stromal cells modify the effects of oxidative stress on endothelial cell functions

Placenta xxx (2017) 1e13

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Human chorionic villous mesenchymal stem/stromal cells modify the effects of oxidative stress on endothelial cell functions M.H. Abumaree a, b, *, M. Hakami a, F.M. Abomaray c, d, M.A. Alshabibi e, B. Kalionis f, M.A. Al Jumah a, A.S. AlAskar a a Stem Cells and Regenerative Medicine Department, King Abdullah International Medical Research Center, King Abdulaziz Medical City, Minstry of National Guard Health Affairs, P.O. Box 22490, Riyadh 11426, Mail Code 1515, Saudi Arabia b College of Science and Health Professions, King Saud Bin Abdulaziz University for Health Sciences, King Abdulaziz Medical City, Minstry of National Guard Health Affairs, P.O. Box 3660, Riyadh 11481, Mail Code 3124, Saudi Arabia c Department of Clinical Science, Intervention and Technology, Division of Obstetrics and Gynecology, Karolinska Institutet, 14186 Stockholm, Sweden d Center for Hematology and Regenerative Medicine, Karolinska Institutet, 14186 Stockholm, Sweden e National Center for Stem Cell Technology, Life Sciences and Environment Research Institute, King Abdulaziz City for Science and Technology, P.O Box 6086, Riyadh 11442, Saudi Arabia f Department of Maternal-Fetal Medicine Pregnancy Research Centre and University of Melbourne Department of Obstetrics and Gynaecology, Royal Women's Hospital, Parkville, Victoria, Australia, 3052

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 February 2017 Received in revised form 28 March 2017 Accepted 3 May 2017

Mesenchymal stem/stromal cells derived from chorionic villi of human term placentae (pMSCs) produce a unique combination of molecules, which modulate important cellular functions of their target cells while concurrently suppressing their immune responses. These properties make MSCs advantageous candidates for cell-based therapy. Our first aim was to examine the effect of high levels of oxidative stress on pMSC functions. pMSCs were exposed to hydrogen peroxide (H2O2) and their ability to proliferate and adhere to an endothelial cell monolayer was determined. Oxidatively stressed pMSCs maintained their proliferation and adhesion potentials. The second aim was to measure the ability of pMSCs to prevent oxidative stress-related damage to endothelial cells. Endothelial cells were exposed to H2O2, then cocultured with pMSCs, and the effect on endothelial cell adhesion, proliferation and migration was determined. pMSCs were able to reverse the damaging effects of oxidative stress on the proliferation and migration but not on the adhesion of endothelial cells. These data indicate that pMSCs are not only inherently resistant to oxidative stress, but also protect endothelial cell functions from oxidative stressassociated damage. Therefore, pMSCs could be used as a therapeutic tool in inflammatory diseases by reducing the effects of oxidative stress on endothelial cells. © 2017 Published by Elsevier Ltd.

Keywords: Placenta Chorionic villous mesenchymal stromal cells Endothelial cells Proliferation Migration Monocyte adhesion Oxidative stress

1. Introduction Mesenchymal stem/stromal cells (MSCs) (also called multipotent stromal cells depending on the source) are isolated from adult and fetal tissues, such as bone marrow, liver, dental pulp, adipose tissue, endometrium, muscle, amniotic fluid, placenta and umbilical cord blood [1e3]. MSCs have self-renewal ability and

* Corresponding author. College of Science and Health Professions, King Saud Bin Abdulaziz University for Health Sciences, King Abdullah International Medical Research Center, King Abdulaziz Medical City, Ministry of National Guard Health Affairs, P.O. Box 22490, Riyadh 11426, Mail Code 1515, Saudi Arabia. E-mail address: [email protected] (M.H. Abumaree).

multipotent differentiation potential that includes cells of multiple organs and systems such as bone, fat, cartilage, muscle, neurons and hepatocytes [3,4]. Previously, we isolated and characterized MSCs from chorionic villi of human term placentae (pMSCs) [3]. pMSCs produce a unique combination of molecules that influence important functions of target cells including proliferation, differentiation, migration and angiogenesis, while concurrently suppressing their immune responses [3]. We showed that pMSCs are capable of self-renewal and differentiation into the three mesenchymal lineages of adipocytes, osteocytes and chondrocytes [3]. Moreover, we demonstrated the immunosuppressive properties of pMSCs [3,5,6]. We showed that pMSCs can induce an antiinflammatory phenotype in human macrophages by shifting the differentiation of human monocytes from M1 inflammatory into

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M2 anti-inflammatory macrophages [6]. In addition, we provided evidence for their inhibitory effects on the differentiation, maturation and function of human dendritic cells, and their ability to inhibit T cell proliferation. Together, these data show the ability of pMSCs to control the immune responses at multiple levels [7]. These characteristics of pMSCs make them an attractive source for cell-based therapies. Cell-based therapies rely on MSC migration to sites of inflammation and injured tissue in response to various stimuli including cytokines, chemokines and growth factors. At these sites, MSCs repair the damaged tissues in a hostile and toxic inflammatory environment, either by engrafting and differentiating into tissuespecific cell types, or more likely through paracrine mechanisms. Paracrine effects of MSCs include various combinations of the following; stimulating endogenous stem cells, preventing cell apoptosis, increasing cell proliferation and modulating the functions of the innate and adaptive immune cells such as antigen presenting cells and lymphocytes [3,5e8]. Oxidative stress is characterized by the imbalance between prooxidant molecules including reactive oxygen and nitrogen species, and antioxidant defenses [9,10]. Oxidative stress plays a key role in the pathogenesis of many diseases [9]. Accumulating evidence supports the notion that acute and chronic uncontrolled overproduction of oxidative stress-related factors, including reactive oxygen species (ROS), are causative agents in cardiovascular diseases, such as atherosclerosis and diabetes [11]. Furthermore, ROS mediate various signaling pathways that underly vascular inflammation in ischemic tissues [11]. pMSCs are derived from the fetal part of the placenta and exposed to the fetal circulation, and thus experience lower levels of inflammation and oxidative stress [12,13]. The exposure of pMSCs to high levels of inflammation and oxidative stress may negatively impact their therapeutic potential, as is the case for MSCs derived from other sources [14]. Endothelium comprises a squamous epithelium that lines the lumen of all blood vessels that is highly metabolically active. Endothelium plays a major role in vascular homeostasis and acts as an endocrine organ by producing variety of molecules including hormones, growth factors, coagulation factors, and adhesion molecules [15]. Moreover, the endothelium is the active biologic interface between blood and tissues that regulates the sensitive balance between vasoconstriction/vasodilatation, coagulation/ fibrinolysis and proliferation/apoptosis. Finally, the endothelium is the interface for the transient adhesion, and subsequent diapedesis, of blood-borne leukocytes [15]. Inflammatory diseases, such as atherosclerosis, are characterized by endothelial cell activation, which results from oxidative stress and increased inflammation [16]. Intense and sustained endothelial cell activation culminates in a damaged endothelium [15]. This damage manifests in major phenotypic changes, such as increased inflammatory marker expression in the endothelium, and endothelial dysfunction as a result of increased proliferation, which is a characteristic of atherosclerosis [17]. Therefore, an essential prerequisite to the use of pMSCs in a cellbased therapy is to determine their effects on the functional characteristics of endothelial cells in a hostile and toxic inflammatory environment. Here, we examined the potential of pMSCs to interact with endothelial cells in a hostile, toxic and inflammatory environment. Our results showed that pMSCs exposed to hydrogen peroxide (H2O2) were able to proliferate and adhere to an endothelial cell monolayer. Furthermore, pMSCs prevent oxidative stress-related damage to endothelial cells. pMSCs were able to reverse the damaging effects of oxidative stress on the proliferation and migration but not on the adhesion of endothelial cells. Together, these data show pMSCs are inherently resistant to oxidative stress

and they can also protect endothelial cell functions from oxidative stress-associated damage. Therefore, pMSCs could be used as a therapeutic tool in inflammatory diseases because of their ability to reduce the effects of oxidative stress on endothelial cells. 2. Materials and methods 2.1. Ethics of experimentation This study was approved by the institutional research board (Reference # IRBC/021/14) at King Abdulla International Medical Research Centre/King Abdulaziz Medical City, Riyadh, Saudi Arabia. All term placentae and umbilical cords were obtained with informed patient consent. 2.2. Placentae Human placentae were obtained from uncomplicated pregnancies following normal vaginal delivery (38e40 weeks of gestation). The gestational age and fetal viability of all pregnancies were confirmed by early ultrasound examination before 20 weeks of gestation. The placentae were used within 2 h of delivery. 2.3. Isolation of pMSCs from chorionic villi of human term placenta in vitro pMSCs were isolated from chorionic villi of human term placenta using our published method [3]. Briefly, placental tissues were dissected and then washed thoroughly with sterile phosphate buffered saline (PBS), pH 7.4. After removing the superficial layer of maternal decidua on the maternal side of the placenta, the underlying fetal chorionic villi were cut into small pieces of approximately 40 mg total wet weight. The tissue was washed with PBS and then incubated with 2.5% trypsin (Life Technologies, Grand Island, USA), which was diluted in DMEM-F12 medium (Invitrogen, Saudi Arabia) containing (271 unit/ml) DNase (Life Technologies, Grand Island, USA), 100 mg/ml streptomycin and 100 U/l penicillin, with gentle rotation overnight at 4
C. Tissues were then washed thoroughly with PBS and allowed to adhere to the plastic in 6 well plates for 1 h at 37
C in a humidified atmosphere containing 5% CO2 (a cell culture incubator). Subsequently, DMEM-F12 medium containing 10% Mesenchymal Stem Cell Certified fetal bovine serum (MSC-FBS) (Life Technologies, Grand Island, USA), 100 mg/ml of Lglutamate, 100 mg/ml streptomycin and 100 U/l penicillin was gently added to the tissues and cultured at 37
C in a cell culture incubator. Every two days, the medium was replaced with fresh medium. On day 14, the tissue pieces were removed and cells that had migrated out from the cut ends of the tissues were then harvested with TrypLE™ Express detachment solution (Life Technologies, Grand Island, USA) and characterized by flow cytometry using MSC positive markers (CD44, CD90, CD146, CD166 and CD105) and hematopoietic negative markers (CD14, CD19, CD45, HLA-DR, CD80, CD83, CD86, and CD40), as described previously [3]. Cells at a density of 1  105 cells in 75 cm2 flasks (Becton Dickinson, New Jersey, USA) were re-cultured until they reached 75% confluency and then used in subsequent experiments. Prior to use in experiments, pMSCs at passage 2 were assessed for differentiation into adipocytes, chondrocytes and osteocytes as previously described [3]. Adipogenic, osteogenic and chondrogenic differentiation was performed by incubating pMSCs in adipogenic (#390415), osteogenic (#390416) and chondrogenic (#390417) media respectively, and were purchased from R&D Systems (Abingdon, UK). Each differentiation medium was supplemented with 10% MSC-FBS, 100 mg/ml of L-glutamate, 100 mg/ml streptomycin and 100 U/l penicillin. Adipocytes, osteocytes and

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chondrocytes were identified using LipidTOX™ Green, Alizarin Red S and Alcian Blue dyes respectively, as described previously [3]. All antibodies were from Beckman Coulter (California, United States). A total of 30 placentae were used in this study. 2.4. Isolation and culture of human umblical vein endothelial cells (HUVEC) Primary human umblical vein endothelial cells (HUVEC) were isolated from umbilical cord veins as previously described, with some modifications [18]. Cannulated umbilical veins were gently massaged and rinsed with sterile PBS, pH7.4 several times to remove clots and blood. The veins were then filled with a digestion solution containing 6 mg/ml collagenase type II (#17101-015, Life Technologies, Grand Island, USA) diluted in PBS, and incubated at 37
C in a cell culture incubator for 25 min. After incubation, the collagenese solution containing the HUVEC was collected by perfusion of the cord with vascular cell basal medium (PCS-100030™, ATCC, USA) and cells were collected by centrifugation at 300 x g for 10 min. Cells were then resuspended in PBS and incubated with red blood cell lysis buffer for 45 min at RT to lyse red blood cells. After centrifugation at 300 x g for 10 min, the cell pellet was resuspended in complete HUVEC growth medium (#ATCC® PCS100-041™, Endothelial Cell Growth Kit-VEGF, ATCC, USA) and cultured in T25 flasks at 37
C in a cell culture incubator. When cells reached 75% confluency, they were harvested with TrypLE™ Express detachment solution and characterized by flow cytometry using CD31 endothelial cell marker (R & D Systems, Abingdon, UK). Samples with purity greater than 95% were used in experiments. Cells from passages 3e5 were used in subsequent experiments. A total of 30 umbilical cords were used in this study. 2.5. Cell proliferation assay pMSCs were treated with various concentrations of (1, 5, 25, 50, 100, 200, 400 and 600 mM) H2O2 during culture. pMSCs were seeded at a density of 5  103 in 96-well tissue culture plates containing complete pMSC culture medium for 24 h at 37
C in a cell culture incubator. Following removal of the culture medium and washing cells with sterile PBS to remove unattached cells, H2O2 was added to pMSC culture at the above concentrations and then cultured in complete pMSC culture medium for 24 h at 37
C in a cell culture incubator. Untreated pMSCs served as a control. The proliferation of HUVEC in response to pMSCs and conditioned medium (CM) prepared as described below was also examined. To produce CM, 1  105 pMSCs were cultured in 75 cm2 flasks containing DMEM-F12 medium with 10% MSCFBS, 100 mg/ml of Lglutamate, 100 mg/ml streptomycin and 100 U/l penicillin. Every two days, the medium was removed and replaced with fresh medium. When cells reached 75% confluency, cells were cultured in fresh medium for two days and CM was then harvested, centrifuged at 500 x g for 10 min and stored at 80
C until use. HUVEC were cultured with different ratios of cells and different concentrations of CM. For HUVEC and pMSC experiments, different HUVEC: pMSC ratios were used ranging from 1:1, 2:1, 4:1 and 10:1. For CM experiments, 1%, 5% and 25% (v/v) CM was diluted in fresh medium, and were used for pMSCs. Briefly, HUVEC were seeded at a density of 5  103 per well in 96-well tissue culture plates containing complete HUVEC culture medium for 24 h at 37
C in a cell culture incubator. Following removal of the culture medium and washing cells with sterile PBS to remove unattached cells, pMSCs or CM were added to HUVEC culture at the indicated ratios of cells and concentrations of CM, and then cultured in a complete HUVEC culture medium with or without 100 mM H2O2 for 24 h at 37
C in a cell culture incubator.

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Cell proliferation was then assessed using a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] kit (#G5421, CellTiter 96® Aqueous Non-Radioactive Cell Proliferation Assay, Promega, Germany) according to the manufacturer's instructions. Briefly, MTS solution was added into each well of the 96 well assay plate containing cells in complete culture medium in the presence or absence of different treatments, and incubated for 4 h. The absorbance at 490 nm was recorded using a plate reader (Spectra MR, Dynex Technologies, Denkendorf, Germany). Results are presented as means (±SD) obtained from triplicate cultures. MTS solution in medium not exposed to cells was used as blank. Before the addition of pMSCs to the coculture experiments, pMSCs were treated with 25 mg/ml Mitomycin C for 1 h at 37
C to inhibit their proliferation and then followed by five extensive washes with culture medium containing FBS as previously described [6]. A range of incubation times (24e72 h) for the culture of HUVEC with different treatments of cells, CM and H2O2 were evaluated. Five experiments were performed in triplicate using HUVEC (passage 3e5) from five independent umbilical cord tissue preparations, and pMSCs (passage 2) from five independent placentae were used. 2.6. Culture of HUVEC with different treatments of pMSCs (direct contact, supernatant and conditioned medium) For intercellular direct contact experiments (ICpMSC), the 0.4 mm pore size transwell chamber membrane culture system (Greiner Bio-One, Germany) was used as previously described [6] and as illustrated in Fig. 1, pMSCs were seeded on the reverse side of the membrane of the chamber until the cells were fully adhered, and HUVEC were seeded on the upper side of the membrane. For soluble factor experiments (SFpMSC), the 8 mm pore size transwell chamber membrane culture system (Greiner Bio-One, Germany) was used as previously described [6] and as illustrated in Fig. 1, pMSCs were physically separated from HUVEC by culturing them on the upper compartments while HUVEC were cultured in the lower compartment. Both culture systems prevent the contamination of HUVEC with pMSCs and facilitate harvesting HUVEC without pMSC contamination. In both systems, cells were cultured at 10HUVEC: 1pMSC ratio in HUVEC culture medium in the presence or absence of 100 mM H2O2 and then incubated for 24 h at 37
C in a cell culture incubator. For conditioned medium (CM) experiments (CMpMSC), 25% supernatant from unstimulated pMSCs, prepared as described above, was added to HUVEC cultured in HUVEC culture medium in the presence or absence of 100 mM H2O2 as illustrated in Fig. 1. After 24 h, HUVEC were harvested with TrypLE™ Express detachment solution and used in proliferation, adhesion and migration experiments as described below. Each experiment was performed in duplicate and repeated with five independent preparations of HUVEC and pMSCs. HUVEC cultured in HUVEC culture medium without pMSCs were included as a negative control for all HUVEC cultured with pMSCs (ICpMSC, SFpMSC and CMpMSC). The viability of cells was determined using Trypan blue and cell counting in a haemocytometer chamber. Five experiments were performed in triplicate using HUVEC and pMSCs as indicated above. HUVEC and pMSCs cultured alone were included as negative controls. 2.7. Adhesion assay of pMSCs to HUVEC The effect of H2O2 on pMSC adhesion to HUVEC was examined by the addition of pMSCs to a monolayer of HUVEC in 96-well tissue culture plates. Two cell groups were examined; 1) the adhesion of H2O2-pretreated pMSCs to H2O2- untreated HUVEC and 2) the

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Fig. 1. HUVEC culture system. CMpMSC culture system consists of HUVEC seeded on a surface of 6 well culture plate in a complete HUVEC culture medium alone (Untreated HUVEC) or with 100 mM H2O2 or with 25% conditioned medium (CM) obtained from unstimulated pMSCs (A); SFpMSC culture system consists of pMSCs seeded in the upper chamber while HUVEC seeded in the lower chamber of transwell membrane culture system (B); and ICpMSC culture system consists of pMSCs seeded on the reverse side of the membrane of the chamber and HUVEC seeded on the upper side of the membrane (C). For SFpMSC and ICpMSC, 8 mm and 0.4 mm pore size transwell chamber membranes were used, respectively. In CMpMSC, cells were cultured with 100 mM H2O2 (D). In SFpMSC and ICpMSC culture systems, cells were cultured at 10HUVEC:1pMSC ratio in HUVEC culture medium in the absence or presence of 100 mM H2O2 (E and F). In all culture systems, cells were incubated for 24 h at 37
C in a cell culture incubator.

adhesion of H2O2-pretreated pMSCs to H2O2-pretreated HUVEC. Following the treatment of pMSCs and HUVEC with 100 mM H2O2 for 24 h at 37
C in a cell culture incubator, H2O2- treated or untreated HUVEC and H2O2-treated or untreated pMSCs were harvested, washed with PBS and then used in the adhesion assay. For groups one and two, H2O2-treated or untreated HUVEC were seeded in 96-well tissue culture plates and allowed to grow to confluency to form a monolayer. H2O2-treated or untreated pMSCs were then fluorescently labelled with 5 mM green fluorescent cell tracker stain (5-chloromethylfluorescin diacetate; CMFDA; Molecular Probes, Life Technologies, Grand Island, USA) in DMEM-F12 medium for 4 h, washed three times with fresh DMEM-F12 medium, and then added to HUVEC culture at a ratio of 5 pMSC:1 HUVEC. Cells were then incuabted at 37
C in a cell culture incubator for 30 min. Non-adherent pMSCs were then removed by gentle washing with sterile PBS, pH 7.4. The fluorescence intensity of the pMSCs adhered to the monolayer of HUVEC was measured at excitation 485 nm and emission 528 nm using a fluorescence microplate reader (Glomax Multi Detection System, Promega, Germany). Results were expressed as relative fluorescence intensity (RFI). Different ratios of HUVEC to pMSCs were evaluated. Five experiments were performed in triplicate using HUVEC and pMSCs as indicated above. 2.8. HUVEC adhesion and proliferation using xCELLigence real-time cell analyser To examine if soluble factors produced by unstimulated (CMpMSC) pMSCs, stimulated pMSCs (SFpMSC) and the intercellular contact between HUVEC and pMSCs (ICpMSC) would have a

reversible or irreversible effect on the functions of endothelial cells, endothelial cells were cultured with different treatments of pMSCs and H2O2 for 24 h as described above, and then their proliferation was examined using the xCELLigence Real-Time Cell Analyser (RTCA-DP version; Roche Diagnostics, Mannheim, Germany), which monitors continuously the cellular events recording labelfree changes in electrical impedance (reported as an arbitrary cell index) [19]. HUVEC initially co-cultured with different treatments of pMSCs were also used in adhesion experiments using the xCELLigence Real-Time Cell Analyser. Briefly, the background impedance was performed using 100 mL HUVEC complete medium per well in 16-well culture plates (#05469813001, E-Plate 16, Roche Diagnostics GmbH, Mannheim, Germany) as per the manufacturer's instructions. In each experiment, 2  104 HUVEC (Untreated HUVEC, HUVEC harvested from the coculture experiments of CMpMSC, SFpMSC and ICpMSC) were seeded in 100 mL of complete HUVEC in quadruplicate wells and left to equilibrate for 30 min at RT before data recording. Culture plates were then incubated at 37
C in a cell culture incubator and the cell index of the HUVEC cultures was automatically monitored for up to 72 h. Data were analysed using the xCELLigence software (version 1.2.1). After two hours, data for cell adhesion was measured and expressed as a cell index and with values expressed as mean ± SD of cell index. Data for cell proliferation was expressed as mean ± SD of the cell index normalized to the cell index recorded after two hours (adhesion time point). The rate of cell growth was determined by calculating the normalized cell index at 24, 48 and 72 h time points. Five experiments were performed in triplicate using HUVEC and pMSCs as indicated above.

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Please cite this article in press as: M.H. Abumaree, et al., Human chorionic villous mesenchymal stem/stromal cells modify the effects of oxidative stress on endothelial cell functions, Placenta (2017), http://dx.doi.org/10.1016/j.placenta.2017.05.001

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Fig. 2. HUVEC migration groups. Group 1 consisted of HUVEC cultured alone (A) or with 25% conditioned medium obtained from unstimulated pMSC culture (CMpMSC) (B) or with pMSCs at 10HUVEC:1pMSC ratio in a paracrine communication culture system (SFpMSC) (C), or with pMSCs at 10HUVEC: 1pMSC ratio in a cellular communication culture system (ICpMSC) (D), or with 100 mM H2O2 (E), or with 100 mM H2O2 and CMpMSC (F), or with 100 mM H2O2 and SFpMSC (G), or with 100 mM H2O2 and ICpMSC (H). Pretreated HUVEC were seeded in HUVEC serum free medium in the upper chamber of CIM migration plate while HUVEC culture medium containing 20% FBS was added to the lower chambers. Group 2 consisted of HUVEC seeded in HUVEC serum free medium alone (A), or with 20% CMpMSC (B), or with 100 mM H2O2 (C), or with 100 mM H2O2 and 20% CMpMSC in the upper chamber of the migration plate, while HUVEC medium with 20% FBS was added to the lower chamber. Group 3 consisted of HUVEC seeded in HUVEC serum free medium in the upper chamber of the migration plate while HUVEC medium with 20% FBS (A), or with 20% CMpMSC (B), or with 100 mM H2O2 (C), or with 100 mM H2O2 and 20% CMpMSC added to the lower chamber of the migration plate.

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2.9. HUVEC migration using xCELLigence real-time cell analyser

centrifuged at 15,000 rpm for 5 min at 4
C to remove the debris, and the supernatant was then collected. Total protein was estimated by the Bradford method. Glutathione reductase activity was then measured using OxiSelect™ Glutathione Reductase Assay Kit (Catalogue # STA-812, Cell Biolabs, Inc., San Diego, USA) as instructed by the manufacturer. This assay is based on the reduction of glutathione disulfide (oxidized glutathione) (GSSG) to reduced glutathione (GSH) by glutathione reductase, using NADPH as a donor for H. Subsequently, the chromogen reacts with the thiol group of GSH to produce a colored compound that absorbs at 405 nm. The glutathione reductase content in HUVEC samples was determined by comparison with the predetermined glutathione reductase standard curve. The assay was performed using 100 ml aliquots of HUVEC supernatant protein (30 mg protein) immediately after preparation, and added to phosphate buffer containing excess GSSG and NADPH. The level of change was determined at 405 nm using a standard curve. Three experiments were performed in triplicate using HUVEC and pMSCs as indicated above.

HUVEC initially co-cultured with different treatments of pMSCs (ICpMSC, SFpMSC and CMpMSC) as described above were used in the migration experiments (Fig. 2). In addition the CM obtained from the cultures of pMSCs initially cultured with and without 100 mM H2O2 for 24 h was also used to examine the migration of HUVEC cultured with CM (see below), or in response to CM (see below) (Fig. 2). All migration experiments were performed using the xCELLigence Real-Time Cell Analyser, and 16-well plates (#05665825001, CIM-16, Roche Diagnostics GmbH, Mannheim, Germany) were used as previously described with modifications [19]. The CIM plates have 16-well migration chambers comprising upper and lower chambers separated by a porous (pore size 8 mm) polyethylene terephthalate (PET) membrane in conjunction with microelectrodes. Treatments (group 1, 2 and 3 as described below) were made to desired concentrations (final volume of 160 ml) and loaded in the lower wells of the plate. Following the addition of 50 ml pre-warmed media to the wells of the upper chamber, the plates were locked in the RTCA DP device at 37
C in a cell culture incubator for 1 h to obtain equilibrium as per the manufacturer's instructions and a measurement step was then performed as a background signal, generated by cell-free media. To initiate the experiment, HUVEC were seeded at a density of 2  104 in the upper chamber in 100 mL growth medium and the plates were then incubated for 30 min at RT to allow the cells to settle onto the membrane as per the manufacturer's instructions. In the migration experiments, three treatment groups were used as illustrated in Fig. 2; Group one: comprised of HUVEC intially co-cultured with pMSCs or cultured alone as described above (Untreated HUVEC, CMpMSC, SFpMSC and ICpMSC). HUVEC were seeded in the upper chamber in HUVEC serum free medium while HUVEC medium supplemented with 20% FBS was added to the lower chamber. Group two: comprised of HUVEC seeded in the upper chamber with 20% CM harvested as described above from the culture of pMSCs or with HUVEC serum free medium (Untreated HUVEC) while HUVEC medium supplemented with 20% FBS was added to the lower chamber. Group three: comprised of HUVEC seeded in the upper chamber with HUVEC serum free medium while HUVEC medium supplemented with 20% CMpMSC or with HUVEC medium supplemented with 20% FBS (Untreated HUVEC) was added to the lower chamber. Each condition was performed in quadruplicate and after equilibration, the analyser was programmed to scan the membrane every 15 min for 24 h. The impedance value of each well was automatically monitored by the xCELLigence system for the duration of 24 h and expressed as a CI value. Migration observed in the presence of 30% FBS, and with medium alone, served as positive and negative controls, respectively. Group one allowed us to determine the migration of HUVEC after their exposure to different treatments of pMSCs. Group two allowed us to determine the migration of HUVEC under the effect of conditioned medium of pMSCs added to the upper chamber of the plate. Group three allowed us to determine the migration of HUVEC in response to conditioned medium of pMSCs added to the lower chambers of the plate. Five experiments were performed in triplicate using HUVEC and pMSCs as indicated above.

All pMSC preparations were derived from normal, healthy term placentae. At passage 2 pMSCs were more than 95% positive for MSC markers (CD44, CD90, CD146, CD166 and CD105) and negative (>95%) for hematopoietic markers (CD14, CD19, CD45, HLA-DR, CD80, CD83, CD86, and CD40), which was consistent with our previous study [3]. pMSCs were successfully differentiated into osteocytes, adipocytes and chondrocytes in vitro using appropriate growth factors and methods as we previously described (data not shown) [3]. Based on the above criteria, we used pMSCs at passage 2 in all subsequent experiments.

2.10. Antioxidant assay

3.2. H2O2 effects on the proliferative potential of pMSCs

Treated and untreated HUVEC harvested from the proliferation experiments described in section 2.6 were washed twice with cold sterile PBS, pH7.4 to remove debris. Following the addition of 100 mL of cell lysis buffer (Cell Signaling Technologies, Beverly, MA) containing protease and phosphatase inhibitors to each well, cells were scrapped using a cell scrapper and lysate was collected,

To evaluate the effect of H2O2 on pMSC proliferation, pMSCs were cultured in pMSC culture medium containing H2O2 and then their proliferation potential was examined using the MTS assay. After 24 h with 1, 5, 25, 50, 100, and 200 mM H2O2, the proliferation of pMSCs did not significantly changed as compared to H2O2-untreated pMSCs, (Fig. 3A). In contrast, treatment with 400 and

2.11. Flow cytometry Cells (pMSCs and HUVEC) were phenotypically characterized by flow cytometry as previously published [1]. Briefly, pMSCs and HUVEC were harvested as described above and 1  105 cells were then stained with monoclonal antibodies described above for 30 min. Cells were washed twice by adding cold PBS, pH 7.4 and centrifuged at 150 x g for 5 min at 8
C. To analyse intracellular expression of proteins, cells were fixed with 4% paraformaldehyde in sterile PBS, pH 7.4 for 10 min at RT and then permeabilized using sterile PBS, pH 7.4 containing 0.1% saponin for 5 min at RT. Unstained and isotype controls were used. Immunoreactivity to cell surface antibody markers or intracellular proteins was assayed by a BD FACS CANTO II (Becton Dickinson, New Jersey, USA) flow cytometer. 2.12. Statistical analysis Data were analysed using the t-test. The analyses were performed using GraphPad Prism 5. Results were considered to be statistically significant if P < 0.05. 3. Results 3.1. Isolation and characterization of pMSCs from human placental chorionic villi

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Fig. 3. The proliferation of pMSCs in response to various concentrations of hydrogen peroxide (H2O2) and the adhesion of pMSCs to a monolayer of HUVEC. In a MTS proliferation assay, the proliferation of pMSCs in response to 5, 25, 50, 100, and 200 mM H2O2 did not significantly change as compared to H2O2-untreated pMSC after 24 h, whereas treatment with 400 and 600 mM H2O2 for 24 h significantly decreased the proliferation of pMSCs as compared to H2O2- untreated pMSCs (A). The adhesion of pMSCs, intially treated with or without 100 mM H2O2 for 24 h and labelled with green fluorescent cell tracker stain (CMFDA) for 4 h, to a monolayer of HUVEC pretreated with or without 100 mM H2O2 for 24 h was evaluated after 30 min using a fluorescence microplate reader to detect the fluorescence intensity of the pMSC adhered to the monolayer of HUVEC. The adhesion of H2O2-pretreated pMSCs to H2O2-untreated HUVEC increased significantly as compared to H2O2-untreated pMSCs (B). The adhesion of H2O2-pretreated pMSCs to H2O2-pretreated HUVEC increased significantly as compared to H2O2-untreated pMSCs (C). Each experiment was performed in triplicate using HUVEC (passage 3e5) from five independent umbilical cord tissues and pMSCs (passage 2) from five independent placentae. *P < 0.05. Bars represent standard errors.

600 mM H2O2 for 24 h, significantly decreased pMSC proliferation as compared to H2O2-untreated pMSCs, P < 0.05 (Fig. 3A). In addition, the viability of pMSCs treated with 5, 25, 50 and 100 mM H2O2 was 90%. In contrast, the treatment of pMSCs with 400 and 600 mM H2O2 reduced the viability to 50% ± 5% and 40% ± 7%, respectively. 3.3. H2O2 effects on pMSC adhesion to a HUVEC monolayer To assess the effect of H2O2 on the adhesion of pMSCs to HUVEC, pMSCs were intially treated with 100 mM H2O2 for 24 h, washed and labelled with green fluorescent cell tracker stain (CMFDA) for 4 h. Then, pMSC adhesion to a monolayer of HUVEC intially pretreated with or without 100 mM H2O2 for 24 h, was then evaluated after 30 min using a fluorescence microplate reader to detect the fluorescence intensity of the pMSC adhered to the monolayer of HUVEC. The adhesion of H2O2-pretreated pMSCs to the H2O2-untreated HUVEC monolayer significantly increased as compared to H2O2-untreated pMSCs, P < 0.05 (Fig. 3B). To assess the effect of H2O2 on the adhesion of pMSCs to H2O2-pretreated HUVEC, HUVEC were intially treated with 100 mM H2O2 for 24 h. The viability of H2O2-pretreated HUVEC was first assesed which was 90%. Exposure of HUVEC to H2O2 higher than 100 mM H2O2 reduced the viability of HUVEC to less than 50%. Therefore, 100 mM H2O2 was chosen in all experiments. The adhesion of H2O2-pretreated pMSCs to the H2O2pretreated HUVEC monolayer significantly increased as compared to H2O2-untreated pMSCs, P < 0.05 (Fig. 3C). 3.4. pMSCs and H2O2 modulate the proliferation potential of HUVEC To gain a better understanding of the effects of pMSCs on endothelial cell functions, the proliferation of HUVEC cultured with pMSCs in the absence or presence of 100 mM H2O2 was examined using the MTS assay. The effect of pMSCs on HUVEC proliferation was maintained with increasing time in culture (up to 72 h) and therefore the culture time used in this study was 24 h. After 24 h, HUVEC proliferation significantly increased at all examined ratios and concentrations of pMSCs and CMpMSC, respectively except at 10HUVEC:1pMSC ratio as compared to untreated HUVEC, P < 0.05 (Fig. 4A and B). Similarly, after 24 h culture with H2O2 in the absence or presence of pMSCs, HUVEC proliferation significantly increased as compared to untreated HUVEC, P < 0.05 (Fig. 4C) except at 10HUVEC:1pMSC and with 25% CMpMSC, (Fig. 4C and D).

In addition, the proliferation of H2O2-treated HUVEC cultured in the presence of pMSCs signficantly increased (P< 0.05) at 2:1 and 1:1 HUVEC: pMSC ratio or with 5% CMpMSC but did not significantly change at 4:1 HUVEC:pMSC ratio or with 1% CMpMSC as compared to H2O2-treated HUVEC (Fig. 4C and D). In contrast, the proliferation of H2O2-treated HUVEC cultured at 10HUVEC:1pMSC or with 25% CMpMSC significantly decreased as compared to H2O2-treated HUVEC, P < 0.05 (Fig. 4C and D). 3.5. Pre-treating HUVEC with pMSCs and H2O2 modulates their proliferation potential To evaluate if the inhibitory effects of pMSCs on the proliferation of H2O2-treated HUVEC cultured at 10HUVEC:1pMSC or with 25% CMpMSC is reversible or not, endothelial cells were intially cultured with different treatments of pMSCs in the absence or presence of 100 mM H2O2 for 24 h and then harvested, washed and used in a proliferation assay using the xCELLigence Real-Time Cell Analyser. After 24, 48 and 72 h, the proliferation of HUVEC pretreated with CMpMSC and ICpMSC significantly decreased and increased, respectively as compared to untreated HUVEC. In contrast, the proliferation of HUVEC pretreated with SFpMSC changed, but not significantly (Fig. 5AeC). In the presence of H2O2, the proliferation of HUVEC pretreated with H2O2 alone, or with CMpMSC or ICpMSC, decreased significantly as compared to H2O2-untreated HUVEC after 24, 48 and 72 h, P < 0.05 (Fig. 5DeF). Similarly, the proliferation of HUVEC pretreated with H2O2 and SFpMSC decreased, but not significantly (Fig. 5DeF). In comparison to H2O2-treated HUVEC, the proliferation of HUVEC pretreated with H2O2 and CMpMSC, decreased significantly after 24 and 72 h of culture (P < 0.05) but not after 48 h (Fig. 5DeF). In contrast, the proliferation of HUVEC pretreated with H2O2 and SFpMSC increased significantly after 48 and 72 h of culture as compared to H2O2-treated HUVEC (P < 0.05) but not after 24 h (Fig. 5DeF). In addition, the proliferation of HUVEC pretreated with H2O2 and ICpMSC significantly increased after 72 h (P < 0.05) but not after 24 and 48 h as compared to H2O2treated HUVEC (Fig. 5DeF). 3.6. pMSC and H2O2 enhance HUVEC adhesion To understand the effects of pMSCs and H2O2 on the adhesion of endothelial cells, endothelial adhesion was examined using the

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Fig. 4. HUVEC proliferation in response to pMSCs with or without H2O2 was examined after 24 h in MTS assay. In cell-cell contact, as compared to untreated HUVEC, pMSCs significantly increased HUVEC proliferation at all examined ratios of cells except at 10HUVEC:1pMSC ratio (A). In response to conditioned medium, pMSCs significantly increased HUVEC proliferation as compared to untreated HUVEC (B). In the presence of 100 mM H2O2 with or without pMSCs at ratios 4HUVEC:1pMSC, 2HUVEC:1pMSC and 1HUVEC:1pMSC, HUVEC proliferation increased significantly as compared to untreated HUVEC (C). As compared to H2O2-treated HUVEC, the proliferation of HUVEC cultured with 100 mM H2O2 and with pMSCs at ratios 2HUVEC:1pMSC or 1HUVEC:1pMSC but not at 4HUVEC:1pMSC increased significantly while with pMSCs at 10HUVEC:1pMSC ratio decreased significantly, but did not change as compared to untreated HUVEC (C). In the presence of 100 mM H2O2, CMpMSCs at 1% and 5% CMpMSC, increased HUVEC proliferation significantly as compared to H2O2-untreated HUVEC (D). As compared to H2O2-treated HUVEC, the proliferation of HUVEC cultured with 100 mM H2O2 and with CMpMSC at 5% increased significantly but not at 1% CMpMSC (D). In the presence of 100 mM H2O2, CMpMSCs at 25% decreased HUVEC proliferation significantly as compared to H2O2-treated HUVEC, but not as compared to untreated HUVEC (D). Each experiment was performed in triplicate using HUVEC (passage 3e5) from five independent umbilical cord tissues and pMSCs (passage 2) from five independent placentae. *P < 0.05. Bars represent standard errors.

xCELLigence Real-Time Cell Analyser. HUVEC were initially cultured with different treatments of pMSCs in the absence and presence of 100 mM H2O2 for 24 h. Then, HUVEC were harvested, washed and re-cultured in a 16 well culture plate and monitored. After two hours, the adhesion of HUVEC pretreated with CMpMSC and

SFpMSC increased significantly as compared to untreated HUVEC, P< 0.05 (Fig. 6A). In contrast, the adhesion of HUVEC pretreated with ICpMSC decreased significantly as compared to untreated HUVEC, P < 0.05 (Fig. 6A). In addition, the adhesion of HUVEC pretreated with H2O2 alone, or pretreated with H2O2 and CMpMSC

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Fig. 5. HUVEC proliferation after removing the effects of pMSCs and H2O2. HUVEC were initially cultured with different pMSC treatments with or without 100 mM H2O2 for 24 h and then used in a proliferation assay using the xCELLigence Real-Time Cell Analyser. After 24 (A), 48 (B) and 72 h (C), the proliferation of HUVEC pretreated with CMpMSC and ICpMSC significantly decreased and increased, respectively as compared to untreated HUVEC. In contrast, the proliferation of HUVEC pretreated with SFpMSC changed, but not significantly, P > 0.05 (AeC). The proliferation of HUVEC pretreated with H2O2 alone, or with CMpMSC, or ICpMSC, but not with SFpMSC decreased significantly as compared to H2O2-untreated HUVEC after 24, 48 and 72 h (DeF). In comparison with H2O2-treated HUVEC, the proliferation of HUVEC pretreated with H2O2 and CMpMSC decreased significantly after 24 and 72 h of culture, but not after 48 h, P > 0.05 (DeF). In contrast, the proliferation of HUVEC pretreated with H2O2 and SFpMSC increased significantly after 48 and 72 h of culture as compared to H2O2-treated HUVEC, but not after 24 h, P > 0.05 (DeF). The proliferation of HUVEC pretreated with H2O2 and ICpMSC increased significantly after 72 h, but not after 24 and 48 h as compared to H2O2- treated HUVEC, P > 0.05 (DeF). Each experiment was performed in triplicate using HUVEC (passage 3e5) from five independent umbilical cord tissues and pMSCs (passage 2) from five independent placentae.*P < 0.05. Bars represent standard errors.

or SFpMSC or ICpMSC, significantly increased as compared to H2O2untreated HUVEC (P< 0.05) while the adhesion of H2O2-treated HUVEC cultured with CMpMSC, SFpMSC and ICpMSC did not significantly change as compared to H2O2-treated HUVEC (Fig. 6B).

3.7. Pretreating HUVEC with pMSCs and H2O2 modulates their migration To further evaluate the effect of the pretreatment with pMSCs and H2O2 on HUVEC migration, HUVEC were treated with pMSCs in the absence or presence of 100 mM H2O2 for 24 h and then harvested, washed and used in a migration assay using the xCELLigence Real-Time Cell Analyser as described above for the migration of group 1 and in Fig. 2. After 24 h, the migration of HUVEC pretreated with CMpMSC, SFpMSC and ICpMSC was not significantly changed as compared to untreated HUVEC (Fig. 7A). The migration of HUVEC pretreated with H2O2 decreased significantly after 24 h as compared to untreated HUVEC, P < 0.05 (Fig. 7B). In contrast, the migration of HUVEC pretreated with H2O2 and CMpMSC increased significantly after 24 h (P < 0.05), while the pretreatment with H2O2 and SFpMSC or ICpMSC had no effect on HUVEC migration as compared to untreated HUVEC (Fig. 7B). In comparison with H2O2-

treated HUVEC, the migration of HUVEC pretreated with H2O2 and CMpMSC or SFpMSC increased significantly after 24 h (P < 0.05) while the pretreatment with H2O2 and ICpMSC had no effect on HUVEC migration (Fig. 7B). We also examined the migration of HUVEC exposed to conditioned medium with or without 100 mM H2O2 during the migration assay as described above for the migration group 2 and in Fig. 2. After 24 h, HUVEC migration was not affected by CMpMSC while the addition of H2O2 to HUVEC culture increased significantly the migration of HUVEC (P < 0.05) as compared to untreated endothelial cells (Fig. 7C and D). Similarly, HUVEC migration in the presence of H2O2 and CMpMSC decreased but not significantly after 24 h as compared to untreated HUVEC (Fig. 7D). In contrast, HUVEC migration in the presence of H2O2 and CMpMSC increased significantly after 24 h as compared to H2O2treated HUVEC (Fig. 7D). In addition, the migration of HUVEC in response to CMpMSC, in the absence or presence of 100 mM H2O2, was also examined by culturing HUVEC on the surface of the upper chamber, while 20% CMpMSC was added to the surface of the lower chamber of the migration plate as described above for the migration group 3 and in Fig. 2. After, 24 h, HUVEC migration in response to CMpMSC did not change significantly as compared to untreated HUVEC (Fig. 7E). In contrast, the migration of HUVEC in response to

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Fig. 6. HUVEC adhesion under the effects of pMSCs and H2O2. HUVEC pretreated with different pMSC treatments in the absence and presence of 100 mM H2O2 were cultured in a 16 well culture plate and their adhesion was then measured using the xCELLigence Real-Time Cell Analyser. After two hours, compared to untreated HUVEC, HUVEC pretreated with CMpMSC and SFpMSC showed a significant increase in their adhesion while the adhesion of HUVEC pretreated with ICpMSC decreased significantly (A). The adhesion of HUVEC pretreated with H2O2 alone, or with CMpMSC, or SFpMSC, or ICpMSC increased significantly as compared to H2O2-untreated HUVEC (B). As compared to H2O2-treated HUVEC, the adhesion of HUVEC pretreated with H2O2 and with CMpMSC or SFpMSC or ICpMSC did not significantly change (B). Each experiment was performed in triplicate using HUVEC (passage 3e5) from five independent umbilical cord tissues and pMSCs (passage 2) from five independent placentae. *P< 0.05. Bars represent standard errors.

H2O2 increased significantly as compared to untreated HUVEC, and the addition of CMpMSC decreased HUVEC migration significantly after 24 h as compared to H2O2-treated HUVEC, but did not change as compared to untreated HUVEC (Fig. 7E). 3.8. Role of antioxidant Cells employ several defense mechanisms to protect themselves from oxidative stress, such as redox cycling of glutathione [20]. To address the possibility that alteration in this defensive pathway might be responsible for the protection induced by pMSCs, we compared this pathway in endothelial cells treated with H2O2 in the presence of different treatments of pMSCs. At baseline, the level of glutathione reductase in HUVEC was 75 mU/mL ±2.87 mU/mL. Exposure of HUVEC to 100 mM H2O2 for 24 h resulted in 13.33% reduction in the level of glutathione reductase. As compared to H2O2- treated HUVEC, glutathione reductase levels were 63.33 mU/ mL ±6.09 mU/mL, 71.67 mU/mL ±1.66 mU/mL and 51.67 mU/mL ±1.66 mU/mL in HUVEC treated with H2O2 and CMpMSC, H2O2 and SFpMSC and H2O2 and ICpMSC, respectively. There were a <1% and 20.5% reduction in the levels of glutathione reductase in HUVEC treated with H2O2 and CMpMSC and H2O2 and ICpMSC, respectively while there was a 9.3% increase in glutathione reductase level in HUVEC treated with H2O2 and SFpMSC as compared to H2O2treated HUVEC. None of these changes were statistically significant, P > 0.05. These data suggest that changes in glutathione reductase level are not of a great magnitude to account for the observed effects of pMSCs on endothelial cells after their exposure to H2O2. 4. Discussion We previously reported that pMSCs have unique phenotypic and functional properties [3,6,7], which could be advantageous in modulating the inflammatory responses associated with inflammatory diseases, such as atherosclerosis [21e23]. In these diseases,

there is an increased level of inflammatory and oxidative stress mediators [14,21e24]. In this oxidative stress environment, MSCs are in close proximity to endothelial cells and can potentially repair oxidative stress-induced damage to them. In this study, we examined the effects of pMSCs on endothelial cell functional responses under a stressful environment. First, we examined the effect of different concentrations of H2O2 on pMSC viability. At 400 and 600 mM, H2O2 was toxic to pMSCs and viability reduced to 50% and 40%, respectively as determined by the Trypan blue assay. The inability of pMSCs to cope with these high H2O2 concentrations could be a consequence of their placental vascular niche, where pMSCs are in close proximity to the fetal circulation that experiences relatively low levels of inflammation and oxidative stress during pregnancy [12,13]. Similarly, bone marrow derived MSCs (BMMSCs) are usually exposed to a low level of oxidative stress in their niche and only experience increased oxidative stress following injury or disease [25]. BMMSCs undergo apoptosis after exposure to H2O2 concentrations ranging from 60 to 500 mM H2O2 [26,27], but survive at a lower concentration (20 mM H2O2) [27]. The viability of pMSCs was further confirmed by their proliferation in response to H2O2 at concentrations up to 200 mM. Above 200 mM H2O2, pMSC proliferation was significantly reduced, P < 0.05 (Fig. 3A). These data indicate that pMSCs can overcome the effects of oxidative stress and maintain their normal proliferation but they can do this only up to a certain level, after which the effects of oxidative stress will be toxic. We further confirmed the resistance of pMSCs to oxidative stress by showing the adhesion of H2O2-treated pMSCs not only to a monolayer of HUVEC, but also to a HUVEC monolayer treated with H2O2 (Fig. 3B and C). These data are consistent with previous reports demonstrating the adhesion of BMMSCs and hematopoietic stem cells to endothelial cells pretreated with TNF-a and H2O2, respectively [28,29]. Our data indicate that the initial stage of a cell-based therapy, where intravenously injected pMSCs interact with the vessel wall in the course of transmigration, is not affected by oxidative stress. This is

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Fig. 7. HUVEC migration was measured using the xCELLigence Real-Time Cell Analyser. After 24 h, the migration of HUVEC pretreated with CMpMSC, or SFpMSC, or ICpMSC, was not significantly changed as compared to untreated HUVEC, P > 0.05 (A). After 24 h, compared to untreated HUVEC, the migration of HUVEC pretreated with 100 mM H2O2 or with H2O2 and CMpMSC significantly decreased and increased, respectively, but not after treatment with H2O2 and SFpMSC or ICpMSC, P > 0.05 (B). In comparison with H2O2-treated HUVEC, the migration of HUVEC pretreated with H2O2 and CMpMSC, or SFpMSC, significantly increased after 24 h but not after treatment with H2O2 and ICpMSC, P > 0.05 (B). The migration of HUVEC cultured with CMpMSC was not affected after 24 h while the migration of HUVEC cultured with 100 mM H2O2 alone or with CMpMSC, increased significantly, and showed no change, respectively, as compared to untreated HUVEC (C and D). As compared to H2O2-treated HUVEC, HUVEC migration in the presence of H2O2 and CMpMSC decreased significantly after 24 h (D). The migration of HUVEC in response to CMpMSC or 100 mM H2O2 added to the lower chamber of the migration plate did not change and increased significantly, respectively, as compared to untreated HUVEC after 24 h (E and F). As compared to H2O2-treated HUVEC, HUVEC migration in the presence of H2O2 and CMpMSC decreased significantly after 24 h (F) but did not change as compared to untreated HUVEC (E). Each experiment was performed in triplicate using HUVEC (passage 3e5) from five independent umbilical cord tissues and pMSCs (passage 2) from five independent placentae.*P < 0.05. Bars represent standard errors.

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an important finding because the adhesion of MSCs is an essential step for successful engraftment during transplantation [30,31]. Next, we examined the effect of pMSCs on endothelial cell functions. Cellular contact with pMSCs (ICpMSC) and secreted factors produced by unstimulated pMSCs (CMpMSC), significantly increased HUVEC proliferation except at a low ratio of pMSCs (10HUVEC:1pMSC), the proliferation increased but not significantly (Fig. 4A and B). Previously, it was shown that H2O2 can induce endothelial cell proliferation [32]. Similarly, we found that H2O2 can induce HUVEC proliferation, and this was reversed by the addition of pMSCs at a low ratio (10HUVEC:1pMSC), and at high concentrations of CMpMSC (25% pMSCs) (Fig. 4C and D). In contrast, at higher ratios of pMSCs and lower concentrations of CMpMSC, the proliferation of endothelial cells treated with H2O2 increased significantly, P < 0.05. Our data suggest that pMSCs have variable effects of either stimulating or inhibiting endothelial cell growth depending on the dose of pMSCs. Therefore, the dose of pMSCs may be a determining factor in activating either their proliferative or anti-proliferative functional activities, and a future study is necessary to elucidate this. The effects of pMSCs on H2O2- treated HUVEC were not associated with changes in glutathione reductase activity (standard antioxidant enzyme). We also examined the reversibility of the induced proliferation in endothelial cells by CMpMSC (25% CMpMSC) or by cellular contact at 10HUVEC:1pMSC ratio (Fig. 4A and B). The pretreatment of HUVEC with 25% CMpMSC or by cellular contact reduced and increased HUVEC proliferation, respectively (Fig. 5AeC). These data demontsrate that the pro-proliferative signal induced in HUVEC by CMpMSC is reversiable, while the pro-proliferative signal induced in HUVEC by ICpMSC is irreversible. In contrast, endothelial cells pretreated with pMSCs in the soluble factor cultured system (SFpMSC) showed no significant changes in their proliferative activity (Fig. 5AeC). This suggests that paracrine communication can maintain HUVEC proliferation at normal levels (Fig. 5AeC). However, this and other mechanisms underlying the pro- and antiproliferative effects of pMSCs on endothelial cells need to be elucidated in future functional studies. We also examined the revesibility of the proliferative effect of H2O2 on HUVEC. After the removal of H2O2, HUVEC proliferation was reduced (Fig. 5DeF). This indicates that the pro-proliferative effect of H2O2 on HUVEC is reversible. In addition, we also found that HUVEC pretreated with H2O2 in the presence of CMpMSC or ICpMSC had reduced and enhanced proliferation activities, respectively (Fig. 5F). These results indicate that the proproliferative and anti-proliferative effects of CMpMSC and ICpMSC on H2O2 induced HUVEC proliferation is reversible. Interestingly, the paracrine communication between pMSCs and HUVEC, in the presence of H2O2, induced a pro-proliferative signal in HUVEC, because after removing the effects of H2O2 and pMSCs, HUVEC proliferation increased significantly (Fig. 5E and F). Importantly, in stress environments induced by H2O2, pMSC effects on HUVEC proliferation were lower than their effects on HUVEC cultured in H2O2 free conditions (Fig. 5DeF). These data indicate that in inflammatory diseases such as atherosclerosis where endothelial cell proliferation is altered due to oxidative stress, pMSCs can maintain and protect the functions of endothelial cells. Thus suggesting the therapeutic potential of pMSCs in inflammatory diseases. However, this requires further investigation using an animal model of atherosclerosis or any other inflammatory diseases. In this study, we also examined the adhesion of HUVEC under the effect of pMSCs. After the removal of pMSCs, HUVEC pretreated with CMpMSC and SFpMSC showed increased adhesion, while pretreatment with ICpMSC decreased HUVEC adhesion (Fig. 6A). Importantly, in a stressful environment induced by H2O2, HUVEC

pretreated with H2O2 showed increased adhesion, and pMSCs could not reverse this effect (Fig. 6B). It is known that H2O2 induces HUVEC adhesion through the upregulation of ICAM-1 by IL-1b, a cytokine which is expressed by pMSCs and therefore, it is possible that pMSCs may use a similar mechanism on HUVEC adhesion [33e35]. However, a future functional study is necessary to elucidate this mechanism. Adhesion of endothelial cells is an important initial step in angiogenesis (blood vessel formation), which is followed by migration [36]. Here, we report that pMSCs had no significant effect on HUVEC migration when CMpMSC was added into the upper or lower chambers (Fig. 7C and E). However, these treatments reversed the stimulatory effect of H2O2 on HUVEC migration (P < 0.05), and this anti-migratory effect of pMSCs on HUVEC was able to reduce HUVEC migration to normal levels (Fig. 7D and F). In addition, after removing the effects of pMSCs, HUVEC migrated normally (Fig. 7A) but after removing the effect of H2O2, HUVEC showed reduced migration ability, and this was reversed by the different treatments of pMSCs (Fig. 7B). However, pMSC secreted factors showed a stronger effect than cellular contact on the reversibility of HUVEC migration (Fig. 7B). These data demonstrate the ability of pMSCs to reduce the stimulatory effect of H2O2 on HUVEC migration, and suggest a protective effect of pMSCs on endothelial cell migration induced by H2O2. In inflammatory diseases, such as atherosclerosis, H2O2 induces endothelial cell migration [37], and therefore, pMSCs could potentially be used as a therapeutic tool to treat atherosclerosis through the inhibition of endothelial cell migration. 5. Conclusion This study shows for the first time that pMSCs can not only maintain their functions in environments of oxidative stress induced by H2O2, but pMSCs can also reverse the effects of H2O2 on endothelial cell proliferation and migration. This suggests that pMSCs could be used as a therapeutic tool in inflammatory diseases to reverse the functional effects of stress on endothelial cells. Authors' contributions Idea and design: MH. Abumaree. Data acquisition: M. Hakami, MA. Alshabibi, and MH. Abumaree. Data analysis and interpretation: MH. Abumaree, FM. Abomaray and B. Kalionis. Laboratory assays: M. Hakami, MA. Alshabibi and MH. Abumaree. Writing of manuscript and revising it critically: MH. Abumaree, FM. Abomaray, B. Kalionis, MA. Al Jumah, AS. AlAskar. All authors read and approved the final manuscript. Conflict of interests The authors declare that there is no conflict of interests regarding the publication of this paper. Acknowledgments We thank the staff and patients of the Delivery Unit, King Abdul Aziz Medical City for their help in obtaining placentae. This study was supported by grants from King Abdullah International Medical Research Centre (Grant No. RC12/117). References [1] F.M. Abomaray, et al., Phenotypic and functional characterization of mesenchymal stem/multipotent stromal cells from decidua basalis of human term placenta, Stem Cells Int. 2016 (2016) 5184601.

Please cite this article in press as: M.H. Abumaree, et al., Human chorionic villous mesenchymal stem/stromal cells modify the effects of oxidative stress on endothelial cell functions, Placenta (2017), http://dx.doi.org/10.1016/j.placenta.2017.05.001

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Please cite this article in press as: M.H. Abumaree, et al., Human chorionic villous mesenchymal stem/stromal cells modify the effects of oxidative stress on endothelial cell functions, Placenta (2017), http://dx.doi.org/10.1016/j.placenta.2017.05.001