Exosomal Signaling during Hypoxia Mediates Microvascular Endothelial Cell Migration and Vasculogenesis

Vasculogenesis and angiogenesis are critical processes in fetal circulation and placental vasculature development. Placental mesenchymal stem cells (pMSC) are known to release paracrine factors (some of which are contained within exosomes) that promote angiogenesis and cell migration. The aims of this study were: to determine the effects of oxygen tension on the release of exosomes from pMSC; and to establish the effects of pMSC-derived exosomes on the migration and angiogenic tube formation of placental microvascular endothelial cells (hPMEC). pMSC were isolated from placental villi (8–12 weeks of gestation, n = 6) and cultured under an atmosphere of 1%, 3% or 8% O2. Cell-conditioned media were collected and exosomes (exo-pMSC) isolated by differential and buoyant density centrifugation. The dose effect (5–20 µg exosomal protein/ml) of pMSC-derived exosomes on hPMEC migration and tube formation were established using a real-time, live-cell imaging system (Incucyte™). The exosome pellet was resuspended in PBS and protein content was established by mass spectrometry (MS). Protein function and canonical pathways were identified using the PANTHER program and Ingenuity Pathway Analysis, respectively. Exo-pMSC were identified, by electron microscopy, as spherical vesicles, with a typical cup-shape and diameters around of 100 nm and positive for exosome markers: CD63, CD9 and CD81. Under hypoxic conditions (1% and 3% O2) exo-pMSC released increased by 3.3 and 6.7 folds, respectively, when compared to the controls (8% O2; p<0.01). Exo-pMSC increased hPMEC migration by 1.6 fold compared to the control (p<0.05) and increased hPMEC tube formation by 7.2 fold (p<0.05). MS analysis identified 390 different proteins involved in cytoskeleton organization, development, immunomodulatory, and cell-to-cell communication. The data obtained support the hypothesis that pMSC-derived exosomes may contribute to placental vascular adaptation to low oxygen tension under both physiological and pathological conditions.

Mesenchymal stem cells are archetypal multipotent progenitor cells that display fibroblastic morphology and plasticity to differentiate into diverse cell types including: osteocytes, adipocytes and endothelial cells. MSCs are isolated from various sources including bone marrow (principal source), adipose tissue and placenta. Within the human placenta, MSC have been isolated from umbilical cord blood and chorionic villi [19,20] displaying phenotypes comparable to those isolated from bone marrow, including surface antigen expression (CD45 2 , CD14 2 , CD19 2 , CD80 + , CD86 + , CD40 + and B7H2 + ) and the capacity to differentiate into multiple linages in vitro. MSC have been implicated in wound healing and display the ability to migrate to sites of injury and engage in tissue repair and regeneration of bone, cartilage, liver tissue or myocardial cells [21,22]. MSC modulate immune responses in collagen disease, multiple sclerosis and transplants bone marrow and contribute to vasculogenesis, angiogenesis and endothelial repair [23], processes that are fundamental for tissue repair. MSC affect tissue repair through the release of paracrine mediators [24][25][26][27] including exosomes [28].
MSCs are present in the first trimester human placenta, however, their role in placental vascular development remains to be established. During early pregnancy, the placental vasculature develops under hypoxic conditions. During the first trimester, placental PO 2 is , 2.6% before placento-maternal perfusion is established. At around 12 weeks of pregnancy, the placenta is perfused with maternal blood and PO 2 increases to , 8% [29,30].
There is a paucity of information about the role of MSC and, in particular, the release of exosomes from MSC during this critical period of vascular development. Of note, however, Hofmann et al., [31], recently proposed that exosomes may function as part of an oxygen sensing mechanism that promotes vasculogenesis and angiogenesis. We hypothesize that: (i) exosomes released by pMSC act paracellularly to promote cell migration and angiogenesis within the placental villus tree; and (ii) that the release of exosomes from pMSC is responsive to changes in oxygen tension.
The aim of this study, therefore, was to establish the effect of oxygen tension on the release of exosomes from pMSC; and the effects of pMSC-derived exosomes on the migration and angiogenic tube formation of human placental microvascular endothelial cells (hPMEC). pMSC-derived exosomes promote hPMEC cell migration and tube formation in vitro. The release and bioactivity of pMSC-derived exosomes is oxygen tension dependent. The data obtained are consistent with the hypothesis that pMSC-derived exosomes are released under hypoxic conditions and promote angiogenesis within the developing placenta.

First Trimester and Term Placental Collection
Tissue collection was approved by the Human Research Ethics Committees of the Royal Brisbane and Women's Hospital, and the University of Queensland (HREC/09/QRBW/14). All experiments and data collection and analyses were conducted with an ISO 17025 and 21 CFR part 11 conforming laboratory environment. Written informed consent was obtained from women for the use of placental tissue for research purposes after clinically indicated termination of pregnancy in compliance with national research guidelines.

Isolation of Placental Mesenchymal Stem Cells
pMSC were isolated, from placental villi by enzymatic digestion using protocols adapted from Steigman & Fauza [32]. Briefly, placental chorionic villi (n = 6; 8-12 weeks gestational age) were separated from the remainder of the placenta unit and were washed in PBS. The villi were minced into small pieces and were transferred in to 50 ml tubes. The tissues were enzymatic digestion with dispase (2.4 U/ml) and collagenase (240 U/ml) made in PBS. The tissues were digested for 1 hr at 37uC on a rocker. The single cell suspension was then filtered through a 100 mm mesh into a new tube. The cells were centrifuged for 15 mins at 5006g at RT and the pellet was resuspended in 10 ml cDMEM. pMSC were cultured in Dulbecco's modified Eagle's medium (DMEM) (Life Technologies TM , Carlsbad, CA), supplemented with 10% fetal bovine serum, 100 IU/mL penicillin, and 100 mg/mL streptomycin (Life Technologies TM ), at 37uC with 5% CO2. pMSC were characterised by well-established cell surface markers. All cells used in this study were passaged less than 6.

MSC Differentiation Assays
The differentiation potential of placental villi MSC was established according to previously published methods [33]. For adipogenesis, 2610 5 pMSC were seeded in 6 well plates until confluent and differentiation was induced by indomethacin (60 mM) dexamethasone (1 mm), insulin (5 mg/ml) and isobutylmethylxanthine (IBMX) (0.5 mM). After 21 days, cells were fixed with 10% formalin and stained with Oil Red O. Adipogenic differentiation was determined by the appearance of Oil Red O. Osteogenic differentiation was induced by culturing 3610 5 cells in 6 well plates in the presence of osteogenic induction media containing dexamethasone (0.1 mM), b-glycerol phosphate (10 mM) and L-ascorbate-2-phosphate (0.2 mM) for 21 days. Cells were fixed in 10% formalin and stained with Alizarin red. Differentiation was determined by the appearance of red deposits, representing areas of mineralized calcium. All reagents were from Sigma-Aldrich. Staining for both adipogenic and osteogenic assays was visualized using bright field phase contrast microscopy.

Flow Cytometry
The expression of cell surface and intracellular antigens was assessed by flow cytometry (FACScalibur TM , Becton Dickinson, San Jose, CA, USA). To identify intra-cellular antigens, cells were detached, blocked with 1% bovine serum albumin (BSA; Sigma, St. Louis, MO) in phosphate buffered saline (PBS, Life Technologies TM ) then fixed with 0.01% paraformaldehyde (PFA) (Sigma) and permeabilized with 0.5% Triton X-100. To characterize the expression of cell surface and intracellular antigens, cells were detached and blocked with 1% BSA and incubated with specific anti-human primary antibodies, either conjugated with PE, FITC or PE-Cy5 or unconjugated. For unconjugated antibodies, cells were subsequently washed with 1% BSA and incubated with secondary goat anti-murine IgM PE (Santa Cruz BiotechnologyH, Santa Cruz, CA, USA). All samples were analyzed in triplicate by FACScalibur TM flow cytometry (Becton Dickinson). Positive controls were hESC and negative controls were IgG or IgM primary antibody-specific isotype controls.

Hypoxia
The effects of oxygen tension on the release of exosomes from pMSC were assessed by incubating cells for 48 h (in exosome-free Figure 2. The level of pMSC-derived exosomes compared to low oxygen tension. Exosomes were isolated from pMSC supernatant exposed to 1%, 3% or 8% oxygen per 48 h. (A) Levels of exosomes are presented as protein concentration from 1610 6 pMSC cell. (B) Same volume of exosome pellet loaded and analyzed by western blot for CD63 and b-actin in exosome from pMSC and cells, respectively.  culture medium) under an atmosphere of 5% CO 2 -balanced N 2 to obtain 1%, 3% or 8% O 2 (pO 2 ,6.75, ,20.25 or ,54 mmHg, respectively) in an automated PROOX 110-scaled hypoxia chamber (BioSpherics TM , Lacona, NY, USA). Cell number and viability was determined after each experimental treatment by Trypan Blue exclusion and CountessH Automated cell counter (Life Technologies TM ). Proliferation data was collected for all the experimental conditions and in particular to assess the effects of proliferation hypoxic conditions using a real-time cell imaging system (IncuCyte TM live-cell ESSEN BioScience Inc, Ann Arbor, Michigan, USA). In all experiments, viability remained at .95%. Incubation media pO 2 and pH were independently confirmed using a blood gas analyzer (RadiometerH, Brønshøj, Denmark) and NeoFox oxygen probe (Ocean Optics TM , Dunedin, FL, USA). HIF expression was used in Western blot analysis as a positive control for hypoxia in MSC (data not show).

Isolation and Purification of pMSC Exosomes
Exosomes were isolated from cell-free pMSC as previously described [35]. In brief, pMSC-conditioned media was centrifuged at 3006g for 15 min, 20006g for 30 min, and 120006g for 45 min to remove whole cells and debris. The resultant supernatant were passed through a 0.22 mm filter sterilize Steritop TM (Millipore, Billerica, MA, USA) and then centrifuged at 100,0006g for 75 min (Thermo Fisher Scientific Ins., Asheville, NC, USA, Sorvall, SureSpin TM 630/36, fixed angle rotor). The pellet was resuspended in PBS, washed and re-centrifuged (100,0006g, 75 min). The pellet was resuspended in PBS, layered on a cushion of 30% (w/v) sucrose and centrifuged at 110,000 g for 75 min. The fraction containing pMSC exosomes (,3.5 ml, 1.1270 density using OPTi digital refractometer (Bellingham + -Stanley Inc., Lawrenceville, GA, USA) was recovered with an 18-G needle and diluted in PBS, and then ultracentrifuged at 110 0006g of 70 min. Recovered exosomes were resuspended in 50 ml PBS and their protein contents were determined using the Bradford assay (Bio-Rad DC) [35]. Exosome samples (5 ml) were prepared by adding RIPA buffer (50 mM Tris, 1% Triton6100, 0.1% SDS, 0.5% DOC, 1 mM EDTA, 150 mM NaCl, protease inhibitor) directly to exosomes suspended in PBS and sonicated at 37uC for 15 s three times to lyse exosome membrane and solubilise the proteins. Bovine serum albumin (BSA) diluted in RIPA buffer and PBS mixture (1:1) were prepared as protein standards (0, 200, 400, 600, 800, 1000, 1500 mg/mL). Standards and samples (exosomes) were transferred to 96-well plates and procedures outlined by the manufacture were followed. In brief, alkaline copper tartrate solution (BIO-RAD, USA) and dilute Folin Reagent (BIO-RAD, USA) were added to the samples and incubated for 15 min. The absorbance was read at 750 nm with Paradigm Detection Platform (Beckman Coulter, USA).

Transmission Electron Microscopy
The exosome fraction isolated by differential and buoyant density gradient centrifugation was assessed by transmission electron microscopy. Exosome pellets (as described above) were fixed in 3% (w/v) glutaraldehyde and 2% paraformaldehyde in cacodylate buffer, pH 7.3. Five microlitres of sample was then applied to a continuous carbon grid and negatively stained with 2% uranyl acetate. The samples were examined in an FEI Tecnai 12 transmission electron microscope (FEI TM , Hillsboro, Oregon, USA).

Migration and Tube Formation Assay
To assess the effect of exosomes on endothelial cell tube formation, hPMEC were cultured in 96 or 48-well culture plates (Corning Life Science, Tewksbury, MA, USA) according to the manufacturer's instructions and visualized using a real-time cell imaging system (IncuCyte TM live-cell ESSEN BioScience Inc, Ann Arbor, Michigan, USA). Cells were imaged every hour to monitor treatment-induced cell migration, tube formation, confluence and morphologic changes. Cell migration was assessed by scratch assays, in which, hPMEC were grown to confluence and then a scratch was made using a 96-pin WoundMaker TM . The wells were  washed with PBS to remove any debris and incubated in the presence of 0 (control) 5, 10 or 20 mg protein/ml of pMSC-derived exosome isolated from cells cultured under 1%, 3% or 8% O 2 . Wound images were automatically acquired and registered by the IncuCyte TM software system. Typical kinetic updates were recorded at 2 h intervals for the duration of the experiment (48 h). The data were then analysed using an integrated metric: Relative Wound density. For the tube formation assay, 48-well culture plates on ice were incubated with 144 ml of chilled BD Matrigel matrix (10 mg/ml) per well at 37uC for 60 min. hPMEC (6610 4 ) were resuspended in culture medium with the indicated concentration of pMSC-derived exosomes (5, 10 or 20 mg/ml) and incubated for up to 24 h at 37uC. The number of networks formed was determined using the IncuCyte TM system.

Proliferation Assay
A real-time imaging system (IncuCyteTM) was used to measure cell proliferation using non-label cell monolayer confluence approach. pMSC confluence was measure before and after the treatment (1%, 3% and 8% O 2 , 48 h). IncuCyteTM provide the capability to acquire high quality, phase-contrast images and an integrated confluence metric as a surrogate for cell number [36]. We used similar approach for to determine the effect of pMSCderived exosomes on hPMEC proliferation during the migration assay.

Proteomic Analysis of Exosomes by Mass Spectrometry (MS)
Isolated exosomes were solubilised in 8 M urea in 50 mM ammonium bicarbonate, pH 8.5, and reduced with DTT for 1 h. Proteins were then alkylated in 10 mM iodoacetic acid (IAA) for 1 h in the dark. The sample was diluted to 1:10 with 50 mM ammonium bicarbonate and digested with trypsin (20 mg) at 37uC for 18 h. The samples were desalted by solid phase extraction using a STAGE tip protocol (Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics). The eluted peptides were dried by centrifugal evaporation to remove acetonitrile and redissolved in Solvent A. The resulting peptide mixture was analysed by Liquid Chromatography (LC)/Mass Spectrometry (MS) LC-MS/MS on a 5600 Triple TOF mass spectrometer (AB Sciex, Framingham, U.S.A.) equipped with an Eksigent Nanoflow binary gradient HPLC system and a nanospray III ion source. Solvent A was 0.1% formic acid in water and solvent B was 0.1% fomic acid in acetonitrile. MS/MS spectra were collected using Information Dependent Acquisition (IDA) using a survey scan (m/ z 350-1500) followed by 25 data-dependent product ion scans of the 25 most intense precursor ions. The data were searched using MASCOT and Protein Pilot search engines.

Functional Analysis of Exosome Proteome
Proteins identified by MS/MS were analyzed by PANTHER (Protein Analysis THrough Evolutionary Relationships; http:// www.pantherdb.org). This software allows the prediction of classify proteins (and their genes) in order to facilitate highthroughput analysis. The classified proteins were classified according to their biological process and molecular function. Differentially expressed proteins were analyzed further by bioinformatic pathway analysis (Ingenuity Pathway Analysis [IPA]; Ingenuity Systems, Mountain View, CA; www.ingenuity. com).

Statistical Analysis
Data are represented as mean 6 SEM, with n = 6 different cells culture (i.e. biological replicates) of pMSC isolated from first trimester pregnancies and n = 4 different cell cultures (i.e biological replicates) of hPMEC isolated from term placenta. Comparisons between two and more groups were performed by means of     Table 2. List of proteins identified in exosomes from pMSC exposed to different oxygen level.

Characterization of Exosome from Placental Mesenchymal Stem Cells
Cell surface protein expression by pMSC was characterized using flow cytometric analysis. pMSC were labelled with monoclonal antibodies specific for markers indicated in each histogram ( Figure 1A). pMSC isolated from first trimester placental villi were positive for CD29 + , CD44 + , CD73 + , CD90 + , CD105 + (top panel) and negative for hematopoietic and endothelial markers: CD11b -, CD14 2 , CD31 2 , CD34 2 , CD45 2 (lower panel). When pMSC were stimulated under adipogenic and osteogenic conditions, they showed characteristic of adipocytes ( Figure 1B1; formation of lipid vacuoles) and osteoblast cells ( Figure 1B2; red deposits, representing areas of mineralized calcium), respectively. The exosomal particulate fraction isolated from pMSC was examined under transmission electron microscopy. Exosomes were identified as small vesicles between 40-100 nm in a cup-shaped form ( Figure 1C). The particulate fraction was further characterized by the expression of specific exosome markers: CD63; CD9; and CD81 by Western blot analysis ( Figure 1D).

Effect of Oxygen Tension on Exosome Release
To determine the effects of oxygen tension on the release of exosomes from pMSC, cells were incubated under atmospheres of 1%, 3% or 8% O 2 and the exosomes released were quantified (as total exosomal protein mg/10 6 pMSC). Under these conditions,  Figure 2A). Furthermore, the relative abundance of the specific exosome marker CD63 in this particulate fraction displayed a similar inverse correlation to oxygen tension, as assessed by Western blot ( Figure 2B). During the time course of these experiments, cell proliferation was not significantly affected by oxygen tension (i.e. 1%, 3% or 8% O 2 ) ( Figure 2C). The effect of oxygen tension on exosome release was not associated with a decrease in cell viability ( Figure 2D).

Effect of pMSC-derived Exosomes on Cell Migration
The effects of exosomes (5, 10 or 20 mg protein/ml) isolated from pMSC cultured under 1%, 3% or 8% O 2 on hPMEC migration are presented in Figure 3 A, C and E. pMSC exosomes significantly increased hPMEC migration in a time-and dosedependent manner (p,0.005, n = 6). In addition, the effect on hPMEC migration was greater when exosomes were prepared from cells cultured under low oxygen tensions. Using the IncuCyte live cell imaging enabled non-invasive system, the cell proliferation based on area metric (confluence) was measurement. Exosomes isolated from pMSC cultures under 1% O 2 increased significantly the hPMEC proliferation in ,1.18-fold and ,1,25-fold with 10 mg/ml and 20 mg/ml, respectively ( Figure 4B). Furthermore, exosomes isolated from pMSC cultured under 3% and 8% O2 increased hPMEC proliferation in ,1.21-fold and ,1,18-fold using 20 mg/ml, respectively. We did not find significant effect of FBS-derived exosome on hPMEC migration and proliferation. Half-maximal stimulatory time (ST 50 ) and half-maximal stimulatory concentration (SC 50 ) values are presented in Table 1.

Effect of pMSC-derived Exosomes on in vitro Tube Formation
In vitro angiogenic tube formation assays were used as a surrogate endpoint to assess the angiogenic effects of pMSCderived exosomes. pMSC-derived exosomes significantly increased tube formation by hPMEC in a dose-and time-dependent manner when compared to vehicle-treated cells (p,0.005, Figure 5A) and inversely correlated to oxygen tension. In addition, exosomeinduced tube formation was significantly greater when exosomes were prepared from cells grown under low oxygen tensions ( Figure 5A). Half-maximal stimulatory time was 4.7160.66, 11.5060.25 and 35.9660.5 mg/ml for exosomes treatment from pMSC exposed to 1%, 3% and 8% oxygen, respectively.

Proteomic Analysis of pMSC-derived Exosome
Mass spectrometry analysis identified over 200 exosomal proteins (Table 2). Data were subjected to ontology and pathway analysis using Panther and Gene Ontology algorithms and classified based on biological process and molecular function ( Figure 6). In biological process, the most clusters identified were: cellular processes, cell communication, developmental and transport ( Figure 6A). In molecular functions, the proteins related to binding and catalytic activity were the greatest recognized ( Figure 6B). IPA analysis identified 157 proteins only present in exo-pMSC-1%O 2 versus 34 and 37 individual proteins present in exo-pMSC-3%O 2 and exo-pMSC-8%O 2 , respectively . ( Figure 7A). Finally, the canonical pathways associated with our proteins defined by IPA Core analysis and related with cell migration were: actin cytoskeleton signaling, growth hormone signaling, clathrinmediated endocytosis signaling, and VEGF signaling ( Figure 7B-E). Furthermore, canonical pathways were associated with highest protein number in exosomes isolated from pMSC exposed to 1% O 2 versus 3% and 8% O 2 .

Discussion
Mesenchymal stem cells are present in the human placenta during early pregnancy. During early pregnancy, placental vasculogenesis and angiogenesis proceed under low oxygen conditions prior to the establishment of a materno-placental perfusion. The role of MSC in directing and promoting placental vascular development remains to be clearly elucidated. The aim of this study was to establish the effects of oxygen tension on the release of exosomes from pMSC and to determine the effects of pMSC exosomes on endothelial cell migration and tube formation. The data obtained in the study are consistent with the hypothesis that the release of exosomes from pMSC is increased in hypoxic conditions and that pMSC exosomes promote endothelial cell migration and tube formation. Based on the data obtained, we suggest that pMSC exosomes contribute to the development of new vessels and promote angiogenesis within the placenta under low oxygen conditions. During early pregnancy this occurs as a physiological and developmental process. In pathological pregnancies characterized by compromized placental perfusion and ischaemia, such as preeclampsia and intrauterine growth restriction, we propose that pMSC may also increase exosome release as an adaptive response.
Germane to any study seeking to elucidate the physiological or pathophysiological role of exosomes is their specific isolation. Several methods for isolating exosomes have been developed and partially characterized. These methods are primarily based on particle size and density. By definition, exosomes are nanovesicles with a diameter of 30-100 nm, a buoyant density of 1.12 to 1.19 g/ml and express characteristic cell-surface markers. In this study, pMSC exosomes were isolated by differential centrifuge and sucrose gradient purification and were characterized by a diameter of 50 nm, a buoyant density of 1.1270 g/ml, and expressed exosome-specific cell surface markers.
Under hypoxic conditions (1% or 3% O 2 ), pMSC exosome release increased by up to 7-fold compared to cells incubated under normoxic conditions (8% O 2 ). These data are consistent with the effects of hypoxia on the release of exosomes from umbilical cord (UC)-derived MSCs, where low oxygen tension increases exosome release by , 5.6-fold [37]. Hypoxia also has been reported to increase the release of exosomes from breast cancer cell lines (MCF7, SKBR3, and MDA-MB 231), squamous carcinoma cells (A431 cells) [26] and cardiac myocytes [38]. The mechanism by which hypoxia induces exosome release remains to be clearly established.
Recent evidence suggests that increased release of exosomes from breast cancer cells under hypoxic condition may be mediated by transcriptional factor HIF-1a [39]. In this study, the authors also observed higher expression of miR-210 in exosomes isolated from cancer cells exposed to hypoxia compared to normaxia cellderived exosomes. Exosomal miR-210 from metastatic cancer cells enhances endothelial cell angiogenesis [40].
In MSCs, HIFs have been reported to promote MSC-mediated angiogenic effect on endothelial cells through the release of interleukin 8, VEGF and other growth factors [41]. It has been demonstrated that the secretion of soluble VEGF requires functional ADP-ribosylation factor 6 (Arf6) [42]. Interestingly, Arf6 is expressed on the membrane of exosomes and may promote exosome release [43]. An association between VEGF and Arf6 within exosomes, however, has not yet been demonstrated. Similarly, HIFs may contribute to the hypoxia-induced release of exosomes from pMSC observed in this study.
Previous studies have established that MSC promote angiogenesis via paracrine mechanisms [44]. The possible contribution of exosomes in mediating such paracrine actions has not been established. It is likely that exosomes were present (and not accounted for) in all conditioned media previously used to establish such paracrine effects. In this study, exosomes were isolated from pMSC, promoting hPMEC cell migration and tube formation. This effect was enhanced when pMSC were cultured under hypoxic conditions. Previously, , demonstrated that exosomes released from UC-MSC are internalized into umbilical cord endothelial cells and enhance in vitro the proliferation and network formation in a dose-dependent manner [37]. Interestingly, pMSC have ,3.2-fold higher than that UC-MSC migration capacity [20]. Recently, Mineo et al., reported that the effect of exosomes on angiogenesis involves the Src family of kinases [45]. In addition, the role of Src family members in angiogenesis, promotion of tube formation and prevention of their regression has been reported [46,47]. Recent commentary, suggests that mesenchymal stem cells-derived exosomes may not only afford therapeutic opportunities in regenerative medicine to repair damaged tissue but also in the cell-specific delivery of anticancer agents [48].
The exosomal content is highly dependent on the cell type and pre-conditioning. One of the first exosomal proteomes characterized was from mesothelioma cells, in which 38 different proteins were identified [49]. Studies in cancer cells show the great variability of proteins expressed in exosomes [50][51][52][53][54]. Supporting our results, exosomes isolated from a human first trimester cell line (Sw71) Atay et al., using an ion trap mass spectrometry approach, identified proteins implicated in a wide range of cellular processes including: cytoskeleton structure, ion channels, lysosomal degradation, molecular chaperones, amino-acid metabolism, carbohy-drate metabolism, lipid metabolism, regulatory proteins, mRNA splicing, immune function and others [55]. Our study provides the first extensive analysis of the proteome of the exosomes derived-MSC primary culture, highlighting the extent of putative functional interactions that may be mediated by exosomes.
Endothelial cell migration requires the initiation of numerous signaling pathways that remodels cytoskeleton. Also, actin and related proteins of cytoskeletal organization are critical for cell motility and migration. From the canonical pathway analysis, we found significantly more proteins associated with actin cytoskeleton, growth hormone, and VEGF signaling in exosomes isolated from pMSC exposed to 1% O 2 compare to 3% or 8% O 2 . Likewise, clathrin-mediated endocytosis signaling was enhanced, possibly increasing the exosome uptake of target cells., Cell migration, however, is the final functional outcome of multiple pathways and the involvement of other regulatory moieties (e.g. miRNA) cannot be negated.
In summary, pMSC isolated from first trimester placenta release exosomes in response to decreased oxygen tension. pMSC exosomes stimulate microvascular endothelial cells migration in a concentration and oxygen-dependent manner, and promote vascular network formation. The data obtained in this study are consistent with the hypothesis that under normal developmental conditions, pMSC promote vasculogenesis and angiogenesis within the early pregnancy placenta via a mechanism(s) involving exosomal trafficking to endothelial cells. We further suggest that in pathological pregnancies associated with under perfusion of the placenta, such as those complicated by pre-eclampsia and intrauterine growth restriction, increased release of exosomes from pMSC may occur as an adaptive response.