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Analysis of the Secretome of Apoptotic Peripheral Blood Mononuclear Cells: Impact of Released Proteins and Exosomes for Tissue Regeneration.

Beer L, Zimmermann M, Mitterbauer A, Ellinger A, Gruber F, Narzt MS, Zellner M, Gyöngyösi M, Madlener S, Simader E, Gabriel C, Mildner M, Ankersmit HJ - Sci Rep (2015)

Bottom Line: Subsequently, in vitro assays showed that the exosome and protein fractions of irradiated and non-irradiated PBMC secretome were the major biological components that enhanced cell mobility; conversely, secreted lipids and microparticles had no effects.We found that the potency for preventing ventricular remodeling was similar with the GMP-compliant and experimentally-prepared PBMC secretomes.Our results indicate that irradiation modulates the release of proteins, lipid-mediators and extracellular vesicles from human PBMCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Thoracic Surgery, Medical University of Vienna, Austria.

ABSTRACT
We previously showed that, when peripheral blood mononuclear cells (PBMCs) were stressed with ionizing radiation, they released paracrine factors that showed regenerative capacity in vitro and in vivo. This study aimed to characterize the secretome of PBMCs and to investigate its biologically active components in vitro and vivo. Bioinformatics analysis revealed that irradiated PBMCs differentially expressed genes that encoded secreted proteins. These genes were primarily involved in (a) pro-angiogenic and regenerative pathways and (b) the generation of oxidized phospholipids with known pro-angiogenic and inflammation-modulating properties. Subsequently, in vitro assays showed that the exosome and protein fractions of irradiated and non-irradiated PBMC secretome were the major biological components that enhanced cell mobility; conversely, secreted lipids and microparticles had no effects. We tested a viral-cleared PBMC secretome, prepared according to good manufacturing practice (GMP), in a porcine model of closed chest, acute myocardial infarction. We found that the potency for preventing ventricular remodeling was similar with the GMP-compliant and experimentally-prepared PBMC secretomes. Our results indicate that irradiation modulates the release of proteins, lipid-mediators and extracellular vesicles from human PBMCs. In addition our findings implicate the use of secretome fractions as valuable material for the development of cell-free therapies in regenerative medicine.

No MeSH data available.


Related in: MedlinePlus

Schematic overview of experimental workflow.PBMCs were either gamma-irradiated with 60 Gy or not irradiated. After culturing for the indicated times, cells were centrifuged to separate the cell pellet and CM supernatant. The pellet was used to extract cellular RNA, which was used for microarray analysis. The CM supernatant was processed to separate and isolate different molecular components. The steps highlighted in blue indicate samples used for in vitro assays. The different methods and bioinformatics tools used for sample analyses are indicated at the appropriate links.
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f1: Schematic overview of experimental workflow.PBMCs were either gamma-irradiated with 60 Gy or not irradiated. After culturing for the indicated times, cells were centrifuged to separate the cell pellet and CM supernatant. The pellet was used to extract cellular RNA, which was used for microarray analysis. The CM supernatant was processed to separate and isolate different molecular components. The steps highlighted in blue indicate samples used for in vitro assays. The different methods and bioinformatics tools used for sample analyses are indicated at the appropriate links.

Mentions: To identify transcripts that encoded secreted proteins, we used three web-based programs: SecretomeP 2.0, SignalP 4.1, and TMHMM 2.0. The workflow of data analysis is shown in Fig. 1. The SignalP program predicts the presence and location of signal peptide cleavage sites in amino acid sequences 26. Based on this information, a specific threshold (D-cutoff score ≥0.45) is generated, which predicts secretory proteins. Currently, the SignalP program shows the best performance and accuracy compared to similar available algorithms27. SecretomeP predicts whether a protein is secreted via a non-classical pathway, based on post-translational and localization information obtained from different protein-prediction servers. The information on protein characteristics is expressed with a neural network score (NN-score), and proteins with a NN-score ≥0.5 (cut-off value) are considered to be secreted via a non-classical pathway. TMHMM 2.0 predicts transmembrane helices in proteins, based on a hidden Markov model. This method discriminates between soluble and membrane proteins with a high degree of accuracy28.


Analysis of the Secretome of Apoptotic Peripheral Blood Mononuclear Cells: Impact of Released Proteins and Exosomes for Tissue Regeneration.

Beer L, Zimmermann M, Mitterbauer A, Ellinger A, Gruber F, Narzt MS, Zellner M, Gyöngyösi M, Madlener S, Simader E, Gabriel C, Mildner M, Ankersmit HJ - Sci Rep (2015)

Schematic overview of experimental workflow.PBMCs were either gamma-irradiated with 60 Gy or not irradiated. After culturing for the indicated times, cells were centrifuged to separate the cell pellet and CM supernatant. The pellet was used to extract cellular RNA, which was used for microarray analysis. The CM supernatant was processed to separate and isolate different molecular components. The steps highlighted in blue indicate samples used for in vitro assays. The different methods and bioinformatics tools used for sample analyses are indicated at the appropriate links.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4645175&req=5

f1: Schematic overview of experimental workflow.PBMCs were either gamma-irradiated with 60 Gy or not irradiated. After culturing for the indicated times, cells were centrifuged to separate the cell pellet and CM supernatant. The pellet was used to extract cellular RNA, which was used for microarray analysis. The CM supernatant was processed to separate and isolate different molecular components. The steps highlighted in blue indicate samples used for in vitro assays. The different methods and bioinformatics tools used for sample analyses are indicated at the appropriate links.
Mentions: To identify transcripts that encoded secreted proteins, we used three web-based programs: SecretomeP 2.0, SignalP 4.1, and TMHMM 2.0. The workflow of data analysis is shown in Fig. 1. The SignalP program predicts the presence and location of signal peptide cleavage sites in amino acid sequences 26. Based on this information, a specific threshold (D-cutoff score ≥0.45) is generated, which predicts secretory proteins. Currently, the SignalP program shows the best performance and accuracy compared to similar available algorithms27. SecretomeP predicts whether a protein is secreted via a non-classical pathway, based on post-translational and localization information obtained from different protein-prediction servers. The information on protein characteristics is expressed with a neural network score (NN-score), and proteins with a NN-score ≥0.5 (cut-off value) are considered to be secreted via a non-classical pathway. TMHMM 2.0 predicts transmembrane helices in proteins, based on a hidden Markov model. This method discriminates between soluble and membrane proteins with a high degree of accuracy28.

Bottom Line: Subsequently, in vitro assays showed that the exosome and protein fractions of irradiated and non-irradiated PBMC secretome were the major biological components that enhanced cell mobility; conversely, secreted lipids and microparticles had no effects.We found that the potency for preventing ventricular remodeling was similar with the GMP-compliant and experimentally-prepared PBMC secretomes.Our results indicate that irradiation modulates the release of proteins, lipid-mediators and extracellular vesicles from human PBMCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Thoracic Surgery, Medical University of Vienna, Austria.

ABSTRACT
We previously showed that, when peripheral blood mononuclear cells (PBMCs) were stressed with ionizing radiation, they released paracrine factors that showed regenerative capacity in vitro and in vivo. This study aimed to characterize the secretome of PBMCs and to investigate its biologically active components in vitro and vivo. Bioinformatics analysis revealed that irradiated PBMCs differentially expressed genes that encoded secreted proteins. These genes were primarily involved in (a) pro-angiogenic and regenerative pathways and (b) the generation of oxidized phospholipids with known pro-angiogenic and inflammation-modulating properties. Subsequently, in vitro assays showed that the exosome and protein fractions of irradiated and non-irradiated PBMC secretome were the major biological components that enhanced cell mobility; conversely, secreted lipids and microparticles had no effects. We tested a viral-cleared PBMC secretome, prepared according to good manufacturing practice (GMP), in a porcine model of closed chest, acute myocardial infarction. We found that the potency for preventing ventricular remodeling was similar with the GMP-compliant and experimentally-prepared PBMC secretomes. Our results indicate that irradiation modulates the release of proteins, lipid-mediators and extracellular vesicles from human PBMCs. In addition our findings implicate the use of secretome fractions as valuable material for the development of cell-free therapies in regenerative medicine.

No MeSH data available.


Related in: MedlinePlus