University of Mississippi eGrove Electronic Theses and Dissertations Graduate School 2014 The Efficacy Of ostaglandinPr Synthase Inhibition On Attenuating Pressure Overload Induced Cardiac Remodeling Krishna T. Nagalla University of Mississippi Follow this and additional works at: https://egrove.olemiss.edu/etd Part of the Pharmacology Commons Recommended Citation Nagalla, Krishna T., "The Efficacy Of Prostaglandin Synthase Inhibition On Attenuating Pressure Overload Induced Cardiac Remodeling" (2014). Electronic Theses and Dissertations. 724. https://egrove.olemiss.edu/etd/724 This Dissertation is brought to you for free and open access by the Graduate School at eGrove. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of eGrove. For more information, please contact [email protected]. THE EFFICACY OF PROSTAGLANDIN SYNTHASE INHIBITION ON ATTENUATING PRESSURE OVERLOAD INDUCED CARDIAC REMODELING. A dissertation presented in partial fulfillment of requirements for the degree of Doctor of Philosophy in the Department of Pharmacology University of Mississippi By Krishna T. Nagalla May 2014 Copyright Krishna T Nagalla 2014 ALL RIGHTS RESERVED ABSTRACT Chronic hypertension has long been known to cause left ventricular remodeling. Although previous studies pointed towards inflammation as the pathological driver of cardiac remodeling, the exact mechanistic pathway associated with pressure overload-induced cardiac remodeling remains to be elucidated. In order to address this issue, we designed this study to determine pathways associated with pressure overload induced cardiac remodeling and identify therapeutic targets to attenuate this maladaptive cardiac remodeling. Rats with surgically constricted abdominal aorta were used as an animal model for pressure overload and associated Left ventricular (LV) structure and functional changes were measured by pressure-volume admittance catheterization. Human cardiac fibroblasts were used to assess the specific mechanistic pathways associated with prostaglandin D2 (PGD2) mediated fibrotic response. At the 14 day period, pressure overloaded animals showed significant changes in LV structural and functional parameters. Cyclooxygenase-2 (COX-2) inhibition with Nimesulide significantly attenuated the structural and functional changes associated with LV remodeling due to pressure overload. Prevention treatment with Nimesulide showed that the beneficial effects seen with acute treatment were still prevalent even after discontinuation of treatment. Intervention treatment with Nimesulide showed that only a few parameters like chamber size and stroke volume can be revered by COX-2 inhibition in rats with already established LV remodeling. Similar results were observed with prevention treatment with HQL-79 a specific H-Prostaglandin D synthase inhibitor, indicating that the PGD2 is a major driver for LV remodeling downstream ii of COX-2. Intervention treatment with HQL-79 showed, selective blockade of H- PGD synthase can significantly reverse maladaptive changes in cardiac structure and function indicated by LV mass index, chamber size, stroke volume and total collagen levels. In vitro studies on human cardiac fibroblasts have shown that both prostaglandin D receptors the DP2 and DP2 receptors down-regulate COX-2 expression. DP1 receptor was shown to up-regulate both Collagen 1A and 3A mRNA expressions, whereas DP2 receptor activation led to elevation of Collagen 1A and reduction in collagen 3A mRNA expression. These results indicate that COX-2 and downstream PGD2 are major drivers for pressure overload induced cardiac remodeling. H-PGD synthase was found to be a potential therapeutic target to prevent and reverse left ventricular remodeling induced by pressure overload. iii AKNOWLEDGEMENTS: 1 would like to thank my advisor Dr. David B Murray for giving me the opportunity to work in his laboratory and guiding me at every step of this work. I greatly appreciate his valuable guidance; constant support and encouragement which helped me advance my education as well as grow as a person. I would also like to thank my committee members, Dr. Anthony J. Verlangieri, Dr. Tracy Brooks and Dr. Rodney Baker for their support, guidance and help in preparing this dissertation. I would like to specially thank Dr. Tracy brooks for letting me access to her lab and teaching me various experimental techniques which were crucial for this study. I would like the express my gratitude to Dr. Matthews and other faculty of the department of Pharmacology for their valuable role in furthering my education. Special thanks to my fellow graduate students for their love and support. I am extremely grateful to my family for the unconditional love and support. iv CONTENTS: ABSTRACT .................................................................................................................................................. ii AKNOWLEDGEMENTS: .......................................................................................................................... iv LIST OF TABLES ..................................................................................................................................... viii LIST OF FIGURES ..................................................................................................................................... ix INTRODUCTION ........................................................................................................................................ 1 Hypertension and heart failure: ................................................................................................................ 1 Left ventricular remodeling: ..................................................................................................................... 2 The role of TNF- cardiac remodeling: .................................................................................................. 4 The role of TGF- on cardiac remodeling: .............................................................................................. 5 Interleukins and cardiac remodeling: ....................................................................................................... 6 Prostaglandins and cardiac remodeling: ................................................................................................... 6 Role of COX-2 and remodeling: .............................................................................................................. 8 Prostaglandin E2 in remodeling: ............................................................................................................... 9 Prostaglandin F2 in remodeling: ............................................................................................................. 10 Prostaglandin I2 in remodeling: .............................................................................................................. 11 Prostaglandin D2 in remodeling: ............................................................................................................. 11 MATERIALS AND METHODS: ............................................................................................................... 13 Drugs used: ............................................................................................................................................. 13 Methods: ................................................................................................................................................. 15 Animals used: ......................................................................................................................................... 15 Surgical induction of pressure overload: ................................................................................................ 15 Assessment of cardiac function: ............................................................................................................. 16 Tissue collection:.................................................................................................................................... 16 Protein Extraction:.................................................................................................................................. 17 Protein Quantification: ........................................................................................................................... 17 Hydroxyproline analysis for total collagen: ........................................................................................... 18 Western blotting: .................................................................................................................................... 18 ELISA (enzyme linked immune sorbent assay): .................................................................................... 18 Cell culture: ............................................................................................................................................ 19 COX-2 cell based ELISA (Human / Mouse Total COX-2 Immunoassay): ........................................... 19 v Collagen gene expression analysis: ........................................................................................................ 20 AIM: 1 ......................................................................................................................................................... 21 Introduction: ..........................................................................................................................................