MAJOR COLLATERAL VESSELS DEVELOP FROM PRE-EXISTING SMALL ARTERIES THROUGH RAC2/NOX2 INDEPENDENT MECHANISMS Matthew Robert DiStasi Submitted to the faculty of the University Graduate School in partial fulfillment of the requirements for the degree Doctor of Philosophy in the Department of Cellular & Integrative Physiology Indiana University January 2009 Accepted by the Faculty of Indiana University, in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Joseph L. Unthank, PhD, Chair Michael Sturek, PhD Doctoral Committee David A. Ingram, MD 8/26/2008 Steven J. Miller, PhD Michael P. Murphy, MD David P. Basile, PhD ii ACKNOWLEDGEMENTS I’d very much like to thank my mentor Joseph L. Unthank for all his guidance in every aspect of my doctoral training. I would also like to thank Jamie Case, Matt Ziegler, Todd Cook, and everyone in the Physiology Department for all your help and support, especially the members of my thesis committee: Michael Sturek, PhD., David A. Ingram, MD., Steven J. Miller, PhD., Michael P. Murphy, MD., and David P. Basile, PhD. iii ABSTRACT Matthew Robert DiStasi Major Collateral Vessels Develop from Pre-existing Small Arteries Through Rac2/Nox2 Independent Mechanisms. There is no consensus on which vascular segment or what size of vessels is most important in the process of collateral growth, the degree to which these vessels can enlarge, or the mechanisms that mediate collateral vessel expansion and its impairment. Chapter I identifies the major collateral vessels that develop in response to femoral arterial occlusion in the pig, rat, and mouse hindlimbs for comparison to humans. Pre- existent small named arteries enlarged ~2-3-fold to become the major collateral vessels in each species, these major collaterals displayed characteristics similar to large arteries experiencing flow-mediated outward remodeling, and important differences in vascular wall thickness were observed between rodents and pigs. Chapter II utilized Rac2-/- and Nox2-/- mice to investigate the hypothesis that Nox2-NAD(P)H oxidase is required for major collateral growth subsequent to femoral arterial occlusion. Previous studies suggest bone marrow cell (BMC)-derived reactive oxygen species (ROS) produced by the Nox2 subunit of NAD(P)H oxidase plays an important role in neovascularization and recovery of hindlimb perfusion subsequent to femoral arterial occlusion; but did not investigate collateral growth. The hematopoietic cell restricted protein Rac2 has been shown to bind to and activate Nox2-NAD(P)H oxidase and Rac2-/- and Nox2-/- leukocytes display impaired ROS related functions. The data demonstrated that Rac2 and Nox2 are not essential for major collateral growth, but both are important for the iv recovery of hindlimb perfusion and preservation of distal tissue morphology. Chapter III investigated BMC and antioxidant therapy in the age-related impairment of collateral growth. Aging, like all cardiovascular disease risk factors is associated with elevated ROS and impaired collateral growth. Studies also suggest BMCs promote collateral growth by secreting paracrine factors but elevated ROS may affect the efficacy of BMCs. The data revealed that neither BMC injection nor antioxidant therapy via apocynin enhanced the process of major collateral artery growth in aged mice. Joseph L. Unthank, PhD, Chair v TABLE OF CONTENTS List of Tables vii List of Figures viii List of Abbreviations xi Introduction 1 Chapter I: Characteristics of Major Collateral Arteries in the Peripheral Circulation of Humans, Pigs, Rats, and Mice 25 Chapter II: Suppressed Hindlimb Perfusion in Rac2-/- and Nox2-/- Mice Does Not Result from Impaired Collateral Growth 57 Chapter III: Pilot Study for Future Experiments: Effect of Bone Marrow Cell and Antioxidant Therapy on Age-Related Collateral Growth Impairment 93 Summary/Discussion of Thesis Work and Future Experiments 122 References 129 Curriculum Vitae vi LIST OF TABLES Chapter I: Table 1: Hindlimb Collateral Vessel Size and Fold Enlargement 46 Chapter II: Table 1: Summary of the Primary Collateral Pathway Locations 76 Table S1: Laser Doppler Flux Values in Control and Experimental Limbs in All Groups 86 Chapter III: Table 1: Collateral Growth in Treated and Untreated Aged Mice 28 Days Post-Ligation 110 vii LIST OF FIGURES Chapter I: Figure 1: Diagram of the hindlimb arterial vasculature in pigs, rats, and mice 50 Figure 2: Major collaterals in human lower extremity angiograms with arterial occlusion 51 Figure 3: Representative porcine hindlimb angiographic series demonstrating major collateral arteries 52 Figure 4: Major collaterals in rat hindlimb with proximal femoral artery occlusion 53 Figure 5: Major collaterals in mouse hindlimb with distal femoral artery occlusion 54 Figure 6: Cross-sections of pig, rat, and mouse control and major collateral arteries 55 Figure 7: Average diameters of hindlimb collateral and control arteries from rats and mice 56 Chapter II: Figure 1: Primary collateral pathways in the mouse hindlimb after 14 days of femoral artery ligation or excision 80 Figure 2: Vascular compensation to femoral arterial ligation in BL6 and Rac2-/- mice 81 Figure 3: Vascular compensation to femoral arterial excision in BL6 and Rac2-/- mice 83 viii Figure 4: HEMAVET and FACS analysis of total white blood cells (WBCs) and CD11b + cells 84 Figure 5: Vascular compensation to femoral arterial excision in Nox2-/- mice 85 Supplemental Figure S1: Schematic of the murine hindlimb vascular anatomy as well as the femoral artery ligation and excision models used in this study 89 Supplemental Figure S2: Control and experimental limb gastrocnemius muscles from BL6 and Rac2-/- mice after 14 days of femoral arterial ligation 90 Supplemental Figure S3: Control and experimental limb gastrocnemius muscles from BL6 and Rac2-/- mice after 14 days of femoral arterial excision 91 Supplemental Figure S4: Representative control and collateral vessels as well as control and experimental limb gastrocnemius muscle micrographs from Nox2-/- mice 92 Chapter III: Figure 1: Hindlimb perfusion in young and old mice 114 Figure 2: Hindlimb perfusion after bone marrow mononuclear cell (BM- MNC) injection therapy 115 Figure 3: Hindlimb perfusion after injection of BM-MNCs at day 1 post- ligation 116 Figure 4: Hindlimb perfusion in aged mice treated with apocynin 117 Figure 5: Hindlimb perfusion in two sets of young and old mice 118 ix Figure 6: Percent increase in collateral vessel diameter in young and old mice at 14 and 28 days post-occlusion 119 Figure 7: Analysis of total white blood cells (WBCs) by HEMAVET and CD11b + cells by FACS 120 Figure 8: Assessment of intimal cell number in young and old mice 121 x [...]... that the major collaterals in all species formed from preexisting small arteries Tortuosity of collaterals varied considerably with major segments displaying limited convolution The pre-existing arteries which formed major collaterals enlarged . members of my thesis committee: Michael Sturek, PhD., David A. Ingram, MD., Steven J. Miller, PhD., Michael P. Murphy, MD., and David P. Basile, PhD. iii ABSTRACT Matthew Robert DiStasi Major. FROM PRE-EXISTING SMALL ARTERIES THROUGH RAC2/NOX2 INDEPENDENT MECHANISMS Matthew Robert DiStasi Submitted to the faculty of the University Graduate School in partial fulfillment. between rodents and pigs. Chapter II utilized Rac2-/- and Nox2-/- mice to investigate the hypothesis that Nox2-NAD(P)H oxidase is required for major collateral growth subsequent to femoral