University of Southern California Dissertations and Theses

Angiotensin (1-7): a novel treatment for diabetes-induced kidney and heart dysfunction

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Angiotensin (1-7): a novel treatment for diabetes-induced kidney and heart dysfunction

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Content ANGIOTENSIN (1-7): A NOVEL TREATMENT FOR
DIABETES-INDUCED KIDNEY AND HEART
DYSFUNCTION

by

Anna Malgorzata Papinska

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)
August 2016
Copyright 2016 Anna Malgorzata Papinska
ii
Dedication
I would like to dedicate this work to my family: my parents – Barbara and
Tadeusz Papinscy, my brother – Maciej Papinski and my grandmother Elzbieta
Wasala. Thank you for always being there for me and believing in me
unconditionally. None of this would have been possible without your love and
endless support for my dreams, however crazy they are. I dedicate this work to
my boyfriend, Dmitry Popov, with whom I have shared every success and failure.
Thank you for spending endless hours listening to all the lab stories and mock
presentations. You give me strength to keep going. I also dedicate my
dissertation to my mentor and role model, Dr. Kathy Rodgers. Thank you for all
the invaluable life lessons, positive feedback and guidance. I could not have
asked for a better PI to lead me though this journey. And last but definitely not
least, I would like to dedicate this work to all my friends, especially Maira Soto,
who has helped me to solve so many problems, not only research-related –
thank you for your love and patience; Alick Tan – for our endless conversations
and your help with troubleshooting – also in personal life; Dr. Sachin Jadhav,
who has thought me so much – not only how to be a good scientist but also a
kind and grateful person. I would not be who I am today without all of you.

iii
Acknowledgements
I would like to thank my committee members: Drs. Kathy Rodgers, Enrique
Cadenas and Curtis Okamoto for their input and support. I would also like to
acknowledge all the past and present members of the Rodgers lab: Maira Soto,
Alick Tan, Dr. Sachin Jadhav, Lila Kim, Tamar Amzaleg, Dr. Roslynn Stone,
Chris Meeks, Josh Dorst, Theresa Espinoza, Norma Roda, Dr. Kevin Gaffney
and Michael Weinberg – thank you all for your help. I would also like to thank
Dr. Nick Mordwinkin for showing me the ropes and allowing me to continue his
project.
iv
Table of contents
1. Chapter 1: Introduction ...................................................................................... 1
1.1. Overview of type 2 diabetes .................................................................................. 1
1.2. Pathogenic mechanisms in type 2 diabetic complications ..................................... 4
1.2.1. Overview of diabetic complications ................................................................ 4
1.2.2. Pathological mechanisms ............................................................................... 5
1.3. The renin-angiotensin system (RAS) ..................................................................... 9
1.3.1. Overview of the RAS ...................................................................................... 9
1.3.2. Pathological effects of Ang-II in T2D ............................................................ 15
1.4. A(1-7) as a novel treatment for diabetic complications ........................................ 22
1.4.1. RAS modifying therapies .............................................................................. 22
1.4.2. A(1-7) for treatment of diabetic heart disease .............................................. 24
1.4.3. A(1-7) for treatment of diabetic kidney disease ............................................ 29
1.4.4. Potential for A(1-7) to become a novel treatment for
diabetes related complications ..................................................................... 33
1.5. Chapter 1 References .......................................................................................... 34
2. Chapter 2: Short-term administration of angiotensin (1-7) ameliorates
diabetic heart and kidney disease. .................................................................. 56
2.1. Background and goals ......................................................................................... 56
2.2. Study design and methods .................................................................................. 60
2.2.1. Animals ......................................................................................................... 60
2.2.2. Pharmacologic agents and inhibitors ........................................................... 61
2.2.3. Echocardiography ........................................................................................ 61
2.2.4. Histology and heart weights ......................................................................... 63
2.2.5. Glomerular area and mesangial expansion .................................................. 65
2.2.6. Apoptosis ...................................................................................................... 66
2.2.7. Gene expression .......................................................................................... 66
2.2.8. Statistical analysis ........................................................................................ 66
2.3. Results ................................................................................................................. 67
2.3.1. Progression of heart dysfunction in db/db mice and effects of A(1- 7) ......... 67
2.3.2. A(1-7) reduces cardiomyocyte hypertrophy through activation of
multiple pathways. ........................................................................................ 68
2.3.3. A(1-7) decreases cardiac damage by improving tissue vascularization
and reducing fibrosis and inflammatory cell infiltration. ................................ 70
2.3.4. Administration of A(1-7) reduces glomerular area and mesangial
expansion in diabetic mice. .......................................................................... 71
2.3.5. A(1-7) reduces inflammation and oxidative stress in kidneys of
db/db mice. ................................................................................................... 71
2.4. Discussion and conclusions ................................................................................. 76
2.5. Acknowledgements .............................................................................................. 81
2.6. Chapter 2 references ........................................................................................... 81

v
3. Chapter 3: Long-term administration of angiotensin (1-7) prevents
progression of diabetic heart disease .............................................................. 87
3.1. Background and goals ......................................................................................... 87
3.2. Study design and methods .................................................................................. 91
3.2.1. Animals ......................................................................................................... 91
3.2.2. Echocardiography ........................................................................................ 92
3.2.3. Histological analysis: .................................................................................... 93
3.2.4. Concentration of cytokines in plasma ........................................................... 95
3.2.5. Isolation of cardiomyocytes .......................................................................... 96
3.2.6. Contractility and calcium imaging ................................................................. 96
3.2.7. Statistical analysis ........................................................................................ 97
3.3. Results ................................................................................................................. 98
3.3.1. A(1-7) has no effect on body mass and hyperglycemia in db/db mice. ........ 98
3.3.2. Treatment with A(1-7) improved heart function as measured by
echocardiography. ........................................................................................ 99
3.3.3. A(1-7) reduced cardiomyocyte hypertrophy, number of apoptotic
cells, fat accumulation, and fibrosis in hearts from diabetic animals. ......... 100
3.3.4. Administration of A(1-7) to diabetic animals results in reduced
oxidative stress in the heart and decreased levels of circulating
proinflammatory cytokines. ......................................................................... 103
3.3.5. A(1-7) treatment for 8 weeks improves calcium handling
and contractility of isolated cardiomyocytes. .............................................. 105
3.4. Discussion and conclusions ............................................................................... 106
3.5. Acknowledgements ............................................................................................ 114
3.6. Chapter 3 references ......................................................................................... 114
4. Chapter 4: Long-term administration of angiotensin (1-7) prevents kidney
dysfunction and decreases oxidative stress in db/db mice. .......................... 120
4.1. Background and goals ....................................................................................... 120
4.2. Study design and methods ................................................................................ 124
4.2.1. Animals ....................................................................................................... 124
4.2.2. Ultrasonographic assessment of blood flow velocity in renal artery ........... 125
4.2.3. Plasma and urine creatinine concentration ................................................ 126
4.2.4. Urine protein concentration ........................................................................ 126
4.2.5. Glomerular hypertrophy and mesangial expansion .................................... 127
4.2.6. Immunohistochemistry for N-Tyr, phospho-eNOS and NOX-4 .................. 127
4.2.7. Gene expression ........................................................................................ 128
4.2.8. Statistical analysis ...................................................................................... 128
4.3. Results ............................................................................................................... 129
4.3.1. Treatment with A(1-7) does not reduce hyperglycemia at any
of the time points ........................................................................................ 129
4.3.2. Treatment with A(1-7) has no effect on kidney weight. .............................. 130
4.3.3. 16 weeks of A(1-7) administration reduces shear stress and
improves kidney function in diabetic mice .................................................. 131
4.3.4. Glomerular structure is improved after treatment with A(1-7) .................... 132
4.3.5. Oxidative stress damage in diabetic kidneys was decreased after
administration of A(1-7) for 12 or 16 weeks. .............................................. 134

vi
4.3.6. Administration of A(1-7) altered gene expression of eNOS
and a subunit of NADPH oxidase but did not have any effect on
expression of pro-inflammatory cytokines. ................................................. 136
4.3.7. Expression of genes associated with oxidative stress was
increased with age. .................................................................................... 138
4.3.8. A(1-7) alters phosphorylation pattern of eNOS. ......................................... 139
4.3.9. Administration of A(1-7) reduces levels of NOX-4 ...................................... 140
4.4. Discussion and conclusions ............................................................................... 141
4.5. Acknowledgements ............................................................................................ 146
4.6. Chapter 4 references ......................................................................................... 146
5. Chapter 5: Discussion and future directions .................................................. 153
5.1. In search for new treatments for T2D ................................................................ 153
5.2. A(1-7) safety profile ........................................................................................... 155
5.3. Alternative formulations of A(1-7) ...................................................................... 156
5.4. Other applications of A(1-7) ............................................................................... 159
5.5. Concluding remarks ........................................................................................... 161
5.6. Chapter 5 references ......................................................................................... 162
6. Comprehensive bibliography ......................................................................... 165

vii
List of Figures

Fig. 1.1: Components of the renin-angiotensin system 11
Fig. 2.1: Mechanisms of A(1-7) signaling 60
Fig. 2.2: Progression of heart dysfunction in db/db mice and
effects of A(1-7) on cardiac output and shortening fraction 67
Fig. 2.3: Effects of short-term A(1-7) administration on
cardiomyocyte hypertrophy 69
Fig. 2.4: Effects of short-term treatment with A(1-7) on vessel density,
fibrosis and inflammatory cell infiltration in cardiac tissue 70
Fig. 2.5: Glomerular area and mesangial expansion in the
kidneys of animals treated with A(1-7) for 2 weeks 72
Fig. 2.6: Short term treatment with A(1-7) reduces oxidative
stress in the kidneys 74
Fig. 2.7: Representative images of immunostaining for nitrotyrosine,
phospho-eNOS Ser1177 and phospho-eNOS Thr495 75
Fig. 3.1: Calcium signaling in cardiomyocytes 90
Fig. 3.2: Long-term treatment with A(1-7) has no effect on
hyperglycemia and body weight 98
Fig. 3.3: Long-term administration of A(1-7) improves physiological
heart function 99
Fig. 3.4: Treatment with A(1-7) for 16 weeks reduces remodeling
of heart tissue 101
Fig. 3.5: Representative histological images of the analysis of
cardiac tissue remodeling 102
Fig. 3.6: Levels of nitrotyrosine in cardiac tissue after long-term
treatment 103
Fig. 3.7: Levels of circulating cytokines in the animals treated for
16 weeks
104
viii
Fig. 3.8: Calcium transients and contractility of cardiomyocytes
is improved after 8 weeks of A(1-7) administration 106
Fig. 4.1: Blood glucose levels over time 129
Fig. 4.2: Kidney weights over time 130
Fig. 4.3: Physiological kidney function in the animals treated for
16 weeks 131
Fig. 4.4: Glomerular function in the animals treated for 12 or
16 weeks 133
Fig. 4.5: Oxidative stress damage in the kidneys from animals
treated for 12 or 16 weeks 135
Fig. 4.6: Formation of nitrotyrosine residues and involvement
of eNOS and NADPH oxidase 136
Fig. 4.7: Gene expression of eNOS and NADPH oxidase in the
kidneys from animals treated for 4 or 12 weeks 137
Fig. 4.8: Effects of aging on gene expression of oxidative stress
markers 138
Fig. 4.9: eNOS phosphorylation in the kidneys from animals treated
for 16 weeks 139
Fig. 4.10: Levels of NOX-4 in the kidneys from animals treated for
16 weeks 141

ix
List of abbreviations

A(1-7) angiotensin (1-7)
ACE angiotensin converting enzyme
ACE2 angiotensin converting enzyme 2
ACEi angiotensin converting enzyme inhibitors
AGE advanced glycation end product
Ang-I angiotensin I
Ang-II angiotensin II
ARBs angiotensin receptor blockers
AT1 angiotensin II type 1 receptor
AT2 angiotensin II type 2 receptor
BDM 2,3-butanedione monoxime
CHF congestive heart failure
CO cardiac output
CVD cardiovascular disease
x
DAB diaminobenzidine
ECM extracellular matrix
eNOS endothelial nitric oxide synthase
EPC endothelial progenitor cell
ERK extracellular-signal regulated kinase
H&E hematoxylin and eosin
HIER heat induced epitope retrival
hzg heterozygous
IL-1β interleukin-1β
IL-6 interleukin-6
JNK c-Jun N-terminal kinase
L-NAME Nω-nitro-L-arginine methyl ester hydrochloride
MCP-1 monocyte chemoattractant protein-1
MrgD Mas related G protein coupled receptor, member D
N-Tyr nitrotyrosine
NCX sodium calcium exchanger
xi
NF-κB nuclear factor kappa-light-chain-enhancer of activated B cells
NFAT nuclear factor of activated T cells
NO nitric oxide
NOS nitric oxide synthase
OS oxidative stress
PAS periodic acid-Schiff
PBS phosphate buffered saline
PKC protein kinase C
PMN granulocytes
RAGE advanced glycation end product receptor
RAS renin-angiotensin system
ROS reactive oxygen species
RyR ryanodine receptor
SEM standard error of the mean
SERCA sarco/endoplasmic reticulum calcium ATPase
SF shortening fraction
xii
SOD superoxide dismutase
SR sarcoplasmic reticulum
SV stroke volume
T2D type 2 diabetes
TGF-β transforming growth factor-β
TNF-α tissue necrosis factor α
VEGF vascular endothelial growth factor

xiii
Abstract
The goal of this dissertation is to evaluate the effects of administration of
angiotensin-(1-7), a component of the renin-angiotensin system, on type 2 diabetes-
related kidney and heart dysfunction. Increased activation of the pathological arm of the
renin-angiotensin system, specifically via enhanced signaling though angiotensin-II and
its receptor AT1, has been implicated in progression of diabetic heart and kidney
diseases through increased inflammation, oxidative stress, fibrosis and tissue
remodeling. Angiotensin-(1-7) is a short peptide that attenuates pathological effects of
angiotensin-II. We hypothesize that treatment with angiotensin-(1-7) ameliorates diabetic
complications through restoration of the balance between components of the renin-
angiotensin system. We investigated reno- and cardio- protective effects of short-term (2
weeks) and long-term (16 weeks) treatment with angiotensin-(1-7) on kidney and heart
function in a mouse model of severe type 2 diabetes (db/db). Angiotensin-(1-7)
prevented renal and cardiac dysfunction. Several mechanisms of this protective action
were identified, including reduction of fibrosis and tissue remodeling, decrease in
systemic inflammation and immune cell infiltration, amelioration of oxidative stress
damage, and enhancement of calcium signaling in the hearts. These effects were
observed in animals with uncontrolled hyperglycemia and obesity. Short-term treatment
studies allowed for identification of potential mechanisms and long-term administration
studies further delineated actions of angiotensin-(1-7). Angiotensin-(1-7) is effective in
the amelioration of kidney and heart dysfunction in a mouse model of severe type 2
diabetes. Data presented in this dissertation supports potential therapeutic benefits of
angiotensin-(1-7) in clinical use.
1
1. Chapter 1: Introduction
1.1. Overview of type 2 diabetes
Diabetes is a metabolic disorder that affects nearly 10% of the US
population(Centers for Disease Control, 2014; Cowie et al., 2006). One fourth of
these cases are due to predicted, but undiagnosed diabetes. Among people 65
years or older, every fourth American suffers from diabetes (Centers for Disease
Control, 2014; Cowie et al., 2006). The prevalence of diabetes continues to grow.
It is estimated that by 2050, as high as one third of US population will have
diabetes (Boyle et al., 2010). The cost of diagnosed diabetes is estimated to
have reached $245 billion in 2012, which is 41% more than $174 billion reported
in 2007 (American Diabetes Association, 2013). This includes $176 billion in
direct costs and $69 billion in decreased productivity. Diabetic patients spend on
average 2.3 times more money on health care than diabetes-free individuals.
Cost of prescription medications to treat symptoms of diabetes and diabetes-
related complications was 12% and 18% of total direct patient’s health care
costs, respectively (American Diabetes Association, 2013). In addition to high
economic burden on the society, diabetes is also associated with decreased
quality of life, as well as pain and suffering that affect not only patients but also
all family members. With the continued growth in number of diabetic patients,
deaths related to diabetic complications and increasing costs of medications,
2
new technologies and health care practices will be invaluable in prevention and
treatment of diabetes.
Diabetes stands for a group of metabolic diseases characterized by a
pathologically increased level of blood glucose (hyperglycemia). Three types of
diabetes are now recognized based on the mechanisms causing hyperglycemia:
type 1 diabetes mellitus, caused by insulin deficiency; type 2 diabetes mellitus
(T2D), caused by insulin resistance; and gestational diabetes, dysfunction of
insulin receptors due to pregnancy-related factors. Type 1 diabetes, also called
insulin-dependent diabetes or juvenile-onset diabetes, develops due to
pancreatic dysfunction and insufficiency in insulin production. Even though exact
mechanisms causing type 1 diabetes are not entirely understood, the loss of beta
cell function is commonly ascribed to autoimmune response (Atkinson et al.,
2014). The most common treatment for patients with this type of diabetes is
insulin that can be delivered by injections or a pump. Gestational diabetes is also
characterized with insulin insufficiency, however the exact mechanism of this
pathology is still unknown. Gestational diabetes is diagnosed when a pregnant
woman without a previous diabetes diagnosis develops hyperglycemia during
pregnancy. Gestational diabetes occurs in approximately 5% of pregnancies
worldwide (Ferrara, 2007). The occurrence of gestational diabetes is considered
a risk factor for developing T2D (Ben-Haroush et al., 2004).
3
T2D, which is the focus of this dissertation, also called non-insulin-dependent
diabetes or adult-onset diabetes, accounts for 90-95% of all diabetes cases
(Centers for Disease Control, 2014; Zimmet et al., 2001). T2D usually occurs
later in life than type 1 diabetes and is characterized with insulin resistance.
Resulting hyperglycemia causes pancreatic beta-cell dysfunction, which leads to
deficiency in insulin secretion. As a result, not only cells are deprived of glucose,
but also sugar concentration in blood remains abnormally high leading to
activation of many pathological pathways that increase the risk of developing
various complications such as cardiovascular disease (CVD), nephropathy,
diabetic foot ulcer, retinopathy, neuropathy, and others. T2D has been
associated with many risk factors such as obesity, age, physical inactivity and
family history. Ethnicity is also a risk factor for developing T2D; African
Americans, Hispanics and Native Americans have particularly high risk of
diabetes (Blackwell et al., 2014; Centers for Disease Control, 2014). T2D is
currently managed with a complex treatment plan; it primarily includes strict diet
and exercise, supported by blood glucose and cholesterol control medications.
Even though weight loss is the most successful way to control T2D, many
patients remain incompliant with their treatment plan (Diabetes Prevention
Program Research Group, 2002; Wing et al., 2011). Lack of compliance in T2D
makes it one of the most prevalent and difficult to control diseases in the US.
4
1.2. Pathogenic mechanisms in type 2 diabetic complications
1.2.1. Overview of diabetic complications
T2D is associated with increased risk for developing several serious
complications such as CVD, nephropathy, retinopathy, stroke, coronary artery
disease and myocardial infarction (Grundy et al., 1999; Kannel and McGee,
1979; Stratton et al., 2000; The Hypertension in Diabetes Study Group, 1993a;
1993b). Recent evidence also shows that T2D is a major risk factor for
Alzheimer’s disease (Peila et al., 2002; Xu et al., 2004). Most complications
develop secondary to prolonged hyperglycemia, obesity and hypertension.
Cardiovascular complications are the main cause of deaths in patients with T2D
(Morrish et al., 2001). Other complications such as diabetic kidney disease
significantly contribute to progression of heart disorders (Sarnak et al., 2003).
Diabetes is now recognized as the main cause of dialysis and kidney failure in
the US (Centers for Disease Control, 2014; Sarnak et al., 2003). Cardiac and
renal health is the main cause of deaths associated with T2D and is the main
focus of this dissertation. However, other complications also significantly
decrease quality of life for diabetic patients and may even result in death.
Damage caused to the small blood vessels in the retina due to diabetic milieu
may result in loss of vision (Hammes et al., 2011). T2D is also the main cause of
amputations. Persistent foot sores associated with diabetes-induced nerve
5
damage and impaired wound healing, account for more than 60% of all non-
traumatic lower-limb amputations (Centers for Disease Control, 2014).
1.2.2. Pathological mechanisms
The mechanisms leading to diabetic complications are complex and not entirely
understood. Below are some of the well-recognized pathological mechanisms
that lead to development and progression of diabetic complications:
Hyperglycemia
Hyperglycemia is thought to be the main pathological mechanism leading to
progression of T2D. High blood glucose levels cause increase in activity of the
polyol pathway that results in reduction of glucose to sorbitol, which can be later
used to form fructose. This reduction is catalyzed by aldose reductase, which
consumes NADPH in this process (Lee and Chung, 1999). NADPH is also used
to reduce glutathione, which acts to lower oxidative stress (Brownlee, 2001). The
increased flow though polyol pathway results in decreased availability of reduced
glutathione and ability of the cell to fight oxidative stress. Increased production of
advanced glycation end products (AGEs) is also associated with hyperglycemia
and is thought to contribute to pathogenesis of diabetes. AGEs not only modify
intracellular proteins such as transcription factors and cause their dysfunction but
they can also diffuse out of the cell and modify extracellular matrix and circulating
proteins such as albumin (Giardino et al., 1994; McLellan et al., 1994; Shinohara
et al., 1998). These modifications caused by AGEs change function of the
6
proteins and activate many signaling pathways leading, for example, to release
of pro-inflammatory cytokines and growth factors (Abordo and Thornalley, 1997;
Charonis et al., 1990; Li et al., 1996; Vlassara et al., 1988). Hyperglycemia
activates different forms of protein kinase C (PKC), which results in changes in
gene expression patterns (Derubertis and Craven, 1994; Koya and King, 1998;
Kuboki et al., 2000). For example, activation of PKC, by increased levels of
glucose inside the cell, leads to decreased expression of endothelial nitric oxide
synthase (eNOS) that produces nitric oxide (NO), a potent vasoconstrictor
(Kuboki et al., 2000). It may also result in increased production of transforming
growth factor-β (TGF-β) that leads to increased fibrosis (Koya et al., 1997).
Lastly, hyperglycemia can directly increase production of reactive oxygen
species (ROS) through dysregulation of mitochondrial function. Elevated levels of
glucose inside the cell cause increased flow of electron donors through the
electron transport chain and result in increased voltage gradient across the
membrane. This causes the electrons to back up to coenzyme Q, which donates
the electrons to molecular oxygen resulting in production of superoxide, a potent
oxidant (Rolo and Palmeira, 2006).
Control of hyperglycemia is still the main goal of anti-diabetic therapies. Several
classes of glucose lowering agents are available on the market. These include
thiazolidinediones and metformin, which focus on improving insulin resistance, as
well as sulfonylureas, glucagon-like peptide agonists and dipeptydyl peptidase-IV
inhibitors that stimulate insulin secretion. Even though hyperglycemia remains
7
the main target of anti-diabetic therapies, recent clinical trials such as ADVANCE
and ACCORD, show that intensive blood glucose control does not necessarily
decrease the risk of CVD (Gerstein et al., 2008; Patel et al., 2008). Similarly,
intensive control of hyperglycemia in a group of veterans did not reduce the risk
of CVD or microvascular complications, with the exception of albuminuria
(Duckworth et al., 2009). In addition, many glucose lowering medications may be
associated with hypoglycemia, which was shown to increase the risk of
macrovascular complications such as myocardial infarction (Zoungas et al.,
2010).
Obesity
Obesity and fat accumulation is another factor that contributes to development of
diabetic complications. Adipose tissue produces high levels of adipokines that
induce a number of effects. Adiponectin, a member of the adipokine family,
through binding to its receptors, participates in regulation of glucose homeostasis
and fatty acid metabolism. It was also shown to play a role in the amelioration of
oxidative stress (Tao et al., 2007). Leptin is another adipokine that plays a role in
regulating the metabolism. Reduced leptin signaling is known to cause
uncontrolled eating (Woods et al., 1998). This effect has been used in
development of two popular type 2 diabetes mouse models – ob/ob that has a
leptin deficiency and db/db that carries a dysfunctional leptin receptor (Belke and
Severson, 2012; Pelleymounter et al., 1995). Recently, leptin has been shown
not only to play a role in the development of metabolic dysregulation but also to
8
contribute to diabetic complications. Leptin stimulates ischemia-induced retinal
neovascularization (Suganami et al., 2004), and causes renal disease (Wolf and
Ziyadeh, 2006). Obesity is usually associated with hyperlipidemia. Increased
levels of lipids cause enhanced uptake of fatty acids by cells. Statins are most
commonly prescribed to target hypercholesterolemia but they were also shown to
reduce levels of triglycerides (Stein et al., 1998). Many diabetic patients are given
statins as a part of their overall anti-diabetic therapy, usually aimed to slow the
progression of vascular disease, including diabetic heart disease. However
dyslipidemia was also shown to play a role in development and progression of
several other diabetic complications. Serum lipids and body mass are
independently associated with diabetic neuropathy (Sone et al., 2005). Increased
levels of lipids are also thought to be a risk factor for developing diabetic
nephropathy (Ansquer et al., 2005).
Hypertension
Hemodynamic factors play a crucial role in development and progression of key
diabetic complications such as CVD, nephropathy and retinopathy. Hypertension
is common even in newly diagnosed diabetics and is associated with obesity
(The Hypertension in Diabetes Study Group, 1993a). High blood pressure is also
recognized as a major risk factor for premature morbidity and mortality in patients
with T2D. Hypertension causes mechanical stress in some of the key organs
such as the heart and kidneys. This shear stress contributes to activation of
many pathological pathways including fibrotic and growth factors (Gnudi et al.,
9
2003; Gruden et al., 1997; 2000). High blood pressure is considered to be an
independent risk factor for developing end-stage renal disease, a condition that
requires dialysis or kidney transplant for survival (Klag et al., 1996). It was also
associated with cardiovascular complications even in newly diagnosed diabetics
(The Hypertension in Diabetes Study Group, 1993a; 1993b). Elevated blood
pressure is also a known risk factor for developing diabetic retinopathy (Kohner
et al., 1996).
One of the most important factors contributing to hypertension is dysregulation of
the renin-angiotensin system (RAS). This system of peptide hormones is
responsible not only for regulation of blood pressure but was also shown to
modulate other pathological mechanisms such as oxidative stress and
inflammation (Benigni et al., 2010; Chabrashvili et al., 2003; Suzuki et al., 2003;
Zimmerman et al., 2004).
1.3. The renin-angiotensin system (RAS)
1.3.1. Overview of the RAS
In the classical view, the RAS is considered to be primarily responsible for
regulation of blood pressure and fluid balance. Recent advances revealed that
components of the RAS play various roles in regulation of function and
homeostasis in different organ systems and disease states (Carey and Siragy,
2003; Chappell, 2007; Chappell et al., 2004).
10
The RAS consists of several biologically active and inactive components.
Angiotensinogen is the precursor of all the active peptides in the RAS. It is mainly
produced in the liver and released to the blood stream as an inactive peptide.
Even though angiotensinogen does not have any biological function per se,
increased levels of this peptide associated with M235T polymorphism were
shown to play a role in hypertension (Gumprecht et al., 2000; Winkelmann et al.,
1999). Higher levels of angiotensinogen are hypothesized to increase production
of angiotensin II (Ang-II), a downstream peptide, well known to cause
vasoconstriction and elevated blood pressure.
Renin is an enzyme secreted in response to low blood pressure that catalyzes
conversion of angiotensinogen to angiotensin-I (Ang-I). This enzyme is
particularly important in the regulation of function of the RAS, as its activity is the
rate-limiting factor in the production of all the active peptides (Weber, 2001;
Zaman et al., 2002). Ang-I is cleaved by angiotensin-converting enzyme (ACE) to
form Ang-II, the central player in the RAS (Fig. 1.1). Ang-II not only causes
vasoconstriction but also increases secretion of aldosterone – a hormone
stimulating reabsorption of sodium and water in kidneys, therefore increasing
blood volume and pressure. ACE was shown not only to be involved in
generation of Ang-II but it also catabolizes angiotensin-(1-7) [A(1-7)], another
active peptide of the RAS that has vasodilatory properties; this further
strengthens the role of ACE in the hypertension (Deddish et al., 1998).
11
ACE2 is another important enzyme in the RAS that primarily catalyzes
conversion of Ang-II to A(1-7) (Rice et al., 2004). Alternatively, ACE2 can also
play a role in formation of angiotensin-(1-9) from Ang-I (Donoghue et al., 2000).
As both ACE and ACE2 are involved in formation of the two vasoactive peptides,
namely Ang-II and A(1-7), it is now recognized that many of the actions of the
RAS are controlled by the ACE/ACE2 balance (Chappell, 2007; Santos and
Ferreira, 2007; Simões e Silva et al., 2006; Warner et al., 2007).
Fig. 1.1: Components of the renin-angiotensin system.
12
Ang-II, the key player in the RAS was first isolated in 1940 by Braun Menendez
et al. and Page and Helmer (Basso and Terragno, 2001). At first, Ang-II was
identified as a potent modulator of blood pressure, however the role of this
peptide extends far beyond that. The use of ACE inhibitors (ACEi) and Ang-II
type 1 (AT1) receptor blockers (ARBs) proved to have positive effects beyond the
control of blood pressure (Ferrario et al., 2004). Increased signaling through the
ACE/Ang-II/AT1 axis was shown to contribute to inflammation (Muller et al.,
2000; Suzuki et al., 2003), oxidative stress (Chabrashvili et al., 2003;
Rajagopalan et al., 1996; Zimmerman et al., 2004) and insulin resistance
(Henriksen, 2007; Olivares-Reyes et al., 2009). In addition to AT1 receptor,
Ang-II is also known to bind to another receptor called Ang-II type 2 receptor
(AT2). While AT1 is widely expressed in mature tissues such as endothelium,
heart, kidney and liver, AT2 is mostly present in fetal tissue. Actions of this
receptor oppose the effects of activation of AT1 (Touyz and Schiffrin, 2000).
A(1-7), another active member of the RAS consisting of 7 amino acids
(DRVYIHP), is also thought to oppose the actions of Ang-II/AT1 signaling. A(1-7)
is formed from Ang-II primarily through the activity of ACE2 but other enzymes
such as prolyendopeptidase and prolylcarboxypeptidase may also be involved.
Alternatively A(1-7) can be produced through direct enzymatic cleavage of Ang-I.
Angiotensin-(1-5) is the product of A(1-7) metabolism by ACE. Thus, ACEi
increase levels of A(1-7) not only through increasing Ang-I but also by preventing
degradation of A(1-7). Some studies suggest that protective effects of ACEi may
13
be partially due to increased levels of A(1-7) (Santos and Ferreira, 2007; Simões
e Silva et al., 2006). A(1-7) action is primarily mediated through activation of a
G-protein coupled receptor called Mas (Santos et al., 2003). Signaling cascades
downstream from Mas are not fully elucidated. However, it has been shown that
activation of Mas causes arachidonic acid release and intracellular Akt activation
(Sampaio et al., 2007; Santos et al., 2003). A(1-7) has been shown to counteract
many of Ang-II actions; for example, it decreases oxidative stress and increases
production of NO (Mordwinkin et al., 2012; Sampaio et al., 2006). NO is involved
in vasodilatory response and also protects from organ failure in diabetes (Kosugi
et al., 2010). In fructose-fed rat model of metabolic syndrome, A(1-7) was found
to decrease inflammation, improve insulin sensitivity and decrease fat deposition
(Marcus et al., 2012). This heptapeptide was also shown to stimulate proliferation
of hematopoietic progenitor cells (Heringer-Walther et al., 2009).
Even though Ang-II and A(1-7) are the main effectors of the RAS, other peptides
are also thought to play a role in the physiology. Recently identified alamandine
was shown to be present in human blood (Lautner et al., 2013). Physiological
effects of alamandine resemble those of A(1-7), including vasodilation and
reduction of fibrosis. In addition, same group identified that effects of alamandine
are mediated through a newly discovered receptor called Mas-related G-protein-
coupled receptor, member D (MrgD). Some studies also report biological activity
of angiotensin-(1-9) through enhanced release of NO and arachidonic acid
(Jackman et al., 2002).
14
The function of other biologically active peptides of the RAS such as
angiotensin-III and angiotensin-IV, which are not the focus of this dissertation,
will only be discussed in brief. Effects of angiotensin-III are though to be similar
to the effects of Ang-II but less potent (Cesari et al., 2002), whereas
angiotensin-IV is thought to be involved in cognitive function and memory
(Braszko et al., 2006; Chai et al., 2004).
Several recent discoveries had shed a new light on the structure and function of
the RAS peptides. The RAS was initially categorized as an endocrine system,
however today we know that local or tissue RAS also exists. Components of the
RAS can be detected not only in circulation but also in tissues such as heart,
blood vessels, kidney, pancreas, adipose tissue and central nervous system
(Lavoie and Sigmund, 2003; Nielsen et al., 2000; Sernia, 2001; Spat and
Hunyady, 2004). The local RAS is also regulated independently of the systemic
RAS, which suggests that it can be adjusted by local regulatory mechanisms
(Miyazaki and Takai, 2006; Paul et al., 2006). This emphasizes the importance of
the local RAS for the homeostasis of the organ systems.
Another important discovery showed functional and structural association of the
RAS receptors. Recent evidence shows that Mas receptor can form heterodimers
with AT2 receptor (Castro et al., 2005). This observation is supported by the fact
that these receptors mediate similar actions. Even though this interaction may
not be necessary for the activity of the receptors, addition of PD123319, an AT2
15
inhibitor, results in antagonism of function of both AT2 and Mas. Interaction of
Mas and AT1 receptor has also been noted. This interaction is thought to play a
role in inhibition of the actions of AT1 receptor (Villela et al., 2015). However, the
function and effects of these relations are thought to be species- and/or tissue-
specific (Castro et al., 2005; Villela et al., 2015).
1.3.2. Pathological effects of Ang-II in T2D
The effectiveness of the RAS modifying therapies such as ACEi and ARBs in
amelioration of both hypertension and end organ injury in diabetic patients, in
particular kidney disease, suggests a significant role of Ang-II in the
pathogenesis of diabetic complications. However, most clinical studies show no
escalation of levels of circulating Ang-II in diabetic patients. In contrast, evidence
suggests increase in production of the organ specific RAS (Anderson et al.,
1993; Carey and Siragy, 2003; Danser et al., 1994). It has also been shown that
hyperglycemia increases activity of the pathological arm of the RAS
(ACE/Ang-II/AT1) and decreases activity of the protective arm
(ACE2/A(1-7)/Mas) (Lavrentyev and Malik, 2008; Toma et al., 2008).
Even though one of the best-described effects of Ang-II is vasoconstriction, blood
pressure regulation is not the main focus of this dissertation. Ang-II has been
shown to induce a variety of effects that are independent of blood pressure
control. A(1-7) is also well known for its vasodilatory effects, however in
proposed treatment regimen the half-life of A(1-7) in blood stream is circa 30 min,
16
which implies that the effects on blood pressure are only transient and are not
the main mechanism of the protective action of this peptide. This review focuses
on pathological effects of Ang-II beyond blood pressure regulation.
Oxidative stress
Hyperglycemia is known to induce excessive production of ROS and contribute
to cellular damage, but Ang-II also adds to oxidative stress in diabetes. Ang-II is
well known to activate NADPH oxidase, one of the most potent producers of
superoxide (Touyz et al., 2002). Although the exact mechanism of NADPH
activation by Ang-II is not entirely understood, several molecules such as
phospholipases/PKC and Rac1 are thought to be involved in this signaling
(Cheng et al., 2006; Karathanassis et al., 2002; Touyz and Schiffrin, 1999). Ang-
II not only causes activation of NADPH oxidase but it also enhances expression
of this enzyme (Hitomi et al., 2006). Ang-II was shown to increase expression of
several NADPH oxidase subunits. Ang-II-induced production of ROS by NADPH
oxidase is the main source of oxidative stress in cardiovascular system (Hitomi et
al., 2007).
Oxidative stress is thought to play a major role in the development and
progression of diabetic complications through causing damage to proteins, lipids
and DNA (Pham-Huy et al., 2008). Under normal conditions, hydrogen peroxide
is needed for activation of insulin receptors (Hayes and Lockwood, 1987). ROS
generated by NADPH oxidase are thought to inhibit tyrosine phosphatase (PTP-
17
1B), which leads to increased phosphorylation and promotes activation of insulin
receptor (Mahadev et al., 2001). Even though low levels of ROS are required for
proper function of insulin signaling, chronic increase in radicals may result in
insulin resistance (Evans et al., 2005; Paolisso et al., 1994). Oxidative stress was
shown to chronically activate stress kinases such as c-Jun N-terminal kinase
(JNK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)
that are implicated in progression of insulin resistance (Bloch-Damti and Bashan,
2005). In addition, increased levels of superoxide may react with NO, which
leads to formation of peroxynitrite, a potent oxidant capable of protein
modification by addition of nitrate group. Nitration of tyrosine residues can
change structure and function of the proteins (Radi, 2013). It is also thought to be
a second mechanism of ROS-induced insulin resistance, as peroxynitrite reacts
with tyrosine residues on insulin receptors and leaves the proteins dysfunctional
(Kaneki et al., 2007). Increased production of ROS also has other implications in
T2D. Oxidative stress caused by Ang-II is known to impair endothelial function
(Rajagopalan et al., 1996). Chronic overproduction of ROS can also stimulate
inflammatory response through promotion of expression of NF-κB and
degradation of IκB, an inhibitor of NF-κB (Pueyo et al., 2000).
Inflammation
Injury caused by the oxidative stress attracts immune cells and may contribute to
increases inflammation. Chronic low-grade inflammation is one of the main
highlights of T2D (Stehouwer et al., 2002).
18
Ang-II also has a direct effect on the inflammatory response and it has been
implicated in all three major steps involved in the inflammatory response:
increased vascular permeability, leukocyte migration and scarring.
Ang-II was shown to increase vascular permeability through two mechanisms.
Some studies suggest that effect of Ang-II on endothelial permeability is
mediated by blood pressure control (Williams et al., 1995); others showed that
Ang-II induced secondary messengers such as prostaglandins and vascular
endothelial cell growth factor (VEGF) play a more significant role in this process
(Guazzi et al., 1997; Pupilli et al., 1999; Reddy et al., 1995).
Another important mechanism leading to inflammation is migration of
inflammatory cells. Ang-II was shown to increase the adhesion of leukocytes to
endothelium and mesangial cells in the kidneys (Kim et al., 1996; Mene et al.,
1995). Diabetic complications are characterized by infiltration of inflammatory
cells into organs (Graves and Kayal, 2008). Ang-II increases expression of
monocyte chemoattractant protein-1 (MCP-1) and contributes to immune cell
infiltration (Ruiz-Ortega et al., 1998). Studies also show that RAS blockade with
ACEi and ARBs in diabetic rats has renoprotective effects through decreased
expression of MCP-1 (Kato et al., 1999). Ang-II has also been shown to promote
expression of other chemokines such as RANTES and interleukin-8 (IL-8) as well
as its homologues (Dol et al., 2001; Suzuki et al., 2002).
19
Ang-II also has an indirect effect on immune cell infiltration by altering the
production of proinflammatory mediators (Ruiz-Ortega et al., 1998; 2001a;
2001b; Wolf et al., 2002). Many studies have shown that Ang-II increases activity
of NF-κB, which controls expression of many proinflammatory genes such as
MCP-1 and RANTES, adhesion molecules (VCAM-1, ICAM-1) and
proinflammatory cytokines such as tissue necrosis factor-α (TNF-α), interleukin-
1β (IL-1β) and interleukin-6 (IL-6) (Guijarro and Egido, 2001; Pueyo et al., 2000;
Ruiz-Ortega et al., 1998). In addition, increased levels of proinflammatory
cytokines have been observed in both experimental models of T2D and patients.
Elevated levels of IL-1β are a marker of T2D (Spranger et al., 2003). Blocking IL-
1 activity has been shown to reduce inflammatory response both in patients and
animal models (Ehses et al., 2009; Larsen et al., 2007). Excess of adipose tissue
and fatty liver associated with obesity and T2D is known to significantly
contribute to production of pro-inflammatory cytokines such as IL-6 and TNF-α
(Xu et al., 2003). Increased levels of cytokines may lead to progression of insulin
resistance and impairment of pancreatic β-cell function (Cai et al., 2005; Maedler
et al., 2002).
Fibrosis and remodeling
The final steps of an inflammatory response include tissue repair by replacement
with new cells or connective tissue, leading to fibrosis and pathological
remodeling in organs affected by T2D. The role of RAS in wound healing has
been well described (Rodgers et al., 2001). Ang-II is considered a potent growth
20
signal that regulates healing and fibrosis. Ang-II through increased expression
and activation of growth factors such as TGF-β, and platelet-derived growth
factor (PDGF), has been shown to activate expression of extracellular matrix
(ECM) in kidneys, contributing to remodeling of the glomeruli and leading to
impaired filtration function (Mezzano et al., 2001; Nakamura et al., 2000). TGF-β
is one of the strongest fibrogenic cytokines and its levels and activity are
stimulated by Ang-II (Border and Noble, 1998; Kagami et al., 1994). In addition,
treatment with ACEi and ARBs was shown to decrease levels and activity of
TGF-β in various models of kidney disease (Ishidoya et al., 1996; Pimentel et al.,
1995). Ang-II is also known to stimulate several mitogen-activated protein (MAP)
kinases such as extracellular-signal regulated kinase (ERK) and JNK. Activation
of these pathways may be in part responsible for hypertrophic effects of Ang-II
(Sugden and Clerk, 1998; Wang et al., 1998; Zou et al., 2004).
Increased production of ECM plays a role in development and progression of
several diabetic complications. The increased interstitial fibrosis leads to
ventricular stiffness and may result in impaired pumping function of the heart
(Weber et al., 1994). Ang-II has been also shown to induce cardiomyocyte
hypertrophy, one of the highlights of diabetic heart disease (Liu et al., 1998;
Ritchie et al., 1998; Wada et al., 1996). In addition to stimulation of hypertrophic
pathways such as ERK and JNK, Ang-II through activation of PKC pathway and
subsequent mobilization of intracellular calcium in the cardiomyocytes is involved
in activation of nuclear factor of activated T cells (NFAT), a potent hypertrophic
21
signal (Molkentin et al., 1998; Wilkins and Molkentin, 2004). Increased ECM
deposition due to activation of the RAS is mainly attributed to stimulation of
TGF-β (Sopel et al., 2011). RAS also regulates cell proliferation and apoptosis.
Ang-II was shown to induce programmed cardiomyocyte death in diabetic hearts
(Singh et al., 2008). All of these changes in structure of the heart lead to impaired
contractility and cardiac function.
Diabetic kidney disease is characterized with impaired filtration function and
overgrowth of mesangial cells. Excessive accumulation of ECM and mesangial
hypertrophy result in structural changes in the glomeruli leading to impaired
glomerular filtration rate and renal failure (Wesson, 1998). Ang-II contributes to
kidney dysfunction not only though increasing glomerular capillary pressure but
also through non-hemodynamic factors. Even though the exact mechanism of
Ang-II induced mesangial expansion is not entirely understood, activation of ERK
and JNK pathways as well as formation of superoxide may play a role (Anderson
et al., 1996; Huwiler et al., 1998; Jaimes et al., 1998). It is however well
established that Ang-II induced production and activation of TGF-β significantly
contributes to renal fibrosis and remodeling (Ishidoya et al., 1996; Pimentel et al.,
1995).
22
1.4. A(1-7) as a novel treatment for diabetic complications
1.4.1. RAS modifying therapies
Current treatments for T2D-related hypertension and kidney dysfunction focus on
reduction of Ang-II levels and activity. While ACEi and ARBs are commonly
prescribed to reduce blood pressure, they were also shown to be more effective
in amelioration of kidney and heart diseases than other anti-hypertensive
medications (American Diabetes Association, 2002).
ACEi are small molecule compounds that inhibit formation of Ang-II. Short-term
administration of ACEi results in virtually complete inhibition of Ang-II production
in the circulation. However, it has been shown that administration of ACEi does
not completely eliminate intra-renal Ang-II, which can be formed through
activation of other enzymes and in the endosomes (Imig et al., 1999; Rabelo et
al., 2011). Ang-II eventually returns to pretreated levels after chronic
administration of ACEi (Juillerat et al., 1990; van de Wal et al., 2006). What is
more, effectiveness of ACEi has been shown to depend on patients’ ethnicity and
genetics (Exner et al., 2001; Weekers et al., 2005). Also, in patients with renal
disorder, ACEi are known to cause hypotension and hyperkalemia (Ljungman et
al., 1992; Palmer, 2004). Drastic decrease in Ang-II levels in some cases may
contribute to renal hypoperfusion and cause acute kidney failure in patients with
nephropathy. Other common adverse effects of ACEi include dry cough,
angioedema, and rash. Even though related to adverse effects, ACEi are still the
23
first line treatment for diabetic nephropathy. Several studies showed that this
class of drugs is also effective in amelioration of diabetic heart disease (Heart
Outcomes Prevention Evaluation HOPE Study Investigators, 2000). Treatment
with ACEi is thought not only to decrease levels of Ang-II but also to increase
levels of A(1-7). In fact, studies suggest that some of the protective effects of
ACEi are mediated through increased activation of protective arm of the RAS
(Santos and Ferreira, 2007; Simões e Silva et al., 2006).
ARBs are a relatively new option for treatment of hypertension and diabetic
kidney disease. ARBs act through blocking AT1 signaling. This class of drugs
was shown to cause minor side effects. Similarly to ACEi, some cases of
angioedema and acute renal failure in patients on ARBs have been reported
(Julius et al., 2006; Saine and Ahrens, 1996). In addition, in most cases, patients
suffering from severe coughing caused by ACEi, develop this side effect after
treatment with ARBs as well (Cicardi et al., 2004; Julius et al., 2006). Inhibition of
AT1 receptor is also associated with increase in plasma Ang-II. This excess of
Ang-II can bind to unblocked AT2 receptors, which have been recently shown to
mediate many adverse effects (Ruiz-Ortega et al., 2001a; Wolf et al., 2002). In
general, ARBs are considered safer than ACEi but the full mechanism of action
of this class of drugs is poorly understood.
We hypothesize that use of A(1-7) can become a safer alternative to ACEi and
ARBs, as it shows virtually no adverse effects in patients and is effective in
24
amelioration of several complications associated with T2D, including
cardiomyopathies, kidney disease, lung dysfunction and neuropathies.
1.4.2. A(1-7) for treatment of diabetic heart disease
1.4.2.1. Overview of diabetic heart disease
One third of all deaths in the US is attributed to CVD. Heart disease is now
considered not only the deadliest, but also one of the most expensive conditions,
with $312.6 billion spent on treatment and hospitalization in 2009 (Go et al.,
2013). Patients suffering from diabetes have up to 4 times higher risk of
developing CVD than adults without diabetes (Sarnak et al., 2003). Some of the
conditions that contribute to higher risk for developing heart disorders include
elevated cholesterol and triglycerides levels. Dyslipidemia contributes to vascular
dysfunction and hypertension that causes mechanical stress and leads to cardiac
hypertrophy. Chronic inflammation and oxidative stress that can cause cellular
damage to cardiac tissue are also consequences of hyperglycemia,
hyperlipidemia and increased release of growth factors such as Ang-II.
Myocardial performance in diabetic patients is decreased, partially because
cardiac cells are not supplied with sufficient amount of glucose as they become
insulin resistant. Hyperglycemia also leads to overproduction of AGEs, which
have proinflammatory, profibrogenic and mitogenic properties in the heart
(Marwick, 2006). Moreover, chronic kidney disease, one of the most prevalent
diabetic complications, is one of major risk factors for developing CVD. Patients
25
with kidney dysfunctions are actually more likely to die of a heart failure than to
develop kidney failure; they are now considered the highest risk group for CVD
(Sarnak et al., 2003).
One of the most prevalent heart disorders associated with T2D is congestive
heart failure (CHF) characterized with remodeling of cardiac tissue. Due to
increased levels of growth factors and mechanical stress, the heart becomes
hypertrophied and fibrotic, and the ventricular walls thickened. Increased
stiffness of the muscle and reduced volume of the ventricles may eventually lead
to heart failure. Uptake of fatty acids in cardiac cells exceeds their oxidation rate,
which leads to accumulation of fat and promotes lipotoxicity. This in
consequence, might induce apoptosis and contribute to abnormal remodeling of
cardiac tissue (McGavock et al., 2006). Organs exposed to chronic
hyperglycemia produce abnormally high levels of ROS. This can lead to protein,
lipid and DNA damage, which result in apoptosis and scaring; leading to
cardiomyopathies (Cai and Kang, 2001; Wold and Ren, 2004). Diabetes-induced
impairment in calcium signaling, a major regulator of cardiomyocyte contractility,
also contributes to progression of heart dysfunction. Some of the experimental
models of T2D show altered expression of calcium channels in cardiac cells and
impaired calcium homeostasis. As a result, cells lose their ability to contract
(Boudina and Abel, 2007).
26
1.4.2.2. Effects of A(1-7) on heart diseases
Ang-II activates many signaling pathways including those leading to cardiac
fibrosis, stiffness and eventually impaired contractility. Increased levels of Ang-II
found in tissues of patients with T2D cause overproduction of ROS and may lead
to apoptosis and pathological cardiac remodeling. It has been shown that ACEi
and ARBs improve heart function in T2D animal models as well as in patients, by
reducing levels and activation of Ang-II (Deedwania, 1990; Henriksen, 2007;
Konstam et al., 2009). As A(1-7) is capable of antagonizing effects of Ang-II, it is
hypothesized that treatment with A(1-7) improves heart function in experimental
models of T2D and in diabetic patients.
Recent evidence suggests an important role of tissue RAS in controlling organ
function (Carey and Siragy, 2003). A(1-7) is locally expressed in the cardiac
tissue of animal models, (Averill et al., 2003; Santos et al., 1990) and in intact
human heart (Zisman et al., 2003), which suggests its involvement in cardiac
tissue homeostasis. These findings were later supported with studies using Mas
deficient mice, which led to observations that inhibition of A(1-7)/Mas axis
signaling causes impaired ventricular function, shown by both physiological and
morphological changes (Santos et al., 2006). Rats overexpressing A(1-7) show
decreased cardiac hypertrophy and improved systolic function after induction of
myopathies, indicating cardioprotective role of this peptide (Santos et al., 2004).
Further importance of both protective (ACE2/A(1-7)/Mas) and pathological
(ACE/Ang-II/AT1) arm of the RAS on heart function was demonstrated in the
27
study by the Oudit laboratory. Pressure-overloaded ACE2-null mice treated with
either ARB or exogenous A(1-7) had improved heart function (Patel et al., 2012).
Extensive studies of the effects of A(1-7) administration in model of myocardial
infarction demonstrated decreased remodeling, improved heart function and
increased mobilization of endothelial progenitor cells (EPCs), as well as
preserved aortic endothelial function and coronary perfusion in treated animals
(Loot et al., 2002; Wang et al., 2010). In animal model of streptozotocin-induced
type 1 diabetes, endogenous A(1-7) has been found crucial in preventing diabetic
heart failure (Yousif et al., 2012). Treatment with exogenous peptide further
improved cardiac function in this model of diabetes (Benter et al., 2007). Finally,
infusion of A(1-7) in fructose-fed rats, a model of insulin resistance significantly
reduced cardiac hypertrophy and remodeling (Giani et al., 2010).
Even though effects of A(1-7) on heart function have been thoroughly studied,
the mechanisms of the protective actions of A(1-7) haven’t been fully
characterized. Work from other groups shows that A(1-7) exhibits protective
effect on cardiomyocytes through anti-hypertrophic, anti-inflammatory and anti-
oxidative actions. Acute A(1-7) treatment of isolated cardiomyocytes leads to
activation of PI3K/Akt/eNOS/cGMP pathway and results in reduction of Ang-II
induced pathological remodeling (Dias-Peixoto et al., 2008; Gomes et al., 2010).
A(1-7) was also shown to inhibit activation of NFAT, a nuclear factor involved in
hypertrophic response to elevated levels of Ang-II (Gomes et al., 2010). This
control occurs upstream of NFAT and focuses on modulation of GSK-3β activity.
28
A(1-7) restores activity of GSK-3β and this in turn inhibits translocation of NFAT
to the nucleus and subsequent activation of hypertrophic genes (Gomes et al.,
2010). Ang-II, through binding to AT1 receptor, activates many growth-promoting
pathways including RAF1-MEK1/2/-ERK1/2, which in turn activates NFAT and
contributes to hypertrophic response (Heineke and Molkentin, 2006). Stimulation
of cultured neonatal rat cardiomyocytes with A(1-7) reduced Ang-II induced
activation of ERK1/2 (Tallant et al., 2005); proving that A(1-7) decreases
hypertrophy through action on several pathological signaling pathways.
As calcium plays a crucial role in regulation of heart contractility, it is worth
considering A(1-7) effects on calcium transients in cardiomyocytes.
Cardiomyocytes from db/db mice, a model of severe T2D, demonstrate impaired
contractility and calcium handling (Belke et al., 2004). Heart cells from Mas
deficient mice also show decreased calcium transients, a phenotype consistent
with overall impaired heart function (Dias-Peixoto et al., 2008; Santos et al.,
2006). This suggests a role of A(1-7) in maintaining proper contractility function
of the heart cells.
A(1-7) was also shown to decrease expression of proinflammatory cytokines
such as TNF-α and IL-6 in cardiomyocytes of treated rats (Qi et al., 2011). In
addition to its anti-inflammatory properties in the cardiomyocytes, A(1-7) also has
anti-oxidative effects. Cardiomyocytes expressing recombinant ACE2 are
protected against the Ang-II induced production of superoxide, an effect that was
29
reversed by treatment with Mas inhibitor. This suggests that A(1-7) plays a role in
reducing oxidative stress in these cells (Zhong et al., 2010).
Interestingly, some reports show that overexpression of ACE2 and Mas may lead
to severe cardiac hypertrophy and dysfunction (Masson et al., 2009). The
authors argue that this is attributed to much higher expression of the enzyme,
however the mechanism causing this effect remains unknown. Nonetheless,
administration of exogenous A(1-7) presents a promising approach to support
treatment of diabetes-related cardiomyopathies.
1.4.3. A(1-7) for treatment of diabetic kidney disease
1.4.3.1. Overview of diabetic kidney disease
Kidney disease is still one of the most prevalent and severe complications
associated with T2D. Uncontrolled kidney disease leads to kidney failure. 44% of
all kidney failure cases are attributed to diabetes (Blackwell et al., 2014). Dialysis
and kidney transplantation are the only currently available treatments for kidney
failure. Medical care of patients with kidney failure in the US cost nearly $32
billion in 2005 (U.S. Renal Data System, 2007). This slowly developing disorder
manifests in decreased glomerular filtration rate and proteinuria, leading to failure
of blood filtration. One of the major factors causing chronic kidney disease is high
blood pressure. Hypertension is not only seen as the cause but also a
consequence of kidney disease. Initially, increased blood pressure causes
hyperfiltration and mechanical stress leading to increased permeability and
30
proteinuria (Metcalfe, 2007). Shear stress then activates signaling pathways
involved in overproduction of ECM and mesangial expansion (Yasuda et al.,
1996).
Mesangial hypertrophy and overproduction of ECM are also induced by other
mechanisms. Hyperglycemia plays a crucial role in progression of kidney
disorders. High blood glucose induced production of cytokines, ROS and other
cellular mediators such as Ang-II also stimulates expression of ECM and
thickening of the glomerular basal membrane (Schena, 2005). This increases the
permeability and compromises the quality of the filtration function. Hyperglycemia
also activates overexpression of growth factors such TGF-β and components of
ECM such as fibronectin and collagen though activation of PKC signaling (Koya
et al., 1997). Overproduction of AGEs also contributes to glomerular sclerosis
and tubulointerstitial damage through binding to receptor for AGE (RAGE) and
activation of profibrogenic and proinflammatory pathways (Bohlender et al.,
2005).
Among many growth factors that are involved in diabetic kidney pathogenesis,
Ang-II was shown to play an important role. Initially Ang-II was thought to be
involved in mesangial expansion by increasing the blood pressure. Later
however, in vitro experiments showed that, even in the absence of the
hemodynamic factor, inhibition of Ang-II reduced injury hallmarks (Kagami et al.,
31
1994). Treatment with ACEi and ARBs also proves that suppression of Ang-II is
renoprotective (Wolf and Ritz, 2005).
The local renal RAS is able to produce most of the components of the RAS. It
has been shown that mechanical stress can stimulate expression of
angiotensinogen and the enzymes, leading to increased levels of Ang-II (Becker
et al., 1998). Enhanced signaling through the Ang-II/AT1 axis activates various
signaling pathways involved in production of ECM and leading to kidney
dysfunction. As A(1-7) reverses many of the Ang-II actions, we hypothesize that
treatment with this peptide improves kidney function in models of T2D and in
diabetic patients. Studies also show that A(1-7) as well as Mas receptor are
expressed in renal tissue in relatively high amounts and the peptide can be
detected in urine (Gwathmey et al., 2010; Li et al., 2005; Pendergrass et al.,
2008). This suggests that A(1-7) plays a role in kidney homeostasis.
1.4.3.1. Effects of A(1-7) on kidney disease
A(1-7) has been shown to act renoprotective is several models of kidney
dysfunctions. In addition, some of the protective actions of ACEi and ARBs are
ascribed to increased levels of A(1-7) (Santos and Ferreira, 2007; Simões e Silva
et al., 2006). A(1-7) improved kidney function and reduced insulin resistance in
models of hypertensive rats (Benter et al., 2006; Giani et al., 2010). Treatment
with A(1-7) is also effective in experimental models of diabetes. A(1-7) lowered
proteinuria and improved kidney function in streptozotocin-induced diabetic rats,
32
a model of type 1 diabetes (Benter et al., 2007). It was also effective in reducing
fibrosis, oxidative stress and inflammation in Zucker diabetic rats, a model of T2D
(Giani et al., 2012). In KK-A
y
/TA mice, another model of T2D due to obesity that
results from reduction in hypothalamic norepinephrine and dopamine, A(1-7)
improved kidney function though reduction of oxidative stress, inflammation and
mesangial expansion (Moon et al., 2011).
Studies suggest that imbalance of Ang-II and A(1-7) may play a significant role in
diabetic nephropathy not only in animal models but also in patients. In rodent
models of diabetes it has been shown that ACE/ACE2 expression ratio in
glomeruli is increased, suggesting upregulation of Ang-II and downregulation of
A(1-7) expression (Soler et al., 2007; Ye et al., 2006). Analysis of biopsies of
kidney tissue obtained from diabetic patients also showed increased ACE/ACE2
ratio which suggests enhanced formation of Ang-II and decreased levels of
A(1-7) (Mizuiri et al., 2008).
Even though effects of A(1-7) have been studied in several models of kidney
disease, its benefits are still a subject to debate. Some studies suggest
detrimental effects of A(1-7) on kidney function. In streptozotocin-diabetic rats
and in non-diabetic model of unilateral ureteral obstruction(Esteban et al., 2009;
Shao et al., 2008), treatment with A(1-7) was not beneficial. The reason for these
discrepancies is still unknown, however some of them are being ascribed to
dosage and route of administration. It is therefore crucial to further study the
33
effects of A(1-7) on diabetic nephropathy to elucidate the mechanisms of
possible protective action of this treatment.
1.4.4. Potential for A(1-7) to become a novel treatment for diabetes
related complications
A(1-7) may represent a novel, safer treatment for diabetic complications. In
contrast to ACEi and ARBs, A(1-7) activates protective arm of the RAS. A(1-7)
acts on multiple levels of the disease. It was shown to decease fibrosis and
remodeling in many crucial systems such as cardiovascular and renal tissues
(Giani et al., 2010; 2012). On the molecular level it decreases inflammation and
oxidative stress, two pathological mechanisms that have been recently described
as the main targets in new diabetic therapies (Maritim et al., 2003; Wellen, 2005).
Even though A(1-7) is readily digested in the gastrointestinal tract, new orally
available formulations are currently under development to improve translational
potential of this short peptide (Feltenberger et al., 2013; Marques et al., 2011;
Santos et al., 2014). NorLeu3-A(1-7), an analogue of A(1-7) formulated into a
topical gel, was also shown to accelerate wound healing in animal models and
stimulate diabetic foot ulcer healing in patients (Balingit et al., 2012; Rodgers et
al., 2002). What is more, A(1-7) is currently undergoing several clinical trials in
oncology and hematopoiesis and was shown to be safe in patients (Rodgers et
al., 2006). This allows for the rapid translation of the preclinical results into
potential clinical evaluation.
34
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56
2. Chapter 2: Short-term administration of angiotensin (1-7)
ameliorates diabetic heart and kidney disease.
2.1. Background and goals
T2D affects nearly 10% of the US population and is associated with a number of
long-term complications including nephropathy, retinopathy, stroke, and
cardiovascular disease, ultimately leading to a decreased quality of life and
reduced life expectancy (Centers for Disease Control, 2014; Rabelo et al., 2011).
Diabetes is now the 7
th
leading cause of death in the US (Heron, 2013).
Patients with T2D have 2-4 times the risk of developing heart disease as
compared to healthy individuals (Centers for Disease Control, 2014). Diabetes is
associated not only with severe hyperglycemia but also dysregulation of lipid
metabolism. In diabetic patients, the uptake of fatty acids in cardiac cells exceeds
their oxidation rate, which leads to accumulation of fat and promotes lipotoxicity.
Consequently, this can induce apoptosis and abnormal remodeling of cardiac
tissue (McGavock et al., 2006). Hyperglycemia contributes to activation of many
pathological pathways including generation of ROS and fibrosis (for more
information refer to chapter 1). Hyperglycemia induced overproduction of AGEs
also has pro-inflammatory, pro-fibrogenic and mitogenic properties in the heart
(Centers for Disease Control, 2014; Marwick, 2006; Rabelo et al., 2011). In
addition, micro- and macrovascular complications, resulting from increased
endothelial dysfunction and decreased numbers of progenitor cells, as well as
57
reduced density of vessels in the heart tissue, contributes to a 3- to 5-fold
increased risk of death in diabetic patients (Benter et al., 2006; Centers for
Disease Control, 2014).
The RAS has been implicated in the pathophysiology of many of the diabetes-
related complications, including hypertension and subsequent CVD (McGavock
et al., 2006; Weekers et al., 2005). Ang-II, the main effector of the pathological
arm of the RAS, though binding to its cognate receptor –AT1, activates various
signaling pathways that lead to development of diabetic complications. Increased
signaling through Ang-II/AT1 axis results in cardiomyocyte hypertrophy (Mehta
and Griendling, 2006). Elevated Ang-II promotes chronic inflammation and the
formation of oxygen radicals, which also contribute to tissue fibrosis and
remodeling (Mehta and Griendling, 2006; Zablocki and Sadoshima, 2013). Ang-II
was shown to induce apoptosis in diabetic hearts, which further affects heart
function (Singh et al., 2008). All of these mechanisms lead to severe cardiac
remodeling, decreased contractility and heart failure.
Diabetic heart disease is strongly associated with renal complications. Clinical
data shows that patients with kidney disease have a 20- to 30-fold higher risk for
developing a heart condition (Sarnak et al., 2003). One of the major factors
causing chronic kidney disease is high blood pressure. Hypertension is seen not
only as the cause but also a consequence of kidney disease and it may
contribute to multi-organ dysfunction including heart failure. Initially, increased
58
blood pressure causes hyperfiltration and mechanical stress leading to increased
permeability and proteinuria. Shear stress then activates signaling pathways
involved in overproduction of ECM and mesangial expansion. In addition, high
blood glucose levels stimulate cells to produce increased amounts of growth
factors, cytokines and other cellular mediators that lead to glomerular dysfunction
(Schena, 2005). Ang-II was shown to play an important role in diabetic kidney
pathogenesis. Inflammation and oxidative stress due to elevated Ang-II add to
tissue fibrosis, increased endothelial permeability, and glomerular damage
(Kashihara et al., 2010; Navarro and Mora, 2006).
Studies have shown that blockade of the RAS, specifically via the inhibition of
Ang-II synthesis or AT1 receptor, with the use of pharmacological agents such as
ACEi and ARBs, may decrease the risk or development of cardiovascular and
renal complications in diabetic patients (Burnier and Zanchi, 2006; Wolf and Ritz,
2005). Some of the beneficial effects of ARBs and ACEi may be in part due to
their ability to increase the levels of A(1-7), a heptapeptide of the RAS which has
been shown to oppose many of the actions of Ang-II (Iyer et al., 2000; 1998).
A(1-7) actions are thought to be mediated primarily through binding to a
G-protein coupled receptor called Mas. In addition, several studies have
suggested that A(1-7) can also activate other receptors such as AT2 receptor
(Bosnyak et al., 2011; Castro et al., 2005; Walters et al., 2005). In contrast to
Ang-II, A(1-7) acts through anti-inflammatory, anti-oxidant and vasodilatory
59
mechanisms. Some of the effects of A(1-7) are ascribed to release of NO and
bradykinin, following activation of Mas receptor (Fig. 2.1).
More recently, evidence has surfaced demonstrating the cardioprotective effects
of A(1-7) in various animal models, e.g. streptozotocin-induced type 1 diabetes,
spontaneously hypertensive rats, and fructose-fed rats (a model of metabolic
syndrome) (Ebermann et al., 2008; Giani et al., 2010; Yousif et al., 2012). In
addition to its effects on the heart, A(1-7) has been also shown to act reno-
protective in streptozotocin-diabetic rats, Zucker diabetic fatty rats, and in
spontaneously hypertensive rats (Benter et al., 2007; Giani et al., 2010; 2012).
Due to the counter-regulatory role of A(1-7) in opposition of Ang-II in diabetes,
this study was designed to investigate the extent of cardiorenal dysfunction in
young type 2 diabetic mice (db/db), as well as the effects of A(1-7) treatment on
diabetes-related renal and cardiovascular dysfunction. The potential mechanisms
of action were studied using various inhibitors of the RAS and A(1-7) signaling
mediators (NO, bradykinin). The inhibitors included (Fig. 2.1):
• A-779 – a Mas receptor antagonist
• losartan – an AT1 receptor inhibitor
• PD123319 – an AT2 receptor antagonist
• Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) an inhibitor of
nitric oxide synthase
• icatibant – an inhibitor of bradykinin B2 receptors.
60
2.2. Study design and methods
2.2.1. Animals
The NIH Principles of Laboratory Animal Care were followed, and the
Department of Animal Resources at the University of Southern California
approved this study. Eight- to nine- week old BKS.Cg-Dock7
m
+/+ Lepr
db
/J
(db/db) mice and age-matched heterozygous controls (non-diabetic) were
purchased from Jackson Laboratories (n=7/group) (Bar Harbor, ME, USA). Food
Fig. 2.1: Mechanisms of A(1-7) signaling. A(1-7) acts primarily on the Mas receptor
but it is also thought to activate the AT2 receptor. Activation of Mas causes
subsequent activation of nitric oxide synthase (NOS) and release of nitric oxide (NO).
Bradykinin acts through activation of B2 receptor. Actions of this receptor are also
partially mediated through NOS. A(1-7) is thought to increase activation of B2. Ang-II
acts primarily on the AT1 receptor but it is also known to bind to AT2. The signaling
pathways activated by A(1-7) were studied using various inhibitors: losartan for
inhibition of AT1, PD123319 for inhibition of AT2, A-779 for inhibition of Mas, icatibant
for inhibition of B2 and L-NAME for inhibition of NOS.
AT1 AT2 MAS B2
NOS
NO
Bradykinin A(1-7) Ang-II
Vasoconstriction
Inflammation
Oxidative stress
Fibrosis
Vasodilation
Anti-inflammatory
Anti-oxidant
Anti-fibrotic
ica.bant A-779 PD123319 losartan
L-NAME
61
and water were available ad libitum. Mice were kept on a 12 hour light/dark cycle.
db/db mice and the heterozygous controls were administered either saline
(vehicle) or A(1-7) at 0.5 mg/kg/day via subcutaneous injections, daily for 14
days. Heart function was assessed at three different time points: 57/58 days of
age, 69/70 days of age, and 82/83 days of age. Renal function was measured at
the 82/83 day-old time point.
The effect of inhibitors was evaluated at the 82/83 days-old time point. Mice were
administered either saline (vehicle), A(1-7) alone (0.5 mg/kg/day), or A(1-7)
(0.5 mg/kg/day) combined with an inhibitor (losartan, PD123319, A-779 or
L-NAME at 10 mg/kg/day or icatibant at 0.4 mg/kg/day) via subcutaneous bolus
injection, daily for 14 days. Mice were weighed three times weekly, and the
doses adjusted accordingly.
2.2.2. Pharmacologic agents and inhibitors
A(1-7) and A-779 were purchased from Bachem (Torrance, CA, USA). Losartan,
PD123319, and L-NAME were purchased from Sigma-Aldrich (Saint Louis, MO,
USA). Icatibant was purchased from Tocris Bioscience (Ellisville, MO, USA).
2.2.3. Echocardiography
Heart function was assessed noninvasively using a high frequency, high-
resolution two-dimensional echocardiography system consisting of Vivid 7
Dimension ultrasound machine equipped with a 6-13MHz linear transducer (GE
62
Healthcare, Little Chalfont, UK). Anesthesia was induced with 3% isoflurane in an
induction chamber. The mouse was then placed in a supine position on a heating
pad to maintain body temperature at 36.5-37
o
C. Anesthesia was maintained
through a nosecone and adjusted to maintain heart rate at 450-550 beats per
minute. The images of the left ventricle were acquired and the shortening fraction
(SF) and cardiac output (CO) were calculated in accordance with the American
Society of Echocardiography guidelines (Lang et al., 2006). CO was calculated
using a modified Simpson’s rule.
To facilitate measurements, mice were injected with a microbubble contrast
agent through the tail vein as described previously (Walton et al., 2011). Briefly,
microbubble stock was prepared from 1,2-dipalmitoyl-sn-glycero-3-
phosphatidylcholine (20 mg/mL), 2-dipalmitoyl-sn-glycero-3-
phosphatidylethanolamine (5 mg/mL) and glucose (100 mg/mL) in PBS. The
mixture was then heated in a water bath for 30 minutes with pipette mixing every
5 minutes. Solution was stored at 4
o
C. A sample of the microbubble stock was
incubated at 40
o
C for 15 minutes and mixed with glycerol (5 parts microbubble
stock, 1 part glycerol). The air in the vial was replaced with octafluoropropane
gas and the tube was shaken in a Bullet Blender Tissue Homogenizer (Next
Advance, Inc., Averill Park, NY, USA). Contrast was then loaded into a syringe
using an 18G needle. Contrast was prepared immediately before use.
63
2.2.4. Histology and heart weights
Hearts and kidneys were collected at the necropsy. Hearts were trimmed and
weight. Tibias were also removed, cleaned and the length was measured using a
digital caliper. Heart weight was normalized to tibia length. The isolated organs
were then formalin-fixed, paraffin embedded and cut at 5 µm.
Cardiomyocyte hypertrophy
Heart samples were sectioned longitudinally and stained with H&E. Ten random
images of each heart section were taken at 10x magnification along the left
ventricular wall, avoiding the septal wall for consistency. Cardiomyocyte area and
width was determined by a blinded observer using ImageJ version 1.47v
(National Institutes of Health, USA) at positions without visible nuclei exposing
substantial lengths of uniform width to avoid large standard error (Tracy and
Sander, 2011).
Heart fibrosis
Heart samples were sectioned longitudinally and stained using standard
picrosirius red method. Twenty pictures of left ventricle were taken at 40x
magnification and evaluated using a macro developed by Hadi et al (Hadi et al.,
2011). The septal wall was avoided for consistency. The fibrosis was expressed
as percentage of cardiomyocyte area stained red.
64
Inflammatory cell count
Pictures of heart sections stained with H&E were also used to assess number of
inflammatory cells in the tissue. Ten random images per heart taken at 10x
magnification were evaluated for neutrophil number by a blinded observer. The
results are expressed as number of inflammatory cells per image.
Immunostaining for CD31
The slides were rehydrated and subjected to heat induced epitope retrieval
(HIER) in Antigen Retrieval Citra Plus (Biogenex, San Ramon, CA, USA). Goat
anti-mouse monoclonal antibody directed against CD31 (BD Biosciences, San
Jose, CA, USA) was used at a 1:250 dilution. After incubation with biotinylated
secondary antibody, a streptavidin conjugated horseradish peroxidase was
added. The sections were then incubated in diaminobenzidine (DAB) for 8-10
minutes and counterstained with Harris hematoxylin. After mounting,
enumeration of CD31-positive vessels in the heart sections was performed by a
blinded observer using light microscopy.
Immunostaining for nitrotyrosine (N-Tyr)
The same procedure was used for N-Tyr staining with few exceptions. The
primary antibody was rabbit anti-mouse polyclonal antibody directed against
nitrated tyrosine residues (EMD Millipore, Billerica, MA, USA) that was used at
1:500 dilution. Ten random images of kidney cortex at 10x were evaluated for
65
extent of N-Tyr staining by a blinded observer using ImageJ version 1.47v and
expressed as percentage of area positively stained.
Immunostaining for phospho-eNOS Ser1177
The same procedure was used for phospho-eNOS Ser1177 staining with few
exceptions. Rabbit anti-mouse polyclonal antibody to eNOS (phospho Ser1177)
(GeneTex Inc., Irvine, CA, USA) was used at 1:100 dilution was used as primary
antibody. Twenty random images of kidney cortex at 40x were evaluated for the
extent of staining by a blinded observer using ImageJ version 1.47v and
expressed as percentage of area positively stained.
Immunostaining for phospho-eNOS Thr495
The same procedure was used for phospho-eNOS Thr495 staining with few
exceptions. The primary antibody was rabbit anti-mouse polyclonal antibody to
eNOS (phospho Thr495) (Bioss Inc., Woburn, MA, USA) that was used at 1:100
dilution. Twenty random images of kidney cortex at 40x were evaluated for the
extent of staining by a blinded observer using ImageJ version 1.47v and
expressed as percentage of area positively stained.
2.2.5. Glomerular area and mesangial expansion
The kidney sections were stained using standard periodic acid-Schiff (PAS)
staining method. Twenty images of random cortical glomeruli were taken at 40x
magnification. The images were analyzed by a blinded observer using ImageJ
66
version 1.47v. Mesangial expansion was expressed as percentage of glomerular
area stained with PAS.
2.2.6. Apoptosis
Apoptosis was evaluated using the Click-iT® TUNEL Assay per manufacturer
instructions (Life Technologies, Grand Island, NY, USA).
2.2.7. Gene expression
Total RNA was extracted from tissue, using TRIzol as described by (Mordwinkin
et al., 2012). Briefly, RNA was reverse-transcribed and real-time PCR was
performed using SYBR green PCR Master Mix (Applied Biosystems by Life
Technologies). Relative expression of each of the genes of interest was
evaluated using ABI 7300 instrument. Abundance of targeted mRNA was
normalized against 18S mRNA.
2.2.8. Statistical analysis
GraphPad Prism version 5.0d for Mac OS X (GraphPad Software, San Diego,
CA, USA) was used to analyze the data. One-way ANOVA followed by Tukey’s
test was used to compare data from more than two groups. The level of statistical
significance was set at 5%. Data are expressed as mean value ± standard error
of the mean (SEM).
67
2.3. Results
2.3.1. Progression of heart dysfunction in db/db mice and effects of
A(1- 7)
Heart function was assessed using non-invasive echocardiography at three
different time points- when mice were 57-58, 69-70 or 82-83 days old. At each
time point animals were treated with either saline (vehicle) or A(1-7) for 14 days.
We did not observe any significant changes in CO or SF at the two early time
points (Fig. 2.2). It was concluded that mice at age of 69-70 days or younger, do
not develop any detectable reduction in physiologic heart function. We also did
0
10
20
30
cardiac output [cm3/min]
**
†
82-83 days old
0
10
20
30
40
50
shortening fraction [%]
**
††
82-83 days old
0
10
20
30
cardiac output [cm3/min]
57-58 days old
0
10
20
30
40
50
shortening fraction [%]
69-70 days old
0
10
20
30
40
50
shortening fraction [%]
57-58 days old
hzg saline
hzg A(1-7)
db/db saline
db/db A(1-7)
0
10
20
30
cardiac output [cm3/min]
69-70 days old
A. B. C.
D. E. F.
Fig. 2.2: Progression of heart dysfunction in db/db mice and effects of A(1-7) on
cardiac output and shortening fraction. No significant changes in heart function
were observed in any of the treatment groups at age of 57-58 days and 69-70 days
(A-B, D-E). Decrease in cardiac output and shortening fraction was observed in
diabetic animals from the control group compared to non-diabetic mice (hzg –
heterozygous) (C, F). Treatment with A(1-7) improved both of those parameters.
(**p<0.01; † or †† significantly increased compared to db/db saline (p<0.05 or 0.01);
calculated using one-way ANOVA; plotted as mean with SEM.
68
not observe any effects of A(1-7) treatment on heart function at these early time
points. However, at age of 82-83 days, db/db mice had significantly decreased
CO and SF, which suggest decrease in the pumping ability and reduction in
contractility of the hearts in these animals. In addition, treatment with A(1-7)
significantly improved both of the measured parameters.
2.3.2. A(1-7) reduces cardiomyocyte hypertrophy through activation of
multiple pathways.
There were no significant differences in heart weight, a measure of cardiac
hypertrophy, between any of the groups (data not shown). In order to investigate
the cardiomyocyte hypertrophy, cell dimensions were measured in H&E stained
sections. In diabetic mice, mean cardiomyocyte area and width were significantly
increased compared to non-diabetic controls (Fig. 2.3). Following A(1-7)
administration, both measurements were decreased to sizes similar to those
observed in non-diabetic mice. This effect was blocked by co-administration of
A-779, PD123319, L-NAME and icatibant suggesting complex signaling involving
Mas, AT2 and bradykinin receptors as well as NOS activity. One of the
mechanisms leading to cardiac remodeling and hypertrophy is uncontrolled
apoptosis. However, in this study, no difference in the number of apoptotic cells
in the heart tissue was found between any of the groups as measured by TUNEL
assay (data not shown).

69

Fig. 2.3: Effects of short-term A(1-7) administration on cardiomyocyte
hypertrophy. Cardiomyocyte area (B) and width (C) were measured in cardiac
sections stained with hematoxylin and eosin. Representative images are shown in
panel A. Cardiomyocyte size was increased in db/db mice compared to healthy
controls (hzg – heterozygous). Treatment with A(1-7) prevented this change. Co-
administration of A(1-7) with either PD123319, A-779, icatibant or L-NAME, but not
losartan, blocked the effects of treatment. (a.u. – arbitrary units; *p<0.05; † or ††
significantly increased compared to db/db A(1-7) (p<0.05 or 0.01); calculated using
one-way ANOVA; plotted as mean with SEM. (Papinska et al., 2015)
hzg saline
db/db saline
db/db A(1-7)
db/db A(1-7) + losartan
db/db A(1-7) + A-779
db/db A(1-7) + PD123319
db/db A(1-7) + L-NAME
dbdb A(1-7) + icatibant
0.0
0.2
0.4
0.6
0.8
1.0
Cardiomyocyte area (a.u.)
*
*
†
†
††
††
hzg saline
db/db saline
db/db A(1-7)
db/db A(1-7) + losartan
db A-(1-7) + A-779
db/db A(1-7) + PD123319
db/db A(1-7) + L-NAME
dbdb A(1-7) + icatibant
0.0
0.2
0.4
0.6
0.8
Cardiomyocyte width (a.u.)
*
*
†
† †
†
Hz saline db saline db + Ang-(1-7) db Ang-(1-7) + losartan
db Ang-(1-7) + A-779 db Ang-(1-7) + L-NAME db A-ng(1-7) + PD123319 db Ang-(1-7) + icatibant
A.
B. C.
70
2.3.3. A(1-7) decreases cardiac damage by improving tissue
vascularization and reducing fibrosis and inflammatory cell infiltration.
Cardiac blood vessel density was significantly decreased in db/db mice
compared to non-diabetic controls (Fig. 2.4A). Treatment with A(1-7) increased
cardiac CD31-positive blood vessel density in db/db mice to levels comparable to
those seen in heterozygous controls. Co-administration of A(1-7) with A-779,
PD123319, L-NAME and icatibant blocked the effects of A(1-7), while co-
administration with losartan had no effect on A(1-7) action. Treatment with A(1-7)
Fig. 2.4. Effects of short-term treatment with A(1-7) on vessel density, fibrosis
and inflammatory cell infiltration in cardiac tissue. Density of CD31-positive blood
vessels was assessed as a measurement of tissue vascularization. Fibrosis is
expressed as percentage of cell area stained with picrosirius red. Inflammatory cell
infiltration was measured by enumerating PMNs. A decrease in blood vessel density,
increase in tissue fibrosis and increase in PMN number was observed in the heart
tissue of db/db mice compared with heterozygous controls. A(1-7) treatment reversed
all of these changes. Co-administration of A(1-7) with either PD123319, A-779,
icatibant or L-NAME, but not losartan, blocked the effects of A(1-7) on blood vessel
density and PMN count. Co-administration of losartan enhanced anti-fibrotic effects of
A(1-7), whereas A-779 blocked the action of A(1-7). (hzg – heterozygous; PMN –
granulocytes; *p<0.05; **p<0.01; † or †† or ††† significantly different compared to
db/db A(1-7) p<0.05 or 0.01 or 0.001); calculated using one-way ANOVA; plotted as
mean with SEM. (Papinska et al., 2015)
hzg saline
db/db saline
db/db A(1-7)
db/db A(1-7) + losartan
db/db A(1-7) + A-779
db/db A(1-7) + PD123319
db/db A(1-7) + L-NAME
db/db A(1-7) + icatibant
0
10
20
30
40
50
Heart section PMN counts
**
††
††
††
††
**
hzg saline
db/db saline
db/db A(1-7)
db/db A(1-7) + losartan
db/db A(1-7) + A-779
db/db A(1-7) + PD123319
db/db A(1-7) + L-NAME
db/db A(1-7) + icatibant
0
2
4
6
8
CD31+ vessels/10x field
*
*
††
††
††
††
hzg saline
db/db saline
db/db A(1-7)
db/db A(1-7) + losartan
db/db A(1-7) + A-779
db/db A(1-7) + PD123319
db/db A(1-7) + L-NAME
db/db A(1-7) + icatibant
0
5
10
15
% area fibrotic
*
**
†††
††
A. B. C.
71
also reduced fibrosis in diabetic animals as measured by picrosirius red staining
(Fig. 2.4B). Co-administration of A(1-7) and losartan further reduced fibrosis in
db/db mice, whereas A-779 blocked the effects on A(1-7). Co-administration of
either PD123319, L-NAME or icatibant did not have an effect on A(1-7) action.
Furthermore, an increase in the number of granulocytes (PMN) in the cardiac
muscle was found in db/db mice compared to heterozygous controls (Fig. 2.4C).
This increase was not observed in the cardiac tissue of db/db mice administered
A(1-7). Co-administration with A-779, PD123319, L-NAME and icatibant, but not
losartan, blocked the anti-inflammatory effects of A(1-7).
2.3.4. Administration of A(1-7) reduces glomerular area and mesangial
expansion in diabetic mice.
Heart disease can be accelerated by kidney dysfunction. To assess glomerular
function, glomerular size and mesangial expansion were measured. A significant
increase in glomerular area and mesangial expansion was seen in vehicle
treated diabetic animals compared to non-diabetic controls (Fig. 2.5). Treatment
with A(1-7) significantly reduced both of these parameters suggesting improved
kidney structure and function.
2.3.4.1. A(1-7) reduces inflammation and oxidative stress in kidneys
of db/db mice.
Oxidative stress can lead to severe kidney damage by activating hypertrophic,
fibrotic and remodeling signals. To assess the extent of damage in the kidneys
72

Hz saline db saline db + Ang-(1-7) db Ang-(1-7) + losartan
db Ang-(1-7) + A-779 db Ang-(1-7) + L-NAME db A-ng(1-7) + PD123319 db Ang-(1-7) + icatibant
hzg saline
db/db saline
db/db A(1-7)
db/db A(1-7) + losartan
db/db A(1-7) + A-779
db/db A(1-7) + PD123319
db/db A(1-7) + L-NAME
db/db A(1-7) + icatibant
0
1000
2000
3000
4000
glomerular area [um2]
***
**
†† †† †
hzg saline
db/db saline
db/db A(1-7)
db/db A(1-7) + losartan
db/db A(1-7) + A-779
db/db A(1-7) + PD123319
db/db A(1-7) + L-NAME
db/db A(1-7) + icatibant
0
5
10
15
20
mesangial expansion
[% glomerular area stained]
****
****
††††
††
†
A.
B. C.
Fig. 2.5. Glomerular area and mesangial expansion in the kidneys of animals
treated with A(1-7) for 2 weeks. Panel A shows representative images of kidney
cortex stained with PAS. Glomerular area and mesangial expansion were increased
in diabetic mice treated with vehicle (B-C). Treatment with A(1-7) significantly reduced
both of these parameters. Addition of A-779, PD123319, and L-NAME but not
losartan or icatibant resulted in inhibition of the effects of A(1-7). (hzg – heterozygous;
**p<0.01; ***p<0.001; ****p<0.0001; † or †† or †††† significantly increased compared
to db/db A(1-7) p<0.05 or 0.01 or 0.0001); calculated using one-way ANOVA; plotted
as mean with SEM. (Papinska et al., 2015)
73
due to oxidative stress, expression of eNOS, p22-phox (a subunit of NADPH
oxidase), and superoxide dismutase (SOD) was measured. In the kidneys of
diabetic animals treated with vehicle, expression of eNOS mRNA was highly
increased (Fig. 2.6A). A(1-7) administration significantly decreased eNOS
expression in diabetic mice. This effect was blocked by co-administration of
A-779, PD123319, L-NAME and icatibant but not losartan. No significant
changes in expression of p22-phox or SOD were observed between
heterozygous or diabetic control animals and any other treatment groups (data
not shown). The abundance of phosphorylated forms of eNOS was assessed
using immunohistochemistry (Fig. 2.6B-C, Fig. 2.7). eNOS phosphorylation on
Ser1177 (activated form) was more abundant in kidneys from diabetic animals.
Staining was further increased by administration of A(1-7). This action of A(1-7)
was mediated specifically through Mas receptor. Administration of A-779 resulted
in increased phosphorylation of eNOS on Ser1177. None of the remaining
inhibitors had an effect on this marker. Deactivated form of eNOS
(phosphorylation on Thr495) was more abundant in db/db mice. Treatment with
A(1-7) decreased levels of this form of eNOS in kidneys from diabetic mice. This
effect was reversed by co-administration of A-779. Diabetic kidneys also had
increased extent of N-Tyr staining, which was reversed by A(1-7) treatment (Fig.
2.6D, Fig. 2.7). A-779, PD123319 and L-NAME blocked the effects of A(1-7) on
this marker.
74

Fig. 2.6. Short term treatment with A(1-7) reduces oxidative stress in the kidneys.
Expression of eNOS was highly increased in diabetic animals (A). Treatment with A(1-7)
reduced levels of eNOS mRNA. Co-administration with A-779, PD123319 , L-NAME and
icatibant inhibited effects of A(1-7). Level of eNOS phosphorylated on both Ser1177
(active form) (B) and Thr495 (inactive form) (C) was increased in diabetic animals.
Treatment further elevated levels of eNOS phosphorylation at Ser1177. Co-
administration of A-779 increased levels of this marker. A(1-7) reduced abundance of
the deactivated form of eNOS. This effect was reversed after treatment with A-779.
A(1-7) decreased nitrotyrosine staining in kidney sections from diabetic animals (D). Co-
administration of A-779, PD123319 or L-NAME reversed the effects of A(1-7) on
nitrotyrosine formation. (hzg – heterozygous; * p<0.05; **p<0.01; ***p<0.001;
****p<0.0001; † or †† or †††† significantly different compared to db/db A(1-7) p<0.05 or
p<0.01 or p<0.0001); calculated using Kruskal-Wallis (A) or one-way ANOVA (B-D);
plotted as mean with SEM. (Papinska et al., 2015)
hzg saline
db/db saline
db/db A(1-7)
db/db A(1-7) + losartan
db/db A(1-7) + A-779
db/db A(1-7) + PD123319
db/db A(1-7) + L-NAME
db/db A(1-7) + icatibant
0.0
0.5
1.0
10
20
30
40
eNOS mRNA fold-change
*
††††
† †
††
†
A.
B.
C. D.
hzg saline
db/db saline
db/db A(1-7)
db/db A(1-7) + losartan
db/db A(1-7) + A-779
db/db A(1-7) + PD123319
db/db A(1-7) + L-NAME
db/db A(1-7) + icatibant
0
2
4
6
p-eNOS Ser1177 [% area stained]
*
****
†
hzg saline
db/db saline
db/db A(1-7)
db/db A(1-7) + losartan
db/db A(1-7) + A-779
db/db A(1-7) + PD123319
db/db A(1-7) + L-NAME
db/db A(1-7) + icatibant
0.0
0.5
1.0
1.5
2.0
2.5
p-eNOS Thr 495 [% area stained]
**
*
††††
hzg saline
db/db saline
db/db A(1-7)
db/db A(1-7) + losartan
db/db A(1-7) + A-779
db/db A(1-7) + PD123319
db/db A(1-7) + L-NAME
db/db A(1-7) + icatibant
0
10
20
30
40
nitrotyrosine [% area stained]
***
*
† † †
75

Hz saline db saline
db + Ang-(1-7)
phospho-eNOS Ser1177 phospho-eNOS Thr495 nitrotyrosine
db Ang-(1-7)
+ losartan
db Ang-(1-7)
+ A-779
db Ang-(1-7)
+ PD 123319
db Ang-(1-7)
+ L-NAME
db Ang-(1-7)
+ icatibant
Fig. 2.7: Representative images of immunostaining for nitrotyrosine,
phospho-eNOS Ser1177 and phospho-eNOS Thr495.
76
2.4. Discussion and conclusions
A(1-7) has been recently shown to improve heart function in different animal
models of heart failure including myocardial infarction, diabetic rats with
cardiomyopathy and metabolic syndrome rat model (Giani et al., 2010; Marques
et al., 2011; Singh et al., 2011). These studies demonstrated reduced cardiac
hypertrophy and fibrosis, which are also hallmarks of T2D related
cardiomyopathies. Recently, Oudit group also showed improvement of heart and
kidney function in db/db mice with advanced disease (Mori et al., 2014a; 2014b).
In these studies, 5-month-old mice were implanted with an osmotic pump and
treated for 28 days. In agreement with these findings, we have shown that short-
term treatment (2 weeks) with A(1-7) prevents progression of heart and kidney
dysfunction even in the early stage of diabetes (Papinska et al., 2015). We have
also recently demonstrated that long-term administration of A(1-7) improves
heart function in db/db mice (Papinska et al., 2016).
db/db mice are known to develop hyperglycemia and obesity early in life –
between 6
th
(42 days) and 8
th
(56 days) week of life. Our results show that heart
function in db/db mice remains unchanged in animals 70 day old or younger.
However, we have detected a significant loss of heart function in mice that were
82-83 days old. This shows that, even though hyperglycemia develops before 56
day of life, significant heart function reduction is not detectable before 70th day of
life. In addition, administration of A(1-7) resulted in significant improvements in
cardiac function in the animals from the last time point. The increase in CO
77
observed in diabetic mice treated with A(1-7) appears to be in part due to an
increase in contractility expressed as SF.
In patients with T2D, left ventricle hypertrophy is often a major cause of the long-
term decrease in cardiac function. In this study, young diabetic mice had a
significant increase in cardiomyocyte area and width, without increase in heart
weight or apoptosis, which suggests an early stage of cardiomyopathy. Two of
the main mechanisms leading to cardiomyocyte hypertrophy are Ang-II activation
of the AT1 receptor and increase in inflammation (van Empel and De Windt,
2004). This may in part help to explain the mechanism by which A(1-7) reduced
cardiomyocyte hypertrophy in db/db mice. First, A(1-7) signaling through the Mas
receptor opposes many of the actions of Ang-II, which could result in decreased
signaling via AT1 receptor. Evaluation of the inflammatory cell infiltration in the
heart confirmed an increase in number of neutrophils in the diabetic mice with a
reversal upon treatment with A(1-7).
Cardiac function is also regulated by the vascular system. The changes in
cardiomyocyte size and blood vessel density after A(1-7) treatment may, in part,
be due to its effects on the bone marrow. Previously, we showed that treatment
with A(1-7) decreased oxidative stress, increased NO production, and decreased
nitration in the bone marrow of db/db mice treated for 2 weeks (Mordwinkin et al.,
2012).
78
We also show that treatment with A(1-7) improves renal function in db/db mice.
Oxidative stress significantly contributes to diabetic nephropathy. Damage to
proteins, DNA and lipids due to increased production of ROS leads to glomerular
fibrosis and mesangial expansion, as well as an increase in endothelial
permeability and overall impairment of filtration function (Kashihara et al., 2010).
Increased levels of eNOS expression have been described in multiple disease
models including high glucose-exposed human aortic cells (Cosentino et al.,
1997) and a rat model of myocardial infarction (Bauersachs et al., 1999). Even
though NADPH oxidase is usually the primary source of superoxide, several
studies suggest that eNOS can be involved in production of superoxide under
certain pathological conditions, including diabetes (Cosentino et al., 1997; Guzik
et al., 2002). Overexpression of eNOS may result in uncoupling of this enzyme
and formation of superoxide rather than NO, contributing to increase in oxidative
stress. However, physiological significance of the ROS produced by the
uncoupled eNOS remains controversial. The amount of superoxide produced by
uncoupled eNOS is thought to be much lower than that produced by NADPH
oxidase, which is still described as the main source of superoxide in diabetic
kidney (Forbes et al., 2008; Gill and Wilcox, 2006; Thallas-Bonke et al., 2008).
Nonetheless, peroxynitrite, which can be formed from superoxide and NO, reacts
with tyrosine residues causing irreversible damage to proteins (Yang et al.,
2009). In fact, Bouloumie and colleagues showed that impaired endothelial
79
function is associated with increased eNOS expression, superoxide production,
and N-Tyr appearance (Bouloumié et al., 1997).
Phosphorylation of Ser1177 on eNOS is known to activate the enzyme and
increase production of NO. The levels of this form of eNOS were increased in
diabetic animals treated with A(1-7), which suggests enhanced production of NO
that might be responsible for some of the renoprotective actions of the peptide
such as decrease in blood pressure. As Akt pathway is the main source of
Ser1177 phosphorylation, increased levels in diabetic animals treated with A(1-7)
might be associated with increased activation of this pathway. Akt pathway is
known be involved in insulin signaling and to act protective in diabetes
(Kobayashi et al., 2004; Mackenzie and Elliott, 2014). In contrast, in the kidneys
from diabetic mice from the control group, the levels of eNOS phosphorylated on
Thr495 were increased compared to healthy controls and diabetic mice treated
with A(1-7). These increased levels of inactivated eNOS might be responsible for
production of superoxide rather than NO, however effects of phosphorylation on
uncoupling of eNOS are not entirely understood (Chen et al., 2008). In addition,
phosphorylation on Thr495 is mediated by PKC pathway, which has been
identified as detrimental in T2D (Koya and King, 1998). While losartan,
PD123319, L-NAME and icatibant did not alter the effects of A(1-7) treatment,
co-administration with A-779 resulted in increase of both phosphorylated forms of
eNOS in db/db mice, which suggests that this signaling occurs primarily through
activation of Mas receptor. We believe that the increase of phosphorylation on
80
Thr495 in diabetic animals treated with saline or combination of A(1-7) and A-779
might be a compensatory response to oxidative stress. Consistent with decrease
in eNOS expression, A(1-7) also reduced extent of tyrosine nitration and thus
contributed to decrease in oxidative stress-induced damage.
In this study we showed beneficial effects of A(1-7) treatment on cardiovascular
and renal function in a murine model of type 2 diabetes (db/db) (Papinska et al.,
2015). Even though the mice were relatively young and the treatment lasted only
2 weeks, the effects on physiological heart and kidney function were profound.
Kidney tissue showed oxidative stress injury perhaps through eNOS uncoupling,
which was attenuated with A(1-7). We also identified multiple possible
mechanisms of A(1-7) action. Treatment decreased cardiomyocyte hypertrophy,
fibrosis and inflammatory cell infiltration in the heart. Vascularization of the heart
was also improved. We identified several receptors and pathways mediating
A(1-7) action. The main receptor involved in A(1-7) is Mas but other receptors
such as AT2, might also play a role.
Because T2D is a chronic condition, we anticipate that the treatment in clinic will
last much longer than the therapy presented here. Heart and kidney dysfunction
is also much more profound in older animals. For these reasons, the effects of
long-term A(1-7) administration on cardiorenal function in db/db mice were
examined (chapters 3 and 4). We hypothesized that prolonged A(1-7) therapy
results in even more profound improvement of heart and kidney function. In the
81
studies described in further chapters, we also established safety of long-term
treatment in this animal model.
2.5. Acknowledgements
This study was performed in collaboration with Dr. Nicholas Mordwinkin. Some of
the data presented in this chapter was produced by Dr. Mordwinkin including:
echocardiography at 82-83 day-old time point (Fig. 2.2), cardiomyocyte width and
area, PMN count, vessel density and kidney nitrotyrosine for groups hzg saline,
db/db saline, db/db A(1-7) and db/db A(1-7)+A-779 (Fig. 2.3, 2.4, 2.6). Dr.
Mordwinkin has also performed TUNEL experiments (data not shown). The
author would like to thank Dr. Mordwinkin for his contribution.
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87
3. Chapter 3: Long-term administration of angiotensin (1-7)
prevents progression of diabetic heart disease
3.1. Background and goals
T2D is a metabolic disease that has reached epidemic proportions in the US and
throughout the globe. The number of individuals diagnosed with T2D increases
every year and is estimated to double over the next 25 years (American Diabetes
Association, 2013; Centers for Disease Control, 2014). The treatment plan for
T2D includes diet and exercise, supported by blood glucose and cholesterol
control medications. However, even under optimal conditions, these strategies
are insufficient to eliminate life-threatening diabetic complications that arise due
to a number of other underlying pathological mechanisms, such as increases in
inflammation, oxidative stress, and fibrosis. Due to their critical role in tissue
damage, inflammation and oxidative stress are promising targets in T2D
treatment.
One of the hallmarks of T2D is dysregulation of the RAS. Ang-II, the central
player in the pathological arm of the RAS, causes vasoconstriction as well as
increased blood volume and pressure. Through interaction with the AT1 receptor,
Ang-II was shown to cause hypertension (Romero and Reckelhoff, 1999;
Zimmerman et al., 2004), inflammation (Muller et al., 2000; Romero and
Reckelhoff, 1999; Suzuki et al., 2003), oxidative stress (Chabrashvili et al., 2003;
Rajagopalan et al., 1996; Zimmerman et al., 2004), fibrosis (Sadoshima and
88
Izumo, 1993), and insulin resistance (Henriksen, 2007; Olivares-Reyes et al.,
2009); all these factors contribute to and exacerbate pathogenesis of diabetes.
Counterbalancing the pathological arm of the RAS is the protective or
regenerative arm. This protective arm is comprised of the Mas receptor, activated
by another member of RAS, A(1-7) (Bader et al., 2012). A(1-7) opposes Ang-II
primarily through activation of a G-protein coupled receptor called Mas (Santos et
al., 2003). Dysregulation of RAS in the diabetic patients results in increased
levels of Ang-II and chronic activation of AT1 contributing to many of the diabetic
complications. In contrast, A(1-7) induces vasodilation, decreases oxidative
stress, and reduces inflammation. These effects can be highly beneficial in the
treatment of diabetic complications such as heart disease, and may contribute to
the enhancement of overall welfare of diabetic patients.
Two thirds of diabetic patients die due to cardiovascular dysfunction (Geiss et al.,
1995). CHF is a common cardiovascular complication associated with T2D.
Decreased heart function causes fluid build-up in different parts of the body and
may contribute to impaired function of other systems, including lung. CHF is
frequently characterized with cardiac hypertrophy and fibrosis, reduced
ventricular volume, and decreased contractility. Due to the excess of cytokines,
growth factors and oxidative stress, as well as increased blood pressure and
mechanical stress, cardiac tissue becomes fibrotic and loses its elasticity. Severe
cardiac remodeling may result in heart failure.
89
Activation of pathological arm of the RAS, through the Ang-II/AT1 axis, is well
recognized for its role in the heart disease progression (Kim and Iwao, 2000).
Increased inflammation and oxidative stress, which result from hyperglycemia
and Ang-II/AT1 signaling, add to tissue damage, abnormal remodeling, fibrosis
and hypertrophy. Obesity also leads to accumulation of fat in the heart, which
may cause lipotoxicity and result in cell death and impaired cardiac function.
Ang-II, through activation of the AT1 receptor, is responsible not only for
regulation of blood pressure and osmotic balance; it was also shown to control
cell growth (Baker and Aceto, 1990; Sadoshima and Izumo, 1993; Zhao et al.,
2009), and is considered a hypertrophic factor (Boudina and Abel, 2007; Masuda
et al., 2012; Mordwinkin et al., 2012; Papinska et al., 2015; Patel et al., 2012).
Ang-II activates many signaling pathways including those leading to cardiac
fibrosis, stiffness and eventually impaired contractility. Although diabetic heart
disease is a well-recognized problem, the underlying mechanisms are still not
completely understood.
Contractility of the heart is compromised not only because of the pathological
remodeling but also because of dysregulation of some of the molecular
mechanisms. It has been shown that db/db mice have impaired calcium signaling
that weakens contractility of the cardiomyocytes and has an effect on the function
of the heart (Pereira et al., 2006). The contraction is initiated when depolarization
of the membrane activates voltage-dependent calcium channels that allow small
amounts of calcium ions to enter the cytoplasm. This in turn, leads to opening of
90
ryanodine receptors (RyR) in the sarcoplasmic reticulum (SR) and release of
much larger amounts of calcium into the cytoplasm. This rapid increase in
calcium concentration activates myofilaments to contract. Relaxation occurs
when calcium is resequestered to SR by sarco/endoplasmic reticulum calcium
ATPase (SERCA). In addition, small amount of calcium is also transported
outside the cell through sodium calcium exchanger (NCX) (Fig. 3.1).
In Chapter 2 we established that db/db mice develop physiological heart
dysfunction at age between 70 and 82 days old, in part due to increased
cardiomyocyte hypertrophy, fibrosis, immune cell infiltration and impaired
vascularization of cardiac tissue. We also demonstrated that two weeks of daily
Fig. 3.1: Calcium signaling in cardiomyocytes. When electrical impulse reaches
the cardiomyocyte, a small amount of calcium is imported to the cytoplasm through a
voltage gated calcium channel (VGCC). This triggers release of a large concentration
of calcium from the sarcoplasmic reticulum to the cytoplasm through a ryanodine
receptor (RyR). The calcium then binds to sarcomeres and triggers the contraction.
Cytoplasmic calcium is then reuptaken back to the sarcoplasmic reticulum through
SERCA channel. A small amount is also eliminated through the sodium calcium
exchanger (NCX). Reuptake of calcium initiates relaxation phase of the contraction.
91
A(1-7) administration prevented heart dysfunction and ameliorated all of the
aforementioned pathological mechanisms. Because T2D is associated with long-
term complications we anticipate that, once in clinic, A(1-7) would be
administered for a prolonged period of time. Here we demonstrate that long-term
administration of A(1-7) is safe and efficacious in preventing pathological
remodeling of cardiac tissue and improving physiological heart function. We also
identify some of the mechanisms associated with this cardioprotective action.
3.2. Study design and methods
3.2.1. Animals
Eight-week old male BKS.Cg-Dock7
m
+/+ Lepr
db
/J (db/db) mice and age-matched
heterozygous controls (non-diabetic) were purchased from Jackson Laboratories
(Bar Harbor, ME, USA). Mice were randomized by weight into four treatment
groups (n=6/group). Animals were kept on a 12h light/dark cycle and food and
water were available ad libitum.
Animals were administered either vehicle (saline) or A(1-7) (0.5 mg/kg/day)
subcutaneously, daily for 8 or 16 weeks. All the analyses, with the exception of
cardiomyocyte contractility and calcium transients, were performed on mice
treated for 16 weeks. As the survival of the cardiomyocytes isolated for calcium
signaling and cell contractility studies decreases with the age of the donor, mice
older than 8 weeks could not be used for these analyses. Previous dose finding
92
studies performed in this laboratory revealed the optimal dosing to be 0.5
mg/kg/day with no further benefit at 1 mg/kg/day (Mordwinkin et al., 2012;
Papinska et al., 2015). Pharmaceutical grade A(1-7) was purchased from
Bachem (Torrance, CA, USA).
Body weight was assessed at necropsy. Fasting blood glucose level was
measured after animals were fasted overnight, using a hand-held blood glucose
meter from a drop of blood obtained from the saphenous vein. At the necropsy
mice were overdosed with ketamine/xylazine and the blood was collected by
cardiac puncture into EDTA coated tubes. Immediately after collection plasma
was isolated by centrifugation and stored at -80
o
C until analysis.
3.2.2. Echocardiography
Heart function was assessed noninvasively using a high frequency, high-
resolution two-dimensional echocardiography system consisting of Vivid 7
Dimension ultrasound machine equipped with a 6-13MHz linear transducer (GE
Healthcare, Little Chalfont, UK) after 16 weeks of treatment. Anesthesia was
induced with 3% isoflurane by placing mice in the induction chamber. The mouse
was then placed in a supine position on a heating pad to maintain body
temperature at 36.5-37
o
C. Anesthesia was maintained through a nosecone.
Isoflurane levels were adjusted to maintain heart rate at 450-550 beats per
minute. The images of the left ventricle were acquired and the shortening fraction
(SF), stroke volume (SV), and cardiac output (CO) were calculated in accordance
93
with the American Society of Echocardiography guidelines (Lang et al., 2006).
CO was calculated using a modified Simpson’s rule.
To facilitate measurements, mice were injected with a contrast agent through the
tail vein. Contrast was prepared as described previously (Walton et al., 2011).
Briefly, microbubble stock was prepared from 1,2-dipalmitoyl-sn-glycero-3-
phosphatidylcholine (20 mg/mL), 2-dipalmitoyl-sn-glycero-3-
phosphatidylethanolamine (5 mg/mL) and glucose (100 mg/mL) in PBS. The
mixture was then heated in a water bath for 30 minutes with pipette mixing every
5 minutes. Solution was stored at 4
o
C. A sample of the microbubble stock was
incubated at 40
o
C for 15 minutes and mixed with glycerol (5 parts microbubble
stock, 1 part glycerol). The air in the vial was replaced with octafluoropropane
gas and the tube was shaken in a Bullet Blender Tissue Homogenizer (Next
Advance, Inc., Averill Park, NY, USA). Contrast was then loaded into a syringe
using an 18G needle. Contrast was prepared immediately before use.
3.2.3. Histological analysis:
At the necropsy hearts were rapidly excised and weighed. The weights were
normalized to tibia length. The heart tissue was cut in half; one half was formalin-
fixed and paraffin-embedded and cut at 5 µm, and other half was embedded in
OCT and cut at 7 µm.
94
Cardiomyocyte hypertrophy
Heart sections were stained using H&E. Cross-area of 30-40 cardiomyocytes
located in the left ventricle was measured by a blinded observer using freehand
selection in ImageJ (1.47v, NIH, USA).
Apoptosis
Apoptosis in the heart was evaluated using Click-iT TUNEL Alexa Fluor from
Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA) with modifications:
tissue was permeabilized using proteinase K solution (Thermo Fisher Scientific,
Waltham, MA, USA) at 20 µg/ml for 12 minutes and washed thrice with PBS for 5
minutes each. Number of apoptotic cells was quantified in the left ventricles.
Fibrosis
Fibrosis was evaluated in sections stained using standard Masson’s trichrome
method. Ten pictures at 40x magnification were acquired for each slide and the
collagen was quantified using color-deconvolution plugin and threshold function
in ImageJ (1.47v, NIH, USA). Results are expressed as percentage of total tissue
area stained for collagen.
Fat accumulation
Heart samples embedded in OCT were cut at 7 µm and stored at -80
o
C until
used. Lipid accumulation was measured using standard Oil-Red-O staining
method. Five random views of the tissue were acquired at 60x magnification for
each slide. Oil-Red-O staining was quantified using color-deconvolution plugin
95
and threshold function in ImageJ (1.47v, NIH, USA). Results are expressed as
percentage of total tissue area stained for lipids.
Immunohistochemistry staining of N-Tyr
The paraffin-embedded heart sections were treated using a standard HIER
procedure in Antigen Retrieval Citra Plus (Biogenex, San Ramon, CA, USA). The
slides were incubated with rabbit anti-mouse polyclonal antibody directed against
nitrated tyrosine residues (EMD Millipore, Billerica, MA, USA) at 1:250. After
incubation with biotinylated secondary antibody, a streptavidin conjugated
horseradish peroxidase was added. The sections were then incubated in DAB for
2-3 minutes and counterstained with Harris hematoxylin. Sections were scanned
using an Aperio CS2 slide scanner (Leica Biosystem, Wetzlar, Germany).
Vessels positive and negative for N-Tyr were quantified in left ventricle. Only
vessels larger than 10 µm in diameter were counted.
3.2.4. Concentration of cytokines in plasma
Multi-Array technology by Meso Scale Diagnostics (Rockville, MD, USA) allows
quantitative assessment of expression levels of proteins using
electrochemiluminescence. To evaluate concentration of proinflammatory
cytokines, a Mouse Proinflammatory 7-Plex kit (Meso Scale Diagnostics,
Rockville, MD, USA) was used. Assay was performed in accordance to
manufacturer's manual. The plate was read using a Sector S 600 analyzer (Meso
Scale Diagnostics, Rockville, MD, USA).
96
3.2.5. Isolation of cardiomyocytes
Isolation of cardiomyocytes from mice treated for 8 weeks was performed as
described previously (O'Connell et al., 2007). Mice were injected with 250 µL of
heparin diluted in sterile PBS to 200 U/mL. After 10 min, the animals were
overdosed with isoflurane and the hearts were rapidly excised, cannulated and
perfused with a buffer consisting of 113 mM NaCl, 4.7 mM KCl, 0.6 mM KH
2
PO
4
,
0.6 mM Na
2
HPO
4
, 1.2 mM MgSO
4
-7H
2
O, 12 mM NaHCO
3
, 10 mM KHCO
3
, 10
mM HEPES buffer solution, 30 mM Taurine, 10 mM 2,3-butanedione monoxime
(BDM), and 5.5 mM glucose. The buffer was maintained at 37
o
C and was
constantly oxygenated. The heart was perfused with the buffer at 3 mL/min for 4
min. The buffer was then switched to digestion buffer (perfusion buffer containing
digestion enzymes). Two digestion enzymes were used: collagenase II – 25 mg
(cat. 17101-015, Gibco by Thermo Fisher Scientific, Waltham, MA) and protease
– 2.5 mg (cat. P5147, Sigma, St. Louis, MO). The hearts were perfused with
digestion buffer at 2.5 mL/min for 10 min. Then the tissue was homogenized
using scissors to release single cardiomyocytes. After reintroduction of calcium to
the cell suspension, cells were resuspended in M199 media (Invitrogen for
Thermo Fisher Scientific, Carlsbad, CA).
3.2.6. Contractility and calcium imaging
Contractility and calcium transient studies were performed as described
previously (Belke et al., 2004). Briefly, after isolation cardiomyocytes were
97
resuspended in a solution of Fluo-4AM, a fluorescent calcium indicator (Thermo
Fisher Scientific, Waltham, MA) in Tyrode’s solution. To facilitate dissolution of
the dye, Fluo-4AM was first dissolved in 20% pluronic acid F-127 in DMSO. The
cells were then plated on the 12 well plates containing coverslips pre-coated with
laminin (BD Biosciences, San Jose, CA) and allowed to attach for 30 min at room
temperature. The wells were washed with Tyrode’s solution to allow de-
esterification of the dye and imaged within 2h. Cells were imaged using a Zeiss
LSM 510 Meta inverted confocal microscope. Coverslips were placed in a field
stimulation microscope chamber with temperature control (37
o
C) and constantly
perfused with Tyrode’s solution at 0.5 ml/min. Cells were stimulated with an
electrical impulse at 1Hz, 5ms, 20V to contract. Changes in cytosolic
fluorescence were measured using a line scan mode. Nuclei were avoided for
consistency. A macro developed by Greensmith was used to analyze calcium
transient data (Greensmith, 2014). Only quiescent, non-contracting, rod-like cells
were selected for functional studies. At least ten beats of ten cells isolated from
each heart were analyzed. LSM Image Browser was used to assess length of the
cells in systole and diastole from the images acquired for calcium signaling
analysis. Contractility was expressed as percentage of cell length change.
3.2.7. Statistical analysis
GraphPad Prism version 6.0c for Mac OS X (GraphPad Software, San Diego,
CA, USA) was used to analyze the data. One-way ANOVA followed by Dunnett’s
98
multiple comparisons test were used to compare data. The level of statistical
significance was set at 5%. Data are expressed as mean value ± SEM.
3.3. Results
3.3.1. A(1-7) has no effect on body mass and hyperglycemia in db/db
mice.
db/db mice develop hyperglycemia at age of 6-8 week-old primarily due to
obesity. Both blood glucose levels and body mass were highly increased in
diabetic animals compared to heterozygotes, which suggests a severe metabolic
dysfunction in db/db mice (Fig. 3.2). Administration of A(1-7) did not have any
effect on the body mass and hyperglycemia in db/db mice. In addition A(1-7) had
no effect on these parameters in non-diabetic mice.
A) B)
hzg saline
hzg A(1-7)
db/db saline
db/db A(1-7)
0
100
200
300
400
500
blood glucose [mg/dL]
****
****
hzg saline
hzg A(1-7)
db/db saline
db/db A(1-7)
0
20
40
60
body weight [g]
****
****
Fig. 3.2: Long-term treatment with A(1-7) has no effect on hyperglycemia and
body weight. Diabetic animals from both treatment groups demonstrated increased
hyperglycemia (A) and body weight (B) compared to non-diabetic controls. Daily
treatment with A(1-7) did not have any effect on these parameters. (hzg –
heterozygous; ****p<0.001 compared to hzg saline); calculated using one-way
ANOVA; plotted as mean with SEM. (Papinska et al., 2016)
99
3.3.2. Treatment with A(1-7) improved heart function as measured by
echocardiography.
One of the hallmarks of heart disease is decreased CO that results in decreased
volume of blood pumped from the left ventricle. Diabetic mice had significantly
decreased CO compared to heterozygous controls. Treatment with A(1-7)
partially prevented the reduction in CO in db/db mice (Fig. 3.3A). Like CO, SV is
a measurement of ability of the heart to effectively circulate the blood. Similar to
Fig. 3.3: Long-term administration of A(1-7) improves physiological heart
function. Treatment with A(1-7) prevented progression of heart dysfunction in
diabetic animals. Parameters of heart function were assessed using
echocardiography. Daily administration of A(1-7) prevented loss in cardiac output (A),
stroke volume (B), and shortening fraction (C) in diabetic animals, which suggests
protection against progressive heart dysfunction (hzg – heterozygous; *p<0.05;
**p<0.01; ***p<0.001; ****p<0.0001); calculated using one-way ANOVA; plotted as
mean with SEM. (Papinska et al., 2016)
hzg saline
hzg A(1-7)
db/db saline
db/db A(1-7)
0
10
20
30
cardiac output [cm3/min]
****
***
hzg saline
hzg A(1-7)
db/db saline
db/db A(1-7)
0.00
0.01
0.02
0.03
0.04
0.05
stroke volume [cm3]
**
***
hzg saline
hzg A(1-7)
db/db saline
db/db A(1-7)
0
10
20
30
40
shortening fraction [%]
*
***
A) B)
C)
100
CO, SV was decreased in diabetic animals compared to non-diabetics.
Treatment with A(1-7) improved this parameter, which suggests a
cardioprotective role of the peptide in this model of T2D (Fig. 3.3B). SF is an
echocardiographic measurement of heart contractility. SF of the left ventricle in
diabetic mice was decreased compared to age-matched, non-diabetic animals
suggesting impaired contractility (Fig. 3.3C). Daily administration of A(1-7)
improved this measurement in diabetic mice, which suggests enhanced
contractility of the left ventricle in db/db mice.
3.3.3. A(1-7) reduced cardiomyocyte hypertrophy, number of apoptotic
cells, fat accumulation, and fibrosis in hearts from diabetic animals.
Cardiac hypertrophy, which results from growth signals, is thought to be one of
the mechanisms that compensates for hemodynamic stress. Ventricular
hypertrophy is one of the characteristics of diabetic heart disease. Heart
hypertrophy is often associated with increased myocyte size. Cardiomyocytes
become enlarged in order to keep up with increased functional demand. Even
though no changes were observed in absolute (data not shown) and normalized
heart weight between any of the treatment groups (Fig. 3.4A), significant
increases in cardiomyocyte area were seen in hearts from diabetic animals,
compared to non-diabetic controls. Treatment with A(1-7) normalized the
cardiomyocyte size in db/db mice (Fig. 3.4B, 3.5A). The increase of myocyte size
without increased organ weight suggests that growth of some of the cells might
be associated with loss of others. An increased number of apoptotic cells was
101
observed in db/db mice. A(1-7) reduced the number of cells undergoing
apoptosis in hearts of diabetic mice (Fig. 3.4C). Number of apoptotic cells
correlated with cardiomyocyte hypertrophy (Fig. 3.4D). One mechanism that
causes cardiomyocyte apoptosis is lipotoxicity due to accumulation of lipids. Lipid
Fig. 3.4: Treatment with A(1-7) for 16 weeks reduces remodeling of heart tissue.
No significant changes were detected in normalized heart weights between the
treatment groups (A). Cardiomyocyte cross-area was increased in db/db mice
compared to non-diabetic controls. Treatment with A(1-7) reduced myocyte hypertrophy
in diabetic animals (B). Activation of hypertrophic signals is one of the mechanisms
compensating for cell loss. Daily administration of A(1-7) reduced number of apoptotic
cells in hearts from diabetic animals (C). Number of apoptotic cells correlated with the
size of cardiomyocytes (D) (r
2
=0.52, p=0.001). Accumulation of lipids in hearts may also
contribute to increased apoptosis and remodeling of the tissue. Fat deposition was
highly increased in hearts from db/db mice, compared to healthy controls. Treatment
with A(1-7) reduced lipid accumulation in diabetic animals (E). Fibrosis in the heart was
assessed using Masson’s trichrome staining. Hearts from diabetic mice had
significantly higher levels of collagen (blue) than hearts from heterozygous animals.
A(1-7) reduced fibrosis in hearts of db/db mice (F). (hzg – heterozygous; *p<0.05;
**p<0.01; ****p<0.0001); calculated using one-way ANOVA; plotted as mean with SEM.
(Papinska et al., 2016)
hzg saline
hzg A(1-7)
db/db saline
db/db A(1-7)
0
2
4
6
8
10
normalized heart weight
hzg saline
hzg A(1-7)
db/db saline
db/db A(1-7)
0
200
400
600
cardiomyocyte cross area [um2]
****
****
hzg saline
hzg A(1-7)
db/db saline
db/db A-(1-7)
0.0
0.5
1.0
1.5
apoptotic cells/field
**
*
300 400 500 600
0.0
0.5
1.0
1.5
2.0
cardiomyocyte cross area [um2]
apoptotic cells/field
A) B) C)
D)
hzg saline
hzg A(1-7)
db/db saline
db/db A(1-7)
0.0
0.5
1.0
1.5
% fibrotic area
****
*
E) F)
htz saline
htz A(1-7)
db/db saline
db/db A(1-7)
0.0
0.5
1.0
5
10
15
% area stained with Oil-Red-O
****
****
102
accumulation was increased in diabetic animals and treatment with A(1-7)
decreased this effect (Fig. 3.4E, 3.5B), which may contribute to reduced cell
death and tissue remodeling. Many of the pathways activated by Ang-II
contribute to increased fibrosis that may severely affect heart function. Fibrosis
not only compromises elasticity of the heart but also interferes with excitation-
contraction mechanisms and may result in impaired contractility. Interstitial
fibrosis was significantly increased in hearts of diabetic animals compared to
non-diabetic controls. Daily treatment with A(1-7) reduced fibrotic area in hearts
of db/db mice (Fig 3.4E, 3.5C).
db/db saline hzg A(1-7) db/db A(1-7) hzg saline
heart, H&E
cardiomyocyte
hypertophy
heart,
Masson's trichrome
fibrosis
lung,
immunofluorescence
macrophage infiltration
lung,
immunohistochemistry
oxidative stress damage
A)
B)
D)
E)
C)
heart,
Oil-Red-O
lipid accumulation
Fig. 3.5: Representative histological images of the analysis of cardiac tissue
remodeling. Representative images of H&E staining (A), Oil-Red-O staining (B), and
Masson’s trichrome staining (C). (Papinska et al., 2016)
103
3.3.4. Administration of A(1-7) to diabetic animals results in reduced
oxidative stress in the heart and decreased levels of circulating
proinflammatory cytokines.
Two factors that contribute to enhanced scarring and fibrosis are increased
inflammation and oxidative stress. Damage due to oxidative stress was assessed
in heart sections stained for N-Tyr, an irreversible protein modification that is
commonly used as an indirect marker of superoxide production contributing to
the formation of peroxynitrite. N-Tyr levels in the cardiomyocytes were virtually
undetectable. However, significant levels of staining were observed in the
vessels (Fig. 3.6A). Number of vessels that were positive for N-Tyr was higher in
db/db mice, indicating increased oxidative stress, which may result in endothelial
dysfunction. A(1-7) reduced the fraction of positively stained vessels in hearts of
diabetic mice (Fig. 3.6B). ROS produced as a result of hyperglycemia and
Fig. 3.6: Levels of nitrotyrosine in cardiac tissue after long-term treatment.
Staining positive for nitrated tyrosine residues was used as a marker of oxidative
stress. Nitrotyrosine was detected in vessels but not in the cardiomyocytes (H).
Fraction of vessels positively stained for nitrotyrosine was significantly higher in diabetic
mice compared to non-diabetics. A(1–7) treatment prevented oxidative stress damage
in the vessels (G). (hzg—heterozygous; *p < 0.05; **p < 0.01; ****p < 0.0001);
calculated using one-way ANOVA; plotted as mean with SEM. (Papinska et al., 2016)
hzg saline
hzg A(1-7)
db/db saline
db/db A-(1-7)
0.0
0.2
0.4
0.6
fraction of vessels
positive for N-Tyr
**
*
A)
positive negative
B)
104
mitochondrial dysfunction are well-characterized markers of diabetes. Increased
oxidative stress contributes to activation of proinflammatory mechanisms that
may in turn lead to excessive fibrosis and remodeling. Epidemiological studies
also suggest strong correlation between systemic inflammation and cardiac
hypertrophy (Palmieri et al., 2003). Increased levels of TNF-α and IL-1β were
observed in plasma of diabetic animals, whereas no changes were seen in levels
of IL-6 in any treatment group (Fig. 3.7). A(1-7) significantly reduced levels of
hzg saline
hzg A(1-7)
db/db saline
db/db A(1-7)
0
5
10
15
20
TNF alpha concentration [pg/ml]
**
A) B)
C)
hzg saline
hzg A(1-7)
db/db saline
db/db A(1-7)
0
2
4
6
IL-1beta concentration [pg/ml]
***
*
hzg saline
hzg A(1-7)
db/db saline
db/db A(1-7)
0
50
100
150
200
IL-6 concentration [pg/ml]
Fig. 3.7: Levels of circulating cytokines in the animals treated for 16 weeks.
Levels of circulating cytokines were measured using electrochemiluminescence
assay. Levels of two proinflammatory cytokines, TNF-α and IL-1β, were significantly
increased in diabetic mice (A, B). Treatment with A(1-7) reduced concentration of IL-
1β in plasma of db/db mice. The reduction of TNF-α levels was not as profound and
has not reached statistical significance levels. No significant changes in IL-6
concentration were detected between any of the groups (C). (hzg – heterozygous;
*p<0.05; **p<0.01; ***p<0.001); calculated using one-way ANOVA; plotted as mean
with SEM. (Papinska et al., 2016)
105
circulating IL-1β. The reduction of TNF-α levels was not as profound and has not
reached statistical significance levels, possibly because the overall increase in
the diseased animals is minimal. This data suggests reduction of systemic
inflammation after administration of A(1-7).
3.3.5. A(1-7) treatment for 8 weeks improves calcium handling and
contractility of isolated cardiomyocytes.
After 8 weeks of treatment the cardiomyocytes were isolated and the calcium
transients and contractility of single cells were measured. Changes in calcium
concentration were detected by probing the intracellular calcium with a
fluorescent indicator. When the cells were stimulated with an electrical impulse, a
rapid release of calcium to the cytoplasm was observed, followed by a reuptake
to the SR phase (Fig. 3.8A). No significant changes were detected in the
amplitude of calcium signal in the cytoplasm between diabetic and non-diabetic
group (data not shown). However, the rate constant of reuptake of calcium was
significantly decreased in diabetic animals treated with vehicle compared to non-
diabetic controls (Fig. 3.8B). Treatment with A(1-7) improved rate of reuptake of
calcium after contraction. Contractility of the cardiomyocytes was measured as
percentage of cell length change during contractions stimulated with an electrical
impulse (Fig. 3.8C). Cardiomyocytes isolated from diabetic animals treated with
saline had significantly impaired contractility compared to healthy controls.
Administration of A(1-7) for 8 weeks significantly improved contractility of isolated
cardiomyocytes.
106
3.4. Discussion and conclusions
We have previously shown that 2 weeks of A(1-7) administration improves heart
and kidney function in young db/db mice (Papinska et al., 2015). As a
continuation of these findings, we designed and executed a study to show that
long-term (16 weeks) therapy with A(1-7) is safe and efficacious in prevention of
heart dysfunction associated with T2D (Papinska et al., 2016). We also identified
a novel mechanisms of cardioprotective action of A(1-7), namely regulation of
calcium signaling in the cardiomyocytes.
Fig. 3.8: Calcium transients and contractility of cardiomyocytes is improved
after 8 weeks of A(1-7) administration. Calcium signaling and cell contractility was
measured in cardiomyocytes isolated from animals treated for 8 weeks. Panel A
shows representative graphs of calcium transients in heterozygous (htz) animals
(blue) and in diabetic animal (red) treated with saline. Rate constant of reuptake of
calcium to the sarcoplasmic reticulum was decreased in diabetic animals from the
control group compared to healthy mice (B). Treatment with A(1-7) improved this
parameter. Cardiomyocytes isolated from db/db group treated with saline also had
reduced percentage of cell length change when stimulated with an electrical impulse
(C). A(1-7) prevented loss of contractility of cardiomyocytes isolated from diabetic
animals. (hzg – heterozygous; *p<0.05; ***p<0.001; ****p<0.0001); calculated using
one-way ANOVA; plotted as mean with SEM.
hzg saline
hzg A(1-7)
db/db saline
db/db A(1-7)
0.000
0.001
0.002
0.003
0.004
0.005
rate constant [1/ms]
***
*
hzg saline
hzg A(1-7)
db/db saline
db/db A(1-7)
0
2
4
6
% of cell length change
****
****
0.5
1
1.5
2
2.5
3
3.5
4
0 1
F/F
0

%me [s]
hzg saline
db/db saline
A. B. C.
107
Heart disease has been well described in various mouse models of T2D,
including ob/ob mice, Zucker fatty rats and db/db mice (Christoffersen et al.,
2003; Daniels et al., 2010; Papinska et al., 2015; Zhou et al., 2000). db/db mice
develop more severe hyperglycemia than ob/ob mice early in life (6-8 weeks old),
which results in more profound changes in heart function. In addition, strain used
in this study also known as BKS db (BKS.Cg-Dock7
m
+/+ Lepr
db
/J) is
characterized with more severe pathologies than similar B6 db strain
(B6.BKS(D)-Lepr
db
/J) and is described by the supplier to survive only up to 40
weeks. Here, we showed that 24 week-old db/db mice develop severe heart
disease dysfunction due to increased inflammation, oxidative stress and fibrosis
(Papinska et al., 2016).
A(1-7) counteracts effects of Ang-II and has been shown to improve
cardiovascular function in some models of type 1 and type 2 diabetes (Benter et
al., 2007; Marcus et al., 2012; Mori et al., 2014; Papinska et al., 2015; Yousif et
al., 2012). However, the exact mechanism of its protective action is not entirely
understood. Safety studies showed that A(1-7) is not associated with any severe
adverse effects. A(1-7) has completed a Phase II clinical trial demonstrating the
efficacy of the peptide in the amelioration of side effects of chemotherapy and the
safety in human populations (Pham et al., 2013). This peptide has been tested in
several clinical populations exposing over 200 patients to up to 1 mg/kg/day with
no safety signals noted to date (personal communication). In this study, in
contrast to profound effects observed in diabetic animals, A(1-7) did not affect
108
any of the studied markers in heterozygous mice (Papinska et al., 2016). This is
consistent with safety studies, where no severe adverse effects of A(1-7) were
observed in patients and toxicology studies (Rodgers et al., 2006).
First observations that A(1-7) is locally expressed in the cardiac tissue of animal
models (Averill et al., 2003; Santos et al., 1990), and in intact human heart
(Zisman et al., 2003), suggested a role in controlling cardiovascular function.
Since then, several animal studies have shown beneficial effect of A(1-7) on
heart function in models of diabetes. A recent study by Oudit group showed
improved diastolic dysfunction in 5 month-old B6 db/db strain (B6.BKS(D)-
Lepr
db
/J) implanted with micro-osmotic pump containing A(1-7) (Mori et al.,
2014). The strain of db/db mice used by Mori is characterized with less severe
diabetes than the model used in studies described here. In addition, we show
that daily subcutaneous injections are sufficient to prevent diabetes-related heart
dysfunction, which has a better translational potential than constant infusion
using mini-pumps.
Echocardiography revealed significantly improved heart function in diabetic
animals treated with A(1-7), which is consistent with our previous findings in
younger animals (chapter 2) (Papinska et al., 2015). Longer treatment resulted in
even more profound changes in CO and SF in db/db mice treated with A(1-7)
(Papinska et al., 2016). T2D has been previously described as an independent
factor associated with cardiac hypertrophy in patients (Eguchi et al., 2008). Even
109
though no change was seen in heart weight between treatment groups,
increased cardiomyocyte size suggests presence of cardiac hypertrophy in
diabetic mice. In addition, myocyte cross-area correlates with number of
apoptotic cells. Activation of hypertrophic signals may be one of the mechanisms
compensating for increased numbers of dying cells in diabetic hearts.
We also demonstrated that A(1-7) reduces fat accumulation in the cardiac tissue
from diabetic animals, which may contribute to reduction of lipotoxicity (Papinska
et al., 2016). Lipid accumulation is known to induce apoptosis, endothelial
reticulum stress, mitochondrial dysfunction and overproduction of ROS not only
in animal models of obesity but also in patients (Wende and Abel, 2010).
Increased oxidative stress and levels of circulating proinflammatory cytokines
also contribute to activation of fibrotic and hypertrophic signals. Tissue damage
due to oxidative stress may be one of the factors leading to increased cell
apoptosis in the hearts of db/db mice.
Oxidative damage can activate immune response and partially contribute to
systemically elevated inflammation. Epidemiological studies suggest strong
correlation between systemic inflammation and cardiac hypertrophy (Palmieri et
al., 2003). We observed increased concentration of TNF-α and IL-1β in plasma
from diabetic animals, whereas no changes in expression of IL-6 were seen
(Papinska et al., 2016). This suggests presence of a low grade of chronic
inflammation that was attenuated by treatment with A(1-7) in diabetic animals.
110
Both oxidative stress and inflammation can activate growth and fibrotic signals.
Extensive fibrosis observed in diabetic hearts contributes not only to impairment
of overall cardiac function but might also directly affect the elasticity and
contractility of the heart. In this study we observed significant effects of A(1-7) on
all of the discussed parameters. Our findings are also consistent with data from
short-term treatment studies described in chapter 2 (Papinska et al., 2015). Here
we further identify that administration of A(1-7) prevents pathological remodeling
of the heart tissue, in part through amelioration of systemic inflammation,
oxidative stress and endothelial dysfunction (Papinska et al., 2016). We
hypothesize that A(1-7) antagonizes actions of Ang-II and therefore contributes
to decreased oxidative stress, inflammation, hypertrophy, lipid accumulation and
fibrosis in the heart, and results in improved cardiac function in db/db mice.
Cellular mechanisms that were investigated in this study may aid in
understanding of the underlying pathologies in diabetic patients and support
transition of A(1-7) to clinic.
In addition, we identified a previously unknown mechanism of the
cardioprotective action of A(1-7). In this study we showed that 8 weeks of
administration of A(1-7) improves calcium handling and contractility of isolated
cardiomyocytes. Even though A(1-7) is known to have positive effects on heart
function in several different disease models, little is known about its effects on
calcium signaling. Dias – Peioxoto and colleagues demonstrated that
cardiomyocytes isolated from Mas knock-out mice have impaired calcium
111
transients and decreased expression of SERCA2a (a cardiac isoform) channel
(Dias-Peixoto et al., 2008). The same study showed no effects of A(1-7) on
calcium handling in cardiomyocytes isolated from wild type mice. These results
are consistent with our observations that A(1-7) has little or no effects in healthy
wild type mice, perhaps due to an up-regulation of the Mas receptor in
pathological conditions or that the disease environment is permissive to Mas
signaling. In a model of streptozotocin induced T2D (streptozotocin infused in
neonatal animals) altered calcium handling was attributed to dysfunctional NCX
and plasmalemmal calcium ATPase (Allo et al., 1991). Another study showed
that transgenic rats that overexpress A(1-7) in plasma had reduced
cardiomyocyte damage and preserved calcium transients after infusion of Ang-II
(Gomes et al., 2010). Here, we identified that enhancement of calcium signaling
might be one of the mechanisms by which A(1-7) improves heart function in
db/db mice.
Calcium signaling in cardiomyocytes is a highly regulated pathway that consists
of various channels and signaling molecules. The exact mechanism of impaired
calcium signaling in cardiomyocytes from db/db mice is still not fully understood.
Belke and colleagues showed that action of SERCA2a channels in db/db mice is
impaired, which may contribute to SR calcium leakage (Belke and Dillmann,
2004). Another study demonstrated that SERCA2a expression in hearts of db/db
mice is decreased compared to wild type mice (Stolen et al., 2009). Decreased
112
activity of SERCA is also thought to be a consequence of either decreased
expression of the channel or increased activity of its inhibitor called
phospholamban (PLN). Only unphosphorylated form of PLN binds to SERCA and
inhibits its activity. Several studies investigated effects of PLN on calcium
handling in hearts of db/db mice. Belke and colleagues showed that 12-week-old
diabetic mice had significantly higher levels of PLN but the levels of
phosphorylated PLN were unchanged as measured by Western blot (Belke and
Dillmann, 2004). In contrast, Stolen et al. demonstrated that expression of PLN in
20-week-old db/db mice was not different from that in wild type mice (Stolen et
al., 2009). However, same study showed decreased levels of phosphorylated
PLN in diabetic animals. Thus, A(1-7) action resulting in the enhancement of
calcium signaling might be attributed to increased activation or expression of
SERCA and/or reduced levels or altered phosphorylation of PLN.
NCX is another means of elimination of calcium from the cytoplasm. The role of
NCX in heart disease is rather controversial. In human heart biopsies from
patients with heart failure expression of NXC was increased (Studer et al., 1994).
It is believed to be a compensatory mechanism for the impaired function of
SERCA. Increased NCX current was also observed in db/db mice (Pereira et al.,
2006). On the other hand, Hattori and colleagues showed that impaired function
of NCX is caused by decreased expression of this channel (Hattori et al., 2000).
Even though the exact function of NCX in diabetic heart disease is not
113
established, the role of this channel in the cardioprotective action of A(1-7)
shouldn’t be disregarded.
Contractility of the heart depends not only on the availability of the calcium in the
cytoplasm but also on calcium sensitivity of the myofilaments (Lovelock et al.,
2012; Morgan, 1991; Westfall, 2002). Here we showed that administration of
A(1-7) is associated with improved contractility of the heart (16 weeks of
treatment) and single cardiomyocytes (8 weeks of treatment). Since myofilament
structure is altered in diabetes (Falcão-Pires et al., 2009; Ramirez-Correa et al.,
2008; Ward and Crossman, 2014), we hypothesize that this improvement of
contractility might be in part due to alteration in expression or post-translational
modifications of filaments.
Reducing hyperglycemia in diabetic patients can decrease some of the
complications but does not fully eliminate them. Targeting other mechanisms that
contribute to the disease state may further improve the outcomes for diabetic
patients. We demonstrated that daily administration of A(1-7) improved heart
function even in the face of uncontrolled hyperglycemia and obesity in a severe
model of T2D. We hypothesize that A(1-7) used in conjunction with a blood
glucose lowering agent might have even more profound effects.
114
3.5. Acknowledgements
Author would like to thank Maira Soto for her help with Masson’s trichrome and
circulating cytokine analyses, Christopher Meerks for his assistance with
echocardiography, as well as Dr. Sachin Jadhav, Alick Tan, Maira Soto, Lila Kim
and Tamar Cohen-Amzaleg for their help with animal handling and necropsies.
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4. Chapter 4: Long-term administration of angiotensin (1-7)
prevents kidney dysfunction and decreases oxidative
stress in db/db mice.
4.1. Background and goals
One of the most dangerous and prevalent complications of T2D is the diabetic
nephropathy, which may lead to kidney failure, an end-stage kidney disease
resulting in shut down of the filtration function. Over 100,000 US citizens are
diagnosed with kidney failure yearly, 44% of them are due to diabetes (Blackwell
et al., 2014; Centers for Disease Control, 2014). Diabetic kidney disease
manifests in decreased glomerular filtration rate and proteinuria. A central
therapeutic strategy to prevent complications in patients with T2D is to reduce
the activity of the pathological arm of the RAS, namely levels and actions of
Ang-II. Thus, the current treatment for diabetic nephropathy focuses on ACEi,
which reduce the production of Ang-II, and ARBs, which block the actions of
Ang-II through its cognate receptor, AT1. However, both therapies are associated
with high risk of adverse effects (Kostis et al., 1996; Lacourciere et al., 2000),
and do not completely eliminate kidney damage. A(1-7), another member of the
RAS, is a promising new treatment for diabetic nephropathy. It has been shown
to decrease hypertension and oppose pathological actions of Ang-II (Ferrario et
al., 1997; Sampaio et al., 2007). In chapter 2 we showed that short-term therapy
with A(1-7) improves glomerular health in young db/db mice, in part through
reduction of hypertrophy and mesangial expansion, as well as decreased
121
oxidative stress damage (Papinska et al., 2015). Here we investigated the effects
of A(1-7) treatment on kidney function over time (up to 16 weeks). In this study
we showed that long-term (16 weeks) administration of A(1-7) acts as a
renoprotective agent, perhaps through amelioration of oxidative stress.
Abnormal cellular responses to hyperglycemia are one of the leading
mechanisms of kidney disease. High blood glucose levels stimulate cells to
produce increased amounts of growth factors, cytokines and ROS; leading to
overproduction of ECM and thickening of glomerular basal membrane (Schena,
2005). As a consequence, the permeability of the basal membrane is increased
and the filtration quality is compromised. High blood glucose also activates PKC,
which is associated with overexpression of TGF-β, fibronectin, and collagen type
IV; over time, leading to mesangial expansion (Koya et al., 1997). Ang-II has
been implicated as a key factor in T2D pathology, however the exact mechanism
is not fully understood. Nonetheless, Ang-II has been shown to cause
hypertension (Romero and Reckelhoff, 1999; Zimmerman et al., 2004),
inflammation (Muller et al., 2000; Suzuki et al., 2003), oxidative stress
(Chabrashvili et al., 2003; Rajagopalan et al., 1996; Zimmerman et al., 2004),
and contribute to insulin resistance progression (Henriksen, 2007; Olivares-
Reyes et al., 2009); all of which add to diabetes pathogenesis.
Hyperglycemia, as well as high levels of cytokines and other growth factors
contributes to increased oxidative stress, which leads to many diabetic
122
complications including nephropathy. Elevated levels of ROS may result in
promotion of fibrosis, inflammation and endothelial dysfunction in diabetic
kidneys (Avogaro et al., 2008; Gorin et al., 2005; Thallas-Bonke et al., 2008; Tojo
et al., 2007). Studies show increased circulating levels of ROS in diabetes in both
animals and patients (Tojo et al., 2007; Yun et al., 2006). The main source of
ROS in diabetic kidneys is thought to be NOX-4, a member of NADPH oxidase
family. NADPH oxidase is known to overproduce superoxide in T2D affected
tissues. Superoxide when combined with NO results in formation of peroxynitrite
- a very potent ROS that has the capability to modify proteins to form N-Tyr.
Addition of the nitrate groups to the proteins changes their structure and function
and may result in pathologies (Pacher et al., 2005; Radi, 2013).
Studies also show that A(1-7) (Pendergrass et al., 2008), as well as its receptor –
Mas (Gwathmey et al., 2010), are expressed in renal tissue in relatively high
amounts and the peptide can be detected in urine (Li et al., 2005). This suggests
that A(1-7) may play a role in kidney homeostasis. The effects of A(1-7)
treatment have been studied in various models of kidney dysfunction. A(1-7)
reduced hypertension and proteinuria, and acted renoprotective in spontaneously
hypertensive rats (SHR) treated with L-NAME (eNOS inhibitor) (Benter et al.,
2006). In stroke prone SHR (SHRSP), A(1-7) treatment reduced insulin
resistance and structural damage in kidneys (Giani et al., 2010). More insight into
the mechanism of how A(1-7) affects kidney function has been gained from
studies focusing on the role of ACE2. In rodent models of diabetes it has been
123
shown that ACE/ACE2 expression ratio in glomeruli is increased, suggesting
upregulation of Ang-II and downregulation of A(1-7) formation (Soler et al., 2007;
Ye et al., 2006). These results are supported by studies of renal biopsies from
patients with diabetic nephropathy, showing increased ACE/ACE2 ratio (Mizuiri
et al., 2008). This suggests that imbalance in expression of Ang-II and A(1-7)
might play an important role in diabetic nephropathy. Further experiments show
that treatment with exogenous A(1-7) have been proven efficient in: lowering
proteinuria in streptozotocin-induced diabetic rats, a model of type 1 diabetes
(Benter et al., 2007); and decreasing renal fibrosis, oxidative stress and
inflammation in Zucker diabetic fatty rats, a model of T2D (Giani et al., 2012).
A(1-7) also reduced nephropathy in KK-A
y
/TA, a mouse model of T2D (Moon et
al., 2011). However some studies show progression of kidney disease with
A(1-7) treatment. For example, in STZ-diabetic rats and in non-diabetic model of
unilateral ureteral obstruction (Esteban et al., 2009), treatment with A(1-7) was
not beneficial. These controversies have still not been fully explained, however
some of the discrepancies are being ascribed to dosage and route of
administration. It is therefore crucial to further study effects of A(1-7) on diabetic
nephropathy to elucidate the mechanisms of possible protective action of this
treatment.
The purpose of this study was to further delineate effects of A(1-7) on diabetic
kidney disease in db/db mice. These mice are known to develop a severe renal
dysfunction especially later in life. In this study we showed that long-term
124
administration of A(1-7) has no adverse effects in the healthy animals and that it
is effective in amelioration of kidney disease in diabetic mice. As diabetes and its
complications are a chronic disease, we anticipate that once in clinic, A(1-7)
therapy will be used for a prolonged period of time. This study confirmed that
extended treatment with A(1-7) is both safe and efficacious for prevention of
nephropathy in diabetic animals.
4.2. Study design and methods
4.2.1. Animals
All animal procedures were carried out in accordance with the Guide for the Care
and Use of Laboratory Animals as adopted and promulgated by the US National
Institutes of Health.
Eight-week old male BKS.Cg-Dock7
m
+/+ Lepr
db
/J (db/db) mice and age-matched
heterozygous controls (non-diabetic) were purchased from Jackson Laboratories
(Bar Harbor, ME, USA). Mice were randomized based on the initial body weight
into four treatment groups (n=6/group). Animals were kept on a 12h light/dark
cycle and food and water were available ad libitum.
Animals were administered either vehicle (saline) or A(1-7) (0.5 mg/kg/day)
subcutaneously, daily for 16, 12, 8 or 4 weeks. Previous dose finding studies
performed in this laboratory revealed the optimal dosing to be 0.5 mg/kg/day with
125
no further benefit at 1 mg/kg/day (Mordwinkin et al., 2012; Papinska et al., 2015).
Pharmaceutical grade A(1-7) was purchased from Bachem (Torrance, CA, USA).
Just before necropsy animals were weighed. The kidneys were isolated, and the
organ weights were normalized to tibia length. Fasting blood glucose level was
measured in animals fasted overnight, using a hand-held blood glucose meter
from a drop of blood obtained from the saphenous vein. Hyperglycemia was
assessed at the beginning of the experiment to ensure that the animals are
diabetic, and the day before the necropsy. Body weight, normalized kidney
weight and blood glucose was assessed at each time point.
4.2.2. Ultrasonographic assessment of blood flow velocity in renal artery
Blood flow velocity in renal arteries was assessed noninvasively using a high
frequency, high-resolution ultrasound system consisting of Vivid 7 Dimension
ultrasound machine equipped with a 6-13MHz linear transducer (GE Healthcare,
Little Chalfont, UK) after 16 weeks of treatment. Fur from the abdomen was
removed using a hair removal cream. Anesthesia was induced with 3% isoflurane
in an induction chamber. The mouse was then placed in a supine position on a
heating pad to maintain body temperature at 36.5-37
o
C. Anesthesia was
maintained through a nosecone and adjusted to maintain heart rate at 450-550
beats per minute. The probe was positioned on the side on the abdomen to allow
visualization of the cross section of the right kidney. Peak systolic blood flow
velocity was measured using pulsed-wave Doppler in the renal artery just before
126
it enters the kidney, as described previously (Boesen et al., 2012). Doppler
measurements were than analyzed by a blinded observer.
4.2.3. Plasma and urine creatinine concentration
Blood was collected from each animal at the necropsy through a cardiac
puncture into EDTA coated tubes. Creatinine concentration was assessed in
samples collected from animals treated for 16 weeks. Immediately after
collection, plasma was isolated by centrifugation and stored at -80
o
C until
analysis. Urine samples were collected in the morning (6-8 am) of the day
preceding the necropsy. Creatinine concentration in the plasma and urine
samples was measured using a Mouse Creatinine Kit (Crystal Chem, Downers
Grove, IL) per manufacturer instructions.
4.2.4. Urine protein concentration
Protein concentration in urine samples was assessed using standard Bradford
assay after 16 weeks of treatment. BSA was used as a standard. The samples
were incubated with Bradford reagent (Bio-Rad, Hercules, CA) for 10 min at
room temperature and spectrophotometrically analyzed using a BioTek Synergy
H1 Hybrid plate reader (BioTek, Winooski, VT). Protein concentration was
normalized to urine creatinine levels.
127
4.2.5. Glomerular hypertrophy and mesangial expansion
After 12 and 16 weeks of treatment the kidneys were isolated, formalin-fixed,
paraffin-embedded and cut at 5µm. To assess glomerular hypertrophy and
mesangial expansion, the sections were stained using standard PAS method.
Twenty images of random cortical glomeruli were obtained at 40× magnification.
The images were analyzed in a blinded fashion using ImageJ (1.47v, NIH, USA).
Glomerular area was measured using a free-hand selection tool and staining was
assessed using color-deconvolution plugin and threshold function. Mesangial
expansion was expressed as percentage of glomerular area stained with PAS.
4.2.6. Immunohistochemistry for N-Tyr, phospho-eNOS and NOX-4
Kidney sections were treated using a standard HIER procedure in Antigen
Retrieval Citra Plus (Biogenex, San Ramon, CA, USA). The slides were then
incubated with: a) rabbit polyclonal antibody directed against nitrated tyrosine
residues (EMD Millipore, Billerica, MA) at 1:250 dilution, b) rabbit polyclonal
antibody to phospho-eNOS Ser1177 (GeneTex Inc., Irvine, CA) at 1:100 dilution,
c) rabbit polyclonal antibody to phospho-eNOS Thr495 (Bioss Inc. Woburn, MA)
at 1:100 dilution, and d) rabbit polyclonal antibody to NOX-4 (EMD Millipore,
Billerica, MA) at 1:200. After incubation with a proper biotinylated secondary
antibody, a streptavidin conjugated horseradish peroxidase was added. The
sections were then incubated in DAB for 2-3 minutes and counterstained with
Harris hematoxylin. Twenty random images of renal cortex at 40x were evaluated
128
for the extent of staining in a blinded fashion using ImageJ (1.47v, NIH, USA)
and expressed as percentage of area positively stained. Immunohistochemistry
was performed only in the kidneys isolated from mice treated for 16 weeks.
4.2.7. Gene expression
qRT-PCR was performed as described previously (Mordwinkin et al., 2012).
Briefly, RNA was isolated using Trizol reagent (Invitrogen by Thermo Fisher
Scientific, Waltham, MA) and reverse transcribed. Real- time PCR was
performed using SYBR green PCR Master Mix (Applied Biosystems by Life
Technologies, Thermo Fisher Scientific, Waltham, MA). Relative expression of
each of the genes of interest was evaluated using an ABI 7300 instrument
(Applied Biosystems by Life Technologies, Thermo Fisher Scientific). Abundance
of targeted mRNA was normalized against 29S mRNA. Gene expression
analysis was performed using tissues collected from animals treated for 4 and 12
weeks. To compare gene expression between these two time points, values
collected using tissues from animals treated for 12 weeks were normalized to
values obtained for heterozygous animals treated with saline for 4 weeks.
4.2.8. Statistical analysis
GraphPad Prism version 6.0c for Mac OS X (GraphPad Software, San Diego,
CA, USA) was used to analyze the data. One-way ANOVA followed by Dunnett’s
multiple comparisons test were used to compare data. The level of statistical
significance was set at 5%. Data are expressed as mean value ± SEM.
129
4.3. Results
4.3.1. Treatment with A(1-7) does not reduce hyperglycemia at any of the
time points
Diabetic animals from both treatment groups had increased blood glucose levels
at each time point compared to non-diabetic controls (Fig. 4.1). Treatment with
A(1-7) had no effect on the hyperglycemia in neither diabetic nor heterozygous
(healthy) animals.
4 weeks
8 weeks
12 weeks
16 weeks
0
100
200
300
400
500
blood glucose [mg/dL]
hzg A(1-7)
hzg saline
db/db A(1-7)
db/db saline
****
****
****
****
Fig. 4.1: Blood glucose levels over time. Diabetic animals from both treatment groups
were highly hyperglycemic at each assessed time point. A(1-7) did not have any effects
on blood glucose levels. (****p<0.0001 significantly increased compared to hzg saline);
calculated using two-way ANOVA; plotted as mean with SEM.

130
4.3.2. Treatment with A(1-7) has no effect on kidney weight.
Diabetic kidney disease is frequently associated with increased renal weight.
However, in this study we did not detect any significant differences in kidney
weights between any of the treatment groups, with the exception of enlarged
kidneys from db/db mice compared to non-diabetic controls at several time points
(Fig. 4.2).
4 weeks
8 weeks
12 weeks
16 weeks
8
10
12
14
16
normalized left kidney weight [a.u.]
hzg A(1-7)
hzg saline
db/db A(1-7)
db/db saline
*
*
4 weeks
8 weeks
12 weeks
16 weeks
8
10
12
14
16
normalized right kidney weight [a.u.]
*
*
A.
B.
Fig. 4.2: Kidney weights over time. Kidney weight were assessed at each necropsy
and normalized to tibia length. Weights of left (A) and right (B) kidneys were assessed
separately. Significant differences in kidney weights were only detected between db/db
mice from the control group and non-diabetics (hzg – heterozygous) at certain time
points. (*p<0.05 significantly increased compared to hzg saline); calculated using two-
way ANOVA; plotted as mean with SEM.
131
4.3.3. 16 weeks of A(1-7) administration reduces shear stress and
improves kidney function in diabetic mice
Blood flow velocity in the renal artery was measured using ultrasonography after
16 weeks of treatment. A(1-7) reduced blood flow velocity in renal arteries of
diabetic mice (Fig. 4.3A). This may contribute to decreased shear stress in the
kidneys and improved renal function. db/db mice also had decreased kidney
function as measured by creatinine levels in plasma and protein/creatinine ratio
in urine (Fig. 4.3B-C). Increased levels of creatinine in plasma suggest impaired
filtration function of the kidneys, whereas increased protein/creatinine ratio in the
urine implies structural changes in the glomeruli. Both of these parameters were
decreased after treatment with A(1-7). No significant differences were seen
hzg saline
hzg A(1-7)
db/db saline
db/db A(1-7)
0.0
0.2
0.4
0.6
0.8
1.0
creatinine concenration [mg/dL]
*
*
hzg saline
hzg A(1-7)
db/db saline
db/db A(1-7)
0.0
0.1
0.2
0.3
0.4
0.5
protein/creatinine ratio
**
*
hzg saline
hzg A(1-7)
db/db saline
db/db A(1-7)
0
50
100
150
200
peak blood flow velocity [mm/s]
**
*
A. B. C.
Fig. 4.3: Physiological kidney function in the animals treated for 16 weeks. Peak
systolic blood velocity was increased in renal arteries of diabetic animals, which might
indicate increased mechanical stress in the kidneys. Treatment with A(1-7) reduced
this measurement (A). Plasma creatinine and urine protein/creatinine ratio was
assessed after 16 weeks of treatment to determine kidney function. Both parameters
were increased in diabetic mice treated with saline and reduced after administration
of A(1-7) (B,C). Increased plasma creatinine concentration indicates impaired filtration
function, whereas elevated protein/creatinine ratio in the urine reflects structural
changes in the kidneys. (hzg – heterozygous; n=6 animals per group; *p<0.05;
**p<0.01); calculated using one-way ANOVA; plotted as mean with SEM.
132
between heterozygous (non-diabetic) groups, which is also true for all the other
parameters presented in this study.
4.3.4. Glomerular structure is improved after treatment with A(1-7)
Increased presence of growth factors, cytokines and oxidative stress contributes
to glomerular dysfunction in diabetic animals. Glomerular structure was assessed
by measuring glomerular hypertrophy and mesangial expansion after 12 and 16
weeks of treatment. Mesangial expansion is one of the highlights of renal
pathology in T2D. Mesangial cells produce excessive amount of extracellular
matrix, which reduces flexibility of the glomeruli and decreases surface area
available for filtration. Both, glomerular hypertrophy and mesangial expansion
were increased in kidneys from diabetic animals at both time points (Fig. 4.4A-B).
Size of the glomeruli in the diabetic animals treated with saline for 16 weeks was
slightly increased compared to 12 weeks of treatment. Glomerular area in the
kidneys from animals treated for 16 weeks was also somewhat higher in
heterozygous animals from both treatment groups than in the groups treated for
12 weeks. This might be due to aging. Administration of A(1-7) reduced
glomerular hypertrophy in diabetic animals at both time points (Fig. 4.4A-B). The
effects of treatment were more significant in the animals from the later time point.
Mesangial expansion was also increased in diabetic animals from the control
group at both time points (Fig. 4.4C-D). A(1-7) reduced glomerular remodeling in
db/db mice and the effects were similar after 12 and 16 weeks of treatment.
133

Fig. 4.4: Glomerular function in the animals treated for 12 or 16 weeks.
Glomerular health was assessed by measuring glomerular area (A-B) and mesangial
expansion (C-D) in animals treated for 12 (A,C) or 16 weeks (B,D). Mesangial
expansion is expressed as percentage of glomerular area stained for extracellular
matrix. Both glomerular hypertrophy and extent of fibrosis were increased in the
diabetic animals treated with saline at both time points. Glomerular dysfunction was
prevented by treatment with A(1-7). Representative images of glomeruli stained using
Periodic acid-Schiff from the animals treated for 16 weeks taken at 40x magnification
are shown in E. (hzg – heterozygous; n=6 animals per group; **p<0.01;
****p<0.0001); calculated using one-way ANOVA; plotted as mean with SEM.
hzg saline
hzg A(1-7)
db/db saline
db/db A(1-7)
0
1000
2000
3000
4000
5000
glomerular area [um2]
**
*
hzg saline
hzg A(1-7)
db/db saline
db/db A(1-7)
0
1000
2000
3000
4000
5000
glomerular area [um2]
****
**
hzg saline
hzg A(1-7)
db/db saline
db/db A(1-7)
0
5
10
15
20
% of glomerular area stained
****
****
hzg saline
hzg A(1-7)
db/db saline
db/db A(1-7)
0
5
10
15
20
% of glomerular area stained
****
****
12 weeks of treatment 16 weeks of treatment
A. B.
C. D.
saline A(1-7)
db/db hzg
E.
134
Improved glomerular health might be one of the factors contributing to enhanced
kidney function after treatment.
4.3.5. Oxidative stress damage in diabetic kidneys was decreased after
administration of A(1-7) for 12 or 16 weeks.
Oxidative stress can be caused by both hyperglycemia and activation of the
pathological arm of the RAS. NADPH oxidase, one of the main sources of
superoxide in diabetic kidney, is directly activated by Ang-II. We evaluated
oxidative stress-induced damage in the kidney sections using anti-N-Tyr directed
antibody. N-Tyr is formed when peroxynitrite reacts with tyrosine residues. This
irreversible modification of the proteins can result in severe damage and loss of
function. The extent of staining in the kidneys from diabetic mice treated with
placebo was increased compared to non-diabetic animals at both timepoints (Fig.
4.5A-B). Administration of A(1-7) significantly reduced presence of this marker,
which suggests decreased oxidative stress damage in the kidneys after 12 and
16 weeks of treatment. The extent of staining in diabetic mice from the control
groups was similar at both time points, however more N-Tyr was detected in the
diabetic group treated with A(1-7) for 16 weeks than for 12 weeks. This might be
due to prolonged exposure to the oxidative stress and reduced capacity of the
compensatory mechanisms such as superoxide dismutase.
135

hzg saline
hzg A(1-7)
db/db saline
db/db A(1-7)
0
5
10
15
% area stained for nitrotyrosine
****
***
hzg saline
hzg A(1-7)
db/db saline
db/db A(1-7)
0
5
10
15
% area stained for nitrotyrosine
****
***
saline A(1-7)
db/db hzg
12 weeks of treatment 16 weeks of treatment
A. B.
C.
Fig. 4.5: Oxidative stress damage in the kidneys from animals treated for 12 or
16 weeks. Damage due to oxidative stress was assessed using sections
immunostained for nitrotyrosine residues. Significantly more staining was observed in
the kidneys from diabetic animals treated with saline than in non-diabetic groups at
both time points, which suggests increased tissue damage due to oxidative stress (A-
B). 12 or 16 weeks of A(1-7) administration to db/db mice reduced oxidative stress
damage in the kidneys. Representative images of kidney cortex taken at 10x
magnification after 16 weeks of treatment are shown in C. (hzg – heterozygous; n=6
animals per group; ***p<0.001; ****p<0.0001); calculated using one-way ANOVA;
plotted as mean with SEM.
136
4.3.6. Administration of A(1-7) altered gene expression of eNOS and a
subunit of NADPH oxidase but did not have any effect on expression
of pro-inflammatory cytokines.
eNOS is one of the main sources of NO in the kidneys. N-Tyr is formed due to
increased levels of peroxynitrite, which is produced through reaction of NO with
superoxide (Fig. 4.6). We evaluated expression of two main enzymes that
produce these molecules – eNOS and p22-phox (subunit of NADPH oxidase).
Gene expression was performed on tissues collected from animals treated for
either 4 of 12 weeks. Even though we did not observe any changes in the
expression of the genes for these enzymes after 12 weeks of treatment (Fig.
4.7C-D), there was a significantly increased expression of both eNOS mRNA and
p22-phox mRNA in the kidneys of diabetic animals after 4 weeks of treatment
O
2
-
superoxide
ONOO
-
peroxynitrite
tyrosine
nitrotyrosine
NADPH
oxidase
Ang-II
eNOS
A(1-7)
NO

nitric oxide
Fig. 4.6: Formation of nitrotyrosine residues and involvement of eNOS and
NADPH oxidase. Ang-II is able to directly activate NADPH oxidase to produce
superoxide. A(1-7) is known to increase production of NO through activation of
eNOS. Superoxide and NO react with each other to form peroxynitrite – a potent
oxidant. Elevated levels of peroxynitrite cause nitration of proteins resulting in
formation of nitrotyrosine residues that change structure and function of proteins.
137
(Fig. 4.7A-B). Administration of A(1-7) reduced expression of the genes for both
of these proteins. Increased expression of eNOS and p22-phox genes in the
kidneys of diabetic animals might be associated with elevated levels of NO and
superoxide respectively. We did not observe any significant changes in the
expression of genes for proinflammatory cytokines such as TNF-α, IL-1β and IL-6
in either of time points, with the exception of decreased IL-1β mRNA in the
diabetic control group at age of 20 weeks (data not shown). Levels of IL-6 were
Fig. 4.7: Gene expression of eNOS and NADPH oxidase in the kidneys from
animals treated for 4 or 12 weeks. Gene expression of both eNOS and p22-phox
(subunit of NADPH oxidase) was increased in diabetic animals after 4 weeks of
treatment (animals were 12 weeks old) (A, B). Treatment with A(1-7) reduced
expression of both of these markers. No significant differences in gene expression of
eNOS and p22-phox were detected in animals treated for 12 weeks (20 weeks old).
(hzg – heterozygous; n=6 animals per group; *p<0.05; **p<0.01); calculated using
one-way ANOVA; plotted as mean with SEM.
hzg saline
hzg A(1-7)
db/db saline
db/db A-(1-7)
0
1
2
3
eNOS mRNA fold change
*
**
hzg saline
hzg A(1-7)
db/db saline
db/db A-(1-7)
0
1
2
3
p22-phox mRNA fold change
*
*
hzg saline
hzg A(1-7)
db/db saline
db/db A-(1-7)
0
1
2
3
eNOS mRNA fold change
hzg saline
hzg A(1-7)
db/db saline
db/db A-(1-7)
0
1
2
3
p22-phox mRNA fold change
eNOS p22-phox
4 weeks of treatment 12 weeks of treatment
A. B.
C. D.
138
below detection limits for animals treated for 4 weeks. Expression after 16 weeks
of treatment was not evaluated due to unlikely changes in the animals older than
12 weeks.
4.3.7. Expression of genes associated with oxidative stress was
increased with age.
Some studies suggest that expression of oxdiative stress markers increases with
age (Gomes et al., 2009). Expression of eNOS and p22-phox in the kidneys was
increased in animals from heterozygous and db/db groups treated with saline for
12 weeks compared to healthy controls treated with saline for 4 weeks (Fig. 4.8).
The expression of these gene markers was also increased in groups treated with
A(1-7) but it did not reach statistical significance.

Fig. 4.8: Effects of aging on gene expression of oxidative stress markers.
Expression of eNOS and p22-phox was normalized to expression in the kidneys of
heterozygous (hzg) animals treated with saline for 4 weeks. mRNA expression of both
markers was increased in hzg and db/db animals treated with saline for 12 weeks
compared to hzg mice treated with saline for 4 weeks. (*p<0.05 compared to hzg saline
4 weeks); calculated using one-way ANOVA; plotted as mean with SEM.
hzg saline
hzg saline
hzg A(1-7)
db/db saline
db/db A(1-7)
0
5
10
15
20
eNOS mRNA fold change
normalized to hzg saline at 4 weeks
* *
4 weeks 12 weeks
hzg saline
hzg saline
hzg A(1-7)
db/db saline
db/db A(1-7)
0
5
10
15
p22-phox mRNA fold change
normalized to hzg saline at 4 weeks
* *
4 weeks 12 weeks
A. B.
139
4.3.8. A(1-7) alters phosphorylation pattern of eNOS.
eNOS can be activated or deactivated through modification of the two main
phosphorylation sites. Phosphorylation of eNOS on Ser1177 and
dephosphorylation on Thr495 activates eNOS to produce NO. Phosphorylation at
Ser1177 is mainly mediated by activated Akt, while phosphorylation on Thr 495 is
Fig. 4.9: eNOS phosphorylation in the kidneys from animals treated for 16 weeks.
Levels of eNOS phosphorylated on Ser1177 (activating phosphorylation) and eNOS
phosphorylated on Thr495 (deactivating phosphorylation) were assessed using
immunohistochemistry in animas treated for 16 weeks. Representative images of kidney
cortex taken at 40x magnification are shown in A. Levels of both phosphorylation forms
were increased in db/db mice from the control group. The extent of phosphorylation on
Ser1177 was also increased in the diabetic animals treated with A(1-7) (B), whereas
levels of phosphorylation on Thr495 were decreased in this group (C). (hzg –
heterozygous; n=6 animals per group; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001);
calculated using one-way ANOVA; plotted as mean with SEM.

hzg saline
hzg A(1-7)
db/db saline
db/db A(1-7)
0.0
0.5
1.0
1.5
% area stained for
p-eNOS Ser 1177
*
***
hzg saline
hzg A(1-7)
db/db saline
db/db A(1-7)
0
5
10
15
% area stained for
p-eNOS Thr 495
****
**
B.
hzg saline hzg A(1-7) db/db A(1-7) db/db saline
p-eNOS Ser1177 p-eNOS Thr495
A.
B. C.
140
catalyzed by activated PKC (Chen et al., 2008). We evaluated effects of A(1-7)
administration on eNOS phosphorylation using immunohistochemistry in the
kidneys isolated from animals treated for 16 weeks, because the kidney disease
was most severe at this time point. This allowed to further investgate the
mechanisms of the amelioration of oxidative stress with the long-term treatment.
Phosphorylation on Ser1177 was increased in both db/db groups (Fig. 4.9B),
whereas phosphorylation on Thr495 was increased in diabetic animals treated
with saline and reduced after treatment with A(1-7) (Fig. 4.9C). This suggests
deactivation of some of the eNOS molecules in the kidneys of diabetic animals
treated with saline. In the kidneys of db/db mice administered A(1-7) most of the
eNOS molecules are active. This data implies that in the kidneys from both
diabetic groups, there is increased production of NO.
4.3.9. Administration of A(1-7) reduces levels of NOX-4
NOX-4 is the most prevalent form of NADPH oxidase in the kidneys and the main
source of superoxide (Gill and Wilcox, 2006). We evaluated levels of NOX-4 in
kidney sections collected from animals treated for 16 weeks. Expression of
NOX-4 was increased in the kidneys from diabetic animals (Fig. 4.10). A(1-7)
reduced the extent of staining for NOX-4 in db/db mice suggesting that treatment
reduced production of superoxide in the tissue resulting in decreased oxidative
stress damage in the kidneys.
141
4.4. Discussion and conclusions
Here, we show that long-term administration of A(1-7) is safe and effective in the
prevention of oxidative stress damage and renal dysfunction in db/db mice. It is
important to note that, so far, ACEi and ARBs even though able to slow down the
progression of diabetic nephropathy, have not been proven to prevent kidney
dysfunction in diabetic patients. As we have demonstrated previously, the
treatment regimen used in this chapter does not have any effects on blood
glucose levels or body weight in this mouse model (Papinska et al., 2016). Thus,
Fig. 4.10: Levels of NOX-4 in the kidneys from animals treated for 16 weeks.
Levels of NOX-4, a member of NADPH oxidase family, were assessed in the kidney
sections using immunohistochemistry. Representative images taken at 40x are
shown in A. The extent of staining for NOX-4 was increased in the diabetic animals
from the control group compared to non-diabetic mice. Treatment with A(1-7) reduced
levels of NOX-4 in the kidneys of diabetic mice (B). (hzg – heterozygous; n=6 animals
per group; ****p<0.0001); calculated using one-way ANOVA; plotted as mean with
SEM.
hzg saline
hzg A(1-7)
db/db saline
db/db A-(1-7)
0
5
10
15
20
% area stained for NOX-4
****
****
saline A(1-7)
db/db hzg
A.
B.
142
the effects of A(1-7) on kidney health occurred independently of blood glucose or
obesity control. Further, we have also previously demonstrated that 16 weeks of
treatment with A(1-7) reduced systemic inflammation (Papinska et al., 2016).
However, we did not observe any significant changes in the gene expression of
pro-inflammatory cytokines in the kidneys from animals treated for 4 and 12
weeks, with the exception of decreased IL-1β mRNA levels in diabetic control
group at age of 20 weeks (data not shown). Nonetheless, reduction of systemic
inflammation in animals treated for 16 weeks might have partially contributed to
decreased oxidative stress damage in the kidneys. We demonstrated that both
12 and 16 weeks of treatment with A(1-7) improved glomerular health in diabetic
animals. The effects of treatment on glomerular area and mesangial expansion
were similar at both of the assessed time points.
Kidney damage in diabetic patients and animal models is known to occur partially
because of the increased blood pressure and mechanistic stress in the glomeruli.
Our group has shown that this treatment regimen with A(1-7) does not have any
effect on systemic blood pressure in db/db mice (unpublished results). Lack of
assessment of blood pressure in this study is a limitation recognized by the
authors. However, as the half-life of A(1-7) when injected subcutaneously is
around 30 minutes, we anticipate that the effects of treatment on blood pressure
are transient and would not have a major effect on systemic hemodynamics.
143
Peroxynitrite is one of the most potent oxidants. Increased production of this
molecule leads to formation of N-Tyr and results in structural and function
changes in the proteins. Both NO and superoxide are needed to produce N-Tyr.
Increased phosphorylation of eNOS on Ser1177 in both diabetic groups suggests
enhanced production of NO in these animals. We believe that increased
phosphorylation on Thr495 in diabetic group given placebo is associated with a
defense mechanism that acts against the overproduction of ROS. In addition,
phosphorylation on Ser1177 is known to be primarily associated with activation of
Akt pathway, which is involved in insulin signaling and is known to act protective
in T2DM (Kobayashi et al., 2004; Mackenzie and Elliott, 2014). In contrast,
activation of PKC pathway, which is detrimental in diabetes (Koya and King,
1998), has been described as the main source of phosphorylation on Thr495.
Increased phosphorylation on Thr495 in db/db mice from placebo group can be
therefore associated with increased activation of PKC pathway. Further,
reduction of the level of phosphorylation at this amino acid suggests reduction in
PKC activity in diabetic animals after treatment with A(1-7).
In addition to enhanced activation of eNOS, kidneys from diabetic animals also
showed increased expression of NOX-4, the most predominant type of NADPH
oxidase - enzyme responsible for cytosolic production of superoxide anion - in
the kidney (Gill and Wilcox, 2006), in diabetic animals. NADPH oxidase was
initially discovered to be present in neutrophils and play a role in response to
pathogens by producing toxic amounts of ROS. In the kidneys this enzyme has
144
been also found in non-phagocytic cell types such as mesangial cells, proximal
tubules, vascular smooth muscle cells, endothelium and fibroblasts (Sedeek et
al., 2010). It is thought that the role of ROS produced by NADPH oxidase in the
kidneys is primarily to act as a secondary messenger. However, in pathological
states such as T2D, NADPH oxidase is known to produce excessive amounts of
ROS. It has been shown that this enzyme contributes to oxidative stress damage
in various renal pathologies including diabetic nephropathy (Forbes et al., 2008;
Thallas-Bonke et al., 2008). In addition, inhibition of NADPH oxidase with
apocynin reduced kidney damage and mesangial expansion in diabetic
nephropathy (Asaba et al., 2005; Thallas-Bonke et al., 2008). Overexpression of
both p22-phox and NOX-4 subunits has been previously shown to play a role in
diabetic nephropathy (Li and Shah, 2003; Matsunaga-Irie et al., 2004). In db/db
mice, NOX-4 has been found to be involved in molecular mechanisms underlying
fibrosis in the kidneys through increased TGF-β and fibronectin production
(Sedeek et al., 2010). NADPH oxidase can also be activated via phosphorylation
of PKC in diabetic kidneys (Thallas-Bonke et al., 2008). Even though intracellular
ROS may come from various sources, NOX-4 is thought to be the main source of
superoxide in diabetic kidneys (Gill and Wilcox, 2006; Hancock et al., 2001).
Because treatment with A(1-7) was able to decrease levels of this enzyme and
reduce tyrosine nitration, we hypothesize that this is one of the major pathways
targeted by our molecule that contributes to decreased oxidative stress damage
in the kidneys.
145
Aging is also associated with incerase in the oxidative stress. Enhanced gene
expression of both eNOS and p22-phox was observed in the kidneys from
animals treated with saline for 12 weeks when compared to heathy mice treated
with saline for 4 weeks. Interestingly, kidneys from both heterozygous and
diabetic groups treated with the vehicle had increased mRNA expression of
these oxidative stress markers. Difference between heterozygous and diabetic
groups treated with A(1-7) for 12 weeks did not reach stastical significance, even
though gene expression appears to be increased in these animals compared to
the 4 week time point. As the data is quite inconsistent, larger number of animals
should be used to draw proper conclusions. However, this preliminary data
suggests that A(1-7) might be effective in the amelioration of oxidative stress in
aging animals.
A(1-7) may represent a novel, safer treatment for diabetic nephropathy. In
contrast to ACEi and ARBs, A(1-7) activates protective arm of the RAS. A(1-7)
has been shown to decrease hypertension and reduce fibrosis, inflammation, and
oxidative stress in various tissues including kidney (Dilauro and Burns, 2009). In
fact A(1-7) has positive effects on kidney cells even in the absence of
hemodynamic factor (Gava et al., 2009; Su et al., 2006). Here we confirmed that
long-term administration of A(1-7) to a severe model of T2D results in prevention
of kidney damage and improved filtration function. One of the mechanisms
involved in this renoprotective action is amelioration of oxidative stress. We have
also shown that administration of A(1-7) to healthy mice has no effect on any of
146
the measured parameters, suggesting that this treatment is not only efficacious
but also safe. What is more, A(1-7) is currently undergoing several clinical trials
in oncology and hematopoiesis and was shown to be safe in patients (Pham et
al., 2013; Rodgers et al., 2006). This allows for the rapid translation of our
preclinical results into potential clinical evaluation.
4.5. Acknowledgements
Author would like to thank Isabela Galvez for her help with analysis of PAS
staining, Sofia Santos for assistance in gene expression assays and Christopher
Meeks for help with ultrasound measurements. Author would also like to thank
Dr. Sachin Jadhav, Maira Soto, Alick Tan, Lila Kim and Tamar Cohen-Amzaleg
for their help with animal handling and necropsies.
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5. Chapter 5: Discussion and future directions
5.1. In search for new treatments for T2D
Currently available treatments for T2D focus on the amelioration of
hyperglycemia. Medications such as metformin, thiazolidinediones (e.g.
pioglitazone) and gliptins (e.g. sitagliptin) are still the top therapies for patients
suffering from T2D. Even though glucose control may be effective in prevention
of some of the diabetic complications, clinical studies show that it does not
necessarily reduce the risk of CVD (Gerstein et al., 2008; Patel et al., 2008).
Targeting other symptoms of T2D, such as high cholesterol levels and
hypertension, has also become a part of standard diabetes care. As a result,
patients with T2D take a handful of medications every day. Just recently, it has
been recognized that ACEi and ARBs are effective not only in lowering blood
pressure but can also slow down progression of diabetic nephropathy. Even
though hypertension remains the main target of RAS modifying therapies, some
studies showed that inhibition of actions of Ang-II has protective effects in the
kidneys even in the absence of hemodynamic factor (Kagami et al., 1994).
Targeting pathological arm of the RAS has proven to be effective in amelioration
of diabetes-related renal and cardiovascular disease. However, as presented in
chapter 1, ACEi and ARBs can be associated with a variety of adverse events
and lose their effectiveness with time.
154
Data presented in this dissertation shows that targeting the protective arm of the
RAS might be a safer and more efficacious alternative to ACEi and ARBs. A(1-7),
through activation of Mas receptor, reduced diabetes-induced kidney and heart
disease in a mouse model of severe T2D. The action of A(1-7) seems to be
mediated though reduction of tissue remodeling – one of the hallmarks of
diabetic complications. In addition, A(1-7) targets some of the pathways that
have been recently recognized to be strongly involved in diabetes related
pathologies, such as oxidative stress and inflammation.
Research shows that A(1-7), in addition to its effects on oxidative stress and
inflammation, also acts to reduce obesity and hyperglycemia (Marcus et al.,
2013). In these studies, A(1-7) was administered though steady infusion using an
implantable mini-pump. However, constant prolonged administration of large
doses of A(1-7) can alter the expression patterns of the RAS components and
stimulate internalization of Mas receptors, diminishing their availability and
reducing effects of A(1-7) (Gironacci et al., 2011). We believe that A(1-7),
administered through subcutaneous injections, once daily, reduces this effect.
However, the treatment regimen presented in this dissertation did not affect the
blood glucose levels in diabetic animals. This proves that A(1-7) has positive
effects on renal and cardiovascular health even in the face of uncontrolled
hyperglycemia and obesity. Combination treatment with A(1-7) and a blood
glucose lowering agent might prove to be even more effective in amelioration of
diabetic complications than administration of A(1-7) alone.
155
Even though various medications are available on the market, there is still no
cure for T2D. We hypothesize that A(1-7) might become new first-line treatment
for diabetic complications because of its effectiveness and safety profile.
5.2. A(1-7) safety profile
Actions of A(1-7) were shown to be dependent on the availability of the Mas
receptors (Santos et al., 2003). Because Mas is overexpressed only in injured
tissues, A(1-7) has virtually no effects in healthy animals (Papinska et al., 2015;
2016). Even though A(1-7) is a short peptide (7 amino acids), it does not behave
like a traditional biological agent. A(1-7) can be chemically synthetized and is
commercially available. In addition, A(1-7) manufactured under good
manufacturing practices (GMP) is also commercially available. Because the
synthesis methods are already established, A(1-7) is readily available for testing
in clinical trials. Chemical synthesis also warrants higher purity and more
consistent product than synthesis using biological systems. Because A(1-7)
consists of 7 amino acids, it does not induce immune response in patients. A(1-7)
was safe and well tolerated at doses 2.5-100 µg/kg in patients with cancer
(Rodgers et al., 2006). In this treatment regimen, no dose limiting toxicities were
found. A(1-7) was also shown to cause no adverse drug-drug reactions in
combination with chemotherapy agents (Pham et al., 2013; Rodgers et al., 2006).
Preclinical studies performed in this laboratory have also shown an exceptional
safety profile of A(1-7).
156
5.3. Alternative formulations of A(1-7)
One of the major drawbacks in the development of A(1-7) as an anti-diabetic
treatment is the need for injections. Patients often refuse to switch to an
injectable medication, even if it is more effective than its orally available
alternative. However, diabetic patients are somewhat accustomed to giving
themselves injections, as many of them are taking injectable insulin.
Nonetheless, advantages of biologics over small molecule drugs are well
recognized. In fact, out of the top 20 best selling drugs, eight of them are
biologics. Biologics tend to be more specific and have less off-target side effects
than small molecule drugs.
Several of the newly developed biologics, including medications for T2D, are
administered subcutaneously using a preloaded pen that allows for easy
administration by patient. In further steps of A(1-7) development, delivery
solutions like this should be closely considered.
In addition, Santos group has recently developed an oral formulation of A(1-7).
A(1-7) in hydroxypropyl β-cyclodextrin, administered orally was shown to improve
insulin sensitivity and act cardioprotective in myocardial infarction in a rat model
(Marques et al., 2011; Santos et al., 2014). More studies need to be performed to
reassure that this formulation has effects comparable to injectable A(1-7). In
addition, FDA has recently indicated in one of the application reviews (application
number 50-763, Pharmacology Review) that hydrozypropyl β-cyclodextrin can be
157
associated with several toxicities. Nonetheless, cyclodextrin formulation of A(1-7)
might prove to be a suitable alternative to injections and may facilitate
introduction of this peptide to the market.
Other A(1-7) formulations have also been considered. Recently, a cyclic A(1-7)
(residues 4 and 7 linked with a thioether bridge) was shown to have improved
stability. Cyclic A(1-7) is capable of stimulating Mas receptor and it has a
cardioprotective effect in rats with myocardial infarction (Durik et al., 2013). In
addition, cyclic A(1-7) could also be delivered via pulmonary route (de Vries et
al., 2010).
As formulating A(1-7) into new delivery systems is quite challenging, other
strategies to improve translatability have been explored. NorLeu
3
-A(1-7) is an
analogue of A(1-7), where valine at position 3 is substituted for norleucine (an
isomer of leucine). NorLeu
3
-A(1-7) was shown to bind to and activate Mas
receptor. It’s physiological effects are similar to A(1-7). A topical gel formulation
was shown to accelerate wound healing (Rodgers et al., 2003). NorLeu
3
-A(1-7) is
also beneficial in treatment of diabetic foot ulcers and is currently undergoing
phase III clinical evaluation for this indication (Balingit et al., 2012)
Recent advances in mass spectrometry allowed for discovery of a previously
unknown angiotensin peptide – alamandine (Lautner et al., 2013). This peptide is
different from A(1-7) only in that aspartic acid in position 1 is substituted for
alanine. Alamandine was identified in human blood and its actions seem to be
158
similar to A(1-7). However, it binds to a distinct receptor called MrgD. Because of
its effects on cardiovascular system, alamandine might become a strong
competitor for A(1-7) (Lautner et al., 2013). Discovery of this new peptide
suggests that there is much more to explore in the RAS.
Another approach to investigate effects of Mas activation and to improve
translatability of the effects ascribed to A(1-7) is to develop a small molecule
agonist of Mas receptor. Small molecule drugs are easier and cheaper to
produce, do not trigger immune response and are more specific. The latter
characteristic might be considered either an advantage or a disadvantage.
Recent findings demonstrate structural and functional interaction of RAS
receptors, e.g. Mas and AT2 (Villela et al., 2015). The roles of these interactions
are not fully understood, however it is hypothesized that full effects of A(1-7)
might be dependent on activation of multiple receptors. Similarly, as we
demonstrated in chapter 2, some of the effects of A(1-7) can be blocked by
PD123319 – an AT2 antagonist. Recently, this inhibitor has also been shown to
block actions of MrgD (Lautner et al., 2013). This suggests that some of the
effects of A(1-7) might be mediated through binding to AT2 or MrgD. In the light
of these findings, improved specificity of a small molecule Mas agonist could be a
disadvantage.
159
5.4. Other applications of A(1-7)
In this dissertation, we showed that A(1-7) is effective in the amelioration of T2D-
induced complications such and nephropathy and CHF (Papinska et al., 2015;
2016). We also discussed positive effects of A(1-7) and its analogue NorLeu
3
-
A(1-7) on diabetic foot ulcers. However, A(1-7) can also be beneficial in
treatment of other diabetic complications.
T2D affects many systems. It is well known that kidney disease leads to
progression of cardiac dysfunction (Sarnak et al., 2003). Decreased heart
function can result in accumulation of fluid in the lung. Recent findings in this
laboratory show that patients with T2D are at increased risk of developing lung
infections (unpublished data). In addition to renal and cardiovascular function, we
have also investigated effects of A(1-7) on lung health in db/db mice treated for
16 weeks. Lungs from diabetic mice treated with A(1-7) showed reduced fibrosis,
congestion, oxidative stress damage and macrophage infiltration; all consistent
with our findings in other systems (Papinska et al., 2016). Effects of A(1-7) on
pancreatic health and diabetic neuropathy in db/db mice are currently under
investigation in this laboratory.
A(1-7) also has regenerative properties (Rodgers et al., 2016). Observation that
A(1–7) stimulates hematopoietic stem cells led to hypothesis that it can also
mobilize endothelial progenitor cells (EPCs). Many studies have shown that
administration of A(1–7) improves endothelial function (Beyer et al., 2013; Iusuf
160
et al., 2008; Xu et al., 2008). We also demonstrated that administration of A(1–7)
in young db/db mice increases numbers of circulating EPCs, enhances
mobilization of EPCs from bone marrow and improves vascularization of the
heart (Papinska et al., 2015). This suggests a potential of A(1-7) for treatment of
myocardial infarction and heart tissue regeneration. Recent study by Wang and
colleagues showed improved heart function and increased number of c-kit
positive cells in the hearts of treated mice, suggesting regenerative potential of
A(1–7) (Wang et al., 2010). The same study also demonstrated enhanced
proliferation of human CD34-positive cells and mononuclear cells in vitro after
treatment with A(1–7). In addition, in a recently published abstract, Qi et al.
reported that administration of cardiac progenitor cells overexpressing A(1–7) to
infarcted heart improved heart function and enhanced engraftment compared to
progenitor cells alone (Qi et al., 2014). Together, this data suggests that localized
increase in A(1–7) might contribute to enhanced heart repair and regeneration.
In addition, regenerative potential of A(1-7) could be used in other diseases.
A(1-7) through its anti-fibrotic properties combined with regenerative actions was
shown to improve muscle function in mdx mouse model of Duchenne muscular
dystrophy (Acuña et al., 2014). Potential of A(1-7) to stimulate regeneration of
hematopoietic cells was explored in cancer patients (Pham et al., 2013; Rodgers
et al., 2006). In these studies A(1-7) attenuated multi-lineage cytopenias resulting
from chemotherapy.
161
In summary, recent discoveries show that components of the RAS have a great
potential to become novel therapies for a battery of different disease states.
5.5. Concluding remarks
In this thesis we explored cardio- and reno- protective effects of A(1-7) in a
mouse model of severe T2D. We also further characterized development and
progression of heart and kidney disease in db/db mice. A(1-7) acted through
several important mechanisms. Some of the most important actions of A(1-7) in
these systems were reduced fibrosis, inflammation and oxidative stress. We also
identified a novel mechanisms of cardioprotective action of A(1-7), namely
enhancement of calcium signaling in the cardiomyocytes.
Even though the treatment had no effect on hyperglycemia and obesity, A(1-7)
was still able to ameliorate diabetes-induced heart and kidney dysfunction. This
brings new insight to future therapies for T2D. In addition to targeting blood
glucose and cholesterol levels, more emphasis should be put on reduction of
inflammation and oxidative stress.
Here we validated that A(1-7) is safe and efficacious in an animal model of T2D.
Its safety was also previously demonstrated in patients. A(1-7) has a good
translational potential and may soon become the first-line treatment for
complications associated with T2D.

162
5.6. Chapter 5 references
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Abstract (if available)

Abstract The goal of this dissertation is to evaluate the effects of administration of angiotensin-(1-7), a component of the renin-angiotensin system, on type 2 diabetes-related kidney and heart dysfunction. Increased activation of the pathological arm of the renin-angiotensin system, specifically via enhanced signaling though angiotensin-II and its receptor AT1, has been implicated in progression of diabetic heart and kidney diseases through increased inflammation, oxidative stress, fibrosis and tissue remodeling. Angiotensin-(1-7) is a short peptide that attenuates pathological effects of angiotensin-II. We hypothesize that treatment with angiotensin-(1-7) ameliorates diabetic complications through restoration of the balance between components of the renin-angiotensin system. We investigated reno- and cardio-protective effects of short-term (2 weeks) and long-term (16 weeks) treatment with angiotensin-(1-7) on kidney and heart function in a mouse model of severe type 2 diabetes (db/db). Angiotensin-(1-7) prevented renal and cardiac dysfunction. Several mechanisms of this protective action were identified, including reduction of fibrosis and tissue remodeling, decrease in systemic inflammation and immune cell infiltration, amelioration of oxidative stress damage, and enhancement of calcium signaling in the hearts. These effects were observed in animals with uncontrolled hyperglycemia and obesity. Short-term treatment studies allowed for identification of potential mechanisms and long-term administration studies further delineated actions of angiotensin-(1-7). Angiotensin-(1-7) is effective in the amelioration of kidney and heart dysfunction in a mouse model of severe type 2 diabetes. Data presented in this dissertation supports potential therapeutic benefits of angiotensin-(1-7) in clinical use.

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Creator Papinska, Anna Malgorzata (author)

Core Title Angiotensin (1-7): a novel treatment for diabetes-induced kidney and heart dysfunction

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Pharmaceutical Sciences

Publication Date 07/05/2016

Defense Date 05/06/2016

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

Tag angiotensin (1-7),diabetic heart disease,diabetic kidney disease,OAI-PMH Harvest,renin-angiotensin system,type 2 diabetes

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Language English

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Advisor Rodgers, Kathleen (committee chair), Cadenas, Enrique (committee member), Okamoto, Curtis (committee member)

Creator Email annapapinska@gmail.com,papinska@usc.edu

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