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Proquest Dissertations INFORMATION TO USERS This manuscript'has been raproducad from tha microfilm mastar. UMI films the text directly from the original or copy sutxnitlad. Thus, some thesis and dissertation copies are in typewriter face, wftNe otfiers may t>e from any type of computer printer. Tha quality of this reproduction is dépendant upon tha quality of the copy submitted. Broken or indistinct print, oolorad or poor qualify illustrations and photographs, print Meadthrough, sut)standard margins, and improper alignment can adversely affect reproduction. In the unlikely event ttiat the autfior did not send UMI a complete manuscript and there are missing pages, tfiese will be noted. Also, if unauttKXized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced tfy sectioning the original, beginning at the upper left-hand corner and continuing from left to right in equal sections with small overlaps. Photographs included in tfie original manuscript have twen reproduced xerographically in this copy. Higfier qualify 6" x 9" black arxf wfiite photographic prints are availat>le for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. Bell & Howell Information and Leaming 300 North Zeeb Road, Ann Arbor, Ml 48106-1346 USA 800-521-0600 UMI" ROLES AND MECHANISMS OF OXIDATIVE PATHWAYS IN CARDIOVASCULAR DISEASE DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University By Suvara Kimnite Wattanapitayakul, M.S. * * * * The Ohio State University 2000 Dissertation Committee: Approved by Associate Professor John A. Bauer, Advisor Professor Lane J. Wallace Adviser Professor Anthony P. Young College o f Pharmacy Assistant Professor Dale G. Hoyt UMI Number 9962459 Copyright 2000 by Wattanapitayakul, Suvara Kimnite All rights reserved. UMI UMI Microform9962459 Copyright 2000 by Bell & Howell Information and Leaming Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. Bell & Howell Information and Leaming Company 300 North Zeeb Road P.O. Box 1346 Ann Aitor, Ml 48106-1346 Copyright by Suvara Kimnite Wattanapitayakul 2000 ABSTRACT The primary goals of this thesis work were to further define the roles and mechanisms of oxidant related events during cardiac and vascular disease, particularly with respect to nitric oxide related actions, and to define potential anti-oxidant therapeutic strategies. In initial studies angiotensin II (ANGII) was administered to rats with a short term (3-day) sub-pressor dose and vascular tissues were then studied. Selective reductions in NO-dependent endothelial dysfunction was observed in ANGII treated animals, and this dysfunction was associated with endothelium specific prevalence of protein 3-nitrotyrosine (3-NT, a stable biomarker of tissue peroxynitrite formation). A novel image analysis technique was developed to quantify the extent of endothelial immunostaining, and a significant inverse correlation between endothelium mediated relaxant response and 3-NT immunoreactivity was observed. Endothelial peroxynitrite formation may therefore participate in (or initiate) early vascular effects of ANGII. Isolated endothelial cells showed that ANG II induced concentration and time dependent increases in protein nitration, demonstrating the direct in vitro action of ANGII in eliciting this response. Time dependent protein nitration was also observed in isolated endothelial cells in the absence of ANG II stimulation, and endothelial cell lysates were shown to possess enzymatic activity in modifying nitrated protein requiring a serum cofiactor(s). These data suggest that nitration of protein tyrosine residues may be a regulated process through nitration/denitration mechanisms rather than a contemporary view of it being a cytotoxic phenomenon. Cardiac remodeling is a key event in progressive heart disease involving selective myocyte dropout and fetal gene expression, but the initiating events are not well defined. Short-term (3-day) administration ANG II or TNF alone at physiologically relevant concentrations induced significant TUNEL positive nuclei (apoptosis marker) and 4-fold increases in LV atrial natriuretic peptide mRNA (fetal gene marker). In contrast, the combination of ANG II and TNF caused only changes in fetal gene expression. These changes in vivo were independent of hemodynamic or cardiac size changes, and not related to cardiac NOS II induction. Our findings suggest that important interactions between ANGII and TNF exist in vivo, and that induction of NOS II is not associated with these early hormonal influences. Ill In memory o f my mother and my father... who have fathomed me to the true meanings of love, loss, and .... life IV ACKNOWLEDGMENTS Many people have participated in assisting me building up my dissertation the way it is now. They are also being part of my appreciation of certain elements and aspects of sciences and the world during my long joumey in the graduate school. First of all, I wish to express my special gratitude to my adviser, Dr. John Anthony Bauer, for his valuable guidance, intellectual support, zealous inspiration, and encouragement. These not only made this dissertation possible but also provided me a life-long foundation of becoming a scientist. My parents have given me imperiled strength and supports. Their encouragement would remain in my mind eternally. Many thanks go to my sisters and brothers for their consistent supports throughout all these years. I am in debt to Dr. Anthony Young for transfection experiment and his co­ advice on the cardiac gene therapy project. I also thank Dr. Terri Riffe and Shawn Pierson for their assistance and companionship at the Neurobiotech. Center on west campus. I wish to thank Or. Dale Hoyt for his kind advice and assistance in endothelial cell culture. Assistance from Dr. Cynthia Carnes in preparing Langendorff hearts is greatly appreciated. I am also in debt to Dr. Bethany Holycross who provided me an initial guidance and assisted me to become proficient in rat surgery and Northem blot analysis. V Thank all the pharmacology colleagues and friends (Alysia Chaves. Mike Mihm, Dave Weinstein, Brandon Schanbacher, and others) who provided a very favorable environment and enchantment during my graduate school years. Special appreciation for ourfrierxtship is sent to Rosario Rojas who always resurrects my passion on sciences. I am grateful to the Royal Thai Government for providing me the opportunity to study abroad, and for the major financial support in my graduate school training. VI VITA September 11,1968 ..............................Bom - Bangkok. Thailand 1991 ................ ................. ..................... B.S. Pharmacy (Hon.), Mahidol University 1991 -1 9 9 4 .......................................... Medical Scientist, National Institute of Health, Nonthaburi, Thailand 1994 - 1998 ........................................... The Royal Thai Government Scholar 1998 .................................................... M.S. Pharmacy Administration, The Ohio State University 1998 - present ...................................... Graduate Teaching and Research Associate, The Ohio State University PUBLICATIONS 1. SK. Wattanapitayakul, DM. Weinstein, BJ. Holycross, and JA. Bauer, “Endothelial dysfunction and peroxynitrite formation are early events in angiotensin-induced cardiovascular disorders”. FASEBJ 14(2):271-278, (2000). VII 2. S. Wattanapitayakul, DM Weinstein, BJ. Holycross, and JA. Bauer, “Cardiac apoptosis and iNOS expression following angiotensin II, TNF-a, or their combination” FASEB J 13(5), Pt II, A753, (1999). 3. S. Wattanapitayakul and JO. Schommer, “The human genome project: benefits and risks to society”. Drug Information J.33(3),729-35, (1999). 4. S. Wattanapitayakul, DM. Weinstein, BJ. Holycross, and JA. Bauer, “Angiotensin II induced peroxynitrite formation in rat vasculature”. Circulation 98(17), SuppI 1395-1396, (1998). FIELDS OF STUDY Major Field: Pharmacy VIII TABLE OF CONTENTS Page Abstract........................................................................................................................ii Dedication .................................................................................................................. Iv Acknowledgments ..................................................................................................... v V ita ............................................................................................................................ vii List of Tables.............................................................................................................xi List of Figures ........................................................................................................... xii Chapters; 1. Introduction: Oxidative pathways in cardiovascular disease ............................. 1 2. Endothelial dysfunction and peroxynitrite formation are early events in angiotensin ll-induced cardiovascular disease ................................................ 49 3. Digital image analysis for immunohistochemical evaluation ........................... 80 4. Angiotensin II induces endothelial cell protein nitration in vitro ...................... 93 5. Interaction of angiotensin II and TNF-a in vivo .............................................. 118 Part I: Cardiac TUNEL staining and
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