Effects of Enhanced External Counterpulsation (EECP) on Glycemic Control and Arterial Function in Patients with Abnormal...

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Effects of Enhanced External Counterpulsation (EECP) on Glycemic Control and Arterial Function in Patients with Abnormal Glucose Tolerance
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english
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Martin,Jeffrey S
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Doctorate ( Ph.D.)
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University of Florida
Degree Disciplines:
Health and Human Performance, Applied Physiology and Kinesiology
Committee Chair:
Braith, Randy W
Committee Members:
Powers, Scott K
Criswell, David S
Sumners, Colin
Aranda-Amador, Juan M

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Subjects / Keywords:
capillarity -- diabetes -- eecp -- flow -- fmd -- glucose -- insulin -- muscle -- nitric -- oxide -- signaling -- vascular -- vop
Applied Physiology and Kinesiology -- Dissertations, Academic -- UF
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Health and Human Performance thesis, Ph.D.
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Abstract:
Enhanced external counterpulsation (EECP) is a noninvasive modality used to treat patients with refractory angina. The salutary benefits of EECP treatment include increases in peripheral vascular function and nitric oxide bioavailability. Nitric oxide (NO) is also known to upregulate the translocation and expression of the major glucose transporter in muscle, glucose transporter 4 (GLUT-4). Of critical importance, is the fact that this NO signaling pathway is distinct from and/or additive to insulin-stimulated skeletal muscle glucose uptake, which is notoriously impaired in humans with abnormal glucose tolerance (AGT). Moreover, the effects of EECP on skeletal muscle morphology and glucose uptake signaling are largely unknown. Therefore, the purpose of this study was to investigate the effects of EECP treatment on glycemic control and arterial function in patients with AGT. Eighteen (n = 18) patients with AGT were recruited for the study. They were randomly assigned to receive either 35 1-hour sessions of EECP (n = 12) or 7-weeks of standard care (n = 6). Peripheral vascular function, vasoactive balance, oxidative stress, inflammation, skeletal muscle morphology, skeletal muscle protein expression, fasting glycemic control, and dynamic glucose tolerance were measured before and after 35 1-hour sessions of EECP or Time-Control. EECP resulted in an increase in normalized brachial artery (27%) and popliteal artery (52%) flow-mediated dilation as well as peak (26%) and total (37%) forearm hyperemic blood flow. Plasma nitrite/nitrate (NOx) increased (30%) and asymmetric dimethylarginine (ADMA) decreased (11%) following 35 sessions of EECP. Additionally, 8-isoprostane-PGF-F2-alpha (8-iso-PGF2-alpha), a marker of lipid peroxidation in the plasma, decreased (23%) with a concurrent decrease (28%) in high sensitivity C-reactive protein (hsCRP), a marker of inflammation. Analysis of vastus lateralis skeletal muscle biopsies revealed significant increases in endothelial nitric oxide synthase (eNOS) (87%) and GLUT-4 protein expression (47%) following EECP. Furthermore, there was a significant elevation in humoral concentrations of vascular endothelial growth factor (VEGF) (75%) and the capillary-to-fiber ratio (8%) following EECP. Fasting plasma glucose (FPG) was reduced by 16.9 mg/dL and the homeostasis model assessment of insulin resistance (HOMAIR) decreased (31%) following EECP. Moreover, plasma glucose concentrations 120 minutes after initiation of an oral glucose test (OGTT) decreased (224.4 mg/dL vs. 196.1mg/dL) and the whole-body composite insulin sensitivity index (ISI composite) increased (21%). The improvements in NO bioavailability and endothelial function observed in the present study, as well as capillary to fiber ratios, likely mediate greater delivery of nutrients to ?nutritive? tissues (i.e. skeletal muscle) at rest and during glycemic challenge. Additionally, NO signaling been has implicated in regulation of GLUT-4 translocation and glucose uptake. The multifaceted nature of vascular function and glycemic control makes it difficult to isolate a single mechanism responsible for these adaptations. However, novel evidence from the present study supports the hypothesis that decreasing nitric oxide bioavailability contributes to the pathology of AGT. Interventions that improve NO bioavailability may be attractive treatment strategies for patients with AGT.
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In the series University of Florida Digital Collections.
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Statement of Responsibility:
by Jeffrey S Martin.
Thesis:
Thesis (Ph.D.)--University of Florida, 2011.
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Adviser: Braith, Randy W.
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EFFECTS OF ENHANCED EXTERNAL COUNTERPULSATION (EECP) ON GLYCEMIC CONTROL AND ARTERIAL F UNCTION IN PATIENTS WITH ABNORMAL GLUCOSE TOLERANCE By JEFFREY S. MARTIN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORID A IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2011 1

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2011 Jeffrey S. Martin 2

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This dissertation is dedicated to my wife, A llison Marie Martin, and my family. Their love and support have carried me through this very rewa rding chapter of my life. I am truly blessed to have so many wonderful people in my corner. I would not be who I am today without them. They provide me with the c onfidence to succeed in wherever the road ahead may take us. 3

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ACKNOWLEDGMENTS I would first like to thank my mentor, Dr. Randy W. Braith, for the opportunity to pursue my Ph.D. in his laborator y. I am thankful for Dr. Brai th giving me the freedom to pursue my own research ideas, developing me to become an independent scientist, and his unwavering and continued support in my career development. My success as a doctoral candidate has been driven by his guidance and tutelage. I would also like to thank the members of my committee, Dr. David Criswell, Dr. Scotty Powers, Dr. Colin Sumners, and Dr Juan Aranda Jr. for their suggestions and guidance. They were instrumental in my education at the University of Florida and frequently offered invaluable guidance for this project and my career. A special thanks to Dr. Juan M. Aranda Jr. for graciously agreeing to perform the muscle biopsies associated with this project. Thank you to Darren T. Beck, Ph.D. Jenna Harty, Blaze D. Em erson, William E. Motch, and Joshua Lucas, for their friendship and assistance in the completion of this research project. Their cont ributions to the development and execution of this project were invaluable. I would also like to thank the many students in the Applied Physiology and Kinesiology department that were alwa ys willing to provide guidance and lend a hand when needed. In particular, Matt Hudson, Erin Talbot, and Linda Ngyuen were imperative to the success of this project. I would also like to thank Kim Hatch and Ka ren Kleis for all of their help with the completion of this project. Their patience and effectiveness in welcoming subjects to the facility each day and the never-ending laboratory orders were vital. I would like to thank all of the subjects who graciously dedicated their time to participate in this study. Their friendshi p and genuine interest in the project is 4

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something that I will carry with me. Their par ticipation and recruitment of subjects for this project was essential to the timely completion of this study. Finally, I would like to thank my wife, and my family, for their love and support. I would not be who I am today, nor had the sa me successes without them. I would also like to thank my friends for thei r support throughout this process. 5

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ..................................................................................................4 LIST OF TABLES ............................................................................................................9 LIST OF FIGURES ........................................................................................................10 LIST OF ABBREVIATIONS ...........................................................................................13 ABSTRACT ...................................................................................................................18 CHAPTER 1 INTRODUC TION....................................................................................................21 Background .............................................................................................................21 Antihyperglycemic Treatment Options ..............................................................23 Glucose Uptake Pathways in Skeletal Muscle ..................................................24 Insulin mediated glucose uptake (Pathway #1) ..........................................24 Contraction mediated glucose uptake (Pathway #2) ..................................25 Nitric oxide (NO) mediated glucose uptake (Pathway #3) ..........................25 Principles of Enhanced Exte rnal Counterpulsation (EECP) ..............................27 Central angiogenesis mechanism of EECP ...............................................28 Peripheral vascular mechanism of EECP ..................................................29 EECP and skeletal muscle glucose metabolism ........................................30 Specific Aims and Hypotheses ...............................................................................33 2 LITERATURE REVIEW..........................................................................................36 Abnormal Glucose Tolerance (AGT) .......................................................................36 Measures of Glycemic Control ................................................................................37 Insulin Mediated Glucose Uptake ...........................................................................39 Contraction Mediated Glucose Uptake ...................................................................40 5-Adenosine Monophosphate-Activa ted Protein Kinase (AMPK) ....................40 Calcium-Calmodulin Dependent Signaling .......................................................42 Nitric Oxide Mediated Glucose Uptake ...................................................................42 Nitric Oxide .............................................................................................................44 Endothelial Nitric Ox ide Synthase (eNOS) .......................................................45 Neuronal Nitric Oxide Synthase (nNOS) ..........................................................47 Calcium-Insensitive Nitric Oxide Synthase (iNOS) ...........................................48 Insulin and Nitric Oxide .....................................................................................48 Reactive Oxygen Species .......................................................................................49 Anti-Oxidants ..........................................................................................................50 Peroxisome Proliferator-Activated ReceptorCoactivator 1 (PGC-1 ) ................51 Asymmetric Dimethylarginine (ADMA) ....................................................................52 6

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ADMA Metabolism ..................................................................................................54 Inflammation ...........................................................................................................56 Prostacyclin (PGI2) ..................................................................................................56 Vasoconstrictors .....................................................................................................57 EECP ......................................................................................................................58 Shear Stress ...........................................................................................................59 Mechanical Effects of EECP ...................................................................................60 Preliminary Data .....................................................................................................62 3 MATERIALS A ND METHOD S................................................................................64 Group Assignment ..................................................................................................64 Eligibility Criteria .....................................................................................................64 Inclusion Criteria ...............................................................................................64 Exclusion Criteria .............................................................................................65 EECP Methods .......................................................................................................65 Screening (Fasting Plasma Glucose and Blood Pressure) .....................................66 Dual Energy X-Ray Absorptiometry (DEXA) ...........................................................67 Skeletal Muscle Biopsies ........................................................................................67 Oral Glucose Tolerance Tests ................................................................................68 Blood Sampling .......................................................................................................68 Peripheral Flow Mediated Dilation (FMD) ...............................................................69 Venous Occlusion Plethysmography (VOP) ...........................................................70 Forearm and Calf Blood Flow ...........................................................................70 Forearm Flow During Reactive Hyperemia .......................................................71 Calf Flow During Reactive Hyperemia ..............................................................72 Western Blotting .....................................................................................................72 Immunostaining for Capillary Density .....................................................................74 Biochemical Assays ................................................................................................75 Vasodilator Measurements ...............................................................................75 Vasoconstrictor Measurements ........................................................................75 Lipid Peroxidation .............................................................................................75 Antioxidant Capacity .........................................................................................76 ADMA ...............................................................................................................76 Inflammatory Markers .......................................................................................77 Glycosylated Hemoglobin (HbA1c) ...................................................................77 Vascular Endothelial Gr owth Factor (VEGF) ....................................................77 Insulin ...............................................................................................................78 Glucose ............................................................................................................78 Statistical Considerations ........................................................................................79 Statistical Analysis ............................................................................................79 Power Analysis .................................................................................................79 4 RESULT S...............................................................................................................82 Subject Characteristics before EECP and Time-Control .........................................82 Brachial Artery Endothelial Function after EECP or Time-Control ..........................82 7

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Popliteal Artery Endothelial Function after EECP or Time-Control .........................83 Forearm and Calf Resistance Artery Blood Flow after EECP or Time-Control .......83 Markers of Angiogenesis/Vasculogenes is after EECP or Time-Control ..................84 Vasoactive Balance after EECP or Time-Control ....................................................85 Lipid Peroxidation, Antioxidant Capacity, and Endog enous Nitric Oxide Inhibition by ADMA after EECP or Time-Control .................................................85 Plasma Markers of Inflammati on after EECP or Time-Control ................................86 Fasting Markers of Glycemic Cont rol after EECP or Time-Control .........................86 Dynamic Indicies of Glucose Toler ance Derived from the Oral Glucose Tolerance Test after EECP or Time-Control ........................................................87 Western Blot Analysis of Protein Expression in Vastus Lateralis Skeletal Muscle Biopsy Homogenate after EECP or Time-Control ................................................87 5 DISCUSSI ON.......................................................................................................114 Peripheral Conduit Artery Endothelial Function and EECP ...................................114 Peripheral Resistance Artery Endothelial Function and EECP .............................116 Angiogeneisis/Vasculogenesis and EECP ............................................................117 Vasoactive Balance and EECP .............................................................................118 Redox Balance and EECP ....................................................................................121 Inflammation and EECP ........................................................................................123 Fasting Glycemic Control and EECP ....................................................................124 Dynamic Measures of Gl ucose Tolerance and EECP ..........................................126 Potential Mechanisms for Improvement in Fasting Glycemic Control and Dynamic Indices of Glucose Toler ance: Evidence for the Nitric Oxide Pathway? ...........................................................................................................127 Conclusions ..........................................................................................................130 LIST OF REFERENCES .............................................................................................132 BIOGRAPHICAL SKETCH ..........................................................................................148 8

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LIST OF TABLES Table page 3-1 EECP and glycemic control protocol; time and events .......................................81 4-1 Baseline subject characteristics before enhanced external counterpulsation (EECP) or Time-Control .....................................................................................89 4-2 Brachial artery flow-mediated dilati on (FMD) at baseline and after EECP or Time-Control .......................................................................................................89 4-3 Popliteal artery flow-mediated dilation (FMD) at baseline and after EECP or Time-Control .......................................................................................................89 4-4 Forearm venous occlusion plethy smography parameters at baseline and after EECP or Time-Control ................................................................................90 4-5 Calf venous occlusion plethysmography parameters at baseline and after EECP or Time-Control ........................................................................................90 4-6 Vascular endothelial growth factor (VEGF) and capillary density parameters at baseline and after EECP or Time-Control ......................................................90 4-7 Vasoactive balance at baseline and after EECP or Time-Control .......................90 4-8 Lipid peroxidation, antioxidant capacity, and endogenous nitric oxide (NO) inhibition at baseline and after EECP or Time-Control .......................................91 4-9 Biomarkers of inflammation at baseline and after EECP or Time-Control ..........91 4-10 Fasting markers of glycemic control at baseline and after EECP or TimeControl ................................................................................................................91 4-11 Dynamic indices of glucose toler ance at baseline and after EECP or TimeControl ................................................................................................................91 4-12 Percent change in protein expression from vastus lateralis skeletal muscle biopsy homogenate after EECP or Time-Control ................................................92 9

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LIST OF FIGURES Figure page 4-1 Brachial artery flow-mediated dila tion (FMD) at baseline and after enhanced external counterpulsation (EECP) or Time-Control .............................................92 4-2 Brachial artery absolute diameter dilation at baseline and after EECP or Time-Control .......................................................................................................93 4-3 Normalized brachial artery flow-medi ated dilation (FMD) at baseline and after EECP or Time-Control ........................................................................................93 4-4 Popliteal artery flow-mediated dilation (FMD) at baseline and after EECP or Time-Control .......................................................................................................94 4-5 Popliteal artery absolute diameter dilation at baseline and after EECP or Time-Control .......................................................................................................94 4-6 Normalized popliteal arte ry flow-mediated dilation (FMD) at baseline and after EECP or Time-Control ................................................................................95 4-7 Resting forearm blood flow (FBF ) at baseline and after EECP or TimeControl ................................................................................................................95 4-8 Peak forearm blood flow (FBF) at baseline and after EECP or Time-Control .....96 4-9 Total area under curve (AUC) forearm bl ood flow (FBF) at baseline and after EECP or Time-Control ........................................................................................96 4-10 Resting calf blood flow (CBF) at baseline and after EECP or Time-Control .......97 4-11 Peak calf blood flow (CBF) at baseline and after EECP or Time-Control ...........97 4-12 Total area under curve (AUC) calf bl ood flow (CBF) at baseline and after EECP or Time-Control ........................................................................................98 4-13 Vascular endothelial growth factor (VEGF) at baseline and after EECP or Time-Control .......................................................................................................98 4-14 Capillary density (CD) of human vastus lateralis biopsy samples at baseline and after EECP or Time-Control .........................................................................99 4-15 Capillary per fiber ratio of human vast us lateralis biopsy samples at baseline and after EECP or Time-Control .........................................................................99 4-16 Representative photomicrogr aph of skeletal muscle morphology .....................100 4-17 Nitrite/Nitrate (NOx) at base line and after EECP or Time-Control ....................100 10

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4-18 Six (6)-keto prostaglandin F1 (6-keto-PGF1) at baseline and after EECP or Time-Control .....................................................................................................101 4-19 Endothelin-1 (ET-1) at baseline and after EECP or Time-Control ....................101 4-20 Eight (8) iso-prostaglandin-F2 (8-iso-PGF2) at baseline and after EECP or Time-Control .....................................................................................................102 4-21 Asymmetric dimethylarginine (ADMA) at baseline and after EECP or TimeControl ..............................................................................................................102 4-22 High sensitivity C-reactive protein (hsCRP) at baseline and after EECP or Time-Control .....................................................................................................103 4-23 Tumor necrosis factor(TNF) at baseline and after EECP or TimeControl. .............................................................................................................103 4-24 Fasting plasma glucose (FPG) at bas eline and after EECP or Time-Control ...104 4-25 Fasting plasma insulin (FPI) at baseline and after EECP or Time-Control .......104 4-26 Homeostatic model assessment of insulin resistance (HOMAIR) at baseline and after EECP or Time-Control .......................................................................105 4-27 Quantitative insulin se nsitivity check index (QUI CKI) at baseline and after EECP or Time-Control ......................................................................................105 4-28 Glycosylated hemoglobin (HbA1c) at baseline and after EECP or TimeControl. .............................................................................................................106 4-29 Plasma glucose at 120 minutes after initiation of oral glucose tolerance testing (PPG120) at baseline and after EECP or Time-Control ..........................106 4-30 Oral glucose insulin sensitivity index (OGIS120) at baseline and after EECP or Time-Control .....................................................................................................107 4-31 Composite whole-body in sulin sensitivity index (ISI) at baseline and after EECP or Time-Control ......................................................................................107 4-32 Percent change in phosphoryl ation state of pr otein kinase B (p-Akt/Akt) from baseline after EECP and Time-Control .............................................................108 4-33 Percent change in phosphorylation state of 5-adenos ine monophosphateactivated protein kinase (p-AMPK/AMPK) from baseline after EECP and Time-Control .....................................................................................................108 4-34 Percent change in phosphor ylation state of TBC1 domain family member 4 (p-TBC1D4/TBC1D4) from baseline after EECP and Time-Control ..................109 11

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4-35 Percent change in glucose transporter-4 (GLUT-4) protein expression from baseline after EECP and Time-Control .............................................................109 4-36 Percent change in endothelial nitr ic oxide synthase (eNOS) protein expression from baseline after EECP and Time-Control ..................................110 4-37 Percent change in neuronal nitric ox ide synthase (nNOS) protein expression from baseline after EECP and Time-Control ....................................................111 4-38 Percent change in manganese superoxide dismutase (MnSOD) protein expression from baseline after EECP and Time-Control ..................................111 4-39 Percent change in copper-zinc super oxide dismutase (CuZnSOD) protein expression from baseline after EECP and Time-Control ..................................112 4-40 Percent change in selenium-dependent cellular glutathione peroxidase (GPx) protein expression from baseline after EECP and Time-Control ......................113 4-41 Percent change in 4-hydroxynonenal (4-HNE) adducts from baseline after EECP and Time-Control ...................................................................................113 12

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LIST OF ABBREVIATIONS 4-HNE 4-hydroxynoneal 6-keto-PGF-1 6-keto-prostaglandin 1 8-iso-PGF2 8-iso-prostaglandin 2 AAPH 2,2-Azobis-2-methyl-propani midamide, dihydrochloride ACE angiotensin converting enzyme ADMA asymmetric dimethylarginine AGEs advanced glycation endproducts AGT abnormal glucose tolerance AICAR aminoimidazole carboxamide ribonucleotide Akt protein kinase B AMP adenosine monophosphate AMPK 5-adenosine m onophosphate-activated protein kinase ARB angiotensin-II receptor blocker AS160 Akt substrate of 160 kDa ATF-2 activating transcription factor-2 ATP adenosine tri-phosphate AUC area under the curve BH 4 tetrahydrabiopterin CAD coronary artery disease CAMKII calcium/calmodulin-dependent protein kinase II CAMKK calcium/calmodulin-dependent protein kinase kinase CBF calf blood flow CDC Centers for Disease Control cGMP cyclic guanosine monophosphate 13

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CREB cAMP response element-binding protein CRP C-reactive protein DDAH dimethylarginine dimethlaminohydrolase DEXA dual energy x-ray absorptiometry DM Diabetes Mellitus EDTA ethylenediaminet etraacetic acid EDV endothelium dependent vasodilatation EECP enhanced external counterpulsation ELISA enzyme-linked immunosorbent assay EMG electromyography eNOS endothelial nitr ic oxide synthase ERK extracellular signal-related kinases ET-1 endothelin-1 FAD flavin adenine dinucleotide FBF forearm blood flow FGF2 basic fibroblast growth factor FMD flow mediated dilation FPG fasting plasma glucose FPI fasting plasma insulin GPX glutathione peroxidase GLUT-4 glucose transporter 4 H 2 O 2 hydrogen peroxide HbA1c glycosylated hemoglobin HDAC histone deacetylase 5 HEC hyperinsulinemic euglycemic clamp 14

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HGF hepatocyte growth factor HOMA IR homeostasis model assessment of insulin resistance hsCRP high sensitivity C-reactive protein IFG impaired fasting glucose IGT impaired glucose tolerance IL-6 interleukin 6 iNOS inducible nitric oxide synthase IPC intermittent pneumatic compression IRS1/2 insulin receptor substrate 1/2 ISI composite whole-body insulin sensitivity index JNK c-Jun-NH 2 terminal kinase L-NAME L-NG nitroarginine methyl ester L-NMMA N G -monomethyl-L-arginine LDL low density lipoprotein LKB1 serine/threonine kinase 11 MAPK mitogen-activated protein kinase MCP-1 monocyte chemotactic protein 1 MEF2 myocyte enhancer factor-2 mRNA messenger ribonucleic acid NAD(P)H nicotinamide adenine dinucleotide phosphate NF B nuclear factor B NIDDM non-insulin dependent Diabetes Mellitus nNOS neuronal nitric oxide synthase nNOS mu neuronal isoform of nitric oxide synthase NO nitric oxide 15

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NOS nitric oxide synthase NOx nitrite/nitrate OGIS 120 oral glucose insulin sensitivity index OGTT oral glucose tolerance testing ORAC oxygen radical absorbance capacity P160myb myb-binding protein 1A PDK1 phosphoinositide 3-kinase dependent kinase PGC-1 peroxisome proliferat or-activated receptorcoactivator 1 PGI 2 prostacyclin PI3K phosphoinos itide 3-kinase PIP2 phosphatidylinosit ol 4,5-bisphosphate PIP3 phospatidylinositol (3,4,5)-triphosphate PKC protein kinase C PKG cyclic GMP-dependent kinase/protein kinase G PPG 120 post-prandial glucose at 120 minutes PRMTs protein-arginine-N-methyltr asferase family of enzymes QUICKI quantitative insulin sensitivity check index RabGAP RabGTPase activating protein SDH succinate dehydrogenase SNAP S-nitroso-N-penicillamine SNP sodium nitroprusside SOD superoxide dismutase sVCAM soluble vascular cell adhesion molecule T2DM Type II Diabetes Mellitus TAK1 TGFactivated kinase 1 16

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TBC1D1 TBC1 domain family member 1 TCA tricarboxylic acid cycle TMB tetramethylbenzadine TNFtumor necrosis factor alpha TZD thiazolidinedione VEGF vascular endothelial growth factor VOP venous occlusion plethysmography WHO World Health Organization 17

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Abstract of Dissertation Pr esented to the Graduate School of the University of Florida in Partial Fulf illment of the Requirements for t he Degree of Doctor of Philosophy EFFECTS OF ENHANCED EXTERNAL COUNTERPULSATION (EECP) ON GLYCEMIC CONTROL AND ARTERIAL F UNCTION IN PATIENTS WITH ABNORMAL GLUCOSE TOLERANCE By Jeffrey S. Martin August 2011 Chair: Randy W. Braith Major: Health and Human Performance Enhanced external counterpulsation (EECP) is a noninvasive modality used to treat patients with refractory angina. The salutary benefits of EECP treatment include increases in peripheral vascular function and nitric oxide bioavaila bility. Nitric oxide (NO) is also known to upregulate the trans location and expression of the major glucose transporter in muscle, glucose transporter 4 (GLU T-4). Of critical impor tance, is the fact that this NO signaling pathway is distinct from and/or additive to insulin-stimulated skeletal muscle glucose uptake, which is not oriously impaired in humans with abnormal glucose tolerance (AGT). Moreover, the effects of EECP on skeletal muscle morphology and glucose uptake signaling are la rgely unknown. Therefore, the purpose of this study was to investigate the effe cts of EECP treatment on glycemic control and arterial function in patients with AGT. Eighteen (n = 18) patients with AGT were recruited for the study. They were randomly assigned to receive either 35 1-hour sessions of EECP (n = 12) or 7-weeks of standard care (n = 6). Peripheral vascular function, vasoactive balance, oxidative stress, inflammation, skelet al muscle morphology, skeletal muscle protein expression, 18

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fasting glycemic control, and dynamic gluc ose tolerance were measured before and after 35 1-hour sessions of EECP or Time-Control. EECP resulted in an increase in normalized brachial artery (27%) and popliteal artery (52%) flow-mediated dilation as we ll as peak (26%) and total (37%) forearm hyperemic blood flow. Plasma nitrite/nitr ate (NOx) increased (30%) and asymmetric dimethylarginine (ADMA) decreased (11% ) following 35 sessions of EECP. Additionally, 8-isoprostane-PGF-F2 (8-iso-PGF2 ), a marker of lipid peroxidation in the plasma, decreased (23%) with a concurrent decrease (28%) in high sensitivity Creactive protein (hsCRP), a marker of inflamma tion. Analysis of vastus lateralis skeletal muscle biopsies revealed significant incr eases in endothelial nitric oxide synthase (eNOS) (87%) and GLUT-4 protein expressi on (47%) following EECP. Furthermore, there was a significant elevation in hum oral concentrations of vascular endothelial growth factor (VEGF) (75% ) and the capillary-to-fiber ratio (8%) following EECP. Fasting plasma glucose (FPG) was reduc ed by 16.9 mg/dL and the homeostasis model assessment of insulin resistance (HOMA IR ) decreased (31%) following EECP. Moreover, plasma glucose concentrations 120 minutes after initiation of an oral glucose test (OGTT) decreased (224.4 mg/dL vs. 196.1mg/dL) and the w hole-body composite insulin sensitivity index (ISI composite) increased (21%). The improvements in NO bioavailabilit y and endothelial functi on observed in the present study, as well as capillary to fiber ratios, likely mediate greater delivery of nutrients to nutritive tiss ues (i.e. skeletal muscle) at rest and during glycemic challenge. Additionally, NO signaling been has implicated in regulation of GLUT-4 translocation and glucose uptake. The mult ifaceted nature of vascular function and 19

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glycemic control makes it difficult to isol ate a single mechanism responsible for these adaptations. However, novel evidence from the present study supports the hypothesis that decreasing nitric oxide bioavailability contributes to the pathology of AGT. Interventions that improve NO bioavailability may be attracti ve treatment strategies for patients with AGT. 20

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CHAPTER 1 INTRODUCTION Background An estimated 23.6 million people in the Un ited States suffer from Diabetes Mellitus (DM). 1 Of the estimated 23.6 million with DM, Type II DM (T2DM) accounts for 90-95% of all diagnosed cases. 1 After the age of 60, the preval ence of T2DM climbs to 23.1% of all people. The prevalence of DM has more than doubled since 1990 leading to the designation of DM as an epidemic in the Unit ed States. T2DM is often associated with the co-morbidities; obesity, hypertension, and hyperlipidemia. The co mbination of these risk factors is known as metabolic syndrome. Cardiovascular disease is the leading cause of morbidity and mortality in patients with T2DM, with heart disease and stroke noted as cause of death on 84% of death certificates. 1, 2 The sequela of vascular disease extends to amputations, blindness, my ocardial infarction, and stroke. Chronic exposure to hyperglycemia is the primary cause of cardiovascular disease in patients with T2DM and the risk of microvascular co mplications is considerably reduced by better glycemic control. Another 56 million Americans are estima ted to be pre-diabetic, and among people aged 20 years or more in Amer ica a staggering 37% of the popul ation is classified as pre-diabetic or having T2DM. 1 Furthermore, aging is a signi ficant risk factor for the development of pre-diabetes. Indeed, among American adults aged 40-74 years, 40.1% had prediabetes. 1 Pre-diabetes is a condition in which blood glucose levels are higher than normal, but not high enough to be classified as T2DM. Pre-diabetes markedly increases the risk for progression to T2DM as well as cardiovascular disease and stroke. 3-5 21

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Impaired glucose tolerance (IGT) is defined as a plasma glucose concentration of 110 to 125 mg/dL after overnight fast and a plasma glucose concentration of 140 to 199 mg/dL after a 2-hour oral gluc ose tolerance test (OGTT). 6 Individuals with plasma glucose concentrations that are greater than the highest r ange of IGT for fasting and 2hour OGTT plasma glucose concentrations are classified as having T2DM. The term abnormal glucose tolerance (AGT) includes individuals with IGT and T2DM. T2DM, previously referred to as non-insulin depende nt or adult onset diabetes, and AGT is characterized by the marked decline in insu lin mediated glucose transport in skeletal muscle and adipose tissue. This phenomenon, by which the cells do not respond appropriately to endogenous circulating insulin, is referred to as insulin resistance. A consequence of insulin resistance is the inabi lity to clear circulating plasma glucose which is reflected by the abnormally elevated fasting and/or post-prandial plasma glucose concentrations. Because skeletal muscle accounts for 65-90% of the clearance of a hyperglycemic challenge, 2, 7, 8 interventions that increase skeletal muscle glucose uptake are known to improve glucose homeostasis in patients with typeT2DM. 8-10 Treatment and managem ent of T2DM can range from alteration of diet and physical activity habits to pha rmacotherapy. However, comp lications often associated with DM, such as neuropathy and obesity, can often limit the ability of persons to increase their physical activity in an effort to manage their condition. Additionally, a lifetime of refusal to be physically active may be an indication that there is little willingness to change physical activity habits Physical activity can help with the management of glycemic control in a variety of ways, but in situations where it may not 22

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be possible or pertinent, enhanced external counterpulsation (EECP) may be an effective means by which to increase insuli n sensitivity and/or gl ucose metabolism. Antihyperglycemic Treatment Options Current treatment strategies to achieve adequate glycemic cont rol in patients with AGT include exercise training and antihyperglycemic drug regimens. However, compliance with home-based exercise guidelines in patients with AGT is abysmally low. Indeed, data from two very recent populati on based surveys (a total of 345 patients) revealed that only 25% of patients with T2DM participate in exercise a minimum of 1 hour per week, despite being under the care of physicians who recommended exercise as treatment. 11 This finding is consistent wit h earlier studies reporting very low compliance with exercise recomm endations among pati ents with T2DM. 12-15 Relevant also, is the fact that 60-70% of diabetic patients have mild to severe nervous system damage. 1 Neuropathy in either upper or lower body extremities can be a contraindication for participation in exer cise therapy for patients with diabetes. Patient compliance with drug therapy for glycemic control is only slightly better than exercise compliance. Daily et al. 16 studied a very large cohort consisting of 37,431 patients with T2DM to determine compliance with anti-hyperglycemic drug regimens. At the 1-year follow-up, only 49% of patients receiving monotherapy and just 36% of patients receiving polytherapy were compliant with anti-hyperglycemic drug regimens. At the 2-year follow-up, compliance deterio rated even further and only 42% of patients receiving monotherapy and 29% of patients re ceiving polytherapy remained compliant with their drug regimens. Considering the results of exercise and drug compliance studies, it is clear that a high percent age of patients with AG T do not achieve adequate 23

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glycemic control. Consequently, alternative methods that improve glucose homeostasis should be considered. In this proof-of-concept study desi gn, we explored an alternative antihyperglycemic therapy for patients with AGT t hat is feasible, noninvasive, exerciseindependent and perhaps more cost-effective than metabolism enhancing medications. The goal of this application was to determine the efficacy and underlying mechanisms responsible for improving glycemic contro l in patients with AGT following 35 1-hr sessions of EECP. Glucose Uptake Pathways in Skeletal Muscle Glucose uptake in skeletal muscle cells is regulated by 3 known pathways: 1) insulin-mediated cell signaling; 2) contracti on-mediated cell signali ng; 3) Nitric Oxide (NO)-mediated cell signaling. A brief outline of each pathway and the potential relevance of EECP as a modulator of Pathway #3 are presented below. Insulin mediated glucose uptake (Pathway #1) The insulin mediated pathway of glucose uptake is dependent upon phosphoinositide 3-kinase (PI3K) activation via receptor associated tyrosine kinase activity and ultimately glucose transporter 4 (GLUT-4) translocati on to the plasma membrane. PI3K activation recruits both protein kinase B (Akt) and phosphoinositide 3kinase dependent kinase (PDK1) to the ce ll membrane where PDK1 phosphorylates and activates Akt. Subsequently, Akt phosphorylates Akt substrate of 160 kDa (AS160) at its RabGTPase activating domain (RabGAP) which inhibits its activity and removes inhibition of Rab protein activity. As a consequence of greater Rab protein activity, GLUT-4 translocation to the cell membrane is realized. This pathway is notoriously impaired in humans with AGT. 17, 18 24

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Contraction mediated gluc ose uptake (Pathway #2) The insulin/PI3K independent pathway, commonly referred to as the contraction/hypoxia pathway, mediates glucose uptake in skeletal muscle due to perturbations in the vasculature and skeletal muscle when compared to the basal state. One mechanism by which contraction mediated glucose uptake is realized is through 5adenosine monophosphate-activated kinase (AMPK) activity. An increase in the adenosine monophosphate (AMP)/adenosine triphos phate (ATP) ratio within the cell (e.g. as during contractions) is known to be an activator of AMPK activity, and correlates strongly with glucose uptake. 19 AMPK phosphorylates AS160 relieving Rab protein inhibition and resulting in GLUT-4 translo cation. AMPK activity also increases expression of peroxisome pro liferator-activated receptorcoactivator 1 (PGC-1 ), a master regulator of cellular metabolism, which leads to an increase in GLUT-4 protein transcription and translation. 19 Therefore, AMPK signaling mediates acute changes in GLUT-4 translocation from existing pools and a later increase in GLUT-4 protein amount. The insulin and contraction-medi ated glucose uptake pathways are additive, supporting the idea that they are two distinct pathways. However, a point of convergence in the two pathways appears to occur at AS160 phosphorylation. This pathway is the therapeutic target of exercise in patients with AGT. Nitric oxide (NO) mediated glucose uptake (Pathway #3) A third signaling pathway uses NO to stim ulate glucose transport in muscle. NO increases glucose uptake through a mechanism that is distinct from the insulinmediated (Pathway #1) and contraction-medi ated (Pathway #2) pathways in muscle. NO increases GLUT-4 protein expressi on and GLUT-4 translocation to the cell membrane via a cyclic guanosine m onophosphate (cGMP) and AMPK-dependent 25

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mechanism. 20 It is well established that muscle tissue expresses significant NO production at rest from both endothelial NO synthase (eNOS) and neuronal NO synthase (nNOS). 21 However, eNOS is typically found in highest concentrations around the mitochondria and is thought to play a gr eater role in cell glucose metabolism. Indeed, in eNOS knockout mice both basal and insulin-stimulated glucose uptake in skeletal muscle are reduced by 40%, when compared to wild-type controls. 22 Critically relevant to this proposal, is the fact that patients with AGT are more reliant upon the NO pathway for glucose uptake than age-matched euglycemic controls. Kingwell et al. 23 found that nitric oxide synt hase (NOS) inhibition with N G -monomethyl-L-arginine (LNMMA) reduced leg glucose uptake by 75% in patients with T2DM, but only by 34% in non-diabetic control subjects. In contrast, sodium nitroprusside (SNP ), a NO donor, significantly increases glucose uptake in a dose-dependent manner and the responsible mechanism appears to be translocation of GLUT-4 to the cell membrane. 20 NO donors significantly increase the activity of AMPK by acting on the 1 catalytic subunit and AMPK can act in positive feedback loop with NOS furt her augmenting NO produc tion and AMPK activity. 20 The increase in AMPK activity could explain t he increase in NO m ediated glucose uptake due to increasing GLUT-4 translocation and PGC-1 transcription. Lira et al. has previously shown that the NO donor S-nitrosoN -penicillamine (SNAP) increased GLUT4 protein expression by 65% in L6 myotubes via a cGMP and AMPK-dependent mechanism. 24 Moreover, L6 myotubes exposed to aminoimidazole carboxamide ribonucleotide (AICAR), an AM PK agonist, experienced a nine-fo ld increase in GLUT-4 mRNA. 24 In vivo, GLUT-4 messenger ribonucle ic acid (mRNA) was increased nearly 26

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two-fold in the rat plantaris muscle 12 hrs after AICAR injection. 5 Thus, evidence from this assisting laboratory corroborates that in skeletal muscle, NO concentration likely influences GLUT-4 expression via control of cellular activity of AMPK kinases. Lastly, NO donors significant ly increase skeletal muscle glucose uptake in the presence of wortmannin, a PI3K inhibitor, indicating that the NO signaling pathway is not a PI3K dependent mechanism (i.e. Pathway #1 above). 20 Clearly, the contraction and NO mediated glucose uptake pathways over lap downstream of AMPK. However, L-NG-nitroarginine methyl ester (L-NAME), another NOS i nhibitor, does not abolish contraction mediated glucose uptake in skeletal muscle (i.e. Pathway #2 above). 20 Thus, there is excellent evidence indicati ng that the NO medi ated glucose uptake pathway in muscle is independent of both the insulin and contraction mediated mechanisms, making NO bioavai lability an attractive target for circumventing impaired insulin sensitivity and augmenting exercise effects. In summary, incr easing the skeletal muscle NO concentration has an antiglycemic e ffect, whereas blocking the role of NO results in marked hyperglycemia and insulin resistance. Principles of Enhanced Exte rnal Counterpulsation (EECP) Intermittent external compre ssion of skeletal muscle is known to upregulate eNOS protein and NO bioavailability in animal studies. 25, 26 Our lab (Braith et al.), 27 and others, 28, 29 have demonstrated that external comp ression upregulates NO bioavailability in humans. We hypothesized that external compression of the large muscle groups of the lower body using EECP would increase muscle perfusion, increase intramuscular eNOS and NO expression, increase GLUT-4 protein, and chronically improve glycemic control in patients with AGT. EECP is an established, Food and Drug Administration and Medicare-approved, non-invasive therapy for patients with coronary artery disease 27

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(CAD) who experience persistent refractory angina, despite standard revascularization procedures and/or aggressive anti-angina medication. EECP has proven to be especially efficacious in patients with CAD who are not amenable to percutaneous coronary intervention or bypass surgery, because of unsuitable coronary anatomy, multiple previous revascularization atte mpts, age, additional co-morbid conditions, or patient preference. 30 Data from most clinical trials and the International Patient Registry demonstrate that EECP is effective in reducing anginal symptom s and nitroglycerin usage, 31-34 increasing exercise tolerance, 31, 34-36 and decreasing the need for hospitalization. 31, 33, 37 The clinical benefits of EECP have been sustained for 2, 3, and 5 years after treatment in most patients. 38-41 EECP utilizes equipment to inflate and deflate 3 compressive cuffs enclosing the lower extremities. During an EECP treat ment session, the patient lies on a padded table in which sets of electronically controlle d inflation and deflation valves are located. These valves are connected to the 3 pneuma tic cuffs, wrapped around the calves, lower thighs, upper thighs and buttocks. The cuffs ar e sequentially inflated to approximately 300 mmHg with compressed air from distal to proximal in early diastole and rapidly deflated at the onset of systol e. Inflation and deflation of the cuffs are triggered by events in the cardiac cycle via microproc essor-interpreted ECG signals. A standard course of EECP treatment consists of 35 1-hour sessions distributed over a 7-week period. Central angiogenesis mechanism of EECP The central hypothesis, in most investigations conducted to elucidate the mechanism of action, was that EECP may promote coronary angiogenesis through robust diastolic pressure augmentation during rapid cuff inflation, analogous to intra28

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aortic balloon counterpulsation. However, th is understanding of EECP is only a theory and remains unconfirmed in clinical trials. 28, 35 In an international trial (7 Centers; 175 chronic stable angina patients) EECP failed to elicit improved cardiac perfusion in 46% of study subjects. 42 More recently, a multicenter trial involving 6 United States University Hospitals showed that EECP failed to improve myocardial blood flow to and within ischemic regions of the myocardium either at rest or during exercise stress in 37 patients with CAD, as assessed by quantitat ive gated single photon emission computed tomography. 43 However, despite negligible impr ovements in myocardial perfusion, approximately 85% of patients in EECP clinical trials ex perience reduction in angina. 31, 42 Consequently, Dr. Braith and coworker s reasoned that the traditional coronary angiogenesis understanding of EECP required extensive r evision and was inadequate to explain the efficacy of therapy (i.e. angi na reductions) in patients with CAD. They found that peripheral endothelial function was the primary therapeutic target for EECP. 27 Peripheral vascular mechanism of EECP Our laboratory recently completed the fi rst randomized, sham-controlled study to investigate the extra-cardiac effects of EECP on endothelial func tion in CAD patients. 27 In that study, the primary hypothesis was that improvement in peripheral arterial stiffness and endothelial function are the mechanisms underlying the chronic antiischemic clinical benefits of EECP by reduci ng myocardial oxygen demand. To test this hypothesis, 42 symptomatic pati ents with CAD were randomized (2 :1 ratio) to either 35 1-hour sessions of EECP (n=28) or Sham EECP (n=14). In brief, overwhelming biochemical and functional evidence that EECP elicits systemic improvements in vascular biology was found. EECP increas ed plasma levels of NO by 36% and decreased endothelin-1 (ET-1) by 25%. NO bioavailability wa s enabled through the 29

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anti-inflammatory effects of EECP, as evi denced by significant reductions in the proinflammatory cytokines tumor necrosis factor alpha (TNF: 16%), C-reactive protein (CRP: 9%), monocyte chemotac tic protein-1 (MCP-1: 13%), and soluble vascular cell adhesion molecules (sVCAM: 6%). NO bi oavailability was further enabled through improvement in systemic redox balance, as evidenced by a 21% decrease in plasma levels of 8-iso-prostaglandin F2 (8-iso-PGF2 ), which is viewed as the most valid plasma marker to assess systemic oxidat ive stress. Endothelial-dependent flow mediated dilation (FMD) in the brachial and femoral arteries was increased by 51% and 30%, respectively and peak blood flow in t he forearm and calf was increased by 18% and 16%, respectively. 27 Improved endothelial function and reduced afterload following EECP treatment resulted in decreased angina symptoms. Patients reduced their Canadian Cardiovascular Society angina classi fication from 3.1 to 1.2 (mean SEM; 4 point scale), daily angina episodes by 72%, and daily nitrate usage by 81%. EECP and skeletal muscle glucose metabolism The primary mechanism responsible for upregulation of eNOS and NO following EECP therapy is related to the intermittent bouts of hemodynamic shear stress created with each inflation-deflation cycle of the 3 pneum atic cuffs. Shear stress is a primary stimulus for the synthesis and release of endothe lial-derived NO. 26 The blood flow shear stress stimulus is transduced into t he endothelial cell via the integrin/cytoskeleton mechano-transduction pathway. 44 It is well documented t hat sequential inflation (~ 300 mm Hg) of the 3 pneumatic EECP cuffs from ca lf to buttocks during diastole produces a robust retrograde pressure wave in the c entral aorta that subsequently increases coronary artery perfusion pressure, analogous to intraaortic balloon counterpulsation. 45 However, inflation of the EECP cuffs also produces high-pressure retrograde blood flow 30

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in the femoral arteries and simultaneous moderate-pressure antegrade flow in the brachial arteries. 26, 46 Using color Doppler imaging in a porcine EECP model, Zhang and coworkers 26 found that brachial artery blood flow velocity increased by 132% (59 versus 24 cm/second; p < 0.001) and brachial artery wall shear stress increased by > 200% (49 versus 23 dyne/cm 2 p < 0.001) during lower body compression of EECP pneumatic cuffs. Recent evidence suggests that arterial exposure to this type of pulsatile, hemodynamic shear stress, compar ed to steady laminar flow, may be the most critical factor af fecting endothelial function. 47 Data from a randomized, shamcontrolled study suggest that the significant in creases in pulsatile and oscillatory flow during EECP treatment, as re corded by color Doppler, may provide a form of massage on the endothelium and im prove its function. 27 That is, each session of EECP may be thought of as providin g a direct dose of vascular medicine. Support for our EECP hypothesis can be f ound in an animal model of external compression. Tan et al. 25 applied intermittent pneumatic compression (55 mmHg pressure achieved in <1 second) to the hind limbs of 16 male Sprague-Dawley rats for the durations of 0.5, 1.0, and 5 hours. Following intermi ttent compression, eNOS in skeletal muscle homogenate was upregulated to 120%, 180%, and 270% from baseline, respectively. Similarly, nNOS expression was up-regulated, but to a lesser degree. Furthermore, EECP increased plasma levels of NO by 36% in CAD patients following 35 1-hour sessions in a sham controlled study. 27 Indices of arterial and endothelial function were equally impressive after 35 1-hour sessions of EECP as endothelialdependent flow mediated dilation (FMD), a non-invasive measure of NO bioavailability, in the brachial and femoral arteries was increased by 51% and 30%, respectively. 27 31

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These results, from this animal model resembling EECP, and fr om data in our own laboratory, serve to confi rm our hypothesis that compre ssion-induced improvement in vascular function is mediated by modulation of NO bioavailability and by regulating expression of NOS isoforms in particular eNOS. While it has been well established that EECP causes an acute increase in blood flow to the muscle tissue, the effects of EECP on skeletal muscle signaling and metabolism have not been well characterized. A previous study by Crenshaw et al. has shown that application of exte rnal pressure to human cadaver limbs there is a linear relationship between external and intramuscular pressures (1988). 48 Furthermore, in a rodent model of intermittent pneumatic comp ression (IPC), pressure underneath the cuffs closely resembles the pressure programmed on the compression unit. 49 L6 myotubes that have been pressurized to mimi c intramuscular pressures during walking or running showed a significant increase in succinate dehydrogenase (SDH) activity and glucose uptake with a decrease in lactate release. 50 Furthermore, a single 30 minute bout of EECP therapy in humans has been s hown to significantly decrease plasma glucose concentration. 51 Although the mechanism by which external pressure augments metabolism has yet to be elucidat ed, available evidence suggests that mechanical pressure, devoid of active c ontraction, can increase metabolism and potentially alter AMP/ATP ratios. Indeed, in an acute rodent model of intermittent pneumatic compression, the arterial-venous oxygen difference was shown to increase during compressions. This is indicative of oxygen consumption and supports an increase in tissue metabolism during external compression. Furthermore, a studies by Grayson et al., demonstrated a significant increase in somatic oxygen consumption in 32

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humans during 30 minutes of EECP therapy. 52 Another potential mechanism for the perturbation in skeletal muscle metabolism du ring IPC is stretch of the muscle tissue directly under and in the immediate vicini ty of cuff edges. Indeed, NO production increases approximately 20% in isolated so leus muscle subjected to passive stretch. 53 Furthermore, myotubes subjected to cyc lic stretching for 2 hours showed a 42% increase in NOS activity from basal rates. Perhaps of long-term consequence of EECP compression, mechanical loading may upregul ate nNOS expression as a result of membrane associated proteins responsive to mechanical disruption. Mechanical loading has been shown to regulate expr ession of talin in C2C12 myotubes 53 and nNOS protein expression is slightly elevated fo llowing IPC in underlying tissue following 1 hour of treatment. 25 Specific Aims and Hypotheses Specific aim 1: Determine whether 35 1-hour sessi ons of EECP therapy will elicit changes in patient fasting glycemic control, HbA1c levels, and insulin sensitivity as measured by oral glucose tolerance testing (OGTT) in subjects with AGT. Hypothesis 1: 35 1-hour sessions of EECP in subjects with AGT will decrease fasting plasma glucose concentrations (relati ve to plasma insulin concentrations), glycosylated hemoglobin (HbA1c) levels, and indi ces of insulin sensitivity as determined by oral glucose tolerance testing (OG TT) compared to time matched controls. Specific aim 2: Determine whether EECP and accompanying changes in skeletal muscle NO bioavailability will indu ce upregulation of the AMPK-PGC-1 cell signaling pathway and ultimately increase GLUT-4 expression when assessed in muscle biopsy samples. 33

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Hypothesis 2: 35 1-hour sessions of EECP in subjects with AGT will upregulate the AMPK-PGC-1 cell signaling pathway as assessed by PGC-1 GLUT-4, AMPK, AS160, and Akt protein concentrations and phos phorylation state in western blots of vastus lateralis biopsy homogenate co mpared to time matched controls. Specific aim 3: Determine whether EECP, as compared to standard care, improves endothelial function in peripheral mu scular arteries (brachial and popliteal) and resistance arterioles (forearm and calf ) in patients with AGT after 35 1-hour sessions. Hypothesis 3: 35 1-hour sessions of EECP in subjects with AGT will increase endothelial function in the peripheral muscular arteries and resistance arterioles, as determined by flow mediated dilation (b rachial and popliteal) and venous occlusion plethysmograpy (forearm and calf) respec tively, compared to time controls. Specific aim 4: Determine whether changes in endothelial function are accompanied by commensurate changes in hum oral levels of vasoactive agents including nitric oxide (nitrate/nit rite), prostacyclin (6-keto-PGF-1 ), and ET-1. Hypothesis 4: 35 1-hour sessions of EECP in subjects with AGT will have beneficial effects on the plasma levels of va soactive agents compared to time matched controls. Specific aim 5: Determine whether systemic vascular changes are accompanied by commensurate changes in skeletal mu scle capillarity, eNOS and nNOS protein concentration, and humoral concentration of vascular endothelial growth factor (VEGF) when assessed in biopsy samples taken from the vastus lateralis and blood draws. 34

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Hypothesis 5: 35 1-hour sessions of EECP in subjects with AGT will increase eNOS and nNOS protein concentration measured in skeletal muscle biopsy homogenate, capillary density in serial sections of skeletal muscle biopsy samples, and humoral concentration of VEGF co mpared to time matched controls. 35

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CHAPTER 2 LITERATURE REVIEW Abnormal Glucose Tolerance (AGT) A hallmark of insulin resistance, a disor der in which the cells do not use insulin properly, is abnormal glucose tolerance (AGT ). Abnormal glucose tolerance includes individuals with impaired glucose tolerance (IGT) and Type II Diabetes Mellitus (T2DM). Individuals with IGT, but not T2 DM, are classified as pre-diabet ics. Pre-diabetes is a condition in which blood glucose levels are higher than normal, but not high enough to be classified as diabetes. Pre-diabetes mark edly increases the risk for progression to T2DM as well as cardiovascular dis ease and stroke. The 2006 World Health Organization (WHO) recommendation for diagnosis of pre-diabetes is the presence of either impaired fasting glucose (IFG) and/or IGT. 6 IFG is defined as a plasma glucose concentration of 110 to 125 mg/dL after overni ght fast. IGT is defined as a plasma glucose concentration of 110 to 125 mg/dL after overnight fast and a plasma glucose concentration of 140 to 199 mg/dL after a 2-hour OGTT. 6 According to the most recent data from the Centers for Disease Control (CDC), amongst adults aged 40-74 years in the United States, 33.8% had IFG, 15. 4% had IGT, and 40.1% had pre-diabetes. 1 The WHO estimates that about 10% of people worldwide have IGT. 6, 54 In addition, the CDC estimate of the prevalence of T2DM in the United states was 23.6 million people, or 7.8% percent of the population. 1 T2DM is defined as a fasting plasma glucose concentration of >125 mg/dL or a plasma gluc ose concentration of > 200 mg/dL after a 2-hour oral glucose tolerance test (OGTT). 6 The total (direct and indirect) costs of diabetes in the United Stat es for 2007 was estimated to be 174 billion dollars. 1 The WHO projects the prevalence of diabetes to more than double in the next 30 years, 36

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placing a significant burden on not only the population as a whole, but also health care costs. 6, 54 Measures of Glycemic Control AGT, defined here as IGT or T2DM, is a ssociated with insulin resistance as well as cardiovascular morbidity and all cause mortality. 55 Insulin resistance is typically defined as decreased sensitivit y to metabolic actions of insulin, such as insulinmediated glucose disposal and inhi bition of hepatic glucose production. 56 Insulin has concentration dependent saturable actions to increase whole body glucose disposal, and several surrogates of insulin sensitivit y are available for evaluation of glycemic control in humans. The gold standard, and most direct measure of insulin sensitivity, is the hyperinsulinemic euglycemic cl amp (HEC). With this tec hnique, glucose disposal in skeletal muscle and adipose tissue is maximi zed while hepatic glucose production is suppressed. Although the glucose infusion ra te determined via HEC provides the most direct measure of peripheral insulin sensitivity, one still has to assume that hepatic glucose production is completely abolished. 2 In addition, clamps are high risk, invasive procedures that are not normally used in clinical practice. The OGTT has been used to diagnose IGT and T2DM in clinical practice fo r many years. A major criticism of the OGTT is that the gastrointestinal influence on glucose disposal cannot be ignored. However, the OGTT may be the most clinica lly and physiologically relevant as oral ingestion of food stuffs is the primary m eans of nourishment. Se veral surrogates of insulin sensitivity have been derived from th e OGTT that correlate very strongly with HEC testing. Simple measures of fasting plasma glucose and insulin concentrations can provide an estimate of insulin resistance. Indices of insulin sensitivity from these measures 37

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include the quantitative insuli n sensitivity check index (QUICKI) and the homeostasis model assessment of insulin resistance (HOMA IR ). The HOMA IR correlates well with the glucose disposal rate derived from HEC 57, 58 although the correlation appears to weaken with higher levels of glycemia. 59 Among the critic isms of the HOMA IR are a large coefficient of variation and the pulsatile nature of insulin secretion. However, 3 separate samples, 5 minutes apart, of plasma insulin concentration were measured to determine a true average fasting plasma insulin concen tration. The mathematical difference between the QIUCKI and the HOMA IR is simply that the former uses the reciprocal of the logarithm of both glucos e and insulin to account for the skewed distribution of fasting insulin values. Both i ndices correlate well with the HEC, 60 but some argue that the QUICKI may be applied to wider ranges of insulin sensitivity. 61, 62 Stronger surrogates of insulin sensitivity can be derived from the multiple sampling of plasma insulin and glucose during an OG TT. During an OGTT fasting blood samples are taken prior to the ingestion of a bever age containing 75 grams of glucose (sugar water). Four additional samp les of plasma insulin and glucose are then taken at 30, 60, 90, and 120 minutes after ingestion of the bev erage. There are a plethora of indices based on the OGTT, but to highlight a few, the Matsuda, 58 Belfiore, 63 Stumvoll, 64 and Mari, 65 indices have all been shown to correlate si gnificantly with the HEC with r values of 0.73, 0.96, 0.80, and 0.73 respectively. 59 Each index uses data from the entire OGTT, including area under the glucose and insulin curves, to determine insulin sensitivity. Importantly, these indices hav e been validated in a wide variety of lean and obese subjects with IGT, T2DM and/or normal glucose tolerance. An inherent limitation to indices of insulin sensitivity derived fr om the OGTT is that it is impossible to 38

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differentiate between whole-body, peripheral, or hepatic insulin sensitivity. However, since this method of glucose ingestion is t he most physiologically relevant, improvement in indices of insulin sensitivity from the OGTT are encouraging signs of improved glucose tolerance in subjects with AGT. Finally, glycosylated hemoglobin (HbA1c) provides a long term index of glycemic control. Since the half life of a red blood cell is about 120 days, a single determination of HbA1c reflects the average blood glucose control level during the preceding 8 to 12 weeks. Therefore, HbA1c is used primar ily to identify the average plasma glucose concentration over prolonged periods of time (2 to 3 months). It is formed in a nonenzymatic glycation pathway by hemoglobins exposure to plasma glucose. In diabetics, higher HbA1c values, indica ting worse glycemic control, have been associated with cardiovascular di sease nephropathy, and retinopathy. 66 HbA1c is reported as percentage, and the equation used to determine estimated average glucose is the following: estimated average glucose (mg/dL) = 28.7 x Hb A1c (%) 46.7. Therefore, a small change in Hb A1c values could indicate a large improvement in long term glycemic control. For ex ample, exercise intervention has been shown to decrease HbA1c percentage by more than one half a percent 67 indicating at least a 15 mg/dL change in average plasma glucose concentration. Insulin Mediated Glucose Uptake Insulin binding to its specific receptor promotes glucose transporter-4 (GLUT-4) trafficking to the plasma membrane. The pr imary, and well established pathway in the literature, is the phosphoinositide 3-kinase (PI3K) pathway. Insulin binding to the specific insulin receptor causes the insulin re ceptor substrate (IRS1/2) to recruit PI3K to the plasma membrane via receptor asso ciated tyrosine kinase activity. Subsequent 39

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PI3K activation catalyses the conversion of phosphatidylinositol 4,5-bisphosphate (PIP2) to phospatidylinosit ol (3,4,5)-triphosphate (PIP3) which recruits both protein kinase B (Akt) and phosphoinositide 3-kinase dependent kinase (PDK1) to the cell membrane where PDK1 phosphorylates and ac tivates Akt. Activated Akt then phosphorylates Akt substrate of 160 kDa (AS160) at its RabGTPase activating protein (RabGAP) domain which inhibits its activity and removes inhibi tion of Rab protein activity. As a consequence of greater Rab prot ein activity, GLUT-4 translocation to the cell membrane is realized. Therefore, AS160 functions as a negative regulator of GLUT-4 trafficking under non-stimulated cond itions where the active GAP domain of AS160 suppresses the activity of Rab pr oteins that are required for GLUT-4 translocation to occur. In individuals with AGT/insulin resistance, this glucose uptake pathway is notoriously impair ed at one or several levels of the insulin signaling cascade in skeletal muscle. 17, 18 Contraction Mediated Glucose Uptake The insulin/PI3K independent pathway, commonly referred to as the contraction/hypoxia pathway, mediates glucose uptake in skeletal muscle due to perturbations in the vasculature and skeletal muscle compared to the basal state. Two of the major players in contraction stim ulated glucose uptake include 5-adenosine monophosphate-activated protein kinase (AMPK) and calcium/calmodulin associated kinases. 5-Adenosine Monophosphate-Activat ed Protein Kinase (AMPK) Contraction mediated glucose uptake is st rongly related to energy status of the cell. 68 An increase in the adenosine monophosphate (AMP)/adenosine triphophate (ATP) ratio within the cell is known to be an activator of AMPK activity. AMPK is a 40

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heterotrimer consisting of alpha ( ), beta ( ), and gamma ( ) subunits. The subunit has an auto-inhibitory and catalytic domain, the subunit binds glycogen which represses activity, and the subunit contains Batemans domains which binds AMP or ATP. The , and subunits can be found in di fferent isoforms: the subunit can exist as the 1 or 2 isoforms; the subunit can exist as the 1 or 2 isoform; and the subunit can exist as the 1 2 or 3 isoforms. The dominant AMPK isoform expressed in human skeletal muscle tissue contains an 2 catalytic subunit and the non-catalytic 2 and 3 subunits. 69-71 Activation of AMPK is achieved in many ways including, but not limited to, allosteric activation by AMP, allosteric activation of upstream kinases by AMP, AMP binding directly to AMPK, allo steric prevention of dephosphorylation by protein phosphatases, glycoge n depletion, and nitric oxide (NO) activation of the subunit. Activating factors include seri ne/threonine kinase 11 (LKB1), TGFactivated kinase 1 (TAK1), calcium/calmodulin-d ependent kinase kinase (CAMKK), NO, and higher AMP/ATP ratios while glycogen and protein phosphatase A act to depress activity. 69 Activated AMPK, like Akt, phosphor ylates AS160 and is a point of convergence between the insu lin and contraction mediated pathways. Acutely polyphosphorylation of the RabGAP domain increas es translocation of existing GLUT-4 protein stores. AMPK also exerts a chronic effect on glucose uptake on skeletal muscle. The chronic effect (late phase: 24-48 hours) of AMPK on glucose uptake is realized through an increase in the number of GLUT-4 pr oteins available for translocation. 19 That is, increased transcription of GLUT-4 proteins via direct phosphorylation of peroxisome proliferator-activated receptorcoactivator 1 (PGC-1 ) by activated AMPK. 19 41

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Chronologically, there is an increase in GLUT-4 messenger ribonucleic acid (mRNA), GLUT-4 protein, and insulin sensitivity. 72 These effects are usually short lived, as within a week GLUT-4 protein number may be back to baseline. 72 However, the effects are additive in that exercise mu ltiple times throughout the week continues to increase the signal. 72 Therefore, there is a cumulative effect of multiple stimuli. Calcium-Calmodulin Dependent Signaling Increases in intracellular calcium c oncentration have also been implicated in glucose uptake via downstream activati on of targets calcium-calmodulin dependent protein kinase II (CAM KII), CAMKK, and protein kinase C (PKC). 70 In skeletal muscle, increases in intracellular concentrations of calcium are fundamental to normal contraction physiology. The increase in in tracellular calcium concentration facilitates greater binding of calcium to calmodulin, a protein when bound with calcium is capable of acting as second messenger and activating downstream targets such as CAMKK and CAMKII. CAMKII inhibition 73 and CAMKK activation 74 have both been shown to impact glucose uptake during muscle contraction. However, the role of CAMKK remains controversial as it can also activate AMPK which facilitates GLUT-4 mobilization and glucose uptake. 19 In addition, AS160 contains a calmodulin biding domain that, when mutated, blunts contraction mediated glucose uptake. 75 Nitric Oxide Mediated Glucose Uptake It has been proposed that there is also a th ird pathway for glucose uptake that is independent of both insulin and c ontraction mediated mechanisms. 20 Sodium nitroprusside (SNP), a NO donor, significantly increases glucose uptake in the presence of wortmannin, a PI3K inhibitor, indicati ng that it is not a PI3K dependent mechanism. 20 In addition, L-NG nitroarginine methyl este r (L-NAME), a nitric oxide synthase (NOS) 42

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inhibitor, administration does not abolish contraction mediated glucose uptake in skeletal muscle. 20 This suggests that the effects of NO are independent of both insulin and contraction and appear to augment glucos e uptake systemically. Indeed, SNP treatment in rat skeletal muscle incr eases glucose uptake in a dose dependent manner 76 and the mechanism responsible for the increase in glucose uptake has been attributed to translocation of GLUT-4. 77 NO donors significantly increase the activity of AMPK by acting on the 1 catalytic subunit and AMPK can act in positive feedback loop with NOS. 24 The increase in AMPK activity coul d explain the increase in NO mediated glucose uptake due to downstream signali ng increasing GLUT-4 translocation and PGC-1 transcription. In addition, aminoi midazole carboxam ide ribonucleotide (AICAR)-induced (AMPK agonist) GLUT-4 protein expression is NO dependent indicating a role for downst ream NO signaling in AMPK mediated GLUT-4 protein expression as well. In resting muscle, NO stimulates glucose uptake through a cyclic guanosine monophosphate (cGMP )-dependent pathway that may involve cGMPdependent kinase/protein ki nase G (PKG) activation. 78, 79 Nitric oxide can also directly activate G protein subunits, specifically the proto-oncogene p21ras for glucose transport, which increases glucos e uptake through nuclear factor B (NF B) signaling. 80 NF B activity is increased transiently from treatment with NO donors and reacts rapidly to increase the activity of the extracel lular signal-related kinases (ERK), c-Jun-NH 2 terminal kinase (JNK), and p38 subgroups of the mitogen-activated protein kinase (MAPK) family. 81 However, the exact mechanism by which the MAPK subfamily elicits glucose uptake has not been elucidated. In addition to the acute effects of p38MAPK on glucose uptake, p38MAPK may also play a role in the chronic glycemic control as it 43

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phosphorylates the transcription factor my ocyte enhancer factor-2 (MEF2) which is implicated in the expression of GLUT-4 protein synthesis from the PGC-1 gene in skeletal muscle. 82 p38MAPK may also play an acute role in glucose uptake, although the exact mechanism by which this increase is realized has not been elucidated. The role of nitric oxide in glucose uptake has been further supported by studies in which the administration of L-NAME results in ma rked hyperglycemia and insulin resistance. These findings support a role for NOS expre ssion and activity in skeletal muscle as a major player in glucose handling. An in vitro study by Lira et al. found that the NO donor Snitroso-N-penicillamine (SNAP) increased GLUT-4 protein expression by 65% in L6 myotubes via a cGMP and AMPK-dependent mechanism. 24 Moreover, L6 myotubes ex posed to AICAR, an AMPK agonist, experienced a nine-fold increase in GLUT-4 mRNA. 24 In vivo, GLUT-4 mRNA was increased 1.8 fold in t he rat plantaris muscle 12 hrs after AICAR injection. Thus, evidence corroborates that in skeletal muscle, NO concentration likely influences GLUT4 expression via control of cellu lar activity of AMPK kinases. Nitric Oxide The free radical NO is a ubiqu itous signaling molecule that participates in virtually all cellular function in the human body. NO regulates cell metabolism, insulin signaling and secretion, vascular tone, and immune system function through interactions with thiol or transition metal c enters in proteins or both. 83 NO is a liable, cytotoxic molecule that acts primarily in a paracrine fashion wi th a half life of approximately 10 seconds in vivo. 84 NO is rapidly oxidized forming nitr ite, and subsequently nitrate by oxygenated hemoglobin, and ultimately excreted in the urine. NO is also susceptible to scavenging by other reactive oxygen species. NO is formed from the enzym e NOS. 3 different 44

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isoforms of NOS exist: endothelial NOS (e NOS), neuronal NOS (nNOS), and inducible NOS (iNOS). All three isofo rms share a similar catalyti c scheme in which the amino acid L-arginine is converted to NO. NOS a gonists mediate NO release by activation of tyrosine kinase, PI3K activator. The activated PI3K then activates Akt which phosphorylates the eNOS Ser-1179 amino ac id in a calcium/calmodulin dependent manner. The resulting reaction yields one NO molecule and the byproduct L-citrulline. NOS activity can be regulated at transcr iptional, translational, and posttranslational levels. Therefore, bioavailability of NO is determined not only by reaction with other molecules (e.g. NO reaction with superoxide radical producing peroxynitrite) but also by NO synthesis from NOS. In humans, impr oved glucose tolerance and insulin sensitivity as seen with exercise intervention studies may be related to augmented expression of NOS in skeletal muscle. It has been well established that muscle tissue expresses NOS and there is significant NO production at rest from bot h constitutively activated forms found in skeletal muscl e, nNOS and eNOS. Endothelial Nitric Oxide Synthase (eNOS) Within skeletal muscle eNOS is typically found in highest concentrations around the mitochondria and is thought to play the gr eater role in cell oxidative metabolism. Importantly, eNOS appears to play the greates t role in glucose handling as isolated skeletal muscle in eNOS knockout mice, in the absence of muscle perfusion, were found to have a 40% reduction in basal and insulin stimulated glucose uptake when compared to wild-type controls. 22 This is supported by evidence that eNOS knockout animals are insulin resistant in addi tion to hypertensive and hyperlipidemic. 22 The importance of NO production may be even more pronounced in T2DM as NOS inhibition decreases glucose uptake during exercise to a significantly greater degree in T2DM 45

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compared to healthy controls. 23 This suggests a greater reliance on NO mediated glucose uptake in these individuals. Another site of NO produc tion is eNOS in the endothelial cells of the vasculature. NO produced in the vasculature is essentia l for relaxation of vascular smooth muscle cells, and in the regulation of angiogenesis, smooth muscle cell proliferation, leukocyte adhesion, platelet aggregation, and thro mbosis. Endothelium derived NO is continuously synthesized from one of the gu anidine-nitrogen atoms of the amino acid Larginine in two successive reactions. 85 The principle mechanis m for the production of NO from eNOS is shear stress along the endothel ial layer in the wall of the vasculature. eNOS agonists, such as acetylcholine and br adykinin, can also bind to receptors on endothelial cells leading to the release of NO. 86-88 Both shear stress and agonistmediated production of NO function by incr easing endothelium intracellular calcium concentrations, which binds to the eNOS comp lex releasing an active eNOS protein. Controversy still exists as to whether or not NO of vascular origin affects skeletal muscle activity and function, but it is plausible. Support for our enhanced external counterpulsation (EECP) hypothesis can be found in an animal model of exte rnal compression. Tan et al. 25 applied intermittent pneumatic compression (IPC) (55 mmHg pressure achieved in <1 second) to the hind limbs of 16 male Sprague-Dawley rats for the durations of 0.5, 1.0, and 5 hours. Following IPC, eNOS in skeletal muscle homogenate was upregulated to 120%, 180%, and 270% from baseline, respectively. Similarly, nNOS expre ssion was up-regulated, but to a lesser degree. Furthermore, EECP in creased plasma levels of NO by 36% in coronary artery disease (CAD) patients following 35 1-hour sessions in a sham 46

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controlled study. 27 Indices of arterial and endothe lial function were equally impressive after 35 1-hr sessions of EECP as endothelialdependent flow mediated dilation (FMD), a non-invasive measure of NO bioavailability, in the brachial and femoral arteries was increased by 51% and 30%, respectively. 27 These results, from this animal model resembling EECP, and from data in our own l aboratory, serve to confirm our hypothesis that compression-induced impr ovement in vascular function is mediated by modulation of NO bioavailability and by regulating expression of NOS isoforms, in particular eNOS. Neuronal Nitric Oxide Synthase (nNOS) The mu neural isoform of NOS (nNOS) is the primary is oform found within skeletal muscle. In adult skeletal muscle, it is often found localized around sarcolemmal proteins that react to mechani cal perturbations of the muscl e. For example, nNOS is found in greatest quantities in the neuromuscular junction wher e the greatest strain is realized in skeletal muscle tissue. nNOS is constitutively active and is associated with the integrin and dystrophin complexes and is thought to be activated by movement of these cytoskeletal protein complexes. nNOS activity, and subsequently NO production, can be increased as much as 2-fold with contraction. 76 nNOS appears to be the largest contributor to NO production in skeletal muscle (in non-inflammatory conditions). In humans, nNOS is expressed in both Type I and Type II muscle fibers L-Arginine is the substrate used for NO production and NOS activity is regulated by several mechanisms. L-arginine is competitively inhibited by asymmetric dimethylarginine (ADMA) and ADMA is degraded by dimethylarginine dimethlaminohydrolase (DDAH). DDAH is a redox sensitive protein that decreases its activity allowing for greater asymmetric dime thylarginine (ADMA) competition in more oxidized environments. BH4 is another redox sensitive cofact or for NOS. In addition, 47

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calcium is a major player in NOS activi ty as increasing calcium concentrations contribute to greater NO production. nNOS protein expression is slightly elevated following IPC in underlying tissue following 1 hour of treatment. 25 Calcium-Insensitive Nitric Oxide Synthase (iNOS) iNOS is a calcium insensitive isoform of NOS. iNOS is thought to only be activated when there is a great inflammato ry response. In humans, iNOS mediated NO production plays the largest role in heart fa ilure patients, auto-immune disorders, and acute inflammatory responses. iNOS appears to be upregulated in the presence of high concentrations of inflammatory cytokines. The activation of iNOS results in a high volume of NO production which is generally detrimental to the cell. However, in humans, iNOS appears to play a minimal ro le in cell signaling under normal physiological conditions. It has been s uggested that iNOS downregulates GLUT-4 expression, but insulin has also been imp licated in downregulation of iNOS at the posttranscriptional level. 89, 90 Insulin and Nitric Oxide The vascular endothelium is the first or gan that insulin encounters upon release from the beta cells of the pancreas. Not onl y is the endothelium in volved in regulating the delivery of insulin and glucose to target tissues, it responds directly to insulin by producing NO. In vivo studies in human and animal models have shown that insulin stimulated production of NO from eNOS is a major player in insulin stimulated glucose uptake via increases in capillary recruitm ent and total limb blood flow in skeletal muscle. 91 The capillary beds are more sensitive to insulin mediated NO production than the large conduit artery vessels greatly enhanc ing the delivery of insulin and glucose to the tissues that are responsible for whole body glucose uptake and disposal. Indeed, 48

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the insulin effect to induce vasodilatation in the resistance arterioles in skeletal muscle is directly proportional to its ab ility to stimulate glucose uptake. 92, 93 Braith et al. have shown that the reactivity of small the small resistance arterioles is improved in CAD patients following 35 1-hour sessions of EECP as peak forearm and calf blood flow increased 22% and 19% from pre-intervention values. 94 Although speculative, increasing basal and insulin -mediated nitric oxide prod uction in these resistance arterioles via EECP therapy may increase t he potential for glucose uptake and disposal. Moreover, a rodent model of IPC acutely in creases expression of vascular endothelial growth factor (VEGF), a prot ein that stimulates the growth of new blood vessels and potentiates an increase in capillary density and nutrient delivery. 49 Reactive Oxygen Species Increased oxidative stress is an establis hed contributor to the development and progression of diabetes. 95 Diabetes, both type I and type II, is associated with increased production of free r adicals and/or impaired antio xidant defenses, shifting redox balance toward greater oxidative stress. 96, 97 Hyperglycemia has been shown to promote free radical generation through gluc ose autotoxidation 98 and lipid peroxidation of low density lipoprotein (LDL) in a superoxide-dependent pathway. 99 Free radical production due to hyperglycemia can also occu r as glucose interacts with proteins ultimately resulting in the formation of advanced glycation endproducts (AGEs). These AGEs can promote free radical formation and quench and block anti-proliferative effects of NO through their binding with specific AGEs receptors. Davy et al. have shown that glycemic control correlated well with a marker of lipid peroxidation, 8-iso-prostaglandin 2 (8-iso-PGF-2 ), 100 but others have reported no correlation in Type I diabetics. 101 Oxidative stress may play an important role in glycemic control as bioavailability of NO 49

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may mediate NO mediated glucos e uptake. Reduced availability of NO may occur due to an increase in breakdown by reactive ox ygen species, especially superoxide which combines with NO to form peroxynitrite, a highly reactive molecule that markedly increases oxidative stress. EECP has been shown to decrease plasma levels of 8-isoPGF-2 viewed as the most valid plasma mark er to assess systemic oxidative stress, by 21% in CAD patients. Al though markers of oxidative stress following EECP therapy in patients with abnormal glucose tolerance have not been specifically investigated, improvements in angina and cardiac events in diabetics with CAD are similar to those with CAD alone. 102 We hypothesized that systemic ox idative stress, as measured by 8iso-PGF-2 would be decreased in subjects with AGT following 35 1-hour sessions of EECP. Furthermore, the measure of 4-hydroxynoneal (4-HNE) pr otein conjugates in skeletal muscle biopsy samples was used as a marker biomarker of oxidative damage. Anti-Oxidants Anti-oxidants are substances, when pres ent in a low concentration, capable of significantly delaying or preventing oxidatio n relative to an oxidizable substrate. Superoxide dismutase (SOD), catalase, glutathione perox idase (GPx), and glutathione reductase are the major enzymatic antioxidants present in cells. Vitamins A, C and E as well as glutathione are some of t he non-enzymatic endogenous enzymatic defenses. These anti-oxidants work in synergy with each other to defend against different types of free radicals. The most common antioxidant deficiencies reported in diabetics are lower levels of vitamin C, glutathione peroxidase and SOD. 98 While the role of antioxidant defense in the treatment and pathology remains controversial, treatment of type T2DM patients with vitamin C or vitamin E decreas ed HbA1c levels, improved insulin action, decreased plasma insulin concentrations, and decreased indicators of oxidative 50

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stress. 103 Measures of total anti-oxidant capac ity in plasma (oxygen radical absorbance capacity assay), as well as quantificati on of SOD and GPX were performed to determine changes in anti-oxidan t defense. We hypothesized that following 35 1-hour sessions of EECP there would be an increase in overall antioxidant capacity and/or a decrease in markers of oxidation. We hy pothesized that these changes would correlate with indices of insulin sensitivity derived from the OGTT. Peroxisome Proliferator-Activated ReceptorCoactivator 1 (PGC-1 ) PGC-1 is a nuclear encoded transcriptional co-activator that increases the expression of several genes in skeletal muscl e, including those involved in skeletal muscle metabolism and glycemic control. PGC-1 expression plays a large role in the metabolic phenotype as its expression is asso ciated with production of proteins involved in oxidative metabolism, mitochondrial density, and fiber type. It also appears to be a site of integration of me tabolic demand and adaptation as its expression is increased with cold exposure, fasting, and exercise. 104 Additionally, PGC-1 expression has been shown to increase GLUT-4 and antioxidant enzyme expression. In diabetics, genes of PGC-1 were reduced and reductions in PGC-1 have been associated with elevated fasting insulin concentrations (indicati ve of decreased insulin sensitivity). 105, 106 In humans, PGC-1 is highly expressed in skeleta l muscle tissue, although expression varies considerably between individuals and between fiber types, but is most highly expressed in slow oxidative fibers. 107, 108 PGC-1 is regulated by the transcription factors cAMP response element-binding protein (CREB), MEF2, and activating transcription factor-2 (ATF-2). PGC-1 upregulation in response to exercise is thought to be mediated, in part, by augmented CAMK II and AMPK activity due to increased in calcium and glycolytic flux respectively CAMKII and AMPK are capable of repressing 51

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histone deacetylase 5 (HDAC5) associati on with the promoter MEF-2 and attenuating HDAC5s repression of PGC1alpha transcription. p38MAPK activity, which can be increased through calcium, AMPK, and NO -dependent signaling can also increase PGC-1 transcription through interaction with the MEF-2, CREB, and ATF-2 transcription factors. Not only does p38MAPK increases PGC-1 transcription, but it also enhances stability and increases the half life of the protein via dislodging the repressor myb-binding protein 1A (p160myb ). Although numerous studies have shown an increase in PGC-1 expression due to exercise or st imulated muscle contraction, the effects of EECP have not been elucidated. We hypothesized that through alterations in nitric oxide mediated signali ng and/or mechanical perturbations of skeletal muscle tissue in response to intermittent cuff compression PGC-1 expression would be upregulated. Ultimately, an increase in PGC-1 expression will increase GLUT-4 protein expression and potentia te a greater GLUT-4 protei n pool for translocation to the plasma membrane to facilitate glucose uptake. Indeed, modest over-expression of PGC-1 (~25%) increased GLUT-4 protein expr ession and insulin stimulated glucose transport in fast and slow rat skeletal muscle tissue. 109 A single bout of exercise has been shown to increase PGC-1 expression as much as 2-fold in rodent skeletal muscle tissue. 110, 111 Therefore, it is not unreasonab le to conclude that EECP may increase PGC-1 expression through perturbations in metabolic flux, mechanical signaling, and/or nitric oxide bioavailability. Asymmetric Dimethylarginine (ADMA) Evidence suggests that ADMA is associ ated with endothelial dysfunction in a number of disorders, includ ing, but not limited to, dys lipidemia, hyperhomocysteinemia, hypertension, CAD, heart failure renal dysfunction, and T2DM. 112, 113 ADMA, like NO, is 52

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derived from the amino acid Larginine. The production of ADMA from L-arginine can decrease NO production due to substrate utiliz ation. L-arginine concentration may be even more important for glucose clearance as McConnel et al. have shown that Larginine infusion during cycling exercise increases glucose uptake in a NO dependent manner. 114 Since type II diabetics are even more reliant on nitric oxide mediated glucose uptake, this may be of significant phy siological relevance. ADMA biosynthesis is catalyzed by the protein-arginine-N-meth yltransferase (PRMTs) family of enzymes. These enzymes utilize S-adenosylmethionine as a methyl group donor for methylation of L-arginine residues on nuclear protei ns. This process and the subsequent proteolysis from nuclear protei ns can result in three different free derivatives: N G monomethyl-L-arginine (L-NMMA), symmetric di methylarginine, and ADMA. While all of these arginine derivatives are present in the human body, only ADMA appears to biologically relevant. 115, 116 Following formation, free ADMA is released into the plasma where it can inhibit NOS and decrease NO bioavailability leadi ng to the development of endothelial dysfunction. 117 A study by Boger et al. demonst rated the negative linear relationship between flow-mediated dilation (a measure of NO bioavailability) and ADMA plasma concentrations in human subjects. 116 In addition, inhibi tion of NOS impairs microvascular recruitment and blunts insulin stimulated glucose uptake. 118 In a study by Steinberg et al., insulin resistance was associated with blunted endothelium-dependent vasodilatation, but not endot helium-independent vasodilatati on, during intrafemoral artery infusions of sodium nitroprussi de and metacholine chloride under euglycemic hyperinsulinemic conditions. 119 In addition, acute glucose challenge in mice and 53

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exposure of cultured endothelial cells to high glucose increases the accumulation of ADMA. 120, 121 ADMA Metabolism Changes in plasma levels of ADMA in humans are mediated by an increase in production and/or a decrease in degradation. Increased ADMA concentrations in cultured endothelial cells, and in patients with endothelial dysfunction, are associated with increased reactive oxygen species production in supernatants and human plasma. 122, 123 One of the mechanisms by which ADMA concentration is increased is by oxidative stress. Oxidative stress is associated with an increase in PRMT activity and gene expression in the cell leading to an increase in ADMA formation. 116, 122-124 Boger et al., has shown that in human endothelia l cells PRMT gene expression is upregulated in the presence of native LDL and oxidized LDL resulting in elevated levels of ADMA. 122 Furthermore, treatment with ant i-oxidants abolished the rise in ADMA levels providing further evidence for the role of redox balance in the cell. 122 Tetrahydrabiopterin (BH 4 ), a cofactor for NOS, is also redox sensitive. Oxidation of BH 4 to its inactive form BH 2 renders it inactive and can lead to NOS uncoupling. 113, 115, 125 When uncoupled, electrons flowing from the reductase domain to the oxygenase domain in NOS are diverted to oxygen leading to the formation of superoxide. High levels of superoxide in the cell can react with NO leadi ng to formation of peroxynitri te and further contributing to an increase in oxidants and a disturbance in redox balance. Evidence suggests that ADMA itself may also act to uncouple NOS 2, 4, 20 or exhibit its effect purely through competition with L-Arginine. 113, 115, 125 This uncoupling of NOS would magnify oxidative stress in a positive feedback fashion in t he cell contributing further to endothelial and cell dysfunction in a viscous cycle. 54

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While an increase in ADMA production due to oxidation of PRMTs is likely at times of high oxidative stress, elimination also plays a large role in explaining the elevation seen in those at cardiovascular risk. Humans produce about 300 mol/day of ADMA during normal protein turnover with about 50 mol/day excreted in the urine and 250 mol/day metabolized by DDAH. 113, 115 ADMA is metabolized to form L-citrulline and dimethlyamine by DDAH and impairment of DD AH activity may explain the potential for elevated levels of ADMA. Two isoforms of DDAH exist, DDAH I and DDAH II, with DDAH II being the most prevalent in tiss ues expressing endothelia l NOS. DDAH is susceptible to inhibition/attenuation due to oxidative stress from oxidized LDL cholesterol, inflammatory cytokines hyperhomocysteinemia, hyperglycemia and infectious agents. 123, 126, 127 The sensitivity of DDAH to oxi dative stress is conferred by reduction of a sulfahydryl group at the CYS-249 residue in the active site of the enzyme which inhibits ADMA metabolism. 126 There is little doubt am ongst researchers that the most common mechanism leading to accu mulation of ADMA involves impaired metabolism by DDAH. 116, 123, 128 In transgenic mice overex pressing DDAH, insulin sensitivity was enhanced during glucose challenge attributabl e to decreased plasma or tissue levels of ADMA. 121 Osanai et al. showed that ADMA levels in human endothelial cells in vitro decreased with15 dynes/cm 2 of shear stress and DDAH ac tivity increased at shear stress levels of > 25 dynes/cm 2 129 Braith et al. showed that following 35 1-hour sessions of EECP in CAD patients that plas ma concentrations of ADMA decreased by 28%. 27 It is not unreasonable to conclude that DDAH activity may be increased with the magnitude of shear stress in voked with EECP (>200% of bas eline, 49 vs. 23 dynes/cm 2 55

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in a porcine model). 26 Unfortunately, to date, there is not a reliable assay for humoral concentration of DDAH in humans. The mechanism by which EECP alters ADMA levels may be related to not only a decrease in production of ADMA, but an increase in degradation via preventing the ox idation of DDAH, eNOS and BH 4 Inflammation Epidemiological evidence for an associat ion of Type II diabetes and inflammation goes back as far as the 1950s. Increased levels of markers and mediators of inflammation such as high sensitivity C-reac tive protein (hsCRP), interleukin 6 (IL-6), and tumor necrosis factor(TNF) correlate well with incident T2DM. 130 In addition, TNFhas been shown to cause insulin resistance in experimental models. 131 Following EECP therapy, Braith et al. demonstrated plasma reductions in levels of TNF(16%) hsCRP (32%), monocyt e chemotactic protein 1 (MC P-1) (13%) and soluble cell adhesion molecules (sVCAM) (6%). 27 Increased bioavailability of NO after EECP therapy is the likely mechanism responsible for the reduction in plasma inflammatory markers. NO serves an anti-inflammatory role by inhibiting the expression of MCP-1 and reducing VCAM-1 expression. 132 Prostacyclin (PGI 2 ) In addition to NO, endothelial cells pr oduce other vasodilators to regulate vascular tone and blood flow. Prostaglandins are a family of eicosanoids derived from endothelial cells. Shear stress has been shown to be a major stimulus for prostacyclin (PGI 2 ) production. 133 Once produced, PGI 2 diffuses into vascular smooth muscles and induces relaxation and ultimately vasodilata tion via elevation of cyclic AMP levels. Decreased NO and PGI 2 release are well established in diabetics. This ultimately results in increased endothelial dysfunction. The potent endothelial-derived vasodilator 56

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6-keto-prostaglandin 1 (6-keto-PGF1 ), the stable me tabolite of PGI 2 was increased by 71% following 35 1-hour sessions of EECP therapy in CAD patients. 27 Vasoconstrictors Among the vasoconstrictors that are produced by endothelial cells is endothelin-1 (ET-1). Human ET-1 is derived from a r eaction catalyzed by endothelin converting enzyme from the substrate big ET-1. 134 ET-1 exerts its major vascular effects, vasoconstriction and cell proliferat ion, through activation of ET A and ET B receptors on vascular smooth muscle cells. ET-1 is chronically upregulated in type II diabetes and hyperinsuliemia may be a stimulus for ET-1 production from the endothelium. 135, 136 In a lean model of T2DM, Elgebaly et al. demonstrated that ET A and ET B blockade increased insulin sensitivity during hyperinsulinemic euglycemic clamps. They also provided evidence that both ET receptors c ontribute to a decreased insulin-mediated vasodilatation in T2DM. The opposing effect of ET-1 to NO is likely the culprit. EECP has been shown to decrease this pot ent vasoconstrictor by 25% in CAD patients. B 137 137 27 Under normal physiological condit ions, eicosanoids produced via the prostaglandin H synthase( PGHS)/Cyclooxygenase(COX) pathway generally induce vasodilatation. However, with endothelial dysfunction, PGHS-dependent vasoconstrictor production, specifically thromboxane A 2 may become more abundant. Thromboxane A 2 can act on their specific receptors in the vascular smooth muscle to produce vasoconstriction. Thromboxane A 2 has been indicated as a contributor to insulin resistance. Wang et al. demonstrated that exposure of cultured endothelial cells exposed to Thromboxane A 2 inhibited both basaland insulin-stimulated Akt-Ser473 57

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phosphorylation, Akt activity, the serine 1177 phosphorylation of endothelial NOS (eNOS-Ser1177), and angiogenesis. 138 Angiotensin II (Ang-II) is another potent vasoconstricto r found in the blood. In addition to its role in salt and water retenti on in the kidneys, Ang-II acts to directly influence endothelial function. The endothelium is capable of producing Ang-II via renin and/or angiontensin I uptake from the bl ood and conversion by locally active angiotensin converting enzyme (ACE). Once synthesized, Ang-II acts as a potent vasoconstrictor opposing the action of NO and PGI 2 Furthermore, Ang-II is a major stimulator of NAD(P) H oxidase, a primary source of superoxide production that can contribute to oxidative st ress. Although the mechanism of action has not been elucidated, angiotensin receptor blockers have been associated with a decrease in new onset diabetes. 139 EECP EECP is a noninvasive, atraumatic, outpat ient therapy that consists of three pneumatic compression cuffs applied to the ca lf, lower thigh, and upper thigh of each leg. These cuffs are sequentially inflated, from distal to proximal, with compressed air during the diastolic phase of the cardiac cycle and rapidly deflated in early systole. Inflation and deflation of the cuffs is tr iggered by events in the cardiac cycle via microprocessor interpreted electrocardiogram (ECG) signals. Acutely, EECP increases diastolic augmentation, reduces systolic afterload, and promotes venous return with a subsequent increase in cardiac output. 140 EECP is traditionally used to treat symptomatic CAD patients who are not readily amendable for interventional procedures. Although initial theory as to the mechanism of action for angina reduction in CAD patients focused on angiogenesis the literature points overwh elmingly to augmentation 58

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of peripheral vascular func tion decreasing cardiac demand. 27 The peripheral vascular nature of adaption to EECP therapy has lead to further indications for EECP treatment including, but not limited to, peripheral vascu lar disease and venous insufficiency. A standard course of EECP treatment is compri sed of 35 1-hour sessions over a 7-week period. Shear Stress Among the primary mechanisms proposed fo r the salutary benefits of EECP therapy is shear stress. Shear stress is an eNOS agonist resulting in the synthesis and release of NO. 26 The shear stress stimulus is transduced into the endothelial cell via the integrin/cytoskeleton mechanotransduction pathway. 44, 141 The sequential inflation (~300 mm Hg) of the 3 pneumatic EECP cuffs from calves to buttocks during a bout of EECP therapy produces a robust retrograde pressure wave in the femoral arteries and simultaneous moderate-pre ssure antegrade flow in the brachial arteries. 26, 46 In a porcine EECP model brachial artery blood flow was velocity was shown to increase by 132% and brachial artery wall shear stress increased by >200% 26 during lower body compression of pneumatic EECP cuffs. It is not unreasonable to conclude that a significant alteration of blood flow velocity and shear stress is realized throughout the entire arterial tree, although it likely varies with location. Exercise is known to create pulsatile, hemodynamic shear forces that improve endothelial function, 142 but exercise prescription adherence is low amongst Type II diabetics. 12-15 In a sham controlled study of symptomatic CAD patients, Braith et al. have demonst rated that 35 1-hour sessions of EECP improve brachial and femoral artery flow mediated dilation (FMD), increases peak forearm and calf blood flow during ven ous occlusion plethysmography, increases plasma nitrate/nitrite (NOx) and 6-keto-P GF1a, and decreases ET-1, 8-iso-PGF2a, 59

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TNF-a, hsCRP, MCP-1, and sVCAM. 27 These results suggest that EECP improves peripheral arterial function by a shear stress mediated mechanism. Mechanical Effects of EECP While it has been well established that EECP causes an acute increase in blood flow to the muscle tissue, the effects of EECP on skeletal muscle have not been well characterized. A previous study by Crens haw et al. has shown that application of external pressure to human cadaver limbs there is a linear relationship between external and intramuscular pressures (1988). 48 The intramuscular pressure was not significantly different at different depths of the tissue, and limb circumference did not affect change in intramuscular pressure. 48 Furthermore, in a rodent model of IPC, pressure underneath the cuffs closely resemb les the pressure set on the compression unit. 49 L6 myotubes that have been pressuriz ed to mimic intramuscular pressures during walking or running s howed a significant increase in succinate dehydrogenase (SDH) activity and glucose uptake with a decrease in lactate release. 50 SDH enhancement, an indicator of tricarboxylic ac id cycle (TCA) activity, increases the capacity to degrade lactate and glucose. Al though the mechanism by which external pressure induces aerobic metabolism has ye t to be elucidated, this suggests that mechanical pressure devoid of active contraction can increase metabolism and potentially alter AMP/ATP ratios. Indeed, in an acute rodent model of IPC, the arterial venous difference was shown to increase duri ng compressions. This is indicative of oxygen consumption and supports an increase in tissue metabolism during external compression. Furthermore, a study by Gr ayson et al., demonstrated a significant increase in somatic oxygen consumpti on during 30 minutes of EECP therapy. 52 The observed increase in oxygen consumption wa s not significantly different between CAD 60

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and healthy subjects. Interestingly, all NOS isoforms may be regulated by hypoxia. 83 The intermittent hypoxia experienced duri ng cuff compression may upregulate NOS and contribute to greater metabolism through nitric oxide mediated cell signaling. Calcium transients may also be induced during EECP therapy as C2C12 myotubes subjected to compression demonstrate an increase in intracellular calcium concentrations. 143 The exact mechanism by which calcium is rel eased is unclear, but pressure sensitive calcium channels or molecular signaling co uld play a role. Another potential mechanism for the perturbation in skeletal muscle metabolism during IPC is stretch of the muscle tissue directly under and in the imm ediate vicinity of cuff edges. Indeed, NO production increases approxim ately 20% in isolated soleus muscle from a single stretch. 53 Furthermore, myotubes subjected to cyclic stretching for 2 hours showed a 42% increase in NOS activity from basal rates. Perhaps of long-term consequence of EECP compression, mechanical loading may upregulate nNOS expression as a result of membrane associated proteins responsive to mechanical disruption. Mechanical loading has been shown to regulate expr ession of talin in C2C12 myotubes 53 and nNOS protein expression is slightly elevated fo llowing IPC in underlying tissue following 1 hour of treatment. 25 The skeletal muscle response to 35 1-hour se ssions of EECP is uncharacterized. However, potential mechanisms for alteration of skeletal muscle metabolism in the short and long term exist. EECP should elicit an increase in intramuscular pressure and stretch the compressed limbs. In addition, acute aerobic metabolism appears to be augmented as demonstrated by an increase in SDH activity, glucose uptake, and oxygen consumption following a single session of IPC. Indeed, Lira et al. examined the 61

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regulation of PGC-1 in muscle cells and found t hat NO donors induce PGC-1 expression via activation of the 1 isoform of AMPK. 144 Further, chronic NO treatment causes increased mitochondrial volume and increased rates of basal and uncoupled aerobic respiration in muscle cells; an effect that is prevented by inhibition of AMPK activity. These data support the conclusion that increased NO availability in skeletal muscle increases AMPK-PGC-1 signaling, resulting in im proved metabolic function. Preliminary Data Given the strong evidence that NO bioavailability and limb perfusion is dramatically improved by EECP, we designe d a pilot study to determine if 35 1-hour sessions of EECP would have a beneficial chroni c effect on fasting glycemic control in patients with T2DM. To the best of our knowledge, the only previous study to measure the salutary effects of EECP on blood glucose was an acute investigation performed immediately following a single EECP session. 51 In that study, blood glucose was acutely reduced following one session of EECP in 18 men and 4 women with diabetes (148 to 129 mg/dL; p<0.0001). We recruited 10 subjects with T2DM (n=10 males; age = 65 9 years; weight = 215 40 lbs) and measured fasting blood glucose levels and performed oral glucose tolerance tests (OG TT; 75 grams of glucose) before and after 35 sessions of EECP. Subj ects fasted and withheld anti glycemic medications for 12 hours prior to measurement of bl ood glucose. Post-intervention laboratory studies were performed between 48 to 72 hours after the last EECP session in an attempt to capture the chronic effect. We found that EECP signific antly reduced fasting glucose by 20% (198 20 to 159 16 mg/dL; p = 0.02). EECP also significantly improved the 2hour changes in plasma glucose (blood sa mples at 30, 60, 90, and 120 minutes) during the OGTT (185 35 to 146 20 mg/dL; p = 0.04). We interpret our pilot data as 62

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strong support for the hypothesis that EECP may improve ch ronic glycemic control in patients with T2DM. We s peculated that improved NO bioavailability and skeletal muscle perfusion were the mechanisms unde rlying sustained improvements in glycemic control. 63

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CHAPTER 3 MATERIALS AND METHODS This proof-of-concept study was design ed to be a single-center, prospective, randomized, and controlled investigation to det ermine the efficacy of enhanced external counterpulsation (EECP) as therapy to im prove glycemic control in patients with abnormal glucose tolerance (AGT). Eighteen (n=18) patients with AGT were recruited by advertisement. Subjects were randomized to receive either 35 1-hour sessions of EECP with target inflation pressure of 300 mm Hg per cuff (EECP group; n=12) or continued medical care with no EECP inte rvention (Time-Control group; n=6). Laboratory testing was perform ed at study entry, and afte r 35 1-hour EECP sessions (7 weeks) or matched control period. All te sting and EECP intervention sessions occurred in the Center for Exercise Science at the Un iversity of Florida. A time and events table is outlined in Table 2-1. Group Assignment After signing an informed consent document, patients who were eligible, based on inclusion and exclusion criteria and scr eening, were randomized in a 2:1 ratio between a group that received 35 1-hour sessions of EECP (n=12) or to a Standard Care control group (n=6). Subjects were inst ructed to refrain from initiating structured exercise training programs during the study. Eligibility Criteria Inclusion Criteria All subjects enrolled in t he study were required to m eet the following inclusion criteria: 1) written informed c onsent for participation in the study was given; 2) presence of abnormal glucose tolerance (fasting pl asma glucose >110 mg/dL and a plasma 64

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glucose concentration of 140 to 199 mg /dL after a 2-hour OGTT or T2DM as determined through clinical diagnoses and screening). Exclusion Criteria Potential subjects were ex cluded from the study for any of the following reasons: 1) insulin dependence for glycemic control; 2) any major illness in the prior 3 months; 3) previous treatment with EECP; 4) participation in moderate intensity exercise for 20 minutes, 2 or more times per week; 5) histor y of deep vein thrombosis, phlebitis, stasis ulcer and/or pulmonary embolism; 6) aged less than 21 years; 7) a ged greater than 75 years; 8) pregnancy; 9) uncontrolled hypertension (defined as a systolic blood pressure of 180 mmHg or more and/or a diastolic blood pressure of 110 mmHg or more, measured as the average of at least two readings, obtained at different occasions); 10) systemic hypotension; 11) any medical, psyc hological, cognitive, social or legal condition that would interfer e with the ability of the subjec t to give informed consent and/or his or her capacity to comply with al l study requirements, including the necessary time commitment; 12) cardiac arrhythmia that would significantly interfere with the triggering of the EECP device; 13) acute coronary syndrome such as unstable angina or acute myocardial infarction. EECP Methods Patients in the EECP group (n=12) were treated with EECP for 1 hour daily on Monday through Friday for 7 consecutive weeks, resulting in a total of 35 hours of EECP. All patients were monitored clinically and hemodynamically, by plethysmography, oximetry and electrocardiographic monitoring, during EECP treatment. EECP involves sequential infl ation and deflation of compressible cuffs wrapped around the patients calves, lower thighs, and upper thighs. Compressed air 65

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pressure is applied via the cuffs to the lower extremities in a sequence synchronized with the cardiac cycle via microprocessor-inter preted electrocardiogram (ECG) signals. The diastolic augmentation pressure is progr essively increased by increasing external compression. In this study, the pressure applied to the cuffs during EECP was set at 300 mmHg. Blood pressure changes and diasto lic augmentation were continuously monitored by finger plethysmography. To a ssess the hemodynamic e ffect of EECP, two ratios were computed electronically, using the systolic and diastolic peak pressures or the area under the systolic and diastolic curves Ratios greater than 1.0 correspond to diastolic values greater than systolic values An effectiveness ratio of 1.5 to 2 is associated with an optimal increase in diastolic femoral artery and aortic retrograde flow, and brachial artery antegrade flow. 145 Optimal pressure fo r modulation of glucose metabolism is unknown. Screening (Fasting Plasma Glucose and Blood Pressure) After informed consent was provided, screening consisted of a simple finger-stick measurement of plasma glucose concentra tion using an at-home blood glucose meter (Accu-Check Advantage, Roche Diagnostics) following an overnight fast. Participants who had a fasting blood glucose of >110 mg/ dL were given the opportunity to continue in the study. Additionally, resting blood pr essure measurements were performed by an experienced technician using a standar d manual mercury sphygmomanometer. Subjects with uncontrolled hypertension defi ned as a systolic blood pressure of 180 mmHg or more and/or a diastolic blood pressu re of 110 mmHg or mo re were excluded from the study. 66

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Dual Energy X-Ray Absorptiometry (DEXA) Subjects underwent a DEXA scan at study entry and after 35 1-hour sessions of EECP or the control period. The DEXA is a low radiation device which measures body composition and bone mineral density. There is a small x-ray dosage during the body composition measurement. The effective radiatio n dose equivalent (HE) is about 2.5 uSv during a total body scan. This exposure is approximately 0.3% of th e average annual per capita background radiation exposure in the U.S (equival ent to 1.1 days of natural background exposure). DEXA scans were performed by Je ffrey S. Martin (Florida Department of Health License #BMO 68895) in the Center for Exercise Science at the University of Florida. Skeletal Muscle Biopsies Subjects were asked to report to Dr. Braiths Cardiovascular Laboratory in the Center for Exercise Science at the Universi ty of Florida where skeletal muscle biopsies were performed. Biopsies were performed at study entry and after 35 1-hour sessions of EECP or the control period. Muscle biopsies commenced within 24-48 hours after the last EECP therapy session, if subjects were in the treat ment group, in an effort to capture the chronic adaptation to EECP therapy as opposed to the acute effect of the last session. The muscle biopsies were perfo rmed by Juan Aranda Jr., M.D. Skeletal muscle tissue (approximately 150 mg) was extrac ted from the right va stus lateralis of each subject using a percutaneous needle u nder local subcutaneous anesthetic (1% lidocaine) using a modification of the Bergstrom technique. 146, 147 Two-thirds (approximately 100 mg) of the muscle biopsy sample was immediately snap frozen in liquid nitrogen and stored at -80 C for quantitative protein anal ysis via western blotting. The other third of the muscle biopsy samp le (approximately 50-60 mg) was separated 67

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for cryo-sectioning. After a ssuring alignment of muscle fi bers at resting length, this portion was frozen in optimal cutting tem perature (OCT) medium by liquid nitrogen cooled isopentane and stored at 80C until cryo-sectioning. Oral Glucose Tolerance Tests Oral glucose tolerance tests (OGTT) were performed in all subjects at study entry and after 35 1-hour sessions of EECP or Time -Control. During the 3 days prior to each clamp, subjects were instructed to consum e a standardized diet, consisting of at least 200 grams of carbohydrate per day, while abstaining from caffeine and alcohol. Subjects reported to the laboratory in t he morning following an overnight fast and withheld vasoactive medications for 10-12 hour s and glycemic control medications for at least 24 hours. A catheter was placed in a vein in the anticubital space. 6 mL blood samples were drawn at -10, -5, and 0 minutes to account for the pulsatile nature of insulin secretion. An average of the 3 baseline time-points was used to determine fasting insulin and glucose concentrations. Following ingestion of a glucose beverage (Fisherbrand Glucose Tolerance Test Beverage, 7.5g glucose/fl oz., 10 oz. beverage, 75 grams of glucose ingested) 6 mL blood samples were ta ken at 30, 60, 90, and 120 minutes from the time the dr ink was finished. Subjects were instructed to drink the entire beverage as quickly as possible and in a time not to exc eed 3 minutes. Blood samples were evaluated by commercially available assays for plasma glucose and insulin concentrations. Blood Sampling In addition to the blood samples collected for plasma insulin and glucose concentrations; approximately 20 mL of blood samples were collected from all subjects at study entry, and after 35 1-hour sessions of EECP (7 weeks) or matched control 68

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period. Venous blood was collected from a vein of the anticubita l space, using the same Teflon catheter as described in the or al glucose tolerance testing. These blood samples were drawn at the -10 minute time-poi nt prior to the star t of OGTT. Blood was collected in tubes containing ethylenediaminetetr aacetic acid (EDTA) and immediately underwent centrifugation at 3,000 rpm for approximately 15 minutes. Plasma was immediately aliquoted into st erile tubes and stored at -80 C for analysis at completion of the study. Peripheral Flow Mediat ed Dilation (FMD) The FMD technique was used to determi ne endothelial-dependent reactivity in the brachial and popliteal arteries. At study entry and within 24-48 hours after the final EECP therapy treatment or ti me matched control, subjects fasted for at least 8 hours and withheld all vasoactive medications fo r 10-12 hours and reported to Dr. Braiths Vascular Laboratory. Afte r lying quietly for 15 minutes, a 10.5MHz linear phase array ultrasound transducer (ATL HDI 3000; Advanced Technologies) was used to image the right brachial artery longitudinally. 148 Resting baseline end diastolic brachial diameters and blood velocity were obtained with the transducer placed 3-5 cm above the anticubital fossa. After obtaining baseline di ameter measures, reactive hyperemia was produced by inflating a blood pressure cuff pl aced on the upper forearm, 1-2 cm below the elbow, for 5 minutes at 200 mmHg. The transducer was manually held in the same position for the duration of cuff inflation. Immediately following cuff release, brachial artery blood flow velocity was measured for 20 seconds. Brachial artery diameter was then imaged and recorded for an additional 2 minutes. Ultrasound images were recorded directly to a digital storage device via video interface (Pinnacle, Avid 69

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Technology) for off-line electronic im age analysis using aut omated FMD software (Vascular Research Tools; Medical Imaging Applications LLC, Iowa). Brachial artery diameters were det ermined during end-diastole (gated with electrocardiogram R wave) by measuring the distance between the near and far wall of the intima. Brachial FMD was calculated in absolute (mm) and relative (FMD%) peak change in brachial artery diameter in response to the hyperemic stimulus. 149 Brachial measurements are normalized to the mean shear rate calc ulated from the first 10 seconds following cuff dilation. With the use of artery diameter and mean velocity doppler measurements, blood flow in the br achial artery was calculated using the following equation: blood flow (mL/min) = mean velocity (diameter/2) 2 60. Additionally, in the absence of blood viscosity shear rate is measured by the following equation: shear rate (s -1 ) = 4 mean blood velocity (cm/s) diameter (cm -1 ). Popliteal artery FMD was performed at t he popliteal fossa, 2 to 3 cm above the bifurcation, using the approximate FMD protocol described above for brachial FMD. Venous Occlusion Plethysmography (VOP) Forearm and Calf Blood Flow To determine EECP-mediated adaptation in small resistance arteries, calf blood flow (CBF) and forearm (FBF ) responses were determined independently by venous occlusion plethysmography (EC-6, D.E. Ho kanson, Inc.) using calibrated mercury strain-gauges. 150 For CBF, strain gauges were app lied to the widest part of the nondominant calf. Patients rested supine for 20 minutes with legs elevated above the right atrium to achieve stable basel ine measurements of CBF. To measure CBF, a thigh cuff was inflated to 50 mmHg for 7 seconds ever y 15 seconds for 3 minutes using a rapid cuff inflator to prevent venous outflow. 151, 152 One minute before measurements, an 70

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ankle cuff was inflated to constant pr essure 50 mmHg above systolic pressure to occlude ankle circulation dur ing CBF measurements. T he CBF output signal was transmitted to NIVP3 software program loaded on a desktop computer and expressed as milliliters (mL) per minute per 100 mL of calf tissue (mLmin -1 mL -1 tissue). CBF for one minute is the average of one plethy smographic measurement every 15 seconds for one minute. 151, 152 To assess FBF the mercury strain gauge was placed 5mm below the anitcubital space and cuffs were placed on the upper arm and wrist. Patients rested supine for 20 minutes with the arm elevated to level of heart, parallel with the body. The upper arm cuff was inflated to 50 mmHg for 7 seconds every 15 seconds using the rapid cuff inflator to prevent venous outflow. One mi nute prior to measurem ents, the wrist cuff was inflated to a constant pressure of 50 mmHg above systolic pressure to occlude hand circulation during CBF m easurements. The output signal is again transmitted to the NIVP3 software program and calculations are performed in the same manner as CBF. Forearm Flow During Reactive Hyperemia Endothelium-dependent FBF wa s measured following 5 minutes of upper arm arterial occlusion during reacti ve hyperemia of the forearm. 151, 152 Endotheliumdependent vasodilation (EDV) during reacti ve hyperemia in the forearm has been shown to correlate highly with acetylchol ine-induced EDV in patients with essential hypertension. 148, 153 Therefore, it is a good non-in vasive measurement of EDV of resistance vasculature. Blood pressure cu ffs were placed on the upper arm, 5 cm above the anticubital fossa, but below the ve nous occlusion cuff. After baseline FBF was confirmed to be stable for 2 minutes and recorded, the upper ar m cuff was rapidly 71

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inflated to 200 mmHg for 5 minutes and then released. At 4 minutes of occlusion, the wrist cuff was rapidly inflated to 200 mmHg. Peak FBF was recorded as the highest FBF observed immediately following releases of the cuff, and total FBF was recorded as the area under the time-curve after baseline FBF is subtracted. 150, 153 Cuff inflation pressures and timing following occlusion were identical to those used for resting blood flow measurements. Calf Flow During Reactive Hyperemia Endothelium-dependent CBF we re measured following 5 minutes of upper leg arterial occlusion during reactive hyperemia of the calf. A blood pressure cuff was inflated on the upper thigh above the knee, but below the venous occlusion cuff. After baseline CBF was confirmed to be stable for 2 minutes, the thigh cuff was rapidly inflated to 200 mmHg for 5 minutes and then released. Peak CBF was recorded as the highest CBF observed immediately following re lease of the cuff, and total CBF recorded as the area under the time-curve after baseline CBF is subtracted. 150, 153 Cuff inflation pressures and timing following occlusion were identical to those used for resting blood flow measurements. Western Blotting Frozen muscle samples were homogenized using procedures as reported by Sakamoto et al. 154 Briefly, samples were weighed and placed in the appropriate volume of homogenate buffer containi ng protease inhibitor (The rmo Scientific, 78415) and phosphatase inhibitor (Thermo Scientific, 78420) cocktails and rotated end over end for 1 hour at 4C. Samples were then sonicated three times for 10 seconds each. Following sonication, samples were centri fuged at 14,000 rcf for 10 minutes at 4C and supernatant was transferred to polypropylene tubes and stored at -80C. Protein 72

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concentrations were measured using the DC Protein Assay Kit (Bio-Rad, 500-0116). Aliquots of muscle homogenate (50g and 75g) were separ ated in 4-20% SDS-PAGE gels, transferred to nitrocellulose memb ranes, and stained with Ponceau S to verify transfer. Separate protein blots were pr obed for proteins of interest along with -actin as a loading control. The primar y antibodies used were: goat anti-Akt1/2 (N-19) (Santa Cruz Biotechnology; sc-1619), rabbit antiphospho-AKT1/2/3 (Ser 473) (Santa Cruz Biotechnology; sc-7985R), mouse anti-AMPK (Cell Signaling Technology; 2793), rabbit anti-phospho(Thr172)-AMPK (Millipore;07-681), rabbit anti-TBC1D4 (AS160) (Abcam; ab24469), rabbit anti-phospho (Thr642)-T BC1D4 (AS160) (Novus Biologicals; NBP1-44074), goat anti-GLUT-4 (Santa Cruz Biotechnology;sc-1608), mouse antieNOS(6H2) (Cell Signaling Technology; 5880), rabbit anti-nN OS (Cayman;160870), mouse anti-SOD-2 (B-1) (Santa Cruz Bi otechnology; sc-133254), mouse anti-SOD-1 (G-11) (Santa Cruz Biotechnology; sc-17767); mouse anti-GPx-1/2 (E-7) (Santa Cruz Biotechnology; sc-74498), rabbit anti-4HNE (Abcam;ab46545), and rabbit anti--actin (Abcam;ab8227). Blots were blocked with Odyssey blocking buffer (LI-COR Biosciences; 927-40000) before incubation with primary ant ibodies. Incubations with secondary antibodies IRDye 680CW donkey anit-goat (LI-COR Biosciences; 92632224), IRDye 680CW donkey anti-mouse (LI-COR Biosciences; 926-32222), IRDye 800CW donkey anti-mouse (LI-COR Biosciences; 926-32212), IRDye 800CW donkey anti-rabbit (LI-COR Biosciences; 926-32213 ) we re performed. Protein blots were scanned and proteins of intere st detected using the Odyssey infrared imaging system (LI-COR Biotechnology, Lincoln, NE). Some membranes were stripped using 10 mL of Restore TM western blot stripping bu ffer (Thermo Scientific; 210 59) at room temperature 73

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for 60 minutes. Following st ripping, membranes were washed in TBS 4 times for 5 minutes each wash. After washing, membr anes were read using the Odyssey infrared imaging system to assure complete stripping of previous signals/proteins before probing for additional proteins of interest. Immunostaining for Capillary Density Frozen sections of muscle biopsy tissue were cut (8 m) in a cryostat on fixed on microscope slides. Slides were then allowed to come to room temperature, and fixed in Carnoys fixative (60% ethyl alcohol, 30% chloroform, and 10% glacial acetic acid) for 10 minutes. After rinsing wit h several exchanges of dH 2 O, slides were incubated in 1% amylase at 37C for 60 minutes. Following another rinse, slides were oxidized in 0.5% periodic acid (Sigma-Aldrich; P-7875) for 10 minutes. Again, slides were rinsed with several exchanges of dH 2 O, followed by incubation in Sc hiffs reagent (Sigma-Aldrich; 3952016) for 5 minutes. Slides were then rinsed and dehydrated in ascending concentrations of ethyl alcohol and mount ed with aqueous mounting medium. Images of the muscle sections were captured with an inverted microscope (Olympus America; Center Valley, PA). Stained sections we re analyzed by magnifying and projecting numerous artifact-free secti ons of approximately 0.20 mm 2 areas onto a screen. The number of fibers within the known ar ea were counted and capillary density (capillaries/mm 2 ), capillary/fiber ratio, and mean fiber area was calculated. Number of fibers and capillaries was determined on 166 13 fibers per biopsy. Areas were assessed by manual drawing of the perimeter using the National Inst itutes of Healths public software (Image J, NIH, USA). 74

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Biochemical Assays Vasodilator Measurements Because NO is rapidly converted to nitrite and nitrate (NOx) in plasma, measurement of these metabo lites can be used to estimate NO production. Since plasma NOx can be influenced by dietary ni trates, subjects were asked follow the National Institutes of Health low nitrate diet guidelines for 36-48 hours prior to blood sampling. 155 Plasma NOx was measured using a commercially available assay kit (Cayman Chemical, Inc.) that converts a ll nitrate to nitrit e using NADH-dependent nitrate reductase. Spectrophotometric analysis of total nitrite was then performed using Greiss reagent and the absorban ce measured at 540 nm. The major metabolite of the vaso dilator prostacyclin, 6-keto-PGF1 was also measured by commercially available enzyme-linked immunosorbent assay (ELISA) (Cayman Chemical, Inc.). Vasoconstrictor Measurements Commercially available competitive ELISA kits were used to determine plasma endothelin (ET-1) (R&D Systems, Inc.). The ET-1 ELISA ki ts utilize a microplate that has been pre-coated with an anti body specific for ET-1. ET-1 present in plasma samples is sandwiched by the immobiliz ed antibody and the enzyme-linked antibody specific for ET-1. Tetramethylbenzadine (TMB) is added to the microplate wells and absorbance at 450 nm is proportional to the amount of ET-1 present in plasma samples. Lipid Peroxidation Isoprostanes are important products of lipid peroxidation, and their measurement has emerged as one of the most reliable approaches to assess oxidative stress in vivo. Limited amounts of isoprostanes can be absorbed and diet has a limited effect on 75

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plasma levels of these compounds. 156 Therefore, oxidative stress induced lipid peroxidation was assessed by measuring pl asma levels of 8-iso-prostaglandin 2 (8iso-PGF 2 ) using a commercially available ELISA ki t (Enzo Life Sciences). This assay is based on the compet ition between 8-iso-PGF 2 and an 8-iso-PGF 2 acetylcholinesterase conjugate for a limited number of 8-iso-PGF 2 -specific rabbit antiserum binding sites. The amount of 8-iso conjugate that is able to bind is inversely proportional to the amount of free 8-iso present in the sample. The plate is read at 405 nm on a spectrophotometer. Antioxidant Capacity Total antioxidant capacity was measured using a Trolox-equivalent antioxidant capacity (oxygen radical absor bance capacity, ORAC) assay kit provided by ZenBio, Inc. This assay relies on the ability of antio xidants in the plasma sample to inhibit the oxidation of 2,2-Azobis-2-methyl-propanimi damide, dihydrochloride(AAPH) by peroxylradical formation. The ORAC assay is a ki netic assay measuring fluroescein decay and antioxidant protection over time The antioxidant activity is normalized to Trolox units to quantify antioxidant capacity. ADMA Serum levels of the endogenous eNOS competitive inhibitor, asymmetric dimethylarginine (ADMA) were measured usi ng an ELISA kit (Alpco, Inc.). Samples were treated with a derivatizatoin-r eagent for ADMA coupling and incubated in microplates pre-coated with ADMA -derivative tracer. ADMA present in plasma samples competes with the tracer immobilized in the wells for binding of the polyclonal antibodies. Therefore, the concentration of tracer-bound antibody as measured by 76

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spectrophotometry at 450 nm, is inversely pr oportional to ADMA concentration in the sample. Inflammatory Markers High sensitivity CRP (hsCRP) was determined by a commercially available ELISA (BioQuant). Th e hsCRP assay is a solid phase direct sandwich assay. The samples and anti-CRP antibodi es conjugated with horseradi sh peroxidase are added to wells pre-coated with monoclona l antibodies for CRP. CRP in the serum binds to the anti-CRP monoclonal antibodi es and the second antibody then binds to CRP. Ultimately, after addition of substrate, the plate is read at 450 nm on a spectrophotometer with the absorbance being proportional to the CRP in the serum samples. Similarly, tumor necrosis factor(TNF) (Cayman Chemical, Inc.) was measured using a sandwhich ELISA and its absorbance read at 405 nm using a spectrophotometer. Glycosylated Hemoglobin (HbA1c) Plasma samples were transported to S hands Hospital Clinic Laboratories at University of Florida CORE lab for HbA1c analysis by standard procedures. Samples were kept on ice and transported to the l ab within 30 minutes of blood sampling. Analysis was performed using convent ional clinical techniques. Vascular Endothelial Growth Factor (VEGF) VEGF was measured by commercially avai lable ELISA kits (R&D Systems). Microplates pre-coated with a monoclona l antibody specific for VEGF will bind and immobilize VEGF present in plasma samples. Ultimately, the substrate is added to microplate wells after the addi tion of a secondary antibody which will increase color in 77

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proportion to the amount of VEGF present in the plasma sa mples. Absorbance is read on a spectrophotometer at 540 nm. Insulin Plasma insulin was measured by commercially available ELISA kits (Alpco Diagnostics). The insulin ELISA is a sandw ich type assay in which a microplate is precoated with a monoclonal antibody specific fo r insulin. After the addition of samples with a horseradish peroxidase enzyme labeled monoclonal antibody to the microplate wells, the microplate is incubated and washed with wash buffer. TMB is added to the microplate, incubation commences, and finally stop solution is added to the wells. Absorbance is read on a spectrophotometer at 450 nm and is proportional to plasma insulin concentration present in the sample. Glucose Plasma glucose was quantified using a commercially available assay kit (Cayman Chemical, Inc.). The glucose assay utilizes the glucose oxidase-peroxide reaction. Glucose is oxidized to -gluconolactone with concomitant r eduction of the flavin adenine dinucleotide (FAD)-dependent enzyme glucose ox idase. The reduced form of glucose oxidase is regenerated to its oxidized form by molecular oxygen to produce hydrogen peroxide (H 2 O 2 ). Finally, with horseradish peroxidase as a catalyst, H 2 O 2 reacts with 3,5-dichloro-2hydroxybenzenesulfonic acid a nd 4-aminoantipyrine to generate a pink dye with absorption read at 515 nm on a spectrophotometer. Absorbance is proportional to the amount of plasma gluc ose concentration present in samples. 78

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Statistical Considerations Statistical Analysis This study was designed as an open l abel, unbalanced, randomized study of EECP vs. Standard of Care fo r patients with AGT. All data were tested for normal distribution using the Shapiro-Wilk test for normality. An alpha level of P < 0.05 was required for statistical significance. Satte rthwaite corrected two sample t-tests of baseline subject characteri stics were performed, and P -values reported for each variable. A repeated measures 2-way analysi s of variance was used to evaluate the continuous primary dependent variables associated with, brachial artery FMD, popliteal artery FMD, forearm VOP, ca lf VOP, capillarity, plasma markers, fasting indices of glycemic control, and dynamic measures of glucose tolerance. When a significant group-by-time interaction was observed, wit hin-group comparisons between time points and between-group comparis ons at each time point were performed using Bonferronis post hoc test for pairwise comparison. To achi eve an overall family error rate of 5% for between-group comparisons, was adjusted for multiple comparisons. When comparing between groups at each time point, was adjusted for 2 comparisons, 0.05/2 = 0.025. Western blot proteins of in terest were analyzed using the Satterthwaite corrected two sample t-test of percent change from baseline to week 7 as our dependent variable, and study group (EECP vs. usual care) as our independent variable. All statistical analyses were performed using IBM SPSS Statistics 19 for Windows (Chicago, IL). All dat a are reported as mean SEM. Power Analysis A power analysis based on a sample size of 15 (n=10 EECP Group and n=5 Control Group) was performed to estimate the statistical power related to testing the 79

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following hypothesis (stated as null): 1) T he distribution of change in average insulin sensitivity determined from oral glucos e tolerance test using the Matsuda Index, 58 when compared to standard care from Baseline to Week 7 is the same for EECP and Standard of care and 2) The di stribution of change in GLUT-4 protein in muscle from Baseline to Week 7 is the sa me for EECP and standard care. Based on the data of OGorman et al., 157 we projected differences for Hypothesis 1 of 4.1 [SD=SE* (n)=2.0] and 2.6 mg/kg/min (SD=1. 0=about half of 2.0 as changes in controls will be more stable) for EECP and cont rol subjects, respectively. A study of 10 evaluable EECP subjects and 5 evaluable controls will have 80% power, based on the Satterthwaite corrected t-test to have a P -value below 5% two-sided. For Hypothesis 2, the means were antici pated to be 0.35 and 0.2 U for EECP and standard care subjects, respectively 157 Using calculations as above for Hypothesis 2, the anticipated standard deviation were about 0.17 for the EECP group and about 0.085 for the control group. The study of 10 evaluable EECP subjects and 5 evaluable controls will have 90% power, based on the Satte rthwaite corrected t-test to have a P value below 5% two-sided. 80

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Table 3-1. EECP and glycemic control protocol; time and events Events Consent and Screening (V1,V2) V3 V4 V5V39 V40 V41 Randomization X Medical history review X Informed consent X Fasting Glucose Measurement X Blood Pressure X X X X DEXA X X Blood Sample X X X X OGTT X X Skeletal Muscle Biopsy X X VOP X X Peripheral FMD X X EECP X V = Visit; DEXA = Dual energy x-ray absorpt iometry; OGTT = oral glucose tolerance test; VOP = venous occlusio n plethysmography; FMD = flow mediated dilation; EECP = enhanced external counterpulsation 81

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CHAPTER 4 RESULTS A total of eighteen (n=18) subjects with abnormal glucose tolerance (AGT) were recruited and randomly assigned (2:1 ratio) to receive either 35 1-hour sessions of enhanced external counterpulsation (EECP) with target inflation pressure of 300 mm Hg per cuff (EECP group; n=12) or continued medical care with no EECP intervention (Time-Control group; n=6). Subject Characteristics befo re EECP and Time-Control The baseline characteristics for t he EECP and Time-Control subjects are presented in Table 4-1. The EECP and Time -Control groups did not differ with respect to age, body weight, body height, body mass index, body fat percentage, trunk to limb fat mass ratio, metformin t herapy, lipid-lowering therapy, beta-blocker therapy, calciumchannel blocker therapy, angiotensin converting enzyme (ACE) inhibitor/angiotensin-II receptor blocker (ARB) therapy, or diuretic therapy. There were no changes in body weight, body mass index, and body fat percentage in either the EECP or Time-Control group following 35 sessions or 7 weeks respectively. Brachial Artery Endothelial Func tion after EECP or Time-Control Brachial artery flow mediated dilation (FMD) results are presented in Table 4-2 and Figures 4-1, 4-2, and 4-3. There was no significant change in baseline brachial artery diameter in the EECP or Time-Contro l groups. As shown in Figures 4-1 and 4-2 there was a significant increase in brachial artery FMD (2.90 0.31% vs. 4.01 0.59%, P < 0.05) and absolute change in diameter (0.13 0.01mm vs. 0.18 0.02mm, P < 0.05) in the EECP group following 35 sessi ons compared to baseline. There was no significant change in brachial artery FMD (3.22 0.34% vs. 3.16 0.22%, P = NS) or 82

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absolute change in diamet er (0.14 0.01 mm vs. 0.13 0.02 mm, P = NS) in the TimeControl group at 7 weeks following baseline. As shown in Figure 4-3, there was a significant increase in the normalized brachial FMD response (0.15 0.02 s -1 vs. 0.19 0.02 s -1 P < 0.05) in the EECP group following 35 sessions compared to baseline, but there was no significant change in the normalized FMD response (0.16 0.03 s -1 vs. 0.15 0.02 s -1 P = NS) in the Time-Control group. Popliteal Artery Endothelial Function after EECP or Time-Control Popliteal artery FMD result s are presented in Table 43 and Figures 4-4, 4-5, and 4-6. There was no significant change in bas eline popliteal artery diameter in the EECP or Time-Control groups. There was also no significant change in popliteal FMD or absolute change in diameter in the EECP or Time-Control groups following 35 sessions or 7 weeks, respectively. As shown in Figur e 4-6, there was a signi ficant increase in the normalized popliteal FMD response (0.23 0.03 s -1 vs. 0.35 0.04 s -1 P < 0.05) in the EECP group following 35 sessions compared to bas eline, but there wa s no significant change in the normalized FM D response (0.26 0.05 s -1 vs. 0.26 0.05 s -1 P = NS) in the Time-Control group. Forearm and Calf Resistance Artery Bloo d Flow after EECP or Time-Control Forearm resistance artery blood flow va lues during reactive hyperemia are presented in Table 4-4 and Figures 4-7, 4-8, and 4-9. There was no significant change in resting forearm blood flow (FBF) for the EECP or Time-Control groups. Peak FBF significantly increased 26% (15.68 1.47 mL/min/100mL vs. 19.71 1.56 mL/min/100mL, P < 0.05) in the EECP group after 35 sessions compared to baseline, but there was no significant change in the Time-Control group (16.49 0.94 mL/min/100mL vs. 16.86 1.12 mL/min/100mL, P = NS). Total FBF area under the 83

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curve (AUC 3min ) increased significantly 37% (5.04 0.63 mL/min/100mL vs. 6.91 0.74 mL/min/100mL, P < 0.05) in the EECP group after 35 sessions compared to baseline, but there was no significant change in the Time-Control group (5.53 0.88 mL/min/100mL vs. 5.63 0.96 mL/min/100mL, P = NS). Calf resistance artery blood flow values during reactive hyperemia are presented in Table 4-5 and Figures 4-10, 4-11, and 4-12. There were no significant changes in resting calf blood flow (CBF), peak CBF, or total CBF AUC 3min in the EECP or Time control groups after 35 sessions or 7 weeks respectively, compared to baseline. Markers of Angiogenesis/Vasculoge nesis after EECP or Time-Control Plasma levels of vascular endothelial gr owth factor (VEGF) mean fiber area, skeletal muscle capillary density, and capillary to fiber rati o values are presented in Table 4-6 and Figures 4-13, 4-14, and 4-15. Additionally, a representative picture of capillary visualization from periodic acid-Schiffs base stai ning is presented in Figure 416. Mean fiber area did not change from base line in the EECP or Time-Control groups. There was a significant increase in plasma VEGF (9.74 1.59 pg/mL vs. 17.04 2.65 pg/mL, P < 0.05) in the EECP group after 35 sessions, but no change (9.94 2.24 pg/mL vs. 8.99 3.75 pg/mL, P = NS) in the Time-Control group compared to baseline. There was no significant difference in skele tal muscle capillary density in either the EECP or Time-Control groups, respective to basel ine. However, capillary to fiber ratio was significantly increased (1.71 0.11 vs. 1.84 0.11, P < 0.05) in the EECP group following 35 sessions, but did not change signif icantly in the Time-Control group (1.65 0.16 vs. 1.63 0.16, P = NS). 84

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Vasoactive Balance after EECP or Time-Control Plasma levels of nitrite/nitrate (NOx), 6-keto-prostaglandin 1 (6-keto-PGF1 ), and endothelin (ET-1) are presented in Table 4-7 and Figures 4-17, 4-18, and 4-19. There was a significant increase in plasma NOx ( 26.47 1.58 mol/L vs. 34.46 1.78 mol/L, P < 0.05) in the EECP group following 35 sessions, but did not change significantly in the Time-Control group (26.40 2.34 mol/L vs. 26.87 2.63 mol/L, P = NS). Furthermore, plasma concentrations of NOx following EECP were si gnificantly greater than the Time-Control group at the same time-point ( P < 0.05). There was a significant increase in plasma levels of 6-keto-PGF1 (112.1 15.3 pg/mL vs. 163.4 11.7 pg/mL, P < 0.05) in the EECP group following 35 sessions, but did not change significantly in the Time-Control group (130.3 21. 6 pg/mL vs. 126.3 16.6 pgl/L, P = NS). There was no significant change in plasma ET-1 or t he NOx/ET-1 ratio in either group. Lipid Peroxidation, Antioxidant Capacity and Endogenous Nitr ic Oxide Inhibition by ADMA after EECP or Time-Control Plasma levels of 8-isoprostane-F 2 (8-iso-PGF 2 ), oxygen radical absorbance capacity (ORAC), and asymmetric dimethylargi nine (ADMA) are presented in Table 4-8 and Figures 4-20, and 4-21. There was a signifi cant decrease in plasma levels of 8-isoPGF 2 (838.2 61.2 pg/mL vs. 644.0 70.7 pg/mL, P < 0.05) in the EECP group following 35 sessions, but no significant ch ange in the Time-Cont rol group (749.9 86.6 pg/mL vs. 811.9 99.9 pg/mL, P = NS). There was no significant change in the ORAC of plasma in either group. There was a signi ficant decrease in plasma levels of ADMA (0.47 0.01 mol/L vs. 0.42 0.01 mol/L, P < 0.05) in the EECP group following 35 sessions, but no significant change in the Ti me-Control group (0.45 0.02 mol/L vs. 0.46 0.02 mol/L, P = NS). In addition, plasma concentrations of ADMA following 85

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EECP were significantly less than the Time-Control group at the same time-point ( P < 0.05). Plasma Markers of Inflammation after EECP or Time-Control Plasma levels of high sensitivity C -reactive protein (hsCRP) and tumor necrosis factor(TNF) are presented in Table 4-9 and Fi gures 4-22 and 4-23. There was a significant decrease in plasma levels of hs CRP (2.67 0.38 mg/L vs. 1.91 0.33 mg/L, P < 0.05) in the EECP group following 35 sessions, but did not change significantly in the Time-Control group (2.62 0.51 mg/L vs. 2.52 0.44 mg/L, P = NS). There was no significant change in TNFplasma concentrations in either group. Fasting Markers of Glycemic Cont rol after EECP or Time-Control Fasting plasma glucose (FPG), fasting pl asma insulin (FPI), homeostasis model assessment of insulin resistance (HOMA IR ), quantitative insulin sensitivity check index (QUICKI), and glycosylated hemoglobin (HbA1c) values at baseline and after 35 sessions of EECP or 7 weeks of Time-Contro l are presented in Table 4-10 and Figures 4-24, 4-25, 4-26, 4-27, and 4-28. There was a signific ant decrease in FPG (143.9 8.5 mg/dL vs. 127.0 6.6 mg/dL, P < 0.05) in the EECP group following 35 sessions, but no significant change in the Time-Control group (138.3 12.0 mg/dL vs. 140.2 9.3 mg/L, P = NS). FPI did not change in either t he EECP or Time-Control group following 35 sessions or 7 weeks of standard care respectively. The HOMA IR was significantly decreased (3.02 0.55 vs. 2.08 0.39, P < 0.05) in the EECP group following 35 sessions, but did not change significantly in the Time-Control group (3.25 0.78 vs. 3.38 0.56, P = NS). The QUICKI was significantly increased (0.322 0.009 vs. 0.335 0.009, P < 0.05) in the EECP group following 35 sessions, but did not change significantly in the Time-Control group (0.321 0.013 vs. 0.321 0.013, P = NS). 86

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HbA1c did not change in the EECP (6.63 0.29% vs. 6.59 0.26%, P = NS) or TimeControl (6.50 0.41% vs. 6.53 0.37%, P = NS) groups. Dynamic Indicies of Glucose Tolerance Deri ved from the Oral Glucose Tolerance Test after EECP or Time-Control Plasma glucose concentrations 120 minut es after initiation of oral glucose tolerance testing (PPG 120 ), the oral glucose insulin se nsitivity index (OGIS), and the composite whole-body insulin se nsitivity index (ISI) at baseline and after EECP or TimeControl are presented in Table 4-11 and figu res 4-29, 4-30, and 4-31. There was a significant decrease in PPG 120 (224.4 24.6 mg/dL vs. 196.1 24.7 mg/dL, P < 0.05) in the EECP group following 35 sessi ons, but did not change significantly in the TimeControl group (246.0 34.8 mg/dL vs. 249 34.9 mg/L, P = NS). The OGIS significantly increased (271.9 10.5 mLmin -1 m -2 vs. 311.5 12.3 mLmin -1 m -2 P < 0.05) in the EECP group following 35 sessions, but did not change significantly in the Time-Control group (287.4 14.8 mLmin -1 m -2 vs. 282.4 17.4 mLmin -1 m -2 P = NS). Moreover, The ISI (composite) significantly increased (2.77 0.40 vs. 3.36 0.42, P < 0.05) in the EECP group following 35 sessions, but did not change significantly in the Time-Control group (2.69 0.57 vs. 2.50 0.59, P = NS). Western Blot Analysis of Protein Expression in Vastus Lateralis Skeletal Muscle Biopsy Homogenate after EECP or Time-Control Western blot analysis of p-protein kinase B (Akt) 1/2/3 /Akt 1/2 p-5-adenosine monophosphate-activated protein kinase (AMPK) 2 /AMPK p-TBC1 domain family member 4 (TBC1D4)/TBC1D4, glucose transpor ter-4 (GLUT-4), endothelial nitric oxide synthase (eNOS), neuronal nitric oxid e synthase (nNOS), manganese superoxide dismutase (MnSOD), copper-zinc superoxid e dismutase (CuZnSOD), glutathione peroxidase (GPx), and 4-hydroxynonenal (4-HNE) expression in vastus lateralis skeletal 87

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muscle biopsy homogenate are presented in Tabl e 4-12 and Figures 4-32, 4-33, 4-34, 4-35, 4-36, 4-37, 4-38, 4-39, 4-40, and 4-41. To assess phosphorylation state of Akt, AMPK, and TBC1D4, phosphorylated protein is expressed relati ve to total protein as a ratio. All other protein expression was normalized to -actin loading control. p-Akt 1/2/3 /Akt 1/2 p-AMPK 2 /AMPK and p-TBC1D4/TBC1D4 did not change in either the EECP or Time-Control groups. There was a significant increase in expression of GLUT-4 in the EECP group (+47.24 13.92%, P < 0.05). There was no change in GLUT-4 expression in the Time-C ontrol group (+3.79 11.64%, P = NS). eNOS protein expression increased significantly in the EECP group (+87.32 20.30%, P < 0.01), but there was no change in the Time -Control group (+1.53 26.64%, P = NS). There was a trend for increased expression of nNOS in the EECP group (+27.39 11.12%, P < 0.10), however it did not reac h statistical significance. There was no change in nNOS expression in the Time-C ontrol group (-4.05 9.79%, P = NS). There was no change in MnSOD expression in either group. Ther e was a trend for increased expression of CuZnSOD in the EECP group (+33.37 11.89%, P < 0.10), and there was no change in CuZnSOD expression in the Time -Control group (-2.70 14.55%, P = NS). In addition, there was a trend for increased expression of GPx in the EECP group (+25.79 7.06%, P < 0.10), however it did not r each statistical significance There was no change in GPx expression in the Time-C ontrol group (+7.90 9.94%, P = NS). Finally, there was no change in 4-HNE, a marker of oxidative modification of pr oteins, in either group. 88

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Table 4-1. Baseline subject characteristics before enhanced external counterpulsation (EECP) or Time-Control EECP (n=12) Time-Control (n=6) P -value Age (years) 64.31 1.86 64.00 3.03 0.933 Body weight (kg) 97.20 4.94 99.22 7.46 0.666 Body height (cm) 178.7 19.8 179.1 18.5 0.893 Body mass index (kg/cm 2 ) 30.33 1.21 30.83 1.93 0.961 Body fat (%) 32.05 1.71 32.67 2.41 0.628 Trunk-to-limb fat mass ratio 1.61 0.07 1.71 0.16 0.678 Metformin therapy, no. (%) 3 (25) 1 (17) 0.709 Sulfonylurea therapy, no. (%) 2 (17) 1 (17) 0.999 Lipid-lowering therapy, no. (%) 7 (58) 3 (50) 0.755 Beta-blocker therapy, no. (%) 2 (17) 1 (17) 0.990 CCB therapy, no. (%) 3 (25) 1 (17) 0.709 ACE-I/ARB therapy, no. (%) 3 (25) 2 (33) 0.729 Diuretic therapy, no. (% ) 3 (25) 1 (17) 0.709 Values are mean SEM; CCB = calcium channel blocker; ACE-I = angiotensin converting enzyme inhibitor; ARB = angiotensin receptor blocker Table 4-2. Brachial artery flow-mediated dilation (FMD) at baseline and after EECP or Time-Control EECP (n=12) Time-Control (n=6) Baseline Final Baseline Final Baseline diameter (mm) 4.49 0.15 4.55 0.13 4.33 0.20 4.27 0.17 Absolute dilation (mm) 0.13 0.01 0.18 0.02* 0.14 0.01 0.13 0.01 Brachial FMD (%) 2.90 0.31 4.01 0.59* 3.22 0.34 3.16 0.22 Normalized FMD (s -1 ) 0.15 0.02 0.19 0.02* 0.16 0.03 0.15 0.02 Values are mean SEM; P < 0.05 vs. baseline within-gr oup. FMD = flow-mediated dilation Table 4-3. Popliteal artery flow-mediated dilation (FMD) at baseline and after EECP or Time-Control EECP (n=12) Time-Control (n=6) Baseline Final Baseline Final Baseline diameter (mm) 6.28 0.39 6.25 0.42 6.49 0.30 6.39 0.24 Absolute dilation (mm) 0.09 0.01 0.15 0.02 0.12 0.01 0.13 0.01 Popliteal FMD (%) 1.50 0.15 2. 43 0.24 1.73 0.12 1.92 0.14 Normalized FMD (s -1 ) 0.23 0.03 0.35 0.04* 0.26 0.05 0.26 0.05 Values are mean SEM; P < 0.05 vs. baseline within-gr oup. FMD = flow-mediated dilation 89

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Table 4-4. Forearm venous occlusion plethysmography paramet ers at baseline and after EECP or Time-Control EECP (n=12) Time-Control (n=6) Baseline Final Baseline Final Resting FBF 1.90 0.15 1.86 0.12 2.25 0.19 2.12 0.26 Peak FBF 15.68 1.47 19.71 1. 56* 16.49 0.94 16.86 1.12 Total FBF AUC 3min 5.04 0.63 6.91 0.74* 5.53 0.88 5.63 0.96 Values are mean SEM; P < 0.05 vs. baseline within-group. Units are mL/min/100mL tissue; FBF = forearm blood fl ow; AUC = area under flow time curve Table 4-5. Calf venous occlusion pleth ysmography parameters at baseline and after EECP or Time-Control EECP (n=12) Time-Control (n=6) Baseline Final Baseline Final Resting CBF 1.65 0.19 1.61 0.17 1.84 0.36 1.53 0.29 Peak CBF 16.36 1.77 18.03 1.50 16.81 3.64 14.50 2.66 Total CBF AUC 3min 4.35 0.62 4.94 0.59 4.91 1.60 3.92 0.94 Values are mean SEM; P < 0.05 vs. baseline within-group. Units are mL/min/100mL tissue; CBF = calf blood flow ; AUC = area under flow time curve Table 4-6. Vascular endothelial growth factor (VEGF) and capillary density parameters at baseline and after EECP or Time-Control EECP (n=12) Time-Control (n=6) Baseline Final Baseline Final VEGF (pg/mL) 9. 74 1.59 17.04 2.65* 9.94 2.24 8.99 3.75 EECP (n=8) Time-Control (n=4) Baseline Final Baseline Final Fiber area (m 2 ) 4883 514 5390 544 4669 727 4786 769 CD (capillaries/mm 2 ) 341.5 20.7 360.1 21.5 329.0 29.2 336.5 30.4 C/F (ratio) 1.71 0.11 1.84 0.11* 1.65 0.15 1.63 0.16 Values are mean SEM; P < 0.05 vs. baseline within-group. VEGF = vascular endothelial growth factor; CD = capillary density; C/F = capillaries per fiber Table 4-7. Vasoactive balance at baseline and after EECP or Time-Control EECP (n=12) Time-Control (n=6) Baseline Final Baseline Final NOx (mol/L) 26.47 1.58 34.46 1.78* # 26.40 2.34 26.87 2.63 6-keto-PGF 1 (pg/mL) 112.1 15.3 163.4 11. 7* 130.3 21.6 126.3 16.6 ET-1 (pg/mL) 2.54 0.34 2.28 0.32 2.66 0.52 2.62 0.50 NOx/ET-1 (A.U.) 8.89 1.40 12. 58 2.05 10.39 1.87 8.86 2.75 Values are mean SEM; P < 0.05 vs. baseline within-group; # P < 0.05 vs. TimeControl at same time-point. NOx = nitrite/nitrate; ET-1 = endothelin-1; 6-keto-PGF 1 = 6-keto prostaglandin F 1 90

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Table 4-8. Lipid peroxidation, antioxidant capacity, and endogenous nitric oxide (NO) inhibition at baseline and after EECP or Time-Control EECP (n=12) Time-Control (n=6) Baseline Final Baseline Final 8-iso-PGF 2 (pg/mL) 838.2 61.2 644.0 70.7* 749.9 86.6 811.9 99.9 ORAC (M trolox) 462.2 1.66 465.7 1.19 463.3 2.35 462.8 1.69 ADMA (mol/L) 0. 47 0.01 0.42 0.01* # 0.45 0.02 0.46 0.02 Values are mean SEM; P < 0.05 vs. baseline within-group; # P < 0.05 vs. TimeControl at same time-point. PGF 2 = prostaglandin F 2 isoprostanes; ORAC = oxygen radical absorbance capacity; ADMA = asymmetric dimethylarginine Table 4-9. Biomarkers of inflammation at baseline and after EECP or Time-Control EECP (n=12) Time-Control (n=6) Baseline Final Baseline Final hsCRP (mg/L) 2.67 0.38 1.91 0.33* 2.62 0.51 2.52 0.44 TNF(pg/mL) 4.10 0.33 3.84 0.36 4.11 0.46 4.15 0.51 Values are mean SEM; P < 0.05 vs. baseline within-group. hsCRP = high sensitivity C-reactive protein; TNF= tumor necrosis factor; IL-6 = interleukin-6 Table 4-10. Fasting markers of glycemic c ontrol at baseline and after EECP or TimeControl EECP (n=12) Time-Control (n=6) Baseline Final Baseline Final FPG (mg/dL) 143.9 8.5 127.0 6.6* 138.3 12.0 140.2 9.3 FPI (IU/mL) 10.52 1.45 8.72 1.21 9.71 1.96 9.87 1.63 HOMA IR (A.U.) 3.02 0. 55 2.08 0.39* 3.25 .78 3.38 0.56 QUICKI (A.U.) 0.322 0.009 0.335 0.009* 0.321 0.013 0.321 0.013 HbA1c (%) 6.63 0.29 6.59 0.26 6.50 0.41 6.53 0.37 Values are mean SEM; P < 0.05 vs. baseline within-gr oup. FPG = fasting plasma glucose; FPI =fasting plasma insulin; HOMA IR = homeostatic model assessment insulin resistance; QUICKI = quantitative in sulin sensitivity check index; HbA1c = glycosylated hemoglobin Table 4-11. Dynamic indices of glucose to lerance at baseline and after EECP or TimeControl EECP (n=12) Time-Control (n=6) Baseline Final Baseline Final PPG 120 (mg/dL) 224.4 24.6 196.1 24. 7* 246.0 34.8 249.4 34.9 OGIS 120 271.9 10.5 311.5 12.3* 287.4 14.8 282.4 17.4 ISI (composite) 2.77 0.40 3.36 0.42* 2.69 0.57 2.50 0.59 Values are mean SEM; P < 0.05 vs. baseline within-group. PPG 120 = post-prandial glucose at 120 minutes; OGIS 120 = oral glucose insulin sens itivity index (Mari et al. index), 65 measured in mLmin -1 m -2 ; ISI (composite) = composite whole-body insulin sensitivity index (Ma tsuda et al. index), 58 measured in A.U. 91

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Table 4-12. Percent change in protein expre ssion from vastus lateralis skeletal muscle biopsy homogenate after EECP or Time-Control EECP (n=12) Time-Control (n=6) p-Akt 1/2/3 /Akt 1/2 (%) -4.41 12. 14 2.42 1.80 p-AMPK 2 /AMPK (%) -4.40 7.38 0.17 2.50 p-TBC1D4/TBC1D4 (%) 2.81 12.40 5.38 7.34 GLUT-4/ -actin (%) 47.24 13. 92* 3.79 11.64 eNOS/ -actin (%) 87.32 20.30* 1.53 26.64 nNOS/ -actin (%) 27.39 11.12 -4.05 9.79 MnSOD/ -actin (%) 9.59 12.37 19.96 13.33 CuZnSOD/ -actin (%) 33.37 11.89 -2.70 14.55 GPx/ -actin (%) 25.79 7.06 -4.08 11.05 4-HNE/ -actin (%) -12.29 4. 76 7.90 9.94 Values are mean SEM; P < 0.05 vs. Time-Control group; P < 0.10 vs. TimeControl group. Akt = protein kinase B; AMPK = 5-adenosine monophosphateactivated protein kinase alpha; TBC1D4 = TB C1 domain family member 4; GLUT-4 = glucose transporter 4; eNOS = endothelial nitric oxide synthase; nNOS = neuronal nitric oxide synthase; MnSOD (SOD -2) = manganese superoxide dismutase; CuZnSOD (SOD-1) = copper-zinc superoxid e dismutase; GPx = selenium-dependent cellular glutathione peroxidase Figure 4-1. Brachial artery flow-mediated di lation (FMD) at baseli ne and after enhanced external counterpulsation (EECP) or Time-Control. Data are mean SEM. P < 0.05 vs. baseline within-groups. 92

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Figure 4-2. Brachial artery absolute diameter dilation at baseli ne and after EECP or Time-Control. Data are mean SEM. P < 0.05 vs. baseline within-groups. Figure 4-3. Normalized brachial artery flow-mediated dilation (FMD) at baseline and after EECP or Time-Control. Data are mean SEM. P < 0.05 vs. baseline within-groups. 93

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Figure 4-4. Popliteal artery flow-mediated di lation (FMD) at baseline and after EECP or Time-Control. Data are mean SEM. Figure 4-5. Popliteal artery absolute diameter dilation at baseline and after EECP or Time-Control. Data are mean SEM. 94

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Figure 4-6. Normalized poplitea l artery flow-mediated dilation (FMD) at baseline and after EECP or Time-Control. Data are mean SEM. P < 0.05 vs. baseline within-groups. Figure 4-7. Resting forearm blood flow (FBF) at baseline and after EECP or TimeControl. Data are mean SEM. 95

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Figure 4-8. Peak forearm blood flow (FBF) at baseline and after EECP or Time-Control. Data are mean SEM. P < 0.05 vs. baseline within-groups. Figure 4-9. Total area under curve (AUC) forearm blood flow (FBF) at baseline and after EECP or Time-Control. Data are mean SEM. P < 0.05 vs. baseline withingroups. 96

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Figure 4-10. Resting calf blood flow (CBF) at baseline and after EECP or Time-Control. Data are mean SEM. Figure 4-11. Peak calf blood flow (CBF) at baseline and after EECP or Time-Control. Data are mean SEM. 97

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Figure 4-12. Total area under curve (AUC) calf blood flow (CBF) at baseline and after EECP or Time-Control. Data are mean SEM. Figure 4-13. Vascular endothelial growth factor (VEGF) at baseline and after EECP or Time-Control. Data are mean SEM. P < 0.05 vs. baseline within-groups. # P < 0.05 vs. Time-Control gr oup at same time-point. 98

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Figure 4-14. Capillary density (CD) of human vastus lateralis biopsy samples at baseline and after EECP or Time-C ontrol. Data are mean SEM. Figure 4-15. Capillary per fiber ratio of human vastus lateralis biopsy samples at baseline and after EECP or Time -Control. Data are mean SEM. P < 0.05 vs. baseline within-groups. 99

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Figure 4-16. Representative photomicrograph of skeletal muscle morphology. Vastus lateralis skeletal muscle biopsie s were obtained and histomounts were stained using an amylase-periodic acid Sc hiff histochemical stain. Fibers and capillaries run perpendicular to the page. The insert bar represents 50 m. Values of representative photomic rograph: Mean fiber area = 4825 m 2 ; Capillary Density (CD) = 396 capillaries/mm 2 ; Capillary to Fiber Ratio (C/F) = 1.79. Figure 4-17. Nitrite/Nitrate (N Ox) at baseline and after EECP or Time-Control. Data are mean SEM. P < 0.05 vs. baseline within-groups. # P < 0.05 vs. TimeControl group at same time-point. 100

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Figure 4-18. Six (6)-keto prostaglandin F1 (6-keto-PGF 1 ) at baseline and after EECP or Time-Control. Data are mean SEM. P < 0.05 vs. baseline within-groups. Figure 4-19. Endothelin-1 (ET-1) at baseline and after EECP or Time-Control. Data are mean SEM. 101

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Figure 4-20. Eight (8) iso-prostaglandin-F 2 (8-iso-PGF 2 ) at baseline and after EECP or Time-Control. Data are mean SEM. P < 0.05 vs. baseline within-groups. Figure 4-21. Asymmetric dime thylarginine (ADMA) at ba seline and after EECP or TimeControl. Data are mean SEM. P < 0.05 vs. baseline within-groups. # P < 0.05 vs. Time-Control group at same time-point. 102

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Figure 4-22. High sensitivity C-reactive prot ein (hsCRP) at baseline and after EECP or Time-Control. Data are mean SEM. P < 0.05 vs. baseline within-groups. Figure 4-23. Tumor necrosis factor(TNF) at baseline and after EECP or TimeControl. Data are mean SEM. 103

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Figure 4-24. Fasting plasma glucose (FPG ) at baseline and after EECP or TimeControl. Data are mean SEM. P < 0.05 vs. baseline within-groups. Figure 4-25. Fasting plasma insulin (FPI) at baseline and after EECP or Time-Control. Data are mean SEM. 104

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Figure 4-26. Homeostatic model asse ssment of insulin resistance (HOMA IR ) at baseline and after EECP or Time-Control. Data are mean SEM. P < 0.05 vs. baseline within-groups. Figure 4-27. Quantitative insuli n sensitivity check index (QUI CKI) at baseline and after EECP or Time-Control. Data are mean SEM. P < 0.05 vs. baseline withingroups. 105

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Figure 4-28. Glycosylated hemoglobin (HbA1c ) at baseline and after EECP or TimeControl. Data are mean SEM. Figure 4-29. Plasma glucose at 120 minutes a fter initiation of oral glucose tolerance testing (PPG 120 ) at baseline and after EECP or Time-Control. Data are mean SEM. P < 0.05 vs. baseline within-groups. 106

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Figure 4-30. Oral glucose in sulin sensitivity index (OGIS 120 ) at baseline and after EECP or Time-Control. Data are mean SEM. P < 0.05 vs. baseline within-groups. Figure 4-31. Composite whole-bo dy insulin sensitivity index (ISI) at baseline and after EECP or Time-Control. Data are mean SEM. P < 0.05 vs. baseline withingroups. 107

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Figure 4-32. Percent change in phosphorylation state of protein kinase B (p-Akt/Akt) from baseline after EECP and Time-Contro l. Data are mean percent change SEM. Figure 4-33. Percent change in phosphorylat ion state of 5-adenosine monophosphateactivated protein kinase (p-AMPK /AMPK ) from baseline after EECP and Time-Control. Data are mean percent change SEM. 108

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Figure 4-34. Percent change in phosphorylation state of TBC1 domain family member 4 (p-TBC1D4/TBC1D4) from baseline a fter EECP and Time-Control. Data are mean percent change SEM. Figure 4-35. Percent change in glucose transpor ter-4 (GLUT-4) prot ein expression from baseline after EECP and Time-Control. Representative bands from western blot analysis picture. Data are mean percent change SEM. P < 0.05 vs. Time-Control group. T1 = baseline time -point; T2 = post time-point; CTL = Time-Control group. 109

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Figure 4-36. Percent change in endothelial nitric oxide synthase (eNOS) protein expression from baseline after EECP and Time-Control. Representative bands from western blot analysis pict ured. Presence of band in standard (STD) lane act as positive control for identification of eNOS. Data are mean percent change SEM. P < 0.05 vs. Time-Control group. MWM = molecular weight marker; T1 = baseline time-poi nt; T2 = post time-point; CTL = TimeControl group. 110

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Figure 4-37. Percent change in neuronal ni tric oxide synthase (nNOS) protein expression from baseline after EECP and Time-Control. Representative bands from western blot analysis pict ured. Data are mean percent change SEM. P < 0.10 vs. Time-Control group. T1 = baseline time-point; T2 = post time-point; CTL = Time-Control group. Figure 4-38. Percent change in manganese s uperoxide dismutase (MnSOD) protein expression from baseline after EECP and Time-Control. Data are mean percent change SEM. 111

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Figure 4-39. Percent change in copper-zinc superoxide dismutase (CuZnSOD) protein expression from baseline after EECP and Time-Control. Representative bands from western blot analysis pict ured. Data are mean percent change SEM. P < 0.10 vs. Time-Control group. T1 = baseline time-point; T2 = post time-point; CTL = Time-Control group. 112

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Figure 4-40. Percent change in seleniu m-dependent cellular glut athione peroxidase (GPx) protein expression from base line after EECP and Time-Control. Representative bands from western bl ot analysis pictured. Data are mean percent change SEM. P < 0.10 vs. Time-Control group. T1 = baseline timepoint; T2 = post time-poin t; CTL = Time-Control group. Figure 4-41. Percent change in 4-hydroxyno nenal (4-HNE) adducts from baseline after EECP and Time-Control. Data are mean percent change SEM. 113

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CHAPTER 5 DISCUSSION To our knowledge, this is the first st udy to evaluate the effects of enhanced external counterpulsation (EEC P) on arterial function, fa sting measures of glycemic control, dynamic measures of glucose tolerance, capillary density, and skeletal muscle protein expression in patients with abnormal glucose tolerance (AGT). The main findings of this study are that, in patient s with AGT: 1) EECP t herapy elicits similar changes in arterial function to those obser ved in coronary artery disease (CAD) patients; 2); increases the c apillary to fiber ratio in skeletal muscle 3) increases endothelial nitric oxide synt hase (eNOS) protein expres sion; 4)increases fasting measures of glycemic control; and 5) increases dynamic measures of glucose tolerance. Peripheral Conduit Artery E ndothelial Function and EECP The present study demonstrated that 35-sessions of EECP in patients with AGT improved brachial artery flow mediated dila tion (FMD) 38% from baseline and popliteal artery FMD 62% from baseline. Similarly, brachial and poplitea l artery normalized FMD increased 27% and 52%, respectively from basel ine. This is in agreement with EECP previous studies in other patient groups. Br aith et al. have previously demonstrated in a sham controlled study of CAD patients that brachial and femoral artery FMD increases 51% and 30% respectively. 27 The distal occlusion method of assessing FMD of the peripheral conduit arteries, as used in the present study, has been shown to be a valid and reliable surrogate of nitric oxide (NO)-mediated endothelial function. 158 Although non-normalized popliteal artery FMD wa s not significant between groups, the normalized FMD response was significantly different (0.23 0.03 vs. 0.35 0.04, P < 114

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0.05). The disparity in the two measures is likely due to the small sample size recruited for the present study. Absolute changes in popliteal artery FMD are similar to those observed in the brachial artery despite larg er baseline diameters and lesser increases in hyperemic shear stress responses. Hyperemic shear stimulus is greater in small arteries due to the dependence of post-ischemic flow on radius squared. 159 However, when normalized to the shear stimulus, the popl iteal artery exhibits an FMD response at least equal to that of the brachial artery. 160, 161 Therefore, without significant changes within or between groups in the post-ischemic shear stimulus, as observed in this study, the power to detect significant changes in normalized popliteal artery FMD is greater due to an increase in the magnitude of change and a decrease in variability. The mechanism responsible for the observed changes in peripheral artery FMD is likely blood flow shear stress. During a bout of EECP therapy, t he sequential inflation (~300 mm Hg) of the 3 pneumatic EECP cuff s from calves to buttocks produces a robust retrograde pressure wave in the femoral arteries and simultaneous moderatepressure antegrade flow in the brachial arteries. 26, 46 In a porcine EECP model brachial artery blood flow was velocity was shown to increase by 132% and brachial artery wall shear stress increased by >200% 26 during lower body compression of pneumatic EECP cuffs. It is not unreasonable to conclude that similar alterati on of blood flow velocity and shear stress is realized throughout the entire ar terial tree, including the popliteal artery. Consideration should also be given to the compression and relaxation of the pneumatic cuffs that occur repeatedly throughout a 60 minute session of EECP therapy. For example, in a patient with a heart rate of 60 bpm there are approximately 3600 cycles of compression and relaxation inducing systolic reactive hyperemia in the legs. 115

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In summary, EECP increases upper and lower body peripheral arterial function, as measured by FMD, following 35-sessions in patients with AGT. The FMD technique is a non-invasive bioassay of NO bioavailability. Therefore, the robus t changes in arterial blood flow and shear stress during EECP therapy may mediate changes in FMD response due to an increase in NO producti on and/or a decrease in NO degradation. Indeed, in the present study, markers of oxidative stress were depressed, markers of NO plasma concentrations were improved, and inhibitors of NO were depressed. These alterations in NO bioavailability ar e discussed in greater detail later in this chapter. Peripheral Resistance Artery Endothelial Function and EECP Our results are similar to previous studies that have demonstrated improvements in peak forearm blood flow (FBF) and calf blood flow (CBF) with reactive hyperemia. 34 We observed a 26% increase in peak FBF and a 10% increase in peak CBF following 35-sessions of EECP in subjects with AGT. This increase in the maximal dilatory capacity in response to reactive hyperemia provides support for our hypothesis that the repetitive inflation and deflation cycles of cuff compression, and resulting alterations in blood flow velocity and shear stress during EECP therapy, improves endothelial function in the peripheral resistance arteries. Increased post-occlusion blood flow suggests increased capillary or arteriolar proliferation, or improved re sistance vessel endothelial functi on. In the present study, the peripheral conduit artery FMD results support the potential for an improvement in endothelial function. EECP has been shown to acutely increase blood flow in various vascular beds which translates to enhanced shear stress, a key factor in endothelial function. 44 A major contributor to the maximal vasodilatory capacity of the peripheral 116

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resistance arteries is nitric ox ide bioavailability. As discu ssed in the previous section, EECP manifested changes indicative of increased potential for NO production and decreased inhibition of its production. However, the potential effects of angiogenesis/vasculogenesis after EECP and t he maximal vasodilatory response must be considered, as they cannot be directly measured using the non-invasive venous occlusion plethysmography (VOP) technique employed in the present study Angiogeneisis/Vasculogenesis and EECP Plasma levels of vascular endothelial growth factor (VEGF) were elevated to 175% of baseline levels following 35-sessions of EECP in subjects with AGT. VEGF is a potent mediator of both angiogenesis and vasculogenesis that is released in response to hypoxia. In this model of EECP, it is plausible that several thousand brief, intermittent bouts of hypoxia are induced by the high pressure compression cuffs. During a single 60 minute session of EECP ther apy in a subject with an average heart rate of 60 bpm, approximately 3600 high pr essure compressions are realized. VEGF can also be produced in response to circulating cytokines which may be acutely elevated during a bout of EECP, si milar to an exercise response. 162 Although VEGF is not the only promoter of angiogenesis, basic fibroblast growth factor (FGF2) and hepatocyte growth factor (HGF) have also be en implicated, the near two-fold increase in VEGF observed following EECP supports a role for angiogenesis/vasculogenesis in the adaptive mechanisms to EECP therapy. Shear stress has also been proposed as a major physiological signal in the stimulation of capillary growth. 163 Hoier et al. have demonst rated that two weeks of passive leg movement training, with negligible vastus lateralis electromyography (EMG) activity, induced significant increases in interstitial VEGF, endothelial nitric oxide 117

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synthase (eNOS) messenger ribonucleic acid (mRNA), and capillary to fiber ratios. 164 In that study, blood flow was increased to about 3-times baseline values during training, similar to changes observed with EECP. 26 In the present study, we observed a significant, 8% increase in the number of capi llaries per muscle fiber in biopsy sections of the vastus lateralis following 35-sessions of EECP. Although not statistically significant, there was also a trend for increased capillarity density following EECP therapy. The lack of statistical significance may be due to variation fiber area, an effect termed capillary dilution. In the present study, a non-significant increase in mean fiber area was observed, and the analysis of samples using a ratio of capillaries to fibers likely decreased the variability observed in capillary density measures. Muscle analysis was performed on one sm all muscle sample from only one skeletal muscle. Although one can speculate on gl obal changes in capillarity, only a site under direct compression was analyzed. However, increased eNOS expression has been demonstrated in tissue upstream from intermittent mechanical compression in rats. 25 Variation due to the fiber type distri bution amongst the sa mples analyzed could also present an additional source of variation, and in the present study, capillarity was not normalized to fiber type. Despite these li mitations, efforts were made to achieve the least heterogeneity amongst samples as biopsy sections were taken within 1-2 cm of the baseline site and at the same recorded dept h for each procedure. Furthermore, an average of 166 13 fibers were analyzed per samp le by selection of sections of tissue with little artifact. Vasoactive Balance and EECP The present study demonstrated that EEC P resulted in a 30% increase in plasma nitrite and nitrate (NOx) levels. Braith et al. have previously demonstrated that plasma 118

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NOx concentrations are elevated 36% fo llowing EECP therapy in CAD patients. 27 Although the increase observed in patients with AGT followin g 35-sessions of EECP is slightly less in magnitude, this is likely due to greater baseline plasma NOx concentrations in patients with AGT, but without CAD. Increas es in NOx as a result of EECP therapy have been reported in both the short and the long term. Akhtar et al. have shown that NOx is incr eased in the first hour following a single bout of EECP and increases progressively as the course of treatment progresses. 29 Importantly, the elevated plasma levels are maintained for at least one month follo wing the culmination of 35-sessions of EECP. 28 The increase in plasma NOx observed in the present study could be due, in part, to an increase in eNOS and/or neuronal ni tric oxide synthase (nNOS) protein expression. We observed a highly significant ( P < 0.01) 87% increase in eNOS protein expression from baseline in vastus lateralis skeletal muscle biopsy homogenate following 35-sessions of EECP therapy. In addition, there was a trend ( P = 0.067) for increased nNOS protein expression as pr otein concentration increased 27% from baseline after 35-sessions of EECP. The increase in nitric oxide synthase (NOS) protein expression with compressive therapy is supported by the findings of Tan et al. who demonstrated that following a singl e 1 hour bout of intermittent pneumatic compression (IPC) on the hind limbs of Sp rague Dawley rats, eNOS and nNOS protein expression was upregulat ed 120% and 30% from baseline, respectively. 25 Importantly, increases in NOS expression were also obs erved in the upstream cremaster muscles flowing 1-hour of intermi ttent pneumatic compression. 25 119

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NOx is influenced by exogenous source s such as food, saliva formation, gastrointestinal microorganism s, and cigarette smoking and is only a gross index of endothelial NO formation in vivo. 165 However, since all of the subjects in the present study were non-smokers and followed a strict National Institutes of Health (NIH) low nitrate diet guideline fo r 48 hours prior to their lab visits we are confident that plasma NOx levels were influenced minimally by exogenous sources. Therefore, the improvements in plasma levels of NOx with concomitant increases in eNOS and nNOS protein expression following 35-sessions of EECP therapy likely reflect an improvement in basal endothelial NO production. Further evidence for an increase in eNOS protein expression and/or activity is demonstrated by the 46% increase in 6-keto-prostaglanin 1 (6-keto-PGF 1 ), the stable metabolite of prostacyclin (PGI 2 ), following 35-sessions of EECP therapy. Prostaglandins are a family of eicosanoi ds derived from endothelial cells and shear stress has been shown to be a major stimul us for prostacyclin production from endothelial cells. 133 These results are similar to those previously described by Braith et al. who showed a 71% increase in 6-keto-PGF 1 following 35-sessions of EECP in patients with CAD. 27 Although previous studies have demonstrated a decreas e in endothelin-1 (ET-1) with EECP therapy, 27 we did not observe a signific ant decrease in ET-1 after 35sessions of EECP in patients with AGT. This could be due to lower basal production of ET-1 at baseline compared to the populations ex amined in prior studies. Also, the value of ET-1 measures in human plasma is controve rsial. ET-1 acts in a paracrine fashion 120

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on nearby smooth muscle cells on the adventit ia side of the vessel. Therefore, the relevance of circulating ET-1 measur ed in the plasma remains in question. Redox Balance and EECP The present study demonstrated that EECP resulted in a 23% decrease in the F2-isoprostane 8-iso-prostaglandin 2 (8-iso-PGF 2 ) following 35-sessions of EECP in subjects with AGT. This reduction is sim ilar to that observed in CAD patients following EECP therapy, 27 although the baseline plasma concentrations of 8-iso-PGF 2 observed in the present study were lesser in magnitude. 8-iso-PGF 2 a prostaglandin like compound that is produced by free-radical mediated lipid peroxidation of arachadonic acid, has been suggested to be the most valid pl asma marker to asse ss oxidative stress in human plasma. 166-168 Increased oxidative stress is an establis hed contributor to the development and progression of diabetes. 95 Both type I and type II diabetes are associated with increased production of free r adicals and/or impaired antio xidant defenses, shifting redox balance toward greater oxidative stress. 96, 97 Hyperglycemia has also been shown to promote free radical generation through glucose autotoxidation 98 and lipid peroxidation of low density lipoprotein (L DL) in a superoxide-dependent pathway. 99 Free radical production due to hyperglycemia can also occur as glucose interacts with proteins ultimately resulting in the fo rmation of advanced glycation endproducts (AGEs). These AGEs can promote free radical form ation and quench and blo ck anti-proliferative effects of nitric oxide through their binding with specific AG Es receptors. Davy et al. have shown that glycemic control correlated well with, 8-iso-PGF 2 100 Oxidative stress may play an important role in not only vascu lar function, but also glycemic control as bioavailability of NO may mediate/alter the NO-mediated glucose uptake pathway. 121

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Evidence suggests that asymmetric dimeth ylarginine (ADMA) is associated with endothelial dysfunction in a number of disorders, incl uding, but not limited to, dyslipidemia, hyperhomocysteinemia, hyper tension, CAD, heart failure, renal dysfunction, and Type II Diabetes Mellitus (T2DM). 112, 113 This is the first study to examine the effects of EECP on circulating le vels of ADMA in patients with AGT. We observed a 26% reduction in plasma ADMA concentrations following 35sessions of EECP. This finding is similar to what has been reported following EECP therapy in CAD patients 27 and in Type I Diabetics following exercise training. 169 ADMA, like NO, is derived from the amino acid Larginine. The production of ADMA from L-arginine can decrease NO production due to substrate utiliz ation. L-arginine concentration may be even more important for glucose clearance as McConnel et al. have shown that Larginine infusion during cycling exercise increases glucose uptake in a NO dependent manner. 114 Since type II diabetics are even more reliant on nitric oxide mediated glucose uptake, 20, 23 this may be of significant physiological relevance. Following formation, free ADMA is released into the plasma where it can inhibit NOS and decrease NO bioavailability leadi ng to the development of endothelial dysfunction. 117 Flow mediated dilation, the non-inva sive bioassay of NO bioavailability, also demonstrates a negative linear rela tionship with ADMA concentrations. 116 Furthermore, inhibition of NOS impairs mi crovascular recruitment and blunts insulin stimulated glucose uptake. 118 Steinberg et al. have shown that insulin resistance was associated with blunted endothelium-dependent vasodilatation, but not endotheliumindependent vasodilatation, durin g intrafemoral artery infusi ons of sodium nitroprusside and metacholine chloride under euglyce mic hyperinsulinem ic conditions. 119 122

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Oxidative stress appears to be a key modulator of ADMA levels. Oxidative stress is associated with an increase in protein-argi nine-N-methyltrasferase family of enzymes (PRMT) activity and gene expression in t he cell leading to an increase in ADMA formation. 116, 122-124 Tetrahydrabiopterin (BH 4 ), a cofactor for NOS, and dimethylargininase (DDAH), the major elimi nator of ADMA, are also redox sensitive. 113, 115, 123, 125-127 Although BH 4 and DDAH were not measured in the present study, compelling data from the decrease in 8-iso-PGF 2 following 35-sessions of EECP therapy in subjects with AGT suggest a decrease in oxidative stress and subsequently, possibly decreased oxidation of BH 4 and DDAH. Osanai et al. showed that ADMA levels in human endothelial cells in vitro decreased with15 dynes/cm 2 of shear stress and DDAH activity increased at shear stress levels of > 25 dynes/cm 2 129 It is not unreasonable to conclude that DDAH activity may be increased with the magnitude of shear stress invoked with EECP (>200% of baseline, 49 vs. 23 dynes/cm 2 in a porcine model). 26 We also observed a trend ( P < 0.10) for increased expression of endogenous antioxidant capacity with increases in copper-z inc superoxide dismutase (CuZnSOD) and glutathione peroxidase (G Px). The most common antioxidant deficiencies reported in diabetics are lower levels of vitamin C, GPx and SOD. 98 While the role of antioxid ant defense in the treatment and pathology of glycemic control remains controversial, treatment of type T2DM patients with vitamin C or vitamin E decreased glycocelated hemoglobin (HbA1c) le vels, improved insulin action, decreased plasma insulin concentrations, and decr eased indicators of oxidative stress. 103 Inflammation and EECP Epidemiological evidence for an a ssociation between Type II diabetes and inflammation goes back as far as the 1950s. Increased levels of markers and mediators 123

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of inflammation such as high sensitivity C -reactive protein (hsCRP) and tumor necrosis factor(TNF) correlate well with incident T2DM. 130 In addition, TNFhas been shown to cause insulin resist ance in experimental models. 131 In the present study, following EECP therapy in patients with AGT, significant reductions in plasma levels of hsCRP (28%) were observed. These results are similar to those observed by Braith and colleagues following 35-sessions of EECP in patients with CAD. 27 Although previous studies of EECP in patients with CAD have demonstr ated decreases in plasma TNF, no significant change was detected in the present study (-6%). Although baseline TNFvalues were considerably less than this comparable study, 27 the concentrations observed in the present study were approximated as the 90 th percentile of healthy control values. 170 Increased bioavailability of NO after EECP therapy is the likely mechanism responsible for the reduction in plasma inflammato ry markers. NO serves an anti-inflammatory role by inhibi ting the expression of monocyte chemotactic protein-1 (MCP-1) and reducing vascu lar cell adhesion protein-1 (VCAM-1) expression. 132 Fasting Glycemic Control and EECP To the best of our knowledge, this is the first study to evaluate the changes in fasting glycemic control following EECP ther apy. In the present study, following 35sessions of EECP, subjects with AGT demonstrated marked improvements in fasting indices of glycemic control. Fasting pl asma glucose (FPG) values were decreased nearly 17 mg/dl following EECP (143.9 8.5 mg /dL vs. 127.0 6.6 mg /dL). This 13.3% decline in FPG is similar to that observed with resistance training intervention in older men with T2DM, 171 and aerobic exercise training ol der subjects with non-insulin dependent Diabetes Mellitus (NIDDM) and impaired glucose tolerance (IGT). 172 124

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Importantly, these changes occurred in the absence of any lifestyle modification and subject weight, body mass index (BMI), body fat percentage, and measures of central adiposity did not change following EECP therapy In a large scale population study, each 13 mg/dL increase in FPG is associated with a 13% increase in the relative risk for cardiovascular disease. 173 Furthermore, data from t he Framingham Heart Study has demonstrated that for every 10m g/dL increase in FPG there is an 18% increase in allcause mortality. 174 Although simple measures of fasting pl asma glucose provide an indication of glycemic control, the inclusion of fasting plas ma insulin concentrations allows for a more accurate measure of insulin sensitivity. We observed a 31% decline in homeostasis model assessment of insulin resistance (HOMA IR ) values and a significant increase in the quantitative insulin sensitivity check index (QUICKI). The HOMA IR and QUICKI both correlate well with the glucose disposal ra te derived from hyperinsulinemic euglycemic clamp (HEC), the gold standard for meas uring peripheral insulin sensitivity. 57, 58, 60 Among the criticisms of the HOMA IR are a large coefficient of variation and the pulsatile nature of insulin secretion. However, thr ee separate samples, five minutes apart, of plasma insulin concentration were m easured to determine a true average fasting plasma insulin concentration. The mat hematical difference between the QIUCKI and the HOMA IR is simply that the former uses the reciprocal of the logarithm of both glucose and insulin to account for the skewed distribution of fasting insulin values and some argue that the QUICKI may be applied to wider ranges of insulin sensitivity. 61, 62 However, in the present study, signific ant improvements in both measures were observed. 125

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Despite declines in fasting plasma glucos e and indices of fasting glycemic control, there was not a significant decline in HbA1c values following 35-sessions of EECP in subjects with AGT. Howeve r, the half life of a red blood cell is about 120 days and reflects the average blood glucose control level during the preceding two to three months. Therefore, with a seven week EE CP intervention, the study design may not have allowed us to capture this variable. Further studies should be undertaken to determine the efficacy of EECP therapy for glycemic control as measured with measures of HbA1c at multiple time-point s beyond the completion of the standard 35 1hour sessions of EECP therapy. Dynamic Measures of Glucose Tolerance and EECP Stronger surrogates of insulin sensitivity can be derived from the multiple sampling of plasma insulin and glucose during an oral glucose tolerance test (OGTT). Following 35-sessions of EECP we observed a 28 mg/ dL decrease (224.4 24.6 mg/dL vs. 196.1 24.7 mg/dL) in plasma glucose at 120 minutes (12.6% decline) following the ingestion of a 75 gram sugar water beverage (PPG 120 ). PPG 120 from the OGTT is frequently used by physicians as a test for T2 DM or IGT. In this contex t, the EECP group, on average, moved from a response that was consistent with T2DM to that observed in IGT. While we did observe a significant decline in PPG 120 the effect was modest when compared to the declines in PPG 120 following exercise interventions. 175-177 Although there are a plethora of indices based on the OGTT that can be used to estimate insulin sensitivity, for the present study, two were chosen; the oral glucose sensitivity index (OGIS 120 ) developed by Mari et al. 65 and the composite whole-body insulin sensitivity index (ISI composite) developed by Matsuda et al. 58 The OGIS 120 incorporates glucose load, weight, and body surface area while al so highlighting the 126

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change in plasma glucose during the final 30 minutes of the OGTT relative to insulin concentration, much like the hyperinsulinemic euglycemic clamp. The ISI (composite) is a more commonly used surrogate of insulin s ensitivity that incorporates fasting and mean plasma glucose and insulin concentrations prior to and during the OGTT. Both indices were chosen due to their high corre lation with the HEC (r = 0.73 for both) and because of their validation in a wide vari ety of lean and obese subjects with AGT. 58, 59, 65 In the present study, we observe d 15% and 21% increases in the OGIS 120 and ISI (composite) respectively. Given the strong correlation of these indices with the HEC, the results from the present study suggest improvements in dynamic measures of glycemic control. An inherent limitation to indices of insuli n sensitivity derived fr om the OGTT is that it is impossible to differentiate betw een whole-body, peripheral, or hepatic insulin sensitivity. However, since this method of glucose ingestion is the most physiologically relevant, improvement in indices of insulin sensitivity from the OGTT are encouraging signs of improved glucose tolerance in s ubjects with AGT following 35-sessions of EECP therapy. Potential Mechanisms for Improvement in Fasting Glycemic Control and Dynamic Indices of Glucose Tolerance: Evidence for the Nitric Oxide Pathway? In the present study, we did not obser ve any changes in the phosphorylation states of Akt, AMPK or TBC1D4. Therefore, c hanges in glucose homeostasis may occur independent of changes in skeletal mu scle signaling. Given the increasing evidence that NO bioavailability (and redox balance) contributes to glucose handling, the significant increases in NOx and significant decreases in 8-iso-PGF 2 and ADMA may provide evidence for the nitric oxide -mediated glucose handlin g. Indeed, fasting 127

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plasma NO concentrations are markedly declined with advancing glucose intolerance. 178 Sodium nitroprusside (SNP), a NO donor, significantly increases glucose uptake in the presence of wortmannin, a phosphoinositide 3-kinase (PI3K) inhibitor, indicating that it is not a PI3K dependent mechanism. 20 In addition, L-NG Nitroarginine methyl ester (L-NAME), a NOS inhibitor, administration does not abolish contraction mediated glucose uptake in skeletal muscle. 20 This suggests that the effects of NO are independent of both insulin and contract ion and appear to augment glucose uptake systemically. Although we hypothesized that NO mediat ed glucose uptake may be realized through alterations in AMPK signaling, severa l other mechanism exist by which NO may mediate glucose uptake. In resting muscle, NO stimulates glucose uptake through a cyclic guanosine monophosphate (cGMP)-depend ent pathway that may involve cyclic GMP-dependent kinase/protein kinase G (PKG) activation. 78, 79 NO can also directly activate G protein subunits, specifically the proto-oncogene p21ras for glucose transport, which increases glucos e uptake through nuclear factor B (NF B) signaling. 80 NF B activity is increased transiently from treatment with NO donors and reacts rapidly to increase the activity of the extracel lular signal-related kinases (ERK), c-Jun-NH 2 terminal kinase (JNK), and p38 subgroups of the mitogen-activated protein kinase (MAPK) family. 81 However, the exact mechanism by which the MAPK subfamily elicits glucose uptake has not been elucidated. In addition to the acute effects of p38MAPK on glucose uptake, p38MAPK may also play a role in the chronic glycemic control as it phosphorylates the transcription factor myoc yte enhancer factor-2 (MEF2), which is implicated in the expression of glucose trans porter-4 (GLUT-4) protein synthesis from 128

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the peroxisome proliferat or-activated receptorcoactivator 1 (PGC-1 ) gene in skeletal muscle. 82 Although we were unable to quantify PGC-1 expression, we did observe a 47% in GLUT-4 protein expression following 35-sessions of EECP in subjects with AGT. It is important to note that while we di d not observe any changes in the Akt, AMPK and TBC1D4 skeletal muscle cell signaling pathways, we were only able to evaluate one time-point. In the present study, mu scle biopsies were performed at 48-72 hours following the last session of EECP therapy in an effort to capture the chronic effect of 35-sessions of EECP therapy in subjects with AGT. Transient ch anges in signaling pathways could occur acutely with each bout of EECP, similar to the effects observed with a bout of exercise. Therefor e, it is possible that we were able to capture increases in total protein content, but not necessarily the signal(s) responsible for the increase in transcription and/or translation. Reduced nutrient exposure to the metabolizing tissue may al so play a major role in the continuum of AGT. Insulin receptors are present throughout the vascular tree and act as vasodilators to increase perfusion and delivery of substrates. The adaptations to 35-sessions of EECP included increases in both peripheral conduit and resistance artery function in patients wit h AGT. Furthermore, capillar y density, like nitric oxide bioavailability, decreases progressively with advancing glucose intolerance. 178 The changes observed in capillary to fiber rati o and endothelial function observed in the present study would suggest that improv ements in glucose homeostasis may be realized through increase in vascular reacti vity to insulin and nutrient delivery. 129

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Conclusions AGT is associated with several perturbations in normal human physiology, including, but not limited to, endothelial dysfun ction, oxidative stress, depressed nitric oxide bioavailability, progressive declines in capillary density, and inflammation. In the present study, we observed significant impr ovements in fasting measures of glycemic control and significant improvements in dynamic measures of glucose tolerance and insulin sensitivity. We also observed impr ovements in measures of endothelial function, nitric oxide bioavailability, and capillary to fiber ratios with concurrent decreases in measures of lipid peroxidatio n and competitive inhibition of NOS. This study provides novel evidence for the improvement of vascular function and glycemic control in subjects with AGT follo wing EECP therapy. The multifaceted nature of vascular functi on and glycemic control makes it difficult to isolate a single mechanism responsible for these adaptations. However, increasing evidence indicates that decreasing nitric oxide bioavailability contributes to the pathology of AGT and the progression to T2DM. 23, 77, 114, 178, 179 Moreover, it has been well characterized that the insulin stimulat ed pathway of glucose uptake is impaired with AGT. 17, 18 Therefore, of particular signifi cance is the increased reliance on NOstimulated glucose uptake in patients with AGT and the potential to specifically target this pathway for glycemi c control intervention. 23 The improvements in NO bioavailability and endothelial function observed in the present study, as well as capillary to fiber ratios, likely mediate greater delivery of nut rients to nutritive tissues (i.e. skeletal muscle) during glycemic challenge. Additionally, NO signaling been has implicated in regulation of GLUT-4 translocation. 180 Although we did not observe changes in AMPK signaling in the present study, NO may c ontribute to cellular signaling and GLUT-4 130

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translocation via several pathways, including cyclic guanosine monophosphate (cGMP). 180 Thus, the potential for increased NO-m ediated translocation of GLUT-4 to the cell membrane during glycemic challeng e cannot be ruled out. Further studies should be conducted with multiple muscle biopsy sampling during a HEC to accurately describe cellular signaling a ssociated with glucose uptake. Furthermore, the acute effect of EECP and the impact on skeletal muscle cell signaling should be characterized. 131

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BIOGRAPHICAL SKETCH Jeffrey Steven Martin is the son of D eborah Anne Martin and Paul Wayne Martin, Jr., the brother of Shane Michael Martin, and the husband of Allison Marie Martin. He was born in Attleboro, Massachusetts and was ra ised in several states including, but not limited to, Maine, Ohio, and Pennsylvania. Jeffrey graduated from Saint Marks High School in Wilmington, Delaware and went to the University of Pittsburgh in Pittsburgh, Pennsylvania to pursue his bachelors degree. He received his Bachelor of Science degree in movement science with a concentrati on in exercise science in the year 2004 from the University of Pi ttsburgh. Following undergraduat e school, Jeffrey went to Northeastern University in Boston, Massac husetts to work as a graduate teaching assistant and to pursue his Master of Sci ence degree. Jeffrey received his masters degree in Clinical Exercise Phys iology from Northeastern Un iversity in 2006. Following graduate school, Jeffrey began his work as a doctoral student in Dr. Randy W. Braiths cardiovascular lab at the University of Florid a in Gainesville, FL. As a doctoral student, he taught the undergraduate Clin ical Exercise Physiology course in addition to academic coursework and research endeavors. Jeffrey received his Ph.D. from the University of Florida in the summer of 2011. 148