Collagen I Induces Preeclampsia-Like Symptoms by Suppressing Proliferation and Invasion of Trophoblasts

Preeclampsia is a common obstetric disorder affecting 2-8% of pregnancy worldwide. Fibrosis is an important histological change occurring in preeclamptic placenta, and might depend on the excess deposition of collagen I. However, the role of fibrotic placenta and collagen I in the pathogenesis of preeclampsia remains unclear. Therefore, we analyzed the collagen deposition and the expression of Collagen I in human placenta by Masson staining, Sirius red staining and western blotting. Further, the role of collagen I in preeclampsia pathogenesis was studied in C57BL/6 mice. HTR-8/SVneo cells were used to investigate the mechanisms underlying the effects of collagen I in trophoblasts by transcriptome sequencing and pharmacological agonists. Human preeclamptic placenta exhibited a significantly higher degree of fibrosis in stem villi and terminal villi than normal placenta, and was characterized by collagen I deposition. In vivo, a single injection of collagen I on gestational day 0.5 led to an increase in systolic pressure of pregnant mice from gestational days 4.5–17.5, to a decrease in weight and number of embryos, and to enhanced placental collagen I expression and degree of fibrosis compared with control mice. In vitro, collagen I attenuated the proliferation and invasion of HTR-8SV/neo cells. This effect could be reversed by treatment with agonists of ERK and β-catenin. Moreover, transcriptome sequencing demonstrated that signaling pathways related to cell proliferation and invasion were significantly downregulated in HTR-8SV/neo cells. Thus, we propose that collagen I induced preeclampsia-like symptoms by suppressing the proliferation and invasion of trophoblasts through inhibition of the ERK phosphorylation and WNT/β-catenin signaling pathways. Our findings could pave the way to the discovery of small-molecule inhibitors for preeclampsia treatment and future studies with larger sample size are required.


Sirius red staining
After fixation in 4% paraformaldehyde of placenta, the slices were embedded in paraffin. Section of 3μm were stained by structural identification, with Masson's trichrome staining for collagen fiber observation, and with Sirius red stain for collagen identification.

Masson-trichrome staining
For Masson trichrome staining, the sections were stained using a Masson's trichrome staining kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer's protocol. The slides were incubated in Weigert's iron hematoxylin (5 min), Biebrich Scarlet-Acid Fuchsin Solution (15 min), Phosphomolybdic-Acid Solution (15 min) and Aniline Blue Solution (10 min), all at room temperature. The slides were visualized under the light microscope. Collagen fiber were stained blue, while cytoplasm and red blood cells were stained red and nucleus blue and brown.
Fibrosis area% was calculated in µm digitally using the software NDP.view2 (Hamamatsu Corp, Japan). The area of tubulointerstitial fibrosis was measured in 5 random fields under ×200 magnification.

Sirius red staining
For Sirius Red staining, the Picrosirius Red stain kit (Abcam, Cambridge, UK) was utilized. Sections were stained with Weigert's iron Sumu essence dye for 15 min, rinsed for 5 min, and then washed with distilled water. The sections were covered with 200 μl Sirius red dye for 1 h. Each analyzed field was chosen randomly and the positive red-stained areas and red-yellow density were quantified using computerized image analysis software (NIH, MD, USA).

Western blotting
Placenta tissue and in vitro-treated cells was homogenized and protein extracted as described below.
Homogenized by a Qiacube machine (Qiagen, Courtaboeuf, Fance ) in RIPA lysis buffer (Thermo, Rockford, USA) on ice. The supernatants were collected after centrifugation at 12000×g at 4°C for 20 min. Protein concentration was determined using a BCA protein assay kit (Thermo, Rockford, USA), and whole lysates were mixed with 5×SDS loading buffer (Coolaber, Shanghai,China) at a ratio of 1:4. Protein samples were heated at 98°C for 5 min and were separated on SDS-polyacrylamide gels(Biodragon,Guangzhou,China). The separated proteins were then transferred to a PVDF membrane (Dogesce, Shanghai, China). The membrane blots were first probed with a primary antibody. After incubation with horseradish peroxidase-conjugated second antibody, autoradiograms were prepared using the enhanced chemiluminescent system to visualize the protein antigen. The signals were recorded using SYNGENE BIO IAMGING (GENE GNOME, Shanghai,China). Primary antibodies for Western Blot are rat anti-collagen I, anti-MMP9, anti-, anti-vimentin, anti-E-cadherin, anti-Ncadherin, anti-β -catenin, anti-ERK, anti-p-ERK and rabbit anti-GAPDH (Cell Signaling, San Jose, CA, USA). GAPDH was used as a protein loading control. The secondary antibody was HRP-conjugated anti-rabbit (Cell Signaling, San Jose, CA, USA). Images shown in the figures were representative of 5 individuals. ImageJ software (https://imagej.nih.gov/ij) was used for image acquisition and densitometric analysis of the gels.

Quantitative PCR (RT-qPCR)
Total RNA was extracted with Trizol reagent (Invitrogen, Corporation, USA) according to the manufacturer's instructions. The reverse transcription reaction was carried out with reverse transcription enzyme (Toyobo, Shanghai, China). Quantitative real-time PCR was carried out on an LightCycler96 real-time PCR system (Roche,Basel, Switzerland) and the specific primers for quantitative PCR are shown below (IGE,Guangzhou,China).

CCK-8
HTR-8/SVneo cells were seeded in 96-well plates at the density of 10000 cells per well in 250μl of complete culture medium. After treatments, three methods were utilized for cell proliferation analysis. Cell Counting Kit-8 (Beyotime, Guangzhou, China) analysis: 10 ul of CCK-8 was added to each well. The culture plates were shaken for 90 min and the optical density (OD) values were read at 450 nm.

Cell cycle
After treatment of collagen I, HTR-8SV/neo cells were harvested by trypsinization and library quality was assessed on the Agilent Bioanalyzer 2100 system.

Clustering and sequencing
The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia, CA, USA) according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an Illumina Novaseq platform and 150 bp paired-end reads were generated.