Aim We have previously identified an embryonic stem cell (ESC)-like population within extracranial arterio-venous malformation (AVM). This study investigated the expression of cathepsins B, D and G, and their localization to this ESC-like population.
Methods Immunohistochemical staining was performed on 13 extracranial AVM tissue samples 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 expression of cathepsins B and D proteins. RT-qPCR was performed to demonstrate mRNA expression of cathepsins B, D and G, and enzymatic activity assays were performed to investigate the functional activity of cathepsins B and D, on three AVM-derived primary cell lines. Localization of the cathepsins was investigated on two AVM tissue samples by immunofluorescence dual-staining of cathepsins B and D with ESC markers OCT4 and SOX2; and cathepsin G with mast cell marker chymase.
Results Immunohistochemical staining demonstrated expression of cathepsins B, D and G in all 13 AVM tissue samples. Western blotting confirmed expression of cathepsins B and D proteins in all four AVM tissue samples and all three AVM-derived primary cell lines. RT-qPCR demonstrated mRNA expression of cathepsins B, D and G and enzymatic activity assays showed that cathepsins B and D were functionally active, on all three AVM-derived primary cell lines. Immunofluorescence staining showed expression of cathepsin B on the OCT4+ and the SOX2+ cells on the endothelium, the media and the stroma in AVM nidus. Cathepsin D was expressed on the endothelium of the lesional vessels and OCT4+ cells within the AVM nidus, and cells that expressed cathepsin B. Cathepsin G was expressed on mast cells.
Conclusions Cathepsins B, D and G were present in AVM with cathepsins B and D being expressed by the ESC-like population and cathepsin G by mast cells.