dysfunction.An imbalance in the secretion and release of pro-and anti-angiogenic factors during pregnancy plays a fundamental role in developing this condition, leading to maternal endothelial dysfunction and clinical PE (Kluivers et al. 2023).Therefore, insufficient trophoblast infiltration of the uterine artery plays a crucial role in the pathological mechanisms of PE pathogenesis.
SIRT1 is ubiquitously expressed in the placenta, primarily localised in syncytiotrophoblasts and cytotrophoblasts, and is closely associated with the regulation of pregnancy (Broady et al. 2017;Lappas et al. 2011).Some studies have indicated that SIRT1 is involved in developing PE; however, the results of these studies are not entirely consistent (Broady et al. 2017;Liu et al. 2022;Poidatz et al. 2015;Yin et al. 2017;Zhang et al. 2022).In addition, a recent study showed that serum SIRT1 expression was strongly related to the severity of PE; further, combined with uterine artery haemodynamic parameters, it could predict disease severity in patients with PE (Ge and Kong 2023).In the present study, 80 maternal cases were included, and placental and serum SIRT1 levels were measured.The results revealed that the expression levels of placental and serum SIRT1 in patients with severe PE tended to decrease compared with those in healthy pregnant  women, which is in accordance with previous studies (Broady et al. 2017;Pei et al. 2022;Poidatz et al. 2015;Yin et al. 2017).However, the exact role of SIRT1 deficiency in PE pathology remains unclear.
Several studies have reported that SIRT1 genetic variations are associated with BMI and the risk of obesity (Peeters et al. 2008;Zillikens et al. 2009) and that SIRT1 expression may be affected in obese populations (Arab et al. 2018).In our study, women with severe PE had increased BMI and reduced serum and placental SIRT1 expression compared to healthy pregnant women.We investigated the relationship between BMI and serum and placental SIRT1 expression, and found no significant correlations between BMI and serum and placental SIRT1 levels (Supplementary Table 2), indicating that they may not be dependent on PE pathophysiology.This supports our hypothesis that reduced SIRT1 levels play a major role in the pathogenesis of PE, and our in vitro findings reinforce this hypothesis.However, this does not exclude the possibility that SIRT1 levels are associated with elevated BMI in these pregnancies, and further studies are necessary to clarify this.
The pro-angiogenic molecule PlGF and the anti-angiogenic molecule sFlt-1, which are mainly produced by trophoblasts during pregnancy, serve as vital players in the pathophysiology of PE, and are closely associated with the maintenance of maternal endothelial cell function and fetoplacental angiogenesis (Gu et al. 2008).Maternal angiogenic imbalances, caused by the reduced placental release of PlGF and increased placental release of sFlt-1 into the maternal circulation, are known to be a crucial factor in generalised vascular dysfunction, which is vital to developing the maternal clinical syndrome of PE (Brennecke et al. 2016).In this study, patients with severe PE showed lower serum PlGF concentrations and higher serum sFlt-1 concentrations than healthy pregnant women, consistent with the results of a previous study (Lecarpentier and Tsatsaris 2016).A recent study pumped recombinant SIRT1 protein intra-abdominally into PE rat models and found that maternal plasma levels of sFlt-1 were attenuated, indicating that SIRT1 may affect maternal plasma sFlt-1 levels (Huang et al. 2021).We analysed the correlation between placental SIRT1 levels and serum PlGF and sFlt-1 levels in patients with severe PE.Pearson's correlation analysis showed that placental SIRT1 protein correlated positively with serum PlGF protein concentration and negatively with serum sFlt-1 protein concentration.
To further explore the association between placental SIRT1, serum PlGF, and sFlt-1, SIRT1 silencing or over-expressing lentiviruses were transfected into HTR-8/Svneo cells, which effectively downregulated or upregulated SIRT1 expression, respectively.PlGF and sFlt-1 protein concentrations in the cell culture supernatant of HTR-8/Svneo cells were detected.SIRT1 activation was found to significantly reduce sFlt-1 concentration and increase PlGF concentration, and downregulation of its expression significantly increased sFlt-1 concentration and decreased PlGF concentration.These observations highlighted that placental SIRT1 deficiency may contribute to the pathophysiological process of PE by regulating the trophoblastic release of PlGF and sFlt-1.Overall, our results suggest that SIRT1 deficiency is an important cause of a pro-and anti-angiogenic imbalance.
There is growing evidence that insufficient trophoblast invasion and migration are closely associated with the pathophysiology of PE (Liu et al. 2023;Zhu and Chen 2023).However, the detailed pathological mechanisms of these associations remain largely unknown.Studies have reported that SIRT1 promotes the invasive and migratory activities of breast and prostate cancer cells (Cui et al. 2016;Xu et al. 2018), while silencing SIRT1 reduces the invasive and migratory activities of prostate cancer cells (Cui et al. 2016).However, whether SIRT1 regulates trophoblast migration and invasion remains unclear.Trophoblasts and tumour metastases have similar molecular mechanisms and often display similar trajectories of migration and invasion (Kohan-Ghadr et al. 2016).In this study, we explored the regulatory effects of SIRT1 on the invasive and migratory activities of HTR-8/ Svneo cells by knocking down or overexpressing SIRT1, and found that SIRT1 upregulation significantly promoted the invasive and migratory activities of HTR-8/SVneo cells.
Conversely, the downregulation of SIRT1 expression significantly inhibited the invasive and migratory activities of HTR-8/SVneo cells.These results indicated that SIRT1 deficiency may contribute to the pathological process of PE by reducing trophoblastic migration and invasion.However, another study reported that SIRT1 negatively regulates the invasion of extravillous trophoblasts, and that knockdown of this protein promotes their invasion (Lee et al. 2019).Therefore, the role of SIRT1 deficiency in PE warrants further investigation.

Conclusion
Our data showed that placental SIRT1 expression was reduced in patients with severe PE.Further, we observed that SIRT1 deficiency may inhibit trophoblastic migration and invasion, and promote an imbalance of angiogenic factors, which is closely related to the pathophysiology of PE.Thus, our results provide new insights into the development of this disorder.

Figure 1 .
Figure 1.SIRT1 was decreased in the placentas and serum samples of patients with severe pre-eclampsia (NP group: n ¼ 40, PE group: n ¼ 40).A, qRTPCR analysis of SIRT1 mRNA expression in placentas from NP and PE groups.B, Western blotting analysis of SIRT1 protein expression in placentas from NP and PE groups.C, A of SIRT1 levels in serum samples from NP and PE groups by ELISA.D, Assessment of PlGF levels in serum samples from NP and PE groups by ELISA.E, Assessment of sFlt-1 levels in serum samples from NP and PE groups by ELISA.F, The correlation between placental SIRT1 protein and serum PlGF in PE group was assessed using the Spearman's correlation coefficient.G, The correlation between placental SIRT1 protein and serum sFlt-1 in PE group was assessed using the Spearman's correlation coefficient.NP group indicates normal pregnancy women, PE group indicates patients with severe pre-eclampsia.�� P <.001.

Table 1 .
The baseline characteristics of the study population.
Abbreviation: BMI: body mass index; BP: blood pressure.NP group indicates normal pregnancy women, and the PE group included patients with severe pre-eclampsia.Data are presented as median (interquartile range) or mean ± standard deviation.