Decreased expression of the HDAC2 disrupts the SLIT-ROBO signaling pathway and induced angiogenesis in placental endothelial cells in preeclampsia

Decreased expression of the HDAC2 disrupts the SLIT-ROBO signaling pathway and induced angiogenesis in placental endothelial cells in 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 Research Article Decreased expression of the HDAC2 disrupts the SLIT-ROBO signaling pathway and induced angiogenesis in placental endothelial cells in preeclampsia Xufei Fan, Xiujuan Zheng, Samiullah Malik, Jianyun Yu, Yali Yang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4143819/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 Background: Preeclampsia is characterized by reduced histone deacetylase 2 (HDAC2) expression in placental tissue HDAC2 enrichment positively affects angiogenesis as it helps prevent endothelial cell dysfunction. Additionally, research has demonstrated that the SLIT2-ROBO signaling pathway influences preeclampsia. Bioinformatics analysis has suggested that HDAC2 may have a transcriptional regulatory effect on SLIT2. Consequently, investigations have examined the relationship between low HDAC2 expression and the SLIT-ROBO signaling pathway in placental angiogenesis in patients with preeclampsia. Objective: To investigate how decreased HDAC2 expression disrupts the SLIT-ROBO signaling pathway and induces angiogenesis in placental endothelial cells in preeclampsia. Methods: The study included patients with preeclampsia as the observation group, while the placental tissue of normal pregnant women was used as the in vivo control model. In vitro endothelial models using human umbilical veins and microvascular endothelial cells were also used to examine the effects of interference with the expression of HDAC2 and SLIT2. Cell viability CCK-8, colony formation, and tube formation assays were conducted to evaluate angiogenesis. Furthermore, Immunohistochemistry, RT-qPCR, and Western blot analyses were used to examine the expression of genes in cells and tissues. Results: The expression of SLIT2 and ROBO1 was increased, and the protein and mRNA expression of CD34, HDAC2, and vonWillebrand factor(vWF) were lower in preeclampsia placentas than in normal placentas. Using an in vitro endothelial model, the knockdown of HDAC2 inhibited colony formation and impaired neovascularization by reducing vascular endothelial growth factor A (VEGFA) and vascular endothelial growth factor -2 (VEGFR2) activity, while SLIT2 and ROBO1 were highly expressed. The changes caused by HDAC2 knockdown were reversed by SLIT2 knockdown. Conclusion: Preeclampsia progression is promoted by low HDAC2 expression, which inhibits the SLIT-ROBO signaling pathway and induces angiogenesis in placental endothelial cells. HDAC2 SLIT-ROBO signaling pathway Placental angiogenesis Preeclampsia Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Preeclampsia (PE) is a severe obstetric disease that is a leading cause of maternal death worldwide [1]. Every year, PE results in the death of approximately 7,000-8,000 pregnant women and 50,000 perinatal infants [2]. Although the prevalence of PE is similar in both developing and developed countries, the symptoms tend to be more severe in patients in developing countries, often leading to poorer pregnancy outcomes [3]. Despite numerous studies on the etiology and mechanism of PE, its causes remain unclear and clinical treatment options are limited. The formation of an abnormal placenta is a crucial pathogenic factor in PE [4]. In normal pregnancy, villous trophoblast cells invade maternal spiral arteries, replacing the endothelial cell layer and transforming them from small, high-resistance vessels to vessels of longer diameter. This process provides sufficient blood perfusion to the placenta, allowing fetal growth. However, this process is incomplete in patients with PE because of insufficient trophoblast cell infiltration and impaired vascular remodeling, resulting in inadequate placental blood perfusion and ischemic hypoxia[5]. Trophoblasts secrete vasoactive substances into the maternal circulation, causing endothelial cell damage and inducing clinical manifestations associated with PE[6]. Placental endothelial dysfunction is a significant factor in PE development. An imbalance in placental factors has been identified as contributes to placental endothelial dysfunction [7]. Studies have shown that placental factors associated with an anti-angiogenic state include vascular endothelial growth factor (VEGF), placental growth factor (PLGF), and other growth factors [8-9]. Therefore, elucidating the mechanisms driving this anti-angiogenic state may help us to better understand how PE develops. Histone deacetylase 2 (HDAC2), a member of class I histone deacetylase (HDACs), deacetylates lysine deposits in the N-terminal region of core histones by framing large multiprotein complexes to modify the chromatin [10]. Endothelial gene transcription and vascular function are regulated by HDAC2. [11-12]. HDAC2 overexpression antagonizes oxidized LDL-mediated endothelial dysfunction in human aortic endothelial cells [10] and mice [13]. The disrupted HDAC2/HIF-1α/VEGF axis inhibits angiogenesis in diabetic retinopathy in mice [14]. Recently, studies have found low levels of HDAC2 in the placental tissue of PE patients, and restoring HDAC2 expression can inhibit p53/PUMA signaling, thereby improving the biological behavior of trophoblast cells to prevent the occurrence of PE [15]. Therefore, we hypothesized that low HDAC2 expression might suppress angiogenesis to induce endothelial dysfunction and drive PE development. Studies have demonstrated that histone modifications determine transcriptional activity through chromatin remodeling [16]. Histone deacetylase can reduce histone acetylation to increase DNA histone binding, thereby blocking the entry of DNA transcription factors [10]. Therefore, our pre-feasibility study utilized hTF target to predict genes whose transcription might be negatively affected by HDAC2. The SLIT-ROBO signaling pathway is specifically expressed at the placental interface, and previous research has shown that ROBO1, ROBO4, and SLIT2 can be detected in the capillary endothelium of normal human placental villi [17]. Our bioinformatic predictions showed that HDAC2 mediates SLIT2 transcriptional repression. Therefore, we aimed to clarify how low HDAC2 expression affects SLIT2 expression in PE endothelial dysfunction. SLIT2 is a SLIT protein secreted as a ligand that exerts regulatory effects by binding to its specific transmembrane receptor, ROBO proteins. SLIT-ROBO signaling was initially identified as a regulator of axonal growth [18]. Furthermore, it is indispensable for organogenesis of the heart and vasculature [19]. Notably, SLIT2 and ROBO1 levels were significantly higher in the PE placenta than in the normal placenta [17]. Fundamentally, raised SLIT2 levels in the circulatory system contribute to endothelial cell dysfunction and impaired angiogenesis in systemic sclerosis. [20]. As HDAC2 potentially inhibits SLIT2, we speculated that low HDAC2 expression reduces the blockade of SLIT-ROBO signaling, thereby inducing impaired angiogenesis and endothelial dysfunction in PE. Therefore, this study aimed to determine whether the aberrantly activated SLIT2-ROBO2 signaling pathway in the placentas of PE patients was due to the low expression of HDAC2. Moreover, we aimed to elucidate the correlation between this system and impaired endothelial cell angiogenesis. Hopefully, these findings provide a theoretical basis for the treatment of PE. Material and Methods 1.1 Experimental object A total of 43 pregnant women from Jinhua Hospital of Zhejiang University participated in this study from January 2018 to December 2019. Among these, 23 pregnant women with PE were assigned to the PE group. Another 20 normal pregnant women were assigned to the control group. The ethics committee at Zhejiang University Jinhua Hospital approved this study, and all relevant participants provided written informed consent. 1.2 Diagnostic criteria The diagnostic criteria refer to to the “Guidelines for Diagnosis and Treatment of Hypertension During Pregnancy “ (2020): Systolic blood pressure ≥140 mmHg and diastolic blood pressure ≥90 mmHg after 20 weeks of gestation, companied with any of the following: urinary protein ≥0.3 g/24 hours (h), or urinary protein/creatinine ratio ≥0.3, or Random urine protein ≥ (+); no proteinuria but with involvement of any of the following organs or systems: heart, lung, liver, kidney and other vital organs, or digestive system, nervous system and An abnormal change in the blood of an affected fetus in the placenta. 1.3 Observation indicators Monitoring indicators during pregnancy included gestational age at onset, blood pressure, liver and kidney function, blood routine, urine protein, cardiopulmonary function, fetal ultrasound, and fetal heart rate monitoring. Maternal and infant prognosis-related evaluation indicators included maternal eclampsia, heart failure, liver and kidney failure, postpartum hemorrhage, cesarean section rate, length of hospital stay, pregnancy termination, neonatal birth weight, neonatal score, neonatal asphyxia, neonatal respiratory distress syndrome, neonatal ischemic hypoxic encephalopathy, neonatal pneumonia, and pathological jaundice. 1.4 Specimen Processing All placental tissues were collected within 30 minutes (min) of delivery. These tissues were thoroughly washed with cold polybutylene succinate (EDTA), cut into small pieces (100-500Mg), and stored in liquid nitrogen. Additionally, the central tissue of the placenta was approximately 1 cm×1 cm×1 cm, immediately placed in 10% formaldehyde solution for 24 h, routinely embedded in paraffin, and resectioned to films with a thickness of 4 μm. 1.5 Experimental method Immunohistochemical determination of SLIT2, ROBO1, CD34, HDAC2 and von Willebrand factor (vWF) protein expression After deparaffinization in xylene and rehydration in graded alcohol baths, 4µm thick tissue sections were used. Antigen recovery was performed by warming the tissue in a microwave at medium-high intensity for 8 min, then from low to high intensity for 5 min, and cooling at room temperature for 20 min. Antibodies against SLIT2 (ab134166, Abcam) were used to stain the sections after 10 min of blocking with goat serum, ROBO1 (ab7279, Abcam, UK), CD34 (ab81289, Abcam, UK), HDAC2 (ab32117, Abcam, UK), and vWF (ab6994, Abcam, UK) for 1 h, followed by goat anti-rabbit IgGH&L (HRP) secondary antibody (ab6721, Abcam, UK) overnight at 4°C. Color development was achieved using a DAB substrate kit (ab64238, Abcam, UK), followed by counterstaining of the nuclei with hematoxylin (ab220365, Abcam, UK). An optical microscope (Olympus) was used for double-blind observation of the immunohistochemical results by two experienced pathologists. The 200x high-power lens randomly selected the power fields of view. A comprehensive evaluation was made of the staining intensity, the number of positively stained cells, and the product of the two: the intensity of staining in positive cells (one point for cells that do not stain; one point for pale yellow; yellow gets two points; 3 points for yellowish-brown); the proportion of cells that are positive or unstained; 1 point is 10-100%; 11-50% equals two points; 51%-80% is 3 focuses; 81%-100 percent is 4 focuses). For the final result, the two scores are multiplied together: Negative (one) is a score of zero, a score of 1 to 4 is a feeble positive (+), and a positive (++) score is between 5 and 8. The positive (+++) scores ranged from 9 to 12. The Weidner et al. method was used to count placental MVD detection results. CD34/vWF-stained. The vessels were observed under a low-power light microscope to detect the highest vessel density. Patients with vessel diameters ≥ 100 μm were excluded. Then, five non-repetitive fields were selected under the (×200) field of view, and the number of blood vessels in an average view unit was determined. (×200); that is, MVD was calculated. Cell culture Human microvascular endothelial cells (HMEC-1) and human umbilical vein endothelial cells (HUVEC-12) were purchased from Mingzhou Biotech (MZ-2329 and MZ-0790, respectively; Ningbo, China). Medium 199 (11043023, Thermo Fisher, Waltham, MA, USA) with 20% fetal bovine serum (FBS; 12103C, Sigma-Aldrich, St. Louis. MO, USA), HUVEC-12 cells were culture. HMEC-1 cells were maintained in 10% FBS-containing MCDB131 medium (10372019, Thermo Fisher, USA). Cell culture was performed at 37°C in humidified air containing 5% CO2. Cell transfection The MISSION® pLKO.1-puro control plasmid (SHC016, Sigma-Aldrich, USA) was used to construct short hairpin RNA against HDAC2 or SLIT2 (shHDAC2 or shSLIT2), and an empty plasmid was designated as a negative control (shNC) for these plasmids shRNA. HUVEC-12 and HMEC-1 cells were cultured in 96-well plates at a density of 1×10[4] cells, cultured to 80% confluence, and finally transfected with shNC, shHDAC2, shSLIT2, or shHDAC2 alone and using Lipofectamine3000 transfection reagent (L3000015, ThermoFisher, USA) in conjunction with shSLIT2. Briefly, the reagents were incubated in Opti-MEM medium and P3000 reagent at 37°C for 10 min to generate gene-lipid complexes, were added cells and incubated for 48 h at 37°C. SLIT2 and HDAC2 mRNA levels are detected by real-time quantitative Polymerase Chain Reaction (RT-qPCR). TRIzol reagent was used to extract total RNA from placental tissue and HUVEC-12 and HMEC-1 cells transfected with shHDAC2 and shSLIT2 following kit instructions and DNase treatment (Invitrogen, Carlsbad, CA, USA). Reverse transcription (RT) was performed using 1 mM deoxynucleotide triphosphate, 1.25 pmol/L random primer, 25U ribonuclease inhibitor, 100U reverse transcriptase, and RT buffer ( Toyobo, Osaka, Japan). RNA was added to each reverse transcription reaction and incubated at 30°C for 10 min, 60 min at 42°C, and 5 min at 85°C. The resulting cDNA was amplified by PCR using the primer sequences shown in the table (synthesized by Sangon Biotech, Shanghai, China). PCR was performed in a thermal cycler (PTC-200 DNA motor; MJResearch, Waltham, MA, USA) with initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s . hybridization at 60°C for 30 s and extension at 72°C for 45 s. The final single-cycle extension was performed at 72°C for 10 min. The relative area under each sample band was used to express the intensity of the amplified products and the levels of SLIT2 and HDAC2 were normalized to those of GAPDH. Cell Counting Kit (CCK)-8 Assay HUVEC-12 and HMEC-1 cells were seeded in 96-well plates at a density of 5×10[3] cells/well for adherent culture and transfected with shHDAC2 and/or shSLIT2a. 24, 4,8 or 72 h after transfection, CCK-8 reagent (C0037, Beyotime, Shanghai, China) was added to each well at a ratio of 1:10 and incubated at 37°C for 2 h. Finally, a microplate reader was used at a wavelength of 450 nm to measure the optical density. (EMaxPlus, MolecularDevices, Sunnyvale, CA, USA). Colony formation assay The colony formation assay involved trypsinizing HUVEC-12 and HMEC-1 cells (9002-07-7, Sigma-Aldrich, USA) and seeding them in 12-well plates at a density of 3 × 10 3 cells/well. After transfection with shHDAC2 aandshSLIT2, cells were grown in the medium at 37°C for 14 days. Once visible colonies were observed, the plate was washed with phosphate-buffered saline (PBS; 806552, Sigma-Aldrich, USA), fixed with 4% paraformaldehyde (A500684, Sangon Biotech, China) for 15 min, and stained with purple crystals. Transfections of shHDAC and shSLIT2 cells were replated and cultured in the medium for 14 days at 37°C. When colonies were visible, the plate was washed with phosphate-buffered saline (PBS; 806552, Sigma-Aldrich, USA) ( C8470, Solarbio, Beijing, China) 20 for minutes. Finally, stained colonies were photographed using a camera (E-M5MarkIII; Olympus, Tokyo, Japan). Tube formation assay Capillary-like network formation was performed using a tube formation assay with matrigel to detect the angiogenic capacity of endothelial cells. The HMEC-1 and HUVEC-12 cells were transfected with shHDAC2 and shSLIT2, seeded into 96-well plates with a 104-cell-per-well cell density, covered with 50 µL Matrigel and 100 µL medium per well, and incubated for 12 hours Afterwards, the number of Capillary-like tubes were counted at ×100 magnification using an inverted microscope (IXploreStandard; Olympus, Tokyo, Japan). Western blot HUVEC-12 and HMEC-1 cells that were transfected with shHDAC2 anandhSLIT2 were lysed with RIPA lysis and extraction buffer (89900, ThermoFisher, USA )containing a cocktail of protease inhibitors(P8340, Si gma-Aldrich, USA), and the total protein was obtained. Protein concentration was determined using a BCA kit. (A53227, Thermo Fisher, USA) and then separated by SDS-PAGE (P0012A, Beyotime, China). A polyvinylidene fluoride membrane (FFP36; Beyotime, China) was used to transfer the separated proteins, which were then blocked with 5% w/v bovine serum albumin (Solarbio, China) for 2 h at room temperature. Afterward, primary antibodies against ROBO1 (ab7279, 181kDa, 1:500, Abcam, UK), vascular endothelial growth factor A ab46154, 23kDa, 1µg/mL, Abcam, UK), vascular endothelial growth factor receptor2 (VEGFA; Abcam, UK), vascular endothelial growth factor receptor 2 (VEGFR2; Abcam, UK ab11939,)151kDa, 1 µg/mL; Abcam, UK), and GAPDH and (ab181602, 36kDa, 1:10000, Abcam, UK) were used to incubate membranes overnight at 4 °C. The membrane was then incubated with goat anti-rabbit IgG secondary antibody (31460, ThermoFisher, USA) for 1 h at room temperature. Immunoreactive western blotting was performed using a SuperECLStar kit (S349652, Aladdin, Shanghai, China). Blot density analysis was carried out with the help of the ImageJ software (version 1.8.0, National Institutes of Health, Bethesda, Massachusetts. Statistical Analysis The statistical software SPSS17.0 was utilized for the analysis. The differences in Fig.1, 2A, 3CDEF, and 4 were analyzed using an independent t-test. Differences between multiple groups were compared using the independent t-test. Correlation analysis was performed using linear regression correlation analysis. Statistical significance was set at P<0.05. Results 1. T he Expression of SLIT2, ROBO1and CD34 in preeclampsia or healthy placenta Immunohistochemical assays confirmed that ROBO1, SLIT2, and CD34 were expressed in the placentas of each group. More specifically, the study group displayed an increase in ROBO1 and SLIT2 protein expression (P0.01, Fig.) compared with the control group. 1A, B; P<0.05, Fig. 1C, D), whereas CD34 protein expression decreased significantly (P < 0.01, Fig. 1E, F). 2. HDAC2 and vWF expression in preeclampsia or healthy placent According to RT-qPCR analysis, the study group's HDAC2 expression was lower than that in the control group (P<0.001, Figure 2A). The immunohistochemical assay yielded similar results (Fig. 2B). Immunohistochemical analysis revealed that the vWF-positive area in the study group was smaller than that in the control group (Fig.2C). 3. Effect of HDAC2 knockdown on the proliferation of HUVECs-12 and HMEC-1 cells In HUVEC-12 and HMEC-1 cells, HDAC2 was successfully knocked down by shHDAC2 transfection in RT-qPCR analysis (P < 0.001, Fig. 3A, B). Then, a CCK-8 assay was performed, and the results showed that HUVEC-12 and HMEC-1 cells with shHDAC2, compared to shNC transfection, showed inhibited viability at 24, 48, and 72 h after HDAC2 knockdown or transfection ( P<0.001, Fig. 3C, D). Furthermore, when HDAC2 was knocked down, a decreased colony formation rate by HUVEC-12 and HMEC-1 cells were observed (P < 0.001; Fig. 3E, F). 4. Effects of HDAC2 knockdown on angiogenesis and SLIT2/ROBO pathway in HUVEC-12 and HMEC-1 cells Through the tube formation assay, we found that shHDAC2 transfection reduced the number of vessel branches formed by HUVEC-12 and HMEC-1 cells compared to shNC transfection (P<0.01, Fig. 4A, B, C). At the same time, the expression of angiogenesis-related proteins, VEGFA and VEGFR2 in HUVEC-12 and HMEC-1 cells was downregulated after HDAC2 knockdown (P<0.001; Figure 4D, E, F). Notably, compared with shNC transfection, shHDAC2 transfection increased the expression of SLIT2 and ROBO1in HUVEC-12 and HMEC-1 cells, as shown by western blot results (P<0.01, Fig. 4G, H, I). 5. The effect of HDAC2 knockdown on the proliferation of HUVEC-12 and HMEC-1 cells can be reversed by SLIT2 knockdown RT-qPCR analysis showed that the knockdown of SLIT2 was achieved in HUVEC-12 and HMEC-1 cells by transfecting with shSLIT2 (P<0.001; Fig. 5A, B). Using the CCK-8 assay, we observed enhanced viability of HUVEC-12 and HMEC-1 cells after shSLIT2 transfection compared with shNC transfection at 24, 48, or 72 h (P< 0.05, Fig. 5C, D), and SLIT2 knockdown could resist the reduction of HDAC2 knockdown-induced viability at 48 and 72 h (P<0.01, Fig. 5C, D). In turn, HDAC2 knockdown counteracted the increased viability induced by SLIT2 knockdown at 48 and 72 h (P<0.05, Fig. 5C, D). In addition, colony formation assay results showed that transfection with shSLIT2 increased the number of colonies in HUVEC-12 and HMEC-1 cells compared to shNC transfection (P<0.001, Figure 5E, F, G). In addition, SLIT2 knockdown and HDAC2 knockdown could cancel each other out their effects on the colony formation rate (P<0.001, Fig. 5E, F, G). 6. SLIT2 knockdown counteracts the effects of HDAC2 knockdown on angiogenesis and SLIT2/ROBO pathway in HUVEC-12 and HMEC-1 cells Compared with shNC, the control group and SLIT2 knockdown group promoted the formation of vessel branches constructed by HUVEC-12 and HMEC-1 cells (Fig 6A, P<0.01). The results showed that SLIT2 and HDAC2 knockdown mutually counteracted their effects on vascular branch formation in HUVEC-12 and HMEC-1 cells (P<0.01 Fig 6A,B). Western blot results showed that the expression of ROBO1 was decreased in HUVEC-12 cells (P<0.01, Fig. 6C, D), and the expression of VEGFA and VEGFR2 was increased (P<0.001, Fig. 6C, D) in HMEC-1 cells after SLIT2 knockdown. Likewise, the effects of SLIT2 and HDAC2 knockdown offset each other significantly (P<0.01, Fig.6C,D). Discussion Placental abnormalities are considered the cause of PE. Trophoblastic cells replace maternal spiral artery endothelial cells in thickening blood vessels, providing sufficient blood perfusion to the placenta to maintain fetal growth. However, this process is incomplete in patients with PE. Insufficient placental blood perfusion is the key to the onset of PE [21]. One report found no significant difference between increased and decreased CD34 expression in patients [22]. According to another study, PE patients had significantly lower expression levels of the angiogenesis markers VEGF1 and CD34 in the placenta and decidua than in normal pregnant women. [23]. This study confirmed that the expression of CD34 was significantly decreased in pregnant women with PE., indicating that the formation of microvessels was lower than that in normal pregnant women. Since defective trophoblast invasion that triggers PE may cause inappropriate spiral arterial remodeling and further induce endothelial dysfunction [8], understanding the mechanisms underlying endothelial dysfunction in PE is of great interest. A recent study reported that HDAC2 was poorly expressed in placentas obtained from patients with PE [24], which is consistent with our results. HDAC2 belongs to class I HDACs, and has been shown to protect mice from endothelial dysfunction and block OxLDL-mediated vascular dysfunction upon endothelium-specific overexpression [12,13]. vWF, a blood glycoprotein involved in hemostasis, is an endothelial antigen marker positively associated with MVD and is expressed at low levels in syncytiotrophoblasts in preeclampsia due to endothelial and placental cell damage [25]. Our study detected downregulated vWF expression and low HDAC2 expression in PE placentas. These data, combined with findings from other studies, reconfirm that placental dysfunction is associated with PE and further suggest that low HDAC2 expression in PE placentas may be responsible for endothelial dysfunction in PE. The blocked proliferation of endothelial cells and reduced angiogenic capacity of cells are the main drivers of endothelial dysfunction in PE [26]. In this study, the knockdown of HDAC2 inhibited proliferation of HUVEC-12 and HMC-1 cells. In addition, changes in the levels of angiogenesis-related factors such as VEGFA and VEGFR2 can be detected weeks before PE onset [10]. VEGFA is abundantly expanded in the placenta and plays a significant role in placental vascular development by affecting endothelial cell proliferation, migration, and vascular permeability [27]. VEGFR2 is the main receptor mediating VEGF vasodilation, and its binding to VEGFA triggers angiogenesis, endothelial cell migration, and vasodilation [28,29]. Our results indicate that HDAC2 knockdown also resulted in the downregulation of VEGFA and VEGFR2 in HUVEC-12 and HMEC-1 cells. Our findings suggested that low HDAC2 expression contributes to endothelial dysfunction in PE by inhibiting angiogenesis. Existing evidence demonstrates that endothelial arginase 2, which controls endothelial NO, proliferation, fibrosis, and inflammation, is negatively regulated at the transcriptional level by HDAC2 overexpression, leading to the blockage of oxLDL-mediated vascular dysfunction [11,12], suggesting that HDAC2 can alleviate the regulation of vascular dysfunction by negatively regulating endothelial gene expression. Based on bioinformatics analysis, SLIT2 was predicted to be regulated by HDAC2 transcription. Previous research has demonstrated that PE placentas have high levels of SLIT2 and its receptor ROBO1. [17], consistent with the results of our study. In normal placentas, co-expression of multiple ligands (SLIT2 and SLIT3) and receptors (ROBO1, ROBO2, and ROBO4) may function as an autocrine/paracrine regulatory system to mediate placental development and maintain normal placental function. [30]. Impaired microvascular formation in patients with PE leads to hypoxia. Although SLIT2 and ROBO1 are highly expressed in villous syncytiotrophoblasts, hypoxia (2% O 2 ) can stimulate mRNA expression of the SLIT and ROBO family of molecules, which are mostly found in placental trophoblasts and endothelial cells in vivo. [17]. In our previous study, we found that HDAC2 knockdown inhibited angiogenesis in HUVEC-12 and HMEC-1 cells and increased ROBO1 and SLIT2 expression in endothelial cells. This suggests that in PE placentas, low HDAC2 expression may transcriptionally restore SLIT2 expression, which then upregulates ROBO1 expression in endothelial cells. Challenge with recombinant human SLIT2 may impair the angiogenic properties of healthy explanted dermal microvascular endothelial cells, leading to peripheral microangiopathy [21]. Elevated SLIT2 activity impairs VEGF-induced angiogenesis in the EphA2-deficient endothelium[31]. These studies and our results suggest that endothelium-specific SLIT2 expression restored by low HDAC2 expression is associated with an attenuated endothelial cell angiogenic capacity in PE placentas. Meanwhile, placental hypoxia can increase the expression of ROBO1, which further leads to placental hypoxia and may be one of the possible pathogeneses of PE. Upregulation of SLIT2-ROBO1 signaling promotes angiogenesis in a mouse liver injury model [32]. Furthermore, ROBO1 expression promotes ox-LDL-induced endothelial cell dysfunction [33]. Combined with Zhang's findings, our study suggests that restoration of ROBO1 expression after HDAC2 knockdown in HUVEC-12 and HMEC-1 cells is associated with suppressed endothelial cell angiogenesis. These findings suggest that the ability of low HDAC2 expression to induce impaired angiogenesis and endothelial dysfunction in PE placentas may be attributed to the activation of SLIT2-ROBO1 signaling in endothelial cells. To confirm the validity of this suggestion, we knocked down the expression of SLIT2 in HDAC2-underexpressed HUVEC-12 and HMEC-1 cells. As expected, the knockdown of SLIT2 reversed HDAC2 knockdown-induced inhibition of endothelial cell proliferation and angiogenesis, expression of pro-angiogenic proteins, and counteracted HDAC2 knockdown-induced upregulation of ROBO1 expression, suggesting that low HDAC2 expression activates SLIT2- ROBO1 signaling inhibits angiogenesis and induces endothelial dysfunction in PE placentas. In conclusion, our study demonstrates that in PE placenta, HDAC2 is underexpressed, but SLIT2 and ROBO1 are overexpressed, and that in endothelial cells, HDAC2 knockdown inhibits viability, proliferation, and angiogenesis by activating SLIT2-ROBO1 signaling. Based on these results, it is reasonable to deduce that low HDAC2 expression impairs the blockade of SLIT2-ROBO1 signaling in endothelial cells, thereby inducing impaired angiogenesis and endothelial dysfunction, resulting in abnormal placental development and function in PE. Abbreviations HDAC Histone deacetylase ROBO Roundabout CCK Cell Counting Kit RT-qPCR Real-Time Quantitative PCR vWF vonWillebrand factor VEGF vascular endothelial growth factor PLGF placental growth factor LDL low-density lipoprotein HIF-1 hypoxia-inducible factor 1 PE preeclampsia VEGFA (vascular endothelial growth factor) A VEGFR (vascular endothelial growth factor) PLGF Placental growth factor LDL Low density lipoprotein HIF-1α Hypoxia-inducible Factor-1α PUMA p53 upregulated modulator of apoptosis DNA Deoxyribonucleic acid EDTA Ethylene diamine tetraacetic acid MVD Microvessel density HMEC Human microvascular endothelial FBS Fetal bovine serum RNA Ribonucleic acid HDAC Histone deacetylase OxLDL Oxidized low-density lipoprotein RT-PCR Real-time Polymerase Chain Reaction Declarations Conflict of Interest declaration The authors declare that they have NO affiliations with or involvement in any organization or entity with any financial interest in the subject matter or materials discussed in this manuscript. Consent of patients All patients provided written informed consent for biological studies. The Ethics Board of Zhejiang University Jinhua Hospital approved the current study, and all participants signed a written informed consent form. Funding This work was supported by the Zhejiang Provincial Natural Science Foundation of China (NO.Q19H040012) and Jinhua Municipal Science and Technology Bureau Project (2023-3-147). Author Contribution Fan Xufei and Zheng Xiujuan were involved in study design and compilation. Samiullah Malik performed a critical review and corrections, and drafted the final copy; Yang Yali, Yu Jianyun were involved in the data analysis. All authors reviewed and approved the final version of the manuscript. References Gestational Hypertension and Preeclampsia: ACOG Practice Bulletin Summary, Number 222. Obstetrics and gynecology. 2020;135(6):1492-5. Steegers EA, von Dadelszen P, Duvekot JJ, Pijnenborg R. Preeclampsia. Lancet (London, England). 2010;376(9741):631-44. Yang Y, Le Ray I, Zhu J, Zhang J, Hua J, Reilly M. Preeclampsia Prevalence, Risk Factors, and Pregnancy Outcomes in Sweden and China. JAMA network open. 2021;4(5):e218401. Rana S, Lemoine E, Granger JP, Karumanchi SA. Preeclampsia: Pathophysiology, Challenges, and Perspectives. Circulation research. 2019;124(7):1094-112. Phipps E, Prasanna D, Brima W, Jim B. Preeclampsia: Updates in Pathogenesis, Definitions, and Guidelines. Clinical journal of the American Society of Nephrology : CJASN. 2016;11(6):1102-6. Gathiram P, Moodley J. Pre-eclampsia: its pathogenesis and pathophysiolgy. Cardiovascular journal of Africa. 2016;27(2):71-8. Stepan H, Hund M, Andraczek T. Combining Biomarkers to Predict Pregnancy Complications and Redefine Preeclampsia: The Angiogenic-Placental Syndrome. Hypertension (Dallas, Tex : 1979). 2020;75(4):918-26. Cui L, Shu C, Liu Z, Tong W, Cui M, Wei C, et al. The expression of serum sEGFR, sFlt-1, sEndoglin and PLGF in preeclampsia. Pregnancy hypertension. 2018;13:127-32. Keshavarzi F, Shahrakipoor M, Teimori B, Yaghmaei M, Narooei-Nejad M, Rasooli A, et al. Association of placental VEGF promoter polymorphisms and VEGF mRNA expression with preeclampsia. Clinical and Experimental Hypertension (New York, NY : 1993). 2019; 41(3): 274-9. Hou Q, Hu K, Liu X, Quan J, Liu Z. HADC regulates the diabetic vascular endothelial dysfunction by targetting MnSOD. Bioscience reports. 2018;38(5). Pandey D, Hori D, Kim JH, Bergman Y, Berkowitz DE, Romer LH. NEDDylation promotes endothelial dysfunction: a role for HDAC2. Journal of molecular and cellular cardiology. 2015;81:18-22. Pandey D, Sikka G, Bergman Y, Kim JH, Ryoo S, Romer L, et al. Transcriptional regulation of endothelial arginase 2 by histone deacetylase 2. Arteriosclerosis, thrombosis, and vascular biology. 2014;34(7):1556-66. Hori D, Nomura Y, Nakano M, Han M, Bhatta A, Chen K, et al. Endothelial-Specific Overexpression of Histone Deacetylase 2 Protects Mice against Endothelial Dysfunction and Atherosclerosis. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology. 2020;54(5):947-58. Hu L, Lv X, Li D, Zhang W, Ran G, Li Q, et al. The anti-angiogenesis role of FBXW7 in diabetic retinopathy by facilitating the ubiquitination degradation of c-Myc to orchestrate the HDAC2. Journal of cellular and molecular medicine. 2021;25(4):2190-202. Zhao Y, Zhao G, Li W. MicroRNA-495 suppresses pre-eclampsia via activation of p53/PUMA axis. Cell death discovery. 2022;8(1):132. Morgan MAJ, Shilatifard A. Reevaluating the roles of histone-modifying enzymes and their associated chromatin modifications in transcriptional regulation. Nature genetics. 2020;52(12):1271-81. Liao WX, Laurent LC, Agent S, Hodges J, Chen DB. Human placental expression of SLIT/ROBO signaling cues: effects of preeclampsia and hypoxia. Biology of reproduction. 2012;86(4):111. Guzmán-Palma P, Contreras EG, Mora N, Smith M, González-Ramírez MC, Campusano JM, et al. Slit/Robo Signaling Regulates Multiple Stages of the Development of the Drosophila Motion Detection System. Frontiers in cell and developmental biology. 2021;9:612645. Santiago-Martínez E, Soplop NH, Kramer SG. Lateral positioning at the dorsal midline: Slit and Roundabout receptors guide Drosophila heart cell migration. Proceedings of the National Academy of Sciences of the United States of America. 2006;103(33):12441-6. Romano E, Manetti M, Rosa I, Fioretto BS, Ibba-Manneschi L, Matucci-Cerinic M, et al. Slit2/Robo4 axis may contribute to endothelial cell dysfunction and angiogenesis disturbance in systemic sclerosis. Annals of the rheumatic diseases. 2018;77(11):1665-74. Fisher SJ. Why is placentation abnormal in preeclampsia? American journal of obstetrics and gynecology. 2015;213(4 Suppl):S115-22. Sahin H, Gunel T, Benian A, Onay Ucar E, Guralp O, Kilic A. Genomic and proteomic investigation of preeclampsia. Experimental and therapeutic medicine. 2015;10(2):711-6. Liu H, Li Y, Zhang J, Rao M, Liang H, Liu G. The defect of both angiogenesis and lymphangiogenesis is involved in preeclampsia. Placenta. 2015;36(3):279-86. Zhu H, Wang C. HDAC2-mediated proliferation of trophoblast cells requires the miR-183/FOXA1/IL-8 signaling pathway. Journal of cellular physiology. 2021;236(4):2544-58. Szpera-Goździewicz, Agata; Majcherek, Maciej; Boruczkowski, Maciej; et al. Circulating endothelial cells, circulating endothelial progenitor cells, and von Willebrand factor in pregnancies complicated by hypertensive disorders.[J] AM J REPROD IMMUNOL. 2017-03-01;77(3): Zhang X, Li Q, Jiang W, Xiong X, Li H, Zhao J, et al. LAMA5 promotes human umbilical vein endothelial cells migration, proliferation, and angiogenesis and is decreased in preeclampsia. The journal of maternal-fetal & neonatal medicine : the official journal of the European Association of Perinatal Medicine, the Federation of Asia and Oceania Perinatal Societies, the International Society of Perinatal Obstet. 2020;33(7):1114-24. Huppertz B. Biology of preeclampsia: Combined actions of angiogenic factors, their receptors and placental proteins. Biochimica et biophysica acta Molecular basis of disease. 2020;1866(2):165349. Shibuya M. Vascular Endothelial Growth Factor (VEGF) and Its Receptor (VEGFR) Signaling in Angiogenesis: A Crucial Target for Anti- and Pro-Angiogenic Therapies. Genes & cancer. 2011;2(12):1097-105. Smani T, Gómez LJ, Regodon S, Woodard GE, Siegfried G, Khatib AM, et al. TRP Channels in Angiogenesis and Other Endothelial Functions. Frontiers in physiology. 2018;9:1731. Chen, Cheng-Yi; Tsai, Chin-Han; Chen, Chia-Yu; et al . Human placental multipotent mesenchymal stromal cells modulate placenta angiogenesis through Slit2-Robo signaling. CELL ADHES MIGR. 2016-03-03;10(1-2):66-76. Youngblood V, Wang S, Song W, Walter D, Hwang Y, Chen J, et al. Elevated Slit2 Activity Impairs VEGF-Induced Angiogenesis and Tumor Neovascularization in EphA2-Deficient Endothelium. Molecular cancer research : MCR. 2015;13(3):524-37. Coll M, Ariño S, Martínez-Sánchez C, Garcia-Pras E, Gallego J, Moles A, et al. Ductular reaction promotes intrahepatic angiogenesis through Slit2-Roundabout 1 signaling. Hepatology (Baltimore, Md.). 2022;75(2):353-68. Zhang Y, Li W, Li H, Zhou M, Zhang J, Fu Y, et al. Circ_USP36 Silencing Attenuates Oxidized Low-Density Lipoprotein-Induced Dysfunction in Endothelial Cells in Atherosclerosis Through Mediating miR-197-3p/ROBO1 Axis. Journal of cardiovascular pharmacology. 2021;78(5):e761-e72. Tables Table 1: Primers used in real-time quantitative polymerase chain reaction for related genes Genes Species Forward Reverse SLIT2 human 5’-GCGAAGCTATACAGGCTTGAT-3’ 5’-TGCAGTCGAAAAGTCCTAAGTTT-3’ HDAC2 human 5’-ATGGCGTACAGTCAAGGAGG-3’ 5’-TGCGGATTCTATGAGGCTTCA-3’ GAPDH human 5’-GAGAAGGCTGGGGCTCATTT-3’ 5’-AGTGATGGCATGGACTGTGG-3’ Table 2: Clinical characteristic in study and control groups Study group Control group P value Age, y 33.74±4.36 29.38±3.93 0.001 Gravity 2.22±1.34 1.33±1.49 0.045 Parity 0.78±0.52 0.52±0.60 0.133 Systolic, mmHg 168.61±22.86 119.38±8.96 0.000 Diastolic, mmHg 106.91±14.14 77.52±8.42 0.000 Fetal distress 5（21.74%） 0（0） Apgar score at birth 8.17±2.90 9.95±0.22 0.008 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4143819","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":282918187,"identity":"f657b520-3cb3-45b3-b1de-b0679f1439bd","order_by":0,"name":"Xufei Fan","email":"","orcid":"","institution":"Affiliated Jinhua Hospital, Zhejiang university school of Medication","correspondingAuthor":false,"prefix":"","firstName":"Xufei","middleName":"","lastName":"Fan","suffix":""},{"id":282918188,"identity":"146dd7b8-07ff-4d16-8b49-0599cdb0c7c2","order_by":1,"name":"Xiujuan Zheng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/UlEQVRIie3PsWrDMBCA4RMGeTHJesbBfgUZgcnWPoqEoV1MOmTxEIqKwVnyAIWEPkZndVEWPYDHuIXOWVKSLUmnTLbHQvUNB4L74QTgOH8R/k4aj/3lx06UGCdDEx6ubM52dspTNSwByZr7LGzrUoLuKZJ1ZdpDPRLQgED5hoIor/1sOhKyMY98YumMrF80k+848y9X8qIj8bDIIizp3JtoIS7JnKiARl0JxaefCBmVNQqm5Qal0j1JgAUN9yWVKxSpkmpAgviQRWApx8DmIAzytOr5S/Kaf4en2sR32+X2dFw8x4lftV9dyZUXgLl99qxfkSMsBqw5juP8W2fCJkrR0DjqZQAAAABJRU5ErkJggg==","orcid":"","institution":"Affiliated Jinhua Hospital, Zhejiang university school of Medication","correspondingAuthor":true,"prefix":"","firstName":"Xiujuan","middleName":"","lastName":"Zheng","suffix":""},{"id":282918189,"identity":"7ead0007-9025-4a73-ac14-19600913efd4","order_by":2,"name":"Samiullah Malik","email":"","orcid":"","institution":"Shenzhen University Health Science Center","correspondingAuthor":false,"prefix":"","firstName":"Samiullah","middleName":"","lastName":"Malik","suffix":""},{"id":282918190,"identity":"80a0caf2-efbd-42c6-bfc0-397aa3d43f54","order_by":3,"name":"Jianyun Yu","email":"","orcid":"","institution":"Affiliated Jinhua Hospital, Zhejiang university school of Medication","correspondingAuthor":false,"prefix":"","firstName":"Jianyun","middleName":"","lastName":"Yu","suffix":""},{"id":282918191,"identity":"db789174-6309-4110-bcce-e0efc987f716","order_by":4,"name":"Yali Yang","email":"","orcid":"","institution":"Affiliated Jinhua Hospital, Zhejiang university school of Medication","correspondingAuthor":false,"prefix":"","firstName":"Yali","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2024-03-21 13:21:01","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4143819/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4143819/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53658445,"identity":"e9c10548-8e8f-4b97-8a67-52cb941e8601","added_by":"auto","created_at":"2024-03-28 15:58:41","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1072885,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of SLIT2, ROBO1 and CD34 in the placenta of patients with preeclampsia (A-F). The expression of ROBO1 (A-B), SLIT2 (C-D) and CD34 (E-F) in the placentas of preeclamptic patients or healthy pregnant women was determined by immunohistochemistry (scale: 100 μm, magnification: ×200). *p\u0026lt;0.05; **p\u0026lt;0.01; * vs control group (SLIT2, homologous cleavage 2; ROBO1, highway 1; RT-qPCR, quantitative real-time polymerase chain reaction).\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4143819/v1/5d5200d6df0dc917426a8719.jpg"},{"id":53658449,"identity":"b8cc1acb-c643-4dbe-8ad0-7a42eda4b537","added_by":"auto","created_at":"2024-03-28 15:58:41","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1399250,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of HDAC2 and vWF in preeclampsia (A) tissues patients. HDAC2 mRNA expression in the placentas of preeclamptic patients or healthy pregnant women was measured by RT-qPCR assay. GAPDH served as the reference gene. (B). HDAC2 protein expression was detected by immunohistochemistry (scale: 50 μm, magnification: ×600) in the intestines of preeclamptic patients or healthy pregnant women. (LE). Expression of the vWF protein in blocks of preeclamptic patients or healthy pregnant women was quantified by immunohistochemistry (scale: 50 μm, magnification: ×400). ***p\u0026lt;0.001; * vs control group (HDAC2, histone deacetylase 2; vWF, von Willebrand factor).\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4143819/v1/d74538c7c2bac3f29e672f3f.jpg"},{"id":53658451,"identity":"03a19283-420d-4484-b6ae-4ddf933331bf","added_by":"auto","created_at":"2024-03-28 15:58:42","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":695720,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of HDAC2 knockdown on HUVEC-12 and HMEC-1 cell proliferation (A–B). HDAC2 mRNA expression in HUVEC-12 and HMEC-1 cells transfected with shHDAC2/shNC was determined by RT-qPCR analysis. GAPDH served as the reference gene. (CD). The viability of HUVEC-12 and HMEC-1 cells transfected with shHDAC2/shNC was measured using a CCK-8 assay. (E-F). Colony formation of HUVEC-12 and HMEC-1 cells transfected with shHDAC2/shNC was assessed by colony formation assay. ***p\u0026lt;0.001; * against. shNC (HDAC2, histone deacetylase 2; RT-qPCR, quantitative real-time polymerase chain reaction; shHDAC2, short hairpin RNA versus HDAC2; shNC, short hairpin RNA versus negative control; OD, optical density).\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4143819/v1/e817cb17e4c5be090b09b30b.jpg"},{"id":53658450,"identity":"4f7f6a07-628e-4e43-b07d-eb5d8d30923f","added_by":"auto","created_at":"2024-03-28 15:58:41","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":655301,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of HDAC2 knockdown on angiogenesis and the SLIT2/ROBO pathway in HUVEC-12 and HMEC-1 cells (A-C). Tube formation of HUVEC-12 and HMEC-1 cells transfected with shHDAC2/shNC was assessed by tube formation assay (scale bar: 50 μm, magnification: ×100). (He gave). Western blotting determined the expression of VEGFA, VEGFR2, SLIT2, and ROBO1 proteins in shHDAC2/shNC-transfected HUVEC-12 and HMEC-1 cells. GAPDH served as the reference gene. **p\u0026lt;0.01; ***p\u0026lt;0.001; * against. shNC (SLIT2, cleavage homolog 2; ROBO1, highway 1; VEGFA, vascular endothelial growth factor A; VEGFR2, vascular endothelial growth factor receptor 2; HDAC2, histone deacetylase 2; RT-qPCR, quantitative real-time polymerase chain reaction analysis; shHDAC2, short hairpin RNA against HDAC2; shNC, short hairpin RNA against negative control).\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4143819/v1/1b147ac7ba059bff977d8f29.jpg"},{"id":53658447,"identity":"a6b8227c-1bfb-45aa-88a4-e0826592d2d3","added_by":"auto","created_at":"2024-03-28 15:58:41","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":970115,"visible":true,"origin":"","legend":"\u003cp\u003eSLIT2 knockdown reversed the effect of HDAC2 knockdown on HUVEC-12 and HMEC-1 cell proliferation (A–B). SLIT2 mRNA expression was determined in HUVEC-12 and HMEC-1 cells transfected with shSLIT2/shNC by RT-qPCR analysis. GAPDH served as the reference gene. (CD). The viability of HUVEC-12 and HMEC-1 cells transfected with shHDAC2 and shSLIT2 alone or in combination was measured by CCK-8 assay. (FOR EXAMPLE). Colony formation in HUVEC-12 and HMEC-1 cells transfected with shHDAC2 and shSLIT2 alone or in combination was determined by colony formation assay. *p or +p \u0026lt; 0.05; ** p or p^p \u0026lt;0.01; ***p or +++ p or ^^^p \u0026lt;0.001; * against. SHNK; + compared to wSLIT2; ^ vs. shHDAC2 (SLIT2, cleavage homolog 2; HDAC2, histone deacetylase 2; RT-qPCR, quantitative real-time polymerase chain reaction; shHDAC2, short hairpin RNA versus HDAC2; shNC, short hairpin RNA versus negative control; OD, optical density).\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4143819/v1/fa6effd36d2c79b694185f6f.jpg"},{"id":53658446,"identity":"fdb94e0b-fe45-4f51-9d3e-298f6b17b75a","added_by":"auto","created_at":"2024-03-28 15:58:41","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":556585,"visible":true,"origin":"","legend":"\u003cp\u003eSLIT2 knockdown counteracts the effects of HDAC2 knockdown on angiogenesis and the SLIT2/ROBO pathway in HUVEC-12 and HMEC-1 cells.\u003c/p\u003e\n\u003cp\u003e(A-B). Tubule formation of HUVEC-12 and HMEC-1 cells transfected with shHDAC2 and shSLIT2 alone or in combination was assessed by a tube formation assay (scale bar: 50 μm, magnification: x100). (CD). The expression of ROBO1, VEGFA and VEGFR2 proteins in HUVEC-12 and HMEC-1 cells transfected with shHDAC2 and shSLIT2 alone or in combination was determined by Western blotting. GAPDH served as the reference gene. **p or ++p or ^^p\u0026lt;0.01; ***p or +++ p or ^^^p \u0026lt;0.001; * against. SHNK; + compared to wSLIT2; ^ vs. shHDAC2 (SLIT2, split 2 homolog; ROBO1, highway 1; VEGFA, vascular endothelial growth factor A; VEGFR2, vascular endothelial growth factor receptor 2; HDAC2, histone deacetylase 2; RT-qPCR, quantitative real-time polymerase chain reaction assay ;shHDAC2, short hairpin RNA against HDAC2;shSLIT2, short hairpin RNA against SLIT2;shNC, short hairpin RNA against negative control\u003c/p\u003e","description":"","filename":"Onlinefloatimage6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4143819/v1/177835922065f78959957517.jpg"},{"id":56659452,"identity":"e734ac84-1a37-483f-8152-c1a9e2eb74c4","added_by":"auto","created_at":"2024-05-17 10:58:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5981571,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4143819/v1/984e6b11-ecb0-491f-a0b5-736d1833584a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Decreased expression of the HDAC2 disrupts the SLIT-ROBO signaling pathway and induced angiogenesis in placental endothelial cells in preeclampsia","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePreeclampsia (PE) is a severe obstetric disease that is a leading cause of maternal death worldwide [1]. Every year, PE results in the death of approximately 7,000-8,000 pregnant women and 50,000 perinatal infants [2]. Although the prevalence of PE is similar in both developing and developed countries, the symptoms tend to be more severe in patients in developing countries, often leading to poorer pregnancy outcomes [3]. Despite numerous studies on the etiology and mechanism of PE, its causes remain unclear and clinical treatment options are limited. The formation of an abnormal placenta is a crucial pathogenic factor in PE [4]. In normal pregnancy, villous trophoblast cells invade maternal spiral arteries, replacing the endothelial cell layer and transforming them from small, high-resistance vessels to vessels of longer diameter. This process provides sufficient blood perfusion to the placenta, allowing fetal growth. However, this process is incomplete in patients with PE because of insufficient trophoblast cell infiltration and impaired vascular remodeling, resulting in inadequate placental blood perfusion and ischemic hypoxia[5]. Trophoblasts secrete vasoactive substances into the maternal circulation, causing endothelial cell damage and inducing clinical manifestations associated with PE[6].\u003c/p\u003e\n\u003cp\u003ePlacental endothelial dysfunction is a significant factor in PE development. An imbalance in placental factors has been identified as contributes to placental endothelial dysfunction [7]. Studies have shown that placental factors associated with an anti-angiogenic state include vascular endothelial growth factor (VEGF), placental growth factor (PLGF), and other growth factors [8-9]. Therefore, elucidating the mechanisms driving this anti-angiogenic state may help us to better understand how PE develops. \u0026nbsp;Histone deacetylase 2 (HDAC2), a member of class I histone deacetylase (HDACs), deacetylates lysine deposits in the N-terminal region of core histones by framing large multiprotein complexes to modify the chromatin [10]. Endothelial gene transcription and vascular function are regulated by HDAC2. [11-12]. HDAC2 overexpression antagonizes oxidized LDL-mediated endothelial dysfunction in human aortic endothelial cells [10] and mice [13]. The disrupted HDAC2/HIF-1\u0026alpha;/VEGF axis inhibits angiogenesis in diabetic retinopathy in mice [14]. Recently, studies have found low levels of HDAC2 in the placental tissue of PE patients, and restoring HDAC2 expression can inhibit p53/PUMA signaling, thereby improving the biological behavior of trophoblast cells to prevent the occurrence of PE [15]. Therefore, we hypothesized that low HDAC2 expression might suppress angiogenesis to induce endothelial dysfunction and drive PE development.\u003c/p\u003e\n\u003cp\u003eStudies have demonstrated that histone modifications determine transcriptional activity through chromatin remodeling [16]. Histone deacetylase can reduce histone acetylation to increase DNA histone binding, thereby blocking the entry of DNA transcription factors [10]. Therefore, our pre-feasibility study utilized hTF target to predict genes whose transcription might be negatively affected by HDAC2. The SLIT-ROBO signaling pathway is specifically expressed at the placental interface, and previous research has shown that ROBO1, ROBO4, and SLIT2 can be detected in the capillary endothelium of normal human placental villi [17]. Our bioinformatic predictions showed that HDAC2 mediates SLIT2 transcriptional repression. Therefore, we aimed to clarify how low HDAC2 expression affects SLIT2 expression in PE endothelial dysfunction.\u003c/p\u003e\n\u003cp\u003eSLIT2 is a SLIT protein secreted as a ligand that exerts regulatory effects by binding to its specific transmembrane receptor, ROBO proteins. SLIT-ROBO signaling was initially identified as a regulator of axonal growth [18]. Furthermore, it is indispensable for organogenesis of the heart and vasculature [19].\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Notably, SLIT2 and \u0026nbsp;ROBO1 levels were significantly higher in the PE placenta than in the normal placenta [17]. Fundamentally, raised SLIT2 levels in the circulatory system contribute to endothelial cell dysfunction and impaired angiogenesis in systemic sclerosis. [20]. As HDAC2 potentially inhibits SLIT2, we speculated that low HDAC2 expression reduces the blockade of SLIT-ROBO signaling, thereby inducing impaired angiogenesis and endothelial dysfunction in PE.\u003c/p\u003e\n\u003cp\u003eTherefore, this study aimed to determine whether the aberrantly activated SLIT2-ROBO2 signaling pathway in the placentas of PE patients was due to the low expression of HDAC2. Moreover, we aimed to elucidate the correlation between this system and impaired endothelial cell angiogenesis. Hopefully, these findings provide a theoretical basis for the treatment of PE.\u003c/p\u003e"},{"header":"Material and Methods ","content":"\u003cp\u003e\u003cstrong\u003e1.1 Experimental object\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 43 pregnant women from Jinhua Hospital of Zhejiang University participated in this study from January 2018 to December 2019. Among these, 23 pregnant women with PE were assigned to the PE group. Another 20 normal pregnant women were assigned to the control group. The ethics committee at Zhejiang University Jinhua Hospital approved this study, and all relevant participants provided written informed consent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.2 Diagnostic criteria\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe diagnostic criteria refer to to the \u0026ldquo;Guidelines for\u0026nbsp;Diagnosis and\u0026nbsp;Treatment of\u0026nbsp;Hypertension\u0026nbsp;During\u0026nbsp;Pregnancy \u0026ldquo; (2020):\u0026nbsp;Systolic blood pressure\u0026nbsp;\u0026ge;140 mmHg and diastolic blood pressure\u0026nbsp;\u0026ge;90 mmHg after 20 weeks of gestation, companied\u0026nbsp;with\u0026nbsp;any of the following: urinary protein\u0026nbsp;\u0026ge;0.3 g/24 hours (h), or urinary protein/creatinine ratio\u0026nbsp;\u0026ge;0.3, or Random urine protein\u0026nbsp;\u0026ge;\u0026nbsp;(+); no proteinuria but with involvement of any of the following organs or systems: heart, lung, liver, kidney and other vital organs, or digestive system, nervous system and An abnormal change in the blood of an affected fetus in the placenta.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.3 Observation indicators\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMonitoring indicators during pregnancy included gestational age at onset, blood pressure, liver and kidney function, blood routine, urine protein, cardiopulmonary function, fetal ultrasound, and fetal heart rate monitoring.\u003c/p\u003e\n\u003cp\u003eMaternal and infant prognosis-related evaluation indicators included maternal eclampsia, heart failure, liver and kidney failure, postpartum hemorrhage, cesarean section rate, length of hospital stay, pregnancy termination, neonatal birth weight, neonatal score, neonatal asphyxia, neonatal respiratory distress syndrome, neonatal ischemic hypoxic encephalopathy, neonatal pneumonia, and pathological jaundice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.4 Specimen Processing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll placental tissues were collected within 30 minutes (min) of delivery. These tissues were thoroughly washed with cold polybutylene succinate (EDTA), cut into small pieces (100-500Mg), and stored in liquid nitrogen. Additionally, the central tissue of the placenta was approximately 1 cm\u0026times;1 cm\u0026times;1 cm, immediately placed in 10% formaldehyde solution for 24 h, routinely embedded in paraffin, and resectioned to films with a thickness of 4 \u0026mu;m.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.5 Experimental method\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemical determination of SLIT2, ROBO1, CD34, HDAC2 and von Willebrand factor (vWF) protein expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter deparaffinization in xylene and rehydration in graded alcohol baths, 4\u0026micro;m thick tissue sections were used. Antigen recovery was performed by warming the tissue in a microwave at medium-high intensity for 8 min, then from low to high intensity for 5 min, and cooling at room temperature for 20 min. Antibodies against SLIT2 (ab134166, Abcam) were used to stain the sections after 10 min of blocking with goat serum, ROBO1 (ab7279, Abcam, UK), CD34 (ab81289, Abcam, UK), HDAC2 (ab32117, Abcam, UK), and vWF (ab6994, Abcam, UK) for 1 h, followed by goat anti-rabbit IgGH\u0026amp;L (HRP) secondary antibody (ab6721, Abcam, UK) overnight at 4\u0026deg;C. Color development was achieved using a DAB substrate kit (ab64238, Abcam, UK), followed by counterstaining of the nuclei with hematoxylin (ab220365, Abcam, UK).\u003c/p\u003e\n\u003cp\u003eAn optical microscope (Olympus) was used for double-blind observation of the immunohistochemical results by two experienced pathologists. The 200x high-power lens randomly selected the power fields of view. A comprehensive evaluation was made of the staining intensity, the number of positively stained cells, and the product of the two: the intensity of staining in positive cells (one point for cells that do not stain; one point for pale yellow; yellow gets two points; 3 points for yellowish-brown); the proportion of cells that are positive or unstained; 1 point is 10-100%; 11-50% equals two points; 51%-80% is 3 focuses; 81%-100 percent is 4 focuses). For the final result, the two scores are multiplied together: Negative (one) is a score of zero, a score of 1 to 4 is a feeble positive (+), and a positive (++) score is between 5 and 8. The positive (+++) scores ranged from 9 to 12. The Weidner et al. method was used to count placental MVD detection results. CD34/vWF-stained. The vessels were observed under a low-power light microscope to detect the highest vessel density. Patients with vessel diameters \u0026ge; 100 \u0026mu;m were excluded. Then, five non-repetitive fields were selected under the (\u0026times;200) field of view, and the number of blood vessels in an average view unit was determined. (\u0026times;200); that is, MVD\u0026nbsp;was calculated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman microvascular endothelial cells (HMEC-1) and human umbilical vein endothelial cells (HUVEC-12) were purchased from Mingzhou Biotech (MZ-2329 and MZ-0790, respectively; Ningbo, China). Medium 199 (11043023, Thermo Fisher, Waltham, MA, USA) with 20%\u0026nbsp;fetal bovine serum (FBS; 12103C, Sigma-Aldrich, St. Louis. MO, USA), HUVEC-12 cells were culture. HMEC-1 cells were maintained in 10% FBS-containing MCDB131 medium (10372019, Thermo Fisher, USA). Cell culture was performed at 37\u0026deg;C in humidified air containing 5% CO2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell transfection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe MISSION\u0026reg; pLKO.1-puro control plasmid (SHC016, Sigma-Aldrich, USA) was used to construct short hairpin RNA against HDAC2 or SLIT2 (shHDAC2 or shSLIT2), and an empty plasmid was designated as a negative control (shNC) for these plasmids shRNA. HUVEC-12 and HMEC-1 cells were cultured in 96-well plates at a density of 1\u0026times;10[4] cells, cultured to 80% confluence, and finally transfected with shNC, shHDAC2, shSLIT2, or shHDAC2 alone and using Lipofectamine3000 transfection reagent (L3000015, ThermoFisher, USA) in conjunction with shSLIT2. Briefly, the reagents were incubated in Opti-MEM medium and P3000 reagent at 37\u0026deg;C for 10 min to generate gene-lipid complexes, were added cells and incubated for 48 h at 37\u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSLIT2 and HDAC2 mRNA levels are detected by real-time quantitative Polymerase Chain Reaction (RT-qPCR).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTRIzol reagent was used to extract total RNA from placental tissue and HUVEC-12 and HMEC-1 cells transfected with shHDAC2 and shSLIT2 following kit instructions and DNase treatment (Invitrogen, Carlsbad, CA, USA). Reverse transcription (RT) was performed using 1 mM deoxynucleotide triphosphate, 1.25 pmol/L random primer, 25U ribonuclease inhibitor, 100U reverse transcriptase, and RT buffer ( Toyobo, Osaka, Japan). RNA was added to each reverse transcription reaction and incubated at 30\u0026deg;C for 10 min, 60 min at 42\u0026deg;C, and 5 min at 85\u0026deg;C. The resulting cDNA was amplified by PCR using the primer sequences shown in the table (synthesized by Sangon Biotech, Shanghai, China). PCR was performed in a thermal cycler (PTC-200 DNA motor; MJResearch, Waltham, MA, USA) with initial denaturation at 94\u0026deg;C for 5 min, followed by 35 cycles of denaturation at 94\u0026deg;C for 30 s . hybridization at 60\u0026deg;C for 30 s and extension at 72\u0026deg;C for 45 s. The final single-cycle extension was performed at 72\u0026deg;C for 10 min. The relative area under each sample band was used to express the intensity of the amplified products and the levels of SLIT2 and HDAC2 were normalized to those of GAPDH.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Counting Kit (CCK)-8 Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHUVEC-12 and HMEC-1 cells were seeded in 96-well plates at a density of 5\u0026times;10[3] cells/well for adherent culture and transfected with shHDAC2 and/or shSLIT2a. 24, 4,8 or 72 h after transfection, CCK-8 reagent (C0037, Beyotime, Shanghai, China) was added to each well at a ratio of 1:10 and incubated at 37\u0026deg;C for 2 h. Finally, a microplate reader was used at a wavelength of 450 nm to measure the optical density. (EMaxPlus, MolecularDevices, Sunnyvale, CA, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eColony formation assay\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe colony formation assay involved trypsinizing HUVEC-12 and HMEC-1 cells (9002-07-7, Sigma-Aldrich, USA) and seeding them in 12-well plates at a density of 3 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells/well. After transfection with shHDAC2 aandshSLIT2, cells were grown in the medium at 37\u0026deg;C for 14 days. Once visible colonies were observed, the plate was washed with phosphate-buffered saline (PBS; 806552, Sigma-Aldrich, USA), fixed with 4% paraformaldehyde (A500684, Sangon Biotech, China) for 15 min, and stained with purple crystals. Transfections of shHDAC and shSLIT2 cells were replated and cultured in the medium for 14 days at 37\u0026deg;C. When colonies were visible, the plate was washed with phosphate-buffered saline (PBS; 806552, Sigma-Aldrich, USA) ( C8470, Solarbio, Beijing, China) 20 for minutes. Finally, stained colonies were photographed using a camera (E-M5MarkIII; Olympus, Tokyo, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTube formation assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCapillary-like network formation was performed using a tube formation assay with matrigel to detect the angiogenic capacity of endothelial cells. The HMEC-1 and HUVEC-12 cells were transfected with shHDAC2\u0026nbsp;and shSLIT2, seeded into 96-well plates with a 104-cell-per-well cell density, covered with 50 \u0026micro;L Matrigel and 100 \u0026micro;L medium per well, and incubated for 12 hours Afterwards, the number of Capillary-like tubes were counted at \u0026times;100 magnification using an inverted microscope (IXploreStandard; Olympus, Tokyo, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHUVEC-12 and HMEC-1 cells that were transfected with shHDAC2 anandhSLIT2 were lysed with RIPA lysis and extraction buffer (89900, ThermoFisher, USA )containing a cocktail of protease inhibitors(P8340, Si gma-Aldrich, USA), and the total protein was obtained. Protein concentration was determined using a BCA kit. (A53227, Thermo Fisher, USA) and then separated by SDS-PAGE (P0012A, Beyotime, China). A polyvinylidene fluoride membrane (FFP36; Beyotime, China) was used to transfer the separated proteins, which were then blocked with 5% w/v bovine serum albumin (Solarbio, China) for 2 h at room temperature. \u0026nbsp;Afterward, primary antibodies against ROBO1 (ab7279, 181kDa, 1:500, Abcam, UK), vascular endothelial growth factor A ab46154, 23kDa, 1\u0026micro;g/mL, Abcam, UK), vascular endothelial growth factor receptor2 \u0026nbsp; (VEGFA; Abcam, UK), vascular endothelial growth factor receptor 2 (VEGFR2; Abcam, UK ab11939,)151kDa, 1 \u0026micro;g/mL; Abcam, UK), and GAPDH and (ab181602, 36kDa, 1:10000, Abcam, UK) were used to incubate membranes overnight at 4 \u0026deg;C. The membrane was then incubated with goat anti-rabbit IgG secondary antibody (31460, ThermoFisher, USA) for 1 h at room temperature. Immunoreactive western blotting was performed using a SuperECLStar kit (S349652, Aladdin, Shanghai, China). Blot density analysis was carried out with the help of the ImageJ software (version 1.8.0, National Institutes of Health, Bethesda, Massachusetts.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe statistical software SPSS17.0 was utilized for the analysis. The differences in Fig.1, 2A, 3CDEF, and 4 were analyzed using an independent t-test. Differences between multiple groups were compared using the independent t-test. Correlation analysis was performed using linear regression correlation analysis. Statistical significance was set at P\u0026lt;0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e1. T\u003c/strong\u003e\u003cstrong\u003ehe Expression of SLIT2, ROBO1and CD34 in preeclampsia or healthy placenta\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunohistochemical assays confirmed that ROBO1, SLIT2, and CD34 were expressed in the placentas of each group. More specifically, the study group displayed an increase in ROBO1 and SLIT2 protein expression (P0.01, Fig.) compared with the control group. 1A, B; P\u0026lt;0.05, Fig. 1C, D), whereas CD34 protein expression decreased significantly (P \u0026lt; 0.01, Fig. 1E, F).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. \u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eHDAC2 and vWF expression in preeclampsia or healthy placent\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAccording to RT-qPCR analysis, the\u0026nbsp;\u003c/strong\u003estudy group\u0026apos;s HDAC2 expression was lower than that in the control group (P\u0026lt;0.001, Figure 2A). The immunohistochemical assay yielded similar results (Fig. 2B).\u0026nbsp;Immunohistochemical analysis revealed that the vWF-positive area in the study group was smaller than that in the control group (Fig.2C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eEffect of HDAC2 knockdown on the proliferation of HUVECs-12 and HMEC-1 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn HUVEC-12 and HMEC-1 cells, HDAC2 was successfully knocked down by shHDAC2 transfection in RT-qPCR analysis (P \u0026lt; 0.001, Fig. 3A, B). Then, a CCK-8 assay was performed, and the results showed that \u0026nbsp;HUVEC-12 and HMEC-1 cells with shHDAC2, compared to shNC transfection, showed inhibited viability at 24, 48, and 72 h after HDAC2 knockdown or transfection ( P\u0026lt;0.001, Fig. 3C, D). Furthermore, when HDAC2 was knocked down, a decreased colony formation rate by HUVEC-12 and HMEC-1 cells were observed (P \u0026lt; 0.001; Fig. 3E, F).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4. Effects of HDAC2 knockdown on angiogenesis and SLIT2/ROBO pathway in HUVEC-12 and HMEC-1 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThrough the tube formation assay, we found that shHDAC2 transfection reduced the number of vessel branches formed by HUVEC-12 and HMEC-1 cells compared to shNC transfection (P\u0026lt;0.01, Fig. 4A, B, C). At the same time, the expression of angiogenesis-related proteins, VEGFA and VEGFR2 in HUVEC-12 and HMEC-1 cells was downregulated after HDAC2 knockdown (P\u0026lt;0.001; Figure 4D, E, F). Notably, compared with shNC transfection, shHDAC2 transfection increased the expression of SLIT2 and ROBO1in HUVEC-12 and HMEC-1 cells, as shown by western blot results (P\u0026lt;0.01, Fig. 4G, H, I).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5. The effect of HDAC2 knockdown on the proliferation of\u0026nbsp;\u003c/strong\u003eHUVEC-12 and HMEC-1 cells\u003cstrong\u003e\u0026nbsp;can be reversed by SLIT2 knockdown\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRT-qPCR analysis showed that the knockdown of SLIT2 was achieved in HUVEC-12 and HMEC-1 cells by transfecting with shSLIT2 (P\u0026lt;0.001; Fig. 5A, B). Using the CCK-8 assay, we observed enhanced viability of HUVEC-12 and HMEC-1 cells after shSLIT2 transfection compared with shNC transfection at 24, 48, or 72 h (P\u0026lt; 0.05, Fig. 5C, D), and SLIT2 knockdown could resist the reduction of HDAC2 knockdown-induced viability at 48 and 72 h (P\u0026lt;0.01, Fig. 5C, D). In turn, HDAC2 knockdown counteracted the increased viability induced by SLIT2 knockdown at 48 and 72 h (P\u0026lt;0.05, Fig. 5C, D).\u003c/p\u003e\n\u003cp\u003eIn addition, colony formation assay results \u0026nbsp;showed that transfection with shSLIT2 increased the number of colonies in \u0026nbsp; HUVEC-12 and HMEC-1 cells \u0026nbsp; compared to shNC transfection (P\u0026lt;0.001, Figure 5E, F, G). In addition, SLIT2 knockdown and HDAC2 knockdown could cancel each other out their effects on the colony formation rate (P\u0026lt;0.001, Fig. 5E, F, G).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6. SLIT2 knockdown counteracts the effects of HDAC2 knockdown on angiogenesis and SLIT2/ROBO pathway\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ein\u0026nbsp;\u003c/strong\u003eHUVEC-12 and HMEC-1 cells\u003c/p\u003e\n\u003cp\u003eCompared with shNC, the control group and SLIT2 knockdown group promoted the formation of vessel branches constructed by HUVEC-12 and HMEC-1 cells (Fig 6A, P\u0026lt;0.01). The results \u0026nbsp; showed that SLIT2 and HDAC2 knockdown mutually counteracted their effects on vascular branch formation in HUVEC-12 and HMEC-1 cells (P\u0026lt;0.01 Fig 6A,B). Western blot results showed that the expression of ROBO1 was decreased in HUVEC-12 cells (P\u0026lt;0.01, Fig. 6C, D), and the expression of VEGFA and VEGFR2 was increased (P\u0026lt;0.001, Fig. 6C, D) in HMEC-1 cells after SLIT2 knockdown. Likewise, the effects of SLIT2 and HDAC2 knockdown offset each other significantly \u0026nbsp;(P\u0026lt;0.01, Fig.6C,D).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePlacental abnormalities are considered the cause of PE. Trophoblastic cells replace maternal spiral artery endothelial cells in thickening blood vessels, providing sufficient blood perfusion to the placenta to maintain fetal growth. However, this process is incomplete in patients with PE. Insufficient placental blood perfusion is the key to the onset of PE [21]. One report found no significant difference between increased and decreased CD34 expression in patients [22]. According to another study, PE patients had significantly lower expression levels of the angiogenesis markers VEGF1 and CD34 in the placenta and decidua than in normal pregnant women. [23]. This study confirmed that the expression of CD34 was significantly decreased in pregnant women with PE., indicating that the formation of microvessels was lower than that in normal pregnant women. Since defective trophoblast invasion that triggers PE may cause inappropriate spiral arterial remodeling and further induce endothelial dysfunction [8], understanding the mechanisms underlying endothelial dysfunction in PE is of great interest. A recent study reported that HDAC2 was poorly expressed in placentas obtained from patients with PE [24], which is consistent with our results. HDAC2 belongs to class I HDACs, and has been shown to protect mice from endothelial dysfunction and block OxLDL-mediated vascular dysfunction upon endothelium-specific overexpression [12,13]. vWF, a blood glycoprotein involved in hemostasis, is an endothelial antigen marker positively associated with MVD and is expressed at low levels in syncytiotrophoblasts in preeclampsia due to endothelial and placental cell damage [25]. Our study detected downregulated vWF expression and low HDAC2 expression in PE placentas. These data, combined with findings from other studies, reconfirm that placental dysfunction is associated with PE and further suggest that low HDAC2 expression in PE placentas may be responsible for endothelial dysfunction in PE. The blocked proliferation of endothelial cells and reduced angiogenic capacity of cells are the main drivers of endothelial dysfunction in PE [26]. In this study, the knockdown of HDAC2 inhibited proliferation of \u0026nbsp;HUVEC-12 and HMC-1 cells. In addition, changes in the levels of angiogenesis-related factors such as VEGFA and VEGFR2 can be detected weeks before PE onset [10]. VEGFA is abundantly expanded in the placenta and plays a significant role in placental vascular development by affecting endothelial cell proliferation, migration, and vascular permeability [27]. VEGFR2 is the main receptor mediating VEGF vasodilation, and its binding to VEGFA triggers angiogenesis, endothelial cell migration, and vasodilation [28,29]. Our results indicate that HDAC2 knockdown also resulted in the downregulation of VEGFA and VEGFR2 in HUVEC-12 and HMEC-1 cells. Our findings suggested that low HDAC2 expression contributes to endothelial dysfunction in PE by inhibiting angiogenesis.\u003c/p\u003e\n\u003cp\u003eExisting evidence demonstrates that endothelial arginase 2, which controls endothelial NO, proliferation, fibrosis, and inflammation, is negatively regulated at the transcriptional level by HDAC2 overexpression, leading to the blockage of oxLDL-mediated vascular dysfunction [11,12], suggesting that HDAC2 can alleviate the regulation of vascular dysfunction by negatively regulating endothelial gene expression. Based on bioinformatics analysis, SLIT2 was predicted to be regulated by HDAC2 transcription. Previous research has demonstrated that PE placentas have high levels of SLIT2 and its receptor ROBO1. [17], consistent with the results of our study. In normal placentas, co-expression of multiple ligands (SLIT2 and SLIT3) and receptors (ROBO1, ROBO2, and ROBO4) may function as an autocrine/paracrine regulatory system to mediate placental development and maintain normal placental function. [30]. Impaired microvascular formation in patients with PE leads to hypoxia. Although SLIT2 and ROBO1 \u0026nbsp;are highly expressed in villous syncytiotrophoblasts, hypoxia (2% O\u003csub\u003e2\u003c/sub\u003e) can stimulate mRNA expression of the SLIT and ROBO family of molecules, which are mostly found in placental trophoblasts and endothelial cells in vivo. [17]. In our previous study, we found that HDAC2 knockdown inhibited angiogenesis in HUVEC-12 and HMEC-1 cells and increased ROBO1 and SLIT2 expression in endothelial cells. This suggests that in PE placentas, low HDAC2 expression may transcriptionally restore SLIT2 expression, which then upregulates ROBO1 expression in endothelial cells. Challenge with recombinant human SLIT2 may impair the angiogenic properties of healthy explanted dermal microvascular endothelial cells, leading to peripheral microangiopathy [21].\u0026nbsp;Elevated SLIT2 activity impairs VEGF-induced angiogenesis in the EphA2-deficient endothelium[31]. These studies and our results suggest that endothelium-specific SLIT2 expression restored by low HDAC2 expression is associated with an attenuated endothelial cell angiogenic capacity in PE placentas.\u003c/p\u003e\n\u003cp\u003eMeanwhile, placental hypoxia can increase the expression of ROBO1, which further leads to placental hypoxia and may be one of the possible pathogeneses of PE. Upregulation of SLIT2-ROBO1 signaling promotes angiogenesis in a mouse liver injury model [32]. Furthermore, ROBO1 expression promotes ox-LDL-induced endothelial cell dysfunction [33]. Combined with Zhang\u0026apos;s findings, our study suggests that restoration of ROBO1 expression after HDAC2 knockdown in HUVEC-12 and HMEC-1 cells is associated with suppressed endothelial cell angiogenesis. These findings suggest that the ability of low HDAC2 expression to induce impaired angiogenesis and endothelial dysfunction in PE placentas may be attributed to the activation of SLIT2-ROBO1 signaling in endothelial cells.\u003c/p\u003e\n\u003cp\u003eTo confirm the validity of this suggestion, we knocked down the expression of SLIT2 in HDAC2-underexpressed HUVEC-12 and HMEC-1 cells. As expected, the knockdown of SLIT2 reversed HDAC2 knockdown-induced inhibition of endothelial cell proliferation and angiogenesis, expression of pro-angiogenic proteins, and counteracted HDAC2 knockdown-induced upregulation of ROBO1 expression, suggesting that low HDAC2 expression activates SLIT2- ROBO1 signaling inhibits angiogenesis and induces endothelial dysfunction in PE placentas.\u003c/p\u003e\n\u003cp\u003eIn conclusion, our study demonstrates that in PE placenta, HDAC2 is underexpressed, but SLIT2 and ROBO1 are overexpressed, and that in endothelial cells, HDAC2 knockdown inhibits viability, proliferation, and angiogenesis by activating SLIT2-ROBO1 signaling. Based on these results, it is reasonable to deduce that low HDAC2 expression impairs the blockade of SLIT2-ROBO1 signaling in endothelial cells, thereby inducing impaired angiogenesis and endothelial dysfunction, resulting in abnormal placental development and function in PE.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eHDAC \u0026nbsp; \u0026nbsp;Histone deacetylase\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eROBO \u0026nbsp; \u0026nbsp;Roundabout\u003c/p\u003e\n\u003cp\u003eCCK \u0026nbsp; \u0026nbsp; \u0026nbsp;Cell Counting Kit\u003c/p\u003e\n\u003cp\u003eRT-qPCR\u0026nbsp;\u0026nbsp; \u0026nbsp;Real-Time\u0026nbsp;Quantitative PCR\u003c/p\u003e\n\u003cp\u003evWF \u0026nbsp; \u0026nbsp; \u0026nbsp;vonWillebrand factor\u003c/p\u003e\n\u003cp\u003eVEGF\u0026nbsp;\u0026nbsp;\u0026nbsp;vascular endothelial growth factor\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;PLGF\u0026nbsp;\u0026nbsp; \u0026nbsp;placental growth factor\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;LDL\u0026nbsp;\u0026nbsp; \u0026nbsp;low-density lipoprotein\u003c/p\u003e\n\u003cp\u003eHIF-1\u0026nbsp;\u0026nbsp; \u0026nbsp;\u0026nbsp;hypoxia-inducible factor 1\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;PE\u0026nbsp;\u0026nbsp; \u0026nbsp;\u0026nbsp;preeclampsia\u003c/p\u003e\n\u003cp\u003eVEGFA\u0026nbsp; \u0026nbsp;\u0026nbsp;(vascular endothelial growth factor) A\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eVEGFR\u0026nbsp;\u0026nbsp; \u0026nbsp;(vascular endothelial growth factor)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePLGF \u0026nbsp; \u0026nbsp;Placental growth factor\u003c/p\u003e\n\u003cp\u003eLDL \u0026nbsp; \u0026nbsp; Low density lipoprotein\u003c/p\u003e\n\u003cp\u003eHIF-1\u0026alpha; \u0026nbsp; Hypoxia-inducible Factor-1\u0026alpha;\u003c/p\u003e\n\u003cp\u003ePUMA \u0026nbsp; p53 upregulated modulator of apoptosis\u003c/p\u003e\n\u003cp\u003eDNA \u0026nbsp; \u0026nbsp;Deoxyribonucleic acid\u003c/p\u003e\n\u003cp\u003eEDTA \u0026nbsp; Ethylene diamine tetraacetic acid\u003c/p\u003e\n\u003cp\u003eMVD \u0026nbsp; \u0026nbsp;Microvessel density\u003c/p\u003e\n\u003cp\u003eHMEC \u0026nbsp; Human microvascular endothelial\u003c/p\u003e\n\u003cp\u003eFBS \u0026nbsp; \u0026nbsp; Fetal bovine serum\u003c/p\u003e\n\u003cp\u003eRNA \u0026nbsp; \u0026nbsp; Ribonucleic acid\u003c/p\u003e\n\u003cp\u003eHDAC \u0026nbsp; Histone deacetylase\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOxLDL \u0026nbsp; Oxidized\u0026nbsp;low-density lipoprotein\u003c/p\u003e\n\u003cp\u003eRT-PCR \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Real-time Polymerase Chain Reaction\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of Interest declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have NO affiliations with or involvement in any organization or entity with any financial interest in the subject matter or materials discussed in this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent of patients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll patients provided written informed consent for biological studies. The Ethics Board of Zhejiang University Jinhua Hospital approved the current study, and all participants signed a written informed consent form.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Zhejiang Provincial Natural Science Foundation of China (NO.Q19H040012) and Jinhua Municipal Science and Technology Bureau Project (2023-3-147).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eFan Xufei and Zheng Xiujuan were involved in study design and compilation. Samiullah Malik performed a critical review and corrections, and drafted the final copy; Yang Yali, Yu Jianyun were involved in the data analysis. All authors reviewed and approved the final version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGestational Hypertension and Preeclampsia: ACOG Practice Bulletin Summary, Number 222. Obstetrics and gynecology. 2020;135(6):1492-5.\u003c/li\u003e\n\u003cli\u003eSteegers EA, von Dadelszen P, Duvekot JJ, Pijnenborg R. Preeclampsia. Lancet (London, England). 2010;376(9741):631-44.\u003c/li\u003e\n\u003cli\u003eYang Y, Le Ray I, Zhu J, Zhang J, Hua J, Reilly M. Preeclampsia Prevalence, Risk Factors, and Pregnancy Outcomes in Sweden and China. JAMA network open. 2021;4(5):e218401.\u003c/li\u003e\n\u003cli\u003eRana S, Lemoine E, Granger JP, Karumanchi SA. Preeclampsia: Pathophysiology, Challenges, and Perspectives. Circulation research. 2019;124(7):1094-112.\u003c/li\u003e\n\u003cli\u003ePhipps E, Prasanna D, Brima W, Jim B. Preeclampsia: Updates in Pathogenesis, Definitions, and Guidelines. Clinical journal of the American Society of Nephrology : CJASN. 2016;11(6):1102-6. Gathiram P, Moodley J. Pre-eclampsia: its pathogenesis and pathophysiolgy. Cardiovascular journal of Africa. 2016;27(2):71-8.\u003c/li\u003e\n\u003cli\u003eStepan H, Hund M, Andraczek T. Combining Biomarkers to Predict Pregnancy Complications and Redefine Preeclampsia: The Angiogenic-Placental Syndrome. Hypertension (Dallas, Tex : 1979). 2020;75(4):918-26.\u003c/li\u003e\n\u003cli\u003eCui L, Shu C, Liu Z, Tong W, Cui M, Wei C, et al. The expression of serum sEGFR, sFlt-1, sEndoglin and PLGF in preeclampsia. Pregnancy hypertension. 2018;13:127-32.\u003c/li\u003e\n\u003cli\u003eKeshavarzi F, Shahrakipoor M, Teimori B, Yaghmaei M, Narooei-Nejad M, Rasooli A, et al. Association of placental VEGF promoter polymorphisms and VEGF mRNA expression with preeclampsia. Clinical and Experimental Hypertension (New York, NY : 1993). 2019; 41(3): 274-9.\u003c/li\u003e\n\u003cli\u003eHou Q, Hu K, Liu X, Quan J, Liu Z. HADC regulates the diabetic vascular endothelial dysfunction by targetting MnSOD. Bioscience reports. 2018;38(5).\u003c/li\u003e\n\u003cli\u003ePandey D, Hori D, Kim JH, Bergman Y, Berkowitz DE, Romer LH. NEDDylation promotes endothelial dysfunction: a role for HDAC2. Journal of molecular and cellular cardiology. 2015;81:18-22.\u003c/li\u003e\n\u003cli\u003ePandey D, Sikka G, Bergman Y, Kim JH, Ryoo S, Romer L, et al. Transcriptional regulation of endothelial arginase 2 by histone deacetylase 2. Arteriosclerosis, thrombosis, and vascular biology. 2014;34(7):1556-66.\u003c/li\u003e\n\u003cli\u003eHori D, Nomura Y, Nakano M, Han M, Bhatta A, Chen K, et al. Endothelial-Specific Overexpression of Histone Deacetylase 2 Protects Mice against Endothelial Dysfunction and Atherosclerosis. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology. 2020;54(5):947-58.\u003c/li\u003e\n\u003cli\u003eHu L, Lv X, Li D, Zhang W, Ran G, Li Q, et al. The anti-angiogenesis role of FBXW7 in diabetic retinopathy by facilitating the ubiquitination degradation of c-Myc to orchestrate the HDAC2. Journal of cellular and molecular medicine. 2021;25(4):2190-202.\u003c/li\u003e\n\u003cli\u003eZhao Y, Zhao G, Li W. MicroRNA-495 suppresses pre-eclampsia via activation of p53/PUMA axis. Cell death discovery. 2022;8(1):132.\u003c/li\u003e\n\u003cli\u003eMorgan MAJ, Shilatifard A. Reevaluating the roles of histone-modifying enzymes and their associated chromatin modifications in transcriptional regulation. Nature genetics. 2020;52(12):1271-81.\u003c/li\u003e\n\u003cli\u003eLiao WX, Laurent LC, Agent S, Hodges J, Chen DB. Human placental expression of SLIT/ROBO signaling cues: effects of preeclampsia and hypoxia. Biology of reproduction. 2012;86(4):111.\u003c/li\u003e\n\u003cli\u003eGuzm\u0026aacute;n-Palma P, Contreras EG, Mora N, Smith M, Gonz\u0026aacute;lez-Ram\u0026iacute;rez MC, Campusano JM, et al. Slit/Robo Signaling Regulates Multiple Stages of the Development of the Drosophila Motion Detection System. Frontiers in cell and developmental biology. 2021;9:612645.\u003c/li\u003e\n\u003cli\u003eSantiago-Mart\u0026iacute;nez E, Soplop NH, Kramer SG. Lateral positioning at the dorsal midline: Slit and Roundabout receptors guide Drosophila heart cell migration. Proceedings of the National Academy of Sciences of the United States of America. 2006;103(33):12441-6.\u003c/li\u003e\n\u003cli\u003eRomano E, Manetti M, Rosa I, Fioretto BS, Ibba-Manneschi L, Matucci-Cerinic M, et al. Slit2/Robo4 axis may contribute to endothelial cell dysfunction and angiogenesis disturbance in systemic sclerosis. Annals of the rheumatic diseases. 2018;77(11):1665-74.\u003c/li\u003e\n\u003cli\u003eFisher SJ. Why is placentation abnormal in preeclampsia? American journal of obstetrics and gynecology. 2015;213(4 Suppl):S115-22.\u003c/li\u003e\n\u003cli\u003eSahin H, Gunel T, Benian A, Onay Ucar E, Guralp O, Kilic A. Genomic and proteomic investigation of preeclampsia. Experimental and therapeutic medicine. 2015;10(2):711-6.\u003c/li\u003e\n\u003cli\u003eLiu H, Li Y, Zhang J, Rao M, Liang H, Liu G. The defect of both angiogenesis and lymphangiogenesis is involved in preeclampsia. Placenta. 2015;36(3):279-86.\u003c/li\u003e\n\u003cli\u003eZhu H, Wang C. HDAC2-mediated proliferation of trophoblast cells requires the miR-183/FOXA1/IL-8 signaling pathway. Journal of cellular physiology. 2021;236(4):2544-58.\u003c/li\u003e\n\u003cli\u003eSzpera-Goździewicz, Agata; Majcherek, Maciej; Boruczkowski, Maciej; et al. Circulating endothelial cells, circulating endothelial progenitor cells, and von Willebrand factor in pregnancies complicated by hypertensive disorders.[J] AM J REPROD IMMUNOL. 2017-03-01;77(3):\u003c/li\u003e\n\u003cli\u003eZhang X, Li Q, Jiang W, Xiong X, Li H, Zhao J, et al. LAMA5 promotes human umbilical vein endothelial cells migration, proliferation, and angiogenesis and is decreased in preeclampsia. The journal of maternal-fetal \u0026amp; neonatal medicine : the official journal of the European Association of Perinatal Medicine, the Federation of Asia and Oceania Perinatal Societies, the International Society of Perinatal Obstet. 2020;33(7):1114-24.\u003c/li\u003e\n\u003cli\u003eHuppertz B. Biology of preeclampsia: Combined actions of angiogenic factors, their receptors and placental proteins. Biochimica et biophysica acta Molecular basis of disease. 2020;1866(2):165349.\u003c/li\u003e\n\u003cli\u003eShibuya M. Vascular Endothelial Growth Factor (VEGF) and Its Receptor (VEGFR) Signaling in Angiogenesis: A Crucial Target for Anti- and Pro-Angiogenic Therapies. Genes \u0026amp; cancer. 2011;2(12):1097-105.\u003c/li\u003e\n\u003cli\u003eSmani T, G\u0026oacute;mez LJ, Regodon S, Woodard GE, Siegfried G, Khatib AM, et al. TRP Channels in Angiogenesis and Other Endothelial Functions. Frontiers in physiology. 2018;9:1731.\u003c/li\u003e\n\u003cli\u003eChen, Cheng-Yi; Tsai, Chin-Han; Chen, Chia-Yu; et al . Human placental multipotent mesenchymal stromal cells modulate placenta angiogenesis through Slit2-Robo signaling. CELL ADHES MIGR. 2016-03-03;10(1-2):66-76.\u003c/li\u003e\n\u003cli\u003eYoungblood V, Wang S, Song W, Walter D, Hwang Y, Chen J, et al. Elevated Slit2 Activity Impairs VEGF-Induced Angiogenesis and Tumor Neovascularization in EphA2-Deficient Endothelium. Molecular cancer research : MCR. 2015;13(3):524-37.\u003c/li\u003e\n\u003cli\u003eColl M, Ari\u0026ntilde;o S, Mart\u0026iacute;nez-S\u0026aacute;nchez C, Garcia-Pras E, Gallego J, Moles A, et al. Ductular reaction promotes intrahepatic angiogenesis through Slit2-Roundabout 1 signaling. Hepatology (Baltimore, Md.). 2022;75(2):353-68.\u003c/li\u003e\n\u003cli\u003eZhang Y, Li W, Li H, Zhou M, Zhang J, Fu Y, et al. Circ_USP36 Silencing Attenuates Oxidized Low-Density Lipoprotein-Induced Dysfunction in Endothelial Cells in Atherosclerosis Through Mediating miR-197-3p/ROBO1 Axis. Journal of cardiovascular pharmacology. 2021;78(5):e761-e72.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1: Primers used in real-time quantitative polymerase chain reaction for related genes\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"832\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.903614457831326%\" valign=\"top\" style=\"width: 16%;\"\u003e\n \u003cp\u003eGenes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.951807228915663%\" valign=\"top\" style=\"width: 8%;\"\u003e\n \u003cp\u003eSpecies\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.963855421686745%\" valign=\"top\" style=\"width: 41.2203%;\"\u003e\n \u003cp\u003eForward\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"32.65060240963855%\" valign=\"top\" style=\"width: 32.9492%;\"\u003e\n \u003cp\u003eReverse\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.884476534296029%\" valign=\"top\" style=\"width: 16%;\"\u003e\n \u003cp\u003eSLIT2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.9422382671480145%\" valign=\"top\" style=\"width: 8%;\"\u003e\n \u003cp\u003ehuman\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.91456077015644%\" valign=\"top\" style=\"width: 41.2203%;\"\u003e\n \u003cp\u003e5\u0026rsquo;-GCGAAGCTATACAGGCTTGAT-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.25872442839952%\" valign=\"top\" style=\"width: 35.3898%;\"\u003e\n \u003cp\u003e5\u0026rsquo;-TGCAGTCGAAAAGTCCTAAGTTT-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.884476534296029%\" valign=\"top\" style=\"width: 16%;\"\u003e\n \u003cp\u003eHDAC2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.9422382671480145%\" valign=\"top\" style=\"width: 8%;\"\u003e\n \u003cp\u003ehuman\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.91456077015644%\" valign=\"top\" style=\"width: 41.2203%;\"\u003e\n \u003cp\u003e5\u0026rsquo;-ATGGCGTACAGTCAAGGAGG-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.25872442839952%\" valign=\"top\" style=\"width: 35.3898%;\"\u003e\n \u003cp\u003e5\u0026rsquo;-TGCGGATTCTATGAGGCTTCA-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.884476534296029%\" valign=\"top\" style=\"width: 16%;\"\u003e\n \u003cp\u003eGAPDH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.9422382671480145%\" valign=\"top\" style=\"width: 8%;\"\u003e\n \u003cp\u003ehuman\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.91456077015644%\" valign=\"top\" style=\"width: 41.2203%;\"\u003e\n \u003cp\u003e5\u0026rsquo;-GAGAAGGCTGGGGCTCATTT-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.25872442839952%\" valign=\"top\" style=\"width: 35.3898%;\"\u003e\n \u003cp\u003e5\u0026rsquo;-AGTGATGGCATGGACTGTGG-3\u0026rsquo;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2: Clinical characteristic in study and control groups\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eStudy group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eControl group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eP value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eAge, y\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e33.74\u0026plusmn;4.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e29.38\u0026plusmn;3.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eGravity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e2.22\u0026plusmn;1.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e1.33\u0026plusmn;1.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e0.045\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eParity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e0.78\u0026plusmn;0.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e0.52\u0026plusmn;0.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e0.133\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eSystolic, mmHg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e168.61\u0026plusmn;22.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e119.38\u0026plusmn;8.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eDiastolic, mmHg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e106.91\u0026plusmn;14.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e77.52\u0026plusmn;8.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eFetal distress\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e5（21.74%）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e0（0）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eApgar score at birth\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e8.17\u0026plusmn;2.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e9.95\u0026plusmn;0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e0.008\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\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":"HDAC2, SLIT-ROBO signaling pathway, Placental angiogenesis, Preeclampsia","lastPublishedDoi":"10.21203/rs.3.rs-4143819/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4143819/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003ePreeclampsia is characterized by reduced histone deacetylase 2 (HDAC2) expression in placental tissue HDAC2 enrichment positively affects angiogenesis as it helps prevent endothelial cell dysfunction. Additionally, research has demonstrated that the SLIT2-ROBO signaling pathway influences preeclampsia. Bioinformatics analysis has suggested that HDAC2 may have a transcriptional regulatory effect on SLIT2. Consequently, investigations have examined the relationship between low HDAC2 expression and the SLIT-ROBO signaling pathway in placental angiogenesis in patients with preeclampsia.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eObjective: \u003c/strong\u003eTo investigate how decreased HDAC2 expression disrupts the SLIT-ROBO signaling pathway and induces angiogenesis in placental endothelial cells in preeclampsia.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003eThe study included patients with preeclampsia as the observation group, while the placental tissue of normal pregnant women was used as the in vivo control model. In vitro endothelial models using human umbilical veins and microvascular endothelial cells were also used to examine the effects of interference with the expression of HDAC2 and SLIT2. Cell viability CCK-8, colony formation, and tube formation assays were conducted to evaluate angiogenesis. Furthermore, Immunohistochemistry, RT-qPCR, and Western blot analyses were used to examine the expression of genes in cells and tissues.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e The expression of SLIT2 and ROBO1 was increased, and the protein and mRNA expression of CD34, HDAC2, and vonWillebrand factor(vWF) were lower in preeclampsia placentas than in normal placentas. Using an in vitro endothelial model, the knockdown of HDAC2 inhibited colony formation and impaired neovascularization by reducing vascular endothelial growth factor A (VEGFA) and vascular endothelial growth factor -2 (VEGFR2) activity, while SLIT2 and ROBO1 were highly expressed. The changes caused by HDAC2 knockdown were reversed by SLIT2 knockdown.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e Preeclampsia progression is promoted by low HDAC2 expression, which inhibits the SLIT-ROBO signaling pathway and induces angiogenesis in placental endothelial cells.\u003c/p\u003e","manuscriptTitle":"Decreased expression of the HDAC2 disrupts the SLIT-ROBO signaling pathway and induced angiogenesis in placental endothelial cells in preeclampsia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-28 15:58:36","doi":"10.21203/rs.3.rs-4143819/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":"f5d73652-7e8a-40ac-b539-ccadae2552e7","owner":[],"postedDate":"March 28th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-05-17T10:50:49+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-28 15:58:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4143819","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4143819","identity":"rs-4143819","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}