Analysis of cytotrophoblast cell-derived exosome proteins.

<p>(A) The Venn diagram represents the distribution of common and unique proteins identified by nanospray LC-MS/MS (ABSciex 5600) in exosomes released from trophoblast cells exposed to 1%, 3% and 8% of oxygen tension. Comparison of canonical pathways: (B) HIFα, and (C) IL-8 signaling identified by IPA core analysis. Values are mean ± SEM. In B and C, *p<0.005 versus all condition; †p<0.05 versus 8% O2.</p

Exosomes released from cytotrophoblast cell exposed to different oxygen tension.

<p>Effects of oxygen tension on the release of exosomes from cytotrophoblast cells are presented as ug exosomal protein/106/48 h. Data are presented as a scatter plot with mean ± SEM (n = 6 biological samples and 2 independent duplicate cultures per placenta duplicate). ***p<0.001 versus 8% O2; **p<0.01 versus 1% O2; †p<0.05 versus 3% O2.</p

Effect of oxygen tension on exosome bioactivity.

<p>EVT cell invasion was measurement in presence of exosomes isolated from cytotrophoblast cells exposed to three different oxygen tension (1%, 3% and 8% O2). (A) The graph represents the changes of half-maximal effective concentration (EC50) and (B) half-maximal stimulatory time (ST50) exosomes on EVT invasion in response to oxygen tension (source). Values are mean ± SEM. *p<0.01 versus all conditions; †p<0.05 versus 8% O2.</p

Ingenuity Pathway Analysis of Exosomal Proteins.

<p>Unique proteins identified in exosomes isolated from cytotrophoblast cells exposed to 1% oxygen were submitted to IPA network analysis. Green: signaling involving in cellular movement.</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

<i>SLCA71</i> promoter activity in response to insulin.

<p>(a) Luciferase (<i>Luc</i>) reporter constructs containing serial truncations of <i>SLC7A1</i> promoter (−1606 bp (pGL3-hCAT-1⁻¹⁶⁰⁶) or −650 bp (pGL3-hCAT-1⁻¹⁶⁰⁶) from the transcriptional start point) were transfected in primary cultures of HUVECs incubated (8 hours) without (plain bars) or with (dashed bars) 10 μmol/L nitrobenzylthioinosine (NBTI). Cell transfection was done in the absence or presence of 1 nmol/L insulin and/or ZM-241385 (10 nmol/L), along with <i>Renilla</i> reporter plasmid, and assayed for <i>Firefly</i> and <i>Renilla</i> luciferase activity, respectively. Results depict ratio of <i>Firefly</i>/<i>Renilla</i> luciferase activity. Cells were also transfected with the empty pGL3-basic vector or pGL3-control vector (SV40 pGL3) as negative or positive controls, respectively. (b) Fraction of <i>SLC7A1</i> reporter constructs activity inhibited (sensitive) by ZM-241385 in absence (Basal) or presence of insulin or NBTI as in (a). (c) <i>SLC7A1</i> reporter constructs fraction activity non-inhibited (insensitive) by ZM-241385 as in (a). In (a), *<i>P</i><0.05 versus Control, †<i>P</i><0.05 versus corresponding values in the presence of ZM-241385. In (b), *<i>P</i><0.05 versus corresponding Basal, †<i>P</i><0.05 versus values in pGL3-hCAT-1⁻¹⁶⁰⁶ in the presence of insulin, ‡<i>P</i><0.05 versus values in pGL3-hCAT-1⁻⁶⁵⁰ in the presence of insulin or insulin + NBTI. Values are mean ± SEM (n = 6).</p

Proposed model for insulin action requiring adenosine receptors on L-arginine transport in human fetal endothelial cells.

<p>Human umbilical vein endothelial cells respond to insulin via activation of insulin receptors (IR) leading to a reduced (–) adenosine uptake via the human equilibrative nucleoside transporters (hENTs). Insulin reduced adenosine uptake leading to extracellular accumulation of this nucleoside, which turns into activation of A_2A adenosine receptors (A_2A) at the plasma membrane. Activation of these membrane receptors leads to a mechanism mediated by transcription factors acting as activators (+) between −1606 and −650 bp from the transcription start point of <i>SLC7A1</i> (for hCAT-1) gene promoter. Alternatively, insulin activates transcription factors acting as activators (+) between −650 bp and the transcription start point of <i>SLC7A1</i>. This phenomenon increases (+) by adenosine receptor-activated transcription factors increasing hCAT-1 mRNA expression and protein abundance resulting in stimulation of L-arginine transport by HUVECs in response to insulin.</p

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

<p>All mass spectra were analysed using the Mascot and Protein Pilot search engines against the Swissprot-swissprot database with the species set as human (score over 30). Exosomal proteins identified by mass-spectrometry were analyzed with the Ingenuity Pathway Analysis (Ingenuity Systems, <a href="http://www.ingenuity.com" target="_blank">www.ingenuity.com</a>). False discovery rate (FDR) was estimated using a reversed sequence database. List of total exosomal protein from cytotrophoblast cells exposed to different oxygen level are presented as Protein ID, Symbol, Entrez Gene Name, Location and type.</p

Detection and characterization of Cytotrophoblast cell-derived exosomes.

<p>Cytotrophoblast cells were isolated from chorionic villi obtained from first trimester pregnancies and cultured under different oxygen tension (see Methods). Exosomes were isolated from CTs culture media and characterized morphologically and using specific marker for exosome proteins. (A) Representative Western blot for exosome markers: CD63, CD9, CD81 and PLAP. Sample loading was normalized by protein content. Fractions 1 to 10, represent fractions collected after buoyant density centrifugation. (B) Protein profile of exosomal proteins and cytotrophoblast cells proteins. Exosomal proteins derived from fraction 5 to 8 (positive for exosomal marker) and cellular proteins of trophoblast cells (cells) were separated by SDS-PAGE and stained with SimplyBlueTM SafeStain. (C) Electron micrograph of exosomes isolated by ultracentrifuge and purified with a sucrose gradient (pooled exosomal pellet) from cytotrophoblast cells. In B, Scale bar 100 nm. In A, B and C, none of the experiments performed were significantly different using different oxygen tension.</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