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

Manuel Lasch; Konda Kumaraswami; Simona Nasiscionyte; Susanna Kircher; Dominic van den Heuvel; Sarah Meister; Hellen Ishikawa-Ankerhold; Elisabeth Deindl

doi:10.3389/fphys.2020.576736

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

Front Physiol. 2020 Nov 6:11:576736. doi: 10.3389/fphys.2020.576736. eCollection 2020.

Authors

Manuel Lasch^{1

2

3}, Konda Kumaraswami^{1

2}, Simona Nasiscionyte¹, Susanna Kircher^{1

2}, Dominic van den Heuvel¹, Sarah Meister⁴, Hellen Ishikawa-Ankerhold^{1

5}, Elisabeth Deindl^{1

2}

Affiliations

¹ Walter-Brendel-Centre of Experimental Medicine, University Hospital, Ludwig-Maximilians-Universität München, Munich, Germany.
² Biomedical Center, Institute of Cardiovascular Physiology and Pathophysiology, Ludwig-Maximilians-Universität München, Munich, Germany.
³ Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital, Ludwig-Maximilians-Universität München, Munich, Germany.
⁴ Department of Obstetrics and Gynaecology, University Hospital, Ludwig-Maximilians-Universität München, Munich, Germany.
⁵ Department of Internal Medicine I, Faculty of Medicine, University Hospital, Ludwig-Maximilians-Universität München, Munich, Germany.

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⁺/CitH₃ ⁺ [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.

Keywords: RNase 5; RNase A; angiogenesis; capillary sprouting; extracellular RNA; ischemia; leukocyte recruitment; neutrophil extracellular traps.