Influence of laminar shear stress on transmembrane chemokines and proteases of the ADAM family in endothelial cells

Babendreyer, Aaron; Ludwig, Andreas (Thesis advisor); Zenke, Martin (Thesis advisor); Fabry, Marlies (Thesis advisor)

Aachen (2018)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2018

Abstract

Shear stress generated by blood flow is sensed by endothelial cells and influences the integrity and inflammatory state of the endothelium. This response involves transcriptional and post-transcriptional regulatory mechanisms. Physiological blood flow has a protective effect on the endothelium, while pathologically reduced blood flow can impair endothelial integrity and thus promote the development of vascular inflammatory lesions. The transmembrane chemokine CX3CL1 is one of the contributors, which lead to the formation of these vascular inflammatory lesions. This can be achieved by acting either as surface-expressed adhesion molecule on the endothelial cells or as a soluble chemoattractant for monocytic cells. Soluble CX3CL1 is generated by the cleavage of CX3CL1 by metalloproteinases of the ADAM family. ADAMs can cleave a number of signalling molecules including CX3CL1 from the cell surface of endothelial cells. By that, ADAMs can regulate a variety of endothelial processes such as survival or inflammatory responses. The aim of this PhD study was to investigate whether transmembrane chemokines and proteases of the ADAM family are regulated by shear stress and are thus involved in influencing endothelial functions. For this purpose, a flow culture system for primary endothelial cells from different vascular beds was established. The system was first validated by confirming upregulation of endothelial NO synthase, and the Krüppel like transcription factor 2. In addition, physiological shear stress was found to suppress both the basal and TNF-induced expression of the transmembrane chemokine CX3CL1 on the mRNA level. This led to a reduced surface expression of CX3CL1 on flow exposed endothelial cells. Adhesion experiments with monocytic cells carrying the CX3CL1 receptor CX3CR1 showed that the suppressed CX3CL1 expression on endothelial cells contributes to a reduced monocyte adhesion on endothelial cells cultured under physiological flow. An additionally performed transcriptomic analysis showed that several proteases from the ADAM family are regulated by flow conditions. Of particular interest was the enhanced expression of ADAM15 and ADAM17 under physiological flow conditions. It could be shown in this work that the induction of the ADAM15 expression is mediated by shear stress via the transcription factor KLF2 as shown by pharmacological manipulation, lentiviral overexpression and siRNA-mediated knockdown of KLF2 expression. Through shRNA-mediated knockdown of ADAM15 evidence could be provided that ADAM15 contributes to increased endothelial cell survival. For ADAM17, a more complex regulatory mechanism was observed. ADAM17 is not only regulated on the transcriptional level but especially on the post-transcriptional level. This is due to an altered maturation of ADAM17 caused by the transcriptional regulation of iRhoms. While the mRNA expression of iRhom1 is preferentially induced by shear stress via KLF2, iRhom2 is predominantly induced by the inflammatory stimulus TNF. This leads to increased surface expression of ADAM17 both under physiological flow conditions and under inflammatory conditions, such as stimulation with TNF. Finally, a microfluidic chamber was developed, which allows monitoring the effect on endothelial permeability under flow and over time by continuous impedance spectroscopic measurements. This chamber may be useful in further studies to investigate the function of ADAMs in endothelial permeability regulation under flow. Thus it could be shown in this work that shear stress can have protective effects on the endothelium by downregulation of inflammatory ADAM substrates such as CX3CL1 and by KLF2 mediated upregulation of protective ADAM15. Moreover, ADAM17, which cleaves inflammatory, as well as protective substrates on endothelial cells, can be upregulated on the cell surface by both inflammatory conditions via induction of iRhom2 and flow conditions via induction of iRhom1. The results all indicate that in vitro investigations of inflammatory and protective endothelial cell functions need to consider appropriate flow conditions.