Abstract
Microvascular endothelial cell heterogeneity and its relationship to hemodynamics remains poorly understood due to a lack of sufficient methods to examine these parameters in vivo at high resolution throughout an angiogenic network. The availability of surrogate markers for functional vascular proteins, such as green fluorescent protein, enables expression in individual cells to be followed over time using confocal microscopy, while photoacoustic microscopy enables dynamic measurement of blood flow across the network with capillary-level resolution. We combined these two non-invasive imaging modalities in order to spatially and temporally analyze biochemical and biomechanical drivers of angiogenesis in murine corneal neovessels. By stimulating corneal angiogenesis with an alkali burn in Tie2-GFP fluorescent-reporter mice, we evaluated how onset of blood flow and surgically-altered blood flow affects Tie2-GFP expression. Our study establishes a novel platform for analyzing heterogeneous blood flow and fluorescent reporter protein expression across a dynamic microvascular network in an adult mammal.
Highlights
Angiogenesis involves network level coordination of individual cellular behaviors accomplished through alterations in biochemical and biomechanical signals in the local environment[1,2,3]
Angiogenesis occurred over seven days, and the new vascular network was imaged using multiple imaging modalities
Studies conducted in large vessels and in cultured monolayers of endothelial cells have demonstrated that endothelial cells are highly mechanosensitive, with fluid flow-induced shear stress serving as a regulator of endothelial cell gene expression[47]
Summary
Angiogenesis involves network level coordination of individual cellular behaviors accomplished through alterations in biochemical and biomechanical signals in the local environment[1,2,3]. There is a substantial body of literature that describes hemodynamic control over endothelial cell protein expression in large vessel endothleium[13,14,15,16,17,18,19], and compelling data from in vitro studies on cultured endothelial cells has suggested that shear stress is a regulator of Tie[2] signaling and vascular quiescence[20, 21] This relationship has never before been corroborated by in vivo studies, despite the ongoing clinical evaluation of a drug, VE-PTP, intended to constitutively block this pathway’s inhibitor.
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