Hypoxia Conditioned Mesenchymal Stem Cell-Derived Extracellular Vesicles Induce Increased Vascular Tube Formation  in vitro

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Abstract

Mesenchymal stem/stromal cells (MSCs) display a variety of therapeutically relevant effects, such as the induction of angiogenesis, particularly under hypoxic conditions. It is generally recognized that MSCs exert their effects by secretion of paracrine factors and by stimulation of host cells. Furthermore, there is increasing evidence that some therapeutically relevant effects of MSCs are mediated by MSC-derived extracellular vesicles (EVs). Since our current knowledge on MSC-derived EVs released under hypoxic conditions is very limited, we aimed to characterize MSC-derived EVs from normoxic vs. hypoxic conditions (5% O ₂). Adipose-derived MSCs were grown under normoxic and hypoxic conditions, and EVs were analyzed by flow cytometry using lactadherin as a marker for EVs exposing phosphatidylserine, CD63 and CD81 as EV markers, as well as CD73 and CD90 as MSC surface markers. Particle concentration and size distribution were measured by nanoparticle tracking analysis (NTA), and the EV surface antigen signature was characterized using bead-based multiplex flow cytometry. Furthermore, we evaluated the potential of MSC-derived EVs obtained under hypoxic conditions to support angiogenesis using an in vitro assay with an hTERT-immortalized human umbilical vein endothelial cell (HUVEC) line. Proliferation and viability of MSCs were increased under hypoxic conditions. EV concentration, size, and surface signature did not differ significantly between normoxic and hypoxic conditions, with the exception of CD44, which was significantly upregulated on normoxic EVs. EVs from hypoxic conditions exhibited increased tube formation as compared to normoxic EVs or to the corresponding supernatants from both groups, indicating that tube formation is facilitated by EVs rather than by soluble factors. In conclusion, hypoxia conditioned MSC-derived EVs appear to be functionally more potent than normoxic MSC-derived EVs regarding the induction of angiogenesis.

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Extracellular Vesicle Heterogeneity: Subpopulations, Isolation Techniques, and Diverse Functions in Cancer Progression

Eduard Willms, Carlos Cabañas, Imre Mäger … (2018)

Cells release membrane enclosed nano-sized vesicles termed extracellular vesicles (EVs) that function as mediators of intercellular communication by transferring biological information between cells. Tumor-derived EVs have emerged as important mediators in cancer development and progression, mainly through transfer of their bioactive content which can include oncoproteins, oncogenes, chemokine receptors, as well as soluble factors, transcripts of proteins and miRNAs involved in angiogenesis or inflammation. This transfer has been shown to influence the metastatic behavior of primary tumors. Moreover, tumor-derived EVs have been shown to influence distant cellular niches, establishing favorable microenvironments that support growth of disseminated cancer cells upon their arrival at these pre-metastatic niches. It is generally accepted that cells release a number of major EV populations with distinct biophysical properties and biological functions. Exosomes, microvesicles, and apoptotic bodies are EV populations most widely studied and characterized. They are discriminated based primarily on their intracellular origin. However, increasing evidence suggests that even within these EV populations various subpopulations may exist. This heterogeneity introduces an extra level of complexity in the study of EV biology and function. For example, EV subpopulations could have unique roles in the intricate biological processes underlying cancer biology. Here, we discuss current knowledge regarding the role of subpopulations of EVs in cancer development and progression and highlight the relevance of EV heterogeneity. The position of tetraspanins and integrins therein will be highlighted. Since addressing EV heterogeneity has become essential for the EV field, current and novel techniques for isolating EV subpopulations will also be discussed. Further dissection of EV heterogeneity will advance our understanding of the critical roles of EVs in health and disease.

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Proteomic profiling of exosomes: current perspectives.

Richard J. Simpson, Søren S. Jensen, Justin Lim (2008)

Exosomes are 40-100 nm membrane vesicles of endocytic origin secreted by most cell types in vitro. Recent studies have shown that exosomes are also found in vivo in body fluids such as blood, urine, amniotic fluid, malignant ascites, bronchoalveolar lavage fluid, synovial fluid, and breast milk. While the biological function of exosomes is still unclear, they can mediate communication between cells, facilitating processes such as antigen presentation and in trans signaling to neighboring cells. Exosome-like vesicles identified in Drosophila (referred to as argosomes) may be potential vehicles for the spread of morphogens in epithelia. The advent of current MS-based proteomic technologies has contributed significantly to our understanding of the molecular composition of exosomes. In addition to a common set of membrane and cytosolic proteins, it is becoming increasingly apparent that exosomes harbor distinct subsets of proteins that may be linked to cell-type associated functions. The secretion of exosomes by tumor cells and their implication in the transport and propagation of infectious cargo such as prions and retroviruses such as HIV suggest their participation in pathological situations. Interestingly, the recent observation that exosomes contain both mRNA and microRNA, which can be transferred to another cell, and be functional in that new environment, is an exciting new development in the unraveling exosome saga. The present review aims to summarize the physical properties that define exosomes as specific cell-type secreted membrane vesicles.

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Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms.

T Kinnaird, E STABILE, M. Burnett … (2004)

We recently demonstrated that marrow stromal cells (MSCs) augment collateral remodeling through release of several cytokines such as VEGF and bFGF rather than via cell incorporation into new or remodeling vessels. The present study was designed to characterize the full spectrum of cytokine genes expressed by MSCs and to further examine the role of paracrine mechanisms that underpin their therapeutic potential. Normal human MSCs were cultured under normoxic or hypoxic conditions for 72 hours. The gene expression profile of the cells was determined using Affymetrix GeneChips representing 12 000 genes. A wide array of arteriogenic cytokine genes were expressed at baseline, and several were induced >1.5-fold by hypoxic stress. The gene array data were confirmed using ELISA assays and immunoblotting of the MSC conditioned media (MSC(CM)). MSC(CM) promoted in vitro proliferation and migration of endothelial cells in a dose-dependent manner; anti-VEGF and anti-FGF antibodies only partially attenuated these effects. Similarly, MSC(CM) promoted smooth muscle cell proliferation and migration in a dose-dependent manner. Using a murine hindlimb ischemia model, murine MSC(CM) enhanced collateral flow recovery and remodeling, improved limb function, reduced the incidence of autoamputation, and attenuated muscle atrophy compared with control media. These data indicate that paracrine signaling is an important mediator of bone marrow cell therapy in tissue ischemia, and that cell incorporation into vessels is not a prerequisite for their effects.

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Author and article information

Contributors

Ciarra Almeria: URI : http://loop.frontiersin.org/people/782300/overview

René Weiss: URI : http://loop.frontiersin.org/people/828455/overview

Michelle Roy: URI : http://loop.frontiersin.org/people/828974/overview

Carla Tripisciano: URI : http://loop.frontiersin.org/people/712127/overview

Cornelia Kasper: URI : http://loop.frontiersin.org/people/158026/overview

Viktoria Weber: URI : http://loop.frontiersin.org/people/495593/overview

Dominik Egger: URI : http://loop.frontiersin.org/people/644462/overview

Journal

Journal ID (nlm-ta): Front Bioeng Biotechnol

Journal ID (iso-abbrev): Front Bioeng Biotechnol

Journal ID (publisher-id): Front. Bioeng. Biotechnol.

Title: Frontiers in Bioengineering and Biotechnology

Publisher: Frontiers Media S.A.

ISSN (Electronic): 2296-4185

Publication date (Electronic): 23 October 2019

Publication date Collection: 2019

Volume: 7

Electronic Location Identifier: 292

Affiliations

[1] ¹Department of Biotechnology, University of Natural Resources and Life Science , Vienna, Austria

[2] ²Christian Doppler Laboratory for Innovative Therapy Approaches in Sepsis, Department for Biomedical Research, Danube University Krems , Krems, Austria

Author notes

Edited by: Sandra Hofmann, Eindhoven University of Technology, Netherlands

Reviewed by: Andreina Schoeberlein, University of Bern, Switzerland; Vincenzo Cantaluppi, University of Piemonte Orientale, Italy

*Correspondence: Dominik Egger dominik.egger@ 123456boku.ac.at

This article was submitted to Tissue Engineering and Regenerative Medicine, a section of the journal Frontiers in Bioengineering and Biotechnology

†These authors have contributed equally to this work

Article

DOI: 10.3389/fbioe.2019.00292

PMC ID: 6819375

PubMed ID: 31709251

SO-VID: 81b24e6f-4799-4c38-8942-f0f88de7ba00

License:

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

History

Date received : 29 July 2019

Date accepted : 09 October 2019

Page count

Figures: 6, Tables: 0, Equations: 0, References: 71, Pages: 12, Words: 7840

Funding

Funded by: Christian Doppler Forschungsgesellschaft 10.13039/501100006012

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Hypoxia Conditioned Mesenchymal Stem Cell-Derived Extracellular Vesicles Induce Increased Vascular Tube Formation in vitro

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Most cited references 34

Extracellular Vesicle Heterogeneity: Subpopulations, Isolation Techniques, and Diverse Functions in Cancer Progression

Proteomic profiling of exosomes: current perspectives.

Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms.

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Cited by 84

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