Biomechanical regulation of arteriogenesis : defining critical endothelial-dependent events

Abstract

Coronary heart disease (CHD) is a major health concern for Americans and people worldwide. Arteriogenesis, an adaptive remodeling process in which pre-existing collateral arterioles remodel to form large diameter conductance arteries, has received recent attention for its therapeutic potential in treating CHD, but the mechanisms regulating the process remain incompletely understood. In particular, little is known about how collateral flow, and the resulting effect of shear stress acting along the collateral vessel wall, regulates coronary collateralization. This Thesis combines a series of experimental systems to define the responses evoked in endothelial cells exposed to hemodynamic waveforms characteristic of coronary collateral vessels and the subsequent paracrine effects on smooth muscle cells. Initially, a lumped parameter model of the human coronary collateral circulation was used to simulate normal (NCC) and adaptive remodeling (ACC) coronary collateral shear stress waveforms. These waveforms were then applied to cultured human endothelial cells (EC) and the resulting differences in EC gene expression were assessed by genome-wide transcriptional profiling, identifying genes distinctly regulated by collateral flow, including genes important for endothelial-smooth muscle interactions. In particular, the transcription factor KLF2 was upregulated by the ACC waveform and several of its downstream targets displayed the expected modulation, including the downregulation of Connective tissue growth factor (CTGF). Moreover, delivery of endothelial conditioned medium generated throughout the collateral flow experiments to culture smooth muscle cells (SMC) resulted in the modulation of SMC genes related to vessel maturation and stabilization. In the second part of this Thesis, the effect of endothelial KLF2 expression on SMC migration was characterized using a 3D microfluidic assay capable of monitoring SMC migration in co-culture with EC. Using this 3D system, it was found that KLF2-expressing EC co-cultured with SMC significantly reduce SMC migration compared to control EC and that this reduction can be rescued by delivery of soluble CTGF.

(cont.) Collectively, these results demonstrate that the shear stress generated by collateral flow evokes distinct EC gene expression profiles and functional phenotypes that subsequently influence vascular events important for adaptive remodeling and provides experimental evidence supporting efforts directed at investigating endothelial KLF2 as a molecular target for therapeutic arteriogenesis.