Patients and samples
This study complied with the Declaration of Helsinki and was approved by the Ethical Review Committee of Tianjin Baodi Hospital. Informed consent was obtained from all patients (ethical number: TJBDLL20210013). The current study enrolled patients (2020-2021) diagnosed with preeclampsia per clinical diagnostic criteria20 and normal pregnant women. Detailed baseline patient characteristics are listed in Table S1.
Preparation of AT1-AA
As mentioned in our previous study21, the functional second loop epitope peptide of human AT1R (165-191: IHRNVFFIINTNITVCAFHYESQNSTL, AT1R-ECII) was hypodermically injected into male AT1-AA negative Sprague-Dawley rats. 8 weeks after injection, serum was collected. AT1-AA was purified from serum by Hi TrapTM Protein G Kit (GE Healthcare, 17-0404-01). AT1-AA concentration and purity were measured by Bicinchoninic Acid Assay (Thermo Scientific™, 23225) and SDS-PAGE respectively.
All animal experiments were approved by the Institutional Animal Care and Use Committee and Ethics Committee of Capital Medical University. The model of AT1-AA-positive pregnant rats was established by a passive immunization method as previously described22. Female Sprague-Dawley rats were mated with male rats overnight. Presence of a vaginal plug the next morning was indicative of embryonic day 0.5. Pregnant rats were divided into control (receiving saline) and treatment (receiving AT1-AA) groups. On gestation day 13 and 15, pregnant rats were injected with AT1-AA (20 µg/g) and saline (identical volume) respectively. Blood pressure was determined (standard tail-cuff method) of pregnant rats on gestation day 12, 14, 16, and 18, followed by tail vein sampling to determine AT1-AA titer. On gestation day 18, after anesthetization by intraperitoneal sodium pentobarbital (150 mg/kg), pregnant rats underwent laparatomy, and fetal hearts were collected for further analyses. All rats were housed at room temperature (25°C), under a 12 hour light/12 hour dark cycle.
H&E staining distinguished cardiac morphological changes, and WGA staining aided cardiomyocyte cross-sectional area determination. For H&E staining, fetal heart samples collected on gestation day 18 were fixed with 4% paraformaldehyde, embedded in paraffin, cut into 4-μm-thick sections, and stained per protocol. For WGA staining, the heart paraffin sections were stained with wheat germ agglutinin coupled to Alexa Fluor 488 (Invitrogen, W11261, dilution of 1:100 for 1 hour) and visualized with a laser scanning confocal microscope.
The differential expression of microRNAs between control and AT1-AA group was detected by MiRCURY™ LNA expression array (KangChen Bio-tech). Briefly, total fetal heart RNAs from treatment groups were extracted by TRIzol (Invitgen) and microRNAsy mini Kit (QIAGEN) per manufacturer protocol. After RNA measurement using NanoDrop1000, the samples were labeled with miRCURY™ Hy3™/Hy5™ Power Labeling kit and hybridized on the miRCURY™ quantity LNA array. After the washing step, slides were analyzed by Axon GenePix 4000B microarray scanner. Scanned images were imported into GenePix Pro 6.0 software (Axon) for grid alignment and data extraction. The average value of replicated microRNAs was determined, with the normalization factor calculated by selecting the microRNAs with intensity>30 in all samples. Data was subjected to Median normalization, and differentially expressed microRNAs were identified by volcano map screening. Finally, hierarchical clustering was performed to exhibit distinguishable microRNA expression profiles between the treatment groups.
Western blot analysis
Protein samples from heart tissues and NRCMs were lysed with RIPA buffer and PMSF. Concentrations of different samples were evaluated by BCA Protein Assay Kit (Thermo Scientific, Waltham, MA). Equal amounts of total protein (20-50 µg per lane) were separated on ~6-15% SDS-PAGE gels, and electrotransferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA). The membranes were blocked with 5% milk in TBST for 1 hour at room temperature and incubated with corresponding primary antibodies at 4°C overnight. The employed antibodies were as follows: anti-ANP (ab225844, Abcam), anti-BNP (orb382908, Biorbyt), anti-β-MHC (ab50967, Abcam), anti-PCNA (10205-2-AP, Proteintech), and anti-VCAN (ab177480, Abcam). The next day, after TBST washing, the membranes were incubated with HRP-conjugated secondary antibodies at a 1:1000-5000 dilution range for 1 hour at room temperature, and developed using ECL reagent (Applygen Technologies Inc.). All experiments were performed in triplicate.
Primary neonatal rat cardiomyocytes culture
Cardiomyocytes were isolated from neonatal Sprague-Dawley rats (1-3 days old) using trypsin (without EDTA) and collagenase type II (Gibco). Briefly, cut the skin of the chest and squeeze the back of the neonatal rat firmly to expose the heart. Hearts were excised and placed in precooled PBS, after rinsing 3–4x. Each ventricle was collected and cut into 1-mm3 tissue blocks. The tissue blocks were then digested repeatedly (approximately 10 times) for ~6 minutes in a 1:2 mixture of trypsin (without EDTA) and collagenase type II (Gibco). The suspension (including cells and enzyme) was gently mixed and added to high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS) and a 1% mixture of penicillin and streptomycin to terminate the digestion. After filtration, centrifugation (1000 rpm, 10 minutes, RT), and resuspension, cells were plated on a 10-cm2 petri dish for 1.5-2 hours for cardiac fibroblast removal. Finally, the cardiomyocytes were seeded into 6-well plates or 60-cm2 petri dishes for further experiments.
Fluorescence in situ hybridization
MicroRNA fluorescence in situ hybridization (FISH) was performed via RNA FISH kit (Genepharma, Shanghai). Briefly, NRCMs were fixed with 4% paraformaldehyde for 15 minutes at room temperature. After fixation, the cells were permeabilized with 0.1% Triton X-100 for 15 minutes at room temperature and washed with PBS. Cells were then incubated with 2x SSC at 37℃ for 30 minutes. After incubation, the hybridization was measured using hybridization buffer with fluorescence-labeled probes via overnight incubation in a 37℃ water bath. The next morning, after discarding the hybridization buffer, the cell slides were washed with 42℃ preheated 1% Tween 20 for 5 minutes, and washed sequentially with 42℃ preheated 2xSSC and 1xSSC. Finally, the cell slides were washed with PBS and stained with mounting medium containing DAPI. Relative FISH probe sequences are displayed in Supplemental Table 3.
NRCMs were fixed in 4% paraformaldehyde for 10 minutes at room temperature and washed with PBS. Cells were immersed in 0.5% Triton X-100 3 times (10 minutes each). Goat serum (10%) was applied to the cells for blocking for 30 minutes at room temperature. The cells were incubated with primary antibodies at 4℃ overnight. Employed antibodies were as follows: anti-ki67 (AF0198, Affinity Biosciences), anti-α-actin (23660-1-AP, Proteintech), and anti-VCAN (bs-2533R, Bioss). The next day, after PBS washing, the cells were incubated with fluorescein-conjugated secondary antibodies for 1 hour. After incubation, the cells were washed with 0.5% Triton X-100 and PBS, stained with DAPI, and finally visualized via laser scanning confocal microscopy.
Dual luciferase reporter gene assay
For the luciferase assays, HEK293 cells were transfected with miR-124-3p mimics, miR-181a-5p mimics, and a negative control combined with the corresponding luciferase reporter plasmid of pischeck2-VCAN 5’UTR or psicheck2-VCAN 3’UTR. After co-transfection, the cells were harvested to measure the luciferase activity using a dual luciferase assay kit per manufacturer protocol (DL101-01, Vazyme). The specific target activity was expressed as the relative activity ratio of firefly to renilla luciferase. At least 3 independent transfections were performed for each experimental group.
Isolation of microRNA and PCR
MicroRNAs were extracted from heart tissues and neonatal rat cardiomyocytes via miRNeasy Mini Kit (OMEGA, R6842-01) per manufacturer instructions. After microRNA extraction, reverse transcription and qPCR were measured with a microRNA Detection Kit (GenePharma, Shanghai, China). qPCR was completed using an ABI 7500 Fast Real Time PCR system (Life Technologies). Relative amounts of transcripts were normalized to the internal control U6 and calculated by the 2−ΔΔCt formula. Primer sequences for real time qPCR are displayed in Supplemental Table 2.
Transfection of shRNA adenovirus, microRNAs, and plasmid
shRNA adenovirus targeting rat VCAN was designed and synthesized by Vigenebio (Shandong, China), and the corresponding control was also purchased from Vigenebio. For microRNA, the miR-124-3p and miR-181a-5p mimic, inhibitor, and negative control RNAs were designed and synthesized by Genepharma (Shanghai, China). For plasmid, sequences of the VCAN gene 3’UTR containing miR-181a-5p target sites and the 5’UTR containing miR-124-3p target sites were inserted into the psicheck2 dual luciferase microRNA target expression vector. Transfection was performed with Lipofectamine 2000 per manufacturer instructions. After 4-6 hours of transfection, NRCMs treated with or without AT1-AA were harvested and lysed for Western blot analysis, while HEK 293A cells were used for the dual luciferase reporter assays.
Enzyme-Linked ImmunosorbentAssays (ELISA)
For AT1-AA titer testing, a biotin-avidin enzyme-linked immunosorbent assay (BA-ELISA) was performed as previously described22. Peptides corresponding to the sequence of the second extracellular loop of human AT1R were synthesized at 98% purity. The results were expressed as optical density (OD) and measured at 405 nm via ELISA plate reader (SpectraMax Plus; Molecular Devices, Sunnyvale, CA, USA). For VCAN testing, ELISA was performed per manufacturer instructions (Cloud Clone Corp., SEB817Hu; Cusabio, CSB-EL025810RA).
TMT-labeled quantitative proteomics
Total sample (saline and AT1-AA groups) protein was extracted. A portion of the protein was removed to determine the concentration for SDS-PAGE, while another portion was subjected to trypsin hydrolysis and labeling. The labeled samples underwent chromatographic separation; LC-MS/MS analysis screened credible and differentially expressed proteins. Experimental data was analyzed by Proteome Discoverer TM 2.2 (Thermo) software. The UniProt rat database was used. The false positive rate of peptide identification was controlled <1%.
Heatmaps of microRNA array and proteomics were plotted at http://www.bioinformatics.com.cn, a free online platform for data analysis and visualization.
All data were expressed as mean±SEM. The Student’s t-test analyzed the differences between two groups. One-way ANOVA compared the differences between multiple groups. P<0.05 was considered statistically significant. Drafting and statistical analysis was performed using GraphPad Prism 8.0 software (GraphPad Software, Inc., San Diego, CA, USA).