ASSESSMENT OF THE ROLE OF PLASMALOGEN IN THE MODULATION OF OXIDATIVE STRESS AND INFLAMMATION IN ATHEROSCLEROSIS Aliki Anadena Rasmiena Bachelor of Science (Honours) This thesis is submitted in total fulfilment of the degree of Doctor of Philosophy December 2015 Department of Biochemistry and Molecular Biology Faculty of Medicine, Dentistry and Health Sciences University of Melbourne and Metabolomics Laboratory, Baker IDI Heart & Diabetes Institute i ABSTRACT Background: Oxidative stress is a contributing factor to atherosclerosis. Circulating levels of plasmalogens (phospholipids with potential anti-oxidant properties) have been shown to be negatively associated with coronary artery disease, suggesting an elevated level of oxidative stress in these patients. We hypothesised that: (1) oxidative stress affects lipoprotein lipid composition and function; and (2) regulation of the level of plasmalogen can influence atherosclerosis progression and inflammation. Method/results: Low density and high density lipoproteins (LDL and HDL) were oxidised with copper chloride at different time points. Liquid chromatography combined with tandem mass spectrometry analysis showed a myriad of changes in the lipid composition of lipoproteins during oxidation. Plasmalogen was one of the lipids that were most affected, early in the oxidation of lipoproteins. Incubation of THP1- derived macrophages with HDL of differing levels of oxidative stress showed that the capacity of mildly- and heavily oxidised HDL to efflux cholesterols was significantly reduced as compared to native HDL. Similarly, the ability of HDL to delay LDL oxidation and to accept oxidised lipids from oxidised LDL deteriorated progressively under mild- and heavy oxidative stress. To investigate the effect of plasmalogen enrichment in atherosclerosis, ApoE- and ApoE/glutathione peroxidase 1-deficient (ApoE-/- and ApoE-/-GPx1-/-) mice were fed a high-fat diet with or without 2% batyl alcohol (BA, precursor to plasmalogen synthesis) for 12 weeks. Mass spectrometry analysis of lipids in plasma, heart, liver and adipose tissue showed that plasmalogen concentration was increased in all tissues of the BA-treated ApoE-/- and ApoE-/-GPx1-/- mice. Oxidation of plasmalogen in the treated mice was apparent by the increase in sn-2 lysophosphatidylcholine in circulation. En face analysis showed that compared to the untreated mice, aortic plaque accumulation in the BA-treated ApoE-/- and ApoE-/-GPx1- /- mice was significantly reduced (70%). Immunohistochemistry of the aortic sinus and aorta indicated that the levels of the inflammatory marker, VCAM-1 and the oxidative stress marker, nitrotyrosine were reduced only in the BA-treated ApoE-/-GPx1-/- mice. Treatment with BA also resulted in a decrease in the body weight gain and fasting blood glucose without any effect on the fasting insulin level in these mice. Further lipidomic analysis demonstrated that diacyl- and triacylglycerols in the liver were lowered whereas that in the plasma was increased. Flow cytometry analysis of the peripheral ii whole blood of C57/BL6 mice showed that treatment with an alkylglycerol mix for 12 weeks lowered the levels of total monocytes and neutrophils. Conclusion: Oxidation affected lipids in both the surface layer and core of the lipoproteins and this translated to a deterioration of the lipoprotein function with increasing level of oxidative stress. In addition, the modulation of plasmalogen levels via treatment with alkylglycerol alleviated atherosclerosis in vivo potentially via a plethora of mechanisms involving inflammation and oxidative stress, and the regulation of glucose and body weight. Plasmalogen modulation represents a potential therapy to prevent atherosclerosis and reduce cardiovascular disease risk. iii DECLARATION This thesis comprises only of original work towards the degree of Doctor of Philosophy except where indicated in the preface. Acknowledgements have been made in text to other material that has previously published. This thesis is no more than 100,000 words exclusive of tables, figures, references, and appendices. Aliki Anadena Rasmiena December 2015 iv PREFACE The following technical work was conducted by individuals in the Baker IDI Heart and Diabetes Institute other than myself. I am sincerely thankful for their time to contribute this work to my thesis: 1) Dr. Judy B. de Haan (Oxidative Stress Laboratory) for providing transgenic ApoE/GPx1-deficient mice (Chapter 5). 2) Mr Annas Al-Sharea (Vascular Pharmacology Laboratory) for performing the mice tail bleed and flow cytometry analysis (Chapter 6). 3) Miss Natalie Mellett (Metabolomics Laboratory) for preparing alkylglycerol mix and the animal technicians at the Baker IDI (Miss Samantha Sacca, Miss Elisha Lastavec, Miss Megan Haillay) for performing the daily gavage of the alkylglycerol mix (Chapter 6). v ACKNOWLEDGEMENTS I am indebted to A/Prof. Peter Meikle, the principal supervisor of my PhD candidature, for first taking me on as his PhD student, and for the guidance and support throughout my candidature. His gift for imparting knowledge on lipidomics has been invaluable to me, as has the opportunities he has given to me to travel to various conferences. It has been instrumental in helping me expand my professional network and to continue to fuel my passion for Science. Furthermore, his calm collectedness and perseverance during challenging times have shown me what it takes to be a leader in the scientific field, and has inspired me to have a future career in science. I thank Dr. Dedreia Tull, co-supervisor and Prof. Malcolm McConville, chair of my PhD committee, for their invaluable feedback and discussion as well as words of encouragement throughout my candidature. I thank Dr. Judy de Haan for providing transgenic ApoE/GPx1-deficient mice. I am also grateful for her guidance both in the analysis of immunohistochemistry data and throughout the study. Her optimism during the manuscript submissions and revisions really encouraged us to pull through. To the past and present members of Metabolomics Laboratory, particularly Dr. Christopher Barlow, I thank him for the time he took to discuss concepts with me, to educate me in the use of complex Excel formulas, which proved to be an instrumental skill for handling large lipidomic data and for the training in mass spectrometry. I also acknowledge Dr. Theodore Wai Ng to inspire me to do PhD in the first place and for providing me training in sequential ultracentrifugation early in my PhD. I thank Kevin Huynh and Ricardo Tan for their technical assistance throughout the animal study and for making animal cull days an enjoyable one. I am grateful for the time Jacqui Weir and Natalie Mellett took to answering my technical questions on mass spectrometry and lipidomics, and for making sure that the laboratory was always neat and in order so I could perform my experiments. Anmar Anwar and Kang Yu Peng, fellow PhD students, warrant special mention for being wonderful office mates. Special thanks to Dr. Husna vi Begum, a very good friend out of the lab and practically a surrogate big sister to me. I thank her for her advice and for being there for me through tough times in PhD. To many staff members in the Baker IDI Heart and Diabetes Institute, particularly Nada Stefanovic and Dr. Arpeeta Sharma, I thank them for providing me training in organ dissection, en face, and immunostaining. I thank the University of Melbourne and Baker IDI for funding my PhD scholarship and the travel award I have received. Also I thank the organisers of various conferences including EAS, AAS, ASMR and Royal Society of Victoria for giving me the opportunities to present my work. To student committee members and friends in the Department and Baker IDI, I am grateful to get to know all of them; I thank them for making my candidature a colourful one and for the friendships we have built as we became more matured in our own journeys to completing our PhDs. I am deeply thankful to Kai Lin Giam, a close friend, for her words of wisdom and emotional support throughout our journey together since Honours. Special thanks to Camelia Quek whose cheerful attitude and positive outlook on life is contagious and got me through challenging times in PhD. I wish to acknowledge the emotional support of my close circle of friends, Joe Lim, Kelvin Yong, Joyce Yong, and Jesse Hon throughout my PhD. I thank them for always making my weekends and breaks from PhD a fun one. Special mention to Shou Farn Chung, meeting him towards the late stages of my PhD has been a blessing; I am grateful for the mini adventures he has introduced to me that taught me that sometimes there is more to life than doing a PhD. Most importantly, I acknowledge the incredible support and endless love from my parents, Dedy Djubaedi and Ratna Mediawati as well as my sister, Gratia Anadena. I am forever grateful for them for believing in me, their prayers, and for inspiring me to do my best. I dedicate this thesis to my dad who once said to me, …”go out there and be useful to your community”. Also, I dedicate this thesis to my grandparents, Kwik Ping Hoo and Kang Haw Nie who passed away from the very disease I studied. May these studies I conducted during my PhD contribute however small to the advancement of vii medical science and so there is hope for better treatment and quality of life for our future generation. viii PUBLICATIONS Publications arising from the research conducted for this thesis at the time of submission include the following: 1) A.A. Rasmiena, T.W. Ng, and P.J. Meikle, Metabolomics and ischaemic heart disease. Clinical Science, 2013. 124(5): 289-306. 2) A.A. Rasmiena, C. Barlow, N. Stefanovic, K. Huynh, R. Tan, A. Sharma, D. Tull, J.B. deHaan, P.J. Meikle. Attenuation of atherosclerosis in ApoE- and ApoE/GPx1- deficient mice by plasmalogen modulation. Atherosclerosis, 2015. 243(2): 598-608. 3) A.A. Rasmiena, C. Barlow, T.W. Ng, and P.J.