University of Groningen
Biomechanical Regulation of Endothelial Phenotype
Lee, Ee Soo
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to
cite from it. Please check the document version below.
Document Version
Publisher's PDF, also known as Version of record
Publication date:
2015
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Lee, E. S. (2015). Biomechanical Regulation of Endothelial Phenotype [Groningen]: University of
Groningen
Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the
author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
Take-down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately
and investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the
number of authors shown on this cover page is limited to 10 maximum.
Download date: 17-06-2017
Biomechanical Regulation
of Endothelial Phenotype
Financial support for the printing of this thesis was kindly provided by:
The University of Groningen
University Medical Center Groningen
The studies described in this thesis were financially supported by research
grants from The Willem Johan Kolff Institute for Biomedical Engineering and
Materials Science, and The Jan-Kornelis de Cock Foundation, The University
of Groningen, The Netherlands.
Front cover: Landscape of Jiuzhaigou (Valley of Nine Fortified Villages), China
by Ee Han Lee
Copyright © 2015 by Ee Soo Lee
All rights reserved. No part of this publication may be reproduced, copied,
modified or transmitted in any form or by any means without the prior written
permission of the author.
ISBN 978-90-367-8447-4 (printed version)
ISBN 978-90-367-8448-1 (electronic version)
Book and chapter covers design by Ee Soo Lee
Artwork by Ee Han Lee and Ee Soo Lee
Layout by Rianne Jongman ([email protected])
Printed in The Netherlands
by CPI Koninklijke Wöhrmann B.V., Zutphen
Biomechanical Regulation
of Endothelial Phenotype
PhD thesis
to obtain the degree of PhD at the
University of Groningen
on the authority of the
Rector Magnificus prof. dr. E. Sterken
and in accordance with
the decision by the College of Deans.
This thesis will be defended in public on
Wednesday 23 December 2015 at 12.45 hours
by
Ee Soo Lee
born on 1 March 1982
in Kuala Lumpur, Malaysia
Supervisors
Prof. dr. M.C. Harmsen Prof. dr. R.A. Bank Assessment Committee
Prof. dr. G. Molema Prof. dr. P. ten Dijke Prof. dr. V. Everts
For my parents and sister
献给我的父母及妹妹
Paranymphs
Mrs. Linda A. Brouwer Mrs. Nurul Afiqah binti Multaza
Contents
Page
Aim of the Thesis9
Outline of the Thesis11
Chapter 1
General Introduction15
Chapter 2
Endothelial-to-mesenchymal transition contributes to fibro-proliferative vascular
disease and is modulated by fluid shear stress Chapter 3
Shear stress counteracts the pro-inflammatory effects of oxidative stress and TGF-β on
endothelial cells by suppressing the TAK1
pathway
Chapter 4
Shear stress does not reverse senescence of endothelial cells despite appropriate sensing:
implications for ageing-associated cardiovascular
disease
37
Chapter 5
TGF-β and inhibition of p38 MAPK alter cytoskeletal remodelling of endothelial cells
under shear stress
129
Chapter 6
General Discussion and Future Perspectives
147
65
101
Chapter 7
Summary173
Nederlandse Samenvatting178
Appendices Acknowledgements183
About the Author189
List of Publications193
List of Abbreviations 195
Aim of the Thesis
Aim of the Thesis
Endothelium, which forms the single-celled interior lining in blood vessels,
is the first component that senses and reacts to biochemical and biomechanical
stimuli in the bloodstream. The endothelium is adaptive to changes in the local
environment, which means that it responds to biochemical and biophysical cues
in its surrounding via changes in phenotype and plasticity. This plastic nature
of endothelium ensures optimal vascular homeostasis and function, while it
safeguards the stability of the intracellular environment in different vascular
beds. In the vascular system, endothelium is a key modulator of uptake and
passage of nutrients and waste products, inflammation, as well as contraction,
relaxation and blood vessel formation. However, excessive and/or chronic
exposure of endothelium to extracellular stimuli can result in an adverse
phenotypic alterations which compromise endothelial homeostasis and function.
This is evident upon overexposure to oxidative stress, pro-inflammatory stimuli
and/or pro-fibrotic factors that bring about unwanted inflammation and wound
healing (fibrosis) in the blood vessel. Also, during ageing, the cellular and
genetic machinery of endothelium wears. This alteration may lead to a state
of senescence in which the endothelial cells lose their proliferative capacity
and change into an adverse, pro-inflammatory phenotype. Adverse alteration of
endothelial phenotype is best reflected on endothelial dysfunction, endothelial
activation, endothelial-to-mesenchymal transition (EndMT) and endothelial
senescence, which associate with the development of cardiovascular diseases.
Shear stress elicited by the flowing blood also plays a pivotal role in
regulation of endothelial phenotype and development of cardiovascular diseases.
The mechanisms by which shear stress regulates endothelial phenotype under
oxidative stress and/or pro-inflammatory condition are well characterised.
Intriguingly, little is known about how shear stress modulates endothelial
phenotype upon exposure to excessive and chronic stimulation with pro-fibrotic
factor, transforming growth factor-β (TGF-β) under oxidative stress or during
ageing and senescence. The aim of this thesis is to dissect the mechanisms
that regulate phenotypic changes (and the consequences) of endothelium in
response to the combined effects of shear stress, TGF-β, oxidative stress and
ageing, in order to provide further molecular insights to relate endothelial cell
biology with pathogenesis of cardiovascular diseases.
10
Outline of the Thesis
Outline of the Thesis
Chapter 1 is the general introduction to the research of this thesis. The
function of endothelium in normal physiology is described. The alteration of
endothelial phenotype results from disruption of endothelial homeostasis by
cardiovascular risk factors is reviewed. Lastly, current mechanistic findings on
regulation of endothelial phenotype by TGF-β, oxidative stress and shear stress
are discussed.
Chapter 2 presents the interaction between TGF-β stimulation and shear stress
responses, as well as the effects of this interplay on endothelial phenotype. Our in
vitro results demonstrate that a pro-fibrotic condition, due to TGF-β stimulation,
alters endothelial phenotype. As a result, the endothelial cells transform from a
functional phenotype to a dysfunctional phenotype, namely “EndMT”. We show
that endothelial cells with characteristics of a mesenchymal lineage are present in
neointimal lesions and atherosclerotic plaques. This associates the occurrence of
EndMT with disturbed flow in the arteries. Such in vivo observations corroborate
our mechanistic findings that unlike disturbed flow, laminar flow (through high
shear stress) antagonises the TGF-β-driven EndMT. Our gain-of-function and
loss-of function studies show that shear stress requires the extracellular-signalregulated kinase 5 (ERK5) pathway to inhibit EndMT.
Since endothelial activation contributes to the onset of neointimal hyperplasia
and atherogenesis, we wondered if EndMT associates with endothelial activation.
Of note, the individual roles of the TGF-β-activated kinase 1 (TAK1), ERK5
and TGF-β pathways in regulating endothelial phenotype are well characterised,
yet an overall understanding of the orchestration of these pathways and their
crosstalk with the redox system under shear stress is lacking. We hypothesised
that shear stress counteracts both EndMT and endothelial activation by restoring
redox balance and repressing the TAK1 pathway through ERK5.
Chapter 3 provides molecular evidence that links EndMT with endothelial
activation and disruption of the endothelial redox balance. We show that TGF-β
aggravates endothelial activation and fails to restore the redox balance while
inducing EndMT. Suppression of the TAK1 pathway or oxidative stress represses
both endothelial activation and EndMT. Intriguingly, activation of the ERK5
pathway under static conditions does not counteract EndMT and endothelial
activation, despite the redox balance is restored. Our in vitro intervention
studies show that laminar shear stress suppresses endothelial activation through
down-regulation of the TAK1 pathway, which was, to our surprise, independent
of ERK5. Herein, we postulate that activation of the TAK1 pathway and the
consequences on endothelial phenotype rely on the level of TGF-β in the local
environment.
Endothelial senescence is endowed with a phenotype that is reminiscent of
EndMT, thereby we were prompted to investigate the underlying mechanisms
12
that relates endothelial senescence to EndMT. Next, we assessed how senescence
and ageing influence mechanotransduction in endothelial cells.
Chapter 4 suggests a positive feedback regulation between EndMT and
endothelial senescence. Remarkably, high shear stress could not counteract
senescence-associated EndMT, despite the activation of shear stress-responsive
pathways. Our in vivo data indicate that ageing may not alter phenotype of
endothelium if the protective flow mechanisms are in place.
Both shear stress and TGF-β cause changes in the cytoskeleton of endothelial
cells, as evident by the change in cell morphology and altered expression of
cytoskeletal proteins. However, the interplay between shear stress and TGF-β
in remodelling of endothelial cytoskeleton is not entirely understood. Thus, we
examined the influence of TGF-β stimulation on shear stress-induced cytoskeletal
remodelling and the mechanisms involved.
Chapter 5 shows the results of a study on cytoskeletal remodelling in
endothelial cells under influence of shear stress and pro-fibrotic stimulation.
We showed that activation of p38 mitogen-activated protein kinase (MAPK) by
shear stress was transient and might involve activation of focal adhesion kinase
(FAK). Notably, long-term shear stress repressed p38 MAPK phosphorylation
independent of the FAK signalling. Our data also demonstrated that inactivation
of the p38 MAPK pathway upon stimulation with TGF-β impairs cytoskeletal
remodelling by shear stress.
Chapter 6 is the general discussion and future perspectives for the studies
in this thesis. This chapter is subdivided into three parts. Firstly, it provides a
detailed overview of how extracellular biomechanical and biochemical signals
mediate endothelial plasticity. Then, novel insights into the role of shear stress
and TGF-β in modulation of endothelial phenotype and their association with
the development of cardiovascular diseases are explored. Lastly, it provides
further information on how senescence that associates with ageing alters the
plastic nature of endothelial cells and what is the role of shear stress and TGF-β
in modulation of endothelial phenotype. Here, we explained how our findings
support and improve the existing knowledge on endothelial cell mechanobiology.
Also, we elucidate how biomechanical and biochemical-induced mechanisms
relate alteration of endothelial phenotype with the pathogenesis of cardiovascular
diseases. We analysed and compared our findings with earlier investigations.
Last but not least, the strength, limitation and future plausible experiments were
discussed.
Chapter 7 provides a summary of this thesis and the significance of research
in each chapter.
13
Outline of the Thesis
14