Expression of Cathepsins B, D, and G in Extracranial Arterio-Venous Malformation

Lauren Hansen; Helen D Brasch; Erin Paterson; Josie Patel; Nicholas Bockett; Paul F Davis; Swee T Tan

doi:10.3389/fsurg.2021.676871

Expression of Cathepsins B, D, and G in Extracranial Arterio-Venous Malformation

Front Surg. 2021 Aug 2:8:676871. doi: 10.3389/fsurg.2021.676871. eCollection 2021.

Authors

Lauren Hansen¹, Helen D Brasch¹, Erin Paterson¹, Josie Patel¹, Nicholas Bockett¹, Paul F Davis¹, Swee T Tan^{1

2

3}

Affiliations

¹ Gillies McIndoe Research Institute, Wellington, New Zealand.
² Centre for the Study and Treatment of Vascular Birthmarks, Wellington Regional Plastic, Maxillofacial and Burns Unit, Hutt Hospital, Lower Hutt, New Zealand.
³ Department of Surgery, The Royal Melbourne Hospital, The University of Melbourne, Melbourne, VIC, Australia.

Abstract

Objectives: We have previously identified a population of cells that expressed stemness-associated markers in extracranial arterio-venous malformation (AVM) and demonstrated expression of cathepsins B, D, and G on embryonic stem cell (ESC)-like populations in other vascular anomalies. This study investigated the expression of cathepsins B, D, and G, and their localization in relation to this primitive population in extracranial AVM. Methods: Immunohistochemical staining was performed on AVM tissue samples from 13 patients to demonstrate expression of cathepsins B, D, and G. Western blotting was performed on four AVM tissue samples and three AVM-derived primary cell lines to confirm protein expression of cathepsins B and D proteins. RT-qPCR was performed on three AVM-derived primary cell lines to demonstrate transcript expression of cathepsins B, D, and G. Enzymatic activity assays were performed on three AVM-derived primary cell lines to investigate if cathepsins B and D were active. Localization of the cathepsins was investigated using immunofluorescence dual-staining of the cathepsins with the ESC markers OCT4 and SOX2, and mast cells marker chymase on two of the 13 AVM tissue samples. Results: Immunohistochemical staining demonstrated expression of cathepsins B, D, and G in all 13 AVM tissue samples. Western blotting showed expression of cathepsins B and D proteins in all four AVM tissue samples and all three AVM-derived primary cell lines. RT-qPCR demonstrated transcripts of cathepsins B, D, and G in all three AVM-derived primary cell lines. Enzymatic activity assays showed that cathepsins B and D were active. Immunofluorescence staining showed expression of cathepsins B and D on the OCT4+/SOX2+ endothelium and media of the lesional vessels and cells within the stroma in AVM nidus. Cathepsin G was expressed on the chymase+ phenotypic mast cells. Conclusions: This study demonstrated the novel finding of the expression of cathepsins B, D, and G in AVM. Cathepsins B and D were expressed by the primitive population, and cathepsin G was localized to mast cells, within the AVM nidus.

Keywords: arterio-venous malformation; cathepsin B; cathepsin D; cathepsin G; embryonic stem cells; mast cells.