University of Southern California Dissertations and Theses

The peptide angiotensin-(1-7) as a novel treatment for complications induced by type 2 diabetes mellitus

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The peptide angiotensin-(1-7) as a novel treatment for complications induced by type 2 diabetes mellitus

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THE PEPTIDE ANGIOTENSIN-(1-7) AS A NOVEL TREATMENT FOR

COMPLICATIONS INDUCED BY TYPE 2 DIABETES MELLITUS

by

Nicholas Michael Mordwinkin

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(PHARMACEUTICAL SCIENCES)

May 2012

Copyright 2012 Nicholas Michael Mordwinkin

ii
Dedication

I would like to dedicate my dissertation, a culmination of four years of work,
first and foremost to my family: my mother, father, and sister, who have always
been there for me through the good and bad, with undying support for me and
my dreams. The hard work ethic and dedication they instilled in me throughout
my life allowed me to achieve heights I would have never thought imaginable. My
grandfather, who prior to his passing in 2004, was and still remains my mentor
and hero, and always told me to work hard and be humble. There is not a day
that goes by that I don’t think about the numerous life lessons he taught me as a
child and young adult. My girlfriend, one of the most unselfish people I know, who
has sacrificed her own personal career and quality of life, to guarantee that my
goals and dreams came before hers. I will never forget what you have done for
me, and hope one day I can repay my debt to you. To my friends and colleagues
who have stuck with me since day one, and always supported my decisions and
offered me encouragement. Last but definitely not least, to Dr. Stan Louie, Dr.
Kathy Rodgers, and the rest of my committee members, Dr. Wei-Chiang Shen,
Dr. Curtis Okamoto, Dr. Nouri Neamati, and Dr. Gere diZerega, who provided
infinite mentoring, support, and positive feedback during my years at the
University of Southern California. I could not have wished for a better group of
leaders to provide me with the scientific and personal support a student needs
during this important period of my life.

iii
Acknowledgements

I would like to acknowledge my colleagues in no particular order, for their
constant support, dedication and hard work towards the completion of these
studies: Dr. Jared Russell, Sachin Jadhav, Natasha Sharma, Christopher Meeks,
Nidhi Sharda, Nhat Hyunh, Jeannette Arangua, Alick Tan, Lila Kim, Theresa
Espinoza, and Norma Roda. Without their help, none of this would have been
possible.

iv
Table of Contents

Dedication ii

Acknowledgements iii

List of Tables vii

List of Figures ix

Abbreviations xi

Abstract xvi

Chapter 1: Introduction
1.1 Overview of Diabetes Mellitus 1
1.2 Complications of Diabetes Mellitus 3
1.2.1 Oxidative Stress and Its Molecular Mechanisms in
Diabetes Mellitus 5
1.2.2 Endothelial Progenitor Cell Dysfunction 8
1.2.3 Inflammation and Immune Dysfunction 11
1.3 The Renin-Angiotensin System 14
1.3.1 The Use of Renin-Angiotensin System Antagonists 16
1.3.2 Angiotensin-(1-7) 17
1.3.3 The RAS and Oxidative Stress 20
1.3.4 The RAS and Endothelial Progenitor Cells 21
1.3.5 The Role of the RAS in Inflammation and
Immune Function 23
1.4 Chapter 1 References 25

Chapter 2: Angiotensin-(1-7) Reverses Oxidative Stress in Diabetic Bone
Marrow
2.1 Background 37
2.2 Study Design 39
2.2.1 Animal Protocol 39
2.2.2 Chemicals and Reagents 40
2.2.3 Bone Marrow Isolation 41
2.2.4 Measurement of Bone Marrow Nitrite and ROS
Levels 41
2.2.5 Preparation of Bone Marrow Cells for Flow
Cytometry 42
2.2.6 Analysis of Bone Marrow mRNA and Protein
Expression 42
2.2.7 Statistical Analysis 43

v
2.3 Results 44
2.3.1 Ang-(1-7) Effects on Bone Marrow ROS and
Nitrite Levels 44
2.3.2 Effects of Ang-(1-7) on NOS Isoform
Expression and eNOS Activation 45
2.3.3 Effects of Ang-(1-7) on SOD Isoforms in the Bone
Marrow 48
2.3.4 Ang-(1-7) Treatment Decreases Bone Marrow
p22-phox Expression in Diabetes 49
2.3.5 Ang-(1-7) Treatment Reduces Bone Marrow
Protein Tyrosine Nitration in Diabetes 51
2.4 Discussion and Significance 52
2.5 Conclusion 57
2.6 Chapter 2 References 58

Chapter 3: The Impact of Angiotensin-(1-7) on Inflammation and Immune
System Dysfunction in Diabetes
3.1 Background 63
3.2 Study Design 65
3.2.1 Animal Protocol 65
3.2.2 Chemicals and Reagents 66
3.2.3 Harvesting of Plasma and Bone Marrow 67
3.2.4 Analysis of Bone Marrow mRNA Expression 67
3.2.5 Intracellular Bone Marrow Cytokine Determination 68
3.2.6 Plasma Cytokine Measurements 70
3.2.7 Bone Marrow CFU-GEMM and CFU-GM Assay 70
3.2.8 Bone Marrow-Derived Dendritic Cell Cultures 70
3.2.9 Bone Marrow CFU Pre-B Cell Assay 71
3.2.10 Statistical Analysis 72
3.3 Results 72
3.3.1 Bone Marrow Cytokine Expression 72
3.3.2 Bone Marrow Intracellular Cytokine Expression 73
3.3.3 Plasma Cytokine Levels 74
3.3.4 Bone Marrow-Derived CFU-GEMM and CFU-GM
Colonies 75
3.3.5 Bone Marrow-Derived Dendritic Cells and CFU
Pre-B Cell Colonies 76
3.4 Discussion and Significance 78
3.5 Conclusion 82
3.6 Chapter 3 References 84

Chapter 4: The Effects of Angiotensin-(1-7) on Bone Marrow-Derived
and Circulating EPC, Bone Marrow-Derived MSC, and
Cardiovascular Function in Type 2 Diabetes

vi
4.1 Background 88
4.2 Study Design 93
4.2.1 Animal Protocol 93
4.2.2 Chemicals and Reagents 94
4.2.3 Cardiac Puncture 95
4.2.4 Bone Marrow Isolation 95
4.2.5 Bone Marrow EPC Counts 95
4.2.6 Circulating EPC Counts 96
4.2.7 MSC Cultures 96
4.2.8 Echocardiographic Analysis 97
4.2.9 Thermodilution Methodology 98
4.2.10 Measurement of Cardiomyocyte Hypertrophy 99
4.2.11 Statistical Analysis 99
4.3 Results 100
4.3.1 Bone Marrow-Derived EPC 100
4.3.2 Circulating EPC 101
4.3.3 Bone Marrow-Derived MSC 101
4.3.4 Cardiac Output and Cardiac Index 102
4.3.5 Measurements of Left Ventricular Function 103
4.3.6 Cardiomyocyte Measurements 105
4.4 Discussion and Significance 106
4.5 Conclusion 109
4.6 Chapter 4 References 112

Chapter 5: Alteration of Endothelial Dysfunction and Markers of
Oxidative Stress in Women with Gestational Diabetes Mellitus
and Their Fetuses
5.1 Background 117
5.2 Study Design and Methods 119
5.2.1 Patient Recruitment 119
5.2.2 Preparation of Whole Blood for Flow Cytometry 120
5.2.3 Analysis of mRNA Expression 121
5.2.4 Measurement of Nitrite, sVCAM-1, sICAM-1, and
CRP Levels 121
5.2.5 Statistical Analysis 122
5.3 Results 122
5.3.1 Maternal and Cord Blood EPC Counts 123
5.3.2 Plasma Nitrite Levels and eNOS Expression 124
5.3.3 SOD and p22-phox mRNA Expression 125
5.3.4 Plasma sVCAM-1, sICAM-1, and CRP Levels 126
5.3.5 Correlations with HbA1c 127
5.4 Discussion and Significance 128
5.5 Conclusion 132
5.6 Chapter 5 References 134

vii

Chapter 6: Conclusion
6.1 Introduction 137
6.2 Type 2 Diabetes and Oxidative Stress 138
6.3 Chronic Immune Activation in Type 2 Diabetes 139
6.4 Diabetes and Cardiovascular Disease 140
6.5 Gestational Diabetes 143
6.6 Concluding Remarks 144
6.7 Chapter 6 References 146

Comprehensive Bibliography 148

viii
List of Tables

Table 1. Effects of Ang-(1-7) on measurements of cardiac function in
Diabetic mice.
108

Table 2. Patient characteristics by GDM group. 123

ix
List of Figures

Figure 1. Mechanism of eNOS uncoupling 6
Figure 2. The role of EPC in vasculogenesis 8
Figure 3. The renin-angiotensin system 14
Figure 4. Effect of Ang-(1-7) on bone marrow ROS and nitrite levels 44
Figure 5. Ang-(1-7) effect on NOS isoform expression 46
Figure 6. Ang-(1-7) activates bone marrow eNOS 47
Figure 7. Effects of Ang-(1-7) on bone marrow SOD expression 48
Figure 8. Ang-(1-7) decreases bone marrow p22-phox expression 50
Figure 9. Bone marrow tyrosine nitration and correlation with nitrite
levels 51

Figure 10. Ang-(1-7) effect on bone marrow cytokine expression 72

Figure 11. Bone marrow intracellular cytokine levels 73
Figure 12. Plasma cytokine levels 74
Figure 13. Bone marrow-derived dendritic cell markers 76
Figure 14. Bone marrow CFU Pre-B cell colonies 77
Figure 15. Bone marrow-derived and circulating EPC counts and
bone marrow-derived MSC counts 100

Figure 16. Cardiac output and cardiac index measurements 102

Figure 17. Measurement of dP/dT
max
and dP/dT
min
103
Figure 18. Measurement of fractional shortening 104
Figure 19. Cardiomyocyte measurements 105
Figure 20. Maternal and cord blood EPC % in non-diabetic and
GDM patients 124

x

Figure 21. Maternal and cord blood nitrite levels and eNOS
mRNA expression in non-diabetic and GDM patients 125

Figure 22. Maternal and cord blood SOD and p22-phox mRNA
expression in non-diabetic and GDM patients 126

Figure 23. Maternal and cord plasma sVCAM-1 and sICAM-1
in non-diabetic and GDM patients 127

Figure 24. Correlations with HbA1c 128

xi
Abbreviations

A-779 D-Alanine-Angiotensin-(1-7)
Ac-LDL Acetylated-Low Density Lipoprotein
ACE Angiotensin-Converting Enzyme
ACE2
ADA
Angiotensin-Converting Enzyme 2
American Diabetes Association
AGE Advanced Glycation Endproducts
AGT Angiotensinogen
Ang I Angiotensin I [Angiotensin-(1-10)]
Ang II Angiotensin II [Angiotensin-(1-8)]
Ang III Angiotensin III [Angiotensin-(2-8)]
Ang IV Angiotensin IV [Angiotensin-(3-8)]
Ang-(1-7) Angiotensin-(1-7)
Ang-(1-9) Angiotensin-(1-9)
ANOVA Analysis of Variance
ARB Angiotensin Receptor Blocker
AT
1
Angiotensin Type 1 Receptor
AT
2
Angiotensin Type 2 Receptor
BH
2
Dihyrdobiopterin
BH
4
Tetrahydrobiopterin
BSI lectin Bandeiraea simplicifolia lectin
Ca
2+
Calcium

xii
CAD Coronary Artery Disease
CFU Colony-Forming Unit
CHF Congestive Heart Failure
CRP C-Reactive Protein
CVD Cardiovascular Disease
DAPI 4’,6-diamidino-2-phenylindole
Db Leptin
DC Dendritic Cells
DM Diabetes Mellitus
ELISA Enzyme-Linked Immunosorbent Assay
eNOS Endothelial Nitric Oxide Synthase
EPC
EPIC
Endothelial Progenitor Cell
European Prospective Investigation into Cancer and Nutrition
ET-1 Endothelin-1
FBS Fetal Bovine Serum
FCS Fetal Calf Serum
FFA Free Fatty Acids
Flk-1 Fetal Liver Kinase-1
GDM Gestational Diabetes Mellitus
GEMM Granulocyte, Erythrocyte, Macrophage, Megakaryocyte
GM Granulocyte, Macrophage
GPCR G-Protein Coupled Receptor

xiii

GUSTO

Global Utilization of Streptokinase and Tissue Plasminogen
Activator for Occluded Coronary Arteries
HbA1c Hemoglobin A1c
HSC Hematopoietic Stem Cell
ICAM-1 Intracellular Adhesion Molecule-1
IDDM Insulin-Dependent Diabetes Mellitus
IDNT Irbesartan Diabetic Nephropathy Trial
IL-1β
IL-6
Interleukin-1 Beta
Interleukin-6
iNOS Inducible Nitric Oxide Synthase
L-NAME
LIFE
N (G)-nitro-L-arginine methyl ester
Losartan Intervention For Endpoint reduction in hypertension study
Ly-6 Lymphocyte Antigen-6
MAPK Mitogen-Activated Protein Kinase
MI Myocardial Infarction
MSC
M
Mesenchymal Stem Cell
Molar
Na
+
Sodium
NADPH Nicotinamide Adenine Dinucleotide Phosphate
NEP Neutral Endopeptidase
NIDDM Non-Insulin-Dependent Diabetes Mellitus

xiv
NIH National Institutes of Health
nNOS Neuronal Nitric Oxide Synthase
NO Nitric Oxide
NOS Nitric Oxide Synthase
O
2
Oxygen
Ob Leptin deficient mouse
OGTT Oral Glucose Tolerance Test
PBS Phosphate Buffered Saline
PI3K Phospoinositide 3-Kinase
PKC Protein Kinase C
PLC Phospolipase C
RAGE Receptor for Advanced Glycation Endproducts
RAAS Renin-Angiotensin-Aldosterone System
RAS Renin-Angiotensin System
RENAAL Reduction of Endpoints in Non-Insulin-Dependent Diabetes Mellitus
with the Angiotensin II Antagonist Losartan
RNS Reactive Nitrogen Species
ROS Reactive Oxygen Species
RPM Revolutions Per Minute
RT-PCR Reverse Transcriptase-Polymerase Chain Reaction
SC Subcutaneous
SD Standard Deviation

xv
SEM Standard Error of the Mean
sICAM-1 Soluble Intracellular Adhesion Molecule-1
SO Superoxide
SOD
SPRINT
Superoxide Dismutase
Secondary Prevention Reinfarction Israeli Nifedipine Trial
sVCAM-1 Soluble Vascular Adhesion Molucule-1
T1DM Type 1 Diabetes Mellitus
T2DM Type 2 Diabetes Mellitus
TNF-α Tumor Necrosis Factor-alpha
VCAM-1 Vascular Adhesion Molecule-1
VEGFR-2 Vascular Endothelial Growth Factor Receptor-2

xvi
Abstract

The aim of this dissertation is to evaluate the impact of type 2 diabetes
mellitus on oxidative stress and inflammation in the bone marrow and circulation,
as well as investigate the relationship between these molecular alterations and
long-term complications of diabetes, specifically cardiovascular disease. In
addition, the role of the peptide Ang-(1-7) i as well as the receptor and second
messager systems involved in reversing these molecular alterations and
complications will also be determined.
The research integrates multiple in vivo studies and a clinical study to
provide a comprehensive picture of this disease state. Nitrite levels in the bone
marrow and blood were measured using the Griess reaction. Expression and
protein levels of molecular markers of oxidative stress and cytokines were
determined using RT-PCR, western blot, and ELISA. Levels of oxidative stress,
protein tyrosine nitration in the bone marrow, intracellular cytokine levels, and
EPC counts were measured using flow cytometric methodologies. Tissue protein
nitration was measured by immunohistochemistry. Murine heart function was
determined in vivo using small animal echocardiography and thermodilution
techniques, and histology was used to measure cardiomyocytes in stained heart
sections. Culture of isolated bone marrow cells was used to determine various
progenitor counts.
Our in vivo and clinical data indicate that oxidative stress and systemic
inflammation play a major role in both type 2 diabetes and gestational diabetes.

xvii
In addition, we illustrate a potential link between these pathologies and
endothelial and cardiovascular dysfunction in this disease state. Treatment of
db/db mice with Ang-(1-7) for 14 days resulted in decreases in markers of
oxidative stress and inflammation, increases in bone marrow-derived and
circulating EPC, as well as increases in other bone marrow-derived progenitors
involved in vasculogenesis and immune function. Lastly, Ang-(1-7) treatment
helped to increase measures of cardiac function that were reduced in diabetic
mice.
While a focus on glucose control is still of the utmost importance, more
attention needs to be spent on reversing the pre-existing cellular damage caused
by oxidative stress and inflammation in diabetes. Ang-(1-7) may be one of
multiple promising agents with the ability to work synergistically with currently
FDA-approved therapies; together able to reduce plasma glucose levels,
preventing further damage, and reverse oxidative stress and inflammation in type
2 diabetes. Combined, this therapeutic strategy could potentially significantly
reduce the risk of some of the long-term and deadly complications of diabetes,
including cardiovascular disease.

1
Chapter 1 1
Introduction 2
3
1.1 Overview of Diabetes Mellitus 4
5
Diabetes mellitus (DM) is a serious metabolic disease that affects over 6
171 million individuals worldwide, with an estimated 25.8 million people in the 7
United States living with this disease (World Health Organization, National 8
Institutes of Health). Approximately one-quarter of those people are undiagnosed 9
(National Institutes of Health). DM is currently the seventh leading cause of death 10
in the United States (Centers for Disease Control and Prevention). As of 2007, 11
the national cost associated with DM in the United States alone is over $174 12
billion (American Diabetes Association [ADA]). Health expenditures for an 13
individual with DM are estimated to be over twice of those for a non-diabetic 14
(ADA). DM is a group of metabolic diseases which is divided into three main 15
types, type 1 DM (T1DM), type 2 DM (T2DM), and gestational diabetes mellitus 16
(GDM), each with a differing pathophysiology and treatments. In general, DM is 17
diagnosed by measuring plasma glucose levels, either while the patient is fasting, 18
or with an oral glucose tolerance test (World Health Organization). Measurement 19
of glycated hemoglobin (HbA1c), which can be used to estimate the average 20
plasma glucose levels over approximately 3 months, is also used to help in the 21
diagnosis and treatment of DM (ADA). 22
T1DM, previously known as juvenile or insulin-dependent diabetes 23
mellitus, is characterized by an endogenous insulin deficiency due to a loss or 24
destruction of beta cells of the islets of Langerhans cells in the pancreas, and 25

2
accounts for approximately 10% of all cases of DM worldwide (ADA). While there 26
are various risk factors and genetic dispositions that can lead to this, and the 27
cause of T1DM is not completely understood, the loss of beta cell function is 28
commonly thought to be secondary to an autoimmune response. T1DM is most 29
often treated with various injected insulin replacement therapies, along with 30
lifestyle management including diet and regular exercise. 31
T2DM, previously known as adult-onset or non-insulin-dependent diabetes 32
mellitus, is characterized by insulin resistance in peripheral tissues, and may also 33
be accompanied by a reduction in insulin secretion by the beta cells of the 34
pancreas. T2DM accounts for approximately 85% of all cases of DM worldwide 35
(Robbins Pathologic Basis of Disease 6
th
edition 1999). Genetics is thought to 36
play a much greater role in T2DM compared to T1DM. Unlike T1DM, 37
autoimmunity does not play a role in the initial development of T2DM, while 38
lifestyle factors such as obesity, smoking, alcohol consumption, and a sedentary 39
lifestyle correlate significantly with the development of T2DM (Mozaffarian et al 40
2009). In addition to lifestyle modifications and diet interventions to promote 41
weight loss, T2DM is most often treated with multiple oral medications, and less 42
frequently exogenous insulin. There are multiple classes of approved drugs, each 43
with different mechanisms of action, however ultimately the goal of these 44
therapies is to normalize plasma blood glucose levels and reduce cardiovascular 45
risk factors (Ripsin et al 2009). 46

3
While the exact mechanisms behind its pathophysiology are still unknown, 47
GDM occurs when a woman without a previous diagnosis of DM develops high 48
plasma glucose levels during pregnancy, and this glucose intolerance is a direct 49
result of insufficient insulin secretion (Kjos et al 1999). An oral glucose tolerance 50
test (OGTT) and fasting plasma glucose levels are currently the gold standard for 51
diagnosis of GDM. In the long-term GDM patients may also develop insulin 52
resistance. The incidence of GDM is in approximately 5% of pregnancies 53
worldwide, although estimations are difficult due to a lack of consensus 54
concerning the appropriate diagnostic measures, and the frequency may actually 55
be much higher (Ferrara 2007, National Institutes of Health). Risk factors for 56
developing GDM include ethnicity (African-Americans and Hispanics are at an 57
increased risk), family history of T2DM and obesity, and maternal age (Ross 58
2006). The primary antepartum treatment of GDM involves dietary modifications 59
and exercise in order to reduce plasma glucose levels and prevent fetal 60
complications. If these interventions are insufficient, insulin and/or oral anti- 61
diabetic medications that are safe for use in pregnancy may be used. Postpartum, 62
there is evidence to suggest that both mother and child are at an increased risk 63
for the development of obesity and DM (Pettit et al 1985, Silverman et al 1995, 64
Kim et al 2002). 65
66
1.2 Complications of Diabetes Mellitus 67
68
Epidemiological studies have shown that compared to non-diabetics, 69
diabetic patients have a significantly increased risk for the rate of development of 70

4
a number of complications including nephropathy, retinopathy, peripheral 71
vascular disease, stroke, cardiovascular disease (CVD), cardiomyopathy, 72
coronary artery disease (CAD), angina, myocardial infarction (MI), and cardiac 73
ischemia, which is the leading cause of morbidity and mortality in these patients 74
(Mak et al 1997, Stratton et al 2000, ADA, National Institutes of Health). 75
Importantly, some studies have suggested that strict blood glucose control via 76
dietary changes and/or medication therapies does not necessarily decrease the 77
risk of development of these complications in diabetics, and in fact may further 78
increase the risk of these long-term complications (Brinchmann-Hansen et al 79
1988, Behar et al 1997, Taubes 2008). The exact reason for this increased risk is 80
unknown, but some suggest it is due to potential hypoglycemia secondary to anti- 81
diabetic therapy, or side effects of the medications themselves. For example, 82
while somewhat controversial, the anti-diabetic drugs in the thiazolidinedione 83
class, which includes pioglitazone (Actos) and rosiglitazone (Avandia), both carry 84
a black box warning stating that some studies have found that administration of 85
these drugs may result in fluid retention that may lead to or worsen congestive 86
heart failure (CHF), as well as increase the risk of MI, especially when combined 87
with insulin therapy (United States Food and Drug Administration). More recently, 88
pioglitazone was pulled from the market in Europe after studies demonstrating an 89
increased risk in the development of bladder cancer. 90
91
92
93
94

5
1.2.1 Oxidative Stress and Its Molecular Mechanisms in Diabetes Mellitus 95
96
An increasingly large body of evidence from numerous in vitro and in vivo 97
studies has demonstrated that abnormally high levels of biomarkers of oxidative 98
stress are present in diabetes, and this increase in oxidative stress is one cause 99
of many of the long-term complications observed in diabetic patients (Maritim et 100
al 2003). This oxidative stress may also be one pathological mechanism that 101
leads to the development of diabetes. The increased oxidative stress observed in 102
diabetes is thought to be secondary to a dysfunction of three different but 103
interconnected enzymes: nicotinamide adenine dinucleotide phosphate-oxidase 104
(NADPH oxidase), endothelial nitric oxide synthase (eNOS), and superoxide 105
dismutase (SOD). 106
NADPH oxidase is a membrane-bound enzyme complex consisting of six 107
subunits. While normally dormant, following various physiological circumstances 108
(such as the presence of bacteria) is it responsible for generating superoxide by 109
transferring and coupling electrons to molecular oxygen (O
2
). Notably, a 110
significant increase in the expression and activity of NADPH oxidase is seen in 111
diabetes, which results in the excessive production of reactive oxygen species 112
(ROS) including superoxide (Xia et al 2008). While the exact mechanisms are 113
still unknown, it has been suggested that increased glucose levels and free fatty 114
acids (FFA) seen in diabetes activate protein kinase C (PKC), which in turn 115
results in an increased activity of NADPH oxidase and increased superoxide 116
production (Hink et al 2001, Zhang et al 2010). In addition to directly inactivating 117

6
and reducing the bioavailability of nitric oxide (NO), the excessive ROS 118
generated by NADPH oxidase in diabetes can also affect the function of the 119
enzyme eNOS. 120
Under normal conditions, eNOS catalyzes the production of NO and the 121
amino acid L-citrulline from O
2
and the amino acid L-arginine. Tetrahydrobiopterin 122
(BH
4
), a cofactor required for this enzymatic reaction, can oxidize BH
4
to 123
dihydrobiopterin (BH
2
) in presence of excess ROS (Figure 1; Scott-Burden 1995, 124
Hink et al 2001). Oxidation of BH
4
to BH
2
can lead to the uncoupling of eNOS 125
(Cai et al 2005). In presence of circulating high glucose, this uncoupling leads to 126
an increased production of superoxide and decreased production of NO 127
(Landmesser et al 2003). Further increases in ROS and reactive nitrogen species 128

Figure 1. Mechanism of eNOS uncoupling. Oxidation of BH
4
by ROS can lead to eNOS
uncoupling, which ultimately results in an increased production of ROS and decreased NO
bioavailability.

7
(RNS) such as peroxynitrite secondary to eNOS uncoupling can subsequently 129
result in protein tyrosine nitration, ultimately altering or impairing protein activity 130
and function (Radi 2004). Along with increased protein tyrosine nitration, another 131
common observation of eNOS uncoupling is a paradoxical increase in eNOS 132
expression, which is often seen as an attempt to increase NO levels (Zhang et al 133
2008). Additional studies on oxidative stress in diabetes have also shown that the 134
increased level of superoxide anions increase the expression of neuronal NOS 135
(nNOS) and inducible NOS (iNOS), which results in an increased generation of 136
NO (Wright et al 2006, Edlund et al 2010). However, due to the already high 137
concentration of superoxide present, NO and superoxide react to form additional 138
peroxynitrite, continuing the vicious cycle. At this point in the cycle superoxide 139
and subsequent peroxynitrite production are already increased. The bodies’ last 140
defense is SOD, which is a primary antioxidant enzyme responsible for the 141
removal of superoxide from the system. 142
SOD consists of a family of enzymes that catalyze the dismutation of 143
superoxide into oxygen and hydrogen peroxide. They are classified as SOD1 144
(soluble, located in the cytoplasm), SOD2 (mitochondrial), and SOD3 145
(extracellular). The SOD1 isoform is a dimer, while SOD2 and SOD3 are 146
tetramers. All SOD isoforms use a metal cofactor; SOD1 and SOD3 utilize 147
copper and zinc while SOD2 uses manganese. Combined with the increased free 148
radical production in diabetes, studies have also shown that there is also a 149
simultaneous reduction in the expression and alteration in the activity of SOD, 150

8
one of the bodies’ primary antioxidant defenses (Mizobuchi et al 1993, Kamata et 151
al 1996). Taken together, this decrease in antioxidant defenses, combined with 152
an increase in ROS production, eNOS uncoupling and a subsequent decrease in 153
NO bioavailability results in the increased oxidative stress observed in diabetes, 154
and may lead to many of the complications associated with this metabolic 155
disease. 156
157
1.2.2 Endothelial Progenitor Cell Dysfunction 158
159
Endothelial progenitor cells (EPC) are critical components in the 160
neovascularization process. These cells are more recently referred to as 161
vasculogenesis-related progenitor cells (VRPC) or proangiogenic hematopoietic 162
cells (Richardson et al 2010). Found primarily in the bone marrow and to a lesser 163
extent in the circulation, these progenitor cells are mobilized during injury, and 164

Figure 2. The role of EPC in vasculogenesis. Mobilization of EPC from the bone
marrow into the circulation by factors including NO is the first step in vasculogenesis.

9
can migrate to and differentiate at sites of endothelial cell damage to promote 165
vasculogenesis (Figure 2; Jujo et al 2008). This important process is a primary 166
defense to protect against and prevent vascular and CVD (Asahara et al 1997, 167
Kawamoto et al 2002). However, both the number and function of EPC are 168
reduced in diabetes, and this is believed to be one of the major factors leading to 169
the increased risk of CVD in diabetic patients (Vasa 2001, Waltenberger 2001, 170
Sheetz et al 2002, Tepper et al 2002, Loomans et al 2004). NO has been shown 171
to play an important primary role in its ability to stimulate the mobilization of EPC 172
from niches in the bone marrow into the circulation (Ozuyaman et al 2005). 173
Studies have shown that prolonged exposure of EPC to high glucose, similar to 174
concentrations seen in diabetic patients, increases superoxide production, which 175
in turn results in a reduction in NO bioavailability, as well as a decrease in EPC 176
numbers and proliferation potential (Chen et al 2007, Thum et al 2007). 177
Therefore, the EPC dysfunction observed in diabetes is thought to be a direct 178
consequence of an increase in oxidative stress, secondary to an increase in 179
superoxide levels and a decrease in NO concentrations (Zou et al 2004, Bitar et 180
al 2005, Hamed et al 2009). 181
There are number of means to identify EPC, both in vitro and in vivo. In 182
adults, EPC are hematopoietic in origin, and also express an important 183
component of the renin-angiotensin system (RAS), angiotensin-converting 184
enzyme (ACE). Due to this, these cells express and can often be identified using 185
markers of both hematopoietic and endothelial cells, among others (Roks et al 186

10
2011). Flow cytometry is often used to identify EPC in multiple species, however 187
there is some controversy regarding which cell surface markers to use in some 188
species, specifically in murine models (Khan et al 2005, Brunt et al 2007). In 189
humans, EPC are often defined as cells positive for CD34, CD309 (KDR), and 190
CD133, however there are other markers used. Cells positive for both Flk-1 and 191
Sca-1 are considered to be EPC in mice, where Flk-1 (fetal liver kinase 1, also 192
known as vascular endothelial growth factor receptor 2 or VEGFR-2) is 193
expressed in endothelial cells, while Sca-1 (also known as lymphocyte antigen 6 194
or Ly-6) is expressed on multipotent hematopoietic stem cells (HSC) in mice. 195
Another assay that quite controversial, however is commonly used to identify 196
EPC involves the uptake of acetylated low-density lipoprotein (Ac-LDL, which is 197
taken up by monocytes, macrophages, and endothelial cells) and lectin from 198
Bandeiraea simplicifolia (BSI lectin). Following the staining of cells with these two 199
markers, they are then counterstained with 4’,6-diamidino-2-phenylindole (DAPI) 200
and visualized under a microscope. Cells that are triply stained are considered 201
EPC. The problem with this assay is the specificity of the markers; for example 202
recently it has been identified that platelet microparticle contamination from 203
cultures can result in false-positive BSI lectin staining, and thus these cells may 204
be incorrectly identified as EPC (Prokopi et al 2009). Because of this, flow 205
cytometry is becoming one of the most widely used assays to identify EPC both 206
in vitro and in vivo. 207
208
209

11
1.2.3 Inflammation and Immune Dysfunction 210
211
Increasing evidence suggests that chronic low-grade systemic 212
inflammation, caused by an ongoing inflammatory cytokine-mediated acute 213
phase response, is involved in the pathogenesis of diabetes and its associated 214
complications, including dysfunction of the innate immune system (Pickup et al 215
1997, Pickup 2004). Various pleiotropic cytokines with overlapping functions are 216
responsible for regulation of the immune system, inflammatory response, and the 217
initiation of hematopoiesis (Akira et al 1990). It is now clear that metabolic 218
dysfunction, such as that seen in T2DM, can cause significant changes in the 219
levels of various cytokines and proteins, leading to the chronic inflammation and 220
immune dysfunction widely observed in patients with this disease (Geerlings et al 221
1999, Wellen et al 2005). Increased circulating and tissue-specific levels of 222
interleukin-1β (IL-1β), IL-6, tumor necrosis-α (TNF-α), and C-reactive protein 223
(CRP) have been reported in obese individuals and patients with diabetes, and 224
have also been shown to be predictive of an increased risk for the development 225
of T2DM (Hotamisligil et al 1993, Kado et al 1999, Arnalich et al 2000, Pickup et 226
al 2000, Pradhan et al 2001, Spranger et al 2003). While the exact mechanism 227
behind the increased levels of pro-inflammatory cytokines and dysfunction in 228
innate immunity is not clear, much evidence points to the role of leptin in diabetes 229
(Loffreda et al 1997, Lord et al 1998, Matarese et al 2005). Studies have shown 230
that leptin is a multifunctional molecule, leading to alterations in hematopoiesis, 231
inflammation and immune system function (Ozata et al 1999). For example, in 232

12
vivo studies have shown that leptin plays an inhibitory on monocyte and 233
macrophage-mediated responses, while it increases the lymphocyte-mediated 234
response. Leptin also increases T-lymphocyte, CD34
+
and endothelial cell 235
proliferation. Leptin is significantly reduced in T2DM, and due to its important 236
roles in regulation of T-cells and the immune response, it remains an important 237
cytokine to study in the pathophysiology of diabetes-induced inflammation and 238
immune dysfunction. 239
Another important player in the link between diabetes, oxidative stress, 240
and inflammation is the receptor for advanced glycation end products (RAGE). 241
RAGE is a 35kD trans-membrane receptor of the immunoglobulin family of cell 242
surface receptors, which binds multiple ligands including AGE, leading to altered 243
gene expression and activation of NADPH oxidase as well as an increase in 244
mitochondrial ROS generation in diabetes (Wautier et al 2001, Coughlan et al 245
2009). Increased RAGE expression has been demonstrated in a number of 246
disease states including atherosclerosis, vascular disease, heart failure, 247
Alzheimer’s disease, and diabetes (Yan et al 2009). The AGE/RAGE axis may 248
also play an important role in inhibition of EPC maturation, EPC apoptosis and 249
decreased NO in T2DM (Shen et al 2010). 250
Two common murine models used to study T2DM and its complications 251
are the ob/ob and db/db mouse, which are unable to produce or respond to the 252
gene ob (also known as leptin), respectively (Fantuzzi et al 2000). Due to this 253
defect, these mice are unable to reach satiety, overeat, are significantly 254

13
overweight, and have decreased energy expenditure due to decreased mobility 255
secondary to obesity. These murine models are similar, but there also are some 256
differences between the two. Hyperphagia contributes to the obesity in both 257
models, however unlike db/db mice, when ob/ob mice are restricted to a diet 258
sufficient for normal weight maintenance in lean mice, they continue to gain 259
excess weight and fat deposits. Db/db mice are insulin resistant and become 260
hyperglycemic at approximately 4-6 weeks of age. In both models, the 261
manifestation of the diabetic syndrome is drastically dependent on the genetic 262
background. In db/db and ob/ob mice with the C57BL/6J background, 263
hyperglycemia is only transient, lasting approximately 14-16 weeks, while mice 264
with the C57BLKS background become severely diabetic and have an early 265
death. Exogenously administered leptin has been shown to reverse insulin 266
resistance in mice, however as expected there was no response to leptin in the 267
db/db mouse, due to its truncated leptin receptor, while ob/ob mice do respond to 268
this treatment. These characteristics make these genetic animal models 269
important in the study of diabetes and its related complications (Yamauchi et al 270
2001). 271
272
273
274
275
276

14
1.3 The Renin-Angiotensin System (RAS) 277
278
The RAS, also referred to as the renin-angiotensin aldosterone system 279
(RAAS), is a tissue-specific hormone system that has been shown to play 280
important roles in the regulation of blood pressure, fluid balance, cardiovascular, 281
renal, endocrine, brain, and metabolic function, among many others (Figure 3; 282
Reid 1985, Ribeiro-Oliveira et al 2008). In addition to the commonly referred to 283
circulating or systemic RAS, tissue-specific RAS also exist in the body, including 284
in the pancreas, kidneys, heart, skin, and bone marrow (Haznedaroglu et al 285
2010). These tissue-specific RAS can operate both dependently and 286
independently of the circulating RAS. Dysfunction of the RAS in various tissues is 287
observed in a multitude of disease states, including diabetes. The first peptide in 288
the RAS cascade is angiotensinogen (AGT), which in humans is a 452 amino 289

Figure 3. The RAS. In addition to the regulation of blood pressure and fluid balance, the
RAS also is involved in cardiovascular, endocrine, brain and metabolic function.

15
acid peptide produced primarily in the liver and released into the circulation as an 290
inactive peptide. Patients with increased blood pressure often have a 291
polymorphism of the AGT gene resulting in increased levels of AGT and a long- 292
term increase in the risk of developing CVD (Winkelmann et al 1999). 293
Renin, an enzyme secreted by the kidneys, cleaves the peptide bond 294
between the leucine and valine residues (tenth and eleventh amino acids) of 295
angiotensinogen, forming the ten amino acid peptide, angiotensin I (Ang I). 296
Renin’s precursor, prorenin, is also released by the kidneys, and was always 297
thought to be inactive. However studies have shown that not only does prorenin 298
have a function, but along with renin also has a specific receptor, the 299
renin/prorenin receptor (Nguyen et al 2002). Interestingly, unlike unbound 300
prorenin, when prorenin is bound to its receptor it exhibits proteolytic activity and 301
can cleave AGT to Ang I (Danser 2007). This discovery has led to a new class of 302
renin inhibitors for the treatment of hypertension. 303
Ang I is known to have no biological activity, and is solely a precursor to 304
the eight amino acid vasoactive peptide angiotensin II (Ang II). ACE is found 305
system-wide, but primarily in the lungs, and cleaves the two C-terminal amino 306
acid residues of Ang I (histidine and leucine), forming Ang II. ACE is a popular 307
target for the class of FDA-approved anti-hypertensive medications known as 308
ACE-inhibitors, which decrease the formation of the hypertensive peptide Ang II. 309
In addition to generating Ang II, ACE has also been shown to inactivate 310
bradykinin, a peptide with vasodilatory properties, which also increases its 311

16
vasoconstrictive properties. Ang II plays a large physiological role in 312
hemodynamics, and in addition to its role as a vasoconstrictive hormone also 313
regulates renal sodium (Na
+
) excretion, aldosterone levels and extracellular fluid 314
volume. Ang II has a short half-life of 30 seconds to 30 minutes depending on if it 315
is located in the circulation or peripheral tissues, and is degraded via 316
aminopeptidases forming angiotensin III (Ang III), and angiotensin IV (Ang IV), 317
both of which have less than 50% of the biological vasopressor activity of Ang II. 318
319
1.3.1 The Use of Renin-Angiotensin System Antagonists 320
321
Ang II produces most of its physiological effects such as vasoconstriction 322
by activating the angiotensin type 1 (AT
1
) receptor, a G protein-coupled receptor 323
(GPCR). AT
1
is one of the best-described angiotensin receptors in the RAS, and 324
is expressed constitutively throughout the body. In rodents, the AT
1
receptor 325
exists in two isoforms, AT
1A
and AT
1B
, with the AT
1A
isoform being the most 326
highly expressed. The AT
1
receptor is coupled to G
q/11
and G
i/o
, which in turn 327
activate phospholipase C (PLC) and increases cytosolic calcium (Ca
2+
) levels, 328
resulting in stimulation of PKC, inhibition of adenylate cyclase, and activation of 329
various tyrosine kinases (Higuchi et al 2007). Similar to other receptors, the AT
1
330
receptor is responsive to negative feedback, where decreased circulating Ang II 331
levels result in an up-regulation of the receptor (Lassegue et al 1995). In addition, 332
other non-RAS factors can alter AT
1
receptor expression, including the cytokines 333
IL-1β, IL-6, and TNF-α (Nickenig et al 2002). Importantly, the AT
1
receptor is the 334
target for the class of anti-hypertensive drugs known as angiotensin receptor 335

17
blockers (ARB). The role of the AT
1
receptor is often studied using ARB such as 336
losartan, or AT
1
receptor knockout mice. 337
Ang II can also activate the G protein-coupled angiotensin type 2 (AT
2
) 338
receptor. The AT
2
receptor is a receptor expressed at very low levels in the adult 339
that is primarily expressed in the fetus and neonate (specifically in the skin, 340
kidneys and intestines), but its expression is also increased during stress and 341
injury (Shanmugam et al 1995). In adults, it is highly expressed in the 342
myometrium, as well as the adrenal gland and fallopian tubes. Unlike AT
1
, the 343
AT
2
receptor is coupled to G
i2
/G
i3
. While the exact effects of activation of the AT
2
344
receptor remain controversial, there is emerging evidence to suggest that it plays 345
an important role in apoptosis and vasodilation via opposing signaling of the AT
1
346
receptor, as well as roles in inflammation and oxidative stress, and that it is also 347
upregulated in obesity (Sabuhi et al 2011). A study has also demonstrated that 348
stimulation of the AT
2
receptor results in an increased production of NO (Ewert et 349
al 2003). The compound PD123,319 is commonly used as a antagonist of AT
2
350
receptors. 351
352
1.3.2 Angiotensin-(1-7) 353
354
Once thought to be an inactive metabolite of the vasoactive peptide Ang II, 355
angiotensin-(1-7) [Ang-(1-7)], an endogenous seven amino acid peptide of the 356
RAS, has been shown to be effective in stimulating hematopoietic progenitor cell 357
proliferation, accelerating dermal healing following injury in diabetes, as well as 358
increasing hematopoietic recovery after chemotherapy and irradiation injury in 359

18
both animal and human studies (Rodgers 2001, Rodgers et al 2002, Rodgers et 360
al 2003a, Rodgers et al 2003b, Rodgers et al 2006). Ang-(1-7) is formed primarily 361
by cleavage of the three C-terminal amino acid residues (phenylalanine, histidine, 362
and leucine) from Ang I via neprilysin, a neutral endopeptidase (NEP) enzyme. 363
The C-terminal amino acid residue leucine of Ang I can also be cleaved by the 364
exopeptidase angiotensin converting enzyme 2 (ACE2), which is found primarily 365
in the heart and kidneys, to form the nine amino acid peptide intermediate 366
angiotensin-(1-9) [Ang-(1-9)], which is then converted to Ang-(1-7) through an 367
unknown mechanism. Lastly, Ang (1-7) can also be produced by the cleavage of 368
the C-terminal amino acid residue phenylalanine from Ang II via ACE2 (Turner et 369
al 2002, Turner et al 2004, Katovich et al 2005). Ang-(1-7) can be degraded by 370
the enzyme ACE, therefore ACE inhibitors result in an increase in plasma Ang- 371
(1-7) levels due to an increase in Ang I as well as preventing the degradation of 372
Ang-(1-7). Further, administration of ACE2 inhibitors has been shown to reverse 373
the renoprotective role of Ang-(1-7) in diabetic mice (Ye et al 2006). Some 374
studies have concluded that in part, the beneficial effects of ACE inhibitors may 375
be secondary to increased levels of Ang-(1-7) (Simoes e Silva et al 2006). 376
Unlike Ang II, Ang-(1-7) has anti-hypertensive properties, which is due to 377
antagonism of Ang II-induced vasoconstriction, as well as augmentation of NO 378
and bradykinin-induced vasodilation. These and other counter-regulatory effects 379
of Ang-(1-7) are believed to be mediated through Mas, a G protein-coupled 380
receptor, however there is speculation as to the role of other receptors, such as 381

19
AT
2
(Santos et al 2003, Gembardt et al 2008). In addition, the Mas receptor has 382
been shown to heterodimerize with the AT
1
receptor, which is another way Ang- 383
(1-7) can counterbalance the effects of Ang II (Kostenis et al 2005). It has also 384
been shown that ACE2 protein levels are increased in the kidneys of diabetic 385
mice, suggesting yet another potentially protective role of Ang-(1-7) (Ye et al 386
2004). Lastly, a recent study has demonstrated that Ang-(1-7) can act as an 387
antagonist of AT
1
receptors (Iusuf et al 2008). Since the Ang-(1-7)/Mas axis 388
counteracts the actions of the more well known and “traditional” Ang II/AT
1
/AT
2
389
axis, it has been more recently referred to as the “protective” side of the RAS. 390
A useful model for the study of Ang-(1-7) has been the Mas receptor 391
knockout mouse, which lacks the Mas proto-oncogene. This model has allowed 392
for the study of the Ang-(1-7)/Mas receptor axis without the use of Mas receptor 393
antagonists such as d-Ala
7
-Ang-(1-7) (A-779). These mice exhibit fibrogenic 394
changes in the kidney and renal dysfunction including glomerular hyperfiltration 395
and microalbuminuria, while other studies have reported increases in anxiety as 396
a result of genetic deletion of the Ang-(1-7) receptor Mas (Walther et al 2008, 397
Pinheiro et al 2009). Also, since binding of Ang-(1-7) to Mas results in an 398
increase in NO and bradykinin release, the NOS inhibitor nitro-l-arginine methyl 399
ester (L-NAME) and bradykinin B
2
receptor icatibant are often used to investigate 400
the mechanisms of Ang-(1-7) signaling. 401
402
403
404
405

20
1.3.3 The RAS and Oxidative Stress 406
407
Oxidative stress has been associated with multiple disease states that are 408
also linked to the RAS, including hypertension, renal and cardiovascular 409
dysfunction, however the relationship between RAS activation and oxidative 410
stress is just being elucidated. Ang II upregulates both membrane-associated 411
and cytosolic subunits of NADPH oxidase, ultimately resulting in an increased 412
production of superoxide (Onozato et al 2002). For example, studies have 413
suggested that Ang II/AT
1
receptor-dependent cellular oxidative stress and 414
resultant glomerular damage may be in part to activation of the AGE/RAGE axis 415
in a hypertensive rat model (Bohlender et al 2005). The complex nature of the 416
RAS, specifically the fact that there exists both a circulating and tissue-specific 417
(i.e. local) RAS highlights the importance of this system, and the potential 418
detrimental effects when this system is negatively affected by disease states 419
such as diabetes and metabolic syndrome. For example, multiple studies have 420
shown that hyperglycemia, such as that seen in diabetes, can activate the RAS 421
(Lastra et al 2007). Specifically, isolated human pancreatic cells exposed to 422
levels of high glucose had an increase in expression of multiple components of 423
the RAS, including AGT, ACE, and the AT
1
receptor (Lupi et al 2006). In the 424
same study, there was a significant increase in oxidative stress, combined with 425
significant decreases in insulin secretion. Lastly, it was found that the increase in 426
oxidative stress was secondary to increased expression of p22-phox, a subunit of 427
NADPH oxidase, as well as phosphorylation of PKC. ACE inhibitors showed 428

21
beneficial results on both insulin secretion and oxidative stress in pancreatic cells 429
treated with high concentrations of glucose. Additional studies have also shown 430
beneficial effects of RAS blockade, via blockade of the AT
1
receptor, in diabetes- 431
induced oxidative stress. Due to the strong relationship between diabetes, Ang II, 432
oxidative stress, and long-term complications such as CVD, many groups have 433
investigated the effects of AT
1
receptor blockade on oxidative stress and other 434
diabetes-related complications. The AT
1
receptor antagonists, losartan, valsartan 435
and candesartan, have been shown to decrease markers of oxidative stress in 436
multiple animal models of diabetes, as well as in human diabetic subjects 437
(Kamper et al 2010). These effects have been demonstrated to be independent 438
of their effects on blood pressure, and in addition have shown promising results 439
in decreasing the incidence of long-term diabetes-related complications including 440
CVD and renal dysfunction (Ogawa et al 2006, Arozal et al 2009, Ozdemir et al 441
2009). Much less research has been focused on the effects of other peptides of 442
the RAS, such as Ang-(1-7), which in itself counterbalances many of the 443
detrimental effects of Ang II. 444
445
1.3.4 The RAS and Endothelial Progenitor Cells 446
Many of the largest clinical trials in the diabetes arena have been 447
dedicated to demonstrating the beneficial effects of RAS blockade on diabetes- 448
related vascular complications. Human subject clinical trials such as the 449
Reduction of Endpoints in Non-Insulin-Dependent Diabetes Mellitus with the 450
Angiotensin II Antagonist Losartan (RENAAL) and the Irbesartan Diabetic 451

22
Nephropathy Trial (IDNT) have shown that blocking the effects of Ang II with 452
ARBs translates to a better long-term prevention of diabetes-induced 453
cardiovascular and renal disease compared to glycemic control alone. Additional 454
studies have also demonstrated that RAS blockade can also prevent the onset of 455
T2DM in hypertensive patients (Lindholm et al 2002, Dahlof et al 2002). In 456
tandem with its role in oxidative stress, the activation of AT
1
receptors via Ang II 457
activates mitogen-activated protein kinase (MAPK) pathways and impairs 458
phosphoinositide 3-kinase (PI3K) activity, leading to an increase in the 459
expression of the pro-inflammatory endothelial-associated proteins intracellular 460
adhesion molecule 1 (ICAM-1) and endothelin-1 (ET-1), demonstrated the multi- 461
faceted role of the RAS in diabetes, oxidative stress, and endothelial dysfunction 462
(Caglayan et al 2005). 463
As mentioned previously, EPC play an important role in endothelial 464
dysfunction and cardiovascular complications in diabetes. While the current body 465
of evidence for the role of the RAS and RAS modulation on EPC numbers and 466
function is much smaller than endothelial cells, it is important nonetheless. A 467
recent study investigated the detrimental effects of Ang II through the AT
1
468
receptor on human EPC, and showed that that Ang II induces EPC apoptosis as 469
well as impairs EPC colony formation and migration in vitro (Endtmann et al 470
2011). Clinical studies have clearly demonstrated that administration of ARBs to 471
patients with T2DM results in stimulation and subsequent significant increases in 472
EPC (Bahlmann et al 2005, Reinhard et al 2010). In addition to ARBs, ACE 473

23
inhibitors have also shown positive effects on EPC, although many of these 474
studies, unlike those with ARBs, are in vivo studies performed in animals and not 475
human subjects. 476
Even more recent studies are beginning to demonstrate the important role 477
Ang-(1-7) plays on EPC function and proliferation (van Beusekom et al 2007). 478
Wang and colleagues demonstrated that Ang-(1-7) administration results in a 479
significant increase in EPC function and stimulates cardioprotection post- 480
myocardial infarction (Wang et al 2010). The mechanism of action of Ang-(1-7) in 481
endothelial cells is thought to be multifold, however two major actions include the 482
inhibition of NADPH activation via Ang II as well as activation of eNOS through 483
Akt signaling (Sampaio et al 2007, Langeveld et al 2008). 484
485
1.3.5 The Role of the RAS in Inflammation and Immune Function 486
487
Along with its deleterious effects on endothelial cells and their precursors, 488
Ang II also increases the expression of pro-inflammatory molecules and 489
cytokines, such as IL-6, and it has been implicated in cytokine storm (Han et al 490
1999, Genctoy et al 2005). When this occurs in vessel walls, mononuclear 491
leukocytes are recruited, and the long-term effects can include atherosclerosis 492
and CVD (Brasier et al 2002). In fact, atherosclerotic plaques stain positive for 493
both ACE and Ang II (Diet et al 1996, Schiefffer et al 2000). The importance of 494
Ang II in the regulation of vascular inflammation has also been demonstrated, 495
where administration of the ARB irbesartan resulted in significant decreases in 496
vascular cell adhesion protein 1 (VCAM-1) and TNF-α, in addition to superoxide 497

24
levels (Navalkar et al 2001). Interestingly, the proinflammatory mechanism of 498
action and signaling of Ang II is very similar to that of many proinflammatory 499
cytokines such as IL-1 and TNF-α in that Ang II can activate the DNA-binding 500
protein nuclear factor (NF)-κB (Brasier et al 2002, Tian et al 2002). 501
Along with activating proinflammatory pathways, RAS components are 502
also directly affected by inflammation. It has been shown that after exposure to 503
bacterial endotoxins, AGT mRNA expression is upregulated by approximately 5- 504
fold, which resulted in a 3-fold increase in circulating AGT levels, and similar 505
results have been shown in humans with severe infections, leading some to refer 506
to AGT as an acute-phase protein (Hoj Nielsen et al 1987, Ron et al 1990). 507
In addition to its role in inflammation, studies have also demonstrated a 508
role for Ang II in modulation of the immune response. AT
1
receptors are 509
expressed on T-cells and macrophages, and Ang II has been implicated in 510
immune-mediated glomerulonephritis, splenic lymphocyte proliferation, and 511
allograft rejection (Nataraj et al 1999, Stigant et al 2000, Suzuki et al 2002). 512
These studies have shown that blockade of the RAS using ARB can attenuate 513
the Ang II-mediated immune response. 514
515
516
517
518
519
520
521
522
523

25
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1021

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277(36):32552-32557.
1026
1027
1028
1029
1030
1031
1032
1033
1034
1035
1036
1037
1038
1039
1040
1041

37
Chapter 2 1042
Angiotensin-(1-7) Reverses Oxidative Stress in Diabetic Bone Marrow 1043
1044
2.1 Background 1045
1046
Studies show that oxidative stress is increased in diabetes, which can lead 1047
to long-term complications observed in diabetic patients (Maritim et al 2003). 1048
Oxidative stress may also be one pathological mechanism that leads to the 1049
development of diabetes (Kocic et al 1998, Ceriello et al 2004). Long-term 1050
diabetes-related complications, including hypertension and CAD, may stem from 1051
multiple factors including excessive production of ROS such as superoxide and a 1052
subsequent decrease in NO bioavailability. In addition to directly reducing NO 1053
levels, superoxide can also negatively alter the function of eNOS. Increases in 1054
ROS and RNS, such as peroxynitrite, can result in protein tyrosine nitration, 1055
ultimately modifying or impairing normal protein activity and function (Radi et al 1056
2004, Zou et al 2004, Bitar et al 2005, Gembardt et al 2008). 1057
Ang-(1-7) is an endogenous seven amino acid peptide of the RAS. The 1058
effects of Ang-(1-7) are believed to be mediated through Mas, a G protein- 1059
coupled receptor, however there is speculation as to the role of other receptors, 1060
such as the fetal receptor AT
2
(Santos et al 2003, Sabuhi et al 2011). This is 1061
important because the expression of the AT
2
receptor in adults is primarily up- 1062
regulated following injury, and is also increased in an obese animal model (Li et 1063
al 1997). 1064
Unlike Ang II, the beneficial effects of Ang-(1-7) are due in part to 1065
antagonism of Ang II induced vasoconstriction of the arteries, as well stimulation 1066

38
of NO release from endothelial cells and cardiomyocytes resulting in vasodilation 1067
(Benter et al 1995, Roks et al 1999, Heitsch et al 2001, Ferrario et al 2002, 1068
Schmaier et al 2003, Dias-Peixoto et al 2008, Wang et al 2010). Cross talk 1069
between the RAS and kinin-kallikrein system has been well documented (Gorelik 1070
et al 1998, Shen et al 2006). Following binding to the Mas receptor, Ang-(1-7) 1071
also potentiates the release of bradykinin, a ligand of the bradykinin B
2
receptor 1072
that results in further vasodilatory effects (Fernandes et al 2001, Ueda et al 2001). 1073
Studies have also shown that Ang-(1-7) is effective in stimulating 1074
hematopoietic progenitor cell proliferation and increasing hematopoietic recovery 1075
after chemotherapy and irradiation injury in vivo (Rodgers et al 2002, Rodgers et 1076
al 2003). In addition, Ang-(1-7) can activate NOS and increase NOS activity, as 1077
well as increase the proliferation of bone marrow-derived progenitors (Heringer- 1078
Walther et al 2009, Costa et al 2010). The bone marrow is a primary source of 1079
progenitors responsible for important biological processes such as 1080
vasculogenesis, angiogenesis, and hematopoiesis (Jujo et al 2008). Oxidative 1081
stress and damage, such as that seen in diabetes, can directly impact cell 1082
survival and function. Exposure of bone marrow cells to these insults could lead 1083
to many of the detrimental and irreversible complications of diabetes. The aim of 1084
this current study is to investigate multiple molecular markers of oxidative stress, 1085
including eNOS dysfunction and its resultant sequelae in diabetic bone marrow. 1086
In addition, we examined the effects of in vivo Ang-(1-7) administration on 1087
diabetes-induced oxidative stress in the bone marrow. Lastly, we analyzed the 1088

39
potential pathways through which Ang-(1-7) alters these processes by using 1089
pharmacological antagonists of angiotensin and bradykinin receptors, as well as 1090
pharmacological inhibition of NOS. 1091
1092
2.2 Study Design 1093
1094
2.2.1 Animal Protocol 1095
1096
The NIH Principles of Laboratory Animal Care were followed, and the 1097
Department of Animal Resources at the University of Southern California 1098
approved this study. Six to eight week-old male BKS.Cg-Dock7
m
+/+ Lepr
db
/J 1099
mice and their heterozygous controls were purchased from Jackson Laboratories 1100
(Bar Harbor, ME, USA). Mice homozygous for the diabetes spontaneous 1101
mutation Lepr
db
(BKS.Cg-Dock7
m
+/+ Lepr
db
/J), which is an obese model of type 1102
2 diabetes due to truncation of the leptin receptor, were used in this study (db/db 1103
mice). The mice were quarantined for one week prior to initiation of the study, 1104
and all diabetic mice had verified plasma glucose levels >500mg/dL prior to 1105
initiation of treatment. Food and water were available ad libitum, and all mice 1106
were kept on a 12-hour light/dark cycle. 1107
BKS.Cg-Dock7
m
+/+ Lepr
db
/J mice and their heterozygous controls 1108
(n=7/group) were administered either saline (control), inhibitors alone (losartan, 1109
PD123,319, A-779 or L-NAME at 10 mg/kg/day or icatibant at 0.4 mg/kg/day), 1110
Ang-(1-7) alone (500 µg/kg/day), or Ang-(1-7) 500 µg/kg/day combined with an 1111
inhibitor at the aforementioned doses for two weeks by subcutaneous (SC) 1112
injection. The mice were weighed three times weekly, and the doses adjusted 1113

40
accordingly. The dose of Ang-(1-7) used in this study is based on unpublished 1114
data from our laboratory following multiple dose-response studies in mice, where 1115
Ang-(1-7) 500 µg/kg/day administered as a once-daily SC dose was found to be 1116
the most effective dose in diabetic mice with no observable toxicities. Fasting 1117
plasma glucose levels were measured at necropsy, and while there were slight 1118
decreases among the treatment groups, none of these changes reached 1119
statistical significance. In addition, there were no significant changes in any of the 1120
parameters investigated following treatment with inhibitors alone, or following 1121
Ang-(1-7) administration in heterozygous mice. Following the 14-day treatment 1122
period, the mice were euthanized and parameters investigated as described 1123
below to determine the effect of diabetes and Ang-(1-7) treatment with and 1124
without the co-administration of the various inhibitors. 1125
1126
2.2.2 Chemicals and Reagents 1127
1128
Ang-(1-7) (prepared using Good Manufacturing Practices) and A-779, an 1129
antagonist of the Mas receptor, were purchased from Bachem (Torrance, CA, 1130
USA). Losartan, an AT
1
receptor antagonist, PD123,319, an AT
2
receptor 1131
antagonist, and L-NAME, an inhibitor of NOS, were purchased from Sigma- 1132
Aldrich (Saint Louis, MO, USA). Icatibant, an antagonist of bradykinin B
2
1133
receptors, was purchased from Tocris Bioscience (Ellisville, MO, USA). 1134
CellROX
™
Deep Red Reagent, a fluorogenic probe for measuring cellular ROS 1135
levels, and MitoSOX
™
Red, a fluorogenic mitochondrial superoxide indicator, 1136
were purchased from Invitrogen (Carlsbad, CA, USA). The mouse anti- 1137

41
nitrotyrosine, clone 1A6, Alexa Fluor
®
488 conjugated monoclonal antibody and 1138
IgG2bκ isotype control for flow cytometry was purchased from Millipore (Billerica, 1139
MA, USA). Primers for qRT-PCR were purchased from Integrated DNA 1140
Technologies (San Diego, CA, USA), and antibodies used for western blotting 1141
were purchased from Cell Signaling Technology (Danvers, MA, USA). 1142
1143
2.2.3 Bone Marrow Isolation 1144
1145
The mice were euthanized using forced carbon dioxide and the femurs 1146
from each mouse were collected and the bone marrow was harvested by flushing 1147
with phosphate buffered saline containing 2% fetal calf serum. Following 1148
collection of the bone marrow and aliquoting, red blood cells were lysed with a 1149
hypotonic solution, mixed with 0.04% trypan blue and the number of nucleated 1150
cells was assessed using a hematocytometer under light microscopy. 1151
1152
2.2.4 Measurement of Bone Marrow Nitrite and Reactive Oxygen Species 1153
Levels 1154
1155
Following permeabilization of aliquoted bone marrow cells with 0.1% 1156
Triton-X, NO levels were measured using the Griess Reagent System (Promega, 1157
Madison, WI, USA). Due to the instable and volatile nature of NO, this assay 1158
measures nitrite, one of the stable metabolites of NO. The assay was performed 1159
per manufacturer protocol. 1160
To measure intracellular ROS levels and intracellular mitochondrial 1161
superoxide levels, isolated bone marrow cells were incubated with a 1162
commercially available cell-permeable fluorogenic probes (CellROX
™
Deep Red 1163

42
Reagent or MitoSOX
™
Red mitochondrial superoxide indicator) at 5µM and 1164
incubated for 30 minutes at 37°C per manufacturer protocol. ROS levels were 1165
measured via flow cytometry and values reported as median fluorescent intensity. 1166
1167
2.2.5 Preparation of Bone Marrow Cells for Flow Cytometry 1168
1169
Bone marrow cells were suspended at 10
6
cells/mL in DMEM containing 1170
2% FCS, 1 mM HEPES, penicillin, and streptomycin. For antibody staining, bone 1171
marrow cells were suspended in HBSS containing 2% FCS, 1 mM HEPES, 1172
penicillin, and streptomycin (HBSS+) at 10
8
cells/mL. An aliquot was first labeled 1173
with Alexa Fluor
®
488 conjugated mouse anti-nitrotyrosine antibody added at 1 1174
µg per 1 x 10
6
cells, and then fixed with 4% paraformaldehyde. Flow cytometric 1175
analysis was performed on a LSR II flow cytometer using FACSDiva software 1176
(Becton Dickinson, Franklin Lakes, NJ, USA) at the Flow Cytometry Core facility 1177
located at the University of Southern California School of Pharmacy. 1178
1179
2.2.6 Analysis of Bone Marrow mRNA and Protein Expression 1180
1181
Total RNA was extracted from bone marrow cells using TRIzol (Invitrogen, 1182
Carlsbad, CA, USA). For each sample, approximately 100 ng of RNA was 1183
reverse-transcribed using Maxima Reverse Transcriptase (Fermentas, Glen 1184
Burnie, MD, USA). Real-time PCR was conducted to examine expression of 1185
eNOS, nNOS, iNOS, SOD1, SOD2, SOD3, and p22-phox mRNA in bone marrow. 1186
Amplification of the cDNA was performed using SYBR Green PCR Master Mix 1187
(Applied Biosystems by Life Technologies, Carlsbad, CA, USA) using an ABI 1188

43
7300. Expression of eNOS, nNOS, iNOS, SOD1, SOD2, SOD3, and p22-phox 1189
mRNA were normalized against 18S mRNA and expressed as fold-change 1190
compared to non-diabetic controls. 1191
Protein lysates isolated from bone marrow cells for western blot analysis 1192
(30 µg) were resolved on SDS-PAGE gels, and transferred to nitrocellulose 1193
membranes by electroblotting. The membranes were incubated with monoclonal 1194
rabbit antibodies against eNOS, phospho-eNOS (Ser1177 and Thr495), nNOS, 1195
SOD3, and p22-phox, followed by anti-rabbit horseradish peroxidase-conjugated 1196
antibody. An antibody against β-actin was used to normalize protein loading. 1197
SuperSignal West Pico ECL substrate was used to detect the bands (Thermo 1198
Scientific, Rockfield, IL, USA). The resultant bands were quantified using 1199
densitometry using ImageJ version 1.46h (National Institutes of Health, USA). 1200
The results were expressed as the ratio of target protein band to β-actin band 1201
intensity. 1202
1203
2.2.7 Statistical Analysis 1204
1205
GraphPad Prism version 5.0d for Mac OS X (GraphPad Software, San Diego, 1206
CA, USA) was used to analyze the data. One-way analysis of variance (ANOVA) 1207
followed by Tukey’s test was used to compare data from more than two groups, 1208
and linear regression was used to determine the relationship between bone 1209
marrow nitrite levels and percentage of bone marrow tyrosine nitration. The level 1210
of statistical significance was set at 5%. Data are expressed as mean value ± 1211
standard error of the mean (SEM). 1212

44
1213
2.3 Results 1214
1215
2.3.1 Ang-(1-7) Effects on Bone Marrow ROS and Nitrite Levels 1216
1217
Bone marrow ROS and mitochondrial superoxide levels were significantly 1218
increased in diabetic bone marrow, while administration of Ang-(1-7) for 14 days 1219
significantly reduced these ROS levels (Figure 4A). Co-administration of Ang-(1- 1220
7) with losartan, PD123,319, A-779, L-NAME, or icatibant resulted in a significant 1221
increase in ROS and mitochondrial superoxide levels in diabetic bone marrow 1222
compared to treatment with Ang-(1-7) alone, while co-administration of Ang-(1-7) 1223

Figures 4A-B. Effect of Ang-(1-7) on bone marrow ROS and nitrite levels. Figure legends: * p<0.05; ** p<0.01;
† p<0.05 compared to diabetic + A-(1-7) group; †† p<0.01 compared to diabetic + A-(1-7) group Bone marrow ROS
levels were significantly higher in diabetic mice compared to non-diabetic controls (p<0.01). Treatment of diabetic
mice with Ang-(1-7) resulted in a significant decrease in bone marrow ROS levels compared to saline-treated
diabetic mice (p<0.01). Co-administration of losartan, PD123,319, A-779, or L-NAME with Ang-(1-7) significantly
increased bone marrow ROS levels compared to diabetic mice treated with Ang-(1-7) alone (p<0.01). Bone marrow
NO levels were significantly lower in diabetic mice compared to non-diabetic controls (p<0.01). Treatment of
diabetic mice with Ang-(1-7) for 14 days resulted in a significant increase in bone marrow NO levels compared to
saline-treated diabetic mice (p<0.01). Co-administration of PD123,319, A-779, or L-NAME with Ang-(1-7)
significantly reduced bone marrow NO levels compared to diabetic mice treated with Ang-(1-7) alone (p<0.05),
while co-administration of Ang-(1-7) with either losartan or icatibant did not have a significant effect on bone
marrow NO levels compared to Ang-(1-7) alone.

45
with losartan, PD123,319, A-779, L-NAME, or icatibant, resulted in a significant 1224
increase in ROS in diabetic bone marrow compared to treatment with Ang-(1-7) 1225
alone. Interestingly, while losartan inhibited the ability of Ang-(1-7) to decrease 1226
bone marrow ROS levels, it did not inhibit it’s ability to decrease bone marrow 1227
mitochondrial superoxide levels. 1228
Due to the integral relationship between diabetes-induced oxidative stress, 1229
NO, and the production of RNS leading to protein tyrosine nitration and post- 1230
translational modifications, NO levels were measured in bone marrow cells 1231
isolated from both non-diabetic and diabetic mice. Bone marrow NO levels were 1232
significantly reduced in diabetic mice when compared to non-diabetic controls, 1233
while treatment of diabetic mice with Ang-(1-7) resulted in a significant increase 1234
in bone marrow NO levels (Figure 4B). Administration of Ang-(1-7) to non- 1235
diabetic mice did not significantly affect bone marrow NO levels. Administration 1236
of PD123,319, A-779, or L-NAME in combination with Ang-(1-7) in diabetic mice 1237
resulted in a significant decrease in bone marrow nitrite levels compared to 1238
diabetic mice treated with Ang-(1-7) alone. The combination of Ang-(1-7) with 1239
either losartan or icatibant resulted in a non-significant reduction in bone marrow 1240
nitrite levels. 1241
1242
2.3.2 Effects of Ang-(1-7) on NOS isoform expression and eNOS activation 1243
1244
As other investigators have observed a paradoxical increase in eNOS 1245
expression in endothelial cells despite decreased NO levels in type 2 diabetes 1246
(Cosentino et al 1997, Hohenstein et al 2008), bone marrow eNOS, nNOS, and 1247

46
iNOS mRNA and protein expression were assessed. Bone marrow eNOS and 1248
nNOS mRNA and protein expression were significantly increased in diabetic 1249
mice (Figures 5A-D), while there were no significant changes in bone marrow 1250
iNOS expression in any group (data not shown). Treatment of diabetic mice with 1251
Ang-(1-7) significantly decreased both eNOS and nNOS mRNA and protein 1252
expression, while the co-administration of PD123,319, A-779, L-NAME, or 1253
icatibant with Ang-(1-7) resulted in a significant increase in eNOS and nNOS 1254
mRNA and protein expression compared to diabetic mice treated with Ang-(1-7) 1255
alone, while co-administration of losartan with Ang-(1-7) did not have a significant 1256
effect. 1257

Figures 5A-D. Ang-(1-7) effect on NOS isoform expression. Bone marrow eNOS and nNOS mRNA expression
and protein levels were significantly higher in diabetic mice compared to non-diabetic controls, respectively (p<0.01).
Diabetic mice administered Ang-(1-7) had significantly lower bone marrow eNOS and nNOS mRNA and protein
expression compared to saline-treated diabetic mice following 14 days of treatment (p<0.01). Co-administration of
PD123,319, A-779, L-NAME, or icatibant with Ang-(1-7) resulted in a significant increase in bone marrow eNOS and
nNOS mRNA and protein expression in diabetic mice compared to treatment with Ang-(1-7) alone (p<0.01).

47
To investigate the effect of diabetes and determine the impact of Ang-(1-7) 1258
on the activation of eNOS, we measure phosphorylation of eNOS at the primary 1259
activation (Ser1177) and inactivation sites (Thr495). Bone marrow eNOS protein 1260
isolated from diabetic mice exhibited significantly lower phosphorylation at 1261
Ser1177 and higher phosphorylation at Thr495 when compared to non-diabetic 1262
controls (Figures 6A-B). Ang-(1-7) treatment in diabetic mice resulted in a 1263
significant increase in eNOS phosphorylation at Ser1177, and a significant 1264
decrease in eNOS phosphorylation at Thr495. When PD123,319, A-779, L- 1265

Figures 6A-B. Ang-(1-7) activates bone marrow eNOS. Phosphorylation of bone marrow eNOS at Ser1177 was
significantly decreased in diabetic mice compared to non-diabetic controls (p<0.01). Diabetic mice treated with Ang-
(1-7) for 14 days had significantly increased phosphorylation of bone marrow eNOS at Ser1177 compared to saline-
treated diabetic mice (p<0.01). Co-administration of PD123,319, A-779, L-NAME, or icatibant with Ang-(1-7) resulted
in a significant reduction in phosphorylation of bone marrow eNOS at Ser1177 in diabetic mice compared to treatment
with Ang-(1-7) alone (p<0.01). Phosphorylation of bone marrow eNOS at Thr495 was significantly increased in
diabetic mice compared to non-diabetic controls (p<0.01). Diabetic mice treated with Ang-(1-7) for 14 days had
significantly decreased phosphorylation of bone marrow eNOS at Thr495 compared to saline-treated diabetic mice
(p<0.01). Co-administration of PD123,319, A-779, L-NAME, or icatibant with Ang-(1-7) resulted in a significant
increase in phosphorylation of bone marrow eNOS at Thr495 in diabetic mice compared to treatment with Ang-(1-7)
alone (p<0.01).

48
NAME or icatibant was co-administered with Ang-(1-7) in diabetic mice, the Ang- 1266
(1-7)-mediated effects on eNOS phosphorylation were blocked. No changes in 1267
eNOS phosphorylation were observed when Ang-(1-7) was co-administered with 1268
losartan. 1269
1270
2.3.3 Effect of Ang-(1-7) on SOD isoforms in the bone marrow 1271
1272
Diabetes causes a decreased expression of various SOD isoforms, 1273
resulting in a subsequent increase in oxidative stress (Kamata et al 1996). In this 1274
study, we measured SOD1 (Cu-Zn SOD), SOD2 (mitochondrial SOD), and SOD3 1275

Figures 7A-D. Effects of Ang-(1-7) on bone marrow SOD expression. There were no significant differences in
bone marrow SOD1 mRNA expression between any groups. Both bone marrow SOD2 and SOD3 expression was
significantly reduced in diabetic mice compared to non-diabetic controls (p<0.01). While treatment with Ang-(1-7)
for 14 days did not have a significant effect on SOD2 expression, treatment with Ang-(1-7) significantly increased
bone marrow SOD3 mRNA expression in diabetic mice. Co-administration of PD123,319, A-779, L-NAME, or
icatibant with Ang-(1-7) resulted in a significant decrease in bone marrow SOD3 mRNA expression in diabetic mice
compared to treatment with Ang-(1-7) alone (p<0.01). SOD3 protein expression was significantly reduced in
diabetic mice compared to non-diabetic controls (p<0.01), and the administration of Ang-(1-7) to diabetic mice
significantly increased bone marrow SOD3 protein expression (p<0.01). Co-administration of PD123,319, A-779, L-
NAME, or icatibant with Ang-(1-7) significantly inhibited its effect in diabetic mice (p<0.01).

49
(extracellular SOD) mRNA expression, where no significant changes in SOD1 1276
expression was detected among the groups evaluated. In contrast, both SOD2 1277
and SOD3 mRNA expression were significantly reduced in diabetic mice when 1278
compared to non-diabetic controls (Figures 7A-C). When diabetic mice were 1279
treated with Ang-(1-7), a significant increase in SOD3 expression was seen, 1280
however changes in SOD2 mRNA expression were not statistically significant. 1281
These results were verified using western blotting to determine protein 1282
expression (Figure 7D). Ang-(1-7)’s ability to increase mRNA and protein 1283
expression of SOD3 was completely blocked when co-administered with 1284
PD123,319, A-779, L-NAME, or icatibant (p<0.01), but not with losartan. 1285
1286
2.3.4 Ang-(1-7) treatment decreases bone marrow p22-phox expression in 1287
diabetes 1288
1289
Hyperglycemia resulting from diabetes induces the expression of NADPH 1290
oxidase through protein kinase C (PKC) activation, resulting in an increased 1291
production of superoxide (Inoguchi et al 2003, Xia et al 2008). This can initiate a 1292
cascade that ultimately leads to eNOS uncoupling and dysfunction. Bone marrow 1293
p22-phox (a subunit of the heterodimeric, membrane-bound portion of NADPH 1294
oxidase) was significantly higher in diabetic mice when compared to non-diabetic 1295
controls. When treated with Ang-(1-7), the expression of p22-phox was reduced 1296
when compared to saline treated animals, where the levels were similar to non- 1297
diabetic mice (Figure 8A). Administration of Ang-(1-7) combined with 1298
pharmacological antagonists of the AT
2
, Mas or B
2
receptors using PD123,319, 1299

50
A-779 or icatibant was able to inhibit Ang-(1-7) mediated activity. Similarly, co- 1300
administration with L-NAME, a NOS inhibitor, was able to inhibit Ang-(1-7) 1301
activity with regards to p22-phox mRNA expression. No antagonism was seen in 1302
diabetic mice given both Ang-(1-7) and losartan, an AT
1
receptor antagonist. The 1303
protein expression of p22-phox was consistent with RNA expression (Figure 8B). 1304
1305
1306
1307
1308
1309

Figures 8A-B. Ang-(1-7) decreases bone marrow p22-phox expression. Bone marrow p22-phox mRNA
expression was significantly higher in diabetic mice compared to non-diabetic controls (p<0.01). Treatment of
diabetic mice with Ang-(1-7) for 14 days resulted in a significant decrease in bone marrow p22-phox mRNA
expression, comparable to levels measured in non-diabetic controls (p<0.01). Co-administration of either
PD123,319, A-779, L-NAME, or icatibant with Ang-(1-7) resulted in significantly higher p22-phox mRNA expression
compared to treatment with Ang-(1-7) alone (p<0.01). There was also a significant increase in bone marrow p22-
phox protein expression diabetic mice compared to non-diabetic controls (p<0.01). Administration of Ang-(1-7) for
14 days significantly decreased bone marrow p22-phox protein expression in diabetic mice, while the co-
administration of PD123,319, A-779, L-NAME, or icatibant with Ang-(1-7) significantly blocked this effect compared
to treatment with Ang-(1-7) alone (p<0.01).

51
2.3.5 Ang-(1-7) treatment reduces bone marrow protein tyrosine nitration in 1310
diabetes 1311
1312
The increased production of superoxide and subsequent peroxynitrite 1313
formation following eNOS uncoupling and dysfunction in diabetes leads to protein 1314
tyrosine nitration, resulting in post-translational modifications and alterations in 1315
protein function (Turko et al 2003, Chi et al 2005). Therefore, we measured 1316
nitrotyrosine in bone marrow cells isolated from non-diabetic and diabetic mice. 1317
The percentage of nitrated bone marrow cells in diabetic mice was significantly 1318
higher compared to non-diabetic controls (Figure 9B). Administration of Ang-(1-7) 1319
to diabetic mice for 14 days resulted in a significant reduction in tyrosine nitration 1320
in the bone marrow. Co-administration of Ang-(1-7) with A-779, PD123,319, L- 1321
NAME, or icatibant blocked the Ang-(1-7) mediated decreases of nitrotyrosine 1322

Figures 9A-B. Bone marrow tyrosine nitration and correlation with nitrite levels. Nucleated bone marrow cells
from diabetic mice had a significantly higher percent tyrosine nitration compared to non-diabetic controls (p<0.01).
Following treatment with Ang-(1-7) for 14 days, the percentage of cells nitrated in diabetic bone marrow was
significantly decreased (p<0.01). Co-administration of Ang-(1-7) with A-779, PD123,319, L-NAME, or icatibant
resulted in a significant increase in bone marrow tyrosine nitration (p<0.05), while co-administration of Ang-(1-7)
with losartan did not result in a significant change compared to Ang-(1-7) treatment alone. The scatter plot of the
relationship between bone marrow nitrite levels and percent tyrosine nitration in non-diabetic and diabetic mice
shows a significant negative correlation (p<0.01).

52
found in the bone marrow. However, co-administration of Ang-(1-7) with losartan 1323
did not block the effect seen following Ang-(1-7) administration alone. 1324
To demonstrate the potential role of eNOS uncoupling in diabetic bone 1325
marrow, NO levels measured in all groups were correlated with tyrosine nitration. 1326
There was a significant negative correlation between bone marrow NO levels and 1327
protein tyrosine nitration levels, where lower bone marrow NO levels were 1328
associated with increased bone marrow protein tyrosine nitration (Figure 9B). 1329
Diabetic mice treated with Ang-(1-7) for 14 days showed significantly increased 1330
bone marrow NO levels and decreased bone marrow protein tyrosine nitration, 1331
suggesting a potential reversal of the negative effects of eNOS uncoupling and 1332
dysfunction observed in diabetes, including increased superoxide formation, a 1333
subsequent decrease in NO levels, and increases in protein tyrosine nitration. 1334
1335
2.4 Discussion and Significance 1336
1337
Our study demonstrates the in vivo effects of Ang-(1-7) on markers of 1338
oxidative stress in a murine model of type 2 diabetes. We showed that Ang-(1-7) 1339
administration decreased ROS and mitochondrial superoxide levels in diabetic 1340
bone marrow, coupled by a decrease in p22-phox expression and an increase in 1341
SOD3 expression. In addition, Ang-(1-7) increased NO levels. These changes 1342
were linked with increased eNOS phosphorylation at Ser1177 and reduced 1343
protein tyrosine nitration in diabetic bone marrow. The effects of Ang-(1-7) were 1344
modulated by a Mas, AT
2
and bradykinin B
2
receptor-dependent mechanism. 1345
Previous studies have shown that hyperglycemia can increase NADPH 1346

53
oxidase expression via protein kinase C activation, where increased NADPH 1347
oxidase expression results in an increased production of ROS. Elevated 1348
production of ROS through NADPH oxidase combined with a decrease in 1349
antioxidant mechanisms, specifically SOD3, could further increase oxidative 1350
stress. The SOD family of enzymes is part of a critical defense mechanism for 1351
managing superoxide levels, where down regulation of this system can increase 1352
cytotoxic levels of ROS, ultimately leading to tissue damage and the long-term 1353
complications seen in diabetes. 1354
Similar to studies examining the effects of diabetes in other tissues, we 1355
observed a decrease in NO levels and an increase in eNOS and nNOS mRNA 1356
and protein expression in the bone marrow. This is often a direct result of NOS 1357
uncoupling, which may be caused by reduced BH
4
availability secondary to 1358
decreased production or oxidation by superoxide to BH
2
(Scott-Burden 1995). 1359
The end result is a decrease in the production of NO, and an increase in the 1360
production of superoxide anions via eNOS due to the dissociation of the heme 1361
ferrous-dioxygen complex in the oxygenase domain of eNOS. Phosphorylation of 1362
eNOS was also altered in diabetic bone marrow, where an increase in 1363
phosphorylation at Thr495 and a decrease in phosphorylation at Ser1177 were 1364
observed. Phosphorylation of eNOS at Ser1177 results in activation, while 1365
Thr495 phosphorylation inactivates eNOS. 1366
Lastly, increased oxidative stress in diabetes, specifically an increased 1367
production of superoxide anions, can result in the formation of peroxynitrite, an 1368

54
oxidant and nitrating agent. Peroxynitrite can cause protein tyrosine nitration, a 1369
post-translational modification that can ultimately result in altered protein 1370
structure and function, and may also be the cause of many of the long-term 1371
complications of diabetes. Indeed, we observed an increase in protein tyrosine 1372
nitration in the bone marrow of diabetic mice. In addition to the commonly 1373
referred to circulating or systemic RAS, tissue-specific RAS also exist in the 1374
body, including in the pancreas, kidneys, heart, skin, and bone marrow 1375
(Haznedaroglu et al 2011). Both in vitro and in vivo studies have shown an 1376
important role for the RAS in the bone marrow microenvironment, especially in 1377
hematopoiesis. For example, Ang-(1-7) itself has been demonstrated to stimulate 1378
hematopoietic progenitor cells following chemotherapy in patients diagnosed with 1379
cancer (Rodgers et al 2003, Rodgers et al 2006). The bone marrow plays a vital 1380
role in the generation of progenitor cells responsible for multiple functions 1381
including wound healing, neovascularization, and immune function, all of which 1382
are compromised in diabetes. Evidence points to the role of oxidative stress in 1383
cellular damage, which may result in many of the long-term complications of 1384
diabetes such as cardiovascular and immune dysfunction. 1385
Ang-(1-7) has been shown to bind to the GPCR Mas, which results in an 1386
increased production of both NO and bradykinin. However, there is evidence for 1387
the involvement of the AT
2
receptor. In agreement with our present results, a 1388
recent paper showed that in spontaneously hypertensive rats, Ang-(1-7) 1389
administration up-regulated phosphorylated eNOS (Ser1177)/eNOS protein 1390

55
expression in cardiac tissues, an effect that was blocked by bradykinin B
2
1391
receptor and AT
2
receptor antagonism (Costa et al 2010). Since AT
2
receptors 1392
are up regulated following injury (e.g. damage from increased oxidative stress), 1393
there may be a role for AT
2
receptors coupled with Mas receptors in diabetes. 1394
This role could potentially involve heterodimerization of the two receptors (Castro 1395
et al 2005). In addition, other Ang-(1-7) signaling pathways that could potentially 1396
explain our results have been suggested in previous studies. This includes the 1397
counterregulation of Ang II signaling by Ang-(1-7), showing specifically that the 1398
activation of NADPH oxidase by Ang II is attenuated by Ang-(1-7) treatment in 1399
vitro
42
. Studies have also shown that Ang-(1-7), through binding to the Mas 1400
receptor, activates Akt-dependent pathways including the stimulation of Akt 1401
phosphorylation via Akt kinase (Sampaio et al 2007, Munoz et al 2010). These 1402
pathways ultimately lead to eNOS activation and increases in NO production 1403
through mechanisms similar to those seen in our study, specifically 1404
phosphorylation of eNOS at Ser1177 and dephosphorylation of eNOS at Thr495. 1405
An interesting result was seen when measuring ROS in diabetic bone 1406
marrow, where blockade of the AT
1
receptor using losartan inhibited the effects 1407
of Ang-(1-7). This was the only marker of oxidative stress evaluated in this study 1408
affected by Ang-(1-7) that was blocked by losartan. There are a few potential 1409
explanations for this effect. First, RAS receptor expression may be altered in 1410
diabetic bone marrow, which could change the dynamics of AT
1
receptor 1411
blockade or Mas and AT
2
receptor activation. While we did not investigate bone 1412

56
marrow RAS expression in the current study, other groups have demonstrated 1413
that AT
1
receptor expression can be altered by various factors. Specifically, 1414
decreased circulating Ang II levels have been shown to up-regulate AT
1
receptor 1415
expression (Lassegue et al 1995). Losartan administration itself has also been 1416
shown to decrease myocardial AT
1
receptor expression (Zong et al 2011). In 1417
addition, other non-RAS factors can alter AT
1
receptor expression, including the 1418
cytokines IL-1β, IL-1, and TNF-α, which are increased in diabetes (Nickenig et al 1419
2002). The second explanation could be the dose used for Ang-(1-7) and length 1420
of treatment in this study, which was chosen based on unpublished in vivo dose 1421
escalation studies in our laboratory examining progenitor cell counts in diabetic 1422
bone marrow. In vitro studies from other groups have shown that at higher doses, 1423
Ang-(1-7) may have some agonist activity at AT
1
receptors (Gurzu et al 2005). 1424
Physiological concentrations of Ang-(1-7) are approximately in the 10
-9
molar (M) 1425
range (Pinheiro et al 2012). Our studies were performed using much higher 1426
doses ranging from 100-1000 µg/kg/day for 14 days, where 500 µg/kg/day was 1427
shown to be the most effective at increasing progenitor cell numbers. However, 1428
the effects of various doses of Ang-(1-7) on oxidative stress markers were not 1429
determined. Additional studies will have to be performed to thoroughly investigate 1430
the effects of various Ang-(1-7) doses and study durations on RAS expression 1431
oxidative stress markers in diabetic bone marrow. In addition, Ang-(1-7)- 1432
associated increases in bone marrow nitrite levels were not significantly blocked 1433
by co-administration with icatibant, although icatibant did block the effects of all 1434

57
other changes in oxidative stress markers caused by Ang-(1-7). There was 1435
however a decrease (although not significant) in mean bone marrow nitrite levels 1436
in the Ang-(1-7) group. Based on other published studies and our data, we 1437
believe the bradykinin B
2
receptor plays an integral role in the mechanism of 1438
action of Ang-(1-7), and the results in this study may be a reflection of the high 1439
variation of nitrite levels often seen in tissue samples from in vivo studies. 1440
1441
2.5 Conclusion 1442
1443
In conclusion, our findings demonstrate that SC Ang-(1-7) treatment for 14 1444
days decreased diabetes-induced oxidative stress in the bone marrow and had 1445
significant effects on cellular components, including NADPH oxidase and SOD 1446
(Mordwinkin et al 2012b). While additional in vitro and in vivo studies will need to 1447
be undertaken to further dissect these mechanisms and signaling pathways, 1448
pharmacological treatment with Ang-(1-7) along with other first-line therapies 1449
may prove useful to reduce diabetes-induced oxidative stress and prevent its 1450
associated long-term complications. 1451
1452

58
2.6 Chapter 2 References 1453
1454
1455
Benter IF, Diz DI, Ferrario CM (1995) Pressor and reflex sensitivity is altered 1456
in spontaneously hypertensive rats treated with angiotensin-(1-7). 1457
Hypertension 26(6 Pt 2):1138-1144. 1458
1459
Benter IF, Ferrario CM, Morris M, Diz DI (1995) Antihypertensive actions of 1460
angiotensin-(1-7) in spontaneously hypertensive rats. Am J Physiol 269(1 Pt 1461
2):H313-319. 1462
1463
Bitar MS, Wahid S, Mustafa S, Al-Saleh E, Dhaunsi GS, Al-Mulla F (2005) 1464
Nitric oxide dynamics and endothelial dysfunction in type II model of genetic 1465
diabetes. Eur J Pharmacol 511(1):53-64. 1466
1467
Castro CH, Santos RA, Ferreira AJ, Bader M, Alenina N, Almeida AP (2005) 1468
Evidence for a functional interaction of the angiotensin-(1-7) receptor Mas 1469
with AT
1
and AT
2
receptors in the mouse heart Hypertension 46(4):937-42. 1470
1471
Ceriello A, Motz E. Is oxidative stress the pathogenic mechanism underlying 1472
insulin resistance, diabetes, and cardiovascular disease? The common soil 1473
hypothesis revisited (2004) Arterioscler Thromb Vasc Biol 24(5):816-23. 1474
1475
Chi Q, Wang T, Huang K (2005) Effect of insulin nitration by peroxynitrite on 1476
its biological activity Biochem Biophys Res Commun 330(3):791-6. 1477
1478
Cosentino F, Hishikawa K, Katusic ZS, Luscher TF. High glucose increases 1479
nitric oxide synthase expression and superoxide anion generation in human 1480
aortic endothelial cells (1997) Circulation 96(1):25-8. 1481
1482
Costa MA, Lopez Verrilli MA, Gomez KA, Nakagawa P, Pena C, Arranz C, 1483
Gironacci MM (2010) Angiotensin-(1-7) upregulates cardiac nitric oxide 1484
synthase in spontaneously hypertensive rats. Am J Physiol Heart Circ Physiol 1485
299(4):H1205-11. 1486
1487
Dias-Peixoto MF, Santos RA, Gomes ER, Alves MN, Almeida PW, Greco L, 1488
Rosa M, Fauler B, Bader M, Alenina N, Guatimosim S (2008) Molecular 1489
mechanisms involved in the angiotensin-(1-7)/Mas signaling pathway in 1490
cardiomyocytes. Hypertension 52(3):542-548. 1491
1492
Fernandes L, Fortes ZB, Nigro D, Tostes RC, Santos RA, Catelli De Carvalho 1493
MH (2001) Potentiation of bradykinin by angiotensin-(1-7) on arterioles of 1494
spontaneously hypertensive rats studied in vivo. Hypertension 37 (2 Part 2): 1495
703-709. 1496

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Ferrario CM, Averill DB, Brosnihan KB, Chappell MC, Iskandar SS, Dean RH, 1497
Diz DI (2002) Vasopeptidase inhibition and Ang-(1-7) in the spontaneously 1498
hypertensive rat. Kidney Int 62(4):1349-1357. 1499
1500
Gembardt F, Grajewski S, Vahl M, Schultheiss HP, Walther T (2008) 1501
Angiotensin metabolites can stimulate receptors of the Mas-related genes 1502
family. Mol Cell Biochem 319(1-2):115-123. 1503
1504
Gorelik G, Carbini LA, Scicli AG (1998) Angiotensin 1-7 induces bradykinin- 1505
mediated relaxation in porcine coronary artery. J Pharmacol Exp Ther 286(1): 1506
403-410. 1507
1508
Haznedaroglu IC (2010) Pathobiological aspects of the local bone marrow 1509
renin-angiotensin system: a review J Renin Angiotensin Aldosterone Syst 1510
11(4):205-13. 1511
1512
Heitsch H, Brovkovych S, Malinski T, Wiemer G (2001) Angiotensin-(1-7)- 1513
Stimulated Nitric Oxide and Superoxide Release From Endothelial Cells. 1514
Hypertension 37(1):72-76. 1515
1516
Heringer-Walther S, Eckert K, Schumacher SM, Uharek L, Wulf-Goldenberg 1517
A, Gembhardt F, Schultheiss HP, Rodgers K, Walther T (2009) Angiotensin- 1518
(1-7) stimulates hematopoietic progenitor cells in vitro and in vivo 1519
Haematologica 94(6):857-60. 1520
1521
Hink U, Li H, Mollnau H, Oelze M, Matheis E, Hartmann M, Skatchkov M, 1522
Thaiss F, Stahl RA, Warnholtz A, Meinertz T, Griendling K, Harrison DG, 1523
Fostermann U, Munzel T (2001) Mechanisms underlying endothelial function 1524
in diabetes mellitus Circ Res 88(2):E14-22. 1525
1526
Hohenstein B, Hugo CP, Hausknecht B, Boehmer KP, Riess RH, Schmieder 1527
RE (2008) Analysis of NO-synthase expression and clinical risk factors in 1528
human diabetic nephropathy Nephrol Dial Transplant 23(4):1346-54. 1529
1530
Inoguchi T, Sonta T, Tsubouchi H, Etoh T, Kakimoto M, Sonoda N, Sato N, 1531
Sekiguvhi N, Kobayashi K, Sumimoto H, Utsumi H, Nawata H (2003) Protein 1532
kinase C-dependent increase in reactive oxygen species (ROS) production in 1533
vascular tissues of diabetes: role of vascular NAD(P)H oxidase J Am Soc 1534
Nephrol 14(8 Suppl 3):S227-32. 1535
1536
Jujo K, Ii M, Losordo DW (2008) Endothelial progenitor cells in 1537
neovascularization of infarcted myocardium J Mol Cell Cardiol 45(4):530-44. 1538
1539

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Kamata K, Kobayashi T (1996) Changes in superoxide dismutase mRNA 1540
expression by streptozotocin-induced diabetes Br J Pharmacol 119(3):583-9. 1541
1542
Kocic R, Radenkovic S, Mikic D, Kocic G, Cvetkovic T, Pavlovic D (1998) 1543
Oxidative stress in the development of diabetes during hypothyroidism 1544
Postgrad Med J 74(872):381 1545
1546
Lassegue B, Alexander RW, Nickenig G, Clark M, Murphy TJ, Griendlink KK 1547
(1995) Angiotensin II down-regulates the vascular smooth muscle AT1 1548
receptor by transcriptional and post-transcriptional mechanisms: evidence for 1549
homologous and heterologous regulation Mol Pharmacol 48(4):601-9. 1550
1551
Li P, Chappell MC, Ferrario CM, Brosnihan KB (1997) Angiotensin-(1-7) 1552
augments bradykinin-induced vasodilation by competing with ACE and 1553
releasing nitric oxide. Hypertension 29(1 Pt 2):394-400. 1554
1555
Maritin AC, Sanders RA, Watkins JB (2003) Diabetes, oxidative stress, and 1556
antioxidants: A review. J Biochem and Mol Toxicol 17(1):24-38. 1557
1558
Munoz MC, Giani JF, Dominici FP (2010) Angiotensin-(1-7) stimulates the 1559
phosphorylation of Akt in rat extracardiac tissues in vivo via receptor Mas 1560
Regul Pept 161(1-3):1-7. 1561
1562
Nickenig G (2002) Central role of the AT(1)-receptor in atherosclerosis J Hum 1563
Hypertens 16(Suppl 3):S26-33. 1564
1565
Pinheiro SV and Simoes e Silva AC (2012) Angiotensin converting enzyme 2, 1566
angiotensin-(1-7), and receptor Mas axis in the kidney. Int J Hypertens 1567
2012:1-8. 1568
1569
Radi R. Nitric oxide, oxidants, and protein tyrosine nitration (2004) Proc Natl 1570
Acad Sci USA 101(12):4003-8. 1571
1572
Rodgers K, Xiong S, DiZerega GS (2003) Effect of angiotensin II and 1573
angiotensin(1-7) on hematopoietic recovery after intravenous chemotherapy. 1574
Cancer Chemother Pharmacol 51(2):97-106. 1575
1576
Rodgers KE, Oliver J, diZerega GS (2006) Phase I/II dose escalation study of 1577
angiotensin 1-7 [A(1-7)] administered before and after chemotherapy in 1578
patients with newly diagnosed breast cancer Cancer Chemother Pharmacol 1579
57(5):559-68. 1580
1581

61
Rodgers KE, Xiong S, diZerega GS (2002) Accelerated recovery from 1582
irradiation injury by angiotensin peptides. Cancer Chemother Pharmacol 1583
49(5):403-411. 1584
1585
Roks AJ, van Geel PP, Pinto YM, Buikema H, Henning RH, de Zeeuw D, van 1586
Gilst WH (1999) Angiotensin-(1-7) is a modulator of the human renin- 1587
angiotensin system. Hypertension 34(2):296-301. 1588
1589
Sabuhi R, Ali Q, Asghar M, Al-Zamily NR, Hussain T (2011) Role of the 1590
angiotensin II AT2 receptor in inflammation and oxidative stress: opposing 1591
effects in lean and obese Zucker rats. Am J Physiol Renal Physiol 300(3): 1592
F700-706. 1593
1594
Sampaio WO, Henrique de Castro C, Santos RA, Schiffrin EL, Touyz RM 1595
(2007) Angiotensin-(1-7) counterregulates angiotensin II signaling in human 1596
endothelial cells Hypertension 50(6):1093-8. 1597
1598
Sampaio WO, Souza dos Santos RA, Faria-Silva R, da Mata Machado LT, 1599
Schiffrin EL, Touyz RM (2007) Angiotensin-(1-7) through receptor Mas 1600
mediates endothelial nitric oxide synthase activation via Akt-dependent 1601
pathways 49(1):195-92. 1602
1603
Santos RA, Simoes e Silva AC, Maric C, Silva DM, Machado RP, de Buhr I, 1604
Heringer-Walther S, Pinheiro SV, Lopes MT, Bader M, Mendes EP, Lemos 1605
VS, Campagnole-Santos MJ, Schultheiss HP, Speth R, Walther T (2003) 1606
Angiotensin-(1-7) is an endogenous ligand for the G protein-coupled receptor 1607
Mas. Proc Natl Acad Sci USA 100(14):8258-8263. 1608
1609
Schmaier AH (2003) The kallikrein-kinin and the renin-angiotensin systems 1610
have a multilayered interaction Am J Physiol Regul Integr Comp Physiol 1611
285(1):R1-13. 1612
1613
Shen B, El-Dahr SS. Cross-talk of the renin-angiotensin and kallikrein-kinin 1614
systems (2006) Biol Chem 387(2):145-50. 1615
1616
Turko IV, Li L, Aulak KS, Stuehr DJ, Chang JY, Murad F (2003) Protein 1617
tyrosine nitration in the mitochondria from diabetic mouse heart. Implications 1618
to dysfunctional mitochondria in diabetes J Biol Chem 278(36):33972-7. 1619
1620
Ueda S, Masumori-Maemoto S, Wada A, Ishii M, Brosnihan KB, Umemura S 1621
(2001) Angiotensin(1-7) potentiates bradykinin-induced vasodilatation in man. 1622
J Hypertension 19(11): 2001-2009. 1623
1624
1625

62
Wang Y, Qian C, Roks A, Westermann D, Schumcher SM, Escher F, 1626
Schoemaker R, Reudelhuber TL, van Gilst W, Schultheiss HP, Tschope C, 1627
Walther T (2010) Circulating rather than cardiac angiotensin-(1-7) stimulates 1628
cardioprotection post myocardial infarction. Circulation: Heart Failure 1629
3(2):286-93. 1630
1631
Wang Y, Qian C, Roks AJ, Westermann D, Schumacher SM, Escher F, 1632
Schoemaker RG, Reudelhuber TL, van Gilst WH, Schultheiss HP, Tschope 1633
C, Walther T (2010) Circulating rather than cardiac angiotensin-(1-7) 1634
stimulates cardioprotection after myocardial infarction Circ Heart Fail 1635
3(2):286-93. 1636
1637
Xia L, Wang H, Munk S, Kwan J, Goldberg HJ, Fantus IG, Whiteside CI 1638
(2008) High glucose activates PKC-ζ and NADPH oxidase through autocrine 1639
TGF-β
1
signaling in mesangial cells. Am J Physiol Renal Physiol 295(6): 1640
F1705-F1714. 1641
1642
Zong WN, Yang XH, Chen XM, Huang HJ, Zheng HJ, Qin XY, Yong YH, Cao 1643
K, Huang J, Lu XZ (2011) Regulation of angiotensin-(1-7) and angiotensin II 1644
type 1 receptor by telmisartan and losartan in adriamycin-induced rat heart 1645
failure Acta Pharmacol Sin 32(11):1345-50. 1646
1647
Zou MH, Cohen R, Ullrich V (2004) Peroxynitrite and vascular endothelial 1648
dysfunction in diabetes mellitus Endothelium 11(2):89-97. 1649
1650
1651
1652
1653
1654
1655
1656
1657
1658
1659
1660
1661
1662
1663
1664
1665
1666
1667
1668
1669

63
Chapter 3 1670
The Impact of Angiotensin-(1-7) on Inflammation and Immune System 1671
Dysfunction in Diabetes 1672
1673
3.1 Background 1674
1675
The incidence and severity of infections are significantly increased in 1676
patients diagnosed with diabetes (Shah et al 2003). Increasing evidence 1677
suggests that chronic low-grade systemic inflammation, caused by an ongoing 1678
pro-inflammatory cytokine-mediated acute phase response, is involved in the 1679
pathogenesis of diabetes and its associated complications, including dysfunction 1680
of the innate immune system (Pickup et al 1997, Pickup et al 2004). A common 1681
complication in patients with diabetes is immune dysfunction, which is often 1682
associated with a decreased function in cellular innate immunity and decreased 1683
cytokine response after stimulation, which may a consequence of pre-existing 1684
low-grade systemic inflammation (Geerlings et al 1999). Clinically, this leads to 1685
an increased susceptibility to infections in diabetic patients, as well as these 1686
infections potentially being more complicated, resulting in an increased rate of 1687
hospitalization (Shal et al 2003). 1688
Originally thought to be specific in their function, various multifunctional 1689
cytokines with overlapping functions are responsible for regulation of the immune 1690
system, inflammatory response, and the initiation of hematopoiesis (Akira et al 1691
1990). With an increasingly larger body of evidence indicating the close 1692
relationship between the metabolic and immune systems, it is now clear that 1693
metabolic dysfunction, such as that seen in T2DM, can cause significant changes 1694

64
in the levels of various cytokines and proteins, leading to the chronic 1695
inflammation and immune dysfunction widely observed in patients with this 1696
disease (Wellen et al 2005). Specifically, increased circulating and tissue-specific 1697
levels of IL-1β, IL-6, TNF-α, and CRP have been reported in obese individuals 1698
and patients with diabetes, and have also been shown to be predictive of an 1699
increased risk for the development of T2DM (Hotamisligil et al 1993, Kado et al 1700
1999, Arnalich et al 2000, Pickup et al 2000, Pradhan et al 2001, Spranger et al 1701
2003). While there is an extensive body of evidence for this increased 1702
inflammation in various compartments, such as adipose tissue and the circulation, 1703
there is little published in regards to inflammation and markers of immune system 1704
function in diabetic bone marrow. In addition, the mechanism of action of current 1705
FDA-approved pharmacological therapies for diabetes focus on a reduction in 1706
plasma glucose levels, but not the underlying mechanisms of long-term damage 1707
in diabetes including inflammation. Therefore, this provides a unique opportunity 1708
to study potential novel therapeutic agents that may reduce the chronic 1709
inflammatory state seen in diabetes. 1710
Once thought to be an inactive metabolite of the vasoactive peptide Ang II, 1711
Ang-(1-7) is an endogenous seven amino acid peptide of the RAS that has been 1712
shown to be effective in stimulating hematopoietic progenitor cell proliferation, 1713
accelerating dermal healing following injury in diabetes, as well as increasing 1714
hematopoietic recovery after chemotherapy and irradiation injury in both animal 1715
and human studies (Rodgers 2001, Rodgers et al 2002, Rodgers et al 2003, 1716

65
Rodgers et al 2006). A number of published toxicokinetic studies have also 1717
demonstrated that Ang-(1-7) is safe even at supratherapeutic doses (Petty et al 1718
2009, Mordwinkin et al 2012a). Unlike Ang II, Ang-(1-7) has anti-hypertensive 1719
properties, which is partially due to antagonism of Ang II-induced 1720
vasoconstriction as well as augmentation of NO and bradykinin-induced 1721
vasodilation, which makes it an ideal pharmacological agent with limited side- 1722
effects to investigate for use in patients with diabetes. These and other effects of 1723
Ang-(1-7) are in part mediated through the GPCR Mas, however there is 1724
speculation as to the role of other RAS receptors, such as AT
2
(Santos et al 2003, 1725
Gembardt et al 2008, Mordwinkin et al 2012b). The goal of the present study is to 1726
evaluate the extent of inflammation and immune system dysfunction in the bone 1727
marrow and circulation in a murine model of T2DM, as well as investigate the 1728
role of Ang-(1-7) in decreasing inflammation and reversing immune system 1729
dysfunction in vivo. The roles of various receptors of the RAS, specifically AT
1
, 1730
AT
2
, and Mas in the mechanism of Ang-(1-7) will also be researched, in addition 1731
to the roles of NO and bradykinin, which are downstream of Mas following Ang- 1732
(1-7) binding. 1733
1734
3.2 Study Design 1735
1736
3.2.1 Animal Protocol 1737
1738
The NIH Principles of Laboratory Animal Care were followed, and the 1739
Department of Animal Resources at the University of Southern California 1740
approved this study. Six to eight week-old male BKS.Cg-Dock7
m
+/+ Lepr
db
/J 1741

66
(db/db) mice and their heterozygous controls were purchased from Jackson 1742
Laboratories (Bar Harbor, ME, USA). All mice were quarantined for one week 1743
prior to initiation of treatment, food and water were available ad libitum, and they 1744
were kept on a 12 hour light/dark cycle. 1745
Db/db mice and their heterozygous controls (n=7/group) were 1746
administered by SC injection either saline (control), inhibitors alone (losartan, 1747
PD123,319, A-779 or L-NAME at 10 mg/kg/day or icatibant at 0.4 mg/kg/day), 1748
Ang-(1-7) alone (500 µg/kg/day), or Ang-(1-7) 500 µg/kg/day combined with an 1749
inhibitor at the aforementioned doses for two weeks. The mice were weighed 1750
three times weekly, and the drug doses adjusted accordingly. Following the 14- 1751
day treatment period, the mice were euthanized and parameters investigated as 1752
described below to determine the effect of diabetes and Ang-(1-7) treatment with 1753
and without the co-administration of the various inhibitors. 1754
1755
3.2.2 Chemicals and Reagents 1756
1757
Ang-(1-7), prepared using Good Manufacturing Practices, and A-779, a 1758
Mas receptor antagonist, were purchased from Bachem (Torrance, CA, USA). 1759
Losartan, an AT
1
receptor antagonist, PD123,319, an AT
2
receptor antagonist, 1760
and L-NAME, a NOS inhibitor, were purchased from Sigma-Aldrich (Saint Louis, 1761
MO, USA). Icatibant, a bradykinin B
2
receptor antagonist, was purchased from 1762
Tocris Bioscience (Ellisville, MO, USA). Antibodies and isotype controls for flow 1763
cytometry were purchased from BD Pharmingen (San Diego, CA, USA) and 1764

67
eBioscience (San Diego, CA, USA). Primers for qRT-PCR were purchased from 1765
Integrated DNA Technologies (San Diego, CA, USA). 1766
1767
3.2.3 Harvesting of Plasma and Bone Marrow 1768
1769
Prior to sacrifice, mice were anesthetized with isoflurane, and cardiac 1770
puncture was performed using a 25-gauge needle and 3 mL syringe to collect 1771
blood. The whole blood was centrifuged at 10,000 rpm for 10 minutes, and 1772
plasma was collected and stored at -80°C until further analysis. Following 1773
euthanization, the femurs from each mouse were collected and bone marrow 1774
was harvested by flushing with PBS containing 2% fetal calf serum. Following 1775
collection of the bone marrow and aliquoting, red blood cells were lysed with a 1776
hypotonic solution, mixed with 0.04% trypan blue and the number of nucleated 1777
cells was assessed using a hematocytometer under light microscopy. 1778
1779
3.2.4 Analysis of Bone Marrow mRNA Expression 1780
1781
Total RNA was extracted from bone marrow cells using TRIzol (Invitrogen, 1782
Carlsbad, CA, USA). For each sample, approximately 100 ng of RNA was 1783
reverse-transcribed using Maxima Reverse Transcriptase (Fermentas, Glen 1784
Burnie, MD, USA). RT-PCR was conducted to examine expression of IL-1β, IL-6 1785
and TNF-α mRNA in bone marrow. Amplification of the cDNA was performed 1786
using SYBR Green PCR Master Mix (Applied Biosystems by Life Technologies, 1787
Carlsbad, CA, USA) using an ABI 7300. Expression of bone marrow IL-1β, IL-6 1788

68
and TNF-α mRNA was normalized against 18S mRNA and expressed as fold- 1789
change compared to non-diabetic controls. 1790
1791
3.2.5 Intracellular Bone Marrow Cytokine Determination 1792
1793
Following harvesting, murine bone marrow cells (2 x 10
6
per animal) were 1794
divided into two separate culture tubes (one to be stimulated and one to be left 1795
unstimulated), each containing 1 x 10
6
cells suspended in 100 µL RPMI cell 1796
culture medium. For cells to be stimulated, 2.5 µL of the protein kinase C (PKC) 1797
stimulator phorbol 12-myristate 13-acetate (PMA) at 10 µg/mL and 1 µL of the 1798
calcium ionophore A23187 at 250 µg/mL were added to each tube and mixed. To 1799
both sets of tubes, 3 µL of brefeldin A at 100 µg/mL was added and mixed well. 1800
Brefeldin A inhibits the transport of proteins from the endoplasmic reticulum (ER) 1801
to the Golgi and induces retrograde transport of proteins from the Golgi to the ER, 1802
leading to accumulation of proteins in the ER. All tubes were incubated in a 1803
humid chamber at 37°C containing 95% O
2
and 5% carbon dioxide (CO
2
) for 6 1804
hours. Following incubation, 3 mL of ice-cold wash buffer consisting of 0.5% 1805
bovine serum albumin (BSA) in PBS was added to all tubes and mixed. The cells 1806
were then centrifuged at 250 x g for 5 minutes at 4°C, and the supernatant was 1807
decanted. The cell pellets were gently resuspended in 0.5 mL of ice-cold murine 1808
globulin, gently agitated, and incubated for 15 minutes at 4°C to block. Cells were 1809
then labeled for cell-surface marker antigens by adding 1 µg of each fluorophore- 1810
conjugated rat anti-mouse antibody Flk-1, Sca-1, c-Kit or isotope control per tube, 1811
and incubated for 30 minutes at 4°C in the dark. Following incubation, 3 mL of 1812

69
ice-cold wash buffer was added to all tubes and mixed. The cells were then 1813
centrifuged at 250 x g for 5 minutes at 4°C, the supernatant was decanted, and 1814
this was repeated for a total of two washes. Cells were fixed by adding 500 µL 1815
4% paraformaldehyde to each tube and incubating for 30 minutes in the dark at 1816
4°C. The cells were then washed with 3 mL of ice-cold wash buffer, centrifuged 1817
at 250 x g for 5 minutes at 4°C, and supernatant decanted. To permeabilize the 1818
cells, 1 mL of permeabilizing solution (0.1% w/v saponin) was added to each 1819
tube and cell pellets were gently resuspended. The cells were incubated for 15 1820
minutes in the dark at room temperature, and gently mixed several times during 1821
the incubation period. Three mL of ice-cold permeabilizing solution was then 1822
added to the cells, gently mixed, and cells were centrifuged at 250 x g for 5 1823
minutes at 4°C, supernatant decanted, and repeated for a total of two washed. 1824
Cell pellets were then resuspended in 0.25 mL of 20 µg/mL ice-cold murine 1825
gamma globulin and 0.25 mL permeabilization solution and incubated for 30 1826
minutes at 4°C in the dark. To label intracellular cytokines, 1 µg of each 1827
fluorophore-conjugated rat anti-mouse IL-6 and TNF-α or isotype control was 1828
added to each tube and incubated for 30 minutes at 4°C in the dark. The cells 1829
were then washed with 3 mL of ice-cold wash buffer, centrifuged at 250 x g for 5 1830
minutes at 4°C, supernatant decanted, and this was repeated for a total of three 1831
washes. Cell pellets were resuspended in 0.5 mL of 1% paraformaldehyde and 1832
stored in the dark at 4°C until flow cytometric analysis (Babcock 2004). 1833
1834
1835

70
3.2.6 Plasma Cytokine Measurements 1836
1837
Plasma IL-1β, IL-6, and TNF-α concentrations were determined using 1838
commercially available Mouse Ready-SET-Go! ELISA Kits (eBioscience, San 1839
Diego, CA). The assays were performed according to the manufacturer protocol. 1840
Standard curves and samples were prepared in duplicate, with plasma being 1841
diluted 1:5 with dilution buffer. 1842
1843
3.2.7 Bone Marrow CFU-GEMM and CFU-GM Assay 1844
To determine bone marrow CFU-granulocyte, erythrocyte, monocyte, and 1845
macrophage (GEMM) and CFU-GM colony counts, isolated bone marrow cells 1846
were diluted in Iscove’s MDM with 2% FBS at 5 x 10
5
cells per mL. In sterile 1847
culture tubes, 0.3 mL of diluted bone marrow cells were added to 3 mL 1848
MethoCult
®
medium and vortexed. To dispense the MethoCult
®
medium and 1849
cells into each well, a 16 gauge blunt-end needle was attached to a 3 cc syringe. 1850
Air was expelled from the syringe, and 1.1 mL of methylcellulose medium and 1851
cell mixture was dispensed into each well of a 6-well plate. Cultures were placed 1852
into an incubator maintained at 37°C, 5% CO
2
in air with 95% humidity. After 12 1853
days of culture, CFU-GEMM and CFU-GM colonies were counted under phase 1854
contrast microscopy. 1855
1856
3.2.8 Bone Marrow-Derived Dendritic Cell Cultures 1857
1858
Following isolation, bone marrow cells were seeded at 200,000 cells per 1859
mL, 1 mL per well in 24-well plates in RPMI with 10% fetal calf serum and 20 1860

71
ng/mL recombinant mouse granulocyte-macrophage colony stimulating factor 1861
(rGM-CSF). Medium was replaced every 3 days. At day 9 of culture, dendritic 1862
cells were identified by staining for 30 minutes at 4°C in the dark with 1863
fluorophore-conjugated rat anti-mouse CD11c, anti-MHC II, anti-CD80 and anti- 1864
CD86 antibodies diluted in PBS containing 0.5% BSA and 0.01% sodium azide. 1865
Dead cells and debris were excluded using propidium iodide, and cell pellets 1866
were fixed in 0.5 mL of 4% paraformaldehyde and stored in the dark at 4°C until 1867
flow cytometric analysis (Lutz et al 1999). 1868
1869
3.2.9 Bone Marrow CFU Pre-B Cell Assay 1870
1871
To quantify Pre-B cells from bone marrow, a colony forming unit (CFU) 1872
assay was used according to the manufacturer’s protocol. After isolation of bone 1873
marrow, cells were diluted to 1 x 10
5
per mL in Iscove’s Modified Dulbecco’s 1874
Medium (MDM) with 2% FBS. In sterile culture tubes, 0.3 mL of diluted bone 1875
marrow cells were added to 3 mL MethoCult
®
medium (StemCell Technologies, 1876
USA) and vortexed to ensure all cells and components were thoroughly mixed. 1877
Tubes were allowed to stand for 5 minutes to allow bubbles to dissipate. To 1878
dispense the MethoCult
®
medium and cells into each well, a 16 gauge blunt-end 1879
needle was attached to a 3 cc syringe. Air was expelled from the syringe, and 1.1 1880
mL of methylcellulose medium and cell mixture was dispensed into each well of a 1881
6-well plate. The methylcellulose medium was distributed evenly by gently tilting 1882
and rotating each plate. Cultures were placed into an incubator maintained at 1883

72
37°C, 5% CO
2
in air with 95% humidity. After 7 days of culture, CFU Pre-B cell 1884
colonies were counted under phase contrast microscopy. 1885
1886
3.2.10 Statistical Analysis 1887
1888
GraphPad Prism version 5.0d for Mac OS X (GraphPad Software, San 1889
Diego, CA, USA) was used to analyze the data. One-way ANOVA followed by 1890
Tukey’s test was used to compare data from more than two groups. The level of 1891
statistical significance was set at 5%. Data are expressed as mean value ± SEM. 1892
1893
3.3 Results 1894
1895
3.3.1 Bone Marrow Cytokine Expression 1896
1897
The mRNA expression levels of IL-1β were unchanged in diabetic bone 1898
marrow compared to non-diabetic controls, however there were significant 1899
increases in both IL-6 and TNF-α mRNA expression in diabetic bone marrow 1900

Figures 10A-C. Ang-(1-7) effect on bone marrow cytokine expression. Both IL-6 and TNF-α mRNA expression
were significantly increased in diabetic bone marrow compared to wild-type controls (p<0.01). Ang-(1-7)
administration to diabetic mice resulted in a significant increase in bone marrow IL-1β and IL-6 mRNA expression,
and a significant decrease in bone marrow TNF-α expression (p<0.01). The effects of Ang-(1-7) in diabetic mice
were inhibited by PD123,319, A-779, L-NAME, and icatibant (p<0.01).

73
(Figures 10A-C, p<0.01). Administration of Ang-(1-7) to diabetic mice for 14 days 1901
resulted in a significant increase in bone marrow IL-1β and IL-6 mRNA 1902
expression, while TNF-α mRNA expression was significantly decreased (p<0.01). 1903
Co-administration of Ang-(1-7) with PD123,319, A-779, icatibant, or L-NAME, but 1904
not losartan, resulted in a significant blockade of the effects of Ang-(1-7) 1905
administration alone in diabetic mice (p<0.01). 1906
1907
3.3.2 Bone Marrow Intracellular Cytokines 1908
1909
Since IL-1β mRNA expression was unchanged in diabetic bone marrow, 1910
production of only IL-6 and TNF-α was examined. As these cytokines are 1911
normally secreted, we measured intracellular levels with and without stimulation 1912
for 6 hours with PMA (activates PKC) and A23187 (a calcium ionophore) in the 1913
presence of a Golgi plug to prevent secretion. In unstimulated diabetic whole 1914
bone marrow, intracellular levels of both IL-6 and TNF-α were significantly 1915

Figures 11A-B. Bone marrow intracellular cytokine levels. Intracellular bone marrow IL-6 and TNF-α levels
were significantly increased in diabetic mice prior to stimulation, while stimulation did not result in a significant
increase in these cytokines. Treatment with Ang-(1-7) resulted in not only a significant decrease in baseline
cytokine levels, but also a restoration in response of these cytokines to stimulation.

74
increased compared to non-diabetic controls, consistent with the gene 1916
expression studies (Figures 11A-B, p<0.01). Following stimulation of non-diabetic 1917
bone marrow cells with PMA and A23187, there was a significant increase in 1918
intracellular IL-6 and TNF-α levels in whole bone marrow. However, in diabetic 1919
bone marrow, stimulation did not result in a significant increase in intracellular 1920
levels of either of these cytokines compared to unstimulated bone marrow, 1921
suggesting tolerance was induced by the prior in vivo exposure of high levels of 1922
these cytokines. After treatment with Ang-(1-7) for 14 days in diabetic mice, 1923
intracellular IL-6 and TNF-α levels were significantly reduced prior to stimulation, 1924
and significant increases in TNF-α (but not IL-6) were seen following stimulation 1925
compared to saline-treated diabetic mice. The effects of Ang-(1-7) were blocked 1926
with co-administration of the Mas receptor antagonist A-779, but not with co- 1927
administration of the other antagonists or inhibitors. Since bone marrow-derived 1928
EPC play an important role in diabetes, intracellular cytokines were also 1929
measured in cells expressing Flk-1
+
and Sca-1
+
bone marrow cells. When 1930
intracellular cytokine levels were measured in Flk-1
+
and Sca-1
+
bone marrow 1931
cells (EPC), similar results were seen to those in the whole bone marrow. 1932
1933
3.3.3 Plasma Cytokine Levels 1934

Figures 12A-C. Plasma cytokine levels. Similar to the bone marrow, plasma IL-6 and TNF-α levels were
significantly increased in diabetic mice (p<0.05). Ang-(1-7) treatment resulted in significant decreases in IL-1β,
IL-6, and TNF-α levels in diabetic mice (p<0.05). The effects of Ang-(1-7) were significantly inhibited by
PD123,319, A-779, L-NAME, and icatibant (p<0.05).

75
1935
In order to correlate cytokine expression in the bone marrow with changes 1936
in cytokines in the circulation, ELISAs were performed to measure plasma 1937
cytokine levels. Similar to gene expression and bone marrow intracellular 1938
cytokine levels, IL-1β levels remained unchanged (Figure 12A), while plasma 1939
IL-6 and TNF-α levels were significantly increased in diabetic mice (Figures 12B- 1940
C; p<0.05). Ang-(1-7) administration resulted in significant decreases in IL-1β, IL- 1941
6, and TNF-α levels in diabetic plasma (p<0.05). The effects of Ang-(1-7) in 1942
diabetic plasma were blocked by co-administration with A-779, PD123,319, L- 1943
NAME, and icatibant (p<0.05). 1944
1945
3.3.4 Bone Marrow-Derived CFU-GEMM and CFU-GM colonies 1946
The bone marrow is also a source of multi-potential progenitors and 1947
lineage-restricted progenitors of the megakaryocyte, erythroid, granulocytic, and 1948
monocyte-macrophage pathways, including CFU-GEMM and CFU-GM. CFU- 1949
GEMM is a multipotential progenitor that contributes to all non-lymphocytic 1950
lineages of the hematopoietic system. CFU-GM cells are derived from CFU- 1951
GEMM and are the precursor to CFU-G (differentiates into granulocytes or 1952
neutrophils) and CFU-M (precursor to the monocyte/macrophage lineage). After 1953
12 days of culture, bone marrow CFU-GEMM and CFU-GM colonies were 1954
significantly reduced in db/db mice compared to non-diabetic controls (p<0.01). 1955
There were significant increases in both bone marrow-derived CFU-GEMM and 1956
CFU-GM colony counts mice from Ang-(1-7) treated mice as compared to 1957

76
diabetic mice administered saline (p<0.01). In diabetic mice, Ang-(1-7)-mediated 1958
activity was blocked by A-779, PD123,319, icatibant, or L-NAME, but not losartan, 1959
and resulted in significant decreases in CFU-GEMM and CFU-GM colony counts 1960
compared to db/db mice receiving Ang-(1-7) alone (p<0.01). 1961
1962
3.3.5 Bone Marrow-Derived Dendritic Cells and CFU Pre-B Cell Colonies 1963
1964
Since diabetes has been shown to alter components of the innate immune 1965
system, the number of dendritic cell progenitors in the bone marrow and the 1966
activation state of the dendritic cells produced in culture (by assessment of cell 1967
surface expression of CD80, CD86, MHC II and CD11c) were examined using 1968

Figures 13A-D. Bone marrow-derived dendritic cell markers. Dendritic cells cultured from diabetic bone marrow
had a significant reduction in the number of cell surface functional markers CD80 and CD85 (p<0.01). Treatment
with Ang-(1-7) resulted in significant increases in both the number of CD11c, MHC II, CD80 and CD86-positive
bone marrow dendritic cells (p<0.01), while this effect was significantly inhibited by co-administration with
PD123,319, A-779, L-NAME and icatibant in diabetic mice.

77
flow cytometry. After 9 days of culture, there was no significant differences in the 1969
number of CD11c or MHC II positive cells (Figures 13A-B), while bone marrow 1970
cells from db/db mice had significantly lower numbers of CD80 and CD86 1971
positive cells compared to non-diabetic controls (Figures 13C-D). Treatment with 1972
Ang-(1-7) 500 µg/kg/day in diabetic mice was able to significantly increase bone 1973
marrow cells positive for CD11c, MHC II, CD80, and CD86 (p<0.01). Again, the 1974
effects of Ang-(1-7) were blocked by co-administration with A-779, PD123,319, L- 1975
NAME, and bradykinin. 1976
Due to the interaction between dendritic cells and B cells in the immune 1977
system and the centrality of B cells to adaptive immunity, CFU Pre-B cell 1978
colonies were also quantified in the bone marrow. After 7 days of culture, bone 1979

Figure 14. Bone marrow CFU Pre-B cell colonies. Bone marrow cells cultured from diabetic bone marrow had
significantly less CFU Pre-B cells compared to non-diabetic controls (p<0.01). Treatment with Ang-(1-7) resulted in a
significant increase in CFU Pre-B cells in diabetic bone marrow compared to saline-treated diabetic mice (p<0.01).

78
marrow isolated from db/db mice had significantly lower numbers of CFU Pre-B 1980
cell colonies compared to bone marrow cells isolated from non-diabetic controls 1981
(Figure 14, p<0.01). Cultured bone marrow from db/db mice administered Ang- 1982
(1-7) for 14 days had significantly higher CFU Pre-B cell colony counts compared 1983
to db/db mice treated with saline (2918 versus 1074 colonies per femur, 1984
respectively; p<0.01). When Ang-(1-7) was co-administered with losartan, A-779, 1985
PD123,319, icatibant, or L-NAME for the 14 day treatment period in diabetic mice, 1986
the effects seen with Ang-(1-7) administration alone in diabetic mice were 1987
blocked (p<0.05). 1988
1989
3.4 Discussion and Significance 1990
1991
Diabetes is commonly associated with dysfunction of the immune system, 1992
which can manifest itself in many ways, including a reduction in wound healing 1993
and increased risk and severity of infections. A number of studies evaluating 1994
immune dysfunction and diabetes have demonstrated that while some 1995
disturbances in the adaptive humoral immune response exist, it is the dysfunction 1996
found in components of the innate immune system that may lead to the overall 1997
decreased immune response in diabetic patients. However, there is a lack of 1998
data in the literature in regards to bone marrow-derived progenitors of the innate 1999
immune response, such as CFU-GEMM and CFU-GM, as well as cells that 2000
bridge innate and adaptive immune systems (dendritic cells) and of the adaptive 2001
immune response (B cells). Diabetic patients may have reduced immune 2002
function secondary to inflammatory tolerance of the innate immune system; 2003

79
however immune complications may result from deficits in antigen presentation 2004
and lymphocytes. In addition, there is also limited research on potential 2005
treatments for these deficiencies. 2006
Dendritic cells play an important role as antigen-presenting cells, and are 2007
a critical link between innate and adaptive immunity. Once released from the 2008
bone marrow into the circulation, dendritic cell progenitors mature into dendritic 2009
cells, which are the primary antigen-presenting cell for adaptive responses to 2010
new antigens (Nahmod et al 2010). Impaired yields in numbers, defective 2011
maturation, and reduced function of dendritic cells has been reported in diabetes, 2012
which can lead to immune dysfunction, inflammation, over-activation of various 2013
immune cells, and autoimmune disease (Jansen et al 1995, Takahashi et al 2014
1998). Indeed, in the present study, while markers used to identify bone marrow- 2015
derived dendritic cell numbers were unchanged in diabetic mice, there was a 2016
significant decrease in dendritic cell functional markers CD80 and CD86 in the 2017
bone marrow of db/db mice. Ang-(1-7) treatment significantly increased not only 2018
bone marrow-derived dendritic cell counts in diabetic mice (CD11c and MHC II), 2019
but also increased the number of dendritic cells expressing the functional 2020
markers CD80 and CD86, which are both involved in co-stimulation of T-cells 2021
(van Rijt et al 2004). Since the effects of Ang-(1-7) treatment in diabetic mice 2022
were blocked by A-779, PD123,319, L-NAME, and icatibant this suggests that 2023
these effects of Ang-(1-7) were mediated via both the Mas and AT
2
receptors, 2024
and involved a NO and bradykinin-dependent mechanism. 2025

80
Dendritic cells are also a critical link to the humoral immune response, 2026
including B cells. Pre-B cells are produced in the bone marrow, and then 2027
eventually migrate to the spleen, where they are referred to as transitional B cells 2028
(Allman et al 2004). Some of these cells then differentiate into mature B- 2029
lymphocytes, which play important roles in the production of antibodies, 2030
functioning as antigen-presenting cells (APCs), and thus developing into memory 2031
B cells as components of the adaptive immune system. Studies have 2032
demonstrated important interactions between dendritic cells and B cells, where 2033
dendritic cells directly provide B cells with both proliferation and survival signals 2034
resulting in B cell growth and differentiation (Dubois et al 1999, Wykes et al 2035
2000). In the current study, we have shown that CFU Pre-B cell counts are 2036
significantly lower in the bone marrow of db/db mice compared to bone marrow 2037
isolated from non-diabetic mice. Following the 14-day treatment period with Ang- 2038
(1-7), bone marrow CFU Pre-B cell counts were significantly increased in diabetic 2039
mice compared to diabetic mice treated with saline. These effects of Ang-(1-7) 2040
were again mediated via both the Mas and AT
2
receptors, and involved a NO and 2041
bradykinin-dependent mechanism. Surprisingly, co-administration of Ang-(1-7) 2042
with losartan significantly blocked the effects of Ang-(1-7) when given alone, 2043
suggesting that the AT
1
receptor may also play a role. Blockade of the AT
1
2044
receptor with losartan reduced bone marrow CFU Pre-B cell counts by 35% 2045
compared to Ang-(1-7) administration alone, while the other antagonists and 2046
inhibitors used resulted in reductions ranging from approximately 46% to 73% 2047

81
when combined with Ang-(1-7), suggesting that the AT
1
receptor may not play as 2048
large of a role as Mas and AT
2
. Also, some in vitro studies have shown that at 2049
higher does, Ang-(1-7) may act as an agonist at AT
1
receptors (Gurzu et al 2005). 2050
Additional experiments will need to be performed to further elucidate this 2051
mechanism. 2052
The cellular deficiencies observed in db/db mice were coupled with 2053
significant increases in bone marrow IL-6 and TNF- α mRNA expression, 2054
combined with significant increases in both circulating levels and bone marrow 2055
intracellular levels of pro-inflammatory cytokines. Importantly, there were no 2056
significant increases in either intracellular IL-6 or TNF-α levels after stimulation 2057
for 6 hours with PMA and A23187 in diabetic bone marrow, suggesting that the 2058
abnormally elevated baseline (unstimulated) levels of these intracellular 2059
cytokines may prevent a normal physiological response following stimulation. 2060
This observation may be one mechanism by which immune dysfunction occurs in 2061
diabetic patients. When Ang-(1-7) was administered to diabetic mice for two 2062
weeks, two main observations were noted. First, intracellular bone marrow levels 2063
of IL-6 and TNF-α significantly decreased prior to stimulation (at baseline). 2064
Second, following stimulation, intracellular bone marrow IL-6 and TNF-α levels in 2065
db/db mice returned to levels similar to those seen in non-diabetic controls. While 2066
the effects of Ang-(1-7) on inflammatory cytokine mRNA expression were 2067
mediated through Mas and AT
2
via NO and bradykinin-dependent mechanisms, 2068

82
its effects on circulating pro-inflammatory cytokines were blocked when co- 2069
administered with A-779 only. 2070
2071
3.5 Conclusion 2072
2073
Taken together, the results from this study show that daily SC 2074
administration of Ang-(1-7) for 14 days decreases circulating levels of the 2075
inflammatory cytokines IL1β, IL-6, and TNF-α, as well as bone marrow mRNA 2076
expression of TNF-α in db/db mice. In the present study, Ang-(1-7) appeared to 2077
affect various aspects of the innate and humoral immune system altered by 2078
T2DM, in particular by decreasing baseline expression of pro-inflammatory 2079
cytokines and restoring the response of the bone marrow intracellular cytokines 2080
IL-6 and TNF-α to stimulation. In addition, Ang-(1-7) treatment resulted in 2081
increases in the number of myeloid colonies (which generate neutrophils and 2082
monocyte/macrophages) as well as the expression of functional dendritic cell 2083
surface markers, dendritic cell progenitor and dendritic cell numbers, and CFU 2084
Pre-B cells isolated from the bone marrow of db/db mice. The majority of the 2085
observed effects of Ang-(1-7) were mediated by the GPCR Mas and AT
2
via NO 2086
and bradykinin-dependent mechanisms, while some effects were only inhibited 2087
by antagonism of the Mas receptor. These results agree with other published 2088
studies showing that RAS blockade using ARB, or treatment with Ang-(1-7) can 2089
decrease inflammatory cytokines and immune markers commonly associated 2090
with T2DM and it’s complications (Tsutamoto et al 2000, Pavlatou et al 2011, 2091
Verma et al 2011). Clinical studies such as the Diabetes Control and 2092

83
Complications Trial (DCCT) have also shown that while intensive glycemic 2093
control is somewhat beneficial, the effect on levels of inflammatory markers in 2094
patients with diabetes is not uniform (Schaumberg et al 2005). This demonstrates 2095
the need for the development of additional therapeutic agents such as Ang-(1-7) 2096
that can have a greater effect on diabetes-induced inflammation and immune 2097
dysfunction. 2098
2099

84
3.6 Chapter 3 References 2100
2101
Akira S, Hirano T, Taga T, Kishimoto T (1990) Biology of multifunctional 2102
cytokines: IL 6 and related molecules (IL 1 and TNF). FASEB J 2103
4(11):2860-2867. 2104
2105
Allman D, Srivastava B, Lindsley RC (2004) Alternative routes to maturity: 2106
branch points and pathways for generating follicular and marginal zone B 2107
cells. Immunol Rev 197:147-160. 2108
2109
Arnalich F, Hernanz A, Lopez-Maderuelo D, Pena JM, Camacho J, 2110
Madero R, Vazquez JJ, Montiel C (2000) Enhanced acute-phase 2111
response and oxidative stress in older adults with type II diabetes. Horm 2112
Metab Res 32(10):407-412. 2113
2114
Babcock GF (2004) Intracellular cytokines. Curr Protoc Cytom (Chapter 2115
9):Unit 9.9. 2116
2117
Dubois B, Bridon JM, Fayette J, Barthelemy C, Banchereau J, Caux C, 2118
Briere F (1999) Dendritic cells directly modulate B cell growth and 2119
differentiation. J Leuk Bio 66(2):224-230. 2120
2121
Geerlings SE and Hoepelman AI (1999) Immune dysfunction in patients 2122
with diabetes mellitus (DM). FEMS Immunol Med Microbiol 26(3-4):259- 2123
265. 2124
2125
Gembardt F, Grajewski S, Vahl M, Schultheiss H, Walther T (2008) 2126
Angiotensin metabolites can stimulate receptors of the Mas-related genes 2127
family. Mol Cell Biochem 319(1-2):115-123. 2128
2129
Gurzu B, Costuleanu M, Slatineanu SM, Ciobanu A, Petrescu G (2005) 2130
Are multiple angiotensin receptor types involved in angiotensin-(1-7) 2131
actions on isolated rat portal vein J Renin Angiotensin Aldosterone Syst 2132
6(2):90-5. 2133
2134
Hotamisligil GS, Shargill NS, Spiegelman BM (1993) Adipose expression 2135
of tumor necrosis factor-alpha: direct role in obesity-linked insulin 2136
resistance. Science 259(5091):87. 2137
2138
Jansen A, van Hagen M, Drexhage HA (1995) Defective maturation and 2139
function of antigen-presenting cells in type 1 diabetes. Lancet 2140
345(8948):491-492. 2141
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Kado S, Nagase T, Nagata N (1999) Circulating levels of interleukin-6, its 2144
soluble receptor and interleukin-6/interleukin-6 receptor complexes in 2145
patients with type 2 diabetes mellitus. Acta Diabetol 36(1-2):67-72. 2146
2147
Lutz MB, Kukutsch N, Ogilvie AL, Rossner S, Koch F, Romani N, Schuler 2148
G (1999) An advanced culture method for generating large quantities of 2149
highly pure dendritic cells from mouse bone marrow. J Immunol Methods 2150
223(1):77-92. 2151
2152
Mordwinkin NM, Russell JR, Burke AS, Dizerega GS, Louie SG, Rodgers 2153
KE (2012) Toxicological and toxicokinetic analysis of angiotensin (1-7) in 2154
two species. J Pharm Sci 101(1):373-380. 2155
2156
Mordwinkin NM, Meeks CJ, Jadhav SS, Espinoza T, Roda N, diZerega 2157
GS, Louie SG, Rodgers KE (2012) Angiotensin-(1-7) administration 2158
reduced oxidative stress in diabetic bone marrow. Endocrinology 153(5):1- 2159
10. 2160
2161
Nahmod K, Gentilini C, Vermeulen M, Uharek L, Wang Y, Zhang J, 2162
Schultheiss HP, Geffner J, Walther T (2010) Impaired function of dendritic 2163
cells deficient in angiotensin II type 1 receptors. J Pharmacol Exp Ther 2164
334(3):854-862. 2165
2166
Petty WJ, Miller AA, McCoy TP, Gallagher PE, Tallant EA, Torti FM (2009) 2167
Phase I and pharmacokinetic study of angiotensin-(1-7), and endogenous 2168
antiangiogenic hormone. Clin Cancer Res 15(23):7398-7404. 2169
2170
Pickup JC (2004) Inflammation and activated innate immunity in the 2171
pathogenesis of type 2 diabetes. Diabetes Care 27(3):813-823. 2172
2173
Pickup JC, Chusney GD, Thomas SM, Burt D (2000) Plasma interleukin-6, 2174
tumour necrosis factor alpha and blood cytokine production in type 2 2175
diabetes. Life Sci 67(3):291-300. 2176
2177
Pickup JC, Mattock MB, Chusney GD, Burt D (1997) NIDDM as a disease 2178
of the innate immune system: association of acute-phase reactants and 2179
interleukin-6 with metabolic syndrome X. Diabetologia 40(11):1286-1292. 2180
2181
Pradhan AD, Manson JE, Rifai N, Buring JE, Ridker PM (2001) C-reactive 2182
protein, interleukin 6, and risk of developing type 2 diabetes mellitus. 2183
JAMA 286(3):327-334. 2184
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Rodgers K, Xiong S, Felix J, Roda N, Espinoza T, Maldonado S, Dizerega 2188
G (2001) Development of angiotensin (1-7) as an agent to accelerate 2189
dermal repair. Wound Repair Regen 9(3):238-247. 2190
2191
Rodgers KE, Oliver J, diZerega GS (2006). Phase I/II dose escalation of 2192
angiotensin 1-7 [A(1-7)] administered before and after chemotherapy in 2193
patients with newly diagnosed breast cancer. Cancer Chemother 2194
Pharmacol 57(5):559-568. 2195
2196
Rodgers KE, Roda N, Felix JE, Espinoza T, Maldonado S, diZerega G 2197
(2003) Histological evaluation of the effects of angiotensin peptides on 2198
wound repair in diabetic mice. Exp Dermatol 12(6):784-790. 2199
2200
Rodgers KE, Xiong S, diZerega GS (2002) Accelerated recovery from 2201
irradiation injury by angiotensin peptides. Cancer Chemother Pharmacol 2202
49(5):403-411. 2203
2204
Santos RA, Simoes e Silva AC, Maric C, Silva DM, Machado RP, de Buhr 2205
I, Heringer-Walther S, Pinheiro SV, Lopes MT, Bader M, Mendes EP, 2206
Lemos VS, Campagnole-Santos MJ, Schultheiss HP, Speth R, Walther T 2207
(2003) Angiotensin-(1-7) is an endogenous ligand for the G protein- 2208
coupled receptor Mas Proc Natl Acad Sci USA 100(14):8258-8263. 2209
2210
Shah BR and Hux JE (2003) Quantifying the risk of infectious diseases for 2211
people with diabetes. Diabetes Care 26(2):510-513. 2212
2213
Spranger J, Kroke A, Mohlig M, Hoffmann K, Bergmann MM, Ristow M, 2214
Boeing H, Pfeiffer AF (2003) Inflammatory cytokines and the risk to 2215
develop type 2 diabetes: results of the prospective population-based 2216
European Prospective Investigation into Cancer and Nutrition (EPIC)- 2217
Potsdam Study. Diabetes 52(3):812-817. 2218
2219
Takahashi K, Honeyman MC, Harrison LC (1998) Impaired yield, 2220
phenotype, and function of monocyte-derived dendritic cells in humans at 2221
risk for insulin-dependent diabetes J Immunol 161(5):2629-2635. 2222
2223
van Rijt LS, Vos N, Willart M, Kleinjan A, Coyle AJ, Hoogsteden HC, 2224
Lambrecht BN (2004) Essential role of dendritic cell CD80/CD86 2225
costimulation in the induction, but not reactivation, of TH2 effector 2226
responses in a mouse model of asthma. J Allergy Clin Immunol 2227
114(1):166-173. 2228
2229
2230
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87
Wellen KE and Hotamisligil GS (2005) Inflammation, stress, and diabetes. 2232
J Clin Invest 115(5):1111-1119. 2233
2234
Wykes M and MacPherson G (2000) Dendritic cell-B-cell interaction: 2235
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2238

88
Chapter 4 2239
The Effects of Angiotensin-(1-7) on Bone Marrow-Derived and Circulating 2240
EPC, Bone Marrow-Derived MSC, and Cardiovascular Function in Type 2 2241
Diabetes 2242
2243
4.1 Background 2244
2245
Mature endothelial cells are terminally differentiated, making them a 2246
limited option for the therapeutic treatment of vascular disease (Churdochomjan 2247
et al 2010). However in 1997, Asahara and colleagues identified a class of 2248
circulating blood cells with the ability to proliferate, migrate to, and differentiate at 2249
sites of vascular injury. These cells, known as EPC, are able to initiate de novo 2250
production of endothelial cells and subsequent blood vessel formation, known as 2251
vasculogenesis (Asahara et al 1997). These cells have been more recently 2252
referred to as VRPC or proangiogenic hematopoietic cells (Richardson et al 2253
2010). Since this groundbreaking discovery, other groups have found that there 2254
also exist bone marrow-derived EPC which may play an important role in 2255
vasculogenesis following injury in various tissues and organs (Pacilli et al 2010, 2256
Tongers et al 2010). In fact the bone marrow is a major source of EPC in the 2257
body. Since EPC play a vital role in the formation and maintenance of the 2258
vasculature, disease states that are associated with decreased numbers or 2259
dysfunction of these progenitor cells have an increased risk of developing 2260
complications involving the cardiovascular system. In addition, other studies have 2261
shown that decreased levels of circulating EPC may be predictive of various 2262
disease states, including CVD and CAD (Werner et al 2005, Zeoli et al 2009). 2263
One disease associated with decreases in both circulating and bone marrow 2264

89
EPC levels is diabetes, which is believed to be a major mechanism involved in 2265
the increased incidence of vascular complications in patients diagnosed with this 2266
disease, even in those with good glycemic control (Tepper et al 2002, Loomans 2267
et al 2005). Multiple mechanisms have been implicated in the reduced numbers 2268
and function of EPC in diabetes, including dysfunction of the RAS, increased 2269
oxidative stress leading to decreased NO bioavailability, cellular apoptosis due to 2270
increased levels of AGE, and decreased levels of adipokines such as adiponectin, 2271
among others (Chen et al 2007, Gallagher et al 2007, Thum et al 2007, Shibata 2272
et al 2008, Shen et al 2010). Initial clinical trials focused on the direct 2273
administration of EPC using methods such as intracoronary injection. However, 2274
two major drawbacks in using EPC therapeutically are their low circulating levels 2275
(approximately 1% of all peripheral blood cells in healthy individuals), and 2276
controversy over identification of cell surface markers and culturing techniques 2277
(Richardson et al 2010). Their profiles may change as they differentiate, and they 2278
also share cell surface markers with hematopoietic stem cells. 2279
EPC are not the only class of bone marrow-derived progenitor or stem 2280
cells involved in tissue repair. MSC are a rare population of immunotolerant, 2281
pluripotent, self-renewing fibroblast-like cells that account for approximately 2282
0.001-0.01% of nucleated cells in the bone marrow stroma, and because of their 2283
long-term viability also play an important role in neovascularization and tissue 2284
repair (Pittenger et al 2004, Zangi et al 2006, Brunt et al 2007). MSC were first 2285
identified in vitro and referred to as fibroblast colony-forming units (CFU-F) by 2286

90
Friedenstein and colleagues (Friedenstein et al 1974). MSC are able to 2287
differentiate into a multitude of cells in vitro and in vivo, including osteoblasts, 2288
chondrocytes, cardiac myocytes, bone marrow, fibroblasts, and adipocytes 2289
(Pereira et al 1995, Barbash et al 2003, Caplan 2009). Some also believe that 2290
MSC have the ability to mobilize into the circulation in order to assist in the repair 2291
process (Roufosse et al 2004). However, similar to EPC, identification of MSC 2292
can be difficult, as they share some cell-surface markers with HSC. Recent 2293
research has demonstrated that due to some of their unique properties, MSC can 2294
potentially be used therapeutically as allogeneic or universal donor cells in the 2295
treatment of diabetes, specifically their ability to aid in the regeneration of 2296
pancreatic cells (Rombouts et al 2003). This is due to the fact that MSC express 2297
only MHC class I and not MHC class II cell-surface markers as well as no co- 2298
stimulator molecules, making them unable to function as antigen-presenting cells 2299
and invisible to the host’s immune system (Krampera et al 2003, Le Blanc et al 2300
2003). There is a lack of data on MSC in T2DM, as most studies involving the 2301
use of MSC focus on T1DM. However, there is some evidence to suggest that 2302
similar to EPC, bone marrow-derived MSC are negatively affected by diabetes. 2303
This is believed to be a direct result of alterations in the bone marrow 2304
microenvironment, where one group demonstrated that in diabetes the 2305
accumulation of AGE in the bone marrow inhibited the differentiation potential of 2306
human MSC in vitro (Kume et al 2005). 2307

91
Combined with their small population sizes and the need for clearer 2308
identification methods and culturing techniques, there are many inherent 2309
difficulties involved with the use of EPC and MSC therapeutically. Due to the 2310
critical roles of these cells, and the serious complications that can arise when 2311
they do not function normally, there is a growing need for research focusing on 2312
targeting the specific mechanisms leading to EPC and MSC dysfunction in 2313
diabetes. This includes the investigation of potential therapeutic agents with anti- 2314
oxidant and anti-inflammatory properties, as well as therapies that can modulate 2315
the RAS in order to attempt to prevent or reverse the damage that diabetes 2316
causes to these bone marrow-derived progenitor cells. 2317
Diabetes is also associated with a number of long-term complications 2318
including nephropathy, retinopathy, stroke, and CVD, leading to a decreased 2319
quality of life and life expectancy. In particular, patients with T2DM have 2-4 2320
times the risk of developing heart disease compared to healthy individuals, and in 2321
2004 alone heart disease was noted on 68% of diabetes-related death 2322
certificates among individuals 65 and older (Centers for Disease Control). Also, 2323
micro- and macrovascular complications in diabetic patients have been shown to 2324
lead to a 3- to 5-fold increased risk of death compared to non-diabetic individuals 2325
(Benter et al 2006). One mechanism responsible for the cardiovascular 2326
dysfunction in diabetic patients is cardiomyocyte hypertrophy, which can lead to 2327
cellular apoptosis, decreased contractility and heart failure. 2328

92
The RAS has been implicated in the pathophysiology of multiple diabetes- 2329
related complications, including hypertension and subsequent CVD (Weekers et 2330
al 2005). Studies have shown that blockade of the RAS, specifically inhibition of 2331
Ang II synthesis or activity with the use of pharmacological agents such as ACE 2332
inhibitors and ARB may decrease the risk or development of these potentially 2333
life-threatening cardiovascular complications in diabetic patients (Burnier et al 2334
2006). In fact, in addition to their role in RAS blockade via attenuation of the 2335
physiological activity of Ang II, some of the beneficial effects of ARB and ACE 2336
inhibitors in CVD may be in part due to their ability to increase the formation of 2337
Ang-(1-7), a heptapeptide of the RAS which has been shown to oppose many of 2338
the actions of Ang II (Iyer et al 1998, Iyer et al 2000). 2339
More recently, evidence has surfaced in the literature demonstrating the 2340
cardioprotective effects of Ang-(1-7) in streptozotocin-induced T1DM, in rats 2341
following surgical induction of myocardial infarction, as well as in spontaneously 2342
hypertensive rats (Loot et al 2002, Benter et al 2006, Benter et al 2007, 2343
Ebermann et al 2008). Ang-(1-7) is a seven amino acid peptide whose actions 2344
are thought to be mediated primarily by acting as an agonist of the GPCR Mas, 2345
however recent studies have suggested that Ang-(1-7) may also bind to other 2346
receptors including AT
2
, a signaling pathway that may involve heterodimerization 2347
of the RAS GPCR. Ang-(1-7) can be formed from either Ang I or Ang II via 2348
multiple enzymatic pathways involving ACE, ACE2, and NEP. Once believed to 2349
be an inactive metabolite of Ang II, Ang-(1-7) has been shown to have properties 2350

93
that directly counteract the actions of Ang II signaling through AT
2
, including 2351
opposition of the hypertensive properties of Ang II secondary to its vasodilatory 2352
effects mediated by NO and bradykinin release following binding to the Mas 2353
receptor. Due to the overall counter-regulatory role of Ang-(1-7) in the RAS and 2354
its beneficial effects in opposing the detrimental effects of Ang II in CVD and 2355
diabetes, this present study was designed to investigate the extent of 2356
cardiovascular dysfunction in db/db mice, as well as the potential role(s) and 2357
mechanism(s) of Ang-(1-7) treatment in attenuating diabetes-related 2358
cardiovascular dysfunction in a murine model of T2DM. 2359
2360
4.2 Study Design 2361
2362
4.2.1 Animal Protocol 2363
2364
The NIH Principles of Laboratory Animal Care were followed, and the 2365
Department of Animal Resources at the University of Southern California 2366
approved this study. Six to eight week-old male BKS.Cg-Dock7
m
+/+ Lepr
db
/J 2367
(db/db) mice and their heterozygous controls were purchased from Jackson 2368
Laboratories (Bar Harbor, ME, USA). All mice were quarantined for one week 2369
prior to initiation of treatment, food and water were available ad libitum, and they 2370
were kept on a 12 hour light/dark cycle. 2371
For investigating EPC and MSC, db/db mice and their heterozygous 2372
controls (n=7/group) were administered by SC injection either saline (control), 2373
inhibitors alone (losartan, PD123,319, A-779 or L-NAME at 10 mg/kg/day or 2374
icatibant at 0.4 mg/kg/day), Ang-(1-7) alone (500 µg/kg/day), or Ang-(1-7) 500 2375

94
µg/kg/day combined with an inhibitor at the aforementioned doses for two weeks. 2376
For measures of cardiac function, db/db mice (n=5/group) were administered by 2377
SC injection either saline (control) or Ang-(1-7) 500 µg/kg/day for two weeks. 2378
Heterozygous mice treated with saline were used as controls. Mice were 2379
weighed three times weekly, and the doses adjusted accordingly. Following the 2380
14-day treatment period, the mice were euthanized, hearts weighed, and 2381
parameters investigated as described below to determine the effect of diabetes 2382
and Ang-(1-7) treatment with and without the co-administration of the various 2383
inhibitors on EPC and MSC, as well the potential mechanism(s), and the effect(s) 2384
of Ang-(1-7) treatment on measurements of cardiac function in db/db mice. 2385
2386
4.2.2 Chemicals and Reagents 2387
2388
Ang-(1-7), prepared using Good Manufacturing Practices, and A-779, an 2389
antagonist of the Mas receptor, were purchased from Bachem (Torrance, CA, 2390
USA). Losartan, an AT
1
receptor antagonist, PD123,319, an AT
2
receptor 2391
antagonist, and nitro-l-arginine methyl ester (L-NAME), an inhibitor of NOS, were 2392
purchased from Sigma-Aldrich (Saint Louis, MO, USA). Icatibant, an antagonist 2393
of bradykinin B
2
receptors, was purchased from Tocris Bioscience (Ellisville, MO, 2394
USA). 2395
2396
2397
2398
2399
2400
2401
2402

95
4.2.3 Cardiac Puncture 2403
2404
Following being anesthetized with isoflurane, cardiac puncture was 2405
performed using a 25-gauge needle and 2 mL syringe to collect blood and 2406
assays were performed as described below. 2407
2408
4.2.4 Bone Marrow Isolation 2409
2410
Following cardiac puncture, mice were euthanized by cervical dislocation 2411
while under anesthesia, and the femurs from each mouse were collected and 2412
bone marrow was harvested by flushing with phosphate buffered saline 2413
containing 2% fetal calf serum. Following collection of the bone marrow and 2414
aliquoting, red blood cells were lysed with a hypotonic solution, mixed with 0.04% 2415
trypan blue and the number of nucleated cells was assessed using a 2416
hematocytometer under light microscopy. 2417
2418
4.2.5 Bone Marrow EPC Counts 2419
2420
Isolated bone marrow cells were diluted in DMEM and 2% FCS in culture 2421
tubes to a concentration of 100,000 cells/mL and 1 mL added to each tube. Bone 2422
marrow cells were centrifuged at 1,500 x g at 4°C for 5 minutes. The supernatant 2423
was aspirated and 400 µL of 2% paraformaldehyde was added to each tube and 2424
vortexed gently. After 15 minutes, bone marrow cells were again centrifuged at 2425
1,500 x g at 4°C for 5 minutes. To each tube of 100,000 bone marrow cells, 1 µg 2426
of isotype control or FITC-conjugated rat anti-mouse Flk-1 and PE-conjugated rat 2427
anti-mouse Sca-1 antibodies (eBioscience, USA) in PBS were added (total 2428

96
staining volume of 100 uL) and incubated at 4°C in the dark for 30 minutes. Cells 2429
were then washed three times with staining buffer (PBS with 2% FCS) and 2430
centrifuged at 1,500 x g at 4°C for 5 minutes. The supernatant was then 2431
aspirated, cells re-suspended in 500 µL of PBS and cells stored at 4°C in the 2432
dark until flow cytometric analysis. 2433
2434
4.2.6 Circulating EPC Counts 2435
Following cardiac puncture, 100 µL of whole blood was added to culture 2436
tubes with 1 µg of isotype control or FITC-conjugated rat anti-mouse Flk-1 and 2437
PE-conjugated rat anti-mouse Sca-1 antibodies in PBS (total staining volume of 2438
100 µL) and incubated at 4°C in the dark for 30 minutes. Following incubation, 2 2439
mL of RBC lysis buffer, pre-warmed to room temperature, was added to each 2440
tube, vortexed gently, and incubated in the dark for 10 minutes. Blood samples 2441
were centrifuged at 400 x g at room temperature, supernatant aspirated, and 2442
washed with 500 µL of staining buffer (PBS with 2% FCS). Samples were then 2443
centrifuged at 400 x g at room temperature, supernatant aspirated, re-suspended 2444
in 500 µL of 2% paraformaldehyde fixation buffer, and stored at 4°C in the dark 2445
until flow cytometric analysis. 2446
2447
4.2.7 MSC Cultures 2448
Following isolation, 100 µL of bone marrow cells at 1 x 10
7
cells/mL in 2449
addition to 3.9 mL of Complete MesenCult
®
Mouse Medium (StemCell 2450
Technologies, USA) were added to each well of 6-well plates to obtain 4 mL of 2451

97
cells at a final concentration of 2.5 x 10
5
cells/mL per well. Cells were cultured for 2452
10 days at 37°C in 5% CO
2
, after which mouse bone marrow-derived CFU-F 2453
colonies were enumerated using phase contract microscopy. 2454
2455
4.2.8 Echocardiographic Analysis 2456
In vivo murine cardiac function was assessed noninvasively using a high- 2457
frequency, high-resolution two-dimensional echocardiography system consisting 2458
of a Philips SONOS 5500 ultrasound machine equipped with a 6-15 MHz linear 2459
transducer (Philips Healthcare, Andover, MA, USA). Mice were anesthetized 2460
using 2.5% isoflurane for induction, the anterior chest was shaved, and mice 2461
were placed in a supine position on an imaging stage equipped with 2462
electrocardiography electrodes for continuous heart rate monitoring and a 2463
heating pad to maintain body temperature at 37°C. Anesthesia was maintained 2464
using a nose cone with 0.75-1.5% isoflurane to maintain a heart rate between 2465
400-450 beats per minute (BPM). High-resolution images were obtained in the 2466
right and left parasternal long and short axis and apical orientations. Standard B- 2467
mode images of the heart and pulsed Doppler images of the mitral and tricuspid 2468
inflow were also acquired. LV dimensions and wall thickness were measured at 2469
the level of the papillary muscles in the left and right parasternal short axis at 2470
systole and diastole. LV ejection fraction (EF), LV outflow tract, aortic R-R, 2471
fractional shortening (FS) and LV internal dimensions at both systole and diastole 2472
were measured. All measurements and calculations were done in accordance 2473
with the American Society of Echocardiography (Lang et al 2006). From these 2474

98
measurements, end systolic volume, end diastolic volume, LVEF, stroke volume 2475
(SV), CO (L/min) and CI (L/min/mg) were determined using the modified 2476
Simpson’s rule, which is the most commonly used and currently recommended 2477
method. 2478
2479
4.2.9 Thermodilution Methodology 2480
To assess maximum change in LV pressure (dP/dT
max
) and minimum 2481
change in LV pressure (dP/dT
min
), mice were first anesthetized with an 2482
intraperitoneal (i.p.) injection of 100 mg/kg of ketamine and 10 mg/kg of xylazine. 2483
Following anesthesia, the neck area was aseptically prepared for surgery and 2484
ventral neck hair shaved. A midline incision was made in the exposed area, right 2485
jugular vein and carotid artery exposed, and thermocouple probe introduced into 2486
carotid artery. A PE10 catheter was filled with Lactated Ringer’s (LR) solution 2487
and inserted into the jugular vein. Both the probe and catheter were then tied off, 2488
with the venous line used to infuse the test fluid (cold LR solution) and the 2489
temperature probe used to monitor the temperature of the test fluid and the 2490
animal’s body temperature during the experiment. Changes in blood temperature 2491
were measured using a Cardiomax-II Thermodilution Cardiac Output Computer 2492
(Columbus Instruments, Columbus, OH, USA) connected to the thermocouple 2493
probe. The analog signal was digitalized and processed with a BIOPAC MP150 2494
Data Acquisition and Analysis System using AcqKnowledge software version 4.2 2495
(BIOPAC Systems, Incorporated, Goleta, CA, USA). 2496
2497

99
4.2.10 Measurement of Cardiomyocyte Hypertrophy 2498
After weighing the hearts, they were formalin fixed for 7 days and 2499
embedded in a position to allow for visualization of the LV and left atrium. 2500
Paraffin-embedded samples were sectioned longitudinally at a thickness of 5 µm 2501
and stained with hematoxylin and eosin (H&E). Ten random images of each 2502
heart section were taken at 4x, 10x, and 40x magnifications along the left 2503
ventricular wall, avoiding the septal wall for consistency. Cardiomyocyte area and 2504
width was determined at 10x magnification using elliptical and linear 2505
measurements in ImageJ version 1.46h (National Institutes of Health, USA) at 2506
positions without visible nuclei exposing substantial lengths of uniform width to 2507
avoid large standard error. Cardiomyocyte area and width were expressed as 2508
arbitrary units (Tracy and Sander 2011). Collagen deposition using Trichrome 2509
was also measured, however there were no significant differences between any 2510
groups. 2511
2512
4.2.11 Statistical Analysis 2513
2514
GraphPad Prism version 5.0d for Mac OS X (GraphPad Software, San 2515
Diego, CA, USA) was used to analyze the data. One-way ANOVA followed by 2516
Tukey’s test was used to compare data from more than two groups. The level of 2517
statistical significance was set at 5%. Data are expressed as mean value ± 2518
standard error of the mean (SEM). 2519
2520

100
4.3 Results 2521
2522
4.3.1 Bone Marrow-Derived EPC 2523
2524
Since most research on EPC in diabetes focuses only on circulating levels, 2525
we used flow cytometry to determine the number of EPC in the bone marrow 2526
(Flk-1
+
Sca1
+
cells). Bone marrow-derived EPC were significantly lower in db/db 2527
mice compared to non-diabetic controls (Figure 15A; p<0.01). Following 2528

Figures 15A-C. Bone marrow-derived and circulating EPC counts and bone marrow-derived MSC counts. EPC
from both the bone marrow and circulation and bone marrow-derived MSC in diabetic mice were significantly
decreased (p<0.01). Ang-(1-7) administration significantly increased both bone marrow-derived and circulating EPC
numbers, as well as bone marrow-derived MSC numbers, and these effect was significantly inhibited by PD123,319,
A-779, L-NAME, and icatibant.

101
administration of Ang-(1-7) daily for 14 days to db/db mice, there was a 2529
significant increase in bone marrow EPC counts compared to db/db mice 2530
administered saline as control (p<0.01). Co-administration of Ang-(1-7) with 2531
PD123,319, A-779, L-NAME, or icatibant, but not losartan, resulted in a 2532
significant decrease in bone marrow-derived EPC in diabetic mice compared to 2533
diabetic mice treated with Ang-(1-7) alone (p<0.05). 2534
2535
4.3.2 Circulating EPC 2536
To correlate bone marrow-derived EPC with circulating EPC, flow 2537
cytometry was also performed to measure the percentage of Flk-1
+
Sca-1
+
cells 2538
in whole blood. Similar to results seen in the bone marrow, there were 2539
significantly lower numbers of circulating EPC in db/db mice (Figure 15B; p<0.01), 2540
and administration of Ang-(1-7) for 14 days resulted in a significant increase in 2541
circulating EPC in diabetic mice (p<0.05). Again, co-administration of Ang-(1-7) 2542
with PD123,319, A-779, L-NAME, or icatibant resulted in a significant decrease in 2543
circulating EPC counts in db/db mice compared to diabetic mice treated with 2544
Ang-(1-7) alone (p<0.05). 2545
2546
4.3.3 Bone Marrow-Derived MSC 2547
Isolated bone marrow cells were cultured for 10 days in MesenCult
®
2548
Mouse Medium, a proprietary culture medium optimized to select for MSC growth, 2549
to determine bone marrow-derived MSC colony counts. MSC numbers from bone 2550
marrow cultures isolated from diabetic mice were significantly lower compared to 2551

102
counts from non-diabetic bone marrow (p<0.05). Diabetic mice administered 2552
Ang-(1-7) daily for 14 days had significantly higher bone marrow-derived MSC 2553
colony counts compared to diabetic mice administered saline (p<0.05). Co- 2554
administration of Ang-(1-7) with either PD123,319, A-779, icatibant, or L-NAME 2555
in db/db mice resulted in a significant decrease in bone marrow MSC counts 2556
compared to db/db mice administered Ang-(1-7) alone (p<0.05). 2557
2558
4.3.4 Cardiac Output and Cardiac Index 2559
Since db/db mice weigh approximately twice that of their heterozygous 2560
controls, hearts were weighed to determine if there were any differences 2561
between the groups. There were no significant differences in heart weight 2562

Figures 16A-B. Cardiac output and cardiac index measurements. Both cardiac out put and cardiac index were
significantly decreased in diabetic mice, where administration of Ang-(1-7) for 14 days resulted in significant
increases in both of these parameters.

103
between any of the groups, however as expected, the ratio of heart weight (mg) 2563
to total body weight (g) was significantly lower in db/db mice (data not shown). 2564
Compared to non-diabetic controls, db/db mice had significant reductions in both 2565
CO (43% decrease) and CI (65% decrease) as measured by echocardiography 2566
(Figures 16A-B; p<0.01). Administration of Ang-(1-7) at 500 µg/kg/day for 14 2567
days to db/db mice resulted in a significant improvement in CO (32% increase) 2568
and CI (35% increase) compared to saline-treated db/db controls (p<0.05). 2569
2570
4.3.5 Measurements of Left Ventricular Function 2571

Figures 17A-B. Measurement of dP/dT max and dP/dT min. dP/dT max and dP/dT min were significantly decreased and
increased, respectively, in diabetic mice (p<0.01). Treatment of diabetic mice with Ang-(1-7) for 14 days resulted in
a significant increase and decrease in dP/dT max and dP/dT min, respectively (p<0.01).

104
Multiple measurements of LV function, including dP/dT
max
and dP/dT
min
2572
(mm Hg/s), and FS (%) were performed in saline-treated heterozygous and 2573
saline and Ang-(1-7)-treated db/db mice. Using thermodilution methodology, 2574
dP/dT
max
and dP/dT
min
were significantly decreased by 39% and 64%, 2575
respectively, in db/db mice compared to non-diabetic controls (Figures 17A-B; 2576
p<0.01). Treatment of db/db mice with Ang-(1-7) for 14 days resulted in 2577
significant increases in both of these parameters; specifically a 37% increase in 2578
dP/dT
max
and a 41% increase in dP/dT
min
compared to saline-treated diabetic 2579
controls (p<0.01). 2580
FS was calculated using echocardiography, and measures the difference 2581
between end-diastolic and end-systolic LV dimensions. A FS percent of less than 2582
30% is generally considered to be a mild decrease in LV function. FS was 2583

Figure 18. Measurement of fractional shortening. FS was significantly decreased in diabetic mice, where Ang-
(1-7) resulted in a significant increase after treatment for 14 days (p<0.01).

105
significantly decreased by 21% in db/db mice compared to non-diabetic controls 2584
(Figure 18; p<0.01). After 14 days, db/db mice treated with SC administered Ang- 2585
(1-7) had a significant 48% increase in FS compared to db/db mice treated with 2586
saline alone (p<0.01). 2587
2588
4.3.6 Cardiomyocyte Measurements 2589
In order to investigate the potential mechanism(s) leading to reduced cardiac 2590
function in T2DM murine model and how Ang-(1-7) is able to improve cardiac 2591
function, the cardiomyocyte dimensions of H&E stained heart sections were 2592
measured. In db/db mice, cardiomyocyte mean area and width were significantly 2593
increased by approximately 3-fold and 2-fold, respectively, compared to non- 2594
diabetic controls (Figures 19A-B; p<0.05). Following administration of Ang-(1-7) 2595
to db/db mice for 14 days, both cardiomyocyte area and width were significantly 2596
decreased to sizes similar to those measured in non-diabetic mice, where 2597

Figures 19A-B. Cardiomyocyte measurements. Cardiomyocytes from stained H&E sections of diabetic mice
hearts showed significant signs of hypertrophy, where Ang-(1-7) treatment significantly reversed this.

106
cardiomyocyte area was decreased by 57% and cardiomyocyte width was 2598
reduced by 35% (p<0.05). 2599
2600
4.4 Discussion and Significance 2601
2602
Increasing EPC and MSC counts in diabetic patients could decrease the 2603
risk of vascular disease in diabetics by increasing angiogenesis and repairing 2604
damaged cardiac tissue. This could potentially reduce the risk or prevent the 2605
development of long-term cardiovascular complications, which are leading 2606
causes of death in diabetic patients. The results of this current study agree with 2607
the current literature demonstrating that circulating EPC counts are decreased in 2608
T2DM (Tepper et al 2002, Fadini et al 2005). However, there is a lack of 2609
published data investigating the effects of diabetes on the bone marrow, which is 2610
a primary source of both EPC and MSC. 2611
While the exact mechanism is not clear, NO is known to act as molecular 2612
signal resulting in the proliferation and mobilization of EPC and MSC from the 2613
bone marrow niche and into the circulation. Our data shows that similar to 2614
circulating EPC, bone marrow-derived EPC and MSC are also significantly 2615
reduced in db/db mice. Studies from our laboratory and other groups have shown 2616
that the bone marrow microenvironment is altered in diabetes, specifically via 2617
increased levels of oxidative stress and inflammation, ultimately resulting in 2618
decreased NO bioavailability and cellular damage including an increase in 2619
protein tyrosine nitration and microvascular remodeling (Oikawa et al 2010, 2620
Mordwinkin et al 2012b). Homeostasis of the bone marrow niche is vital for the 2621

107
proliferation and subsequent lineage commitment, differentiation and maturation 2622
of EPC and MSC. Due to the important role of NO in progenitor cell and stem cell 2623
migration, the decreased NO levels observed in diabetes could prevent the 2624
migration of EPC out of the bone marrow and into the circulation. Combined with 2625
the decreased number of bone marrow-derived EPC observed in this study, 2626
these mechanisms could contribute to the reduction in circulating EPC commonly 2627
seen in diabetic patients (Loomans et al 2005). In addition, damage to the bone 2628
marrow in diabetes due to the effects of increased oxidative stress and 2629
subsequent nitration of proteins could lead to a reduction in EPC and MSC. 2630
More importantly, the data from this study demonstrates that the daily 2631
administration of Ang-(1-7) to db/db mice over 14 days results in significant 2632
increases in both bone marrow-derived and circulating EPC counts. This effect 2633
was mediated through the Mas and AT
2
receptors via NO and bradykinin- 2634
dependent pathways. Recently, Huang and colleagues investigated the anti- 2635
diabetic drug and peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist 2636
pioglitazone on endothelial dysfunction in a different murine model of diabetes 2637
(Huang et al 2008). They found that pioglitazone ameliorated endothelial 2638
dysfunction via activation of eNOS activity and eNOS phosphorylation at 2639
Ser1177, and these effects were inhibited by the NOS inhibitor L-NAME. Studies 2640
from our group and others have shown that Ang-(1-7) itself may also act as a 2641
PPAR-γ agonist, and that it also activates eNOS via increased phosphorylation at 2642
Ser1177 and decreased phosphorylation at Thr495 (Dhaunsi et al 2010, 2643

108
Mordwinkin et al 2012b). Therefore, Ang-(1-7)-dependent increases in EPC and 2644
MSC may be secondary to a decrease in oxidative stress and inflammation. This 2645
could ultimately result in decreases in tissue damage secondary to nitration, as 2646
well as increases in NO bioavailability. These effects may be one mechanism by 2647
which Ang-(1-7) increases EPC counts in the circulation and bone marrow. The 2648
ability of Ang-(1-7) to increase EPC and MSC numbers in this study warrants 2649
further study of this peptide as a potential therapeutic treatment option for 2650
patients with diabetes-related complications. 2651
These findings also demonstrate that cardiac function is significantly 2652
decreased in db/db mice, and Ang-(1-7) administration results in significant 2653
improvements in multiple measures of cardiac function in a murine model of 2654
T2DM (Table 1). The increase in cardiac output observed in diabetic mice 2655
following treatment with Ang-(1-7) for 14 days appears to be in part due to a 48% 2656
increase in FS compared to saline-treated diabetic mice. Our data agree with the 2657
findings of two recently published papers, where similar results were seen in 2658

Table 1. Effects of Ang-(1-7) on measurements of cardiac function in diabetic mice.

109
regards to the effects of Ang-(1-7) on measures of cardiac function in different 2659
animal models of heart failure including two models of MI and diabetic rats with 2660
cardiomyopathy (Marques et al 2011, Singh et al 2011). In patients with T2DM, 2661
cardiac hypertrophy, specifically left ventricular hypertrophy, is often a major 2662
cause of the long-term decrease in cardiac function. Indeed, when we 2663
determined the extent of cardiomyocyte hypertrophy in both non-diabetic and 2664
db/db mice in this study, diabetic mice had significant increases in cardiomyocyte 2665
area and width, where treatment with Ang-(1-7) administered daily for 14 days 2666
partially reversed this. Much research has been done to determine the potential 2667
causes of myocyte hypertrophy, where two of the main mechanisms are Ang II 2668
activation of the AT
1
receptor, as well as increases in pro-inflammatory cytokine 2669
signaling such as TNF-α (van Empel et al 2004). This may in part help to explain 2670
the mechanism by which Ang-(1-7) reduced cardiomyocyte hypertrophy in db/db 2671
mice. First, Ang-(1-7) signaling through the Mas receptor opposes many of the 2672
actions of Ang II, which could result in decreased signaling via AT
1
. In addition, 2673
our previous data has shown that Ang-(1-7) has anti-inflammatory effects in vivo, 2674
resulting in decreases in pro-inflammatory cytokine levels including IL-6 and 2675
TNF-α in the bone marrow and circulation. 2676
2677
4.5 Conclusion 2678
2679
This data demonstrates that bone marrow-derived MSC counts as well as 2680
both bone marrow-derived and circulating EPC counts are significantly 2681
decreased in a murine model of T2DM. Diabetics are at an increased risk of CVD, 2682

110
and it has been postulated that this may be secondary to decreased EPC and 2683
MSC counts, limiting the ability to repair damaged heart tissue. There are two 2684
major potential future therapeutic uses for Ang-(1-7) in the treatment of diabetes- 2685
related complications. First, by decreasing oxidative stress in the bone marrow, 2686
this peptide may return the bone marrow niche to normal homeostasis, allowing 2687
EPC and MSC numbers to return to normal levels. In addition, since the 2688
populations of these bone marrow stem cells are relatively small to begin with, 2689
administration of Ang-(1-7) may provide a pharmacologic means to increase EPC 2690
and MSC counts, allowing for isolation and propagation ex vivo. These cells 2691
could then be used as injectable, undifferentiated stem cells or differentiated cells 2692
to directly treat heart disease and other long-term diabetes-related complications. 2693
While additional studies will need to be performed, we can also 2694
hypothesize as to why Ang-(1-7) partially reversed cardiac dysfunction in a 2695
diabetic mouse model. The increase in cardiac function in diabetic mice receiving 2696
Ang-(1-7) may be secondary to it’s role in decreasing oxidative stress and 2697
inflammation, which may slow the rate of cardiac output decline. In addition, 2698
because Ang-(1-7) is a vasodilator via its actions on Mas and subsequent 2699
release of NO, it may result in increased perfusion of the heart and prevent 2700
ischemia. Ang-(1-7) may also increase neovascularization due to its positive 2701
effects on bone marrow-derived and circulating progenitor cells such as EPC. 2702
Lastly, and perhaps most importantly, Ang-(1-7) appears to reduce the 2703
cardiomyocyte hypertrophy that was observed in db/db mice. Future studies 2704

111
should include not only older db/db mice to investigate the effects of Ang-(1-7) on 2705
more advanced cardiac dysfunction in diabetes, but also longer treatment 2706
durations. While 2 weeks of treatment with Ang-(1-7) demonstrated significant 2707
increases in measurements of cardiac output in db/db mice, treatment for 30 2708
days or longer could show greater effects. 2709
2710
2711
2712
2713
2714
2715
2716
2717
2718
2719
2720
2721
2722
2723
2724
2725
2726
2727
2728
2729
2730
2731
2732
2733
2734
2735
2736
2737
2738
2739
2740
2741
2742
2743

112
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Caplan AI (2009) Why are MSCs therapeutic? New data: new insight. J 2773
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Dhaunsi GS, Yousif MH, Akhtar S, Chappell MC, Diz DI, Benter IF (2010) 2788
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Iyer SN, Averill DB, Chappell MC, Yamada K, Allred AJ, Ferrario CM (2000) 2815
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LeBlanc K, Tammik L, Sundberg B, Haynesworth SE, Ringden O (2003) 2841
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Loomans CJ, De Koning EJ, Staal FJ, Rabelink TJ, Zonneveld AJ (2005) 2846
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consequence of oxidative stress? Antiodix Redox Signal 7(11-12):1468-1475. 2848
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Rombouts WJ and Ploemacher RE (2003) Primary murine MSC show highly 2890
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Shibata R, Skurk C, Ouchi N, Galasso G, Kondo K, Ohashi T, Shimano M, 2902
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progenitor cell number and function. FEBS Lett 582(11):1607-1612. 2904
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diabetics exhibit impaired proliferation, adhesion, and incorporation into 2911
vascular structures. Circulation 106(22):2781-2786. 2912
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Thum T, Fraccarollo D, Schultheiss M, Froese S, Galuppo P, Widder JD, 2914
Tsikas D, Ertl G, Bauersachs J (2007) Endothelial nitric oxide synthase 2915
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diabetes. Diabetes 56(3):666-674. 2917
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Tongers J, Roncalli JG, Losordo DW (2010) Role of endothelial progenitor 2920
cells during ischemia-induced vasculogenesis and collateral formation. 2921
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Weekers L, Bouhanick B, Hadjadj S, Gallois Y, Roussel R, Pean F, Ankotche 2928
A, Chatellier G, Alhenc-Gelas F, Lefebvre PJ, Marre M (2005) Modulation of 2929
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Werner L, Deutsch V, Barshack I, Miller H, Keren G, George J (2005) 2934
Transfer of endothelial progenitor cells improves myocardial performance in 2935
rats with dilated cardiomyopathy induced following experimental myocarditis. 2936
J Mol Cell Cardiol 39(4):691-697. 2937
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Zangi L, Rivkin R, Kassis I, Levdansky L, Marx G, Gorodetsky R (2006) High- 2939
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2946
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2950
2951
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2956
2957
2958
2959
2960
2961
2962
2963

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Chapter 5 2964
Alteration of Endothelial Function and Markers of Oxidative Stress in 2965
Women with Gestational Diabetes Mellitus and Their Fetuses 2966
2967
5.1 Background 2968
2969
CVD is the main cause of death in the adult population in the United 2970
States and many countries (Lloyd-Jones et all 2010). CVD is responsible for 70% 2971
of the deaths in patients with DM, whose risk is increased 2-4 times compared to 2972
non-diabetics. Endothelial dysfunction is now considered a key element in the 2973
development of CVD, and has also been strongly associated with obesity and 2974
insulin resistance, which are key features of T2DM (de Jager et al 2006). 2975
Impairment of endothelial function often occurs prior to diagnosis of DM (Calles- 2976
Escandon et al 2001), and endothelial dysfunction is often present years before 2977
any signs of microangiopathy are apparent (Meigs et al 2004). Such findings 2978
have generated the hypothesis that, in T2DM, endothelial dysfunction may 2979
precede the development of chronic hyperglycemia, prompting the suggestion 2980
that the next generation of therapeutic agents should also focus on the 2981
mechanisms of endothelial dysfunction, and not solely on glycemia (Hod et al 2982
2007). 2983
Once endothelial dysfunction has occurred, it may be too late to intervene 2984
to reverse existing damage, whereas early therapeutic interventions may prevent 2985
further damage to the endothelium. In this regard, it is critical to identify 2986
biomarkers that may appear in diabetes prior to permanent and irreversible 2987
damage to tissues. Hyperglycemia has been shown to lead to increased 2988

118
production of ROS via a number of enzymatic pathways (Guizik et al 2002, 2989
Sheetz et al 2002), resulting in a state of oxidative stress. The mechanisms 2990
associated with these pathways are now being elucidated and show early 2991
indicators of heightened oxidative stress (Bitar et al 2005).

Specifically, these 2992
mechanisms are associated with a decreased availability of NO secondary to 2993
eNOS uncoupling. NO is a potent vasodilator, and appears essential for the 2994
function of EPC, which can migrate to and differentiate at sites of injury to 2995
promote angiogenesis and vasculogenesis in healthy individuals (Asahara et al 2996
1997). However, both the number and function of EPC have been shown to be 2997
decreased in diabetes, where EPC proliferation is impaired in hyperglycemic 2998
states (Tepper et al 2002, Loomans et al 2004). 2999
Since the actions of the endothelium are multiple and involve several 3000
systems, endothelial dysfunction has been defined pragmatically, with the most 3001
commonly measured alterations in normal endothelial function pertaining to the 3002
production of specific molecules that have a regulatory role in inflammation, and 3003
abnormalities in the regulation of the lumen of vessels (where NO plays an 3004
important role). Two standard molecules associated with endothelial dysfunction 3005
resulting from inflammation are soluble intercellular adhesion molecule-1 3006
(sICAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM-1). Although 3007
adhesion molecules are vital for the normal development and function of the 3008
heart and blood vessels, they have now been implicated in the pathogenesis of 3009
CVD. Elevations in the levels of these biomarkers have been a consistent finding 3010

119
in studies of patients with T2DM and people at increased risk for DM. The early 3011
mechanisms of endothelial dysfunction have not been thoroughly investigated in 3012
women with GDM. Such evidence would have implications not only for the 3013
diagnosis and treatment of affected mothers and their offspring, but also for 3014
modeling early vascular changes associated with T2DM. The purpose of this 3015
study was to test whether biomarkers of oxidative stress, eNOS uncoupling, and 3016
endothelial dysfunction may be altered in women with GDM and their fetuses. 3017
3018
5.2 Study Design and Methods 3019
3020
5.2.1 Patient Recruitment 3021
3022
Patients were enrolled at two hospitals, both of which are affiliated with the 3023
Keck School of Medicine of the University of Southern California. We conducted 3024
a case-control observational study of women with GDM and their neonates using 3025
pregnant patients without a diagnosis of GDM as controls. Pregnant women with 3026
preterm and multiple gestations, maternal history of smoking, or chronic 3027
hypertension were excluded from the study. Case-patients were eligible for study 3028
enrollment if they had GDM as diagnosed by having two of the following: a 3029
fasting blood glucose >95 mg/dL, a 2-hour oral glucose tolerance test (OGTT) 3030
>180 mg/dL, or a 3-hour OGTT >140 mg/dL. Eligible case-patients were 3031
identified from outpatient clinics in their last month of pregnancy and enrolled 3032
upon hospital arrival prior to delivery. Eligible control-patients were identified 3033
upon hospital arrival for delivery. Maternal and venous cord blood was drawn to 3034
quantify markers of endothelial dysfunction which included sVCAM-1, sICAM-1, 3035

120
CRP, EPC counts, NO levels, and eNOS, p22-phox, and SOD mRNA expression. 3036
Maternal hemoglobin A1c (HbA1c) was tested to determine the level of glycemic 3037
control in both case and control patients. The study was approved by the 3038
University of Southern California Institutional Review Board, and complied with 3039
all patient protection criteria set forth therein. 3040
Maternal venous blood was collected 3-12 hours before delivery. Venous 3041
cord blood was collected from the placental side of the cord after the delivery and 3042
cord clamping. All blood samples were centrifuged within 30 minutes after 3043
collection and processed immediately, or stored at the appropriate temperature 3044
until analyzed. 3045
3046
5.2.2 Preparation of Whole Blood for Flow Cytometry 3047
3048
One hundred µL of whole blood were incubated in test tubes for 30 3049
minutes at room temperature in the dark with APC-, PE- and FITC-conjugated 3050
antibodies for mouse anti-human CD34, CD309 (KDR), and CD133 added at the 3051
appropriate dilutions in staining buffer after mixing. Two mL of red blood cell lysis 3052
buffer (pre-warmed to room temperature) was added to each sample, mixed 3053
gently and incubated in the dark at room temperature for 10 minutes. Samples 3054
were centrifuged at 400 g at room temperature; after the supernatant was 3055
aspirated, it was washed one time with 2 mL of staining buffer. After an additional 3056
centrifugation, the stained cell pellets were re-suspended in 2% formaldehyde 3057
prior to analysis. Flow cytometric analysis was performed on a LSR II flow 3058
cytometer using FACSDiva software (Becton Dickinson, Franklin Lakes, NJ) at 3059

121
the Flow Cytometry Core Facility at the University of Southern California School 3060
of Pharmacy. 3061
3062
5.2.3 Analysis of mRNA Expression 3063
3064
Total RNA was extracted from whole blood using TRIzol (Invitrogen, 3065
Carlsbad, CA). For each sample, approximately 100 ng of RNA was reverse- 3066
transcribed using Maxima Reverse Transcriptase (Fermentas, Glen Burnie, MD). 3067
Real-time PCR was conducted to examine expression of eNOS, superoxide 3068
dismutase isoforms (SOD2 and SOD3), and p22-phox, a subunit of NADPH 3069
oxidase in maternal and cord blood. Quantitative amplification of the cDNA was 3070
performed using SYBR Green PCR Master Mix (Applied Biosystems, Life 3071
Technologies, Carlsbad, CA) for 40 cycles consisting of heat denaturation, 3072
annealing and extension using an ABI 7300 (Applied Biosystems). Expression of 3073
eNOS, SOD2, SOD3, and p22-phox mRNA were normalized against 18S mRNA 3074
and reported as fold-change compared to non-diabetic controls. 3075
3076
5.2.4 Measurement of Nitrite, sVCAM-1, and sICAM-1 Levels 3077
3078
Nitrite was determined using the Griess reaction, Griess Reagent System, 3079
(Promega, Madison, WI). The remaining parameters were measured using 3080
commercial kits according to manufacturer’s protocols (HbA1c: BioQuant, San 3081
Diego, CA; sVCAM-1, sICAM-1, and CRP: R&D Systems, Minneapolis, MN). 3082
3083
3084
3085
3086

122
5.2.5 Statistical Analysis 3087
3088
Relevant patient information was retrieved from medical records using 3089
standardized tools. Data were analyzed using SAS statistical software (Cary, NC). 3090
Laboratory data were combined with the patient data for analysis. Univariate 3091
analyses were performed, and means are expressed ± the standard deviation 3092
(SD), with median and range. Analyses of continuous variables were performed 3093
with Kruskal-Wallis testing. For each marker, and for maternal and cord blood, 3094
data were examined comparing case- and control-patients. Correlations were 3095
determined using linear regression in GraphPad Prism version 5.0d for Mac OS 3096
X (GraphPad Software, San Diego, CA). 3097
3098
5.3 Results 3099
3100
Sixteen patients were enrolled in this study, where nine patients met 3101
criteria classifying them as GDM, and seven were classified as non-GDM 3102
controls. Ethnically, all patients enrolled were Hispanic. Three control patients 3103
and one GDM patient were nulliparous. Only one GDM patient had a previous 3104
history of GDM, while no other patients had a previous history of any glucose 3105
abnormalities. Of the patients with GDM, six (67%) were classified as A1 (fasting 3106
blood glucose >95 mg/dL), and three patients were classified as A2. In the GDM 3107
group, one had a cesarean in early labor, and one had an elective repeat 3108
cesarean (without labor); three of the non-GDM group had elective cesarean 3109
deliveries. All other patients delivered vaginally, with one control patient delivered 3110
by vacuum because of maternal exhaustion. There was no difference in BMI or 3111

123
birth weight (Table 2) between the two groups. A statistically significant 3112
difference in maternal age was detected between the two groups (p<0.05). When 3113
we analyzed the association between maternal age and HbA1c values and 3114
percent of circulating EPC, no significant correlation was detected (data not 3115
shown). Mothers diagnosed with GDM had significantly higher HbA1c as 3116
compared to the control subjects (Table 2; 7.6% versus 5.2%, respectively; 3117
p<0.01). 3118
3119
5.3.1 Maternal and Cord Blood EPC Counts 3120
3121
EPC play an important role in the neovascularization process following 3122
injury, and have been shown to be significantly decreased in diabetes (Tepper et 3123
al 2002, Loomas et al 2004). We therefore evaluated mean circulating EPC 3124
counts from maternal blood. A statistically significant reduction in the percentage 3125
Patient characteristic Among patients
with GDM (N =
9)
Among patients
without GDM (N
= 7)
P value
Maternal Age (years) 32·8 + 6·3
36·2, (21·2-39·7)
25·1 + 5·3
23.7 (19·0-34·8)
0·02
Maternal BMI
(based on pre-pregnancy
weight)
30·1 + 4·8
27·9 (24·4-37·6)
29·8 + 7·7
28·2 (20·6-44·6)
0·63
Gestational age (weeks) 38·2 + 1·2
38·1 (35·9-39·6)
38·9 + 0·9
38·9 (37·3-40·3)
0·37
Birth weight (g) 3,193 + 367
3,041 (2,877-
3,915)
3,496 + 235
3,535 (3,234-
3,908)
0·08
HbA1c% 7.6 + 1.1
7.4 (6.5-10)
5.2 + 0.8
4.8 (4.3-6.2)
0.0002

Table 2. Patient characteristics by GDM group. Results are expressed as mean + SD, median (range).

124
of circulating EPC was seen in GDM versus non-diabetic patients (Figure 20; 3126
0.26% versus 0.41%, respectively; p<0.05). Additionally, we measured EPC 3127
counts from cord blood of fetuses delivered. Umbilical cord EPC were higher 3128
from GDM patients than control patients, however this difference did not reach 3129
statistical significance (1.76% versus 1.46%, respectively). 3130
3131
5.3.2 Plasma Nitrite Levels and eNOS Expression 3132
In diabetes, decreased circulating NO levels are often accompanied by a 3133
paradoxical increase in eNOS expression, a molecular mechanism referred to as 3134
eNOS uncoupling. To determine circulating NO levels, the stable metabolite, 3135
nitrite, was measured using the Griess reaction. Mean nitrite levels from maternal 3136
plasma were significantly lower in women diagnosed with GDM compared to 3137
non-diabetic controls (54 µM versus 28 µM, respectively; p<0.05; Figure 21A). 3138
Mean nitrite levels from umbilical cord plasma of fetuses delivered from women 3139

Figure 20. Maternal and cord blood EPC % in non-diabetic and GDM patients.

125
diagnosed with GDM were higher compared to those of non-diabetic women, 3140
however this difference did not reach statistical significance. In addition, maternal 3141
and cord blood eNOS mRNA expression were significantly higher in women 3142
diagnosed with GDM compared to non-diabetic women (Figure 21B; p<0.01). 3143
3144
5.3.3 SOD and p22-phox mRNA expression 3145
3146
An imbalance in SOD isoform and NADPH oxidase expression is also 3147
observed in diabetes, which may explain increases in oxidative stress and 3148
concomitantly decreasing anti-oxidant mechanisms. SOD2 (mitochondrial SOD) 3149
and SOD3 (extracellular SOD) mRNA expression in the blood was measured 3150
using RT-PCR, where maternal SOD2 and SOD3 expression were significantly 3151
decreased in women with GDM compared to non-diabetic controls (Figures 22A- 3152
B; p<0.01). In cord blood, SOD3 mRNA expression was significantly lower in 3153
women diagnosed with GDM compared to non-diabetic women, while there was 3154
no significant change in cord blood SOD2. Maternal blood p22-phox expression 3155

Figures 21A-B. Maternal and cord blood nitrite levels and eNOS mRNA expression in non-diabetic and GDM
patients.

126
was increased in women with GDM, however this difference did not reach 3156
statistical significance (Figure 23C). Surprisingly, cord blood p22-phox mRNA 3157
expression was significantly decreased from women with GDM compared to non- 3158
diabetic women (p<0.05). 3159
3160
5.3.4 Plasma sVCAM-1, sICAM-1, and CRP Levels 3161
3162
Levels of adhesion molecules such as soluble vascular adhesion molecule 3163
(sVCAM-1), soluble intracellular adhesion molecule (sICAM-1) and C-reactive 3164
protein (CRP) are associated with the inflammatory process, and can occur as a 3165
result of damage to tissue such as the endothelium. Increased levels of these 3166
adhesion molecules are observed in diabetes, and have been correlated with 3167
severity of disease. We evaluated maternal plasma for sVCAM-1 and sICAM-1, 3168

Figures 22A-C. Maternal and cord blood SOD and p22-phox mRNA expression in non-diabetic and GDM patients.

127
where both levels were significantly higher in GDM patients as compared to non- 3169
diabetic women (Figures 23A-B; p<0.05). There was a significant increase in 3170
cord blood sVCAM-1 levels from patients with GDM (p<0.01), however there was 3171
no significant difference in cord sICAM-1 levels. In addition, there were no 3172
significant differences between any groups in regards to plasma CRP levels, a 3173
marker of the overall inflammatory state (data not shown). 3174
3175
5.3.5 Correlation with HbA1c 3176
To determine whether a correlation exists between HbA1c as a measure 3177
of glycemic control and markers of oxidative stress or endothelial dysfunction 3178
exist, linear regression was performed on all parameters measured (Figures 24A- 3179
D). Maternal blood EPC correlated negatively with HbA1c, where an increase in 3180
HbA1c was associated with a decrease in circulating EPC numbers (R
2
=0.55; 3181
p<0.01). In addition, maternal plasma sVCAM-1 and sICAM-1 levels correlated 3182
positively with maternal HbA1c, where increased HbA1c was associated with 3183
increased sVCAM-1 and sICAM-1 levels (R
2
=0.79, p<0.01; R
2
=0.33, p<0.05 3184

Figures 23A-B. Maternal and cord plasma sVCAM-1 and sICAM-1 in non-diabetic and GDM patients.

128
respectively). Maternal blood eNOS mRNA expression also correlated positively 3185
with maternal HbA1c, where increased HbA1c was associated with increased 3186
eNOS mRNA expression (R
2
=0.25; p<0.05). Lastly, cord blood SOD3 mRNA 3187
expression correlated inversely with maternal HbA1c (R
2
=0.60; p<0.01). 3188
3189
5.4 Discussion and Significance 3190
3191
GDM affects 3-8% of pregnancies in the United States and up to 12-14% 3192
in some high-risk populations, and is a form of hyperglycemia, occurring when 3193
the insulin supply cannot meet tissue demands for normal glucose regulation 3194

Figures 24A-D. Correlations with HbA1c.

129
(Jovanovic et al 2001). Robust plasticity of β-cell function in the face of 3195
progressive insulin resistance is a principal feature of normal glucose regulation 3196
during pregnancy. The majority of women with GDM appear to have β-cell 3197
dysfunction that occurs on a background of chronic insulin resistance (Buchanan 3198
et al 2002). Hyperglycemia, as determined by glucose tolerance testing at mid- 3199
pregnancy, has been the mainstay of GDM diagnosis for decades. Maternal 3200
hyperglycemia leads to fetal hyperinsulinemia and can lead to adverse long-term 3201
maternal outcomes (e.g. development of type 2 DM and atherosclerotic CVD), 3202
increased perinatal morbidity (e.g. macrosomia, birth trauma, pre-eclampsia), 3203
and long-term sequelae in offspring (e.g. childhood overweight, and metabolic 3204
factors that may increase risk of CVD). 3205
In this case-control study of women diagnosed with GDM and their fetuses, 3206
we have demonstrated an increase in circulating adhesion molecules (sVCAM-1 3207
and sICAM-1) in patients with GDM. Moreover, GDM subjects had a significantly 3208
decreased circulating EPC counts in maternal blood, SOD isoform mRNA 3209
expression in both maternal and cord blood, and increased eNOS mRNA 3210
expression in both maternal and cord blood. These findings are consistent with 3211
the hypothesized mechanisms where hyperglycemia can lead to increased 3212
oxidative stress and endothelial dysfunction in diabetes. More importantly, our 3213
results suggest that alterations in endothelial function are already present in both 3214
mothers and their fetuses in GDM pregnancies. If these measures remain 3215
elevated, it is possible that these alterations can confer an increased risk for the 3216

130
development of CVD and type 2 DM. Furthermore, if confirmed in ongoing larger 3217
studies, the identification of disturbances at the molecular level (e.g. EPC counts, 3218
NO levels, SOD and NADPH oxidase activity, and other molecular markers for 3219
oxidative stress and NO production) should not only enable the identification of 3220
potential new treatments, (e.g. pharmaceuticals to prevent eNOS uncoupling and 3221
oxidative stress), but also should provide earlier targets for monitoring the 3222
effectiveness of current treatment strategies, including dietary changes, exercise 3223
regimens, and pharmacological management. 3224
In spite of the historical use of hyperglycemia as the diagnostic criterion 3225
for GDM, it is now evident that hyperglycemia may be a late consequence of the 3226
disease process, becoming detectable only after the progressive loss of insulin 3227
secretion due to longstanding demands imposed by chronic insulin resistance 3228
(Buchanan et al 2002). Even with strict glycemic control, women diagnosed with 3229
GDM are still at risk for adverse pregnancy outcomes. Therapeutic strategies that 3230
focus on tight glucose control often have limited success in avoiding accelerated 3231
fetal growth, particularly in pregnancies characterized by maternal obesity 3232
(Schaefer-Graf et al 2005, Ouzounian et al 2011). Furthermore, adverse 3233
pregnancy outcomes have also been documented in women with mild glucose 3234
intolerance who do not meet current diagnostic criteria for GDM (Jensen et al 3235
2001). In 2010, the International Association of Diabetes and Pregnancy Study 3236
Groups created a consensus panel to arrive at diagnostic criteria because there 3237
have been no obvious glucose thresholds at which risks of key outcomes are 3238

131
increased. Controversies regarding diagnostic criteria remain active, impeding 3239
the generation of new approaches to prevention and treatment. 3240
To date, the literature provides limited evidence of endothelial dysfunction 3241
among women with GDM. A few small studies have suggested that 3242
normoglycemic women with a history of GDM demonstrate aspects of endothelial 3243
dysfunction, such as impaired endothelium-dependent vasodilatation and 3244
increased artery stiffness. Multiple biomarkers have also been implicated in the 3245
connection between endothelial dysfunction and GDM. Krauss and colleagues 3246
found evidence for potentially elevated sICAM-1 levels in women with GDM, and 3247
Kautsky-Willer and colleagues demonstrated increased levels of sVCAM-1 in 3248
women with GDM and pregnant women with normal glucose tolerance compared 3249
with non-pregnant controls (Kautsky-Willer et al 1997, Krauss et al 2002). In 3250
women with GDM, these levels remained elevated 12 weeks postpartum, while 3251
correcting in women with normal glucose tolerance. In an evaluation conducted 3252
years after delivery, Bo and colleagues studied women with and without a history 3253
of GDM, and confirmed these findings (Bo et al 2007). Our studies support these 3254
findings. In addition, we found a significant inverse correlation of HgA1C with 3255
EPC. Hyperglycemia promotes an environment prone to oxidative stress, which 3256
can result in tissue damage and also prevent the ability to repair exisiting 3257
damage. Interestingly, a correlation between HgA1C and sVCAM-1 as well as 3258
sICAM-1 has been demonstrated, suggesting that high glucose increases the 3259

132
adhesiveness of the circulating cells, which could explain the endothelial 3260
dysfunction often observed in diabetic patients. 3261
For offspring, in utero exposure to a diabetic environment has been 3262
associated with obesity, type 2 DM, and dyslipidemia in the offspring, consistent 3263
with the concept of “fetal programming,” wherein adult disease may find its 3264
origins in fetal development (Hillier et al 2007). Elevated levels of sICAM-1 and 3265
sVCAM-1 were found in obese, hypertensive, and diabetic children with a mean 3266
age of 15 years, suggesting that endothelial activation appears early in life, and 3267
that adhesion molecules are related to the earliest stages of atherosclerosis 3268
(Glowinska et al 2005). Although the mechanisms by which exposure to a 3269
diabetic environment in utero causes metabolic abnormalities in the offspring are 3270
unknown, it is hypothesized that disturbed endothelial function may play a role 3271
(Sobngwi et al 2003). 3272
3273
5.5 Conclusion 3274
3275
In summary, our results have demonstrated the presence of maternal and 3276
fetal molecular markers of endothelial dysfunction and oxidative stress in women 3277
with GDM and their fetuses. We submit that continued advances in the 3278
understanding of the molecular mechanisms associated with endothelial 3279
dysfunction will have a substantial impact in the field of GDM, where the 3280
dynamics of the perinatal environment are remarkably complex. Given that 3281
current standards for strict glycemic control during pregnancy do not appear to 3282
eliminate the maternal and fetal repercussions of GDM, new treatment avenues 3283

133
are needed. The ability to identify and track early markers of vasculopathy in 3284
women with GDM, as we have demonstrated in this study, may provide a 3285
foundation for an improved understanding of the physiology of the disease 3286
process and its impact on both the mother and fetus at an early, and potentially 3287
reversible stage. 3288
3289
3290
3291
3292
3293
3294
3295
3296
3297
3298
3299
3300
3301
3302
3303
3304
3305
3306
3307
3308
3309
3310
3311
3312
3313
3314
3315
3316
3317
3318
3319
3320
3321

134
5.6 Chapter 5 References 3322
3323
Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, 3324
Witzenbichler B, Schatteman G, Isner JM (1997) Isolation of putative 3325
progenitor endothelial cells for angiogenesis. Science 275(5302):964-967. 3326
3327
Bitar MS, Wahid S, Mustafa S, Al-Saleh E, Dhaunshi GS, Al-Mulla F 3328
(2005) Nitric oxide dynamics and endothelial dysfunction in type II model 3329
of genetic diabetes. Eur J Pharmacol 511(1):53-64. 3330
3331
Bo S, Valpreda S, Menato G, Bardelli C, Botto C, Gambino R, Rabbia C, 3332
Durazzo M, Cassader M, Massobrio M, Pagano G (2007) Should we 3333
consider gestational diabetes a vascular risk factor? Atherosclerosis 3334
195(2):72-79. 3335
3336
Buchanan TA, Xiang AH, Peters RK, Kjos SL, Marroquin A, Goico J, 3337
Ochoa C, Tan S, Berkowitz K, Hodis HN, Azen SP (2002) Preservation of 3338
pancreatic beta-cell function and preservation of type 2 diabetes by 3339
pharmacological treatment of insulin resistance in high-risk Hispanic 3340
women. Diabetes 51(9):2796-2803. 3341
3342
Calles-Escandon J and Cipolla M (2001) Diabetes and endothelial 3343
dysfunction: a clinical perspective. Endocr Rev 22(1):36-52. 3344
3345
de Jagr J, Dekker JM, Kooy A, Kostense PJ, Nijpels G, Heine RJ, Bouter 3346
LM, Stehouwer CD (2006) Endothelial dysfunction and low-grade 3347
inflammation explain much of the excess cardiovascular mortality in 3348
individuals with type 2 diabetes: the Hoorn Study. Arterioscler Thromb 3349
Vasc Biol 26(5):1086-1093. 3350
3351
Glowinska B, Urban M, Peczynska J, Florys B (2005) Soluble adhesion 3352
molecules (sICAM-1, sVCAM-1) and selectins (sE selectin, sP selectin, sL 3353
selectin) levels in children and adolescents with obesity, hypertension, and 3354
diabetes. Metabolism 54(8):1020-1026. 3355
3356
Guzik TJ, Mussa S, Gastaldi D, Sadowski J, Ratnatunga C, Pillai R, 3357
Channon KM (2002) Mechanisms of increased vascular superoxide 3358
production in human diabetes mellitus: role of NAD(P)H oxidase and 3359
endothelial nitric oxide synthase. Circulation 105(14):1656-1662. 3360
3361
Hillier TA, Pedula KL, Schmidt MM, Mullen JA, Charles MA, Pettitt DJ 3362
(2007) Childhood obesity and metabolic imprinting: the ongoing effects of 3363
maternal hyperglycemia. Diabetes Care 30(9):2287-2292. 3364
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Hod M and Yogev Y (2007) Goals of metabolic management of 3366
gestational diabetes: is it all about the sugar? Diabetes Care 30 Suppl 3367
2:S180-187. 3368
3369
Jensen DM, Damm P, Sorenson B, Molsted-Pedersen L, Westergaard JG, 3370
Klebe J, Beck-Nielsen H (2001) Clinical impact of mild carbohydrate 3371
intolerance in pregnancy: a study of 2904 nondiabetic Danish women with 3372
risk factors for gestational diabetes mellitus. Am J Obstet Gynecol 3373
185(2):413-419. 3374
3375
Jovanovic L and Pettit DJ (2001) Gestational diabetes mellitus. JAMA 3376
286(20):2516-2518. 3377
3378
Kautzky-Willer A, Fasching P, Jilma B, Waldhausl W, Wagner OF (1997) 3379
Persistent elevation and metabolic dependence of circulating E-selectin 3380
after delivery in women with gestational diabetes mellitus. J Clin 3381
Endocrinol Metab 82(12):4117-4121. 3382
3383
Krauss T, Emons G, Kuhn W, Augustin HG (2002) Predictive value of 3384
routine circulating soluble endothelial cell adhesion molecule 3385
measurements during pregnancy. Clin Chem 48(9):1418-1425. 3386
3387
Lloyd-Jones D, Adams RJ, Brown TM, Carnethon M, Dai S, De Simone G, 3388
Ferguson TB, Ford E, Furie K, Gillespie C, Go A, Greenlund K, Haase N, 3389
Halipern S, Ho PM, Howard V, Kissela B, Kittner S, Lackland D, Lisabeth 3390
L, Marelli A, McDermott MM, Meigs J, Mozaffarian D, Mussolino M, Nichol 3391
G, Roger VL, Rosamond W, Sacco R, Sorlie P, Stafford R, Thom T, 3392
Wasserthiel-Smoller S, Wong ND, Wylie-Rosett J; American Heart 3393
Association Statistics Committee and Stroke Statistics Subcommittee 3394
(2010) Executive summary: heart disease and stroke statistics--2010 3395
update: a report from the American Heart Association. Circulation 3396
121(7):948-954. 3397
3398
Loomans CJ, de Koning EJ, Staal FJ, Rookmaaker MB, Verseyden C, de 3399
Boer HC, Verhaar MC, Braam B, Rabelink TJ, van Zonneveld AJ (2004) 3400
Endothelial progenitor cell dysfunction: a novel concept in the 3401
pathogenesis of vascular complications of type 1 diabetes. Diabetes 3402
53(1):195-199. 3403
3404
Meigs JB, Hu FB, Rifai N, Manson JE (2004) Biomarkers of endothelial 3405
dysfunction and risk of type 2 diabetes mellitus. JAMA 291(16):1978-1986. 3406
3407
3408
3409

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Ouzounian JG, Hernandez GD, Korst LM, Montoro MM, Battista LR, 3410
Walden CL, Lee RH (2011) Pre-pregnancy weight and excess weight gain 3411
are risk factors for macrosomia in women with gestational diabetes. J 3412
Perinatol 31(11):717-721. 3413
3414
Schaefer-Graf UM, Pawliczak J, Passow D, Hartmann R, Rossi, Buhrer C, 3415
Harder T, Plagemann A, Vetter K, Kordonouri O (2005) Birth weight and 3416
parental BMI predict overweight in children from mothers with gestational 3417
diabetes. Diabetes Care 28(7):1745-1750. 3418
3419
Sheetz MJ and King GL (2002) Molecular understanding of 3420
hyperglycemia’s adverse effects for diabetic complications. JAMA 3421
288(20):2579-2588. 3422
3423
Sobngwi E, Boudou P, Mauvais-Jarvis F, Leblanc H, Velho G, Vexiau P, 3424
Porcher R, Hadjadj S, Pratley R, Tataranni PA, Calvo F, Gautier JF (2003) 3425
Effect of a diabetic environment in utero on predisposition to type 2 3426
diabetes. Lancet 361(9372):1861-1865. 3427
3428
Tepper OM, Galiano RD, Capla JM, Kalka C, Gagne PJ, Jacobowitz GR, 3429
Levine JP, Gurtner GC (2002) Human endothelial progenitor cells from 3430
type II diabetics exhibit impaired proliferation, adhesion, and incorporation 3431
into vascular structures. Circulation 106(22):2781-2786. 3432
3433
3434
3435
3436
3437
3438
3439
3440
3441
3442
3443
3444
3445

137
Chapter 6 3446
Conclusion 3447
3448
6.1 Introduction 3449
3450
T2DM is a model of progressive oxidative stress and chronic inflammation 3451
as caused by systemic hyperglycemia. These events can result in the 3452
modification of proteins by ROS and RNS by multiple mechanisms including 3453
tyrosine nitration, leading to tissue damage. Despite tight glycemic control using 3454
current FDA-approved oral and injectable anti-diabetic pharmacological therapies, 3455
a progression of these molecular alterations continiue to occur, leading to further 3456
damage and long-term complications including vascular disease and 3457
cardiovascular dysfunction. We have focused our research on the use of a novel 3458
peptide of the RAS, Ang-(1-7), to treat and potentially alterating oxidative stress 3459
and inflammation which may be the principal pathogenic mechanism. 3460
The studies presented in this thesis demonstrate the important link 3461
between the RAS, oxidative stress, and inflammation in relationship to long-term 3462
complications associated with diabetes such as immune dysfunction and 3463
endothelial dysfunction leading to infections and CVD. The data presented here 3464
also suggest that oxidative stress, inflammation, and endothelial function may 3465
represent potential biomarkers at early stages of this metabolic disorder. These 3466
biochemical markers may be used alone or in combination with established 3467
laboratory values such as HgA1c or plasma glucose levels. The ability to identify 3468
these high-risk individuals may allow for preemptive interventions that may stop 3469
the progression to irreversible complications. 3470

138
6.2 Type 2 Diabetes and Oxidative Stress 3471
The current diabetes research focuses on afflicted vital organs and tissues 3472
such as the pancreas and kidneys. However, little data has emerged on the 3473
impact of diabetes have on bone marrow cellularity and functionality. This is 3474
surprising considering its central involvement in the production of a vast number 3475
of stem cells and progenitor cells including EPC, MSC, and many other cells 3476
involved in immune system function, the inflammatory response, and 3477
hematopoiesis. Moreover, the bone marrow plays a critical role in response to 3478
tissue injury and reestablishing homoestasis. This is of particular importance in 3479
light of the fact that diabetes is associated with chronic inflammation leading to 3480
the elaboration or excessive ROS causing vascular and other tissue damage. 3481
We have shown that diabetes-induced oxidative stress, caused in part by 3482
eNOS uncoupling, can also affect cells of the bone marrow in a murine model of 3483
T2DM. Changes in molecular markers found in oxidative stress observed in the 3484
bone marrow are similar to those seen in other tissues, including increases in 3485
ROS and superoxide production via NADPH oxidase. Evidence of eNOS 3486
uncoupling were also evident, such as decreased NO bioavailability and 3487
increased expression of eNOS in bone marrow cells isolated from db/db mice. 3488
More critically, the use of Ang-(1-7) was able to ameliorate and even reverse 3489
some oxidative mechanisms. Our findings were limited in that the use of these 3490
agents was only assessed at 14-day timepoints. In the future, it will be critical to 3491
determine whether longer treatment can provide additional benefits. Another 3492

139
outstanding question is whether Ang-(1-7) continues to be effective in animals 3493
with more severe disease. 3494
3495
6.3 Chronic Immune Activation in Type 2 Diabetes 3496
Diabetes-induced immune dysfunction is well established, where low- 3497
grade systemic inflammation is present. This chronic immunoactivated state may 3498
blunt cytokine response to external stimuli. Our data confirms there are also 3499
markers of inflammation present in the bone marrow microenvironment, including 3500
increases in the expression and intracellular levels of pro-inflammatory cytokines 3501
in db/db mice that correlate with circulating levels. In addition, we have found 3502
significant reductions in the number and function of various bone marrow-derived 3503
progenitor cells. In particular, mechanisms that are intimately involved in both 3504
the innate and adaptive immune systems are altered in type 2 diabetes, including 3505
dendritic cells and pre-B cells, among others. We also evaluated the impact of 3506
Ang-(1-7) on chronic immunoactivation, where treatment with this peptide 3507
significantly decreased TNF-α and IL-6 levels, as well as restored their response 3508
to stimulation in diabetic bone marrow. Furthermore, antagonism of Ang-(1-7) 3509
receptors reversed the Ang-(1-7) mediated effect, demonstrating a causal 3510
relationship. 3511
Questions also exist regarding if Ang-(1-7) can also reduce IL-1β, which is 3512
often elevated in the circulation in humans with type 2 diabetes. Since the safety 3513
of Ang-(1-7) has been established in published studies (Petty et al 2009, 3514
Mordwinkin et al 2012a), further studies investigating the impact of this peptide 3515

140
on patients with type 2 diabetes are necessary to verify its ability to block 3516
oxidative stress and return the immune system back into homeostasis. 3517
3518
6.4 Diabetes and Cardiovascular Disease 3519
As aforementioned in the introduction of this thesis, patients with diabetes 3520
have a significantly higher risk for the development of CVD, which is the leading 3521
cause of death in these individuals (ADA). Similar to other groups, we have 3522
observed that this diabetes-induced cardiovascular dysfunction is a result of a 3523
decrease in both CO and LV function (Marques et al 2011, Singh et al 2011). Our 3524
data demonstrates that this LV dysfunction is at least partially due to a decrease 3525
in FS as well as an increase in cardiomyocyte hypertrophy in db/db mice. 3526
Other hypothesized mechanisms thought to be responsible for the cardiac 3527
dysfunction observed in diabetes include tissue and cellular damage secondary 3528
to oxidative stress and inflammation, which we have shown are increased in the 3529
bone marrow and circulation of diabetic mice (Mordwinkin et al 2012b). A 3530
reduction in the number and function of various bone marrow-derived and 3531
circulating progenitor and stem cells such as EPC and MSC, which are 3532
responsible for vasculogenesis and the repair of damaged heart tissue, may also 3533
contribute to diabetes-related CVD. In addition to the existing knowledge that the 3534
number and function of circulating EPC are reduced in diabetes, we have shown 3535
that bone marrow-derived EPC are also significantly decreased in db/db mice. 3536
We also determined that circulating EPC numbers were reduced in our T2DM 3537

141
murine model, which correlates with published results from other groups (Tepper 3538
et al 2002, Loomans et al 2005). 3539
Currently there is limited data on the ability of FDA-approved and 3540
investigational anti-diabetic medications to treat these significant molecular 3541
alterations observed in T2DM. FDA-approved anti-diabetic drugs like pioglitazone 3542
are only indicated for the reduction of blood glucose levels. However glucose 3543
control only slows down the process of progressive organ damage such as seen 3544
in the kidney, heart and even the brain. What may explain the inability to block 3545
organ damage is the inability of glucose controlling agents to block diabetes- 3546
induced oxidative stress. We were able to show that the administration of Ang- 3547
(1-7) was able to block the progression of reduced cardiovascular dysfunction. 3548
What is unclear is the mechanism by which Ang-(1-7) is preventing organ 3549
damage without the siginificant benefits of glucose control. Our data suggest that 3550
Ang-(1-7) can restore circulating EPC, which are significantly reduced in diabetes. 3551
Other outstanding questions regarding the mechanism of Ang-(1-7), including 3552
whether Ang-(1-7) exerts its cardioprotective activity partially through mobilizing 3553
EPC and reducing inflammation have yet to be answered, although this link 3554
seems to be important. 3555
However, unlike Ang-(1-7), pioglitazone is associated with potentially 3556
serious side-effects, including an increased risk for the development of CHF, and 3557
a recent study suggesting that it may increase the risk of bladder cancer (U.S. 3558
Food and Drug Administration). This demonstrates the need for research 3559

142
investigating therapeutic agents that can target the molecular alterations seen in 3560
diabetes that contribute to long-term complications of the disease. In addition, it 3561
is vital that these therapeutic agents must have limited toxicities and side-effect 3562
profiles. As research from our lab as well as other groups show, Ang-(1-7) is able 3563
to reduce diabetes-induced oxidative stress, inflammation, immune dysfunction, 3564
and cardiac dysfunction in a murine model of T2DM (Mordwinkin et al 2012b). 3565
Also, while additional work will need to be done, two published toxicokinetic 3566
studies have also demonstrated that Ang-(1-7) is safe even at supratherapeutic 3567
doses (Petty et al 2010, Mordwinkin et al 2012). 3568
This leads to our future visions for the application of this peptide in 3569
diabetes. While Ang-(1-7) did result in modest reductions in fasting blood glucose 3570
levels in db/db mice, this decrease was not statistically significant (a reduction of 3571
approximately 50 mg/dL after two weeks of treatment). As mentioned previously, 3572
multiple published studies have shown that glucose control alone in diabetic 3573
patients can prevent further damage induced by hyperglycemia, but does not 3574
reverse pre-existing systemic damage. Previous studies have reported that strict 3575
glucose control may even result in harmful side effects due to hypoglycemia 3576
(Brinchmann-Hansen et al 1988, Behar et al 1997, Taubes 2008). The addition of 3577
a second agent to current anti-diabetic pharmacological regimens that has the 3578
ability to reverse pre-existing damage secondary to oxidative stress, 3579
inflammation, and over-activity of the RAS may lead to the next generation of 3580
therapy for the treatment of T2DM. 3581

143
6.5 Gestational Diabetes 3582
One example of where the addition of Ang-(1-7) or a similar therapeutic 3583
agent to a FDA-approved anti-diabetic agent would be useful is in GDM. As our 3584
clinical study demonstrates, despite receiving treatment based on currently 3585
accepted guidelines, pregnant women diagnosed with GDM still exhibit increases 3586
in molecular markers of oxidative stress including evidence of eNOS uncoupling, 3587
reductions in circulating NO levels, and decreases in SOD expression. In addition, 3588
markers of endothelial dysfunction were also present, including a reduction in 3589
circulating EPC counts and increases in plasma sICAM-1 and sVCAM-1 levels. 3590
Importantly, many of these markers correlated significantly with HbA1c. 3591
Additionally, this study shows that temporal hyperglycemia may lead to 3592
permanent disease. Data from this study demostrated that GDM-induced 3593
changes, such as decreased SOD expression, were present in the cord blood of 3594
babies delivered from these mothers. This data may help to explain the results of 3595
other longitudinal studies that have followed children born to mothers diagnosed 3596
with GDM over time. These studies have shown that children born to mothers 3597
diagnosed with GDM have an increased risk of the development of not only 3598
obesity and diabetes, but also diabetes-related complications such as CVD 3599
(Sobngwi et al 2003, Hillier et al 2007). With the diabetes epidemic showing no 3600
sign of slowing, this data suggests that a shift in the current diabetes treatment 3601
paradigm may be necessary to prevent these long-term complications not only in 3602
diabetic patients, but also in their offspring. While a focus on glucose control is 3603

144
still of the utmost importance, more attention needs to be spent on reversing the 3604
pre-existing cellular damage caused by oxidative stress and inflammation in 3605
diabetes. Ang-(1-7) may be one of multiple promising agents with the ability to 3606
work synergistically with currently FDA-approved therapies; together able to 3607
reduce plasma glucose levels, preventing further damage, and reverse oxidative 3608
stress and inflammation in T2DM. Combined, this therapeutic strategy could 3609
potentially significantly reduce the risk of some of the long-term and deadly 3610
complications of diabetes, including CVD. 3611
3612
6.6 Concluding Remarks 3613
In this thesis, we have explored the impact of modulating systemic 3614
inflammation present in T2DM and the impact it would have on disease 3615
progression. Using a murine model of T2DM, we have confirmed that 3616
inflammation and oxidative stress can lead to tissue damage. More importantly, 3617
this process also reduces circulating EPC, which are critical for tissue repair. 3618
Excitingly, we have found that Ang-(1-7) can ameliorate oxidative stress and 3619
restore immune function. Moreover, Ang-(1-7) can also restore circulating and 3620
bone marrow-derived EPC numbers in diabetes, and animals treated with Ang- 3621
(1-7) had reduced cardiovascular dysfunction compared to untreated diabetic 3622
animals. These physiological effects were all accomplished without significant 3623
glucose control, which is the current therapeutic paradigm. The results from this 3624
thesis have also provided insight on potentially important biomarkers that may be 3625
able to detect the emergence of diabetes earlier than currently used biomarkers. 3626

145
The successful validation of these findings may significantly change current 3627
strategies of diabetes detection and the development of preemitive interventions. 3628
3629
3630
3631
3632
3633
3634
3635
3636
3637
3638
3639
3640
3641
3642
3643
3644
3645
3646
3647
3648
3649
3650
3651
3652
3653
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Abstract (if available)

Abstract The aim of this dissertation is to evaluate the impact of type 2 diabetes mellitus on oxidative stress and inflammation in the bone marrow and circulation, as well as investigate the relationship between these molecular alterations and long-term complications of diabetes, specifically cardiovascular disease. In addition, the role of the peptide Ang-(1-7) i as well as the receptor and second messager systems involved in reversing these molecular alterations and complications will also be determined. ❧ The research integrates multiple in vivo studies and a clinical study to provide a comprehensive picture of this disease state. Nitrite levels in the bone marrow and blood were measured using the Griess reaction. Expression and protein levels of molecular markers of oxidative stress and cytokines were determined using RT-PCR, western blot, and ELISA. Levels of oxidative stress, protein tyrosine nitration in the bone marrow, intracellular cytokine levels, and EPC counts were measured using flow cytometric methodologies. Tissue protein nitration was measured by immunohistochemistry. Murine heart function was determined in vivo using small animal echocardiography and thermodilution techniques, and histology was used to measure cardiomyocytes in stained heart sections. Culture of isolated bone marrow cells was used to determine various progenitor counts. ❧ Our in vivo and clinical data indicate that oxidative stress and systemic inflammation play a major role in both type 2 diabetes and gestational diabetes. In addition, we illustrate a potential link between these pathologies and endothelial and cardiovascular dysfunction in this disease state. Treatment of db/db mice with Ang-(1-7) for 14 days resulted in decreases in markers of oxidative stress and inflammation, increases in bone marrow-derived and circulating EPC, as well as increases in other bone marrow-derived progenitors involved in vasculogenesis and immune function. Lastly, Ang-(1-7) treatment helped to increase measures of cardiac function that were reduced in diabetic mice. ❧ While a focus on glucose control is still of the utmost importance, more attention needs to be spent on reversing the pre-existing cellular damage caused by oxidative stress and inflammation in diabetes. Ang-(1-7) may be one of multiple promising agents with the ability to work synergistically with currently FDA-approved therapies

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Asset Metadata

Creator Mordwinkin, Nicholas Michael (author)

Core Title The peptide angiotensin-(1-7) as a novel treatment for complications induced by type 2 diabetes mellitus

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Pharmaceutical Sciences

Publication Date 04/24/2012

Defense Date 03/27/2012

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag angiotensin,Bone Marrow,cardiovascular disease,Diabetes,endothelial dysfunction,immune dysfunction,Inflammation,OAI-PMH Harvest,oxidative stress,progenitor cells,stem cells

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Shen, Wei-Chiang (committee chair), diZerega, Gere S. (committee member), Louie, Stan G. (committee member), Okamoto, Curtis Toshio (committee member), Rodgers, Kathleen E. (committee member)

Creator Email mordwink@usc.edu,nmordwinkin@stanford.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-9565

Unique identifier UC11289124

Identifier usctheses-c3-9565 (legacy record id)

Legacy Identifier etd-Mordwinkin-630.pdf

Dmrecord 9565

Document Type Dissertation

Rights Mordwinkin, Nicholas Michael

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA