Development and application of a flow system to study shear stress-induced genes in primary endothelial cells

Abstract

Atherosclerosis is a chronic lipid-modulated inflammatory disease, which develops preferentially in arterial regions, where blood flow is disturbed. Shear stress is exerted on the endothelium and it has been shown that the tangential force can influence intracellular signalling cascades, modulating transcriptional activation or repression of mechanosensitive genes. The exact mechanisms of mechanotransduction are not entirely understood, but the shear-dependent transcription factor Kruppel-like factor 2 (KLF2) has been considered as the master switch in shear-controlled endothelial gene expression. Induction of its target genes triggers anti-inflammatory and anti-thrombotic signalling cascades, determining an atheroprotective phenotype. Endothelial KLF4 has also recently been shown to be shear-sensitive and that it can compensate for reduced KLF2 transcripts. Another member of the KLF family, KLF6, is known to be present in the endothelium, but has not been further investigated. This suggests that KLF2 might not be a single master regulator in the process of shear-dependent atheroprotection. Since KLFs have been demonstrated to form circuitries in other tissues, the hypothesis of the present study was that shear stress invokes a KLF2-KLF4-KLF6 network, which regulates the atheroprotective transcriptional program in the endothelium. The work aimed at understanding shear-dependent signalling mechanisms to potentially reveal therapeutic targets. Firstly, a flow device allowing the in vitro exposure of primary porcine aortic endothelial cells (PAECs) to shear stress was designed. Gene expression patterns were examined with the consideration of parameters associated with time, shear stress magnitude and interference with short inhibitory ribonucleic acid (siRNA). These studies were carried out using quantitative polymerase chain reaction (qPCR), genome-wide microarray analysis, protein detection and computational modelling. The findings revealed an oscillatory expression pattern for KLF2 and KLF4 in high shear regimes of 20 dyne/cm2. siRNA-mediated silencing of KLF4 resulted in a significant downregulation of KLF2 and this dependency was shown for the first time. Global genome analysis identified a number of novel shared, but also individual downstream targets, suggesting that the transcription factors have important non-compensatory regulatory functions. In conclusion, this work contributes to the understanding of the individual roles of KLF2 and KLF4 and their functional overlap in the mediation of atheroprotection. Further investigations are required to assess whether KLFs could potentially be useful in preventing atherosclerosis or identifying atherosclerosis treatments.