Exosomes from hypoxia increases microvascular tube formation in a dose-dependent manner.

<p>hPMEC were incubated in Matrigel in absence or presence of different exosomal protein concentration from pMSC exposed to 1%, 3% or 8% O₂. (A) Quantitative analysis of the total tube formation. (B) Concentration response from data in A. insert: half-maximal stimulatory concentration (SC₅₀) at 16 h. Values are mean ± SEM. In A, *<i>*p</i><0.001 versus all condition; <i>*p</i><0.005 versus corresponding values in 5 µg/ml, ^†<i>p</i><0.005 versus corresponding values in 10 or 5 µg/ml. In B, <i>*P</i><0.005 versus all values; ^†<i>p</i><0.005 versus values in 8% O₂.</p

Characterisation of exosomes from placental mesenchymal stem cell (pMSC).

<p>Cells were isolated from chorionic villi obtained from first trimester pregnancy and cultured under standard conditions. Exosomes were isolated from pMSC supernatant as was indicated in Methods. (A) Representative flow cytometry histogram of pMSC labeled with positive markers such as CD29, CD44, CD73, CD90 and CD105 (top panel) or negative markers such as CD11b, CD14, CD31, CD34 and CD45 (bottom panel). Black solid peaks represent the isotype controls and the red solid peak represents the marker indicated. (B) Mulit differntiation potential of first trimester placental chorionic villi. 1, Adipogenesis was determined using oil red O staining of lipid droplets after 21 days in adipogenic media. 2, Osteogenesis was determined using alizarin red staining for the mineral matrix deposition after 21 days in osteogenic media. (C) Electron micrograph of exosomes isolated by ultracentrifuge from pMSC. (D) pMSC were exposed to 1%, 3% or 8% O2 during 48 hours and then exosomes proteins were isolated. Samples in each condition were analyzed by western blot after the separation of 20 ug of exosomes protein (same amount of exosome protein lead) for the presence of CD63, CD9 and CD81. In B, <i>Scale bar</i> 100 nm.</p

List of proteins identified in exosomes from pMSC exposed to different oxygen level.

<p>List of proteins identified in exosomes from pMSC exposed to different oxygen level.</p

The level of pMSC-derived exosomes compared to low oxygen tension.

<p>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 1×10⁶ pMSC cell. (B) Same volume of exosome pellet loaded and analyzed by western blot for CD63 and β-actin in exosome from pMSC and cells, respectively. Lower panel: CD63/β-actin ratio densitometries from data in top panel normalized to 1 in 1% O₂. (C) Effect of low oxygen tension on pMSC proliferation. (D) Trypan blue dye exclusion test to show residual pMSC cell viability exposed to 1%, 3% or 8% O₂. Values are mean ± SEM. In A and B, <i>*P</i><0.001 versus all condition; ^†<i>p</i><0.001 versus 8% O₂.</p

Kinetic characteristic of exosome effects on hPMEC migration.

<p>The effect of exosomes isolated from pMSC-conditioned media on hPMEC <i>in vitro</i> migration. Data are expressed as half-maximal Stimulatory Time (<i>ST</i>₅₀ in hours) and represent the mean ± SEM. Primary cultures of endothelial cells were exposed (24 h) to increasing concentration of exosome (0, 5, 10 or 20 µg exosomal protein/ml) obtained from placental mesenchymal stem cell exposed to 1%, 3% or 8% O₂. hPMEC (CD31⁺) were used in passage 3 for migration assay. *<i>p</i><0.005 versus control; ^†<i>P</i><0.005 versus all condition for <i>ST</i>₅₀.</p

Ingenuity pathway analysis of pMSC derived-exosomes proteins.

<p>(A) The Venn diagram depicts the distribution of common and unique proteins identified by nanospray LC-MS/MS (ABSciex 5600) in exosomes released from pMSC exposed to 1%, 3% and 8% oxygen. Comparison of canonical pathways: (B) actin cytoskeleton signaling, (C) growth hormone signaling, (D) VEGF signaling, and (E) clathrin-mediated endocytosis signaling identified by IPA core analysis. Values are mean ± SEM. In B, C, D and E, <i>*p</i><0.005 versus all condition.</p

Concentration response of exosomes on hPMEC migration.

<p>Activation analysis of exosomes effect on hPMEC migration. Concentration response of exosomal protein from pMSC exposed to 1% (•), 3% (▪) or 8% (▴) O₂ on hPMEC migration. <i>Insert</i>: half-maximal stimulatory concentration (SC₅₀) at 6 h. Data represent an n = 6 well each point. Values are mean ± SEM. Insert: <i>*p</i><0.001 versus all condition; ^†<i>p</i><0.005 versus 8% O₂.</p

Exosomes increases cell migration in hPMEC.

<p>hPMEC were grown to confluence in complete media, wound were made using 96 well WoundMaker and culture in absence (○) or presence (• 5, ▴ 10 or ▪ 20 µg/ml) of exosomal protein obtained from pMSC exposed to different oxygen tension. (A) Top: a, hPMEC image immediately after wounding; b, Graphical representation showing the calculation of initial wound width; c, Graphical representation of cell migration at the midpoint of the experiment. Bottom: The time course of the concentration-dependent effect of exosomal protein from 1% O₂ on hPMEC, (C) 3% O₂ or (E) 8% O₂. (B) Area under curves from data in A, (D) from data in C, (F) from data in E. (G) Effect of pMEC-derived exosomes on hPMEC proliferation. Data represent an n = 6 well each point. Values are mean ± SEM. In B, D and F: <i>*p</i><0.005 versus all condition; ^†<i>P</i><0.005 versus 5 µg/ml; ^‡<i>p</i><0.005 versus 10 µg/ml. In G, <i>*p</i><0.005 versus control (−) with exo-pMSC from 1%, 3% or 8% O₂; *<i>*p</i><0.001 versus control (−) with exo-pMSC from 1% O₂.</p

Additional file 1: Figure S1. of The rat placental renin-angiotensin system - a gestational gene expression study

Correlation plots Microarray Signal vs qPCR gene expression. A) to F) displays correlation plots for Ace, Ace2, Mme, Thop1, Anpep, Agtr1a. All the results were significant using Pearson Correlation. (DOCX 59 kb