GENE EXPRESSION PROFILING IN HUMAN ACUTE ISCHEMIC STROKE by Taura Lynn Barr, RN BSN, University of Pittsburgh, 2004 Submitted to the Graduate Faculty of The School of Nursing in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2009 UNIVERSITY OF PITTSBURGH SCHOOL OF NURSING This dissertation was presented by Taura Lynn Barr It was defended on April 20, 2009 And approved by Dissertation Advisor: Yvette P. Conley, PhD, Associate Professor, School of Nursing Sheila A. Alexander, RN, PhD, Assistant Professor, School of Nursing Richard Henker, RN, PhD, Associate Professor, School of Nursing Steven Warach, MD, PhD, Chief, Stroke Diagnostics and Therapeutics, NINDS/NIH Andrew Singleton, PhD, Chief, Laboratory of Neurogenetics, NIA/NIH Jinhui Ding, PhD, Bioinformatician, Computational Biology Core, NIA/NIH ii Copyright © by Taura Lynn Barr 2009 iii GENE EXPRESSION PROFILING IN HUMAN ACUTE ISCHEMIC STROKE Taura Lynn Barr, RN, PhD University of Pittsburgh, 2009 Background: Gene expression profiling of human acute ischemic stroke (AIS) has the potential to identify a diagnostic panel for differential diagnosis of AIS early in the treatment phase. Purpose: The objective of this dissertation was to identify peripheral blood biomarkers that could be further explored for use in differential diagnosis of AIS and the design of stroke therapeutics. Methods: A prospective gene expression profiling study of 39 patients and 25 healthy controls was conducted. Peripheral blood samples were collected in Paxgene Blood RNA tubes from patients who were ≥18 years of age with MRI diagnosed AIS after differential diagnosis and controls who were Non-stroke neurologically healthy. In stroke patients, blood was redrawn 24 hours following onset of symptoms to determine changes in gene expression profiles over time. RNA was hybridized to Illumina humanRef-8v2 bead chips. Validation was performed using Taqman Gene expression polymerase chain reaction on significant targets. Results: A nine gene profile has been identified for AIS. Five of these nine genes were identified in the previously published whole blood gene expression profiling study of stroke and therefore play a likely role in the response to AIS in humans. One of these nine genes (s100A12) was significantly associated with increasing age and therefore may be non-specific for stroke. Three genes were identified as the whole blood expression profile change over time (LY96, IL8, and SDPR). Pathway analysis revealed a robust innate immune response, with toll like receptor iv (TLR) signaling as a highly significant pathway present in the peripheral whole blood of AIS patients. Conclusion: The findings of this study support the claim that gene expression profiling of peripheral whole blood can be used to identify diagnostic markers of AIS. A plausible case for innate immunity through the activation of TLR4 as a mediator of response to AIS has been made from the results of this study. This study and those conducted by Moore et al (2005) and Tang et al (2006) provide the foundation of data that support the use of peripheral whole blood for future blood profiling studies of neurological disease; which significantly opens the door of opportunity. v TABLE OF CONTENTS PREFACE............................................................................................................................... XVII 1.0 INTRODUCTION........................................................................................................ 1 1.1 ISCHEMIC STROKE......................................................................................... 1 1.1.1 The cerebral ischemic response ................................................................... 3 1.1.2 The cerebral genomic response.................................................................... 4 1.2 HYPERINTENSE ACUTE REPERFUSION INJURY MARKER................ 5 1.3 WHOLE GENOME EXPRESSION.................................................................. 6 1.4 PURPOSE............................................................................................................. 7 1.5 SPECIFIC AIMS ................................................................................................. 7 1.6 RESEARCH QUESTIONS/HYPOTHESES..................................................... 8 1.7 DEFINITION OF TERMS ................................................................................. 9 1.7.1 Independent variables .................................................................................. 9 1.7.1.1 Acute ischemic stroke........................................................................... 9 1.7.1.2 Hyperintense acute reperfusion injury marker................................. 9 1.7.2 Dependent variables...................................................................................... 9 1.7.2.1 Whole genome expression .................................................................... 9 1.7.3 Covariates .................................................................................................... 10 1.7.3.1 Environment........................................................................................ 10 vi 1.8 CONCEPTUAL FRAMEWORK..................................................................... 11 2.0 BACKGROUND AND SIGNIFICANCE ................................................................ 12 2.1 ISCHEMIC STROKE....................................................................................... 12 2.1.1 Pathophysiology of Ischemic Stroke ......................................................... 12 2.1.1.1 Reactive oxygen species...................................................................... 16 2.1.1.2 Neuro-inflammatory and immune responses................................... 17 2.1.2 Clinical Diagnosis........................................................................................ 19 2.1.2.1 Imaging Biomarkers........................................................................... 21 2.1.2.2 Blood Biomarkers ............................................................................... 23 2.1.3 Treatment .................................................................................................... 24 2.1.3.1 Thrombolysis....................................................................................... 25 2.1.3.2 Other treatments................................................................................. 27 2.1.3.3 Neuroprotection .................................................................................. 28 2.2 BLOOD BRAIN BARRIER DISRUPTION ................................................... 30 2.2.1 Pathophysiology following ischemic stroke .............................................. 32 2.2.1.1 Age and the BBB................................................................................. 34 2.2.2 Hyperintense acute reperfusion injury marker ....................................... 35 2.3 WHOLE GENOME EXPRESSION................................................................ 39 2.3.1 Ischemic Stroke and Genetics.................................................................... 39 2.3.2 Ischemic stroke and whole genome expression ........................................ 41 2.3.2.1 Animal proof of principle studies...................................................... 47 2.3.2.2 Human proof of principle studies...................................................... 49 2.4 SIGNIFICANCE AND INNOVATION........................................................... 53 vii 3.0 PILOT STUDIES ....................................................................................................... 55 3.1 PILOT STUDY I, MANUSCRIPT I: MMP9 AND HARM .......................... 55 3.2 PILOT STUDY II: KCNK17 AND ISCHEMIC STROKE............................ 57 3.3 PILOT STUDY III, MANUSCRIPT II: AGE AND HARM ......................... 58 3.4 REVIEW, MANUSCRIPT III: GENE EXPRESSION PROFILING IN BRAIN INJURY ................................................................................................................. 60 4.0 METHODS ................................................................................................................. 62 4.1 DESIGN.............................................................................................................. 62 4.2 SAMPLE............................................................................................................. 63 4.3 RATIONAL FOR SAMPLE SIZE................................................................... 65 4.4 SETTING............................................................................................................ 66 4.5 RECRUITMENT............................................................................................... 66 4.6 STANDARD MEDICAL CARE ...................................................................... 67 4.7 DATA COLLECTION...................................................................................... 68 4.8 DATA MANAGEMENT................................................................................... 68 5.0 VARIABLES, MEASURES, AND LEVEL OF MEASUREMENT ..................... 70 5.1 GENE EXPRESSION ....................................................................................... 70 5.1.1 Paxgene Blood RNA system ....................................................................... 70 5.1.2 RNA extraction............................................................................................ 71 5.1.3 RNA amplification