Abstract 12507: Spatial Gradients in Wall Shear Stress Promote Collective Human Lymphatic Microvascular Endothelial Cell Migration and Alignment Through Sphingosine 1-phosphate Receptor 1

Abstract

Introduction: Spatial variations in fluid flow are thought to help trigger the formation of valves within the blood and lymphatic circulatory systems during embryonic development. However, how the physical stimulus drives the collective cell movements and changes in gene expression that underlie valve formation is not known. This knowledge gap is of considerable importance since valve failure within the lymphatic and venous vessels is significant in a wide variety of cardiovascular disease states. Methods: In this study, we developed in vitro fluid flow devices that allow us to replicate key aspects of the fluid flow environment found in the vicinity of developing valves within the lymphatic and venous vasculatures. We developed an impinging flow chamber as a model for vessel bifurcations, applying a spatial gradient in wall shear stress (WSS). We have also developed a device which applies flow through a constriction such as is commonly found at sites of valve initiation. Results: Using these devices, we examined the migration of human lymphatic microvascular ECs (MVECs) subjected to spatial gradients in WSS. We found that lymphatic MVECs subjected to a variety of WSS ranges from 0 to 9, 33, or 72 dynes/cm 2 align and collectively migrate against the flow direction over the course of 20 hours. When subjected to flow through a constriction, lymphatic MVECs turned perpendicular to the flow direction at regions near the site of maximum constriction. We determined that the G-Protein Coupled Receptor, Sphingosine 1-Phosphate Receptor 1 (S1PR1) was required for both lymphatic MVEC alignment and collective migration: a 70% S1PR1 mRNA knockdown relative to scrambled siRNA control was sufficient to prevent the collective migration and perpendicular alignment phenotypes we observed in our in vitro flow devices. In addition, these shear stress gradients resulted in higher nuclear levels of Prospero Homeobox Protein 1, a transcription factor known to be required for valvulogenesis, relative to cells in the absence of flow (Impinging Flow; 9.8 +/- 2.2 fold, Constriction; 5.1 +/- 0.8 fold). Conclusions: Our observations implicate S1PR1 as a key component in both the phenotypic and transcriptional responses of LECs to gradients in WSS, such as those at sites of valve formation.