Investigating the mechanisms by which endothelial cells sense and respond to wall shear stress gradients

The lymphatic vasculature collects the protein-rich fluid that is excluded from the venous ends of blood capillaries. Improper lymph drainage leads to tissue swelling and can contribute to the pathophysiology of cardiovascular diseases. Fluid flow is thought to be essential to controlling the formation and patterning of the lymphatic system. In particular, recent evidence suggests that local variations in flow-induced wall shear stress (WSS) may provide a critical spatial cue that influences the timing and location of the development of lymphatic valves, which promote flow unidirectionality. How exactly the mechanical stimulus provided by spatial gradients in WSS can drive the alterations in morphological behavior, cellular rearrangements and intracellular signaling that lead to valve formation remains poorly understood. More broadly, how the endothelial cells (ECs) that line lymphatic vessels sense and respond to WSS is currently unknown. Previous studies in developing mouse embryos have shown that lymphatic valves preferentially form near sites of vessel constriction and branching, where lymphatic ECs (LECs) are exposed to gradients in WSS. To investigate how LECs specifically respond to spatial gradients in WSS, our labs previously developed an impinging flow chamber that simulates the flow profile found near sites of vessel branches. We used this flow device to determine that human lymphatic microvascular ECs (HLMVECs) migrate against the direction of flow in response to spatial gradients in WSS, a response that is required for proper vessel formation in the developing embryo. However, the molecular mechanisms by which ECs detect spatial patterns in WSS remain poorly understood. In our first study, we performed a screen of signaling pathways which may be implicated in directional EC migration in response to gradients in WSS. We determined that both the G-protein coupled receptor sphingosine 1-phosphate receptor 1 (S1PR1) and its ligand sphingosine 1-phosphate (S1P) were required for this collective upstream migration of HLMVECs in an in vitro setting. These findings are consistent with a model in which signaling via S1P and S1PR1 are integral components in the response of LECs to the stimulus provided by fluid flow. In a second study, we used live-cell calcium (Ca2+) imaging to determine how HLMVECs respond to uniform or spatially varying WSS. Previous work had established that Ca2+ oscillations were strongly triggered by WSS, and that Ca2+ signaling activates downstream cellular responses that include migration, proliferation and alterations in gene expression. We observed that Ca2+ pulses were sensitive to spatial gradients in WSS. In particular, the number of Ca2+ pulses per cell increased with increasing WSS, while the duration of individual Ca2+ pulses increased in the presence of a gradient in WSS. Inhibition of the Ca2+ channel ORAI1 and the Ca2+-dependent phosphatase calcineurin revealed that both were required to set the dynamics of the individual pulses, suggestive of feed-forward and feed-back regulation of Ca2+ dynamics. We speculate that LECs are capable of interpreting both WSS magnitude and spatial variations through intracellular Ca2+ dynamics, which in turn control lymphatic developmental pathways including lymphangiogenesis and lymphatic valve formation.

Keywords: Chemical Engineering.
Advisor: Dunn Alexander Robert 
Fuller Gerald G.
Red-Horse Kristy
Rockson Stanley G.