Exosomal Signaling during Hypoxia Mediates Microvascular Endothelial Cell Migration and Vasculogenesis

Carlos Salomon,Luis Sobrevia,Murray Mitchell,Gregory E Rice,Keith Ashman,Jennifer Ryan,Miharu Kobayashi,Costanza Emanueli

doi:10.1371/journal.pone.0068451

Carlos Salomon, Luis Sobrevia + Show 6 more

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https://doi.org/10.1371/journal.pone.0068451

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Journal: PloS one	Publication Date: Jul 8, 2013
Citations: 350	License type: CC BY 4.0

Affiliation: University of Queensland

Abstract

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.

Highlights

Exosomes are secreted nanovesicles (30–100 nm diameter) formed through the inward budding of multivesicular bodies (MVBs) that traffic and transfect proteins, mRNAs and miRNAs into target cells [1]
Cell surface protein expression by Placental mesenchymal stem cells (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+ and negative for hematopoietic and endothelial markers: CD11b, CD142, CD312, CD342, CD452
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% O2 and the exosomes released were quantified

Summary

Introduction

Exosomes are secreted nanovesicles (30–100 nm diameter) formed through the inward budding of multivesicular bodies (MVBs) that traffic and transfect proteins, mRNAs and miRNAs into target cells [1]. Of particular relevance to this study, exosomes released from progenitor cells stimulate: endothelial cell migration [14]; tissue vascularization and angiogenesis [15,16]; induce cell proliferation [17]; and are cardioprotective of ischemia/reperfusion injury [18]. 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. 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 (CD452, CD142, CD192, CD80+, CD86+, CD40+ and B7H2+) and the capacity to differentiate into multiple linages in vitro. MSC affect tissue repair through the release of paracrine mediators [24,25,26,27] including exosomes [28]

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