Human Umbilical Cord Mesenchymal Stem Cell (HUCMSC)-derived Exosomes as a Cell-Free Therapy for Soluble Fms-like Tyrosine Kinase-1 (St-1)-induced Endothelial Dysfunction in Preeclampsia

Preeclampsia is a unique multisystem disorder that affects 5-8% of pregnancies. A high level of soluble fms-like tyrosine kinase-1 (sFlt-1) is a hallmark of preeclampsia that causes endothelial dysfunction. Exosomes derived from mesenchymal stem cells (MSCs) have been indicated to improve endothelial performances by transporting signals to target cells. We hypothesized that exosomes derived from MSCs have potential effects against preeclampsia. The and morphology of the were examined using nanoparticle to the preeclampsia-like mouse cells were tet-on-sFlt-1 sFlt1. Cell proliferation and migration assays were to measure functions. The exosomes enriched proteins underlying mechanisms were explored by proteomic analysis.


Introduction
Preeclampsia (PE) is characterized by new-onset hypertension at ≥ 20 weeks of gestation with or without proteinuria [1]. This gestation-speci c syndrome is a serious pregnancy complication that remains one of the main causes of maternal, fetal, and neonatal mortality globally and is also related to intrauterine growth retardation (IUGR) and preterm birth [2]. PE is primarily a consequence of an imbalance between proangiogenic and antiangiogenic growth factors, such as soluble fms-like tyrosine kinase-1 (sFlt-1), which causes multisystem endothelial dysfunction [3]. Women who are diagnosed with PE have decreased quality of life and elevated risk of postpartum depression.
Mesenchymal stem cells (MSCs) are de ned as multipotent adult cells with the ability to differentiate into multiple cell types. Human umbilical cord MSCs (HUCMSCs) have become a prominent stem cell type used for allogeneic cell-based therapy because of their advantages in ethical access and rapid renewal properties [4]. In addition, HUCMSCs are considered nonimmunogenic because they express low levels of major histocompatibility complex-II (MHC-II) and have therefore emerged as one of the most promising treatment options for vascular diseases [5]. HUCMSCs have been under investigation in a variety of clinical therapeutic trials including cardiovascular de cits, recovering ovarian function, and immune system diseases. Our previous work indicated that HUCMSCs provided a new avenue for treating placenta-related diseases during pregnancy like PE[6], however, the underlying molecular mechanism remains unknown.
A large body of evidence indicates that HUCMSCs secrete extracellular vesicles containing a number of functional molecules. Exosomes are nanosized extracellular vesicles (30-150 nm) of endocytic origin that are released by most cells [7]. They are cup-shaped phospholipid nanocarriers functioning as transmitting amounts of bioactive molecules for intercellular communication. Exosomes have been well studied in angiogenesis, and our previous research indicated that exosomes from PE patients induced endothelial dysfunction by transferring high levels of sFlt-1 and soluble Endoglin to human umbilical vein endothelial cells (HUVECs) [8]. Therapies with the potential to prevent PE progression or reverse diseaseinduced organ damage are lacking; thus, the pursuit of a treatment option to replace expectant management and instant delivery needs to be investigated. Recently, HUCMSC-derived exosomes (HUCMSC-exos) showed high translational value in anti-aging intervention by enhancing the regenerative capacities in bone formation, wound healing, and angiogenesis [9]. In addition, HUCMSC-exos have been recognized as new candidates for the treatment of vascular diseases because they are selectively enriched in proangiogenic molecules [10]. However, the roles and underlying mechanisms of HUCMSCexos in the treatment of reproductive complications such as PE are only beginning to be understood and appreciated.
Here, we provide new evidence that HUCMSC-exos have the ability to reverse sFlt-1-induced PE-like adverse birth outcomes in vivo. In addition, exosomes can facilitate sFlt-1-imparied endothelial dysfunction in HUVECs. The proteomic analysis revealed that HUCMSC-exos were highly enriched in the proteins involved in regulation of cell communication, cell migration, and angiogenesis, as well as vascular endothelial growth factor receptor signaling pathway. Based on the improvements brought about by HUCMSC-exos treatment in terms of vascular functions and birth quality in PE-like mice model, we propose that HUCMSC-exos may represent a new approach to treat or prevent PE.

Isolation of HUCMSCs
Human umbilical cord tissues were collected from full-term, healthy cesarean births and were donated by consenting mothers at the Department of Obstetrics of Shanghai First Maternity and Infant Hospital from 2018 to 2019. The demographic and clinical characteristics of the participants in this study were reported in our previous study [11]. Umbilical arteries and veins were removed, and the remaining Wharton's jelly was transferred to sterile, ice-cold Hank's balanced salt solution (HBSS, Gibco, Thermo Fisher Scienti c, Inc., Waltham, MA, USA) containing antibiotics (streptomycin/penicillin, Gibco). The tissues were washed multiple times with HBSS to remove excess blood and were subsequently cut into small pieces. The pieces were transferred to 10 cm 2 dishes with 5 mL of minimum Eagle's medium alpha (Gibco) containing 10% fetal bovine serum (FBS, Gibco) and 1% streptomycin/penicillin and were incubated at 37°C in 5% CO 2 . The explants were left undisturbed for 7-10 days to allow cell migration, and the medium was changed every three days. After 2 weeks, the HUCMSCs (passage 0) were grown to 60-70% con uence and passaged by trypsinization. Sample collection was approved by the Research and Ethics  For osteogenic differentiation, passage 3 cells were cultured in differentiation medium (Gibco) for 3 weeks in six-well plates. After the cells were xed with 4% paraformaldehyde (PFA) and stained with alizarin red S (Sigma-Aldrich, Merck, Darmstadt, Germany), the cells were observed by microscopy (Nikon Eclipse Ti, Tokyo, Japan).

Isolation and characteristics of HUCMSC-exos
HUCMSCs at passages 3-6 were cultured in FBS-free medium with 2% bovine serum albumin (BSA, Sigma-Aldrich) for 48 h, and then 500 mL of the supernatant was centrifuged at 3,000 g for 10 min to remove cell debris and passed through a 0.22-µm lter. The cleared supernatant was ultracentrifuged at 110,000 g for 70 min and washed in PBS using the same ultracentrifugation conditions to isolate exosomes. Exosome pellets were suspended in 5 mL of PBS and stored at -80 °C.
The size and morphology of the exosomes were examined using a transmission electron microscope (TEM) at the Laboratory of Electron Microscopy (Chinese Academy of Sciences Shanghai, China). The size distribution of HUCMSC-exos was determined using nanoparticle tracking analysis (NTA, ZetaView, Particle Metrix, Meerbusch, Germany). These procedures were performed as previously described [8].

Mice
All mouse experiments were approved by the Department of Laboratory Animal Science, Tongji University. Adult male (n=40) and female (n=80) CD-1 (ICR) mice were purchased from Jackson Laboratories. Animals were housed in a temperature-and humidity-regulated environment with a 12-h light cycle. For a consistent and accurate assessment of the gestational age of mouse embryos, male and female (1:2) mice were pair-housed for one night. Embryonic day 0.5 (E0.5) was designed as the rst morning at which a vaginal plug was noted.
Tissue preparation for histological analysis Placental and kidney tissues were xed with 4% PFA for 48 h and processed by conventional procedures. Sections 3-5 μm in thickness were cut from the para n-embedded tissues, mounted on poly-L-lysinecoated slides, depara nized in xylene, dehydrated in alcohol and then stained with hematoxylin and eosin (H&E) or periodic acid-methenamine silver (PAS) stain.

Immunohistochemical (IHC) analysis of mouse placental tissues
For IHC analysis, para n sections were depara nized and incubated with citrate buffer for antigen retrieval. The slides were then incubated with rabbit anti-CD31 polyclonal antibody (1:100, Abcam, Cambridge, MA, USA) overnight at 4 °C and developed using the ImmPRESS horseradish peroxidase (HRP) anti-rabbit IgG polymer detection kit (Weiao, Shanghai, China).

Morphological analysis
Using CD31 as an endothelial marker, the densities, diameters, and areas of fetal blood vessels were analyzed as previously described [13]. Four images per tissue section were taken using a Nikon inverted microscope with a 40× objective (vascular density) or 100× objective (vascular diameter). For each image, the number of capillaries was counted, and the lumen area was measured using ImageJ imaging analysis software (NIH, Bethesda, MD). Five capillaries per image were randomly selected for diameter measurements by CaseViewer software (3DHISTECH, Ltd., Hungary).

ELISAs
ELISAs for mouse sFlt-1 and sEndoglin were performed according to the manufacturer's instructions (R&D Systems). Urine albumin/creatinine ratio was measured using Urinary Albumin and Creatinine Assay kits (Abnova, Taipei City, Taiwan, China). Brie y, the various samples were diluted 1:2 in dilutions and were incubated in a 96-well plate pre-coated with capture antibodies. The wells were washed and incubated with a secondary antibody conjugated to horseradish peroxidase. Then substrate solution was added, and optical density was determined at 450 nm. The concentrations were calculated using a standard curve of the respective recombinant proteins.

Western blot
Total protein concentration was measured using the bicinchoninic acid assay (Pierce®, Thermo Fisher Scienti c, Bonn, Germany). Western blot for expressions in exosomes were performed using CD63 Cell culture The HUVECs were isolated from 3 individual donors by a standard collagenase enzyme digestion method and cultured steadily in Endothelial Cell Medium (ECM, ScienCell, San Diego, CA) containing 5% FBS, 1% P/S and 1% ECGS.

Tet-One induced sFlt-1 expression in HUVECs
The human sFlt-1 (ID: NM_001159920.1) cDNA was cloned into Lenti-X Tet-One System expression vector (Clontech, Moutain View, CA) as described previously. The recombinant lentivirus tet-sFlt-1 and the negative control lentivirus (NC-lentivirus; Hanyin Co. Shanghai, China) were prepared and titered to 10 9 TU/ml (transfection unit). HUVECs were infected with lentiviruses expressing Tet-on-sFlt-1 to obtain cells overexpressing sFlt-1 only at the time when tetracycline existing. HUVECs expressed the sFlt-1 in the cellular background with doxycycline (Dox) and the e ciency of overexpression was examined by western blot analysis.

Cell proliferation and migration assays
Cell proliferation and migration ability was performed using modi ed systems as described previously [15]. In brief, 3 x 10 3 cells/well were plated in 96-well plates, later Dox or the vehicle were added to induce overexpression of sFlt-1. After 24 h, HUCMSC-exos with different doses were added. Then cell numbers were assessed using the cell counting kit-8 (CCK-8) at 450 nm. For migration, HUVECs were plated in 6-well plates and treated with Dox or the vehicle to induce overexpression of sFlt-1. Then 3 x 10 4 cells were seeded into the upper chambers and exosomes (100 µg/mL) or NS were added into the lower chambers. Following incubation for 16 h, uorescent stain (calcein-AM) was added to each lower chamber. The migrated cells were counted by uorescence analysis (Nikon, Tokyo, Japan).

Proteomic analysis
The HUCMSCs-exo and HUCMSCs samples were processed for tandem mass tag (TMT)-based quantitative proteomic analysis by Lu-Ming Biotech Co., Ltd. (Shanghai, China). High-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) was used to compare the proteomic content of HUCMSCs-exo and cells. Differentially expressed proteins were identi ed with a cutoff of absolute fold change ≥ 2 and P-value 0.05 Statistics All data were expressed as the mean ± the standard error of the mean (S.E.M) and analyzed using the SPSS 23.0 statistical analysis software (SPSS Inc., Chicago, IL, USA). Statistical signi cance was determined by performing paired Student's t-test, one-way analysis of variance (ANOVA) and Dunett's post-hot test. A p-value < 0.05 was considered statistically signi cant.

Isolation and characterization of HUCMSC-exos
We isolated HUCMSCs from human umbilical cord Wharton's jelly using the tissue adherence method [16].
Observation under an inverted microscope showed that the HUCMSCs (passage 0) had long fusiform shapes ( Figure 1A). After osteogenic induction, alizarin red staining showed multiple calcium nodules in the cells, which indicated that the cells had well-developed osteogenic differentiation functions ( Figure  1B). Flow cytometric analysis showed that the HUCMSCs were positive for CD73, CD90 and CD105 but lacked the hematopoietic marker CD45 (Figure 1C), which was consistent with the characteristics of MSCs.
The characteristics of HUCMSC-exos isolated and puri ed using a well-established ultracentrifuge method are presented [17]. Our hypothesis was supported by transmission electron microscopy analysis, which showed a cup shape ( Figure 1D). NTA identi ed particles with diameters of 30-150 nm ( Figure 1E). Furthermore, exosomes were positive for the exosomal protein markers CD63, CD81 and CD9 ( Figure 1F). These results indicated that we successfully isolated HUCMSC-exos.

HUCMSC-exos ameliorated sFlt-1-induced PE-like adverse birth outcomes in pregnant mice
We rst established a widely used PE-like mouse model by injecting 6×10 8 pfu of the recombinant sFlt-1expressing adenovirus into pregnant ICR mice via the tail vein on E8.5. In accordance with our previous ndings, exosomes from human uids can enter murine organs[8], thus, HUCMSC-exos were administered to examine the potential of exosomes in treating PE. Pregnant mice were injected with exosomes or diluent on E6.5, E9.5, E12.5 and E15.5 (Figure 2A). Hypertension is the most common diagnostic sign of PE, then blood pressure response in the sFlt-1-induced preeclampsia model was examined and showed in Figure 2C. The sFlt-1-injected mice had both increased systolic and diastolic blood pressure. No signi cant differences were observed among the CTL and EXO groups, while dams treated with both sFlt-1 and HUCMSC-exos remained nearly normotensive on E18.5 relative to that of dams treated only with sFlt-1. SFlt-1 injection induced the production of sFlt-1 and increased its serum concentration in mice ( Figure 2E, sFlt-1 vs CTL, P < 0.01) without affecting sEndoglin (sEng) levels. Circulating sFlt-1 concentrations were slightly decreased by exosome treatment (sFlt-1 vs EXO + sFlt-1, P = 0.13).
The effects of exosomes on PE-induced IUGR were evaluated, and the observed decrease in body weight ( Figure 2B) was most likely caused by small-sized fetuses and placentas rather than a reduction in the number of fetuses ( Figure 2F). Fetuses that survived to term had decreased weights and were small in the sFlt-1-treated group, and exosomes prevented this growth restriction in fetuses and placentas ( Figure 2F). The urine albumin/creatinine ratio (ACR) was slightly increased after sFlt-1 injection, but the difference was not signi cant ( Figure 2D, P = 0.19). These results suggest that HUCMSC-exos signi cantly reduce the IUGR that occurs in the fetuses of dams exposed to sFlt-1. Therefore, HUCMSC-exos have the potential to treat PE by improving sFlt-1-induced hypertension and IUGR in mice.

HUCMSC-exos improved placental vascular development in PE-like mice
The sFlt-1 virus-treated mice showed retarded fetal development, which is driven by impaired placental feto-maternal exchange. After exchange of gases and nutrients between the maternal and fetal circulation in the maze of labyrinth (La), deoxygenated blood then moves to the junctional zone (JZ) [18].
Thus, we examined the histology of the placentas, including the La and JZ by H&E and IHC staining ( Figure 3A). The total placental area did not change among the four groups ( Figure 3B), while the thickness of the La layer was notably reduced in sFlt-1-injected mice and rescued by HUCMSC-exo ( Figure  3C-D). Then, we analyzed placental vascularization by staining for CD31, which speci cally binds to endothelial cells. High magni cation views showed extensive vascular damage in the La, which indicated impaired blood ow, in the sFlt-1 group. Importantly, in EXO+sFlt-1 mice, there was a partial reversal of the vascular narrowing observed in placentas from sFlt-1 mice, as indicated by increased lumen diameters and areas ( Figure 3E). The number of lumens per image in the exosome-treated group relative to the sFlt-1 group was slightly increased but was not signi cant ( Figure 3F). Quanti cation of vascular diameter after CD31 staining showed that this decreased from 22.1 ± 1.95 μm in CTL mice to 14.45 ± 1.64 μm in sFlt-1 mice (P < 0.01) and reversed to 21.6 ± 1.44 μm in EXO+sFlt-1 mice (P < 0.01) ( Figure 3G). Furthermore, the fetal vascular area (%) was reduced in sFlt-1 mice (19.49 ± 0.71 vs 25.23 ± 1.38, p < 0.001) and increased to 22.75 ± 1.4 in EXO+sFlt-1-treated mice (p=0.06) ( Figure 3H). There was no signi cant difference in the vascular number, diameter, or area between EXO and CTL mice. Neither glomerular endotheliosis nor mesangial expansion, which are characteristic features of PE [19], was present in any group, as determined by silver staining (Figure 3I).

In vitro proangiogenic effects of HUCMSC-exos
A substantial body of evidence indicates that PE in humans is a consequence of vascular endothelial damage, resulting in multiorgan dysfunction. Therefore, we chose HUVECs as the cell model for studying the effects of HUCMSC-exos on endothelial dysfunction in PE. The Tet-On-sFlt-1 Flag HUVEC model was established to simulate vascular endothelial cells in women with PE. In the absence of Dox, negative control (NC) HUVECs did not express any detectable sFlt-1 protein, and following induction with different doses of Dox, strong signals for sFlt-1 were observed in sFlt-1-overexpressing (OV-sFlt-1) cells, as measured by western blotting ( Figure 4A). HUCMSC-exos labeled with the uorescent dye Dil were incubated with NC and OV-sFlt-1 HUVECs for 24 h, and most recipient cells were positive for Dil uorescence and showed no difference among the groups ( Figure 4B).
A series of cellular functional analyses were performed, and we rst demonstrated that HUCMSC-exos are capable of facilitating angiogenesis because they contain many proangiogenic components. OV-sFlt-1-HUVEC proliferation and migration were signi cantly inhibited, and these attenuated angiogenetic behaviors were reversed after incubation with HUCMSC-exos ( Figure 4C-D). It is suggested that high levels of circulating sFlt-1 lead to suppression of eNOS signaling pathway, which, in turn, enhances sensitivity to vasoconstrictors that induces maternal hypertension in PE [20]. Therefore, we examined the protein expression of eNOS in HUVECs and as expected, found that OV-sFlt-1-HUVECs exhibited decreased eNOS protein expression, which was partially rescued in the presence of HUCMSC-exos ( Figure  4E). These ndings suggest that HUCMSC-exos have the potential protective effects on sFlt-1-induced endothelial and eNOS dysfunction.

Proteomic analysis of HUMSC and HUCMSC-exos
HUCMSC-exos modulated sFlt-1-induced endothelial dysfunction and improved placental angiogenesis in PE-like mice. It was therefore decided to investigate how exosomes enhanced vascular angiogenesis. After successfully extracting proteins from exosomes and parallel cells, proteomics analysis using tandem mass tag (TMT) technology was performed to detect protein expression pro les. Unsupervised hierarchical clustering analysis was used to generate a heat map of the differentially expressed proteins, and a total of 568 proteins were differentially expressed between HUCMSC-exos and HUCMSCs using the cutoff value of a 2-fold change and a P value < 0.05 ( Figure 5A). On the basis of gene ontology (GO) enrichment analysis, both HUCMSC and HUCMSC-exos displayed functional enrichment in biological processes related to vesicle-mediated transport, cell communication, cell migration, and angiogenesis ( Figure 5B). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed the oxidative phosphorylation, focal adhesion, endocytosis, and extracellular matrix-receptor (ECMreceptor) were enriched pathways ( Figure 5C). We next analyzed the differentially expressed genes (DEGs) between these two chambers. Compared with HUCMSCs, the up-expressed proteins in HUCMSCexos were MMP2 (Matrix metalloproteinase 2, fold change = 14.7) and VCAN (versican, fold change = 11.7). Previous study showed that MSC-exo could promote the migration of endothelium cells by delivering MMP2 [21]. VCAN contributes to tissue development and maintenance by participating in cell adhesion, proliferation and angiogenesis via binding to ECM components [22]. Then, we veri ed that VCAN was obviously upregulated in HUCMSC-exos compared with cell lysates (Data were not shown).

Discussion
PE is a unique multisystem disorder that leads to maternal and fetal morbidity and mortality. Therapeutic strategies aimed at reducing blood pressure and improving fetal birth outcomes are being pursued as avenues to prevent PE [23]. In the current study, we showed that HUCMSC-exos provided therapeutic bene ts in animal models of PE by improving birth outcomes and vascular endothelium functions.
Compelling evidence indicates that MSCs and their derived exosomes can be used in many vascular dysfunction-related diseases. Due in part to studying the paracrine effects of stem cell therapy, this speci c therapy has been mechanistically linked to the inherited functions of secreting exosomes [24].
Recently, research suggested HUCMSC-originated exosomes might be an innovative direction for therapeutic approaches against PE by accelerating trophoblast cell invasion and migration [21,25].
Despite the therapeutic effects observed, however, the underlying molecular mechanism, especially how the vascular is regulated, remains unclear. In the current study, we focused on HUCMSC-exos as potential cell-free therapeutics for the treatment of PE by improving angiogenesis. We chose a well-established mouse model of PE that mimics many features of the human pregnancy disease, including upregulated blood pressure and serum sFlt-1 concentration, and is established by a single injection of sFlt-1 adenovirus [20]. Administration of HUCMSC-exos during pregnancy prevented the development of sFlt-1induced preeclamptic complications, decreasing blood pressure and improving fetal and placental weights.
Modi ed vascular responses are prevented in hypertensive pregnancy complications due to an imbalance in angiogenetic and antiangiogenetic factors. Circulating sFlt-1 levels are considered a diagnostic and prognostic marker of PE, and sFlt-1 is a clinically promising therapeutic target for this disease. In our mouse model, administration of HUCMSC-exos slightly decreased the serum sFlt-1 concentration.
Insu cient placental vascularization and reduced blood ow to the feto-placental unit resulted in impaired fetal growth in PE. Placental vascular network density in the labyrinth zone of sFlt-1-treated mice was sparse, and the fetal sinusoids were narrow. The mice treated with HUCMSC-exos had improved fetal and placental weight and size as the result of a decrease in the diffusion distance between the fetal and maternal blood supplies that provided enough nutrition for the developing fetus. Widespread vascular damage is an invariable nding in PE[26], and we indicated the addition of HUCMSC-exos recused cell proliferation and migration abilities of OV-sFlt-1-HUVECs in vitro. NO-mediated reductions in blood pressure are crucial, and decreased NO bioavailability can lead to hypertension by enhancing vascular oxidative stress and endothelial dysfunction. OV-sFlt-1-mediated interference in eNOS expression in HUVECs could be reversed by exosomes. Thus, HUCMSC-exos may serve as a novel medication that rescues endothelial dysfunction in treating PE.
Exosomes have therapeutic potential owing to their roles as carriers to deliver nucleic acids and proteins between human body systems. In addition, genetic or molecular engineering of exosomes can improve target speci city and anti-disease activity with less toxicity. Based on our proteomic analysis, a better understanding of the molecular and cellular properties of HUCMSC-exos contributes to their therapeutic potential as innovative drug delivery systems. Exosomal MMP2 derived from mature osteoblasts was previously demonstrated to promote endothelial angiogenesis via VEGF/Erk1/2 signaling pathway [27]. VCAN, which highly expressed in metabolically active tissues can mediate angiogenesis possibly depends upon interactions with VEGF and in uence the assembly of the ECM [28]. Upon uptake by the vascular endothelium, these proteins accumulated in HUCMSC-exos promoted cell proliferation, migration and angiogenesis to rescue damaged vascular tissues in preeclamptic-like mice.
The colors from green to black and to red represent the expression values of the differentially expressed