Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Master of Science

Program

Medical Biophysics

Supervisor

Pickering, J. Geoffrey

Abstract

BACKGROUND: Endothelial cells (ECs) line the blood vessel lumen and respond to fluid shear stress. ECs are also responsible for initiating the formation of new blood vessels. These new vessels can form either by sprouting angiogenesis or intussusceptive, or splitting, angiogenesis. The latter is poorly understood but recent evidence suggests that intussusception occurs with altered blood flow. However, the biomechanical and biochemical mechanisms of intussusceptive angiogenesis remain largely unknown and in vitro models do not exist.

The purpose of this thesis was to develop a three-dimensional EC culture model capable of forming transluminal pillars, a key step in angiogenesis, and to utilize this model to identify regulators of pillar formation.

METHODS: I developed a novel 3D microvessel model using microfluidic channels of circumferentially lined with a confluent monolayer of endothelial cells that are exposed to range of shear stress from physiological to ultra-low. Cells were immunostained for VE-Cadherin, primary cilia, and phosphorylated VEGFR2 (pVEGFR2). A similar microfluidic model was used to co-culture HUVECs differentially treated with control or VEGFR2 siRNA and subjected to ultra-low shear stress to directly test the role of VEGFR2 in pillar formation.

RESULTS: Endothelial cells exposed to ultra-low shear stress showed marked changes in morphology compared to cells exposed to physiological shear stress, including changes in elongation and alignment in the direction of flow, and cytoskeletal reorganization. Furthermore, abundance of primary cilia was increased in HUVECs exposed to ultra-low shear stress, alongside a decrease in the expression of pVEGFR2. A co-culture of HUVECs differentially transfected with control siRNA or VEGFR2 siRNA demonstrated changes in cell morphology dependent on VEGFR2 content.

CONCLUSIONS: I established a 3D microfluidic culture model of endothelial cells that successfully yields transluminal endothelial pillars. Pillar formation was dependent on both altered shear stresses and expression and/or activities of shear sensors.

Summary for Lay Audience

Peripheral arterial disease (PAD) is a disorder common among older individuals and diabetics wherein the arteries that supply blood to the muscles in the leg become blocked, starving the tissue of oxygen. New therapies for PAD include attempting to promote the growth of new blood vessels to return blood flow to these muscles. However, these therapies have seen little success in patients.

In this thesis, I developed a 3D model of a blood vessel using microfluidics to understand how new blood vessels form. I particularly investigated how a single vessel can split into two. I discovered that endothelial cells that line blood vessels can be made to take on a flow-seeking behaviour that motivates them to stretch across the blood vessel channel. This happens when the blood flow in the vessel is low. The study revealed the critical first steps in blood vessel duplication.

This knowledge could help in developing therapies that stimulate blood vessel development to restore blood flow to damaged leg muscles in patients with PAD.

Available for download on Wednesday, August 31, 2022

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