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Allicin Reverses Diabetes-Induced Dysfunction of Human Coronary Artery Endothelial Cells

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Allicin Reverses Diabetes-Induced Dysfunction of Human Coronary Artery Endothelial Cells Philadelphia College of Osteopathic Medicine DigitalCommons@PCOM PCOM Biomedical Studies Student Scholarship Student Dissertations, Theses and Papers 5-2019 Allicin Reverses Diabetes-Induced Dysfunction of Human Coronary Artery Endothelial Cells Daniel Horuzsko Follow this and additional works at: https://digitalcommons.pcom.edu/biomed Part of the Medicine and Health Sciences Commons Recommended Citation Horuzsko, Daniel, "Allicin Reverses Diabetes-Induced Dysfunction of Human Coronary Artery Endothelial Cells" (2019). PCOM Biomedical Studies Student Scholarship. 187. https://digitalcommons.pcom.edu/biomed/187 This Thesis is brought to you for free and open access by the Student Dissertations, Theses and Papers at DigitalCommons@PCOM. It has been accepted for inclusion in PCOM Biomedical Studies Student Scholarship by an authorized administrator of DigitalCommons@PCOM. For more information, please contact [email protected]. Philadelphia College of Osteopathic Medicine Biomedical Sciences Allicin Reverses Diabetes-Induced Dysfunction of Human Coronary Artery Endothelial Cells A Thesis in Biomedical Science by Daniel Horuzsko Copyright May 2019 Daniel Horuzsko Biomedical Sciences Degree Program Thesis Signatory Page We approve the thesis of Daniel Horuzsko. Richard E. White, Ph.D., F.A.H.A. Date of Signature Professor of Neuroscience, Pharmacology, and Physiology Associate Director of Biomedical Sciences Program Philadelphia College of Osteopathic Medicine-Georgia Thesis Advisor Shu Zhu, M.D., Ph.D. Date of Signature Associate Professor of Neuroscience, Pharmacology, and Physiology Department of Biomedical Sciences Philadelphia College of Osteopathic Medicine-Georgia Committee Member Shafik Habal, M.D. Date of Signature Associate Professor of Microbiology and Immunology Department of Biomedical Sciences Philadelphia College of Osteopathic Medicine-Georgia Committee Member Richard E. White, Ph.D., F.A.H.A. Date of Signature Associate Director of Biomedical Sciences Program Professor of Neuroscience, Pharmacology, and Physiology Philadelphia College of Osteopathic Medicine-Georgia iii Abstract Allicin Reverses Diabetes-Induced Dysfunction of Human Coronary Artery Endothelial Cells Daniel Horuzsko Master’s in Biomedical Science, May 2019 Philadelphia College of Osteopathic Medicine Georgia Richard E. White, Thesis Advisor Cardiovascular disease (CVD) is the leading cause of death in the United States, and is the major source of morbidity and mortality associated with diabetes mellitus. Because the incidence of diabetes continues to increase, reducing the risk of CVD in diabetes will continue to be a major focus of cardiovascular research. An early manifestation of diabetes-induced CVD is dysfunction of the vascular endothelium, as indicated by depressed production of NO. Our findings now demonstrate depressed activity of endothelial nitric oxide synthase (eNOS) in diabetes, and suggest that treating human coronary artery endothelial cells with allicin, the major bioactive organosulfur component in garlic extract, can restore NO production in these cells. Coronary artery endothelial cells (Lonza) were obtained from control (HCAEC) or diabetic (DHCAEC) donors, and NO production was measured by fluorescence microscopy via 4,5- diaminofluorescein diacetate. On average, NO production was depressed by 12.9% in DHCAEC compared to controls. Treating these cells for 20 minutes with 4μM allicin restored NO production by 32.9%. Further, immunoblot studies revealed that diabetes decreased expression of eNOS protein by 20.3%; however, allicin was able to reverse this effect of diabetes. On average, eNOS expression was increased by 26% by overnight exposure to 5μM allicin. Taken together, these data indicate that allicin improves endothelium-dependent NO production in diabetes by enhancing the expression and/or iv activity of eNOS in human coronary artery endothelial cells. These studies further suggest that this improved endothelial function likely contributes to the established health benefits of garlic consumption (e.g., lowering blood pressure), and suggests a natural means of reducing the devastating consequences of diabetes on CVD. Future experiments are needed to identify the mechanism of allicin action on eNOS and in vascular endothelial cells. v Table of Contents Abstract…………………………………………………………………………………..iv List of Figures and Tables…………………………………………………………….viii Figures………………………………………………………………………….viii Tables……………………………………………………………………….…..viii Abbreviations…………………………………………………………...……….ix Acknowledgements……………………………………………………………………...xi Introduction……………………………………………………………………………....1 Cardiovascular Disease………………………………………………………….1 Coronary Arte ry Dise ase………………………………………………………...2 Athe rosclerosis…………………………………………………………………...4 Diabetes…………………………………………………………………………...6 Vascular Endothelium…………………………………………………………...9 Nitric Oxide Production………………………………………………………..11 Therapies and Interventions…………………………………………………...13 Allicin………………………………………………………………...………….15 Materials and Methods……………………………………………………………........18 Thawing Cells for Cell Culture……………………………………………...…18 Cell Culture and Cell Passaging……………………………………………….19 Harvesting Cells for Storage in Liquid Nitrogen……………………………..21 Mechanism of DAF-2DA……………………………………………………….22 Allicin Treatment and EVOS Imaging………………………………………..24 vi L-NAME Treatment and EVOS Imaging…………………………………………….24 Immunoblotting………………………………………………………………....……...25 Immunoprecipitation and Mass Spe ctrome try (MS)…………………………………26 Statistical Analysis……………………………………………………………………...26 Results………………………………………………………………………………...…28 Allicin Increases NO Production and L-NAME Reverses This Effect in DHCAEC-I……………………………………………………………….…......28 Allicin increases eNOS expression in DHCAEC-I……………………………30 MS Analysis of eNOS Proteins Modified by Allicin………………………….32 Discussion……………………………………………………………………………….75 Summary………………………………………………………………………………...80 Bibliography…………………………………………………………………………….82 vii List of Figures and Tables Figures 1. Mechanism of DAF-2DA Fluorescence 2. Mechanism of L-NAME 3. NO production in DHCAEC-I cells detected by DAF-2DA 4. Allicin increased eNOS expression in DHCAEC-I cells 5. Venn Diagram of DHCAEC-I Treatment Groups 6. Proteins Associated with eNOS Immunocomplex After Allicin Treatment Mass Spectrometry Results 7. Proposed Vasodilatory Mechanism of Allicin Tables 1. Media Composition 2. Experimental Design viii Abbreviations Abbreviation Definition ANG-II Angiotensin-II BH2 7,8-Dihydrobiopterin BH4 Tetrahydrobiopterin CAD Coronary Artery Disease CVD Cardiovascular Disease DAF-2 4,5-Diaminofluorescein DAF-2DA 4,5-Diaminofluorescein Diacetate DAF-2T 4,5-Diaminofluorescein Triazole DHCAEC-I Diseased Human Coronary Artery Endothelial Cell-Type I Diabetic DHCAEC-II Diseased Human Coronary Artery Endothelial Cell-Type II Diabetic DM Diabetes Mellitus DMSO Dimethyl Sulfoxide DPP-IV Dipeptidyl-Peptidase IV EDHF Endothelial Derived Hyperpolarizing Factor eNOS Endothelial Nitric Oxide Synthase ET-1 Endothelin-1 GCL Glutamate-Cysteine Ligase GSH Glutathione HCAEC Human Coronary Artery Endothelial Cell HO-1 Heme Oxygenase-1 ICAM-1 Intercellular Adhesion Molecule-1 IL-1β Interleukin-1 Beta IL-4 Interleukin-4 IL-6 Interleukin-6 IL-8 Interleukin-8 ix L-NAME Nitro-L-arginine methyl ester NADH Nicotinamide Adenine Dinucleotide NCDs Non-Communicable Diseases NO Nitric Oxide PGI2 Prostacyclin PGH2 Prostaglandin PKA Protein Kinase A PKB; Akt Protein Kinase B PRFM Phenol-Red Free Media ROS Reactive Oxygen Species SGLT-2 Sodium-Glucose Cotransporter-2 SH Sulfhydydrl SNP Sodium Nitroprusside TNFα Tumor Necrosis Factor Alpha T1DM Type 1 Diabetes Mellitus T2DM Type 2 Diabetes Mellitus x Acknowledgements Undertaking this Master’s degree has been an incredible experience for me and it would not have been attainable without the aid and guidance that I received from faculty, staff, and colleagues at PCOM-GA. I extend my sincere gratitude and appreciation to all who have made this research possible. First and foremost, I would like to thank Dr. Richard E. White for encouraging my research endeavors, guiding me along the way in my academic pursuits, and allowing me the opportunity to grow as researcher. He has been supportive and patient in the past year and I am beyond appreciative for his contributions of time, ideas, expertise, motivation, and encouragement to make my project dynamic and exciting. I would also like to give a big thank you to Ms. Handong Ma for spending countless hours with me in the lab teaching me laboratory techniques and lessons. Another large thank you goes to Ms. Yan Wu who taught me microscopic use and methods along with always providing lab assistance. Without their knowledge, my research would not have been possible. Thank you to my committee members, Dr. Mary Owen, Dr. Shu Zhu, and Dr. Habal, for their flexibility to provide knowledge and input throughout my research project. Thank you to Dr. Lambert Ngoka at the Georgia Cancer Center for his expertise in mass spectrometry analysis. Lastly, I would like to thank all of my thesis colleagues and peers for sharing their support, advice, and motivation throughout my research experience. xi Introduction Cardiovascular Disease Cardiovascular disease is one of the most serious non-communicable diseases (NCDs) and health-related problems in the world (Balakumar, Maung, & Jagadeesh, 2016). This disease actually represents a number of different heart- or blood vessel- related diseases, including coronary artery disease (CAD), stroke, hypertension, heart failure, congenital heart
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