Studies reported in this dissertation examine the biomechanical adaptations of endothelial cells to various fluid shear stress conditions that mimic in vivo vascular conditions, and the biochemical signaling events involved in stimulating these changes. Endothelial cells that line blood vessels are constantly exposed to hemodynamic forces, and their dysfunction leads to the development of atherosclerosis, a cardiovascular disorder characterized by plaque formation in arteries and hardening of the arterial wall. Atherosclerosis is a geometrically focal disorder where endothelial dysfunction and subsequent plaque formation occur in areas of blood re-circulation, such as the outer wall of vessel bifurcations and areas near vascular branching points. These athero-prone regions experience lower shear stress conditions (<4 dynes/cm 2 ), while areas where wall shear stress is relatively high (>15 dynes/cm 2 ), and blood flow is laminar and axial, are protected from plaque formation. Endothelial cells have the capability of sensing differences between low and high fluid shear stress (FSS) conditions (mechanosensing), and translating these mechanical stimuli into biochemical signals (mechanotransduction). These abilities allow them to adapt to the different flow conditions and induce different biological responses, one being the alignment of actin filaments in the direction of flow. In these studies the oscillating optical tweezers (OOT) methodology was developed as a tool to study micro-mechanical properties of endothelial cells, and used to examine micro-mechanical changes in the vicinity of mechanosensors as a response to FSS. FSS-induced structural changes, and mechanotransduction events that translate these mechanical stimuli into biological responses were also studied.
Micro-mechanical properties of cultured bovine aortic endothelial cells (BAECs) were obtained using the oscillating optical tweezers (OOT) technique, where cortical cell properties were probed using protein A-conjugated silica beads functionalized to bind integrin and PECAM transmembrane proteins, and the inhomogeneous intracellular space was probed using endocytosed polystyrene beads as well as intrinsic structures. Measurements obtained with the extracellular bead probes exhibited a power law-dependence on the oscillating frequency, suggesting that the cortical micro-mechanical properties are time-invariant responses. But measurements from the endocytosed beads showed that the intracellular micro-mechanical properties exhibited very weak power law dependence. Intracellular viscoelastic measurements varied from cell-to-cell, and from region-to-region within the same BAEC, and these variations were mostly dependent on the availability of energy, where cell-to-cell and region-to-region reproducibility was achieved by depleting cells of nutrients. Cytoskeletal composition also had a significant role in endothelial cell mechanics, where actin and microtubule depolymerization caused a decrease in cell stiffness as well as in the dynamic viscoelastic changes that the intracellular space undergoes.
The OOT methodology was also used to examine mechanosensing events as a response to different FSS conditions, where mechanical changes in the vicinity of 2 mechanosensors, integrins, and PECAM adhesion receptors, were measured. FSS induced an increase in stiffness in the vicinity of both mechanosensors, and also promoted increased actin associations with these 2 mechanosensors. Actin filament association with these mechanosensors as well as with caveolae mechanosensors, was established using the novel technology of the Duolink(TM) Proximity Ligation Assay. These mechanical and structural changes in the vicinity of the mechanosensors suggested that the increase in stiffness was possibly caused by FSS-induced actin recruitment in their vicinity. Lastly, mechanotransduction events that led to FSS-induced actin reorganization were studied, where the spatial and temporal distributions of active phosphorylated JNK (phospho-JNK) and p38 (phospho-p38) signaling proteins, as well as the actin cytoskeleton, were tracked after exposure to low and high FSS conditions. Both JNK and p38 activities were transiently activated by the higher FSS treatments, and were required to achieve actin alignment in the direction of flow. Phospho-JNK co-localized with stress fibers and cortical actin, and its activity appeared to be required in the EC rounding as well as the elongation phases of the remodeling response, where different pathways were possibly utilized to achieve each phase. Phospho-p38 co-localized at the ends of stress fibers, where its activity seems to be involved in focal adhesion remodeling that is required to achieve EC alignment in the direction of flow.