UNIVERSITY OF CINCINNATI _____________May 18 , 20 00_____ I, Xiuqiong Wang , hereby submit this as part of the requirements for the degree of: Doctor of Philosophy in: Department of Molecular & Cellular Physiology It is entitled: Investigation of the Role of Annexin V in Mouse Placenta: Development of Approaches to Explore the Therapeutic Potential of the Protein Approved by: John R. Dedman, Ph.D., Chairman Robert O. Banks, Ph.D. Maria F. Czyzyk-Krzeska, M.D., Ph.D. Robert F. Highsmith, Ph.D. Gregory S. Retzinger, M.D., Ph.D. Investigation of the Role of Annexin V in Mouse Placenta: Development of Approaches to Explore the Therapeutic Potential of the Protein A Dissertation submitted to the Division of Graduate Studies and Research of the University of Cincinnati in partial fulfillment of the requirement for the degree of DOCTOR OF PHILOSOPHY in the Department of Molecular and Cellular Physiology of the College of Medicine 1999 By XIUQIONG WANG M.D., 1985, West China University of Medical Sciences, Chengdu, China M.S., 1988, West China University of Medical Sciences, Chengdu, China Advisor: John R. Dedman, Ph.D., Ohio Eminent Scholar ABSTRACT Annexin V is a promising candidate as an anticoagulant and anti-inflammatory agent due to its high affinity for anionic phospholipid surfaces. Its abundance and tissue localiza- tion in placental trophoblasts and vascular endothelial cells imply a physiological impor- tance in these tissues. In the first section of this study, I demonstrate that annexin V is critical in maintaining murine placental integrity. I conclude that the protecting role of annexin V appears to be local because I did not detect the protein in culture media of either trophoblasts or endothelial cells. In the next portion of this study, I successfully targeted annexin V to the secretory pathway of mammalian cells and the protein was secreted into the medium. This unnatural targeting of annexin V to the lumen of the endoplasmic reticu- lum did not have significant effect on cellular function. Therefore, I concluded that target- ing secretion of annexin V in transgenic animals should provide a valuable model for evalu- ating the therapeutic potentials of annexin V as anticoagulant and/or anti-inflammatory agent. Acknowledgement I would like to dedicate my thesis to my family for their endless love and support: My father Xijie Wang; My mother Xuehua Chen; My sister Xiuyun Wang; My brothers Jun Wang and Kai Wang; My nephews Jao Yang and Yifan Wang. Very special thanks to Charles William Hall Jr, the love of my life. Table of Contents CHAPTER I .................................................................................................. 4 INTRODUCTION ................................................................................................ 4 CHAPTER II............................................................................................... 17 ANNEXIN V IS CRITICAL FOR THE MAINTENANCE OF MURINE PLACENTAL INTEGRITY .................................................................. 17 INTRODUCTION .............................................................................................. 17 MATERIALS AND METHODS ....................................................................... 18 RESULTS ............................................................................................................ 21 COMMENTS ...................................................................................................... 24 CHAPTER III ............................................................................................. 35 DETECTION OF EXTRACELLULAR ANNEXIN V AND ATTEMPTS TO SECRET RECOMBINANT ANNEXIN V FROM CULTURED CELLS ..................................................................................................... 35 INTRODUCTION .............................................................................................. 35 MATERIALS AND METHODS ....................................................................... 35 RESULTS AND DISCUSSION ......................................................................... 40 CHAPTER IV ............................................................................................. 53 TRANSGENIC MANIPULATION TO TARGET THE SECRETION OF ANNEXIN V INTO THE CIRCULATION OF MICE ....................... 53 INTRODUCTION .............................................................................................. 53 MATERIALS AND METHODS ....................................................................... 55 RESULTS AND DISCUSSION ......................................................................... 58 CHAPTER V ............................................................................................... 66 TARGETING ANNEXIN V TO THE SECRETORY PATHWAY OF THYROID EPITHELIAL CELLS DOES NOT SIGNIFICANTLY ALTER CELLULAR FUNCTIONS ..................................................... 66 INTRODUCTION .............................................................................................. 66 MATERIALS AND METHODS ....................................................................... 68 RESULTS ............................................................................................................ 73 DISCUSSION ...................................................................................................... 88 CHAPTER VI ............................................................................................. 91 CONCLUSIONS ................................................................................................. 91 BIBLIOGRAPHY............................................................................................... 95 1 List of Figures Figure 1.1. Basic structure of annexin V............................................................................ 5 Figure 1.2. The intrinsic, extrinsic and common coagulation pathways. (HMW-K=high molecular-weight kininogen; PL=phospholipid). Adapted from Highsmith, RF, in Speralakis and Banks, 1993)..................... 9 Figure 2.1. Purification of recombinant epitope-tagged annexin V. ............................. 25 Figure 2.2. Mouse placental histology (4X). ................................................................... 26 Figure 2.3. Identification of extracellular binding sites for annexin V in placenta. ........ 27 Figure 2.4. Immunolocalization of endogenous annexin V in placenta. ......................... 28 Figure 2.5. Clotting assays (aPTT) with annexin V and anti-annexin V alone or in combination. ........................................................................................ 29 Figure 2.6. Time course of anti-annexin V in the circulation after tail-vein infusion. .... 30 Figure 2.7. Effects of circulating anti-annexin V antibody on pregnancy: gross morphology. ................................................................................................. 31 Figure 2.8. Effects of circulating anti-annexin V antibody on pregnancy: .......................... histopathology. ............................................................................................. 32 Figure 3.1. Localization of annexin V in rat placental trophoblasts and blood vessel endothelial cells. .......................................................................................... 47 Figure 3.2. Immunofluorescent staining of annexin V in permeabilized BeWo cells and immunoblot detection of annexin V in culture media. ......................... 48 Figure 3.3. Immunofluorescent localization of annexin V in permeabilized HUVEC and immunoblot analysis detection of annexin V in culture media............. 49 Figure 3.4. FITC-annexin V binding and immunofluorescent staining of annexin V on the surface of non-permeabilized BeWo cells. ....................................... 50 Figure 3.5. FITC-annexin V binding and localization of cell surface annexin V in human umbilical vein endothelial cells (HUVEC). ................................. 51 Figure 3.6. Expression and secretion of annexin V from COS-7 cells. ........................... 52 Figure 4.1. Transgene that is under control of PEPCK promoter. ................................... 60 Figure 4.2. PCR identification of transgenic mice. ......................................................... 61 Figure 4.3. Transgene that is under control of the albumin enhancer/promoter. ............. 62 Figure 4.4. PCR analysis of transgenic mice. .................................................................. 64 Figure 4.6. RT-PCR analysis of transgenic mice. ............................................................ 65 2 Figure 5.1. The transgene designed to secrete annexin V via the ER/Golgi secretory pathway......................................................................................... 77 Figure 5.2. Schematic description of tetracycline-inducible system (tet-on). ................. 78 Figure 5.3. Comparison of the localization of ER-targeted and endogenous annexin V in PCrTTA7 cells using indirect immunofluorescent microscopy. .......... 79 Figure 5.4. Western blot analysis of annexin V secretion from PCrTTA7 cells.............. 80 Figure 5.5. Cell growth curves evaluated by MTT assay................................................. 81 Figure 5.6. Equilibrium 45Ca2+ uptake and release by thapsigargin. ............................. 82 Figure 5.7. 45Ca2+ uptake and release in saponin-permeabilized cells. ......................... 83 Figure 5.8. The synthesis of thyroglobulin in non-induced and induced cells. ............... 84 Figure