Angiogenesis-Associated Gene Expression Changes In
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ANGIOGENESIS-ASSOCIATED GENE EXPRESSION CHANGES IN SURGICAL SKIN FLAPS OF DIABETIC RATS By Miao Xian (Cindy) Zhou, MB, DMD Submitted to the Faculty of the School of Graduate Studies of the Medical College of Georgia in partial fulfillment of the Requirements of the Degree of Master of Science July 2010 ANGIOGENESIS-ASSOCIATED GENE EXPRESSION CHANGES IN SURGICAL SKIN FLAPS OF DIABETIC RATS This thesis is submitted by Miao Xian (Cindy) Zhou and has been examined and approved by an appointed committee ofthe faculty of the School of Graduate Studies of the Medical College of Georgia. The signatures which appear below verify the fact that all required changes have been incorporated and that the thesis has received final approval with reference to content, form and accuracy of presentation. This thesis is therefore in partial fulfillment of the requirements for the degree of Master of Science. Date Major Advisor Department Chairperson Dean, School of Graduate Studies iv To my director, Colonel Frederick C. Bisch, for your remarkable leadership and extraordinary dedication to the program. We are very fortunate to have you as our director as I could not have asked for anything more. To Colonel Robert C. Gerlach for your endless optimism and cheerful demeanor that never failed to encourage me to do better. To Colonel Robert W. Herold for your concern in my professional and personal growth. Your balance of family and career is truly admirable and I hope I can learn even half your skills. To Lieutenant Colonel (P) Douglas R. Dixon for all the expertise and time you've put into shaping me to become a better researcher and clinician. I truly appreciate all your effort. Thank you for believing in us. To Major Thomas Johnson for introducing me to this project and for all the work and effort you've put into it to pave the way for me. I certainly would never have been able to tackle this challenging project otherwise. To my assistant, Mrs. Idella Harris, for all your hard work, patience and mentorship. You are truly a delight to work with. To Ms. Vickie Browning for always being there to support us no matter what and for providing outstanding support throughout our Master's and residency program. To Mrs. Lynne McTier for holding all of us together and for exemplifYing such excellent teamwork skills. To my awesome classmates, Dr. Brian Stancoven, Dr. Kimberly Inouye and Dr. Yat-Ho Ma for being such wonderful friends and great individuals! I value the program so much more because of you guys! Thank you! To all the other current and prior residents for your friendship, advice and expertise. To the wonderful staff and assistants at Tingay for making this residency all the more enriching with the vast experience and laughter you bring forth. To Mrs. Millar and the library staff at Eisenhower for the support you've provided and for always responding to my endless requests for article. Last but not least, to my family, for always being there for me. Words cannot express how grateful I am for your unconditional love and support. Thank you so much for all that you have done for me. I know I would not be where I am today without you all. v Major Advisor: Dr. Jill B. Lewis Committee Members: Dr. Douglas R. Dixon Dr. Robert C. Gerlach Dr. Stephen Hsu Dr. Carol A. Lapp Dr. Jose M. Pizarro Dr. UlfM. Wikesjo TABLE OF CONTENTS Acknowledgements iii List of Tables viii List at: Figures ix Introduction Statement of the Problem Significance 2 Review of the Literature 4 Diabetes mellitus, an overview 4 Diabetes and its complications 8 Association between diabetes mellitus and the periodontium II Altered immune function in diabetic patients 13 Investigation of genetic biomarkers for periodontal disease 14 Angiogenesis in diabetes mellitus 14 Molecular mechanisms for altered angiogenesis in diabetes mellitus 16 Surgical wound healing in skin vs. oral soft tissues 19 Angiogenesis in the modified McFarlane skin flap model 20 Purpose 22 Hypothesis 22 Specific Aims 22 Materials and Methods 23 Overview of Methods 23 RNA Isolation 25 RNA Amplification and Labeling 26 vi vii Orthogonal Polarization Spectral Imaging 32 Results 35 Discussion 61 Monitoring of blood flow in diabetic and control rats 61 Temporal and spatial characterization of flaps 62 Genes displaying the greatest up- and down-regulation 63 Baseline Changes 66 Genes changes that may be pertinent to disease entity 66 Correlating gene expression with flap physiology 69 Limitations 73 Future directions 74 Conclusions 75 Appendix A: Cumulative Changes of Significant Gene Changes 78 Appendix B: Volcano plots and scatter plots across all time points 80 Appendix C: Table of angiogenesis-associated genes grouped by function 84 Appendix D: Genes significantly modulated & RNA Checklist 86 Appendix E: Gene modulation across all zones over 7 days 89 Appendix F. Surgical flap model and microarray layout 92 References 93 viii LIST OF TABLES Table I. Microarray gene distribution 30 Table 2. Gene regulation in the A vascular Zone (Zone I) 41 Table 3. Gene regulation in the Static Zone (Zone II) 43 Table 4. Gene regulation in Flow Zone I (Zone III) 46 Table 5. Gene regulation in Flow Zone II (Zone N) 52 Table 6. Significantly under-expressed genes across all zones. 54 Table 7. Significantly over-expressed genes across all zones 54 Table 8. Significantly altered genes across all zones (p<0.05; Fold-change: 2::2 or 9 55 Table 9. Significantly down-regulated genes in Zone I & II but not in Zone III & N 56 Table I 0. Genes significantly down-regulated in only the flow zones (Zone III & IV) 56 Table II. Genes significantly up-regulated in only the flow zones 57 Table 12. Significantly altered genes during baseline 58 ix LIST OF FIGURES Figure I. Overview of methods 23 Figure 2. Surgical skin flap model measuring 3 x I 0 em 25 Figure 3. Division of the surgical flap into 4 equal zones measuring 3 x 2.5 em 25 Figure 4. Isolation ofmRNA 26 Figure 5. Angiogenic genes and their positions on a microarray 29 Figure 6. Sample of a developed microarray. 30 Figure 7. Determination of gene significance with a volcano plot 32 Figure 8. Cytoscan analysis 34 Figure 9. Determination of avascular and static lengths 34 Figure 10. Bar graph ofthe avascular, static and flow zones in diabetics vs. controls 35 Figure II. Mean total affected lengths over time measured) 36 Figure 12. Mean total affected lengths over time measured 37 Figure 13. Control vs. Diabetic Flaps on Day 0, I, 3 and 7 38 Figure 14. Significant gene regulation per zone across all zones 39 Figure 15. Significant gene regulation per day across all zones 40 Figure 16. Real-Time PCR results of significantly up-regulated genes 59 Figure 17. Pooled and unpooled gene expressions ofVEGFA, TGFBRI and ANGPTI 60 INTRODUCTION Statement of the Problem Despite current medical advances, diabetes mellitus remains one of the leading causes of death. Millions of people are affected by diabetes and millions more are either undiagnosed or pre-diabetic. It is well-documented that individuals with this condition have delayed wound healing and an increased susceptibility to infections. Implicated in the pathogenesis of impaired wound healing of diabetics is an abnormal angiogenic response. 1 Diabetic patients have been shown to have less coronary collateral vessel circulation compared to non-diabetic controls? One of the key factors associated with successful wound healing in regenerative periodontal therapy is the ability to achieve an adequate and functional blood supply. Much research has gone into analyzing factors that may enhance the wound healing potential in diabetic patients. However, the underlying genetic basis of these differences in wound healing is still poorly understood. Important questions to be answered in this study are 1) Is angiogenesis altered in diabetic skin flap healing compared to non-diabetic controls; 2) Is the direction of change toward greater or lesser angiogenesis, and 3) Importantly, what genes are most affected? The modified McFarlane flap helps to generate a predictable skin flap model characterized by 1 2 areas of avascularity, which may also include avascular tissue, an area of stasis and an area with blood flow, thereby allowing us to study the pattern of gene expression in these three physiological zones over time, using a streptozotocin-induced type I diabetic rodent model. Significance Angiogenesis, defined as the formation of new blood vessels from the pre-existing blood supply, is a well-orchestrated event fundamental to many physiological and pathological processes such as bone and tissue regeneration, diabetes, ischemic diseases and chronic inflarnmation?· 4 Excessive angiogenesis is a part of the pathology of certain diseases such as diabetic retinopathy and rheumatoid arthritis. Inadequate angiogenesis, on the other hand, occurs in cardiac failure and tissue damage after reperfusion of ischemic tissue. 5 The former group may benefit from therapeutic inhibition of angiogenesis, whereas the latter may see improvement with an enhancement of factors that promote angiogenesis. Anti-angiogenesis therapy is a promising focus of today's research to battle pro-angiogenic diseases, such as in diabetic retinopathy, and is viewed as a desperately needed breakthrough in treatment therapy. Recent studies have shown that angiogenesis is genetically pre-determined. 6 Furthermore, it has been purported that epigenetic changes caused by aberrant DNA methylation, or histone acetylation of anti angiogenic genes, may play an important role not only in angiogenesis but also in the 6 9 development of diabetes. " However, to date, no study has examined expression of genes associated with angiogenesis in skin flap healing of diabetics and compared this to 3 controls. Elucidation of the temporal and spatial expression of skin flap healing at the genetic level may provide valuable information that will impact the effect of these genes on a physiological level.