Abstract
Peripheral blood mononuclear cells (PBMCs) have been shown to produce and release a plethora of pro-angiogenetic factors in response to γ-irradiation, partially accounting for their tissue-regenerative capacity. Here, we investigated whether a certain cell subtype of PBMCs is responsible for this effect, and whether the type of cell death affects the pro-angiogenic potential of bioactive molecules released by γ-irradiated PBMCs. PBMCs and PBMC subpopulations, including CD4+ and CD8+ T cells, B cells, monocytes, and natural killer cells, were isolated and subjected to high-dose γ-irradiation. Transcriptome analysis revealed subpopulation-specific responses to γ-irradiation with distinct activation of pro-angiogenic pathways, cytokine production, and death receptor signalling. Analysis of the proteins released showed that interactions of the subsets are important for the generation of a pro-angiogenic secretome. This result was confirmed at the functional level by the finding that the secretome of γ-irradiated PBMCs displayed higher pro-angiogenic activity in an aortic ring assay. Scanning electron microscopy and image stream analysis of γ-irradiated PBMCs revealed distinct morphological changes, indicative for apoptotic and necroptotic cell death. While inhibition of apoptosis had no effect on the pro-angiogenic activity of the secretome, inhibiting necroptosis in stressed PBMCs abolished blood vessel sprouting. Mechanistically, we identified tumor necrosis factor (TNF) receptor superfamily member 1B as the main driver of necroptosis in response to γ-irradiation in PBMCs, which was most likely mediated via membrane-bound TNF-α. In conclusion, our study demonstrates that the pro-angiogenic activity of the secretome of γ-irradiated PBMCs requires interplay of different PBMC subpopulations. Furthermore, we show that TNF-dependent necroptosis is an indispensable molecular process for conferring tissue-regenerative activity and for the pro-angiogenic potential of the PBMC secretome. These findings contribute to a better understanding of secretome-based therapies in regenerative medicine.
Highlights
Regenerative medicine, aiming at restoring damaged tissues and organs, has become an emerging branch of translational research in the last century worldwide[1]
Canonical pathway analysis of genes upregulated by γ-irradiation in peripheral blood mononuclear cells (PBMCs) suggested activation of death receptors, upregulation of tumor necrosis factor (TNF) receptor 2 (TNFRSF1B) signalling, and induction of apoptosis
Low TNF expression was detectable in PBMCs and all subfractions, with highest mRNA levels in Discussion In the past, stem cell therapies had been praised as a promising therapeutic option for tissue regeneration of a variety of damaged organs[40,41,42,43]
Summary
Regenerative medicine, aiming at restoring damaged tissues and organs, has become an emerging branch of translational research in the last century worldwide[1]. An increasing number of studies suggests that not stem cells themselves, but rather the factors released from stem cells are important and sufficient to promote tissue regeneration[8,9]. In 2005, Thum et al.[10] speculated that stem cells undergo apoptosis while being processed for clinical applications and induce immunomodulatory and tissue-regenerative effects. We were able to show that the application of the PBMC secretome alone causes tissue repair in AMI12–14, stroke[15], spinal cord[16], and skin wounds[17,18,19], in small and clinically relevant large animals. A previous study from our group suggested that γ-irradiation is able to induce apoptosis and necroptosis[20], a contribution of necroptosis to tissue regeneration by the release of paracrine factors has not been investigated so far
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