RNase A Treatment Interferes With Leukocyte Recruitment, Neutrophil Extracellular Trap Formation, and Angiogenesis in Ischemic Muscle Tissue

Abstract

Background: RNase A (the bovine equivalent to human RNase 1) and RNase 5 (angiogenin) are two closely related ribonucleases. RNase 5 is described as a powerful angiogenic factor. Whether RNase A shares the same angiogenic characteristic, or interferes with vessel growth as demonstrated for arteriogenesis, has never been investigated and is the topic of this present study. Methods and Results: To investigate whether RNase A shows a pro-or anti-angiogenic effect, we employed a murine hindlimb model, in which femoral artery ligation (FAL) results in arteriogenesis in the upper leg, and, due to provoked ischemia, in angiogenesis in the lower leg. C57BL/6J male mice underwent unilateral FAL, whereas the contralateral leg was sham operated. Two and seven days after the surgery and intravenous injection of RNase A (50 μg/kg dissolved in saline) or saline (control), the gastrocnemius muscles of mice were isolated from the lower legs for (immuno-) histological analyses. Hematoxylin and Eosin staining evidenced that RNase A treatment resulted in a higher degree of ischemic tissue damage. This was, however, associated with reduced angiogenesis, as evidenced by a reduced capillary/muscle fiber ratio. Moreover, RNase A treatment was associated with a significant reduction in leukocyte infiltration as shown by CD45 + (pan-leukocyte marker), Ly6G + or MPO + (neutrophils), MPO + /CitH 3 + [neutrophil extracellular traps (NETs)], and CD68 + (macrophages) staining. CD68/MRC1 double staining revealed that RNase A treated mice showed a reduced percentage of M1-like polarized (CD68 + /MRC1 −) macrophages whereas the percentage of M2-like polarized (CD68 + /MRC1 +) macrophages was increased. Conclusion: In contrast to RNase 5, RNase A interferes with angiogenesis, which is linked to reduced leukocyte infiltration and NET formation.

Neutrophil Extracellular Traps: A New Player in Cancer Metastasis and Therapeutic Target

“…Though the direct evidence of NETs in angiogenesis and vascular permeability in the context of tumor is scarce, a functional link between NETs and angiogenesis has been implicated in tissue repair [ 63 ] as well as in vascular pathology, including pulmonary hypertension [ 64 ] and corneal neovascularization [ 65 ]. The possible mechanism could be NETs-mediated activation of endothelial cells via TLR-4/ NFκB signalling [ 64 ], inflammation-induced upregulation of proangiogenic factors such as vascular endothelial growth factor (VEGF) [ 65 , 66 ] , and the newly identified mechanism relying on the NETs clearance of senescent endothelial cells [ 67 ]. In addition to angiogenesis, NETs have been found to increase vascular permeability by disrupting the endothelial barrier.…”

RNase A Inhibits Formation of Neutrophil Extracellular Traps in Subarachnoid Hemorrhage

“…Moreover, while a direct inhibition of NETs via DNase has been postulated as a potential mechanistic treatment strategy to reduce NET burden ( Ondracek et al, 2020 ; Korai et al, 2021 ), there is mounting evidence that NET-associated RNA is a relevant component of NET formation and can occur independently of DNA ( Herster et al, 2020 ). Based on this finding and previously published data on the dampening effects of RNase A on immune cells ( Lasch et al, 2020 ), we questioned whether RNase A also modifies NET formation. Indeed, our data show that RNase A significantly reduces NET formation in all compartments of the brain.…”

“…Next to extracellular DNA, other alarmins or so-called danger-associated molecular pattern (DAMP) signals such as extracellular RNA (exRNA) have been described as a key player in the involvement of central system pathologies, including hemorrhagic stroke ( Tielking et al, 2019 ). In this context, RNase is a natural enzyme involved in the regulation of vascular homeostasis that can counteract exRNA when applied exogenously ( Fischer et al, 2011 ; Lasch et al, 2020 ; Preissner et al, 2020 ). Recent studies have shown that NET-associated RNA is a relevant NET component and its formation can occur also independently of DNA, which opened the hypothetical avenue that RNase A (the bovine equivalent to human RNAse1) may also modify NET formation in vivo SAH models ( Herster et al, 2020 ).…”

Extracellular RNA as a Versatile DAMP and Alarm Signal That Influences Leukocyte Recruitment in Inflammation and Infection

“…The different steps of leukocyte recruitment appear to be a blueprint for arteriogenesis taken from the respective steps in innate immunity, whereby exRNA is considered in this context not to be a damaging component but rather a vessel and tissue regenerating factor. Although the aforementioned process of collateral artery growth also entails reactions of vasculogenesis and angiogenesis at distant sites, a definitive role of exRNA here is still speculative (Lasch et al, 2020). Nevertheless, in an ex vivo cellular spheroid model of vasculogenesis, exRNA was found to stimulate the formation of new vessels and leukocyte differentiation in embryonic bodies via increased VEGF-dependent signaling and ROS production (Sharifpanah et al, 2015).…”