G. Chitragari1, S. Y. Shalaby1, B. J. Sumpio1, J. Kurita1, B. E. Sumpio1 1Yale University School Of Medicine,Section Of Vascular Surgery, Department Of Surgery,New Haven, CT, USA
Introduction:
Yes Associated Protein (YAP) regulates cell growth and proliferation based on cell shape and matrix stiffness and has been implicated in the pathogenesis of atherosclerosis. Previous experiments showed that the level of YAP decreases significantly when exposed to disturbed flow (TFF). We hypothesized that the decrease in YAP is due to ubiqutination dependent proteasomal degradation. The aim of this study was to elucidate the mechanism of decrease in the level of YAP under TFF.
Methods:
HUVECs seeded on fibronectin coated glass slides were grown to confluence in culture medium containing 20% fetal bovine serum (FBS). Thereafter, they were serum starved overnight in medium containing 5% FBS and exposed to TFF utilizing a parallel plate flow chamber system controlled by a computerized pump for 4 hours at 37° C in a humidified CO2 environment in the presence or absence of 7.5µM MG132 (a proteasomal inhibitor). HUVECs under static conditions served as control. At the end of experimentation, cell lysates were prepared and immunoblotted with anti-YAP and anti-Ubiquitin antibodies. Percent change compared to static control at 0 hours was calculated and statistical analysis was performed using t-test. ‘p’ value of less than 0.05 was considered statistically significant.
Results:
The addition of MG132 increased ubiquitinated products as shown in Figure 1A. Exposure of HUVECs to TFF for 4 hours significantly decreased the level of YAP to 79.4± 3.7% compared to static control at 0 hours and compared to static control at 4 hours (124.1± 3.7%; p=0.001). However, addition of MG132 failed to significantly inhibit the decrease in the level of YAP when exposed to TFF for 4 hours (84.7± 7.3; p=0.54)
Conclusion:
Exposure of HUVECs to TFF decreased the level of YAP. Since addition of MG132 which blocked proteasomal degradation of ubiquitinated YAP did not lead to accumulation of YAP under TFF, it can be concluded that the observed decrease in the level of YAP is due to degradation via a proteasomal independent mechanism. Further studies have to be done to elucidate the details of this unknown mechanism of degradation and to better understand its role in the pathogenesis of atherosclerosis.
