Characterization of the Angiogenic Potential of Human Regulatory Macrophages (Mreg) after Ischemia/Reperfusion Injury In Vitro

Abstract

Ischemia/reperfusion- (I/R-) induced organ damage represents one of the main causes of death worldwide, and new strategies to reduce I/R injury are urgently needed. We have shown that programmable cells of monocytic origin (PCMO) respond to I/R with the release of angiogenic mediators and that transplantation of PCMO results in increased neovascularization. Human regulatory macrophages (Mreg), which are also of monocytic origin, have been successfully employed in clinical transplantation studies due to their immunomodulatory properties. Here, we investigated whether Mreg also possess angiogenic potential in vitro and could represent a treatment option for I/R-associated illnesses. Mreg were differentiated using peripheral blood monocytes from different donors (N=14) by incubation with M-CSF and human AB serum and stimulation with INF-gamma. Mreg cultures were subjected to 3 h of hypoxia and 24 h of reoxygenation (resembling I/R) or the respective nonischemic control. Cellular resilience, expression of pluripotency markers, secretion of angiogenic proteins, and influence on endothelial tube formation as a surrogate marker for angiogenesis were investigated. Mreg showed resilience against I/R that did not lead to increased cell damage. Mreg express DHRS9 as well as IDO and display a moderate to low expression pattern of several pluripotency genes (e.g., NANOG, OCT-4, and SOX2). I/R resulted in an upregulation of IDO (p<0.001) while C-MYC and KLF4 were downregulated (p<0.001andp<0.05). Proteome profiling revealed the secretion of numerous angiogenic proteins by Mreg of which several were strongly upregulated by I/R (e.g., MIP-1alpha, 19.9-fold; GM-CSF, 19.2-fold; PTX3, 5.8-fold; IL-1β, 5.2-fold; and MCP-1, 4.7-fold). The angiogenic potential of supernatants from Mreg subjected to I/R remains inconclusive. While Mreg supernatants from 3 donors induced tube formation, 2 supernatants were not effective. We suggest that Mreg may prove beneficial as a cell therapy-based treatment option for I/R-associated illnesses. However, donor characteristics seem to crucially influence the effectiveness of Mreg treatment.

Bioinformatic Analysis of lncRNA Mediated Competitive Endogenous RNA Network in Intestinal Ischemia/reperfusion Injury

“…Thus, the role of Klf4 is determined by different genes interacting with Klf4. Hummitzsch et al [21] found that the expression level of Klf4 was decreased in Human Regulatory Macrophages after H/R. Meanwhile, Klf4 could alleviate lipopolysaccharide-induced in ammation and cerebral vascular injury after cerebral ischemic stroke [22,23].…”

Effects of remote ischemic preconditioning (RIPC) and chronic remote ischemic preconditioning (cRIPC) on levels of plasma cytokines, cell surface characteristics of monocytes and in-vitro angiogenesis: a pilot study

“…In the present work, tube formation assays utilizing human umbilical vein endothelial cells (HUVEC) were performed to estimate the pro-angiogenic capacity of RIPC/cRIPC plasma and cell culture supernatants derived from RIPC/ cRIPC treated human monocytes. Although in-vitro tube formation assays do not fully resemble all aspects of in-vivo angiogenesis, they have been used by several groups and represent a reproducible and stable system for the in-vitro analysis of early processes of angiogenesis [3,22,23,42]. Employing in-vitro tube formation assays in combination with computer-assisted analysis [4], several parameters of angiogenesis were significantly increased by RIPC/cRIPC plasma.…”

Strategies to Overcome the Barrier of Ischemic Microenvironment in Cell Therapy of Cardiovascular Disease

“…Therefore, so-called programmable cells of monocytic origin (PCMO) and regulatory macrophages (Mreg) have been described as promising cell types for transplantation into ischemic tissues [ 17 ]. Both cell types can be generated from leukapheresis products and cultured similarly with macrophage colony-stimulating factor (M-CSF) and interleukin 3 (Il-3), respectively, with interferon (IFN) γ [ 18 , 19 ]. PCMO and Mreg were designed to overcome the obstacles of ischemic microenvironment showing a robust phenotype in ischemia/reperfusion in vitro experiments and enhanced pro-angiogenic potential by paracrine secretion of macrophage inflammatory protein α (MIP-1 α), granulocyte-macrophage colony-stimulating factor (GM-CSF), pentraxin-related protein 3 (PTX 3), and monocyte chemoattractant protein-1 (MCP-1) in vitro and in vivo studies [ 17 , 18 , 19 ].…”

“…Both cell types can be generated from leukapheresis products and cultured similarly with macrophage colony-stimulating factor (M-CSF) and interleukin 3 (Il-3), respectively, with interferon (IFN) γ [ 18 , 19 ]. PCMO and Mreg were designed to overcome the obstacles of ischemic microenvironment showing a robust phenotype in ischemia/reperfusion in vitro experiments and enhanced pro-angiogenic potential by paracrine secretion of macrophage inflammatory protein α (MIP-1 α), granulocyte-macrophage colony-stimulating factor (GM-CSF), pentraxin-related protein 3 (PTX 3), and monocyte chemoattractant protein-1 (MCP-1) in vitro and in vivo studies [ 17 , 18 , 19 ]. Moreover, cell lines from monocytic origin may provide further cellular features that could also support or induce tissue regeneration due to the reparative capabilities and phagocytic activity of mononuclear cells.…”

“…The administration of recombinant CCL26 abolished the hypoxia-induced directed migration of human monocytes, while the addition of CCL26 under normoxic conditions resulted in a repulsion of monocytes from the source of CCL26. Due to its chemorepulsive nature, these findings might be directly linked with monocyte migration toward hypoxia and might be a promising target for hypoxia-directed immunotherapy, not only for cancer, but also for cardiovascular therapy [ 19 , 51 ].…”