Early missed abortion is associated with villous angiogenesis via the HIF-1α/VEGF signaling pathway

Institutional review board approval was obtained from GXMU for the collection of villous tissue, and written informed consent was obtained from all patients. Villous tissue was obtained from women who requested elective induced abortions in early pregnancy (n = 30) and women who experienced missed abortions (n = 30), defined by an empty gestational sac or an embryo/fetus without cardiac activity after repeated transvaginal ultrasonographic scanning. The subjects who requested induced abortions showed a normal evolution in clinical and ultrasonographic assessments. All study subjects were at 6–10 weeks of gestation at the time of vacuum aspiration. Prior to inclusion in a study group, all patients underwent routine examinations to rule out any verifiable cause of missed abortion. None of the study participants had a history of pregnancy complications. Villous samples were collected when pregnancies were terminated by vacuum aspiration under mask anesthesia, and were immediately fixed in 10% neutral formalin for further immunohistochemical study.

The HTR8/SVneo cell line was obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences. Previous reports have shown that HTR-8/SVneo cells show endothelial cell-like behavior in their ability to form networks of tube-like structures when grown on matrigel [11, 12]. The cells were maintained in RPMI 1640 Medium (GIBCO, Invitrogen, NY, USA) supplemented with 5% fetal bovine serum (GIBCO) under normoxic or hypoxic conditions. Normoxic culture was performed in a standard incubator in an atmosphere containing 5% CO₂ and 20% O₂. Hypoxic-culture experiments were performed in a multigas incubator (ASTEC, Hukuoka,Japan) at 37 °C in an atmosphere balanced with 1% O₂, 5% CO₂ and 94% N₂. After reaching ~ 80% confluency, the cells were subjected to hypoxic conditions in hypoxic chamber for 24 h. Cells cultured under hypoxic conditions were processed in the chamber itself to avoid any exposure to normoxic conditions. After incubation for 24 h, cells were collected for analysis within 1 min being removed from the chamber to prevent HIF1α protein degradation.

Streptavidin-peroxidase (SP) immunohistochemical staining was adopted to detect the expression of HIF, VEGF, and MVD in villous tissue samples. In accordance with kit instructions (Zhongshan, Beijing, China), rabbit anti-human HIF, VEGF, and CD34 antibodies (dilution 1:200, ABclonal Biotechnology, USA) were used as primary antibodies. PBS buffer instead of the primary antibody was utilized as a negative control. Horseradish peroxidase-conjugated (HRP-conjugated) goat anti-rabbit IgG antibody (dilution1:800, Zhongshan, Beijing, China) was the secondary antibody. We then analyzed the samples using DAB substrate kit (Zhongshan, Beijing, China) according to the manufacturer’s instructions. All specimens were observed at five horizons and the average value was used for analysis. Photomicrographs were taken by light microscopy (Olympus BX-53, Tokyo, Japan) of five discontinuous visual fields under high magnification (400×) in each section in accordance with the conditions necessary for further software analysis. The images were analyzed using Image-Pro plus 6.0 software (Media Cybernetics, Rockville, MD) with respect to mean density (IOD/AIO). MVD was assessed by counting CD34-staining blood vessels at high power (400×) according to the method reported by Foote et al. [13]. Two pathologists determined the MVD score for the five most vascularized areas on the same section, and a mean count was obtained as the MVD.

We used real-time PCR to examine the mRNA expression levels of HIF-1α and VEGF in HTR8/SVneo cells. Total RNA was extracted from HTR8/SVneo cells with TRIzol reagent (Takara, Japan) according to the manufacturer’s protocol. The purity and concentration of RNA were assessed by ultramicrospectrophotometer (NanoDrop 2000, Thermos, USA). We performed reverse transcription in 20 μl of reaction system at 37 °C for 15 min and at 85 °C for 5 min. After reverse transcription of total RNA, the first-strand cDNA was then used as a template for the amplification of mRNA using quantitative real-time PCR with SYBR Green Master Mix (Roche, Germany). Human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control. QRT-PCR was performed using an ABI StepOne system (Applied BioSystems, USA) according to the manufacturer’s instructions, and the primers as follows: HIF-1α (forward: 5′-TTGCTCATCAGTTGCCACTTCC-3′, reverse: 5′-AGCAATTCATCTGTGCTTTCATGTC-3′), VEGF-A (forward: 5′-CGAGGGCCTGGAGTGTGT-3′, reverse: 5′-CGCATAATCTGCATGGTGATG-3′), GADPH (forward: 5′-GAGTCAACGGATTTGGTCGT-3′, reverse: 5′-GACAAGCTTCCCGTTCTCAG-3′). The PCR reaction was performed in 20 μl of reaction system as follows: 95 °C for 30 s, and 40 cycles of 95 °C for 5 s and 60 °C for 30 s. The expression of a specific gene was determined with the 2^−ΔΔct method.

Cells were washed twice with cold PBS and lysed in ice-cold lysis buffer with added protease inhibitor cocktail (Roche, Germany). The protein concentration was then determined with a BCA kit (Thermo Scientific, USA). A volume of 20 μg of each sample was separated on SDS-PAGE, and then transferred to PVDF membranes, and blocked with 5% nonfat dry milk for 1 h. Membranes were incubated overnight at 4 °C to primary antibodies anti-HIF-1α, anti-VEGF, or anti-GADPH at 1:200 dilutions (ABclonal Biotechnology, USA). The membranes were then washed twice for 10 min each in 0.1% Tween phosphate buffered saline solution (PBST), and probed with goat anti-rabbit IR-Dye 670 or 800 cw-labeled secondary antisera (CST, USA) in 0.1% Tween, and 0.01% SDS LiCor blocking buffer for 1 h at room temperature. Washes were repeated after secondary labeling, washing twice for 10 min in PBST, and then the membranes were placed in water. Membranes were imaged using a LiCor Odyssey scanner. Boxes were manually placed around each band of interest, which returned near-infrared fluorescent values of raw intensity, with intra-lane background subtracted using Odyssey 3.0 analytical software (LiCor, Lincoln, NE).

The siRNA transfection was performed using Lipofectamine RNAiMAX transfection reagent (Roche, Germany) according to the manufacturer’s instructions. One day before transfection, 25,000 cells were plated in 250 μl of growth medium without antibiotics per well in a 24-well plate. The cell density reached 30–50% confluency at the time of transfection. Cells were transfected with either siRNA duplexes that targeted HIF-1α or with a negative control siRNA. The cells were incubated for 24 h at 37 °C under hypoxic conditions until the time of the assay for gene knockdown. Knockdown was evaluated using qRT-PCR of HIF-1α, and lead to a > 90% decrease in HIF-1α mRNA expression in HTR8/SVneo cell lines.

Growth factor-reduced matrigel (BD Biosciences, USA) was diluted with serum-free medium at a ratio of 1:2 with cooled pipettes and distributed in a 24-well plate (150 ml per well) on ice. After the matrigel mixture solidified, HTR8/SVneo cells (1.0 × 10⁵ per well) with various pretreatments were gently added to each of the triplicate wells followed by normaxic or hypoxic culture for 12 h. Photomicrographs of three random fields of view were taken using an inverted phase-contrast microscope (Olympus, Tokyo, Japan). To evaluate the angiogenic process, we quantified the number of branching points. The number of branching points that generated at least three tubules was then counted in independent experiments each performed in duplicate.

For statistical analysis, SPSS for Windows version 18.0 (SPSS, Chicago, IL, USA) was used. All data are presented as mean ± SD. Comparisons between groups were analyzed by Student’s t test or one-way ANOVA followed by Tukey’s test for multiple comparisons. For correlational analyses, the Pearson correlation analysis was used. P value < 0.05 was considered to be statistically significant. All experiments in vitro were performed in triplicate.

There were no significant differences in age, gestational age, gravidity, parity, and induced abortion rates between the two groups. As shown in Fig. 1, the expression of HIF-1α and VEGF in missed abortion tissues was significantly lower than in tissues from normal pregnancies. In addition, there were lower levels of MVD with missed abortion than in elective abortion. HIF-1α and VEGF expression were significantly correlated with MVD in both groups (shown in Fig. 2). Therefore, we showed that a positive correlation exists between HIF-1α/VEGF and villous angiogenesis. The HIF-1α/VEGF signaling pathway stimulated angiogenesis in trophoblast cells.

To determine the expression of HIF-1α/VEGF in the HTR8/SVneo cell line, we measured mRNA and protein levels for HIF-1α and VEGF with qRT-PCR and WB. The HIF-1α and VEGF mRNA levels were significantly increased in hypoxia compared to normoxia (Fig. 3). In addition, hypoxic treatment significantly increased HIF-1α and VEGF protein levels compared to normoxic conditions. When we further examined HIF-1α effects on VEGF, we observed that HTR8/SVneo transfection with HIF-1α siRNA reduced both HIF-1α and VEGF mRNA and protein levels with hypoxia.

We found that tube formation in HTR8/SVneo cells was higher under hypoxic relative to normoxic conditions. The results of the in vitro tube formation experiment showed that microtubule density of the siRNA group was significantly lower than that of the negative control group (P < 0.01, Fig. 4). The results for this section confirmed that the ability for vasculogenic mimicry by cell lines was inhibited after HIF-1α silencing.