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 × 103 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.