Previous findings from our laboratory demonstrated that endothelial cells (ECs), when exposed to shear stress (SS), release an unidentified protease with elastase-like characteristics. This protease affects the ECM, leading to the activation of integrin a_vb₃ and, ultimately, to the release of FGF-2 into the supernatant. The current study aimed to further characterise this protease, especially in regard to potential sites of storage.

METHODS: 

Human umbilical cord ECs were exposed for 4 h to laminar SS (16 dyne/cm²) using a cone and plate apparatus. FACS analysis was used to measure intracellular or surface expression of Weibel-Palade-Body (WPB) contents (von Willebrand Factor, P-Selectin). An antibody against neutrophil elastase (HNE) was employed to localise the protease and its activity was measured photometrically with an elastase-specific substrate. LAMP-1 was used as a late endosome/lysosome marker for immunohistochemistry.

RESULTS: 

No quantitative or kinetic relationship between the release of WPB contents and the protease could be observed after exposure to SS. Moreover, protease release was also observed in porcine aortic ECs which do not express WPBs. Immunohistochemistry displayed punctate accumulations of the protease in the cytosol, suggestive of lysosomal storage. Indeed, co-staining revealed a high degree of overlap of the protease with LAMP-1. Though the protease displayed characteristics of HNE, rtPCR did not demonstrate its endothelial expression, nor that of another potential candidate, proteinase 3 (PR-3).

CONCLUSION: 

Our findings suggest a lysosomal storage of the protease, which is not identical with HNE or PR-3. Future investigations will focus on lysosomal trafficking under SS and further biochemical characterisation of the protease.