Abstract 1457: Oscillatory and Laminar Shear Stress Inversely Regulate Cathepsins K and S and Cystatin C in Mouse Aortic Endothelial Cells (MAEC)
Cathepsins, the lysosomal cysteine proteases, have recently been implicated in atherosclerosis, a process known to be regulated by blood flow and shear stress on the endothelium. Recently, we have shown that unidirectional laminar shear stress (LS, 15 dyn/cm2) inhibits cathepsin L activity in MAEC to prevent degradation of extracellular matrix (ECM) proteins such as gelatin and elastin compared to oscillatory shear stress (OS, +−5 dynes/cm2, 1 Hz) found at sites prone to atherosclerosis. Here we investigated the shear regulated activity of cath K and S, powerful cysteine proteases, and that of their endogenous inhibitor, cystatin C. Western blot confirmed that MAEC exposed to OS had higher levels of cath K and S but significantly lower levels of cystatin C, while LS increased cystatin C but decreased cath S and K. MAEC also showed a preference for basal secretion of the cathepsins, suggesting their secretion into the vessel wall for remodeling purposes. A 30% reduction in elastase activity under St and OS and a 43% reduction in OS mediated gelatinase activity after transfection with cath K siRNA supported the hypothesis that OS activates cath K and increases ECM proteolytic activity; those exposed to LS had only 50% of the elastase and gelatinase activity and there was no further reduction by cath K siRNA presumably because of the already significant reduction in cathepsin protein. Immunostaining of MAEC showed a lysosomal staining pattern of cath S, but cath K was present in the cytosol and cell membrane. This was confirmed with Western blotting after cell fractionation. Cystatin C showed strong co-localization with the Golgi apparatus after exposure to LS. Staining of diseased human coronary arteries revealed a strong correlation (r2 =0.74) between intensity of cath K staining in endothelial cells and loss of IEL integrity. The inverse regulation of cath S and K and their inhibitor, cystatin C by OS and LS demonstrate the importance of this family of proteases at sites of disturbed flow to promote or prevent vascular remodeling and atherosclerosis development.