Endothelial Cell Response to Impinging Flow and Aneurysm Formation

Abstract

Flow sensing by endothelial cells (ECs) influences development, disease, and cancer. While EC sensing of uniform shear stress is well understand, little is know about EC response to gradients in shear stress. The goal of this project is to understand the cellular response and determine the mechanisms for flow sensing in response to shear stress gradients produced by impinging flow.  We find that under this flow field, the cells move against the direction of fluid flow, concentrate in regions of maximum shear stress, and orient themselves azimuthally.  In addition to analyzing EC response, we investigate the cellular flow sensing pathways with chemical inhibition.

Introduction

  In the body, blood vessels are lined with a layer of endothelial cells.  Typically, fluid in blood vessels flows in a direction parallel to the vessel wall, resulting in a homogenous laminar flow and high shear stress on the endothelial cells lining the blood vessel.  However when conditions in the body result in “disturbed” flow (such as vessel branches and bifurcations), the blood flow becomes inhomogeneous with low oscillatory shear stress.  Lack of steady shear stress results in unhealthy cells and thus weakens the vessel wall, leading to a spectrum of disease states, including aneurysms (which involve a ballooning of the artery wall).  Endothelial cell response to laminar flow has been assessed but little is known about sensory mechanisms for cellular response to disturbed flow.

Methods and Materials

  To test the effect of disturbed flow on endothelial cells experimentally, fluid was applied to the cells in the perpendicular direction at various flow rates, thus creating a shear stress gradient.  An acrylic design with a viewing window allowed us to visualize the cell migration and alignment using live-cell imaging.  To test which flow sensors and pathways within endothelial cells are involved in the cells’ migration and alignment in response to the shear stress gradient, the cells were exposed inhibitors to known flow sensing pathways. 

Results

  Experimental results have determined that under a shear stress gradient, the cells move against the direction of the flow, towards the center and orient themselves azimuthally (in concentric circles).  Alignment perpendicular to the jet center occurs to a greater extent in regions of maximum shear stress. Responses were characterized by dividing images into concentric rings such that cells within a given ring experienced similar shear stress due to the radial symmetry of the impinging flow.  Cells migrate inward to a greater extent and exhibited more alignment perpendicular to the jet center at higher flow rates and shear stress gradients.  By using inhibitors to methodically block select cell functionalities, the functionalities that are relevant to the cells’ alignment and migration against the direction of flow can be determined.

Conclusions

  Endothelial cells sense and respond to shear stress gradients in unique and unexpected ways.  These shear stress gradients have physiological relevance to diseases such as aneurysms.  Determining the flow sensors responsible for cytoskeleton rearrangement in response to shear stress is an important step towards understanding how aneurysms form and ultimately developing innovative treatment options.