Endothelial cells (ECs) line the lumen of the cardiovascular system and serve as a regulatory barrier to blood-borne molecules allowing the transport of nutrients, oxygen, hormonal agents, waste, and immune cells to and from underlying tissue. Complex blood flow patterns in hemodynamically challenged sites in the circulatory system correlate with sites of atherosclerotic plaque formation, implicating blood flow mechanics in regional endothelial function. This dissertation investigated, at the basic science and cellular level, the mechanosensitivity of endothelial cells in vitro to temporal gradients of shear stress and explored the role the plasma membrane system as a mechanism of mechanotransduction.

A microscope mounted controlled cell shearing device (CCSD) was developed to deliver hydrodynamically-applied shear stress profiles to cultured endothelium similar to blood flow-induced conditions in vivo. To investigate the effects of the temporal gradients of shear stress on EC response, simultaneous changes in intracellular free calcium (Ca²⁺_i) concentrations in response to changes in the initial shear stress rate (or rise time) were monitored.

Upon the onset of the stimulus, an immediate biphasic activation of Ca²⁺_i was elicited and displayed sensitivity in the loading rate and magnitude of shear stress. In these experiments, shear stress stimulated the release of calcium from intracellular storage sites in the endoplasmic reticulum through a phosphatidylinositol (4,5)- bisphosphate-mediated pathway. Extracellular Ca²⁺ modulated the response, while its entry following the stimulatory period was activated and required for sustained Ca²⁺_i. This study has identified either transmembrane proteins or the plasma membrane, or both, as an upstream mechanosensor in this transduction pathway.

To investigate the role of the membrane system in mechanotransduction, EC membranes were successfully enriched with cholesterol and showed a selective up-take that was dependent on the exposure time of the cholesterol solution to static cultures. Experimental work demonstrated that selectively altering the cholesterol composition in the membrane caused a dose-dependent attenuation in the peak [Ca²⁺]_i, at physiologic loading conditions.

Collectively, these data identified a potential role for the membrane system in mechanotransduction of shear stress induced Ca²⁺_i response in vitro. In conclusion, local hemodynamics may act as modulator of endothelium response leading to distinct physiological and pathophysiological states.