The endothelial glycocalyx: recovery, stability and role in electric field-directed cell migration in vitro By Weiqi Li Supervisors: Professor Wen Wang Dr Yiling Lu Submitted for the Degree of Doctor of Philosophy Institute of Bioengineering Queen Mary University of London 2014 I, Weiqi Li, confirm that the research included within this thesis is my own work. I accept that the College has the right to use plagiarism detection software to check the electronic version of the thesis. I confirm that this thesis has not been previously submitted for the award of a degree by this or any other university. Weiqi Li 25-11-2014 Conference: W.Li, Y.Lu and W.Wang. Contribution of endothelial glycocalyx to electric field directed cell migration. Bioelectrochemistry (Gordon Research Seminar). Biddeford, USA. July 5-6, 2014. (Oral presentation) W.Li, Y.Lu and W.Wang. Contribution of endothelial glycocalyx to electric field directed cell migration. Bioelectrochemistry (Gordon Research Conference). Biddeford, USA. July 6-11, 2014. (Poster presentation) Acknowledgement Foremost, I would like to thank my supervisor, Professor Wen Wang, for his encouragement, guidance and support to me throughout my PhD projector. Without his patience, I can’t finish my work in time. I am grateful to my co-supervisor, Dr Yiling Lu, now at University of Derby. I appreciate his detailed and constructive comments on my work. I would like to thank Dr Julien Gautrot in School of Engineering and Materials Science for his help on micropatterning studies and Dr Ann Wheeler in Blizard Institute of Cell and Molecular Science for the training on time-lapse microscopy. I thank engineering technicians Mr Vince Ford and Ms Jun Ma for their help on designing and manufacturing the EF and flow chambers. I thank Professors Qian Wang and Lei Zheng at Southern Medical University, China for their help and support that ensured my PhD studentship award from QMUL and the China Scholarship Council (CSC). I would also like to thank colleagues in the Biofluids and Cell Mechanics Laboratory, Dr Yankai Liu, Dr Ke Bai, Dr Lin Qiu, Dr Devendra Deo, Ms Miao Lin and Mr Xia Chen. Thank you very much for the warmth and happiness you have given me. My deepest gratitude goes to my parents and my wife Shuchen Zhang. I couldn’t have done my PhD without your understanding and support. Finally, I would like to thank all who have supported me during my PhD study. I Abstract Cardiovascular disease is the leading cause of unnatural death worldwide. Damage to the endothelial glycocalyx impairs endothelial functions and thereafter leads to the development of cardiovascular diseases. Despite this, many issues remain to be explored in our understanding of the metabolism and vasculoprotective potential of the glycocalyx. This study focuses on the recovery and structural stability of the glycocalyx, and its role in electric field-directed cell migration in vitro. The integrity of the glycocalyx is compromised following trypsin treatment during cell passages. Results from our study show that cell seeding density affects the recovery speed of the glycocalyx in the first 48h. Higher cell density results in more rapid recovery of the glycocalyx. Regardless of the initial cell seeding density, a well-developed glycocalyx layer is observed when cell confluence is reached. Micropatterning is used to study effects of the cell shape on the recovery of the glycocalyx. Elliptical patterns have been used to conform endothelial cells to torpedo shapes, mimicking their morphology under a shear flow. More rapid development of the glycocalyx on elliptical cells is observed than that on circular shaped cells during the early stage of recovery. Effects of the actin cytoskeleton on the stability of the glycocalyx are investigated, following our interest in shedding of the glycocalyx in abnormal vascular microenvironment. Rapid depolymerisation of the actin cytoskeleton leads to cell retraction within 10mins, with the glycocalyx preserved on the cell surface. This is also seen during 24h persistent actin disruption under static conditions. However, when endothelial cells are subjected to 24h steady laminar shear stress, the glycocalyx is seen to II shift to the downstream of the cell surface in the control group, and with actin depolymerisation, significant shedding of the glycocalyx from the luminal surface of the cell is observed. This happens together with the loss of focal adhesions on the basal membrane. Using a custom designed electric field (EF) chamber, I demonstrate that the cell migration speed increases by 30~40% following 5h of EF exposure. Cells also show preferred movement towards the anode. However, both are abolished after the enzymatic removal of the glycocalyx, indicating that the speedup and the directional cell migration in applied EF require the presence of the glycocalyx. Even distribution of the glycocalyx on the cell surface at the end of the EF stimulation suggests that EF-directed cell migration is not related to the polarization of the glycocalyx on the cell membrane. All these findings provide a better understanding of the glycocalyx, which will help to develop new strategies for protection of the glycocalyx, restoration of endothelial functions and finally prevention of cardiovascular diseases. III Table of contents Acknowledgement .................................................................................................................. I Abstract ................................................................................................................................. II Table of contents .................................................................................................................. IV List of abbreviations ............................................................................................................ IX List of tables ......................................................................................................................... XI List of figures ..................................................................................................................... XII 1. Introduction .................................................................................................................... 1 1.1. Composition of the endothelial glycocalyx ............................................................. 2 1.1.1. Proteoglycans ................................................................................................... 3 1.1.2. Glycoproteins ................................................................................................... 7 1.2. Dimensions of the endothelial glycocalyx ............................................................ 11 1.2.1. Thickness of the endothelial glycocalyx varies within blood vessels ........... 11 1.2.2. Thickness of the endothelial glycocalyx varies according to detection methods 14 1.2.3. Discrepancies between natural endothelium and cultured endothelial cells .. 16 1.2.4. Glycocalyx formation on cultured endothelial cells ...................................... 19 1.3. Functional importance of the endothelial glycocalyx ........................................... 26 1.3.1. Glycocalyx functions as a molecular sieve .................................................... 26 1.3.2. Glycocalyx functions as an immuno-modulator ............................................ 28 1.3.3. Glycocalyx functions as a mechano-sensor and -transduer ........................... 30 1.4. Perturbation of the endothelial glycocalyx ........................................................... 42 IV 1.4.1. Metabolic disorder ......................................................................................... 42 1.4.2. Ischaemic/Reperfusion ................................................................................... 43 1.4.3. Inflammatory stimuli ..................................................................................... 45 1.5. Electric field (EF)-directed endothelial cell migration ......................................... 47 1.6. Potential mechanism for EF-directed endothelial cell migration .......................... 48 1.6.1. Opening of gated Ca2+ channels .................................................................... 49 1.6.2. Redistribution of surface receptors ................................................................ 52 1.6.3. Polarization of charged molecules ................................................................. 54 1.6.4. Determination of moving direction ................................................................ 56 1.7. Aim and objectives ................................................................................................ 59 2. Materials and methods .................................................................................................. 61 2.1. Cell culture and treatment ..................................................................................... 61 2.2. Micropatterning ..................................................................................................... 63 2.3. Apparatus assembly .............................................................................................. 64 2.3.1. Shear stress apparatus .................................................................................... 64 2.3.2. Electric field apparatus .................................................................................. 67 2.4. Immunostaining ....................................................................................................