The Impact of Shear Stress on Aquaporin 1 Expression in the Pulmonary Endothelium

PROJECT SUMMARY Pulmonary endothelial cells (ECs) are in direct contact with laminar blood flow, resulting in exposure to shear stress. Normal blood flow provides a physiologic degree of shear stress at which ECs achieve quiescence. Pathologic changes in shear stress can occur in several conditions ranging from pulmonary embolism, where

shear stress acutely decreases due to vessel occlusion, to pulmonary hypertension (PH), where shear stress in the distal arteries increases due to luminal narrowing. These disease entities carry considerable morbidity and mortality despite available therapeutics. The biochemical derangements that occur when shear stress is altered

are not well-characterized, and elucidating these pathways may provide novel insight into potential therapeutic targets to prevent long-term dysfunction of ECs. In vitro culture of ECs is often performed under static conditions, leading to underappreciation of the effects of physiologic shear stress on normal cellular function as well as the

biochemical and functional impact of shear perturbations. Aquaporin 1 (AQP1), a ubiquitous protein that forms water channels, is known to be expressed in vivo in the pulmonary endothelium, but we noted that AQP1 expression is not observed in human lung microvascular endothelial cells (hLMVECs) grown in static cell culture.

My preliminary data show restored AQP1 expression in cultured hLMVECs with exposure to physiologic shear stress, suggesting a critical role of shear stress in dynamically regulating AQP1 expression. Increased AQP1 has recently been linked to important cellular functions, including angiogenesis and proliferation in certain

malignancies, as well as contributing to vascular remodeling through apoptosis resistance and hyperproliferation in the ECs from rat models of PH. Regulation of AQP1 is not well-described in hLMVECs but is calcium- dependent in pulmonary vascular smooth muscle cells. Aim 1 of this proposal is designed to elucidate the

biochemical signaling that occurs in response to changes in shear stress. In preliminary data, I show intracellular calcium levels increase in response to increased shear stress. I seek to define this signaling pathway focusing on the role of activation of the membrane ion channel, TRPV4, which can increase calcium influx in ECs in

response to mechanical stimuli, in regulating AQP1 levels. Aim 2 will explore the functional outcome of changes in AQP1 expression in response to varying degrees of shear stress, focusing on apoptosis and proliferation. Techniques utilized will include but are not limited to cell culture under shear stress, ratiometric calcium

measurement, protein and mRNA measurement, immunofluorescence microscopy, and measures of apoptosis and proliferation. Completion of this project will provide novel insight into the impact of shear stress on EC function, and how derangements in shear stress may alter cell signaling and cell growth and survival. The skills

acquired in the design and execution of this study and the experimental results obtained will provide the necessary foundation for a K award and an excellent platform on which to start a career as an independently funded clinician-scientist focused on diseases of endothelial dysfunction.