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

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.

SUBMITTER: Hansen L

PROVIDER: S-EPMC8367294 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundCerebral arterio venous malformations (AVM) are a major causal factor for intracranial hemorrhage, which result in permanent disability or death. The molecular mechanisms of AVM are complex, and their pathogenesis remains an enigma. Current research on cerebral AVM is focused on characterizing the molecular features of AVM nidus to elucidate the aberrant signaling pathways. The initial stimuli that lead to the development of AVM nidus structures between a dilated artery and a vein are however not known.MethodsIn order to understand the molecular basis of development of cerebral AVM, we used in-depth RNA sequencing with the total RNA isolated from cerebral AVM nidus. Immunoblot and qRT-PCR assays were used to study the differential gene expression in AVM nidus, and immunofluorescence staining was used to study the expression pattern of aberrant proteins in AVM nidus and control tissues. Immunohistochemistry was used to study the expression pattern of aberrant proteins in AVM nidus and control tissues.ResultsThe transcriptome study has identified 38 differentially expressed genes in cerebral AVM nidus, of which 35 genes were upregulated and 3 genes were downregulated. A final modular analysis identified an upregulation of ALDH1A2, a key rate-limiting enzyme of retinoic acid signaling pathway. Further analysis revealed that CYR61, a regulator of angiogenesis, and the target gene for retinoic acid signaling is upregulated in AVM nidus. We observed that astrocytes associated with AVM nidus are abnormal with increased expression of GFAP and Vimentin. Triple immunofluorescence staining of the AVM nidus revealed that CYR61 was also overexpressed in the abnormal astrocytes associated with AVM tissue.ConclusionUsing high-throughput RNA sequencing analysis and immunostaining, we report deregulated expression of retinoic acid signaling genes in AVM nidus and its associated astrocytes and speculate that this might trigger the abnormal angiogenesis and the development of cerebral AVM in humans.

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

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