ENDOTHELIAL EXPRESSION OF THROMBOMODULIN IS REVERSIBLY REGULATED BY FLUID SHEAR-STRESS

The vascular endothelium, by virtue of its position at the interface b etween blood and the vessel wall, is known to play a critical role in the control of thrombosis and fibrinolysis. Thrombomodulin (TM) is a s urface receptor that binds thrombin and is a potent activator of the p rotein C anticoagulant pathway. Although TM expression is known to be regulated by various cytokines, little is known about its response to ever-present biomechanical stimuli. We have explored the role of fluid shear stress, imparted on the luminal surface of the endothelial cell as a result of blood flow, on the expression of TM mRNA and protein i n both bovine aortic endothelial (BAE) and bovine smooth muscle (BSM) cells in an in vitro system. We report in the present study that TM ex pression is regulated by flow. Subjecting BAE cells to fluid shear str ess in the physiological range of magnitude of 15 (moderate shear stre ss) and 36 (elevated shear stress) dynes/cm(2) resulted in a mild tran sient increase followed by a significant decrease in TM mRNA to 37% an d 16% of its resting level, respectively, by 9 hours after the onset o f flow. In contrast, shear stress at the low magnitude of 4 dynes/cm(2 ) did not affect TM mRNA levels. The sensitivity of TM mRNA expression by flow was found to be specific to endothelium, since it was not obs erved in BSM cells exposed to steady laminar shear stress of 15 dynes/ cm(2). Furthermore, unlike BAE cells, BSM cells did not exhibit altere d cell shape nor align in the direction of flow after 24 hours of shea r stress at 15 dynes/cm(2). In BAE cells, TM protein assessed by Weste rn blot analysis also showed a decrease to 33% after 36 hours of lamin ar shear stress of 15 dynes/cm(2). The downregulation of TM mRNA in re sponse to shear was found to recover completely to its static level wi thin 6 hours after the cessation of the mechanical stimulus. Furthermo re, the process was entirely reversible and without hysteresis, since further shear stimulus after recovery yielded the same behavior. The d ynamic character of the shear was varied, and it was found that steady laminar, turbulent, and pulsatile shear stress with a mean magnitude of 15 dynes/cm(2) all resulted in a similar decrease in TM mRNA. Expre ssion of tissue-type plasminogen activator (TPA) mRNA in the same BAE cells, previously reported to be affected by flow in human umbilical v ein endothelial cells, was found to be increased 3-fold by 15 dynes/cm (2) and 22-fold by 36 dynes/cm(2) at 9 hours. The reciprocal behavior of TM and TPA to shear stress suggests a switch in endothelial phenoty pe from a predominantly antithrombotic state under static conditions, dominated by TM expression, to a fibrinolytic state governed by TPA un der flow conditions, particularly under elevated shear stress (36 dyne s/cm(2)). These findings suggest that TM may be playing a localized pr otective role against thrombosis in regions of stasis and low flow and identify flow as a novel regulator of endothelial TM expression.