Role of cathepsin O variants in intracranial aneurysm formation

Abstract

Introduction

Intracranial aneurysms (IA) are localized dilations of cerebral arteries affecting 3–5% of the global population, with familial forms representing 10% of cases. Rupture of an IA leads to subarachnoid hemorrhage, a condition with a 33% mortality rate and long-term disabilities in half of the survivors. Using whole-exome sequencing, we identified two rare missense variants in the gene encoding cathepsin O (CTSO) (p.Val316Ile and p.Ala43Val) in two large families with multiple affected subjects. CTSO is a cysteine protease, but its physiological role remains undefined.

Objective

The aim of this study is to investigate the expression and function of CTSO in vascular cells to understand how CTSO variants contribute to the predisposition to intracranial aneurysms.

Method

CTSO expression was analyzed by Western blot (WB). CTSO variants (p.Val316Ile, p.Ala43Val) were overexpressed in 3T3 cells. SiRNA knockdown of CTSO was performed in rat aortic vascular smooth muscle cells (VSMCs). Fibronectin expression and localization was analyzed by WB and confocal microscopy. Apoptosis of Human umbilical vein endothelial cells (HUVECs) was assessed by TUNEL and adhesion by impedancemetry (xCelligence) and protein activation (WB).

Results

Western blot analysis revealed that CTSO is expressed and secreted by primary VSMCs, and its secretion was notably enhanced under mechanical stretch. Overexpression of CTSO variants (p.Val316Ile and p.Ala43Val) resulted in reduced secretion of the mutant proteins compared to the WT. In primary VSMCs, we silenced CTSO expression to mimic the loss of secretion induced by CTSO variants and observed an increase of VSMC adhesion, a decrease in VSMC migration, and an accumulation of fibronectin around the cells. To recapitulate the paracrine effect of VSMC-secreted CTSO on endothelial cells, HUVECs were treated with recombinant CTSO (200 ng/mL), which had no effect on adhesion speed or apoptosis but significantly reduced vinculin activation at the Y822 phosphorylation site.

Conclusion

These findings suggest that CTSO may play a key role in regulating endothelial cell-cell junction dynamics. However, further detailed analyses will be necessary to confirm these findings. Additionally, the data indicate that CTSO influences VSMC functions and extracellular matrix composition. The reduced secretion of CTSO variants may disturb these processes, potentially contributing to arterial wall dysfunction.