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Secretome Analysis of Apoptotic PBMCs Suggests Impact of Released Proteins & Exosomes on Tissue Regeneration; First Study to Evaluate Effects of Radiation Stress on Oxidized Lipid Content of Secretome

In a new study, scientists from the Medical University of Vienna in Austria, together with colleageues, have shown that irradiation stress modulates the release of proteins, lipid-mediators, and extracellular vesicles (EVs) from human peripheral blood monocytes (PBMCs). The researchers suggest that their new findings implicate the use of secretome fractions as valuable material for the development of cell-free therapies in regenerative medicine. The research was reported in an open-access article published online on November 16, 2015 in Scientific Reports. The article is titled “Analysis of the Secretome of Apoptotic Peripheral Blood Mononuclear Cells: Impact of Released Proteins and Exosomes for Tissue Regeneration.” The senior and corresponding author of the article is Hendrik Jan Ankersmit, M.D., of the Department of Thoracic Surgery and the Christian Doppler Laboratory for Cardiac and Thoracic Diagnosis and Regeneration at the Medical University of Vienna. In their article introduction, the authors write that they had previously showed that, when PBMCs were stressed with ionizing radiation, they released paracrine factors that showed regenerative capacity in vitro and in vivo. The current study was designed to characterize the secretome of PBMCs and to investigate its biologically active components in vitro and vivo. The scientists say that bioinformatics analysis revealed that irradiated PBMCs differentially expressed genes that encoded secreted proteins. These genes are primarily involved in (a) pro-angiogenic and regenerative pathways; and (b) the generation of oxidized phospholipids with known pro-angiogenic and inflammation-modulating properties. In in vitro assays, the researchers show 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. The scientists tested a viral-cleared PBMC secretome, prepared according to good manufacturing practice (GMP), in a porcine model of closed-chest, acute myocardial infarction (AMI). The researchers say that they found that the potency for preventing ventricular remodeling was similar between the GMP-compliant PBMC secretome and the experimentally-prepared PBMC secretome. In conclusion, the authors state that their results indicate that irradiation modulates the release of proteins, lipid-mediators, and EVs from human PBMCs. In addition they say that their findings suggest that the use of secretome fractions may well be valuable for the development of cell-free therapies in regenerative medicine.

In more detail, the authors state, in their article summary, that they have shown that irradiation induces quantitative and qualitative changes in the secretome of human PBMCs.

Irradiated cells expressed higher amounts of pro-angiogenic proteins, EVs, and oxidized phospholipids than non-irradiated cells. Furthermore, the authors say that, in selected in vitro assays with primary human fibroblasts (FBs) and keratinocytes (KCs), the two main biologically active components of conditioned media (CM) were found in the fractions that contained either proteins or exosomes.

In addition, the researchers repor that validated, viral-cleared GMP-compliant, PBMC secretomes displayed cardioprotective effects comparable to those displayed with experimental-grade CM in an in vivo model of acute myocardial infarction (AMI).

The authors note that their study is the first to evaluate the effect of irradiation on the oxidized lipid content of the secretome, which includes microparticles, exosomes, and soluble lipids.

More specifically, the scientists report that they investigated irradiation-induced changes by performing lipidomics on CM samples from irradiated and non-irradiated PBMCs. They focused on oxidized phosphatidylcholines (oxPCs). High-pressure lipid chromatography-tandem MS analysis (HPLC-MS/MS) of PCs showed that irradiation promoted the formation of oxidized lipid species with pro-angiogenic and immunomodulatory properties.

They say that the lipidomics protocol they used enabled detection of a large number of oxidation products derived from the most abundant cell membrane phospholipids. We found that CM obtained from irradiated PBMCs contained significantly higher concentrations of specific oxPCs than CM from non-irradiated PBMCs.

The authors note that microparticles represent the largest class of EVs, and that microparticle are abundantly present in the CM from irradiated PBMCs. PCs are the most abundant lipid class in microparticles. In contrast, exosomes have less phospholipid content and more ceramide contents than microparticles.

Therefore, the researchers speculated that irradiation-induced changes in oxidation products might be predominantly mediated by the oxidation of PCs incorporated in microparticles.

However, although oxPCs have previously been shown to exert biological activity, to induce expression of CXCL8, and to modulate angiogenesis, the scientists, in this study, could not detect any in vitro effects of soluble CM lipids in their selected experiments.

The authors said that “It is tempting to speculate that probably other cell types than those used in this study might be sensitive to oxPC treatments. These findings should stimulate further research with different functional assays to clarify the role of PBMC-derived lipids as paracrine mediators.”

The image shows two PBMCs (blue) amidst a number of red blood cells.

[Scientific Reports article]