Multiscale imaging of endothelial cell guidance

Abstract

Angiogenesis, the process through which new blood vessels are formed, relies on coordinated endothelial cell behaviours, regulated by key signalling pathways such as vascular endothelial growth factor (VEGF) and integrin pathways. In vitro study of endothelial cell guidance at multiple scales is vital to understand how different environmental cues are integrated at the subcellular level. The aim of this thesis was to develop high content imaging methods to capture temporal information at the cellular, subcellular and molecular scale in controlled microenvironments. To examine the hypothesis that persistent endothelial cell migration is directed by soluble and surface-bound gradients, a method to create steady soluble or immobilised gradients on radiofrequency-plasma-polymerised surfaces to support endothelial cell attachment and migration was developed. A novel method of imaging single molecule protein adsorption by Total Internal Reflection Fluorescence Microscopy (TIRF-M) was developed. This new single molecule counting method may have future applications, particularly the study of how proteins and cells interact with surfaces at the molecular scale. Endothelial cells should not be considered as homogeneous cell populations as they take on different roles during new vessel growth. Single cell live-cell imaging was developed to capture the heterogeneous nature of endothelial cell migration directed by a soluble gradient. Statistical methods for analysing directed cell motion were evaluated. Both circular and Hotelling’s T² statistics provided robust statistics for evaluating the effect of a chemical gradient on endothelial cell migration. It was surprising to discover that a soluble VEGF165 gradient on its own was not sufficient to direct endothelial cell migration, requiring synergy with a sphingosine-1-phosphate gradient to elicit optimal responses. The Rho GTPases are thought to coordinate cell surface signalling with remodelling of cytoarchitecture for directed endothelial cell motion. A Förster resonance energy transfer (FRET) probe (Raichu) was used to visualise Rho GTPase activity in live endothelial cells at subcellular scales in real time using fluorescence lifetime imaging and intensity-based measurements. A fluorescent protein toolbox was developed to quantify spectral bleed-through. A new quantification method has extended the resolution of live cell FRET measurements by statistical modelling of spectral bleed-through and biological noise.