Co‐culture with chorionic villous mesenchymal stem cells promotes endothelial cell proliferation and angiogenesis via ABCA9‐AKT pathway

Human chorionic villous mesenchymal stem cells (CV‐MSCs) are a promising and effective therapeutic option for tissue injury. Vascular dysfunction during pregnancies is significantly involved in the pathogenesis of preeclampsia (PE). This work aims to investigate how CV‐MSCs regulate the function of vascular endothelial cells. In this study, RNA‐seq analysis was used to examine the changes in HUVECs treated with CV‐MSC conditioned medium (CM). We examined the levels of ABCA9 and AKT signaling in human umbilical vein endothelial cells (HUVECs) by immunohistochemistry, western blotting, and qRT‐PCR assays. CCK‐8, colony formation, and tube formation assays were used to understand the role of ABCA9 in HUVEC proliferation and angiogenesis mediated by CV‐MSCs. The CV‐MSC treatment significantly enhanced the HUVEC proliferation and angiogenesis. Furthermore, a significant increase in the ABCA9 expression and AKT pathway activation was observed in CV‐MSCs ‐treated HUVECs. Consistent with these findings, ABCA9 overexpression exhibited the same proliferation‐and angiogenesis‐promoting effect in HUVECs as induced by CV‐MSC CM, also accompanied the AKT signaling activation. In addition, inhibition of ABCA9 inactivated the AKT signaling in HUVECs and reduced the HUVEC proliferation and angiogenesis. Importantly, the elevation of proliferation and angiogenesis induced by ABCA9 overexpression in HUVECs could be reversed by AKT pathway inhibition. Our results suggest that ABCA9‐dependent AKT signaling activation mediated by CV‐MSCs could promote HUVEC proliferation and angiogenesis.


| INTRODUCTION
Vascular endothelial cell is a type of epithelium that composes the blood vessel's the inner surface. It is involved in various aspects of vascular biology, including blood clotting, angiogenesis, and barrier function. 1,2 Therefore, endothelial dysfunction is often regarded as a hallmark for various vascular diseases, including hypertension, atherosclerotic diseases, and PE. 3,4 PE is a pregnancy-specific disorder affecting 2%-8% of all pregnancies. 5 Poorly formed placentas produce anti-angiogenetic factors, inducing generalized systemic inflammation and endothelial cell dysfunction, and resulting in clinical symptoms of preeclampsia, such as hypertension and proteinuria. 6,7 Although the mechanisms underlying the pathogenesis of preeclampsia remain unclear, endothelial cellular injury has recently become a major focus of the pathogenetic research. 8 Derived from BM which adheres to the formed and plastic fibroblast colonies, mesenchymal stem cells (MSCs) are firstly considered as spindle-shaped cells, and are able to turn into derivative cells of the mesenchymal lineage, such as myocytes, osteocytes, adipocytes, and chondrocytes. 9,10 MSCs have been derived from various adult tissues, such as amniotic fluid, and umbilical cord blood, liver, adipose tissue, bone marrow, endometrium, dental pulp, muscle, and placenta. 11 Because of their innumerable biological characteristics connected with the ease of their obtention and in vitro expansion, MSCs have played an important role in the cell therapy-based regenerative medicine field. [12][13][14] With the increasing knowledge about MSC regenerative property, more and more attention is paid to their clinical potential. For example, MSCs significantly reduce cell angiogenesis and proliferation. 15,16 Moreover, MSCs are found to have antimicrobial, antiapoptotic, and immunoregulatory functions. 17,18 More importantly, placental cells have been demonstrated to play an important part in the regulation of peroxidation reactions and recovery of specific damaged tissue. 18,19 Placental MSCs are shown to have an effect on various experimental animal models of diseases, such as cerebral ischemia, spinal cord injury, Parkinson's disease, ischemic brain disease, and Alzheimer's disease, by secreting cytokines and/or neurotrophic factors, as well as angiocrine growth factors. [20][21][22] However, information about the effects of CV-MSCs on vascular endothelial cells involved in placental-umbilical cord circulation, including cellular proliferation, migration, and formation of capillary-like tube structures, is relatively limited. This study used CV-MSCs conditioned medium (CM) to treat HUVECs, and then investigated whether and how HUVECs would be affected by CV-MSCs.
Transcriptome analyses manifested considerable upregulation of ABCA9 in HUVECs after treatment with CV-MSCs CM. In addition, the expression of ABCA9 affected the AKT signaling in HUVECs, which in turn resulted in changes in their proliferation and tube formation. Our results confirmed that CV-MSCs could promote the proliferation of HUVEC and thus tube formation via ABCA9 and AKT regulation.

| Patients and samples
The use of human placentas and all experiments were approved by the Ethical Committee of the Affiliated Hospital of Qingdao University (Shandong, China). Moreover, before enrollment in the research, all the volunteers had signed their written informed consent. With parental permission, the age-matched placentas were collected from severe PE after cesarean section (n = 20), while normal placentas were derived from full-term births (n = 20). All placental specimens were collected through elective cesarean section after induced labor. Besides, placental tissues were collected by getting rid of a vertical plane through the full thickness of an obviously normal and central area and thus contained not only the maternal but also fetal surfaces. However, tissues with clots or calcification were discarded.

| Cell culture and CV-MSC CM preparation
Three healthy placental tissues from the above-mentioned donors were minced and cleared of blood. Then, collagenase (0.1%) and trypsin (0.25%) (type I; Sigma-Aldrich, St. Louis, MO) were used to digest the tissues for 30 min. After filtration through a 100-μm nylon filter and centrifugation, the cells were seeded on culture plates using a stem cell culture medium (SCCM) consisting of 5% UltraGRO™ (Helios, USA) and Stem Cell Basic Medium (Dakewe Biotech Co., Guangzhou, China). Besides, primary cells were cultured at 37°C in an incubator that maintained 5% CO 2 . In this study, all CV-MSCs were harvested at three to six passages. The surface marker testing results using flow cytometry were shown in our previous published research. 23 The medium was replaced with FBS-free DMEM/ F12 (Gibco, Carlsbad, CA), and the cells were cultured for another 24 h, after the CV-MSCs isolated from the placentas reached 80% confluence. The medium was subjected to filtration using a 0.22-μm filter (Millipore, Billerica, MA) after 12-min centrifugation (1200 g). And the medium was collected for later experiments.
Besides, the FBS-free DMEM/F12 medium was adopted as a control.
HUVECs were purchased from the Type Culture Collection China Centre. P1 cells were human umbilical vein endothelial cells. To culture these cells in an incubator that maintained 5% CO 2 at 37°C, DMEM/F12 including 10% FBS was adopted.

| Transient transfection
Transient transfections were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, USA). In order to set up stable transfected cell lines, the transfected cells were isolated as single clones after puromycin treatment.
The sequences of the shRNAs used are as follows: sh- Control lentivirus (Vector), lentiviral constructs expressing full-length ABCA9, were used to establish stably overexpressed cell lines.

| Cell proliferation analysis
Cells (5 × 10 3 /well) were embedded in 96-well plates. CCK-8 reagent (Thermo Fisher Scientific) was applied daily to examine cell proliferation as per the manufacturer's guidance. All samples were tested at 450 nm with a microplate reader. The growth curves were justified in three independent experiments.

| Immunohistochemistry
Placental tissues from PE patients (n = 20) and normal full-term pregnancies (n = 20) were subject to 60-min fixation using 4% paraformaldehyde. The tissues were then embedded in paraffin, sliced into 4-μm sections, and deparaffinized. For antigen retrieval, the slides were boiled in 6.0-pH sodium citrate buffer (10 mM) for 7 min under 120°C. Endogenous peroxidase was blocked by hydrogen peroxide for 10 min. Each slide was washed with TBS containing 0.05% Tween 20 (TBS/T, Merck, Darmstadt, Germany) for 5 min three times. Next, these slides were incubated with anti-P62 antibodies (1:1000) and monoclonal anti-STAT3 antibodies (1:200) for 12 h at 4°C. Moreover, under 37°C, the sections were incubated for 20 min using diluted biotinylated secondary antibodies. The target proteins were visualized by fresh DAB solution. Hematoxylin was subsequently used for counterstaining. The expression of target proteins was independently assessed by two analysts using an optical microscope (Olympus FV500, Tokyo, Japan). The area was analyzed via image-Pro Plus 5.1, with the intensity of staining in five random regions (200× magnification).

| Colony formation assay
A total of 500-1000 HUVECs were incubated in sixwell plates for 14 days. Paraformaldehyde was then used to fix the colonies which were afterward stained with a crystal violet solution for visualization.

| RNA sequencing
Samples were collected using TRIzol (1 ml) (Thermo Fisher Scientific) and were stored at −80°C. The libraries were prepared using an Illumina TruSeq RNA Sample Prep Kit as per the manufacturer's guidance, and the sequencing was conducted on a MiSeq instrument. Analysis of data (from Annoroad Gene Technology, Beijing, China) was performed on the RSEM software. With labeling of no. SRR11493647-no. SRR11493652, all RNA-seq data are available at Sequence Read Archive (SRA).

| Tube formation assay
Tube formation assays were conducted as previously described. 25 The pre-cooled culture plates (Corning; Midland, Michigan, USA) were added with Matrigel and incubated at 37°C for 30 min. Then with different treatments, 2 × 10 4 HUVEC cells were embedded in each well and incubated for 6 h. Furthermore, the formation was evaluated and photographed by a light microscope.

| Matrigel plug assay
Five-week-old male BALB/c mice were purchased from the Shanghai Animal Centre and used for the Matrigel plug assay. All procedures were conducted after being approved by the Animal Care & Use Committee of the Affiliated Hospital of Qingdao University, China. Matrigel (500 μl) containing 20 units of heparin, ABCA9 overexpressing, or vector-transfected HUVECs was injected subcutaneously into the ventral area of the mice. The mice were sacrificed, and the Matrigel plugs were spread from the skin and photographed after 7 days. 26

| Statistical analysis
Statistical analyses were conducted using an unpaired two-tailed Student's t-test. Data are given as mean ± SEM. All experiments were conducted at least in three replicates. GraphPad Prism version 7.00 software (GraphPad; La Jolla, USA) was adopted to analyze those data. Statical significance was determined based on the threshold of p < .05.

| CV-MSCs promote the proliferation of HUVECs and tube formation in vitro via ABCA9
First, we isolated CV-MSCs from healthy placentas, and then, the CCK-8 assay, tube formation assay and colony formation assay were performed to investigate the HUVEC proliferation and angiogenesis after treatment with or without CV-MSC CM. The HUVECs treated with CV-MSC CM showed significantly higher proliferation rates and angiogenic abilities than those untreated cells ( Figure 1A-C, all p < .01). To further elucidate the possible molecular mechanisms of CV-MSCs CM, differentially expressed genes (DEGs) in CM-treated HUVECs were identified using RNA sequencing, and the molecular mechanism for functional changes in HUVEC was explored. We compared the transcriptomes between HUVECs co-cultured with or without CV-MSCs CM, and a total of 4006 upregulated and 4412 downregulated genes showed at least a two-fold change in expression ( Figure 1D). The GO and KEGG results were shown in Figure S1. Based on the results, the expression of ABCA9 in HUVECs treated with CV-MSCs CM increased significantly compared with that in nontreated HUVECs. The result was verified by western blot and qRT-PCR assays ( Figure 1E,F).
Collectively, CV-MSCs promoted HUVEC proliferation and angiogenesis, and ABCA9 was upregulated in this process.

| ABCA9 is downregulated in the placenta of PE pregnancies
To better understand the role played by ABCA9 in placenta of PE patients, we examined its protein and mRNA expression levels in placentas both from normal and PE pregnancies using immunohistochemical, western blotting, and qRT-PCR assays. The immunohistochemical assay revealed a lower expression of ABCA9 in placentas from PE pregnancies (Figure 2A). Our qRT-PCR results confirmed that the mRNA levels of ABCA9 were significant decreased in those placentas ( Figure 2B). In consistence with the mRNA level, the ABCA9 protein levels were also significantly decreased in placenta from PE pregnancies ( Figure 2C). Our results suggested that ABCA9 could be inhibited in placenta during the pathological process of PE.

| ABCA9 promotes HUVEC proliferation and angiogenesis
To better understand the functions contributed by ABCA9 in CV-MSC CM-treated HUVECs, HUVECs were transfected with ABCA9 shRNA and ABCA9overexpressing lentivirus. Transfection efficiency of three ABCA9 shRNA was detected by rt-PCR ( Figure S1). The mRNA levels of ABCA9 were examined, and significant decrease and increase in ABCA9 mRNA levels were, respectively, observed in transfected HUVECs ( Figure 3A). In consistence with the mRNA levels, the ABCA9 protein levels were also significantly changed in transfected HUVECs compared to the untransfected control ( Figure 3B). Moreover, the proliferation of HUVECs was significantly inhibited in the presence of ABCA9 siRNA, and promoted by the ABCA9 overexpression ( Figure 3C). In addition, we examined the colony and tube formations by the transfected HUVECs and found that they shared the same trend of changes as observed in proliferation ( Figure 3D,E). These results suggested that ABCA9 could be promoted by CV-MSCs, which regulated HUVEC proliferation and angiogenesis.

| ABCA9 enhances cell proliferation and angiogenesis through the AKT pathway in vivo and in vitro
AKT is a serine/threonine protein kinase activated by cytokines and various growth factors in a PI3Kdependent manner. 27 It is well known that the AKT signaling pathway regulates vascular homeostasis and angiogenesis through antiapoptotic activity in endothelial cells, 28,29 suggesting that it is involved in ABCA9associated HUVEC regulation. To understand whether the AKT signaling plays a part in ABCA9's effects on HUVECs, protein levels of βcatenin, p-GSK3β, p-AKT, and AKT in HUVEC transfected with ABCA9 siRNA and primary cultured HUVEC transfected with ABCA9 overexpressed plasmids were examined by western blotting analysis ( Figure 3F). Our results confirmed that the AKT signaling pathways could be regulated by ABCA9 in HUVECs.
The ABCA9-overexpressing HUVECs were treated with or without the specific PI3K-AKT pathway inhibitor LY294002 (ab120243) in order to verify the role played by AKT signaling in the ABCA9-mediated HUVEC proliferation and angiogenesis. The AKT and p-AKT expressions were examined by western blotting analysis in ABCA9-overexpressing primary cultured HUVECs treated with or without LY294002. The relative p-AKT expression levels were significantly upregulated in the ABCA9-overexpressing HUVECs, and LY294002 could significantly reversed the elevated p-AKT expression ( Figure 4A). Moreover, the CCK-8 assay revealed that the ABCA9 overexpression markedly promoted cell proliferation, which could be partially reversed by LY294002 ( Figure 4B). As we expected, ABCA9 overexpression resulted in the promotion of tube and colony formations by primary HUVECs, which could be reversed by LY294002 treatment (Figure 4C,D).
These results suggested the possible involvement of ABCA9 in modulating the AKT pathway in CV-MSCstreated HUVECs. Then, we investigated if CV-MSCs could regulate the HUVEC proliferation and angiogenesis via the ABCA9 regulation in vitro.
Metrigel plug angiogenesis assay was performed to further confirm if AKT signaling plays a role in ABCA9-mediated HUVEC promoting effect ( Figure 4E). ABCA9-overexpressing HUVECs showed a higher proliferation rate and thus resulted in faster angiogenesis, while LY294002 could reverse such effect induced by ABCA9 overexpression.
To summarize, these data indicate that CV-MSC-CM may promote HUVEC proliferation and angiogenesis by activating the ABCA9-AKT pathway.

| DISCUSSION
PE and its related complications are one of the main causes of maternal morbidity and mortality during pregnancies, and endothelial cell dysfunction has been shown to be a central contributor to this pathophysiology. 30 When under oxidation and inflammation, endothelial cells will show signs of dysregulation such as increased cell adhesion, resulting in loss of balance between vasodilation and vasoconstriction. 31,32 Due to aberrant placentation and shallow invasion of trophoblast cells in PE, multiple inflammatory cytokines were released in high levels, leading to excessive vascular endothelial injury. 33 Many researches have demonstrated that MSCs could secrete a greater number of chemokines and various growth factors, and could be applied to the treatment of various diseases because of its therapeutic potentials. 34 Despite limited information on the MSC's regulation of functional development in placentas, several studies have indicated that MSC could protect endothelial cells from injury. 35 Human mesenchymal stem cells (hMSCs) are multipotent stem cells that are able to renew themselves and grow into tissues with different functions. 36 hMSCs are sources of trophic factors in vivo and are capable of inducing intrinsic stem cells to fix injured tissues and modulating the immune system. 37 For the present, a great number of clinical trials have been applying hMSCs for therapeutic purposes in many immune diseases, and preliminarily show promising outcomes. 38 The placenta is made up of villous and smooth chorion, the amnion, decidua basalis, umbilical cord, and chorionic plate. 39 The chorionic plate has already been an available source of MSCs, which showed undifferentiation marker gene expression and transdifferentiation, such as TERT, OCT3/4, KLF4, SOX2, and c-MYC. 40 The placenta is a readily reliable and available source of allogeneic cells in addition to other sources such as cartilage, bone marrow, ligament, and adipose tissue in various cases of genetic and degenerative diseases. 41 Isolation of CV-MSCs and subsequent propagation are suitable for industrial scale-up of production of a large number of quality-controlled and affordable cells. 42 To understand the role of CV-MSCs in improving endothelial function, we performed RNA-seq analysis in CV-MSCs treated HUVECs. Then, ABCA9 was found to be significantly increased in those HUVECs, while substantially under-expressed in cells from placental tissue of PE pregnancies. Moreover, activation of AKT signaling induced by ABCA9 overexpression could contribute to the increased angiogenesis and proliferation of HUVEC mediated by CV-MSCs.
The A-subclass of ATP-binding cassette (ABC) transporters is a well-protected family member of potent lipid transporters. 43 The roles of the ABCA6-like subgroup transporters still remain largely unknown. 44 Nevertheless, they have been identified as a unique gene cluster on human chromosome 17q24. Several researches reveal that ABCA9 mRNAs could be isolated from vascular endothelial cells and placenta. This potentially indicates the importance of ABCA9 in regulating the functions of vascular endothelial cells. [44][45][46] In this study, we confirm that ABCA9 could be regulated by CV-MSC and take part in the promotion of vascular endothelial cell proliferation induced by CV-MSCs. ABCA9 overexpression in HUVECs could significantly increase cell proliferation and angiogenesis through the activation of AKT signaling.
Several studies have also confirmed that MSCs could enhance endothelial cell function through AKT activation. 47 AKT and Wnt signaling pathways are of great significance for the survival of cells and angiogenesis through the regulation of endothelial cell migration, proliferation, remodeling, vascular system maturation, and vascular sprouting. [48][49][50] In this study, the elevated protein levels of p-GSK3β and p-AKT are probably associated with the increased cell proliferation in ABCA9-overexpressing HUVECs, and the observation is consistent with previous findings in many functional endothelial cells. 51 In summary, it is revealed in our study that CV-MSCsinduced ABCA9 activation promotes the HUVEC proliferation and angiogenesis in vitro, partially through the activation of AKT pathways in HUVECs. However, it is still uncertain if CV-MSCs also affect other placentabased cells. Further studies shall also be performed to understand how ABCA9 regulates AKT pathways, which will be important for a thorough understanding of PE pathogenesis.