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Introduction

Preeclampsia (PE) is a significant pregnancy-related disorder affecting 3 to 5% of pregnant women globally, marked by hypertension and proteinuria, appearing after 20 weeks of gestation (1). PE has been the third leading cause of maternal and neonatal morbidity and mortality with >60,000 mortalities in pregnant women with PE annually across the world (2,3). Currently, there are still no effective therapies for PE beyond delivery of the placenta (4,5). Despite ongoing research, the etiology of PE is not fully understood. However, studies have linked inappropriate remodeling of spiral arteries, immune dysregulation, inadequate trophoblastic invasion and endothelial damage to this condition (3,6,7).

MSCs display a capacity of potential self-renewal, broad differentiation potential, low immunogenicity and readily accessible properties, which offer MSCs advantages over other cell-based therapies (8,9). Moreover, factors secreted by MSCs have been correlated with angiogenesis and trophoblast formation, and can maintain successful pregnancy (10–12). However, the molecular mechanisms are still unclear. MSCs perform their therapeutic roles through paracrine mechanism. Exosomes are small extracellular vesicles that range from 30–100 nm in size and contain various biomolecules including nucleic acids, lipids, proteins, mRNA, miRNA and other non-coding RNAs (13). Exosomes derived from MSCs (MSC-Ex) can alleviate liver fibrosis, acute and chronic kidney injury, myocardial ischemia/reperfusion damage and acute tubular injury (14–16).

Exosomes have gained a lot of attention as a potential method for delivering therapeutics due to their non-immunogenicity and non-toxicity (17). Exosomes can be genetically engineered or their surface chemically modified to specifically target cells or tissues, allowing them to accumulate in tumor tissues (18). This enables more precise and targeted delivery of therapeutics. The tumor-targeting capability of exosomes was conferred by linking lysosome-associated membrane glycoprotein 2b (LAMP-2B), a well-characterized exosomal membrane protein (19), to internalizing arginine-glycine-aspartic acid (CRGDKGPDC), which is widely recognized as an efficient cell membrane penetration peptide targeting αvβ3 integrins and neuropilin-1 (NRP-1) receptors (20). The CCG ligand (CCGKRK) has also shown selectivity towards placental tissue in mice (21). To the best of our knowledge, there is currently no study that has attempted to conjugate CCGKRK to LAMP-2B in order to enable exosomes target the placenta.

It is hypothesized that uteroplacental ischemia/hypoxia caused by impaired trophoblast invasion and uterine spiral arteriole remodeling is one of the leading causes of PE (6). During the first trimester of pregnancy, trophoblast cells are known to proliferate and survive in a hypoxic environment, which is beneficial for mural trophectoderm proliferation and spiral artery remodeling (22). Thus, during the initial phase of embryonic development, hypoxia-inducible factor-1α (HIF1α), an important transcription factor in placental development, is highly expressed in in trophoblast subpopulations. However, sustained hypoxia or HIF1α expression after 9 weeks of gestation will lead trophoblast cells to fail to differentiate from a proliferative to an invasive phenotype, shallow invasion of the trophoblasts and insufficient myometrial spiral artery transformation, which is strongly associated with early-onset PE (23,24). Therefore, HIF1α deletion might facilitate trophoblast invasion.

The present research is dedicated to exploring whether short hairpin RNA (shRNA)-HIF1α (sh-HIF1α) carried by MSC-Ex can effectively enhance the invasion ability of placental cells by conjugating sh-HIF1α-CCGKRK to LAMP-2B, thus providing a potential new therapeutic option for the prevention or treatment of PE (25). MSCs exosomes were used as a cell-based carrier for sh-HIF1α in the present study.

Materials and methods

JEG-3 cells culture

JEG-3 cells (ATCC; cat. no. HTB-36) were cultured in DMEM (cat. no. 10313039) adding 10% FBS (cat. no. 16140071) as well as 1% penicillin/streptomycin (cat. no. 15140122) (Gibco; Thermo Fisher Scientific, Inc.) under atmospheric oxygen tension (~21%) and 5% CO2 at 37°C.

Vector construction and lentiviral infection

HIF1α-overexpressing lentivirus (GV492-HIF1α) was constructed based on human HIF1α sequences from the Ensembl database (www.ensembl.org, Ensembl gene: ENSG00000100644) and synthesized by Shanghai GeneChem Co., Ltd. A 3rd generation system was used to package the lentivirus. The vectors (100 nM) and packaging plasmids (vector:packaging vector:envelope ratio, 10:3:1) were co-transfected into 1×106 293T cells (The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences) using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 8 h. The lentivirus-HIF1α and negative control lentivirus (empty vector) were collected and filtered through a 0.45 µM filter 3 days after transfection. JEG-3 cells were then seeded in six-well plates at a density of 1×105 cells/well and cultured at 37°C in 5% CO2. The JEG-3 cells were transduced with lentiviral vectors at a multiplicity of infection (MOI) of 10 at 37°C for 8 h, followed by replacement with fresh medium. JEG-3 cells were grown for 48 h and subsequently treated with puromycin (1 µg/ml) for 48 h to select stably transduced cells and 0.5 µg/ml puromycin was used for maintenance. Subsequently, transduction efficiency was determined using immunofluorescence microscopy, and the expression of HIF1α was detected using quantitative PCR and western blotting.

Reverse transcription-quantitative PCR (RT-qPCR)

RNA-iSo PluS (cat. no. 9109; Takara Bio, Inc.) was used to isolate total RNA from JEG-3 cells and MSC-Ex. cDNA was produced by cDNA Synthesis SuperMix (cat. no. 11119ES60; Shanghai Yeasen Biotechnology Co., Ltd.). The reverse transcription procedure was as follows: 25°C for 5 min; 42°C for 30 min; and 85°C for 5 min. The cDNA was subjected to qPCR using SYBR Green qPCR Mix (cat. no. HY-K0501A; MedChemExpress) using the ABI 7500 Real-Time PCR system (Thermo Fisher Scientific, Inc.). The following ingredients were used to a total of 20 µl: cDNA (1 µl), SYBR Premix ex Taq (2X; 10 µl), reverse primer (10 µM; 0.4 µl), forward primer (10 µΜ; 0.4 µl) and double-distilled water (to 20 µl). Amplifications were performed following the procedure of a two-step method (95°C for 30 sec; 1 cycle at 95°C for 10 sec followed by 95°C for 10 sec; 60°C for 30 sec and 40 cycles; and melting curve stage). Relative HIF1α expression was normalized to GAPDH mRNA level and calculated via the 2−ΔΔCq method (26). The sequences of primers used were as follows: GAPDH forward 5′-GGGAGCCAAAAGGGTCAT-3′, and reverse 5′-GAGTCCTTCCACGATACCAA-3′; HIF1α forward, 5′-GGCGCGAACGACAAGAAAAA-3′, and reverse 5′-GGCTGTGTCGACTGAGGAAA-3′.

Western blotting

Proteins were extracted using RIPA lysis buffer (cat. no. HY-K1001; MedChemExpress) from JEG-3 cells and MSCs-Ex. The concentration of the isolated protein was quantified via BCA Protein Assay kit (cat. no. 23225; Thermo Fisher Scientific, Inc.). Isolated proteins (5 µg) were mixed with 5X SDS-PAGE protein loading buffer (cat. no. 20315ES05; Shanghai Yeasen Biotechnology Co., Ltd.). Proteins (20 µg) were separated on 12% SDS-acrylamide gels, followed by transferring onto PVDF membranes, which were incubated with 5% non-fat milk for 1 h at room temperature, and with rabbit anti-HIF-1α (1:1,000; cat. no. ab179483; Abcam), rabbit anti-CD9 (1:1,000; cat. no. ab236630; Abcam), rabbit anti-CD81 (1:1,000; cat. no. ab79559; Abcam), rabbit anti-LAMP-2B (1:1,000; cat. no. ab18529; Abcam), rabbit anti-TSG101 (1:1,000; cat. no. ab125011; Abcam) and rabbit anti-GAPDH (1:10,000; cat. no. 10494-1-AP; ProteinTech Group, Inc.) overnight at 4°C. Subsequently, membranes were incubated with horseradish peroxidase-conjugated mouse anti-rabbit IgG (1:5,000; cat. no. BM2006; Boster Biological Technology) for 1 h at room temperature. Protein bands were determined via ECL western blot detection reagents (Thermo Fisher Scientific, Inc.). The protein gray value was calculated using ImageJ (Version 1.5.3; National Institutes of Health).

Glucose uptake and lactate production

Transfected JEG-3 cells were seeded in six-well plates at the concentration of 3×105/well. After culturing at 37°C for 48 h, the cells were collected and lysed with Cell and Tissue Lysis Buffer for Glucose Assay (cat. no. S3062; Beyotime Institute of Biotechnology). The samples were then centrifuged at 1,200 × g at 4°C for 10 min to obtain the supernatant of cells. The glucose concentration in the supernatant was determined using the glucose oxidase method (Amplex Red Glucose/Glucose Oxidase Assay kit; cat. no. MP 22189; Invitrogen; Thermo Fisher Scientific, Inc.). An appropriate amount of 1X reaction buffer was added to dilute 400 mM glucose to produce a glucose concentration of 0–200 µM to make the standard curve. Subsequently, 50 µl of each standard, controls and samples were added to the individual wells of the microplate in duplicate. The absorbance at OD 590 nm was measured using a microplate reader (LT-4000; Labtech International Ltd,).

The lactate in the supernatant was measured according to the instructions of the Lactate Detection kit (cat. no. K607-100; BioVision; Abcam). A total of 10 µl samples, 90 µl distilled water, 40 µl reagent 2 and 60 µl chromogen solution were added to the well and mixed thoroughly. Then the wells were incubated at 37°C in dark for 30 min followed by measuring the absorbance at 530 nm.

Cell viability assay using Cell Counting Kit-8 (CCK-8)

Transfected JEG-3 cells were passed onto the 96-well plates (1×103 cells/well) and cultured in an incubator for 24, 48, 72, 96 and 120 h at 37°C. Next, CCK-8 regent (10 µl/well; cat. no. A311-01; Vazyme Biotech Co., Ltd.) was added to the cells and kept at 37°C for 3 h. Eppendorf BioPhotometer® D30 (Eppendorf) was used to acquire the values of absorbance at 450 nm. The cell viability was calculated from the absorbance values for five consecutive days. The cell viability percentage was calculated as follows: Cell viability (%)=(sample absorbance/control absorbance) ×100.

Cell invasion assay

Transwell assay was conducted to measure the changes in JEG-3 cells invasion after HIF1α lentivirus transfection. Transwell chambers (cat. no. 3402; Corning Life Sciences) were pre-coated with Matrigel (cat. no. 356234; Corning Life Sciences) for 6 h at 37°C. A 150-µl JEG-3 cell suspension in serum-free DMEM was seeded in the upper layer of the six-well Transwell chamber (7×103 cells/well). In addition, 600 µl DMEM containing 10% FBS was added to the lower chamber. After 48 h incubation at 37°C, JEG-3 cells on the basolateral chamber were washed twice using PBS, and stained with 1% crystal violet for 30 min at room temperature. JEG-3 cells were visualized and images were captured under the light microscope (magnification, ×200; Olympus cX2; Olympus Corporation) after washing using PBS twice.

Mesenchymal stem cells (MSCs) identification

MSCs were obtained from Procell Life Science & Technology Co., Ltd. (cat. no. CP-H166), which were primary human MSCs that have not been immortalized. The cells were kept in DMEM medium adding 10% FBS and 100 U/ml penicillin and streptomycin. Then the surface markers of the cultured MSCs were identified using CD90 and CD44 by immunofluorescence assays. MSCs were plated in a 35-mm confocal dish and cultured for 48 h followed by fixing with 4% paraformaldehyde for 20 min at room temperature. After rinsing with PBS, cells were permeabilized with 0.5% Triton X100 in PBS and blocked with 3% BSA for 1 h at room temperature. Cells were then incubated overnight with anti-CD44 (1:50; cat. no. 15675-1-AP; ProteinTech Group, Inc.), anti-CD90 (1:50; cat. no. 66766-1-Ig; ProteinTech Group, Inc.), anti-CD34 (1:50; cat. no. 60287-1-Ig; ProteinTech Group, Inc.) and anti-CD45 (1:50; cat. no. 14486-1-AP; ProteinTech Group, Inc.) antibodies at 4°C followed by 2 h incubation with goat anti-mouse IgG (H+L) Cy3-conjugated and goat anti-rabbit IgG (H+L) FITC-conjugated secondary antibodies (1:100; cat. no. BA1031 and BA1105, respectively; Boster Biological Technology). After three TBS-0.05% Tween-20 washing steps, cells were incubated with DAPI (cat. no. D1306; Thermo Fisher Scientific, Inc.) for 10 min at 37°C. Finally, cells were observed using Olympus fluorescence microscope BX53 (Olympus Corporation). The positive rate was calculated using the following formula: Positive rate=(number of positive cells/number of total cells) ×100%. The cell number were analyzed using ImageJ software (1.4; National Institutes of Health).

Homing peptide, sh-HIF1α peptide nucleic acid conjugates

The LAMP-2B + 5′-TGTTGTGGTAAACGTAAA-3′ (gene sequence of CCGKRK) gene was amplified from cDNA template by PCR, then purified and recovered with a gel recovery kit (Beijing Solarbio Science & Technology Co., Ltd.). The gene was subsequently cloned and ligated to the GV493 vector (hU6-MCS-CBh-MCS-IRES-puromycin; Shanghai GeneChem Co., Ltd.). A 3rd generation system was used to package the lentivirus. Additionally, an sh-HIF1α sequence (5′-ACGACAAGAAAAAGATAAGTT-3′) was also ligated into the same GV493 vector using an independent promoter. Upon transduction into cells, the lentiviral particles generated in this manner facilitated the overexpression of both LAMP-2B + CGKRK and HIF1α shRNA. The vectors (100 nM) and packaging plasmids (vector:packaging vector:envelope ratio, 10:3:1) were co-transfected into 1×106 293T cells with Lipofectamine 2000 at 37°C for 8 h. The sh-HIF1α-LAMP-2B lentivirus and negative control (empty vector) were collected and filtered through a 0.45 µM filter after 293T cells were cultured at 37°C for 3 days. MSCs were then transduced with lentiviral vectors at a MOI of 10 for 8 h followed by replacement with fresh medium. MSCs were cultured at 37°C for 48 h and subsequently treated with puromycin (1 µg/ml) for 48 h to select stably transduced cells, 0.5 µg/ml puromycin was used for maintenance.

Isolation of exosomes

MSCs supernatant was collected and centrifugated at 2,000 × g for 30 min at 4°C. Following being filtered through 0.22 µm syringe filter (cat. no. SLGVR13SL; MilliporeSigma), which was further centrifuged at 120,000 g overnight at 4°C using Optima XPN-100 high-speed freezing centrifuge (cat. no. CP100MX; Hitachi, Ltd.). Exosomes were precipitated from the supernatants. The sediments of exosomes were resuspended by cold PBS, and ultracentrifuged again at 120,000 × g for 90 min at 4°C. The final sediments of exosomes were resuspended in cold PBS or SDT lysate buffer, and immediately stored at −80°C.

Pep-sh-HIF1α and exosomes binding

Exosomes (30 g) were preincubated with LAMP-2B-CCGKRK-sh-HIF1α peptide (30 g) overnight at 4°C, followed by washing with PBS five times in 2-ml ultracentrifuge tubes and filtration with 100-kDa diafiltration tube (MilliporeSigma) to remove unbound peptides. Subsequently, peptide-exosome complexes (30 g) were incubated with 4-mm aldehyde/sulfate latex beads (Invitrogen; Thermo Fisher Scientific, Inc.) for 15 min at room temperature under rotation followed by washing with PBS for three times. Then recovered beads were subjected to various tests.

Transmission electron microscopy

Purified exosomes supernatant was resuspended PBS. The exosome pellets were fixed with 2.5% glutaraldehyde for 1 h at room temperature, and post-fixed with 1% osmium for 1 h at room temperature. A total of 20 µl exosomes suspensions were added onto copper grid carefully, blotted up and stained with 2% phosphotungstic acid (PTA) for 2 min at room temperature. Sample was imaged using a transmission electron microscope (cat. no. HT-7700; Hitachi, Ltd.).

Nanoparticle tracking analysis

Isolated exosome was diluted to 1 ml in TBS with 0.1% Pluronic F-68 and 2 mmol/l EDTA for the next analysis. Size of exosomes was determined using Nanosight Tracking Analysis by utilizing nanoparticle tracking analysis (NTA; N30E; NanoFCM, Inc.) referring to the documented protocol (17).

Flow cytometry

To characterize individual exosomes by flow cytometry, exosomes were labeled with 1,1′-dioctadecyltetramethyl indotricarbocyanine iodide (cat. no. DL22065, Duolaimi Biotechnology Co., Ltd.), which labels the lipid in exosomes, as per the manufacturer's instruction and subjected to flow cytometry. To measure the percentage of pep-positive exosomes in total exosomes, DiR-labeled exosomes (5 g) were incubated with homing peptide (CCGKRK)-labeled anti-mouse antibody (1:20) generated in collaboration with NovoPro Bioscience, Inc. in 4% BSA for 30 min at 4°C, followed by 1:10 dilution with PBS, and were analyzed with flow cytometry (FACSCalibur; BD Biosciences). Uncoated beads were used as negative controls for gating.

Statistical analysis

Statistical analysis was conducted using GraphPad Prism software (8.0; GraphPad Software, Inc.). All experiments were repeated thrice and data in the present study are presented as with mean ± standard deviation. Kolmogorov-Smirnov testing for normality of distribution was used to determine if variables were parametric or non-parametric. Unpaired Student's t-test was used for two group parametric comparisons, and one-way ANOVA followed by Tukey's post hoc test was used for multiple parametric comparisons. Mann-Whitney U testing was used for nonparametric variables. P<0.05 was considered to indicate a statistically significant difference.

Results

Expression level of HIF1α in trophoblast cells

HIF1α was overexpressed in JEG-3 cells via lentiviral transfection. Fluorescence microscopy confirmed high efficiencies of lentivirus transfection with a large proportion of GFP-positive cells (Fig. 1A). Analysis of both mRNA and protein levels revealed a significant increase of HIF1α in the transfected cells when compared with the control group detected by RT-qPCR and western blotting (Fig. 1B-D).

Figure 1. HIF1α is overexpressed in JEG-3 cells. (A) Transfection efficiency of HIF1α-overexpressing lentivirus was detected using immunofluorescence (magnification, ×100). (B) mRNA and (C) protein levels of HIF1α in JEG-3 cells were determined using reverse transcription-quantitative PCR and western blotting, respectively. (D) Grayscale analysis was performed using ImageJ software. ***P<0.001 compared with NC. HIF1α, hypoxia-inducible factor-1α; NC, negative control; lv, lentiviral.

HIF1α promotes aerobic glycolysis and decreased the invasion capacity of trophoblast cells

Elevated HIF1α in JEG-3 cells led to a significant increase in glucose uptake compared with the NC group (Fig. 2A). Additionally, there was a significant elevation in lactate production in HIF1α-overexpressed JEG-3 cells (Fig. 2B). Overexpression of HIF1α significantly enhanced the proliferation ability of JEG-3 cells (Fig. 2C). Moreover, increased expression of HIF1α significantly suppressed the invasion ability of JEG-3 cells via Transwell invasion assays (Fig. 2D). These observations suggested that HIF1α was associated with the invasion and aerobic glycolysis of JEG-3 cells.

Figure 2. HIF1α promotes aerobic glycolysis and decreases the invasion capacity of JEG-3 cells. (A) Glucose concentration in the supernatant of JEG-3 cells was determined using a Glucose Oxidase Assay kit. (B) Lactate in JEG-3 cells supernatant was measured via Lactate Detection kit. (C) Viability of JEG-3 cells was detected using CCK-8. (D) Invasion capability of JEG-3 cells was determined using Transwell. ***P<0.001 compared with NC. HIF1α, hypoxia-inducible factor-1α; NC, negative control; lv, lentiviral; CCK-8, Cell Counting Kit-8.

Observation and identification of human MSCs

CD44 and CD90 are cell surface markers that are expressed by human MSCs (27). The present study demonstrated that the MSCs bought from Procell Life Science & Technology Co., Ltd. were spindle-shaped (fibroblast-like cells), which is typical morphology of MSCs. Results of immunofluorescence assay indicated that both CD90-positive and CD44-positive rates of MSCs were ~100%, while lacking CD34 and CD45 human leukocyte markers (Fig. 3A). The results revealed that the purity of the isolated MSCs was high enough for the subsequent experiments.

Figure 3. Identification of human MSCs. Representative fluorescence images of CD90-/CD44-positive and CD45-/CD34-negative MSCs. Scale bar, 200 µm. CD90, red; CD44, green; DAPI, blue. MSCs, mesenchymal stem cells.

Characterization of isolated exosomes

The exosomes harvested from the supernatant of MSCs were characterized using electron microscopy, which demonstrated a presence of small membrane-bound vesicles (Fig. 4A). This observation is consistent with the typical morphology of exosomes (28). The majority of the population was in the size range of 50–130 nm in diameter (Fig. 4A), which is consistent with previously reported features of exosomes (29). Western blotting was applied to determine the level of the typical exosomal markers CD9 antigen (CD9), CD81 antigen (CD81), exosomal protein LAMP-2B and tumor susceptibility gene 101 (TSG101) (30). As expected, the exosomal markers, including CD63, CD81, LAMP-2B and TSG101, were enriched in exo-NC and exo-pep-sh-HIF1α compared with those in cells (Fig. 4B). Approximately 69.94% of exosomes were labeled by DiR, and ~87.56% of DiR-labeled exosomes were modified with homing peptide (CCGKRK) demonstrated by flow cytometry exosomes (Fig. 4C). These findings support the conclusion that the homing peptide successfully binds to exosomes LAMP-2B.

Figure 4. Characterization of exosomes after isolation. (A) Electron micrographs of exosomes isolated from MSCs. NTA (NanoSight) was applied to determine the size distribution of isolated exosomes in diameter. (B) Western blotting was performed for the indicated proteins. Grayscale analysis was performed by ImageJ software. (C) Flow cytometry for measuring the modification efficiency of placental homing peptide CCGKRK on exosomes. DiR-labeled exosomes (5 g) and homing peptide (CCGKRK)-labeled anti-mouse antibody were used. *P<0.05 and ***P<0.001. MSCs, mesenchymal stem cells; NC, negative control; sh-, short hairpin; TSG101, tumor susceptibility gene 101; LAMP-2B, lysosome-associated membrane glycoprotein 2b; HIF1α, hypoxia-inducible factor-1α.

HIF1α knockdown of MSCs-derived exosomes increases the invasion capacity of trophoblast cells

Next, HIF1α levels in MSC-Ex-treated JEG-3 cells were tested. The RT-qPCR assay revealed that the HIF1α level in MSC-Ex-treated JEG-3 cells was significantly decreased compared with the exo-NC group (Fig. 5A). In addition, HIF1α protein level in HIF1α-silenced MSC-Ex-treated JEG-3 cells was also significantly suppressed (Fig. 5B). Knockdown of HIF1α significantly enhanced the invasion ability of trophoblast JEG-3 cells (Fig. 5C), which plays an important role in embryo implantation.

Figure 5. Exo-pep-sh-HIF1α facilitates placental invasion. The level of HIF1α in Exo-pep-sh-HIF1α treated JEG-3 cells was determined by (A) reverse transcription-quantitative PCR and (B) western blotting. Grayscale analysis was performed by ImageJ software. (C) Invasion capability of Exo-pep-sh-HIF1α treated JEG-3 cells was determined by Transwell (magnification, ×100). ***P<0.001. HIF1α, hypoxia-inducible factor-1α; NC, negative control; sh-, short hairpin.

Discussion

PE is often linked to reduced invasiveness of fetal trophoblast cells, leading to inadequate uterine placental perfusion (31). Sustained hypoxia leads trophoblasts to fail to differentiate from a proliferative to an invasive phenotype after 9 weeks of gestation, resulting in failed implantation (23,32). The current study conjugated an anchor peptide specific CCG ligand (CCGKRK) to exosomal membrane protein LAMP-2B to target HIF1α deletion exosomes to the placenta, which significantly enhanced the invasion ability of trophoblast. The results unveil new potential therapeutic targets for the prevention and treatment of PE.

At present, there are no effective treatments for PE. The few drugs licensed for pregnancy disorders often lead to serious systemic toxicity as their low molecular weights cross the placenta from mother to fetus (33,34). Thus, developing targeted therapies will be a highly effective therapeutic strategy for PE. HIF1α, a key regulator of intracellular oxygen metabolism, plays an important role in the development of the tumor (35). HIF1α is significantly upregulated to maintain trophoblasts in a proliferative, non-invasive and immature phenotype, which share some similarities with tumor cells in the biological processes, during the initial phase of embryonic development (23,36). After that, oxygen levels in the intervillous spaces increase from 2% O2 before 9 weeks to 8% O2 at 10 to 12 weeks of pregnancy (37). Correspondingly, HIF1α are rapidly reduced after 9 weeks of gestation. However, preeclamptic women have significantly higher oxidative stress and serum HIF1α levels compared with normal pregnant women, which leads the trophoblasts development to remain arrested at an immature stage, causing inadequate trophoblast invasion (38,39). HIF1α-overexpressed pregnant mice have significantly elevated blood pressure, decreased placental weights and histopathological placental abnormalities (40). Serum HIF1α levels have already been used to predict PE (23). In the present study, upregulated HIF1α was confirmed to significantly promote the proliferation while suppressing the invasion of trophoblast cells. This suggested that HIF1α deletion might be a potential therapeutic possibility for PE.

Moreover, PE is implicated in abnormal glucose and lipid metabolism (41–43). Gestational diabetes mellitus is one of the most common and important complications of pregnancy (44). Chronic fetal hypoxia causes deficits in oxidative glucose metabolism and enhancement of glycolytic metabolism, which causes energy production via glucose oxidation to be replaced by glycolysis (43). This abnormal aerobic glycolysis is also called metabolic reprogramming, a phenomenon known as the Warburg Effect, which is a hallmark of cancer, such as breast cancer, gastric cancer and colorectal cancer (45–47). However, a potential role for aerobic glycolysis in PE has not been elucidated. HIF1α facilitates glucose transporters and glycolytic enzymes expression that promote glucose uptake and glycolysis in Th17 cells (48). Thus, elevated levels of HIF1α in the hypoxic microenvironments at 10 to 12 weeks of gestation might mean trophoblasts fail to switch to an invasive phenotype by promoting aerobic glycolysis. The present study revealed that HIF1α overexpression promoted the uptake of glucose and the production of lactate in trophoblast cells. However, HIF-1α overexpression led to increased proliferation of trophoblast cells. The enhanced glucose and lactate production did not definitively exclude the possibility of cell proliferation-induced promotion. Thus, the enhanced glucose and lactate production resulted from increasing cell number or upregulation of HIF-1α overexpression needs further investigation.

Disrupting the expression of disease-causing genes by gene editing is already a well-established technique (49). However, the method to achieving efficient intervention of HIF1α expression in the placenta needs to be investigated. In recent years, several research reports have focused on delivering therapeutic drugs specifically to the placenta via nanoparticles, which exert high chemical stability, high drug loading capacity and low toxicity (50,51). Notably, extensive studies have confirmed the positive effects of MSCs on PE (11,52–54). In various PE rat models, MSC transplantation improves PE symptoms and inhibits inflammatory cytokines such as TNF-α and IL-6, providing evidence that human umbilical cord-derived MSCs may be a possible therapy for preeclampsia (53,55,56). MSCs exert their therapeutic functions via secretion of bioactive products, namely the exosomes, which have a high biocompatibility, low toxicity and low immunogenicity that makes them ideal drug delivery carriers of anti-PE drugs (57). The present study succeeded in conjugating CCG ligand (CCGKRK) to the exosomal membrane protein LAMP-2B, which led MSCs-derived exosomes to target the placenta. In the present study, MSC exosomes were used as a biocompatible drug carrier to target HIF1α siRNA to the placenta. To the best of our knowledge, this is the first attempt to make exosomes target placental tissue by genetic engineering. Notably, silencing HIF1α encouraged an aggressive phenotype that facilitated trophoblasts invasion, suggesting that HIF1α deletion is a potent way to interfere with the progression of PE. Clinically, some factors can be used to predict PE (58). If it poses a high risk of PE, some measures can be taken to intervene PE in advance, such as enhancing the invasion ability of trophoblasts by carrying sh-HIF1α to placenta by exosomes before PE is diagnosed.

There were some pitfalls and drawbacks in present study. Firstly, the experiments in vitro were only conducted in JEG3 cells, a commonly used choriocarcinoma cell line that serves as a model of villous trophoblast cells, whereas immortalized human chorionic trophoblast cells such as HTR-8 cells are considered to be an improved cell model to study trophoblast function. Therefore, further studies should be conducted using HTR-8 cells to confirm the findings presented in this study. Additionally, due to limited experimental conditions, the feasibility and effectiveness of targeted delivery of HIF1α deletion exosomes to the placenta by conjugating CCG ligand (CCGKRK) to LAMP-2B in animal models or human tissue samples has not yet been confirmed. In the present study, knockdown of HIF1α was proposed as a potential treatment to increase trophoblast invasion. However, it is essential to consider the potential effects of HIF1α knockdown on early pregnancy processes, such as implantation and placental development. Therefore, targeted and precise control of HIF1α knockdown is necessary to avoid any adverse effects on these critical events. Further research is required to elucidate the underlying mechanisms and optimize the therapeutic approach to ensure the safety and efficacy of HIF knockdown in the context of pregnancy complications.

In summary, the present study facilitated the targeted delivery of HIF1α deletion exosomes to the placenta by conjugating CCG ligand (CCGKRK) to LAMP-2B, and provided a novel platform for the development of placenta-specific therapeutics. Silencing HIF1α was confirmed to be an effective therapeutic target for PE by promoting trophoblast invasion.

Acknowledgements

Not applicable.

Funding

This study was supported by Hainan Provincial Natural Science Foundation of China (no. 821MS128 and 822MS164) and National Natural Science Fund Cultivating 530 Project of Hainan General Hospital (grant no. 2021MSXM04). Scientific research project of health industry in Hainan Province (grant no. 22A200234).

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Authors' contributions

FC and HC conceived and designed this study. FC and DM carried out the analyses and also participated in the study design. HC wrote the manuscript. RF participated in the analysis of data and helped edit the manuscript. FC and HC confirm the authenticity of all the raw data. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The medical ethics committee of Hainan General Hospital waives the requirement for authors to obtain ethical approval for the use of commercially available cells.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References