Abstract
Extracellular Cold-inducible RNA-binding protein (eCIRP), a damage-associated molecular pattern, is released from cells upon hypoxia and cold-stress. The overall absence of extra- and intracellular CIRP is associated with increased angiogenesis, most likely induced through influencing leukocyte accumulation. The aim of the present study was to specifically characterize the role of eCIRP in ischemia-induced angiogenesis together with the associated leukocyte recruitment. For analyzing eCIRPs impact, we induced muscle ischemia via femoral artery ligation (FAL) in mice in the presence or absence of an anti-CIRP antibody and isolated the gastrocnemius muscle for immunohistological analyses. Upon eCIRP-depletion, mice showed increased capillary/muscle fiber ratio and numbers of proliferating endothelial cells (CD31+/CD45−/BrdU+). This was accompanied by a reduction of total leukocyte count (CD45+), neutrophils (MPO+), neutrophil extracellular traps (NETs) (MPO+CitH3+), apoptotic area (ascertained via TUNEL assay), and pro-inflammatory M1-like polarized macrophages (CD68+/MRC1−) in ischemic muscle tissue. Conversely, the number of regenerative M2-like polarized macrophages (CD68+/MRC1+) was elevated. Altogether, we observed that eCIRP depletion similarly affected angiogenesis and leukocyte recruitment as described for the overall absence of CIRP. Thus, we propose that eCIRP is mainly responsible for modulating angiogenesis via promoting pro-angiogenic microenvironmental conditions in muscle ischemia.
Highlights
The cold-inducible RNA binding protein (CIRP) is a member of the glycine-rich RNAbinding protein family, including several proteins which regulate nucleic acid interactions through their RNA-binding sites [1,2]
In order to investigate whether the depletion of extracellular CIRP (eCIRP) affects angiogenesis, a CD31/ CD45/BrdU/DAPI quadruple immunofluorescence staining on tissue sections collected 7 days after femoral artery ligation (FAL) was performed
The depletion of eCIRP resulted in ameliorated angiogenesis, evidenced by an increased capillary to muscle fiber ratio, as well as a reduction of the total apoptotic area in gastrocnemius muscle of mice treated with an anti-CIRP antibody
Summary
The cold-inducible RNA binding protein (CIRP) is a member of the glycine-rich RNAbinding protein family, including several proteins which regulate nucleic acid interactions through their RNA-binding sites [1,2]. As a reaction to hypoxia, in hemorrhagic shock and other ischemia-related pathologies, iCIRP translocates from the cytoplasm towards the extracellular space (possibly via lysosomal secretion) [8,9,10,11,12]. ECIRP activates a wide range of immune cells, such as neutrophils and macrophages, via the “Toll-like receptor 4” (TLR4)-myeloid differentiation factor 2 (MD2)-complex as well as via the “Triggering receptor expressed on myeloid cells 1” (TREM-1). These interactions result in amplified inflammatory processes through the release of pro-inflammatory cytokines and chemokines [8,13,14]. The modulation of eCIRP’s bioavailability may provide a novel approach in therapeutic drug treatment in the immune regulation of different inflammatory-dependent pathologies
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