Human umbilical cord mesenchymal stem cells restores mTOR-mediated autophagy homeostasis to alleviate placental injury and improve pregnancy outcomes in preeclampsia

doi:10.21203/rs.3.rs-4957657/v1

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Research Article

Human umbilical cord mesenchymal stem cells restores mTOR-mediated autophagy homeostasis to alleviate placental injury and improve pregnancy outcomes in preeclampsia

https://doi.org/10.21203/rs.3.rs-4957657/v1

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Background

Preeclampsia is a hypertensive disorder during pregnancy, which seriously threatens both maternal and infant health. Currently, the only treatment available is to induce infant and placenta delivery, resulting in interest in potential fetal-safe treatment strategies. One such strategy is cell therapy with human umbilical cord mesenchymal stem cells (hUC-MSCs), which possesses immunomodulatory, anti-inflammatory and angiogenic functions that could alleviate pre-eclamptic symptoms. However, the precise effects and underlying mechanisms behind their activities are still largely unknown. In this study, we aimed to elucidate the effect of hUC-MSCs, as well as the pathways involved, on placental function in preeclampsia, thereby highlighting potential novel avenue for stem cell therapy.

Methods

Both an in vivo rat model, involving N-nitro-L-arginine methyl ester (L-NAME) injections in pregnant rats, and an in vitro model, entailing HTR8 trophoblasts/human umbilical cord vein endothelial cells (HUVECs) being stimulated with lipopolysaccharide (LPS), were established to simulate pre-eclampsia. In vivo, maternal blood pressure, renal function, as well as placental and fetal weights, were measured. ELISA was used to measure maternal serum levels of angiogenic, inflammatory, and oxidative stress factors. Placental mitochondrial morphology was evaluated using transmission electron microscopy, while autophagic pathways were analyzed by Western blots. With the in vitro model, cell proliferation, invasion, oxidative stress, and apoptosis were evaluated in a Transwell co-cultured with hUC-MSCs.

Results

hUC-MSC administration was found in the in vivo model to increase fetal weights, along with alleviating hypertension and proteinuria, which are owed to those cells promoting placental angiogenesis and blood perfusion, as well as lowering inflammation, oxidative stress, and apoptosis. These findings were further supported by the in vitro model, where hUC-MSC co-culture with LPS-treated HTR8/HUVECs resulted in increased cell proliferation and invasion, along with lowered apoptosis and reactive oxygen species generation. All of these effects are owed to hUC-MSCs improving placental mitochondrial function by lowering autophagy; this is through activating Akt/mTOR and inhibiting AMPK/mTOR pathways, leading to pro-autophagic LC3 and Beclin1 downregulation, as well as anti-autophagic P62 upregulation.

Conclusion

hUC-MSCs are able to alleviate pre-eclampsia by restoring physiological placental autophagic homeostasis, which could serve as a promising therapeutic strategy for the disease.

preeclampsia

human umbilical cord mesenchymal stem cells

autophagy

mTOR

placenta

Preeclampsia is a pregnancy-specific disease, characterized by hypertension occurring after 20 weeks of gestation; this hypertension may be accompanied by proteinuria or other organ dysfunction, all of which collectively could seriously endangers both maternal and fetal health [1]. It is the leading cause of both maternal and perinatal mortality, causing > 70,000 maternal and > 500,000 fetal deaths yearly worldwide [2, 3]. As a result, treating preeclampsia is regarded as a key approach to improve placental function and pregnancy outcomes [4]. Multiple underlying mechanisms have been postulated to be involved in preeclampsia pathogenesis, such as abnormal placentation stemming from the failure of cytotrophoblasts to transform from the proliferative epithelial to the invasive endothelial subtype, resulting in narrow maternal vessels developing [4]; these vessels are more prone to atherosis, and subsequently placental ischemia [4], a significant precursor condition associated with pre-eclampsia. This connection between placental ischemia and pre-eclampsia, in turn, is bolstered by pre-eclamptic trophoblasts being found to overexpress the hypoxia marker hypoxia-inducible factor (HIF)-1α [4]. Another possible mechanism is the overexpression of soluble fms-like tyrosine kinase 1 (sFLT1) from the placenta, which serves as a “ligand trap” against the pro-angiogenic proteins vascular endothelial (VEGF) and placental growth factors (PIGF); indeed, a number of studies have observed that high sFLT1:PIGF ratios are predictive of adverse pre-eclampsia outcomes [5]. This association is further reinforced by findings demonstrating that VEGF inhibitors yield similar adverse effects as sFLT1 [5], as well as sFLT1 expression being suppressed by the administration of HIF-1α inhibitor 2-methoxyestradiol [4]. Additionally, altered immune system activities, in the form of increased T helper 1 cells, activation of the alternative complement pathway, and oxidative stress, have been found to be linked to pre-eclampsia [4, 5, 6].

Out of those mechanisms, though, one that has been found to play a key role is abnormal autophagy among trophoblasts [7]. During normal pregnancy, trophoblast autophagy is involved in degrading misfolded or damaged proteins and organelles, thereby maintaining placental homeostasis [8, 9]. However, its dysregulation could impair trophoblast invasion and angiogenic capabilities [10], leading to the accumulation of harmful substances, as well as limiting the energy supply to those cells, resulting in preeclampsia onset and development.

Recently, stem cell-based therapies have become a promising novel approach for treating various pregnancy disorders [11], of which human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) have attracted widespread attention, owing to their low immunogenicity, high proliferative capabilities, and multipotency [12, 13]. In fact, a number of studies have shown that hUC-MSCs are able to improve pregnancy outcomes and regulate trophoblast function [14–16], yet the specific mechanisms of action they are involved in to alleviate preeclampsia has still not been fully elucidated. In this study, we aimed to shed more light on those mechanisms of action through which hUC-MSCs treat preeclampsia. We developed both an in vitro co-culture system, as well as a preeclampsia rat model, in order to evaluate hUC-MSC effects on improving trophoblast and placental functions. We found that in vivo, hUC-MSC administration alleviated pre-eclamptic maternal blood pressure increases and renal dysfunction, along with increasing fetal weights. These improvements were likely owed to hUC-MSCs promoting placental angiogenesis via increasing the production of pro-angiogenic and anti-inflammatory factors, along with suppressing oxidative stress and apoptosis. Furthermore, hUC-MSCs promote trophoblast cell proliferation and invasion, as well as lowering apoptosis, via activating Akt/mTOR and inhibiting AMPK/mTOR pathways to alleviate pathological placental autophagy. These findings, particularly in terms of hUC-MSCs being able to counteract against autophagy alterations in pre-eclampsia, would thus aid in developing and validating stem cell therapy-based approaches for treating the disease.

Establishing the pre-eclampsia rat model

All animal procedures were performed under sterile conditions, in accordance with the Guide for the Care and Use of Laboratory Animals (NIH, 8th Edition, 2011) and ARRIVE guidelines 2.0, and received approval from the Institutional Animal Care and Use Committee of Shanxi Medical University (REB# DWYJ-2023-106). Both female and male Sprague-Dawley rats (8–10 weeks old) were obtained from the Animal Center of Shanxi Medical University and reared under specific-pathogen-free (SPF) conditions (12 hr light/dark cycle, 40–60% humidity, 22°C). Males were mated to females in a 2:1 ratio, and day 0 of pregnancy was defined, based on a copulatory plug, or sperm, being present in vaginal smear examinations of female rats. Pre-eclampsia was then induced by daily subcutaneous injections of 200 mg/kg N-nitro-L-arginine methyl ester (L-NAME; Sigma-Aldrich, N5751), from days 13 to 17 of pregnancy, while rats who received an equal volume of saline during the same time period served as the control [17]. Blood pressure and urine protein levels were measured among the pregnant rats, and successful pre-eclampsia inducement was identified based on whether they had increases in blood pressure by > 30 mmHg, as well as in the 24 hr-urine protein level by > 300 mg/day.

Additionally, at days 15, 17, and 19 of pregnancy, pre-eclamptic rats were injected with 100 µL of hUC-MSCs (1×10⁶ cells/rat per injection, suspended in 100 µL PBS) into their tail veins, while control rat had tail vein injection of 100 µL of saline on those days. These hUC-MSCs were obtained from the Shanxi Provincial Clinical Cell Pilot Base [18, 19], after passages 4–8. Flow cytometry analyses showed that hUC-MSCs were positive for mesenchymal cell markers CD90, CD44, CD105 and CD73 (Fig. S1A), and negative for hematopoietic stem cell lineage markers HLA-DR, CD45, CD19, and CD11b (Fig. S1B). These cells were also able to differentiate into adipogenic (Oil Red O; Fig. S1C), osteogenic (Alizarin Red S; Fig. S1D), and chondrogenic lineages (Alcian Blue; Fig. S1E)

At day 20 of pregnancy, all pregnant rats were anesthetized with 2% isoflurane (R510-22, RWD), euthanized by cervical dislocation, followed by removal of fetuses and placentas via caesarian section; their weights, as well as the number of viable fetuses, were measured. Ultimately, a total number of 30 rats (minimum to meet desired statistical constraints), randomized by block randomization into 3 treatment groups, were used in this study, after applying the following exclusion criteria to an initial cohort of 35 rats: blood pressure did not increase by 30 mmHg after L-NAME injection (3 rats), mortality after L-NAME injection (1 rat, whose cause of death was unable to be identified after autopsy), or pre-pregnancy hypertension (1 rat). minimum number of necessary samples to meet the desired statistical constraints.

Measuring blood pressure, urine protein levels, and enzyme-linked immunosorbent assay (ELISA) among pre-eclamptic rats

To measure blood pressure among pregnant rats, the Medlab Non-Invasive Blood Pressure (NIBP) System for Rats and Mice (Nanjing Calvin Biotechnology Co., Ltd) was used at the tail vein, on days 0, 6, 12, 15, 18, and 20 of pregnancy. As for urine protein levels, they were obtained at days 11 and 18 of pregnancy, in which rats were placed in metabolic cages for 24-hr urine collection, followed by measurement using the Total Protein (TP) Colorimetric Assay Kit (Coomassie Brilliant Blue method; Nanjing Jiancheng Bioengineering Institute, A045-2-2).

Blood samples were also collected from pregnant rats, centrifuged at 3500×g for 10 min, followed by ELISA using Beyotime ELISA Kits in accordance with the manufacturer’s instructions. These kits were used to measure serum levels for sFLT-1 (EK0871), soluble endoglin (sENG; EK1120), PIGF (EK0597), VEGF (EK0539), inflammatory factors interleukin (IL)-6 (EK0412), IL-10 (EK0416), tumor necrosis factor (TNF)-α (EK0526), interferon (IFN)-γ (EK0373), as well as oxidative stress-related indicators endothelial nitric oxide synthase (eNOS; EK1167), nitric oxide (NO; S0023), malondialdehyde (MDA; S0131) and superoxide dismutase (SOD; S0109). For blood urea nitrogen (BUN) and creatinine (Cr) levels, they were measured, respectively, by the urease method with the Urea Assay Kit (C013-2-1), and the sarcosine oxidase method with the Creatinine Assay kit (both from Nanjing Jiancheng Bioengineering Institute, C011-2-1).

Histological analyses and transmission electron microscopy (TEM) of pre-eclamptic rat tissues

For histological analyses, pregnant rats were sacrificed, placenta and kidney samples obtained, and fixed in 4% paraformaldehyde (PFA, Biosharp, BL539A). These samples were then paraffin-embedded, sectioned into 5 µm sections. Some sections were sequentially deparaffinized in xylene I, II, and III for 10 min, rehydrated in a series of ethanol solutions with decreasing concentrations, and stained with hematoxylin and eosin (H&E), while some other sections were immuno-histologically stained for endothelial (CD31; MedChemExpress, HY-P80447) and immuno-fluorescence stained for smooth muscle markers (α-SMA; MedChemExpress, HY-P80484). Images were captured (3D HISTECH), and positive areas quantified by ImageJ.

For TEM, freshly excised rat placental tissue was cut into 1–5 mm pieces, fixed in 2.5% glutaraldehyde (Solarbio, P1126) in darkness, at room temperature for 2 hr, then transferred to 4°C for storage. Afterwards, ultrathin sections were prepared from fixed tissues, and a HITACHI HT7700 microscope was used to observe ultrastructure.

Establishing the in vitro pre-eclampsia cell model and functional assays

Human HTR-8/SVneo extravillous trophoblasts (Boster, CX0115) and umbilical vein endothelial cells (HUVECs; Univ-bio, C2517AS) were obtained and cultured in either RPMI-1640 or high glucose DMEM with 10% FBS (Gibco, 10099141C) and 1% penicillin-streptomycin solution (Solarbio, P1400) in an incubator, at 37°C with 5% CO₂. Those cells were then treated with 100 ng/mL lipopolysaccharide (LPS; Solarbio, L8880) to induce pre-eclampsia-like cell states and seeded in the lower chamber of Transwell (Corning, 3470), while hUC-MSCs were seeded in the upper chamber, at a ratio of 5 HTR-8/SVneo or HUVECs to 1 hUC-MSC [20, 21].

hUC-MSC effects on HTR-8/SVneo/HUVEC cell proliferation were evaluated by both colony formation assays and cell cycle analysis, while their effects on apoptosis were evaluated by Annexin V/7-Aminoactinomycin D (7-AAD) staining. The colony formation assay was carried out by seeding HTR-8/SVneo or HUVECs in the lower chamber of Transwell, at 500 cells/well, followed by treatment with 100 ng/mL LPS. hUC-MSCs were then added to the upper chamber, at a 1:5 ratio to HTR-8/SVneo or HUVECs. After 7 days of co-culture, HTR-8/SVneo or HUVECs were stained with crystal violet (Solarbio, G1063) to evaluate their clonogenic ability. For cell cycle analysis, HTR-8/SVneo or HUVECs were washed with PBS, ethanol fixed, and stained with propidium iodide (PI) dye solution containing RNAse for 30 min. PI fluorescence intensity was then measured via flow cytometry (Bioss, BA00205). For apoptosis analysis, HTR-8/SVneo or HUVECs were incubated with Annexin V and 7-AAD for 15 min in darkness, following the manufacturer’s instructions (Bioss, BA00207); the percentages of Annexin V⁺ and 7-AAD⁺ cells were quantified by flow cytometry.

To examine cell invasion capabilities, Transwell invasion analysis was carried out, in which Matrigel (BD, 356234) was diluted in serum-free medium and used to coat the bottom of the Transwell, followed by incubation overnight at 37°C. HTR8/SVneo or HUVECs were then seeded into the upper chamber at 1×10⁴ cells/well, while 500 µL complete medium with 10% FBS was added to the lower chamber. HTR8/SVneo or HUVECs were subsequently treated with 100 ng/mL LPS, while hUC-MSCs were added to the lower chamber. After 24 hr culturing, invading cells from the upper chamber were fixed with 4% PFA, stained with 1% crystal violet, and quantified by randomly selecting 5 microscope fields of view.

To measure ROS levels, a ROS detection kit was used (Beyotime, S0033M) Briefly, HTR8/SVneo or HUVECs were washed with PBS, incubated with 20 µM 2’, 7’-dichlorodihydrofluorescein diacetate (DCFH-DA) at 37°C for 45 min, washed another time with PBS, and intracellular fluorescence measured using a flow cytometer (Beckman, NAVIOS).

Detecting cell autophagy

Cell autophagy was also detected using a monodansylcadaverine (MDC)/PI staining kit, according to the manufacturer’s instructions (Beyotime, C3019M). In short, 1 mL MDC/PI staining solution was added to each well and incubated in light at 37% for 30 min; MDC selectively labels autophagosomes, while PI labels necrotic cells. Afterwards, the MDC/PI staining solution was aspirated, and wells washed 3 times with assay buffer to remove unbound dye. One mL of assay buffer was then added to resuspend the cells, and stained ones detected using the Beckman flow cytometer, at excitation/emission of 335/512 nm.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)

Total RNA was isolated from placental using Trizol reagent (Mei5bio, MF034-01), following the manufacturer’s instructions. One µg total RNA served as the template for cDNA synthesis, using the M5 Super plus qPCR RT kit with gDNA remover (Mei5bio, MF166-plus-01). The accumulation of PCR products during cycling was detected using 2X M5 HiPer SYBR Premix EsTaq (with Tli RNaseH) (Mei5bio, MF787-01), during cycling with the LightCycler® Real-time PCR System (Roche Life Science). All reactions were carried out in triplicate. Fold differences in the expression level of each gene were calculated using the 2^−ΔΔCt method and normalized to the housekeeping gene β-actin (ACTB). All primers were designed using qPCR assay design software (Invitrogen), and their sequences were provided in Table S1.

Western blots

Western blot was conducted by lysing well-grounded rat placental tissues in RIPA buffer containing phosphatase (Boster, AR1183) and protease inhibitors (Boster, AR1178), followed by incubation on ice for 30 min and centrifugation to obtain total protein extract. Total protein concentration was measured by the bicinchoninic acid method (Boster, AR0146), and 30 µg of total protein were loaded onto SDS-PAGE gel for electrophoresis. The separated proteins were transferred to PVDF membrane, which was blocked with 5% bovine serum albumin solution for 1 hr at room temperature. The membranes were subsequently incubated with the following primary antibodies overnight at 4°C: β-actin (Abclonal, AC026; 1:10000), Beclin-1 (Proteintech, 11306-1-AP; 1:1000), LC3 (Proteintech, 14600-1-AP; 1:1000), p62 (Proteintech, 18420-1-AP; 1:10000), Bax (Proteintech, 50599-2-Ig; 1:5000), Bcl-2 (Proteintech, 12789-1-AP; 1:5000), phosphorylated (p)-mTOR (Proteintech, AP0096; 1:2000), mTOR (Proteintech, 20657-1-AP; 1:5000), p-Akt (Cell Signaling Technology, 4060; 1:5000), Akt (Cell Signaling Technology, 4691; 1:5000), p-AMPK (Cell Signaling Technology, 2535; 1:1000), AMPK (Proteintech, 10929-2-AP; 1:2000). Afterwards, horseradish peroxidase-labeled secondary antibodies (Zen-bio, 511203/511103, 1:5000) were added and incubated at room temperature for 2 hr. Protein was detected using ECL luminescent substrate, imaged with an electrophoresis imaging system (Minichemi, 610, Beijing, China), and band density measured by ImageJ.

Statistical Analysis

All analysis was carried out using GraphPad Prism (9.5). All data are expressed as mean ± standard error of the mean (SEM), for at least 3 independent experiments. Comparisons between 3 or more groups were conducted with one way analysis of variance (ANOVA), while for repeated measurements over time, two way ANOVA was used, followed by Tukey's multiple comparison test. P < 0.05 is considered statistically significant.

hUC-MSCs improved fetal growth by lowering maternal blood pressure and correcting kidney function in L-NAME induced-preeclampsia rats

To determine whether hUC-MSCs had any impact on preeclampsia, we first subcutaneously injected rats with 200 mg/kg of L-NAME daily, from days 13–17 of pregnancy to induce preeclampsia, followed by tail vein injection of hUC-MSCs from days 15–19 of pregnancy (Fig. 1A). Three groups, comprising 10 rats each, were thus established: control who only received saline (Ctrl), L-NAME rats who did not receive hUC-MSC (L-NAME), and L-NAME rats who received hUC-MSC (L-NAME + MSC). After 20 days of pregnancy, fetuses, placentas, as well as maternal kidney and serum, were analysed, and representative images of fetuses and placentas from all 3 groups are shown in Fig. 1B. We found that fetal weights were significantly lower among L-NAME compared to control, while L-NAME + MSC was intermediate between the 2 (Fig. 1C). However, no significant difference between the 3 groups was present in terms of fetal survival (Fig. S2A), while a significant difference was only present between Ctrl versus L-NAME and L-NAME + MSC with respect to fetal length, where it was significantly longer in Ctrl compared to the other 2 groups (Fig. S2B). As for placental weight, it was also significantly lower among L-NAME and L-NAME + MSC, compared to Ctrl (Fig. 1D). Therefore, compared to L-NAME, there was an improvement in fetal weight among L-NAME + MSC.

Additionally, maternal blood pressure and urinary protein levels were monitored among the 3 groups, in which for both systolic and diastolic blood pressures, hUC-MSC injection alleviated the L-NAME-associated pressure increases during 15–20 days of pregnancy (Fig. 1E). With respect to urinary proteins, L-NAME had significantly higher 24 hr-urinary protein levels at day 19 of pregnancy, compared to Ctrl and L-NAME + MSC (Fig. 1F), which is likely owed to glomerular dysfunction. As observed by H&E staining, L-NAME had collapsed glomerular capillary loops, disordered structures, and enlarged renal tubule lumens, with some ruptures present. All these alterations are alleviated with hUC-MSC administration (Fig. 1G). These observations are further supported by L-NAME having significantly lower glomerular densities compared to Ctrl, which significantly increased towards that of Ctrl in L-NAME + MSC, indicating that hUC-MSCs could alleviate maternal kidney dysfunction (Fig. 1H).

Furthermore, L-NAME + MSC, compared to L-NAME, had significantly lower BUN levels, similar to that of Ctrl (Fig. 1I); a similar pattern, but without statistical significance, was present for Cr (Fig. 1J). All these findings thus suggest that hUC-MSCs improved pregnancy outcomes by lowering maternal blood pressure and improving kidney function in L-NAME induced-preeclampsia.

hUC-MSCs improved blood perfusion to the fetus in preeclampsia rats by increasing placental angiogenesis

The effects of hUC-MSCs on placental tissue in preeclampsia were analyzed by H&E staining (Fig. 2A), in which the ratios of the labyrinth/junctional zone area, as well as labyrinth/total area, are significantly lower among L-NAME rats; this lowered labyrinth area indicates lowered capabilities to ensure adequate maternal and fetal blood supply exchange (Fig. 2B). However, hUC-MSC administration restored both of those ratios towards that of Ctrl (Fig. 2B). To further examine the possible basis behind the increased labyrinth area among L-NAME + MSC, immuno-histological and immune-fluorescence staining were conducted, in which compared to Ctrl, L-NAME had significantly lower CD31⁺, representing capillaries. On the other hand, L-NAME having higher α-SMA⁺ densities, which is due to the spiral arteries in the placenta failing to undergo proper conversion to highly dilated, thin-walled vessels required for proper uteroplacental blood flow. As a result, the spiral arteries exhibited thickened vessel walls and narrowed vessel lumens, thereby resulting in higher arterial densities to compensate (Fig. 2C-D). L-NAME + MSC, though, had CD31⁺ and α-SMA⁺ densities approaching that of Ctrl (Fig. 2C-D). These observations therefore indicate that hUC-MSCs could improve blood perfusion to the fetus in preeclampsia by increasing placental angiogenesis and attenuating atherogenesis.

hUC-MSCs promoted placental angiogenic and anti-inflammatory factors, along with suppressing oxidative stress/apoptotic protein expression in preeclampsia rats

To further verify the association between hUC-MSCs and the promotion of placental angiogenesis, we examined the levels of angiogenesis-related factors in maternal blood. We found that L-NAME had significantly lower levels of pro-angiogenic factors VEGF and PIGF, which were restored towards that of Ctrl in L-NAME + MSC (Fig. 3A). On the other hand, significantly higher levels of anti-angiogenic factors sFLT-1 and sENG were present in L-NAME, which were reduced towards that of Ctrl in L-NAME + MSC (Fig. 3A).

Other processes involved in preeclampsia pathogenesis are excessive inflammation and oxidative stress. Indeed, we found that the levels of pro-inflammatory factors IL-6, IFN-γ, and TNF-α significantly increased among L-NAME; these increases, however, were reversed towards that of Ctrl among L-NAME + MSC (Fig. 3B). Conversely, the anti-inflammatory factor IL-10 is significantly lower among L-NAME versus Ctrl, while hUC-MSC administration restored IL-10 to similar levels as that of Ctrl (Fig. 3B). Similar patterns were present for oxidative stress indicators, in which the oxidative stress indicator, MDA, significantly increased among L-NAME versus Ctrl. This increase, though, was reversed towards that to Ctrl among L-NAME + MSC (Fig. 3C). On the other hand, anti-oxidative markers eNOS, NO, and SOD were significantly lower in L-NAME than Ctrl; these levels were restored towards that of Ctrl in L-NAME + MSC (Fig. 3C). All these findings regarding increased angiogenesis-related factors, as well as lowered inflammation and oxidative stress associated with hUC-MSC administration, are further supported by L-NAME placentas having increased pro-apoptotic Bax, and lowered anti-apoptotic Bcl-2 expression, compared to Ctrl (Fig. 3D-E). hUC-MSC administration, however, reversed those levels towards that of Ctrl (Fig. 3D-E). In summary, hUC-MSC was able to improve placental angiogenesis by increasing pro-angiogenic factor production, along with lowering inflammation, oxidative stress, and apoptosis.

hUC-MSCs counteracted LPS-induced detrimental effects on HTR8/HUVEC cell proliferation, migration and apoptosis in vitro

To further examine the effects of hUC-MSC on placental cells in preeclampsia, an in vitro preeclampsia model was established, where in a Transwell, HTR-8/SVneo trophoblasts or HUVECs were seeded in the lower chamber, followed by LPS administration to induce cell states similar to that observed in preeclampsia. hUC-MSCs were then seeded into the upper chamber to rescue those cells (Fig. 4A). Under the clonogenic assay, we found that LPS administration significantly lowered HTR-8 and HUVEC cloning efficiencies, reflecting lowered cell proliferation (Fig. 4B-C). These reductions, though, were restored back towards that of control after hUC-MSCs were added in the LPS + MSC group (Fig. 4B-C). This lowered cell proliferation in LPS was further correlated with flow cytometry analysis of cell cycle progression, in which LPS, compared to Ctrl, had significantly higher proportions of arrested cells, at the G1 phase, and lower proliferative S and G2 phase cells, for both HTR-8 and HUVECs. However, these proportions were more similar to Ctrl in LPS + MSC (Fig. 4D-E).

To verify whether hUC-MSC administration was also able to counteract against apoptosis in vitro, flow cytometry of Annexin-V/7AAD stained cells was carried out, in which among both HTR8 and HUVECs, LPS had significantly increased apoptotic cell counts. hUC-MSCs, though, were able to reverse those levels towards that of Ctrl in LPS + MSC (Fig. 4F-G).

We then examined the invasion capabilities of HTR8 and HUVECs, in which the bottom of a Transwell was coated with Matrigel, followed by seeding of HTR-8 or HUVECs in the upper chamber, which were treated with LPS. hUC-MSCs were seeded in the lower chamber, and the extent of cell invasion was examined after 24 hr of co-culture. We observed that compared to Ctrl, LPS-treated HTR8 and HUVECs had significantly lower 24 hr invasion rates, which increased back towards that of Ctrl in LPS + MSC (Fig. 4H-I). All these findings thus indicate that in line with in vivo findings, hUC-MSCs counteracted LPS-induced detrimental effects on HTR8/HUVEC cell proliferation, migration and apoptosis in vitro.

hUC-MSCs corrected pre-eclampsia-associated mitochondrial dysfunction by decreasing pathological autophagy

Increased apoptosis and ROS production has been associated with pathological alterations in autophagic activities. To verify whether this was present in preeclampsia, we first examined ROS levels in our in vitro model among Ctrl, LPS, and LPS + MSC groups. We found that in line with our maternal serum findings, ROS levels were significantly higher among LPS-treated HTR8 and HUVECs, which were lowered towards that of Ctrl upon hUC-MSC administration in LPS + MSC (Fig. 5A-B). We then examined the extent of autophagy among the 3 groups, in which LPS also had significantly higher MDC levels, indicating increased autophagy, among both HTR8 and HUVECs, compared to Ctrl; these levels, though, were lowered towards that of Ctrl in LPS + MSC (Fig. 5C-D). To further verify these observations in vivo, TEM analyses of placental mitochondrial ultra-structures were conducted, in which compared to Ctrl, L-NAME-treated pre-eclamptic rats had mitochondrial swelling and reduced cristae numbers. These pathological alterations, though, are partially reversed upon hUC-MSC administration (Fig. 5E). Furthermore, the density of normal-appearing mitochondria was significantly lower, and autophagosome density higher, in L-NAME compared to Ctrl, while L-NAME + MSC levels for both densities were reverted towards that of Ctrl (Fig. 5F-G). All these findings indicate that pre-eclampsia increased autophagy, which is linked to elevated oxidative stress and mitochondrial dysfunction. By contrast, hUC-MSC administration alleviated mitochondrial dysfunction by decreasing ROS and autophagy.

hUC-MSCs lowered pathological pre-eclamptic placental autophagy by activating Akt/mTOR and inhibiting AMPK/mTOR pathways

To further investigate the ability of hUC-MSC administration to lower pathological placental autophagy, relative mRNA and protein expression levels were measured for various autophagy-related mediators. We found that mRNA expression of pro-autophagic LC3 and Beclin1 significantly increased (Fig. 6A-B), and anti-autophagic P62 decreased (Fig. 6C), in L-NAME versus Ctrl. These changes were reverted towards that of Ctrl upon hUC-MSC administration (Fig. 6A-C). With respect to protein, Western blot analyses similarly found that the elevated levels of LC3 II/I ratio and Beclin1 in L-NAME was decreased upon hUC-MSC treatment (Fig. 6D-E). On the other hand, P62 protein level was significantly higher in L-NAME + MSC compared to L-NAME (Fig. 6D-E).

The mTOR-regulated signaling pathway is crucial for cell invasion and vascular remodeling, along with being involved in placental ischemia and hypoxia. To investigate if this pathway is responsible for the increased pathological autophagy in pre-eclampsia, RT-qPCR was first used to measure mRNA expression levels for mTOR pathway components mTOR, AMPK, and Akt. The results showed that mTOR and Akt mRNA levels were significantly lower in L-NAME but were restored around Ctrl levels in L-NAME + MSC (Fig. 6F-G). Conversely, AMPK expression was significantly higher in L-NAME than the other 2 groups (Fig. 6H). To determine whether these alterations were also present at the protein level, Western blot was conducted, in which the p-mTOR/mTOR and p-Akt/Akt ratios were significantly lower in L-NAME but increased to levels similar to Ctrl in L-NAME + MSC (Fig. 6I-J). The opposite, though, was the case for p-AMPK/AMPK, where expression levels were significantly higher in L-NAME than Ctrl and L-NAME + MSC (Fig. 6I-J). All these analyses thus suggested that hUC-MSCs were able to lower pathological pre-eclamptic placental autophagy, via activating Akt/mTOR and inhibiting AMPK/mTOR pathways.

The treatment of preeclampsia poses a significant challenge, as no effective treatment strategy, aside from inducing the delivery of the baby and placenta, is currently present [5, 22]. As a result, treatment strategies that could alleviate this condition, without harming the fetus, are necessary. One potential strategy is hUC-MSCs, as they are multipotent, self-renewing, and have been observed in previous studies to be able to treat pre-eclamptic symptoms [23–25].

However, the precise effects and underlying mechanisms involved in this treatment are still largely undefined [26]. In this study, we aimed to further confirm the therapeutic capabilities of hUC-MSCs for pre-eclampsia, as well as to identify the underlying mechanisms. We developed both an in vivo L-NAME rat model, as well as an in vitro HTR8 trophoblast/HUVEC cell model with LPS stimulation to simulate pre-eclampsia. In the in vivo model, we found that hUC-MSC administration lowered blood pressures and urinating protein levels, along with improving renal functions among pre-eclamptic L-NAME rats; these improvements, in turn, led to the alleviation of pre-eclamptic symptoms and increased fetal weights. All of these hUC-MSC-linked improvements likely stem from hUC-MSC treatment promoting the expression of pro-angiogenic factors, such as PIGF and VEGF, as well as anti-inflammatory factor IL-10; these were coupled with oxidative stress and apoptosis being suppressed, as detected in maternal serum. As a result, increased placental angiogenesis was present in hUC-MSC-treated L-NAME rats, as indicated by increased CD31⁺ and decreased α-SMA⁺ densities in the labyrinth area. These in vivo findings were further reinforced by in vitro results, where co-culture of hUC-MSC with LPS-treated trophoblasts/HUVECs counteracted against the negative effects of LPS to increase cell proliferation and invasion, along with lowering apoptosis. The positive impact of hUC-MSCs originates from them improving placental mitochondrial function by lowering autophagy, via downregulating pro-autophagic LC3 and Beclin1, along with upregulating anti-autophagic P62, which are owed to the activation of Akt/mTOR and inhibition of AMPK/mTOR pathways. Overall, hUC-MSCs were able to treat pre-eclampsia by reducing pathological autophagy in the placenta, which were coupled with lowered oxidative stress, inflammation, and apoptosis.

The L-NAME-induced preeclampsia rat model has been a widely applied animal model to examine this disease. We found that daily L-NAME administration for 5 days could replicate pre-eclampsia-associated hypertension and proteinuria symptoms, which peak on the 20th day of pregnancy. However, after injecting hUC-MSCs, these hypertensive and proteinuria symptoms were significantly relieved, which, along with increased fetal weights, indicated that this cell therapy could improve maternal health and pregnancy outcomes. Indeed, our findings are in line with Xiong et al., who showed that MSC treatment could reduce blood pressure and proteinuria, as well as improve fetal weight, in pre-eclampsia [15]. This improvement of pre-eclamptic symptoms is likely owed to a series of coordinated events to alleviate placental damage, including increased angiogenesis and cell proliferation, along with lowering inflammation and oxidative stress [11].

Insufficient trophoblast invasion and impaired spiral artery remodeling, which results in lowered placental blood perfusion, have been previously identified as important mechanisms in pre-eclampsia development [2]. Therefore, promoting angiogenesis could serve as a potential approach for alleviating pre-eclampsia. Indeed, our histological analysis confirmed that hUC-MSCs were able to promote placental blood perfusion, via promoting placental capillary development and spiral artery remodeling, as demonstrated by increased CD31⁺ and decreased α-SMA⁺ densities. This may be due to hUC-MSCs providing paracrine support by secreting pro-angiogenic factors, such as VEGF. Subsequently, these pro-angiogenic factors promoted cell proliferation and invasion, as shown in our in vitro model, where there was increased HTR8/SVneo and HUVEC cell proliferation and invasion, along with lowered cell apoptosis; all these processes contributed to the overall improvement in placental angiogenesis.

As for inflammation and oxidative stress, their overactivity in the second stage of pre-eclampsia have been identified as key factors that exacerbate its symptoms, such as hypertension, proteinuria, and endothelial dysfunction [27]. In fact, L-NAME rats were found to have increased levels of pro-inflammatory IL-6, IFN-γ, and TNF-α, as well as MDA at the 20th day of pregnancy. These increases in inflammation, though, have been found in a previous study, using an LPS-induced pre-eclamptic animal model, to be alleviated by MSCs, owing to them suppressing pro-inflammatory cytokine expression [28]. Our findings, though, suggest that the anti-inflammatory effects of hUC-MSCs are more owed to increased anti-inflammatory cytokine production, such as IL-10, rather than inhibition of pro-inflammatory cytokines, which agrees with a previous study reporting that MSCs, by promoting IL-10 production and macrophage polarization, exerted beneficial effects on wound healing [29]. Therefore, hUC-MSCs may also exert their anti-inflammatory effects via promoting anti-inflammatory factor production to achieve favourable macrophage polarization.

In terms of possible underlying mechanisms, we found that hUC-MSCs restored placental mitochondrial morphologies, possibly by lowering pro-apoptotic Bax levels, along with increasing antioxidant enzyme production, such as SOD and NO to reduce ROS. This was further confirmed in vitro, in which hUC-MSCs lowered ROS production and apoptosis among LPS-treated HTR8/HUVECs. The restoration of placental mitochondrial function may be owed to the restoration of proper autophagic homeostasis. Autophagy is an important eukaryotic catabolic system that plays a key role in regulating cellular homeostasis and physiology [8, 30]. In fact, our study suggests that hUC-MSCs protect the placenta by attenuating the level of autophagy dysregulation. Physiological levels of autophagy clears damaged organelles and misfolded proteins to provide the cells with proper nutrients and energy, while low physiological levels of ROS enables cells to adapt to inflammation and oxidative stress, along with stimulating angiogenesis. However, excess autophagy and ROS are detrimental to cells. With respect to our study, we found that hUC-MSC treatment significantly reduced pro-autophagic LC3 and Beclin1, along with increasing anti-autophagic P62 expression, thereby reducing autophagic activities back towards that of physiological levels to maintain placental homeostasis. This regulation of autophagic activity involves the downregulation of the AMPK/mTOR pathway, which is consistent with the studies of Xu et al. and Yang et al., in which its overactivation inhibited trophoblast invasion [31, 32], thereby exacerbating pre-eclamptic manifestations; this indicated that AMPK inhibition played a protective role in the placenta. This downregulation of AMPK/mTOR is coupled with upregulation of Akt/mTOR signaling pathways, both of which, as upstream signals of autophagy, thereby co-ordinate with each other to maintain proper autophagic activity for cellular homeostasis. Based on our observations, these data indicated that hUC-MSCs can regulate autophagy in damaged placental tissue to maintain energy supply and cellular homeostasis. However, placental damage pathogenesis in preeclampsia is complex, involving the interaction of multiple factors, such as dysregulated angiogenesis and autophagy, as well as aggravated inflammatory responses and enhanced oxidative stress. Therefore, future studies will examine additional potential mechanisms contributing to the beneficial effects of MSCs, which may involve paracrine release of cytokines, growth factors, exosomes, and other bioactive substances to exert reparative effects [33–35].

In this study, to simulate pre-eclampsia, both an in vivo L-NAME pregnant rat model, as well as an in vitro HTR8 trophoblast/HUVEC cell model with LPS stimulation, were established. We found that in vivo, hUC-MSC administration improved maternal pre-eclamptic symptoms, particularly in terms of lowering blood pressure and improving kidney function, as well as increasing fetal weights. These improvements are owed to hUC-MSCs bolstering pro-angiogenic and anti-inflammatory factor production, along with lowering oxidative stress and apoptosis, which was further supported by the in vitro model. All of these activities for alleviating pre-eclampsia were further identified as stemming from hUC-MSCs being able to restore physiological placental cell autophagic levels to improve mitochondrial function. This restoration is through activating Akt/mTOR and inhibiting AMPK/mTOR pathways, resulting in pro-autophagic LC3 and Beclin1 being down-regulated, while anti-autophagic P62 is up-regulated. All these observations, both in vivo and in vitro, thus demonstrate that hUC-MSCs could serve as a potential cell therapy for treating pre-eclampsia.

Acknowledgements

We acknowledge the resources provided by the Departments of Obstetrics and Otolaryngology, Head & Neck Surgery at the First Clinical College of Shanxi Medical University. We also thank Alina Yao for her assistance in manuscript preparation and editing.

Author contributions

Miao Xu, Huijing Ma, Yuwen Chen, Xinhuan Zhang, and Mengnan Li designed and conducted the experiments, and were aware of the group allocation at the different stages of the experiment, as well as writing the paper. Hong Yu, Jing Ji, Juanwen Li, Nan Zhang, Fang Wang, Huiniu Hao, Lu Li, Yinmin Chen, Lijun Yang, and Zhuanghui Hao participated in data collection and analyses; they were blinded to the group allocation. Huifang Song,Sheng He and Hailan Yang revised the paper and provided funding support.

Funding

This work was supported by the Science and Technology Innovation Base Project Construction Task of Shanxi Province (#YDZJSX2022B010,YDZJSX2021B008), Basic Research Program of Shanxi Province(202303021221214),National Key Clinical Specialty Construction Project of Shanxi Province (#Y2022ZD001/2,2024-ZZ-001/010/011), Shanxi Province ten billion Project (#2C622024092),Pilot Base Construction Funding of Shanxi Province (#2023-167-15), Fund Program for the Scientific Activities of Selected Returned Overseas Professionals in Shanxi Province (#20240044), and Four “Batches” Innovation Project of Invigorating Medical through Science and Technology of Shanxi Province (#2023XM031).

Data availability

All relevant data are included in the article and its supplementary materials or are available upon request.

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing interests to declare.

AI used declaration

The authors declare that they have not use AI-generated work in this manuscript.

Ethics approval and consent to participate

All procedures were approved by the Research Ethics Board of the First Hospital of Shanxi Medical University.the approved project’s title is miRNA-23b-5p regulates TRAIL intervention in MSCs expression and significance in post-preeclampsia rats(REB#: 2022-K-K0247) ,The ethical approval date was Oct,18,2022.Human samples were collected from the First Hospital of Shanxi Medical University, Taiyuan, China. Written informed consent was obtained from all patients.

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Human umbilical cord mesenchymal stem cells restores mTOR-mediated autophagy homeostasis to alleviate placental injury and improve pregnancy outcomes in preeclampsia

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Abstract

Background

Methods

Results

Conclusion

Figures

Introduction

Materials and Methods

Establishing the pre-eclampsia rat model

Measuring blood pressure, urine protein levels, and enzyme-linked immunosorbent assay (ELISA) among pre-eclamptic rats

Histological analyses and transmission electron microscopy (TEM) of pre-eclamptic rat tissues

Detecting cell autophagy

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)

Western blots

Statistical Analysis

Results

hUC-MSCs improved blood perfusion to the fetus in preeclampsia rats by increasing placental angiogenesis

hUC-MSCs counteracted LPS-induced detrimental effects on HTR8/HUVEC cell proliferation, migration and apoptosis in vitro

hUC-MSCs corrected pre-eclampsia-associated mitochondrial dysfunction by decreasing pathological autophagy

hUC-MSCs lowered pathological pre-eclamptic placental autophagy by activating Akt/mTOR and inhibiting AMPK/mTOR pathways

Discussion

Conclusion

Declarations

References

Supplementary Files

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