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Theses, Dissertations and Capstones
2012
The Use of Cerium Oxide and CurcuminNanoparticles as Therapeutic Agents for theTreatment of Ventricular Hypertrophy FollowingPulmonary Arterial HypertensionMadhukar Babu [email protected]
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Recommended CitationKolli, Madhukar Babu, "The Use of Cerium Oxide and Curcumin Nanoparticles as Therapeutic Agents for the Treatment ofVentricular Hypertrophy Following Pulmonary Arterial Hypertension" (2012). Theses, Dissertations and Capstones. Paper 353.
THE USE OF CERIUM OXIDE AND CURCUMIN NANOPARTICLES AS THERAPEUTIC AGENTS FOR THE TREATMENT OF
VENTRICULAR HYPERTROPHY FOLLOWING PULMONARY ARTERIAL HYPERTENSION
A Dissertation submitted to the Graduate College of
Marshall University
In partial fulfillment of
the requirements for the degree of
Doctor of Philosophy
in Biomedical Sciences
By
Madhukar Babu Kolli, B.V.Sc&A.H, M.S.
Approved by
Dr. Eric R. Blough, Ph.D., Committee Chairperson
Dr. Monica A. Valentovic, Ph.D.
Dr. Todd L. Green, Ph.D.
Dr. Nalini Santanam, Ph.D.
Dr. Robert O. Harris, Ph.D.
Department of Pharmacology, Physiology and Toxicology
Joan C. Edwards School of Medicine,
Marshall University
Aug, 2012
ii
ACKNOWLEDGMENTS
I would like to express my deepest gratitude to my advisor, Dr. Eric R. Blough, for
his guidance, motivation and support during my research project. Dr. Blough’s support
has been invaluable in the successful completion of this dissertation. Dr. Blough was
always there to help me with the research project selection, design and the successful
completion of the project while helping with the troubleshooting of the experiments. Dr.
Blough always gave me confidence and support at each and every phase of my career
at Marshall University and without his support and motivation this journey wouldn’t have
been possible. I’m very thankful to Dr. Blough, for his invaluable support and motivation
throughout the journey of my graduate education at Marshall University.
I would like to convey my sincere thanks to my other committee members, Drs.
Monica Valentovic, Todd Green, Nalini Santanam and Robert Harris for their valuable
suggestions in fulfilling this research project. I am very thankful to my fellow graduate
students, Radhakrishna Para and Siva Nalabotu, for their valuable support in
conducting animal studies and giving me a helpful hand in sacrificing the research study
animals, helping me to complete the project successfully. I would like to thank the
division directors at the Center for Diagnostic Nanosystems, Kevin M. Rice, Miazong
Wu and Arun Kumar, for providing me equipment and supplies needed for this project. I
would also like to thank the members of Dr. Blough’s lab, Sunil Kakarla, Anjiah Katta,
Sriramprasad Mupparaju, Anil Gutta, Satyanarayana Paturi, Sarath Meduru,
Sudarsanam Kundla, Ravi Arvapalli, Gadde Murali, Srinivasarao Thulluri, Jacqueline
Fannin, Hari ddagarla, Nandini Manne, Selvaraj Vellaisamy, Wang Cuifen, Geeta
Nandyala, Sravanthi Bodapati, Hideyo Takatsuki, and Brent Kidd for their friendly
iii
support and cooperation in research. I would like to extend my thanks to Lucy Dornon
for helping me with the echocardiography recordings.
It is the most apt situation to thank my parents, Kolli VenkataSwamy Chowdary
and Kolli Jayamma, who have been an invaluable source of emotional, moral and
financial support throughout my career, and this dissertation wouldn’t have existed
without them. I would like to thank my elder sister’s family, Radhika and Narayan
Konanki, sweet niece Yukti, nephew ChetanSai and my younger sister’s family,
Sreelakshmi and Mahesh Amilineni and nephew Aarush for their love and support. I
would like to extend my thanks to my father-in-law Asundi Venkatesulu and mother-in-
law Asundi Padma for their helpful support. I would like to thank my friends and relatives
Uday Vunnam, Guru Vemula, Pramod Yelavarthi, Anjan Reddy G.B, SubbaNaidu
Darapaneni, VenkatBaineni, Murali Edara and Praveen Yadlapalli for their continuous
moral support. I would like to thank Dr. Kristen King, Dr. Kelly Pinkston, Dr. Mike Dyer,
and Dr. Chad Brown for their help in achieving my veterinary license.
These acknowledgments would be incomplete without recognizing the support
from my wife Soujanya Kolli and my beloved son VenkatSreekar Kolli. It is their love and
encouragement that has always inspired me to do my best in this research project.
Finally I would like to thank Dr. Blough and Marshall University BMS Graduate program
for providing me financial support for this project.
iv
DEDICATION
This dissertation is dedicated to my parents, my wife and to
my son for their continuous support in this journey.
v
TABLE OF CONTENTS
ACKNOWLEDGMENTS ...................................................................................................ii
DEDICATION ..................................................................................................................iv
LIST OF FIGURES ......................................................................................................... xii
LIST OF ABBREVIATIONS ............................................................................................xv
ABSTRACT .................................................................................................................. xvii
CHAPTER I ..................................................................................................................... 1
INTRODUCTION ......................................................................................................... 1
SPECIFIC AIMS ........................................................................................................... 6
Specific aim I: ....................................................................................................... 7
Specific aim II: ...................................................................................................... 7
CHAPTER II .................................................................................................................... 8
REVIEW OF LITERATURE .......................................................................................... 8
2.1 Pulmonary arterial hypertension (PAH) .................................................................. 8
2.2 Epidemiology ....................................................................................................... 11
2.3 Right ventricular hypertrophy ............................................................................... 13
2.4 Animal models ..................................................................................................... 14
2.5 Diagnosis ............................................................................................................. 15
2.6 Current strategies for treatment of PAH ............................................................... 17
vi
2.7 Introduction to Nanotechnology ........................................................................... 19
2.8 Cerium oxide nanoparticles .................................................................................. 21
2.9 Curcumin .............................................................................................................. 25
3.0 Summary .............................................................................................................. 31
CHAPTER III ................................................................................................................. 33
PAPER 1 .................................................................................................................... 33
Cerium oxide nanoparticles attenuate monocrotaline-induced right ventricular
hypertrophy following pulmonary arterial hypertension ................................. 34
Abstract ...................................................................................................................... 35
Introduction ......................................................................................................... 37
Materials and Methods ............................................................................................... 39
Animal model and experimental design .............................................................. 39
CeO2 nanoparticle size analysis by Dynamic Light Scattering (DLS) ................. 40
CeO2 nanoparticle analysis using atomic force microscopy (AFM) .................... 40
Echocardiography ............................................................................................... 40
Tissue processing and immunoblotting .............................................................. 41
OxyBlot analysis ................................................................................................. 43
Histological analysis ........................................................................................... 44
a. Dystrophin staining .................................................................................... 44
b. Picrosirius red staining............................................................................... 44
vii
c. Dihydroethidium staining ............................................................................... 45
d. TUNEL staining ......................................................................................... 45
Serum protein immune assays ........................................................................... 46
Data analysis ...................................................................................................... 47
Results ....................................................................................................................... 48
Characterization of CeO2 nanoparticles. ............................................................. 48
CeO2 nanoparticles treatment decreases right ventricular tissue weight ............ 48
CeO2 nanoparticles treatment decreases MCT-induced changes in pulmonary
arterial pressure and cardiac remodeling. ..................................................... 48
CeO2 nanoparticles treatment decreases MCT-induced increases in oxidative
stress signaling ............................................................................................. 50
CeO2 nanoparticles treatment attenuates MCT-induced increases in AMPK
alpha, ERK1/2 and JNK phosphorylation ...................................................... 51
CeO2 nanoparticles treatment attenuates MCT-induced increases in the
expression of NF-kB-p-50 ............................................................................. 52
Effects of CeO2 nanoparticles treatment on serum protein biomarkers .............. 52
CeO2 nanoparticles treatment decreases MCT-induced cardiomyocytes apoptotic
signaling ........................................................................................................ 53
Discussion .......................................................................................................... 54
Effect of CeO2 nanoparticle administration on monocrotaline-induced pulmonary
hypertension and cardiac remodeling ........................................................... 54
viii
CeO2 nanoparticle administration diminishes indices of oxidative stress during
pulmonary arterial hypertension .................................................................... 56
CeO2 nanoparticle administration diminishes monocrotaline-induced increases in
apoptosis ....................................................................................................... 58
PAPER 3 ....................................................................................................................... 75
Curcumin nanoparticles attenuate cardiac remodeling due to pulmonary arterial
hypertension .................................................................................................. 76
Abstract ...................................................................................................................... 77
Introduction ................................................................................................................ 78
Materials and Methods ............................................................................................... 80
PAH model and experimental design ................................................................. 80
Preparation of curcumin nanoparticles ............................................................... 80
Scanning Electron Microscopy ........................................................................... 81
Atomic Force Microscopy ................................................................................... 81
Echocardiography ............................................................................................... 82
Tissue processing and immunoblotting .............................................................. 82
Histology ............................................................................................................. 84
Determination of cytokine levels in serum by ELISA method ............................. 84
Determination of cardiac TNF-α and IL-1βmRNA levels ..................................... 85
Data analysis ...................................................................................................... 86
ix
Results ....................................................................................................................... 87
Curcumin NP characterization ............................................................................ 87
Curcumin NP treatment decreases PAH induced right ventricular tissue weight 87
Curcumin NP treatment decreases MCT-induced changes in pulmonary arterial
pressure and cardiac remodeling .................................................................. 87
Curcumin nanoparticle treatment decreases serum TNF-α levels ...................... 88
Curcumin nanoparticle treatment decreases AMPK alpha phosphorylation but not
MAPK or apoptotic signaling ......................................................................... 89
Curcumin nanoparticle treatment decreases MCT-induced increases in oxidative
stress signaling ............................................................................................. 89
Discussion.................................................................................................................. 90
CHAPTER IV ............................................................................................................... 102
GENERAL DISCUSSION......................................................................................... 102
Effects of CeO2 nanoparticle administration on monocrotaline-induced RV
hypertrophy in Sprague-Dawley rats ........................................................... 103
Effects of curcumin nanoparticle administration on monocrotaline-induced right
ventricular hypertrophy in Sprague Dawley rats .......................................... 104
SUMMARY ............................................................................................................... 105
FUTURE DIRECTIONS ........................................................................................... 109
References .................................................................................................................. 112
Appendix ..................................................................................................................... 124
x
Letter from Institutional Research Board .................................................................. 124
Curriculum Vitae .......................................................................................................... 125
xi
LIST OF TABLES
Table 2-1. The updated clinical classification of pulmonary hypertension from Dana
point, 2008. .............................................................................................................. 10
Table 2-2. World Health Organization Functional Class of PAH. .................................. 11
Table 3-1. Cerium oxide nanoparticle treatment attenuates monocrotaline-induced
increases in right ventricular remodeling. ................................................................. 59
Table 3-2. Body and organ weight for control, MCT only and MCT + Cur NP rats
collected 4 weeks after injection of MCT (60mg/kg). ................................................ 94
xii
LIST OF FIGURES
Figure 2-1.The illustration shows visual examples of the size and the scale of
nanotechnology........................................................................................................ 20
Figure 2-2.The fluorite structure of cerium oxide. .......................................................... 22
Figure 2-3. Curcuma longa plant. Inserts show the flower (a.), rhizome (b.) and turmeric
powder(c.). ............................................................................................................... 26
Figure 2-4.Structure of curcumin (diferuloylmethane) ................................................... 27
Figure 2-5.Molecular targets of curcumin. ..................................................................... 28
Figure 3-1.Characterization of CeO2 nanoparticles size. .............................................. 60
Figure 3-2.CeO2 nanoparticle treatment improves pulmonary flow. .............................. 61
Figure 3-3.CeO2 nanoparticle administration diminishes pulmonary artery remodeling. 62
Figure 3-4.CeO2 nanoparticle treatment attenuates MCT-induced increases in right
ventricle wall thickness. ........................................................................................... 63
Figure 3-5.CeO2 nanoparticle treatment attenuates MCT-induced cardiac remodeling. 64
Figure 3-6.PAH associated changes in cardiac function in the MCT only animals. ....... 65
Figure 3-7.CeO2 nanoparticle administration attenuates monocrotaline-induced
increases in cardiomyocyte cross sectional area and cardiac fibrosis. .................... 66
Figure 3-8.MCT induced increases in RV fibronectin and MHC-beta are attenuated with
cerium oxide nanoparticle treatment. ....................................................................... 67
Figure 3-9.CeO2 nanoparticle treatment decreases the incidence of superoxide levels in
MCT-induced RV hypertrophy. ................................................................................ 68
Figure 3-10.Cerium oxide nanoparticle treatment decreases MCT-induced increases in
protein nitration and carbonylation. .......................................................................... 69
xiii
Figure 3-11.Cerium oxide nanoparticle treatment decreases the MCT-induced increases
in AMPK-alpha, ERK1/2 and JNK phosphorylation. ................................................. 70
Figure 3-12. Cerium oxide nanoparticle treatment decreases the MCT induced oxidative
stress associated phosphorylation of HSP-27 and NF-kB p50. ............................... 71
Figure 3-13. Alterations in serum protein biomarkers with MCT and MCT + CeO2
nanoparticle treatment. ............................................................................................ 72
Figure 3-14.CeO2 nanoparticles treatment decreases the incidence of TUNEL positive
nuclei in MCT induced RV hypertrophy. ................................................................... 73
Figure 3-15.Cerium oxide nanoparticle treatment is associated with diminished pro-
apoptotic signaling. .................................................................................................. 74
Figure 3-16.Optical characterization of curcumin nanoparticles. ................................... 95
Figure 3-17.Curcumin nanoparticle treatment improves MCT-induced decreases in
pulmonary artery flow. .............................................................................................. 96
Figure 3-18.Curcumin nanoparticle treatment attenuates PAH induced increases in RV
anterior free wall thickness. ..................................................................................... 97
Figure 3-19.Curcumin nanoparticle treatment decreases PAH-induced cardiac
remodeling. .............................................................................................................. 98
Figure 3-20. Curcumin nanoparticle treatment diminished MCT-induced increases in
inflammatory cytokines. ........................................................................................... 99
Figure 3-21. Curcumin nanoparticle treatment alters AMPK-α activation but does not
change MAPK or apoptotic signaling. .................................................................... 100
Figure 3-22.Curcumin nanoparticle treatment attenuates PAH-associated increases in
heat shock protein and protein nitrosylation. .......................................................... 101
xiv
Figure 4-1. Possible role of CeO2 nanoparticles in attenuation of MCT-induced RV
hypertrophy following pulmonary arterial hypertension. ......................................... 107
Figure 4-2. Possible role of Cur NP in cardiac remodeling following pulmonary arterial
hypertension. ......................................................................................................... 108
xv
LIST OF ABBREVIATIONS
AALAC American Association of Laboratory Animal Care
ALK1 Activin receptor like kinase type
AMP Adenosine monophosphate
AMPK Adenosine monophosphate activated kinase
ANOVA One-way analysis of variance
ATP Adenosine triphosphate
Bax Bcl-2-associated X protein
Bcl2 B-cell lymphoma 2
BMPR2 Bone morphogenetic protein receptor 2
BSA Bovine serum albumin
CeO2 Cerium oxide
Cur NP Curcumin nanoparticles
ECL Enhanced chemiluminescence
ELISA Enzyme-linked immunosorbent assay
ERK Extracellular signal-regulated kinase
GAPDH Glyceraldehyde 3-phosphate dehydrogenase
HSP Heat shock protein
i.p. Intra peritoneal
IL-1 Interleukin-1
JNK Jun-N-terminal kinase
MAPK Mitogen activated protein kinase
MCT Monocrotaline
xvi
MHC Myosin heavy chain
NF-kB Nuclear factor kappa-light-chain-enhancer of
activated B cells
NT Nitrotyrosine
PAH Pulmonary arterial hypertension
PBS Phosphate buffered saline
PBST Phosphate buffered saline with 0.5% Tween
PDE-5 Phosphodiesterase
PLAX Parasternal long-axis
PSAX Parasternal short-axis
PVR Pulmonary vascular resistance
ROS Reactive oxygen species
s.c. Subcutaneous
TBS Tris buffered saline
TBST Tris buffered saline with 0.5% Tween
TNF-α Tumor necrosis factor-alpha
TUNEL TdT-mediated dUTP nick end labeling
xvii
ABSTRACT
THE USE OF CERIUM OXIDE AND CURCUMIN NANOPARTICLES AS THERAPEUTIC AGENTS FOR THE TREATMENT OF
VENTRICULAR HYPERTROPHY FOLLOWING PULMONARY ARTERIAL HYPERTENSION
Pulmonary arterial hypertension (PAH) is a progressive and fatal disease
characterized by inflammation, increased pulmonary vascular resistance, right
ventricular failure and premature death. Monocrotaline (MCT) has been used to induce
PAH in laboratory rats. Previous in vitro and in vivo work suggested that cerium oxide
(CeO2)-and curcumin nanoparticles exhibit anti-inflammatory activity; however, it is
unknown if these materials are effective for the treatment of PAH induced cardiac
hypertrophy.
To determine the efficacy of CeO2 nanoparticle treatment in preventing MCT-
induced RV hypertrophy, male Sprague Dawley rats were divided into one of three
groups (control, MCT, or MCT + CeO2 nanoparticle, n=6/group). MCT was administered
at a dosage of 60mg/kg subcutaneously. CeO2 nanoparticle treated animals received
0.1mg/kg via tail vein injection twice a week for two weeks. Echocardiography was
performed before and at the completion of the study. CeO2 nanoparticles treatment
attenuated MCT-induced increases in RV mass, cardiomyocyte cross sectional area,
and diminished cardiac oxidative stress levels. These changes in cardiac structure and
oxidative stress were accompanied by decreased mitogen activated protein kinase
phosphorylation, diminished heat shock proteins expression, improvements in the
Bax/Bcl-2 ratio, and diminished caspase-3 activation.
xviii
To investigate the efficacy of curcumin nanoparticle (Cur NP) administration in
preventing PAH-induced cardiac remodeling after pulmonary arterial hypertension, rats
were divided into one of three groups (control, MCT, or MCT + Cur NP treatment,
n=6/group). MCT was injected at a dosage of 60mg/kg subcutaneously. Nanoparticle
treated animals received Cur NP (50mg/kg i.p.) for seven days. Cur NP treatment
diminished MCT-induced increases in pulmonary arterial pressures, RV thickness and
myocardial fibrosis. These changes in cardiac structure were accompanied by down
regulation of myosin heavy chain (MCH-β) and fibronectin protein. Cur NP treatment
also reduced expression of tissue tumor necrosis factor (TNF-α) and interleukin-1β (IL-
1β) mRNA levels, decreased heat shock protein expression and reduced the amount of
protein nitrosylation.
Taken together, the results of these studies suggest that administration of
CeO2/curcumin nanoparticles may attenuate the degree of cardiac remodeling typically
observed following the development of pulmonary arterial hypertension in the laboratory
rat. Additional studies to investigate the mechanism(s) underlying these changes may
be warranted. (WC: 349)
1
CHAPTER I
INTRODUCTION
Pulmonary arterial hypertension (PAH) is an increase in mean pulmonary arterial
pressure >25 mm Hg at rest, or >30 mm Hg during exertion. PAH is characterized by
vascular proliferation and remodeling of the small pulmonary arteries, which results in a
progressive increase in pulmonary vascular resistance. The increased resistance can
cause pressure overload and lead to right ventricular (RV) hypertrophy, which if allowed
to proceed unchecked can terminate in heart failure and death [1]. The prognosis of
patients with PAH remains poor, with approximately 10%–15% mortality within 1 year of
the diagnosis. The median life expectancy from the time of diagnosis in patients with
idiopathic PAH without targeted treatments is 2.8 years, with 1-year, 3-year, and 5-year
survival rates of 68%, 48%, and 34%, respectively [2]. As of yet, a cure for PAH has not
yet been found. Limitations in evaluating treatment approaches for PAH include its
rarity, the inclusion of small number of patients in clinical trials, and issues regarding the
use of placebo controlled trials in a disease with high mortality if left untreated [3].
The current treatment options for PAH include prostacyclin and prostacyclin
analogues (e.g., Epoprostenol, Treprostinil, Iloprost), Endothelin receptor antagonists
(e.g., Bosentan, Ambrisentan) and phosphodiesterase (PDE-5) inhibitors (e.g.,
Sildenafil, Tadalafil, Vardenafil), as well as traditional background therapy with diuretics
(e.g., Furosemide), calcium-channel blockers (e.g., Dilitiazem, Nifedipine) and
anticoagulation agents (e.g., warfarin) [4]. Even with modern medical therapy, most
2
patients experience progression of their disease and many are referred for lung
transplantation [5].
Increases in oxidative stress due to excessive levels of reactive oxygen species
(ROS), e.g., superoxide anion (•O2-), hydroxyl radical (•OH), and hydrogen peroxide
(H2O2) have been implicated in the pulmonary and cardiac dysfunction [6]. It is well
known that ROS may damage tissues either by direct oxidation of key biological
molecules (i.e., lipid peroxidation, DNA damage) or by inducing the activation of
transcription factors, e.g., nuclear factor-kB (NF-kB) leading to apoptosis [7]. Recent
studies have shown that ROS may be produced in the lung tissue of patients with
severe PAH as a consequence of tissue hypoxia or through the activation of
inflammatory mediators [8]. Indeed, inflammation has been shown to play a significant
role in PAH associated with connective tissue diseases and HIV infection through the
activation of inflammatory cascades and increased production of inflammatory cytokines
(TNF-α,IL-1β,IL-6) [9]. It is thought that excessive oxidative stress and increased tissue
levels of inflammatory mediators can damage small pulmonary arterioles, which can
result in an increase in pressure overload and right heart failure [10].
Monocrotaline (MCT) is a toxic pyrrolizidine alkaloid present in the plant
Crotalaria spectabilis that can cause injury to the vasculature of the lungs and
pulmonary arterial hypertension in animals [11]. Lung injury following MCT exposure is
thought to occur after MCT processing in the liver by cytochrome P450
monooxygenases and the conversion of MCT to pyrrolic metabolites, which travel via
3
the circulation to the lung [12]. In Sprague Dawley rats a single dose of 60 mg/kg MCT
induces a rapid intrapulmonary inflammatory response, which increases inflammatory
monocytes in the adventitia of pulmonary resistance arterioles within 8-16 h after
injection [13]. Muscularization of nonmuscular arterioles leading to increased medial
layer thickness is detectable as early as 3 and 7 days post injection and reaches
significance by 10 and 14 days, resulting in increased pulmonary vascular resistance
and pulmonary arterial hypertension. RV hypertrophy is apparent by 21 days and
becomes progressively more severe if left untreated [14]. Whether strategies designed
to diminish ROS levels and inflammatory mediators are efficacious in improving MCT
induced PAH and RV function are not well understood.
Cerium is a rare earth element of the lanthanide series, having a fluorite lattice
structure in which the unit cell, consisting of cerium atoms arranged face-centered with
oxygen atoms, are located within the cell in the tetrahedral interstices. Most of the rare
earth elements exist in trivalent state (+3), but the cerium atom can exist in either Ce+3
(fully reduced) or +4 (fully oxidized) state [15]. Due to its redox capacity, cerium oxide
(CeO2) is an excellent oxygen buffer causing simultaneous oxidation of hydrocarbons
as well as the reduction of oxides of nitrogen, thus reducing the emissions from the
diesel exhaust as fuel additive [16]. CeO2 can exhibit oxygen vacancies or defects in
lattice structure through the loss of oxygen or its electrons. These defects in CeO2
structure are dynamic and can be altered by changes in temperature, the presence of
ions, or variations in the partial pressure of oxygen [17]. Recent work has demonstrated
that decreases in CeO2 nanoparticle size is associated with the formation of oxygen
4
vacancies [18] and increased catalytic activity [19]. Recently, CeO2 nanoparticles have
been used to modulate oxidative stress in biological systems by acting as free radical
scavengers [20]. In vitro studies have demonstrated that CeO2 nanoparticles are able to
rescue HT22 cells (a hippocampus neuronal cell line) from oxidative stress-induced cell
death [21] and protect normal human breast cells from radiation-induced apoptotic cell
death [22]. In vivo studies have shown that CeO2 nanoparticles treatment can attenuate
progressive cardiac dysfunction and remodeling by inhibition of myocardial oxidative
stress, endoplasmic reticulum stress and inflammation [23]. Whether treatment with
CeO2 nanoparticles can help to prevent cardiac dysfunction following the development
of PAH is currently unclear.
Curcumin is a hydrophobic polyphenol compound extracted from turmeric, the
powdered rhizome of Curcuma longa [24]. Turmeric has been used in Ayurvedic
medicine for centuries to treat inflammatory diseases [25]. Curcumin has been shown to
exhibit anti-oxidant [26], anti-inflammatory [27], anti-microbial, and anti-carcinogenic
activities [28]. The anti-inflammatory activity of curcumin is associated with inhibition of
cyclooxygenase [29] which can block the activation of the NF-kB and activator protein-
1(AP-1) transcription factors [30]. Studies have shown that blockade of NF-kB may
contribute to curcumin dependent-inhibition of the TNF-α, IL-1β, and IL-6 inflammatory
cytokines [31]. Furthermore, curcumin significantly inhibiting the generation of ROS and
nitride radical generation by activated macrophages [32]. The free radical scavenger
activity of curcumin has also been shown to protect against cyclophosphamide and
parquat induced lung injury [33]. It is thought that curcumin treatment is associated with
5
decreased•O2- levels and lipid peroxidases activity. In addition, curcumin exposure has
also been shown to increase the amount antioxidant enzymes, e.g. superoxide
dismutase and catalase, in the heart [34]. Finally, recent work has demonstrated that
curcumin can inhibit the hypertrophy of cultured cardiomyocytes and prevent the onset
of heart failure in rat models of hypertensive heart disease and myocardial infarction
[35].
To date, no studies in either animals or humans have discovered any toxicity
associated with the use of curcumin even at very high doses (500-8000 mg/day for 3
months) [36]. In spite of its therapeutic potential, the utilization of curcumin for the
treatment of various diseases has been hindered due to its fast metabolism and poor
water solubility [37, 38]. Efforts to circumvent these problems include the development
of curcumin nano-formulations using liposomes, polymer nanoparticles, and other
synthetic materials [39]. Recent work has shown that curcumin nano-emulsions
improved oral bioavailability and therapeutic efficacy in targeted cancer therapy [40, 41].
Other work has shown that liposome-encapsulated curcumin is more effective than free
curcumin in suppression of pancreatic carcinoma [42]. Recent studies have also
reported that nano curcumin can inhibit NF-kB activation and down regulate the
expression of the pro-inflammatory cytokines TNF-α, IL-6, and IL-8 [43]. Whether
treatment with curcumin nanoparticles can help to prevent cardiac dysfunction following
the development of PAH is currently unclear.
6
SPECIFIC AIMS
Right ventricular failure is the leading cause of death in patients with PAH [44].
PAH is a progressive, fatal disease characterized by an increased pulmonary vascular
resistance that causes pressure overload of the RV, leading to RV hypertrophy and
premature death [45]. Monocrotaline (MCT) is a pyrrizolidine alkaloid that causes
increases in oxidative stress, inflammation and fibrosis of pulmonary vasculature which
can lead to pulmonary arterial hypertension and RV hypertrophy in laboratory rats [46].
The objective of this study is to develop new strategies for the treatment of
pulmonary arterial hypertension and the resultant RV hypertrophy. Recent studies have
shown that CeO2 nanoparticles exhibit anti-oxidant properties that may be useful for the
treatment of chronic inflammation [47]. CeO2 nanoparticles have shown to increase the
life span of neuronal cell lines [20], and to protect cultured cells against the damaging
effects of radiation [19]. Curcumin has demonstrated anti-oxidant and anti-inflammatory
properties suggesting to some that this molecule may be useful to treat pulmonary and
cardiovascular diseases [48]. In vitro, curcumin nanoparticles (Cur NP) have been
shown to block NF-kB activation and down regulate the expression of the pro-
inflammatory cytokines TNF-α, IL-6, and IL-8 [43].
The central hypothesis of this research is that CeO2 and curcumin nanoparticle
administration will be associated with decreases in inflammatory oxidative stress,
diminished pulmonary arterial hypertension and an attenuation of right ventricular
hypertrophy following animal exposure to MCT.
7
To test our central hypothesis and accomplish the objective of this study two specific
aims will be pursued:
Specific aim I: To investigate if CeO2 nanoparticle administration is able to attenuate
monocrotaline-induced pulmonary arterial hypertension and right ventricular
hypertrophy in Sprague Dawley rats.
Specific aim II: To investigate if systemic administration of Cur NP is able to diminish
monocrotaline-induced pulmonary arterial hypertension and right ventricular
hypertrophy in Sprague Dawley rats.
8
CHAPTER II
REVIEW OF LITERATURE
The following chapter presents a review of the current literature pertinent to the
present study. Specifically, the following areas will be addressed: pulmonary arterial
hypertension, RV hypertrophy, animal models of PAH, diagnosis of PAH, current
strategies for treatment of PAH, introduction to nanotechnology and cerium oxide
(CeO2) nanoparticles, biological effects of CeO2 nanoparticles, toxic effects of CeO2
nanoparticles, curcumin and its applications, the safety and bioavailability of curcumin.
2.1 Pulmonary arterial hypertension (PAH)
Pulmonary arterial hypertension (PAH) is caused by restricted flow through the
pulmonary arterial circulation resulting in increased pulmonary vascular resistance
which can lead to right heart failure [49]. The hemodynamic definition of PAH is rise in
mean pulmonary arterial pressure >25 mm Hg at rest or >30 mm of Hg during exercise
[10]. Elevated pulmonary pressure can result from a decrease in arterial lumen diameter
through a combination of: a) endothelial dysfunction and increased contractility of small
pulmonary arteries, b) proliferation and remodeling of endothelial and smooth muscle
cells, and c) in situ thrombosis [50].
PAH may occur primarily as a disease or secondary to various disease
conditions such as connective tissue diseases, pulmonary and cardiac diseases,
9
infection and inflammation. The nonspecific nature of the symptoms associated with the
disease often delays accurate diagnosis, making it difficult to diagnose and treat.
The 4th World Health Organization symposium on pulmonary hypertension held
in 2008 at Dana Point, California served to update the classification of PAH and is used
as a guide for the clinical assessment and treatment.
WHO Classification of PAH:
1. Pulmonary arterial hypertension 1.1 IPAH 1.2 Heritable
1.2.1 BMPR2 1.2.2 ALK1 (With or without hereditary hemorrhagic telangiectasia) 1.2.3 Unknown
1.3 Drug and Toxin induced 1.4 Associated with
1.4.1 Connective tissue diseases 1.4.2 HIV infection 1.4.3 Portal hypertension 1.4.4 Congenital heart diseases 1.4.5 Schistosomiasis 1.4.6 Chronic hemolytic anemia
1.5 Persistent pulmonary hypertension of the newborn 1.6 Pulmonary veno-occlusive disease (PVOD) and/ or Pulmonary capillary
hemangiomatosis (PCH) 2. Pulmonary hypertension owing to left heart disease
2.1 Systolic dysfunction 2.2 Diastolic dysfunction 2.3 Valvular disease
3. Chronic obstructive pulmonary disease 3.1 Chronic obstructive pulmonary disease 3.2 Interstitial drug disease 3.3 Other pulmonary diseases with mixed restrictive and obstructive pattern 3.4 Sleep disordered breathing 3.5 Chronic exposure to high altitude 3.6 Developmental abnormalities
4. Chronic thromboembolic pulmonary hypertension (CTEPH) 5. Pulmonary hypertension with unclear multifactorial mechanisms
10
5.1 Hematologic disorders : Myelo-proliferative disorders, splenectomy 5.2 Systemic disorders: Sarcoidosis, pulmonary Langerhans cell histiocytosis:
lymphangioleiomyomatosis, neurofibromatosis, vasculitis 5.3 Metabolic disorders: glycogen storage disease, Gaucherdisesase, thyroid
disorders 5.4 Others: tumoral obstruction, fibrosingmediastinitis, chronic renal failure on
dialysis ALK1 = activin receptor-like kinase type-1; BMPR2 = bone morphogenetic protein receptor type=2; HIV = human immunodeficiency virus; IPAH=Idiopathic pulmonary arterial hypertension Table 2-1. The updated clinical classification of pulmonary hypertension from
Dana point, 2008. Adapted from Simonneau et al. 2009 [45], Ryan JJ et al., 2012 [51].
Symptoms associated with PAH:
The early phases of PAH may be asymptomatic or associated with non-specific
symptoms. Patients usually present with dyspnea exacerbated by exertion, fatigue,
chest pain, and palpitations. Advanced PAH may present as right side heart failure,
dizziness, syncope, edema or cyanosis [52].
The normal pulmonary vasculature is a low resistance, high flow system. In PAH,
the vessel occlusion leads to a persistent elevation in pulmonary pressure that can lead
to right heart failure. The progressive reduction in cardiac output results in exercise
intolerance, shortness of breath, fluid retention, and possible death from right heart
failure [53].
11
Clinical presentation
Class I: Patients with PAH that causes no limitations on physical activities. Routine
physical activity does not cause increased dyspnea, chest pain, fatigue, or pre-syncope.
Class II: Patients with PAH that causes mild limitations on physical activities. Patients
are comfortable at rest but routine physical activity results in increased dyspnea, chest
pain, fatigue, or syncope.
Class III: Patients with PAH that have marked limitations on physical activities. Patients
are comfortable at rest, but less than routine physical activity results in dyspnea, chest
pain, fatigue, or palpitations.
Class IV: Patient with PAH that results in the inability to perform any physical activity
without symptoms. These patients may have signs of right heart failure. Dyspnea with or
without fatigue may be present at rest, and symptoms are increased by any physical
activity.
Table 2-2. World Health Organization Functional Class of PAH. Adapted from
Jeanne Houtchens et al., 2011[5].
2.2 Epidemiology
A national study examining PAH patients between 1980 to 2002 [54] indicated
that PAH affects just over 15 per million people [55]. The mean age of those affected
was 53±14 years at the time of diagnosis, of which 79.5% were women. The mean
12
duration between onset of symptoms and disease diagnosis was 2.8 years [56]. The
Registry to EValuate Early And Long-term PAH disease management (REVEAL
Registry) reported that IPAH that occurs with neither a family history of PAH nor an
identified risk factor associated clinical condition and that IPAH accounts for > 40% of all
PAH diagnoses in USA [56].
PAH associated with underlying systemic disease is present in approximately
50% of those diagnosed with PAH. PAH oftentimes develops as a consequence of
chronic obstructive pulmonary diseases, hypoxia, congenital heart disease and portal
hypertension. Connective tissue diseases such as scleroderma account for 11-35% of
PAH cases [57] while 1 to 10% of patients with portal hypertension go on to develop
PAH [55].
Although there are many factors involved in the pathogenesis of PAH, recent
data has suggested that increases in oxidative stress and inflammation may be a
primary driver of PAH development and progression of PAH [9]. Oxidative stress is most
commonly defined as an imbalance between the production and scavenging of reactive
oxygen species (ROS) and reactive nitrogen species (RNS) [58]. This imbalance can
alter cellular homeostasis through either direct oxidative and often irreversible damage
of basic cellular components (proteins, lipids, and nucleic acids), which can impair cell
function and lead to cellular apoptosis, tissue damage, and vasculopathy.
13
2.3 Right ventricular hypertrophy
Myocardial hypertrophy is a compensatory mechanism where the cardiac tissue
adapts to increased work load. In PAH, elevations in pulmonary vascular resistance
(PVR) lead to the development of pressure overload and RV hypertrophy [59].
Depending on the degree or duration of increased work load, ventricular hypertrophy
may progress from a compensatory state to impaired systolic or diastolic function and
heart failure. This transition is characterized by alterations in extracellular matrix
composition, energy metabolism, changes in myofilament protein expression,
dysregulation of Ca+2 handling, and the activation of different signal transduction
pathways [60].
Molecular markers of cardiac hypertrophy
Myosin and myofibrillar ATPase activity are reduced in the hypertrophied
myocardium. In the pathological hypertrophy there is a shift from α-MHC to β-MHC and
from cardiac to skeletal actin isoform expression [61]. The α-MHC form has a three to
seven fold greater ATPase activity than β-myosin. It is thought that an increase in the
abundance in β-MHC increases the efficiency of force development as it requires less
ATP to develop the same amount of muscle tension [62]. RV myocyte hypertrophy also
stimulates the expression of thyroid degrading enzyme D3 which functions to reduce
cellular metabolism [63].
14
Oxidative stress in RV hypertrophy
It is well established that elevations in ROS may be related to the development of
the cardiac hypertrophy, changes in blood vessel structure and function, and
inflammation seen with PAH [64]. Increased ROS levels have been shown to stimulate
myocardial growth, matrix remodeling and induce cellular apoptosis [65]. It is thought
that increases in NADPH oxidase, xanthine oxidase and nitric oxide synthase activity
are a major source of ROS in PAH [66]. ROS also have potent effect on the
extracellular matrix, stimulating increased accumulation of fibronectin and collagen and
activating the matrix metalloproteinases [67, 68].
2.4 Animal models
The etiology and molecular events associated with PAH have been widely
studied using animal models. The two most common animal models are the use of
monocrotaline to induce PAH and the chronic hypoxia model of PAH.
2.4.1 Monocrotaline injury model
Monocrotaline (MCT) is a pyrrolizidine alkaloid from the seeds of Crotalaria
spectabilis. The MCT model was introduced more than 40 years ago by Lalich and co-
workers [69]. In this model, PAH is typically induced by a single injection of MCT (60
mg/kg intraperitoneal or subcutaneous injection). The reactive metabolite MCT pyrrole
is bioactivated in the liver by CYP3A4 [70], which cause pulmonary mononuclear
vasculitis and severe pulmonary vascular disease [71]. The MCT rat model is widely
15
used given its technical simplicity, reproducibility, and low cost compared to other
models.
2.4.2 Chronic hypoxia model
Exposure of animals to low levels of oxygen results in alveolar hypoxia. A
reduction of the alveolar oxygen pressure to <70 mm Hg elicits strong pulmonary
arterial vasoconstriction [72]. In the laboratory chronic exposure of animals to hypobaric
hypoxia has been used to induce pulmonary vascular remodeling leading to PAH [73].
This model is widely used because it is very predictable and reproducible within a
selected animal strain. One limitation of this model is that the responses are significantly
affected by age as the response is much greater in immature animals than in adults
[74].
2.5 Diagnosis
A high index of suspicion, a meticulous history and a careful physical
examination are paramount to the diagnosis of PAH. Suspicion is increased by the
presence of increasing dyspnea on exertion in a patient [75]. A comprehensive
evaluation of pulmonary function is necessary for the accurate diagnosis of PAH. Other
diagnostic tools include echocardiography, and serological tests to examine for markers
of connective tissue remodeling [76].
16
2.5.1 Six-Minute-Walk Test
The 6-min-walk test is a sub-maximal exercise test in which the patient walks as
far as possible in 6 min. During this test, oxygen saturation, heart rate, and distance
walked are measured. The 6-min-walk test is oftentimes used in the initial patient
workup to assess exercise performance and predict prognosis [77].
2.5.2 Echocardiography
If PAH is suspected, transthoracic echocardiography is used to evaluate the right
heart hemodynamics including pulmonary arterial pressure, tricuspid regurgitation
velocity, right heart chamber dimensions, interventricular septum width, RV wall
thickness, and whether the pulmonary artery is dilated [78]. ECG may provide
suggestive or supportive evidence of PAH by demonstrating RV hypertrophy and right
atrial dilatation.
2.5.3 Cardiac magnetic resonance and high resolution computed tomography
In addition to echocardiography, cardiac magnetic resonance imaging is often
employed for the assessment of PAH since it can provide a direct evaluation of RV size,
morphology, and function, while it also allows for a non-invasive assessment of stroke
volume, cardiac output, pulmonary artery distensibility, and RV mass [79]. Similar to
magnetic resonance imaging, high-resolution computed tomography is another non-
invasive technology that is used to provide detailed views of the lung parenchyma for
the diagnosis of interstitial lung disease and emphysema [80].
17
2.5.4 Right heart catheterization
Right heart catheterization is required to confirm the diagnosis of PAH and to
assess the severity of the hemodynamic impairment [81]. Right-heart catheterization is
used to directly measure pulmonary-artery pressure and for the estimation of pulmonary
vascular resistance [57].
2.6 Current strategies for treatment of PAH
The first line therapy for PAH is supportive health care consisting of oxygen
supplementation, administration of calcium channel blockers and diuretics, and
recommendations to avoid physical exertion [82].
Prostacyclin therapy
Prostaglandin I-2 is a metabolite of arachidonic acid that is produced by the
vascular endothelium. It is a potent vasodilator, inhibits platelet aggregation, and
exhibits anti proliferative effects [83]. One of the most successful strategies for PAH
treatment has been to augment endogenous prostacylin production with exogenous
prostanoids [10]. Intravenous prostacylin (epoprostenol) for the treatment of PAH was
approved by the FDA in 1995 and has been shown to decrease pulmonary vascular
resistance, increase cardiac output, and improve life expectancy [84]. Other drugs
similar to epoprostenol include Iloprost, which is inhaled, treprostinil which is taken
subcutaneously, and the oral agent beraprost.
18
Endothelin-A receptor antagonists
Endothelin-1 is a vasoconstrictor and smooth muscle mitogen that is over
expressed in the lungs of patients with PAH [85]. Bosentan and ambrisentan are FDA
approved endothelin receptor antagonists commonly used for the treatment of PAH.
Bosentan was approved in 2001 and functions as a combined Endothelin-A and
Endothelin-B receptor antagonist. Bosenten has been found to decrease pulmonary
vascular resistance and increase cardiac output in PAH patients [86]. Ambrisentan was
FDA approved in 2007 and is available as once a day oral therapy only for idiopathic
PAH, heritable PAH and connective tissue disease-PAH [87]. Ambrisentan blocks only
the Endothelin-A receptors and similarly to bosentan has been shown to significantly
decrease pulmonary vascular resistance in PAH patients [87].
Phosphodiesterase-5 (PDE-5) inhibitors, calcium channel blockers and
antioxidants
Inhibition of the cGMP-degrading enzyme phosphodiesterase type-5 results in
vasodilatation through the NO/cGMP pathway [88]. Since the pulmonary vasculature
contains substantial amounts of phosphodiesterase type-5 enzyme, the potential clinical
benefit of phosphodiesterase type-5 inhibitors for the treatment of PAH appear to be
well justified [89]. Currently sildenafil and tadalafil are FDA approved oral PDE-5
inhibitors for the treatment of PAH. Sildenafil, approved in 1998, is typically taken three
times daily orally [90]. Tadalafil, approved in 2003, is available as once daily oral
therapy for idiopathic and connective tissue disease PAH [91].
19
McLaughlin and colleagues demonstrated that the calcium channel blockers
nifedipine and diltiazem can be used to diminish pulmonary vascular resistance and
prolong PAH patient survival [92]. In rats, compounds with antioxidant properties such
as N-acetylcysteine [93], resveratrol [94], superoxide dismutase [95, 96] SOD mimetic
tempol [97] and EUK-134 [98] have been found to inhibit PAH and or/ RV dysfunction.
2.7 Introduction to Nanotechnology
Nanotechnology is study of manipulating matter on an atomic and molecular
scale [99]. The National Nanotechnology Initiative (NNI) defines nanoparticles as
particulate matter that range in size between 1 and 100 nm (Figure 2-1). Nanomaterials,
unlike their bulk counterparts, oftentimes exhibit unique electronic, optical and catalytic
properties [100].
20
Figure 2-1.The illustration shows visual examples of the size and the scale of
nanotechnology. (Adopted from www.nano.gov )
Recent advances in nanotechnology have resulted in the development of various
engineered nanomaterials including carbon-based nanotubes, C60 -Bucky balls, metal
oxide nanoparticles, and the nano-encapsulation of drugs [101]. Nanoparticles, given
their large surface to volume ratio, are characterized by increased surface reactivity
[102].
Nanomedicine is the application of nanotechnology to medicine. Nanomedicine
includes the development of nanoparticles and nanostructured surfaces as well as
nanoanalytical techniques for the diagnosis and treatment of disease. Nanomaterials
21
used in medicine include quantum dots, liposomes, multifunctional constructs and gold
nanoparticles [103]. It is thought that nanotechnology will contribute significantly to the
“find, fight, and follow” concept of early diagnosis, therapy and follow-up in medicine
and that nanotechnology will be a key contributor to the future of medical care [104].
2.8 Cerium oxide nanoparticles
Cerium has several unique properties among the rare earth elements and is used
in the production of oxide fuel cells, ultraviolet absorbents, oxygen sensors, and
automotive catalytic converters [105]. The cerium atom can easily transition from either
the +3 (fully reduced) or +4 (fully oxidized) state in several types of redox reactions [15],
which allows it to act as a catalyst [16].
Ce+3 Ce+4 + e-
Ce+3+OH. Ce+4+OH-
Ce+4+O2.- Ce+3 +O2
The oxidation state of CeO2 can change in response to alterations in
environmental temperature, the presence of other ions, and oxygen partial pressure
[106]. Investigations of nanocrystalline cerium oxide (CeO2) have demonstrated an
increase in the Ce+3valance state and radical scavenging ability with decreasing particle
size [102].
22
Figure 2-2.The fluorite structure of cerium oxide. (Adopted from Ali Bumajdad et al.,
2009).
Recently, several researchers have begun to investigate the use of CeO2
nanoparticles for biomedical purposes. Rzigalinski and coworkers demonstrated that
CeO2 nanoparticles smaller than 20 nm in size were able to prolong the lifespan of
cultured neuronal cells, with some cultures remaining still viable even after 6-8 months
[107]. Chen and colleagues reported that nanoceria particles prevented increases in
cellular ROS levels in primary rat retina cells and that the use of nanoceria in vivo
prevented loss of vision due to light induced degeneration of photo receptor cells in
albino rats [106]. Taken together, these data suggested that nanoceria may be useful
for preventing ocular degeneration. Schubert and coworkers showed that cellular
treatment with cerium or yttrium oxide nanoparticles was able to rescue HT22 cells from
oxidative stress induced cell death by functioning as antioxidants [21].
23
Recent in vivo experiments conducted by Niu and colleagues reported that CeO2
nanoparticles diminished the degree of cardiac dysfunction and remodeling in MCP-1
transgenic mice by attenuating the development of myocardial and ER oxidative stress
[23]. Similarly, CeO2 nanoparticles have also been capable of protecting lung fibroblast
cells from radiation induced damage in vitro and in vivo using athymic nude mice [108].
It is thought that CeO2 nanoparticles function to protect cells and tissues through
their ability to scavenge ROS. Indeed, Hirst and coworkers using murine macrophage
cells (J774A.1) showed treatment with CeO2 nanoparticles reduced superoxide anion
levels and that this reduction in superoxide was associated with decreased expression
of inducible nitric oxide synthase (iNOS) and inflammation [22]. Likewise, Estevez and
colleagues, using an in vitro model of brain ischemia, demonstrated that nanoceria
treatment was associated with neuroprotective effects and that this effect was
associated with increased scavenging of peroxynitrite.
Kong and coworkers showed that nanoceria could protect against retinal
degeneration in tubby mice by decreasing ROS production, up-regulating the
expression of neuroprotective genes, and by inhibiting the activation of caspase-
induced apoptotic signaling pathways [109]. CeO2 nanoparticles have also be shown to
protect against monocrotaline-induced hepatic toxicity by virtue of their ability to
increase the amount of glutathione reductase, glutathione, and glutathione peroxidases
[110].
24
Toxic effects of CeO2 nanoparticles
The potential hazardous health effects of nanoparticles are an emerging issue
amongst toxicologists and regulatory authorities. Given the extensive use of cerium
oxide nanoparticles in the glass polishing, the electronics industry, and use as a diesel
fuel additive, interest regarding the potential toxicity of these particles has intensified
[111]. Indeed, although the literature has demonstrated the CeO2 nanoparticles can
promote cell survival under conditions of oxidative stress it is likely that these
nanoparticles also exhibit toxic effects if cellular exposure is excessive.
In vitro studies by Lin and colleagues using human branchoalveolar cells (A549
cells) demonstrated that exposure to 20-nm CeO2 nanoparticles was associated with
the generation of free radicals, increased lipid peroxidation, and cell membrane damage
that led to reduced cell viability [112]. Park and coworkers showed that CeO2
nanoparticles exposure caused the induction of ROS in cultured human lung epithelial
cells (BEAS-2B) and that increases in cellular ROS were associated with decreased
levels of intracellular glutathione (GSH) and reduced cell viability [113].
Similar findings have been demonstrated in vivo. Using male Sprague Dawley
rats, Ma and colleagues demonstrated that the intratracheal instillation of cerium oxide
nanoparticles induced acute pulmonary inflammation and chronic lung injury [114].
Follow up studies by these same researchers showed that lung injury caused by cerium
oxide nanoparticles was due to increased ceria deposition in the lung, increased
pulmonary fibrosis and an up-regulation of fibrogenic cytokines [111].
25
More recently, Nalabotu and co-workers found that the intratracheal instillation of
CeO2 nanoparticles was associated with the accumulation of ceria in the liver, the
hydropic degeneration of hepatocytes, dilatation of sinusoids, and elevations in serum
liver enzyme level [115].
2.9 Curcumin
Curcumin is a constituent of turmeric, a curry powder extracted from the plant
Curcuma longa that belongs to the Zingiberaceae family (Figure 2-3). Turmeric is native
to India and Southeast Asia, and is used in cooking to add color. The ability of curcumin
to preserve food is well known. Indian Ayurvedic medicine has used turmeric for
centuries to treat biliary disorders, anorexia, cough, diabetic wounds, liver disorders,
rheumatism, and respiratory conditions [116]. Turmeric also exhibits haemostatic-like
activity and is also anti-inflammatory in nature [117].
26
Figure 2-3. Curcuma longa plant. Inserts show the flower (a.), rhizome (b.)
and turmeric powder (c.). (Adopted and modified from Seema Singh 2007).
Curcumin is a hydrophobic polyphenol compound with a characteristic yellow
color. The chemical structure of curcumin is a bis-α, β-unsaturated β-diketone. It is
characterized by two oxy-substituted aryl moieties that are linked together through by a
seven carbon chain [118] (Figure 2-4).
27
Figure 2-4.Structure of curcumin (diferuloylmethane)
Applications of curcumin
Curcumin possesses several pharmacological properties including anti-
inflammatory [119], antioxidant [120], antimicrobial [121], chemo sensitizing [122], anti-
neoplastic [123], and wound healing activities [124]. Curcumin has proven to be
beneficial in the prevention and treatment of a number of inflammatory diseases
possibly due to its ability to inhibit the inflammatory mediators cyclooxygenase (cox-2)
and lipoxygenase-5 at the transcriptional level [125]. The free radical scavenging activity
of curcumin due to the presence of H-atom donation and resonance stabilization from
two phenolic OH groups and from the electron transfer capacity of β-diketone moiety. In
addition, curcumin also down-regulates the expression of several pro-inflammatory
cytokines including tumor necrosis factor (TNF-α), the interleukins (IL-1, IL-2, IL-6, IL-8,
IL-12) and some chemokines, most likely through inactivation of the nuclear
transcription factor NF-κB [126, 127] (Figure 2-5).
The transcription factor NF-κB is thought to play a critical role in the resistance of
cancer cells to chemo and radiation therapy as it can prevent the induction of apoptosis
[128]. In recent experiments Deeb and coworkers demonstrated that curcumin
28
sensitizes prostate cancer cells to TNF-related apoptosis inducing ligand (TRAIL)-
induced apoptosis through the inhibition of NF-κB [122]. Other data has shown that
curcumin can induce cell cycle arrest and/or apoptosis in human cancer cell lines
derived from a variety of solid tumors including colorectal, lung, breast, pancreatic and
prostate carcinoma [129].
Figure 2-5.Molecular targets of curcumin. (Adopted and modified from Bharat
B. Aggarwal et al., 2009 [28] ).
In addition to its anti-cancer properties, curcumin is also widely known to possess
potent antioxidant activity as it is able to scavenge several different types of ROS
including O2 −, OH , NO and ONOO radicals [126]. In addition, Manikandan and
colleagues have shown that exposure to curcumin can also increase the activity of
29
superoxide dismutase, catalase and glutathione peroxidases [34]. It is thought that the
antioxidant activity of curcumin is attributed to its phenolic and methoxy groups [130].
The antibacterial effects of curcumin have been reported by K.S. Parvathy and
colleagues where they showed that curcumin suppressed the growth of Streptococcus,
Staphylococuus, E.coli, and Y.enterocolitica. Ronita De and coworkers using a mouse
model demonstrated that curcumin also inhibited the growth of Helicobacter pylori both
in vitro and in vivo. Whether curcumin exhibits antibacterial activity against other strains
is currently under investigation.
Safety
Animal and human clinical studies have shown that curcumin is extremely safe
even at doses as high as 12gm/day [131]. Recent phase-1 clinical trials have shown
that curcumin is not toxic to humans when administered at 8gm/day for up to 3 months
[132]. On the basis of these studies and others [131, 133], curcumin is generally
recognized as safe (GRAS) by the United State’s FDA and has been granted an
acceptable daily intake level of 0.1–3 mg/kg body weight by the Joint FAO/WHO Expert
Committee on Food Additives, 1996 [134].
30
Bioavailability
A major limiting factor of curcumin use for biomedical purposes is its solubility
and low oral bioavailability [135, 136]. The factors responsible for the low bioavailability
of curcumin are not fully understood but are likely related to the poor absorption from
the gut, the rapid metabolism and its rapid systemic elimination [137]. The serum
concentration of curcumin has been found to peak at 1–2 h after oral intake and to
gradually decline thereafter [138]. Curcumin undergoes glucuronidation and sulfation in
the liver [139]. The metabolites of curcumin for the most part appear to lack any
pharmacological activity [28]. The addition of piperdine, which functions to inhibit the
glucronidation of curcumin, has been shown to increase curcumin bioavailability in rats
and in human subjects by at least one order of magnitude [140].
In addition to piperdine supplementation, the bioavailability of curcumin could
also be increased by protecting it from degradation. Various nano-formulations have
been formulated for this purpose including PLGA nanoparticles (Nano-CUR) [141]
liposomal nano-curcumin [42], curcumin loaded magnetic nanoparticles [142], curcumin
nano-emulsions [40], and solid lipid nanoparticles [39]. Indeed, the entrapment of
curcumin into nanoparticles led to a 9-fold increase in oral bioavailability when
compared to that observed when curcumin was administered orally in bulk form [143].
This increase in bioavailability is thought to be due to improved water solubility, a higher
release rate in the intestinal juice, enhanced permeability and absorption by the small
intestine, and increased residence time in the intestinal cavity [144]. Wang and
coworkers demonstrated that liposomal encapsulated curcumin suppressed NF-κB
31
activation and attenuated the proliferation of human pancreatic carcinoma cells [145].
Supporting this contention, Bisht and colleagues showed that nano curcumin, like its
bulk counterpart, inhibited the activation of the transcription factor NF-κB, and that this
inhibition was associated with reduced levels of pro-inflammatory cytokines (IL-6, IL-8)
and TNF-α in human pancreatic cancer cell lines [43].
3.0 Summary
In summary, this review of literature described pulmonary arterial hypertension
and RV hypertrophy. PAH is an increase in pulmonary arterial pressure due to
obliteration of small pulmonary arteries causing an increase in pressure overload on the
RV leading to RV failure and death [1, 52]. Currently, there is no cure for the patients
suffering from PAH [5]. Increased markers of oxidative stress have been found in
human as well as in animal models of PAH supporting the evidence that ROS and RNS
play a major role in the development and progression of PAH [50]. Thus far, several
antioxidants including N-acetylcysteine, resveratrol and the synthetic antioxidant EUK-
134 have been tested for their efficacy in preventing the progression of PAH in animal
models.
Cerium is a rare earth element of the lanthanide series, having a fluorite lattice
structure with oxygen vacancies or defects in the lattice structure by loss of oxygen or
its electrons. At the nanometer scale, the atomic and molecular properties of nano
materials will change. Studies have demonstrated that decreases in CeO2 nanoparticles
size are associated with the formation of more oxygen vacancies [18]. Recent studies
32
have shown that CeO2 nanoparticles have been shown to protect against cardiac
dysfunction by attenuating myocardial oxidative stress and the inflammatory process in
transgenic mice through the autoregenerative antioxidant capacity.
Curcumin, a polyphenolic compound with neuroprotective, cardio protective, anti-
oxidant and anti-inflammatory properties, has been used for centuries in Ayurveda, an
Indian traditional medicine, to treat a diversity of disorders including rheumatism, body
aches, skin diseases, wounds, intestinal worms, diarrhea, intermittent fevers, hepatic
disorders, urinary discharges, dyspepsia, and inflammation. For the past two decades,
curcumin has been extensively studied as a potent anti-oxidant due to its ability to
scavenge ROS and an anti-inflammatory agent due to its down regulation of pro
inflammatory cytokines and transcription factors [126].
Whether ceria or curcumin nanoparticles are effective for the treatment of PAH is
currently unclear.
33
CHAPTER III
The following chapter includes three different research papers describing in detail
the research experiments conducted to test our hypotheses set forth for the specific
aims I and II of this dissertation project.
PAPER 1
The following paper corresponds to the specific aim I
34
Cerium oxide nanoparticles attenuate monocrotaline-induced right ventricular
hypertrophy following pulmonary arterial hypertension
Key words:
Monocrotaline, pulmonary arterial hypertension, right ventricular hypertrophy,
cerium oxide nanoparticles
Running title:
Efficacy of cerium oxide nanoparticles on right ventricular hypertrophy following
pulmonary arterial hypertension.
35
Abstract
Introduction: Cerium oxide (CeO2) nanoparticles have been shown to exhibit
potent antioxidant activity and recent data has suggested that these materials may
attenuate oxidative stress-induced cardiomyopathy. Here, we investigate whether CeO2
nanoparticle administration can diminish the amount of right ventricular hypertrophy
developed following monocrotaline (MCT)-induced pulmonary arterial hypertension
(PAH) in the laboratory rat. Methods: Male Sprague Dawley rats (175-200 gm) were
randomly divided into control, MCT only and MCT + CeO2 nanoparticle groups (n=6).
MCT animals received a single injection of MCT 60 mg/kg subcutaneously to induce
PAH. CeO2 treated animals were given 0.1 mg/kg CeO2 nanoparticles intravenously
twice a week for 2 weeks. Echocardiography was done at the start of the study and 28
days after MCT injection to assess pulmonary, cardiac structure and function. Results:
Compared to control rats, the right ventricle weight to body weight ratio was 45% and
22% higher in the MCT and MCT + CeO2 rats, respectively (p<0.05). Echocardiography
data demonstrated that CeO2 nanoparticle treatment appeared to attenuate
monocrotaline-induced changes in pulmonary flow and increases in right ventricle wall
thickness. Immunoblotting analysis demonstrated that CeO2 nanoparticle treatment
diminished MCT-induced increases in the amount of β-myosin heavy chain and
fibronectin. CeO2 nanoparticle treated rats also exhibited diminished expression of the
MAP kinases, decreased TUNEL positive nuclei. These changes were accompanied by
improvements in the Bax/Bcl2 ratio, and diminished caspase-3 activation. Conclusion:
Taken together, these data suggest that CeO2 nanoparticle administration may
36
attenuate MCT-induced PAH associated RV cardiomyocytes apoptosis possibly via
diminishing indices of oxidative stress. (WC: 250)
37
Introduction
Right ventricular failure is the leading cause of death in patients with pulmonary
arterial hypertension (PAH) [146]. PAH is defined as an increase in mean pulmonary
arterial pressure (PAPm) >25mm Hg at rest, or >30mm Hg during exercise [55]. If
improperly managed, the median life expectancy of patients with idiopathic PAH is 2.8
years, with 1-year, 3-year, and 5-year survival rates of 68%, 48%, and 34%,
respectively [2]. Even with modern medical therapy, PAH patients oftentimes
experience disease progression and eventual referral for organ transplantation [5]. The
rat monocrotaline (MCT)-PAH model is thought to recapitulate many of the pathological
features of PAH seen in humans [147]. Recent data has suggested that the cardiac
remodeling associated with MCT induced-PAH may be mediated, at least in part, by
increases in oxidative stress [98]. Whether treatments aimed at diminishing oxidative
stress levels effective in preventing the cardiac hypertrophy seen with PAH is currently
unclear.
Cerium is a rare earth element of the lanthanide series that can cycle between
the Ce+3 (fully reduced) or Ce+4 (fully oxidized) state [15]. Recent data has suggested
that CeO2 nanoparticles can act as free radical scavengers [15] and protect H9C2
cardiomyocytes from cigarette smoke extract induced oxidative damage [148]. In vivo
studies using the monocyte chemo-attractant protein-1 (MCP-1) transgenic mouse
model of oxidative stress-induced cardiac hypertrophy have shown that CeO2
nanoparticle administration can attenuate the development of cardiac dysfunction and
remodeling [23]. Supporting these data, other work has shown that the antioxidant
38
activity of CeO2 nanoparticles can protect against monocrotaline-induced hepatic toxicity
[110]. Whether CeO2 nanoparticle administration can attenuate the development of the
RV hypertrophy seen during PAH is currently unclear.
Monocrotaline (MCT) is a toxic pyrrolizidine alkaloid present in the plant
Crotalaria spectabilis that selectively injures lung endothelial cells causing the infiltration
of monocytes, a thickening of the pulmonary arterioles and the development of PAH in
the laboratory rat [12, 13]. Here we examine if the administration of CeO2 nanoparticles
is associated with the attenuation of right ventricular hypertrophy, oxidative stress
induced ventricular remodeling following monocrotaline induced PAH. Our data suggest
that CeO2 nanoparticle treatment may be effective for counteracting the hypertrophy
seen following PAH in the laboratory rat.
39
Materials and Methods
Animal model and experimental design
All procedures were performed in accordance with the Marshall University
Institutional Animal Care and Use Committee (IACUC) guidelines, using the criteria
outlined by the Assessment and Accreditation of Laboratory Animal Care (AAALAC).
Seven week old, 175-200 gm, male Sprague Dawley rats were obtained from Hilltop
laboratories (Scottdale, PA). Rats were housed two per cage with a 12:12H dark-light
cycle and temperature maintained at 22 ± 2°C. Rats were provided food and water ad
libitum and allowed to acclimatize for at least two weeks before initiating the study.
Periodic body weight and feed intake measurements were taken throughout the
duration of the study. Rats were randomly assigned to one of three different groups:
Control (n=6), MCT only (n=6), MCT + CeO2 nanoparticle (n=6). PAH was induced by a
single injection of MCT (60mg/kg S.C.) (Sigma-Aldrich, St.Louis, MO) as described
previously [149]. Animals injected with MCT were given either CeO2 nanoparticles
(0.1mg/kg via tail vein) (NanoactiveTM CeO2 nanoparticles, NanoScale materials Inc.,
Manhattan, KS) or vehicle (deionized water) twice a week for the 1st and 2nd week of the
study. Importantly, there was no toxicity or accumulation observed in vivo at therapeutic
low doses 0.1 mg/kg and 0.5 mg/kg of CeO2 nanoparticles injected via tail vein as
reported previously [47].
40
CeO2 nanoparticle size analysis by Dynamic Light Scattering (DLS)
The measurement of particle size distribution was done using a LB-550 dynamic
light scattering (DLS) particle size analyzer (Horiba scientific, Edison, NJ) at Marshall
University. Briefly, CeO2 nanoparticles were sonicated in deionized water for two
minutes to ensure equal dispersion. Particle size was performed as outlined by the
manufacturer and measured in triplicate.
CeO2 nanoparticle analysis using atomic force microscopy (AFM)
Twenty micro liters of CeO2 nanoparticles (1µg/µl) was placed on freshly cleaved
mica (V1 mica, SPI Inc, West Chester, PA). After 10 min of incubation, the surface was
gently rinsed with deionized water to remove unbound CeO2 nanoparticles and the
surface was dried under nitrogen. Particles were imaged in noncontact mode at a
frequency of 319 kHz and a scan speed of 0.5 Hz using a Nano-R microscope (Pacific
Nanotechnology Inc., Santa Clara, CA) equipped with a TM300A noncontact probe
(SensaProbes Inc., Santa Clara, CA) at MBIC, Marshall University.
Echocardiography
Echocardiography was performed prior to the start of the study and at day 28 of
the study at an ICAEL accredited echocardiography laboratory with help of trained
echocardiography technician in University Cardiovascular services, Marshall University
as outlined previously [150]. Rats were anesthetized with an intraperitoneal injection of
41
ketamine and xylazine cocktail mixture (50 mg/kg; 4:1) and the ventral thorax area
shaved. ECG analysis was performed during the echocardiography procedure to
monitor heart rate. Echocardiography measurements were obtained with a Philips
Sonos 5500 echocardiogram system using 12-MHZ transducer. M-mode and 2-D
modalities were used to measure RV and LV wall thickness at the end of diastole using
the right parasternal long axis view with the ultrasonic beam positioned perpendicular to
the ventricular wall. Papillary muscles were used as reference point for measurements
and to assure proper position in subsequent studies. Pulmonary artery flow was
measured using pulsed wave Doppler at the pulmonary valve level while pulmonary
artery diameter was measured at the level of pulmonary out flow tract during systole
using the parasternal short axis view. An apical four-chamber view was employed to
measure end-diastolic right ventricular area. Pulsed-wave Doppler was used to
measure pulmonary artery acceleration time and flow.
Tissue processing and immunoblotting
Animals were anesthetized with ketamine-xylazine (4:1) cocktail mixture
(50mg/kg, i.p) and supplemented as necessary to achieve loss of reflexive response
before tissue collection. After mid ventral laparotomy, the heart was removed and
placed in Krebs–Ringer bicarbonate buffer (KRB) containing 118 mM NaCl, 4.7 mM
KCl, 2.5 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 24.2 mM NaHCO3, and 10 mM D-
glucose (pH 7.4) equilibrated with 5% CO2 / 95% O2 and maintained at 37°C. The right
ventricle was separated from the left ventricle and septum, weighed and immediately
42
flash frozen in liquid nitrogen and stored at -80°C. Lungs were weighed as a measure of
the degree of pulmonary vasculitis and as an indication of the increase in pulmonary
arterial pressure [98].
Frozen right ventricles were homogenized in buffer (T-PER, 10 ml/g tissue;
Pierce, Rockford, IL, USA) containing protease (P8340, Sigma-Aldrich, St. Louis, MO,
USA) and phosphatase inhibitors (P5726, Sigma- Aldrich, St. Louis, MO, USA). After
incubation on ice for 30 min, the homogenate was collected by centrifuging at 12,000 g
for 5 min at 4°C. Protein concentration of homogenates was determined via the
Bradford method (Fisher Scientific, Rockford, IL). Homogenate samples were boiled in a
Laemmle sample buffer (Sigma-Aldrich, St. Louis, MO, USA) for 5 min. Forty
micrograms of total protein for each sample was separated on a 10% PAGEr Gold
Precast gel (Lonza, Rockland, ME) and then transferred to nitrocellulose membranes
using standard methods as detailed elsewhere [151]. Membranes were blocked with 5%
milk in Tris Buffered Saline (TBS) containing 0.05% Tween-20 (TBST) for 1 h and then
probed with primary antibodies for the detection of β-myosin heavy chain (M 8421,
sigma Aldrich, St. Louis, MO), fibronectin (AB23750, Abcam, Cambridge, MA), (AMPK-
α (#2532), p-AMPK-α (#2535), Bax (#2772), Bcl2 (#2870s), Caspase-3 (#9662),
Cleaved caspase-3 (#9661s), ERK 1/2 (#9106s), p-ERK 1/2 (#4377s), p-P-38 (#9216L),
P-38 (#9212), p-HSP-27 (#24068), HSP-27 (#2442s), HSP-70 (#4872), p-HSP-90
(#3488), p-JNK (#9251s), JNK (#9252), glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) (#2118), (from Cell Signaling Technology, Inc., Beverly, MA)) and p-NFkB-
p50 (SC-33022), NFkB-p50 (SC-8414), and 3-nitrotyrosine (SC-55256) (from Santa
43
Cruz Biotechnology, Inc., Santa Cruz, CA). After an overnight incubation at 4°C in
primary antibody buffer (5% BSA in TBS-T, primary antibody diluted 1:1000)
membranes were washed with TBST, and then incubated with HRP-conjugated
secondary antibodies (anti-rabbit (#7074) or anti-mouse (#7076), Cell Signaling
Technology, Danvers, MA) for 1 h at room temperature. After removal of secondary
antibody, membranes were washed (3 x 5 min) in TBS-T and protein bands were
visualized following reaction with ECL reagent (Amersham ECL Western Blotting
reagent RPN 2106, GE Healthcare Bio-Sciences Corp., Piscataway, NJ). Target protein
levels were quantified by AlphaEaseFC image analysis software (Alpha Innotech, San
Leandro, CA) and normalized to (GAPDH).
OxyBlot analysis
OxyBlot™ Protein Oxidation Detection kit (#S7150, Millipore, MA) was used for
the immune blot detection of carbonyl groups. Carbonyl groups are introduced into
proteins due to oxidative reactions by oxygen free radicals and other reactive oxygen
species [152]. Briefly, 5 μg total protein of each sample was derivatized by reaction with
2, 4-dinitrophenylhydrazine (DNPH) at room temperature for 15 min before separation
using 10% PAGEr Gold Precast gels. Oxidized proteins were detected by
immunoblotting using the anti-DNP antibody provided in the kit [29]. Bands were
visualized following reaction with ECL reagent (Amersham ECL Western Blotting
reagent RPN 2106, GE Healthcare Bio-Sciences Corp., Piscataway, NJ). Target protein
44
levels were quantified by AlphaView SA image analysis software (Alpha Innotech, San
Leandro, CA).
Histological analysis
a. Dystrophin staining
RV tissues were serially sectioned (8 μm) using an IEC Microtome cryostat and
the sections were collected on poly-L-Lysine coated slides. Sections were stained for
dystrophin immunoreactivity and used to evaluate the cardiomyocyte cross sectional
area (CSA) as outlined elsewhere [153]. Briefly, sections were incubated for 30 min in a
blocking solution (5% bovine serum albumin [BSA] and phosphate-buffered saline [PBS]
containing 0.5% Tween 20 (PBS-T), pH 7.5 and then incubated with specific anti sera
diluted in PBS-T (anti-dystrophin 1:100, rat-igG 1:100) for 1 h at 37°C in a humidified
chamber. After washing in PBS-T (3 x 5 min), sections were incubated with the
appropriate FITC labeled secondary antibody (1:200) for 1 h at 37°C in a humidified
chamber. Muscle cross sections were visualized by epi-fluorescence using an Olympus
BX51 microscope (Olympus, Center Valley, PA), fitted with 20x and 40x objectives.
b. Picrosirius red staining
Picrosirius red staining was used to assess the collagen content deposition in RV
sections as detailed elsewhere [154]. Briefly, RV frozen tissue sections were fixed with
95% alcohol for 1 min, washed three times with running tap water, and then stained with
45
hematoxylin for 8 min, and picrosirius red for 20 min. After dehydrating sections and
mounting, collagen content was visualized at 20x magnification using an Olympus BX51
microscope (Olympus, Center Valley, PA).
c. Dihydroethidium staining
RV cardiac muscles were serially sectioned (8 μm) with an IEC Microtome
cryostat, and the sections were collected on poly-L-lysine coated glass slides. The
fluorescent superoxide marker dihydroethidium (hydro ehidine) was used to evaluate
superoxide levels as outlined elsewhere [155]. Upon oxidation, dihydroethidium is
intercalated within DNA exhibiting a bright fluorescent red [156]. Briefly, RV frozen
tissue sections were washed with phosphate-buffered saline (PBS) for 5 min and then
incubated with 200 μl of 10 μM of dihydroethidium (Molecular Probes, Eugene, OR) for
1 h at room temperature. After washing with PBS (3 x 5 min), fluorescence was
visualized under a Texas red filter using an Olympus BX-51 microscope (Olympus
America, Melville, NY, USA) equipped with Olympus WH 20x wide field. Images were
recorded digitally using a CCD camera and the integrated optical density determined
using the Alpha view image analysis software (Cell Biosciences, Inc., Santa Clara, CA).
d. TUNEL staining
DNA fragmentation was determined by TdT-mediated dUTP nick end labeling
(TUNEL) according to the manufacturer’s recommendations (In Situ Cell Death
46
Detection Kit, Roche Diagnostics, Mannheim, Germany) as described elsewhere [157].
Briefly, TUNEL staining was performed on RV tissue sections (8 μm) obtained from
control (n=3), MCT only (n=3), and MCT + CeO2 nanoparticle treated rats (n=3), which
were fixed with 4% paraformaldehyde, washed with PBS (pH 7.4), and then
permeabilized with 0.1% sodium citrate and 0.1% Triton-X. Cross-sections from each
muscle were treated with DNase I to induce DNA fragmentation as a positive control.
Heart sections were blocked with 3% BSA and the sections were incubated with the
TUNEL reaction mixture containing TdT and fluorescein dUTP. Nuclei were counter
stained using DAPI (VECTASHIELD HardSet Mounting Medium, Vector Laboratories,
and Burlingame, CA). Fluorescence images were visualized using an Olympus BX-51
microscope (Olympus America, Melville, NY) equipped with Olympus WH 20x and 40x
wide fields.
Serum protein immune assays
Sera collected during exsanguations were pooled from all six rats in each group
and sent to Myriad RBM (Austin, TX) for RodentMAP® v 2.0 Antigen analysis using a
Luminex Lab MAP instrument as detailed elsewhere [19, 115]. Each analyte was
quantified using 4 and 5 parameter, weighted and non weighted curve fitting algorithms
using proprietary data analysis software developed at Rules-Based Medicine.
47
Data analysis
Results are presented as mean ± SEM. Data were analyzed using SigmaPlot
11.0 computer software. One-way analysis of variance (ANOVA) was used for overall
comparisons. The Student Newman Keuls post hoc test was used to determine
statistical significance. The level of significance accepted a priori was p ≤ 0.05.
48
Results
Characterization of CeO2 nanoparticles
CeO2 nanoparticle diameter as determined by DLS and AFM were 300 ± 58 nm
and 300 ± 25 nm, respectively (Figure 3-1 Panels A, B).
CeO2 nanoparticles treatment decreases right ventricular tissue weight
Compared to the control animals, body weight in the MCT and MCT +
CeO2animals was 14% and 13% less, respectively (P< 0.05, Table 3-1). Conversely,
the ratio of heart weight to body weight was 22% and 4% higher in the MCT and MCT +
CeO2 animals (P< 0.05). Similarly, the ratio of right ventricle (RV) weight to body weight
ratio was 83% and 29% higher in the MCT and MCT + CeO2 nanoparticle animals (P<
0.05), while the wet lung weight to body weight ratio was 48% and 36% higher in the
MCT and MCT + CeO2 nanoparticle animals (P< 0.05).
CeO2 nanoparticles treatment decreases MCT-induced changes in pulmonary
arterial pressure and cardiac remodeling
Compared to the MCT + CeO2 nanoparticle animals, echocardiographic analysis
of pulmonary arterial flow demonstrated a mid-systolic notch in the MCT animals (Figure
3-2, Panel B). Consistent with this finding, pulmonary arterial pressure (PA Pr) was 18%
higher in MCT while PA Pr was equal to the base line measurement in the MCT + CeO2
49
animals (Figure 3-3, Panel A P<0.05). Mean pulmonary arterial diameter was 27% and
1% higher in MCT and MCT + CeO2 nanoparticle animals, respectively (Figure 3-3,
Panel B, P<0.05). Supporting these data, the mean pulmonary arterial area was 46%
higher in the MCT animals but only 3% greater in the MCT + CeO2 nanoparticle animals
(Figure 3-3, Panel C, P<0.05). Similarly, the right ventricular outflow tract diameter was
14% and 1% higher in MCT and MCT + CeO2nanoparticle animals (Figure 3-3, Panel D,
P<0.05). Similar to our findings at the whole tissue level, echocardiography analysis
demonstrated that CeO2 nanoparticle treatment attenuated MCT-induced increases in
RV anterior free wall thickness (Figure 3-4). Likewise, the interventricular septum
diameter was 16% higher in MCT animals but only 3% greater in the MCT + CeO2
animals (Figure 3-5, Panel B, P<0.05).
In the MCT only animals, the echocardiography analysis showed that the right
atrium septum tended to bow toward the left atrium (Figure 3-6, Panel A). Similarly, in
MCT animals a D-shaped inter-ventricular septum, septal flattening and tricuspid
regurgitation was observed (Figure 3-6, Panels B, C), which are all consistent with
advanced PAH and RV hypertrophy.
To examine the molecular alterations that might accompany PAH-induced
changes in RV thickness we next performed dystrophin immunofluoresence staining to
determine the average cardiomyocyte cross sectional area (CSA) in the right ventricle.
Compared to the control animals, ventricular CSA was 34% higher in the MCT animals
but only 1% greater in the MCT + CeO2 group (Figure 3-7, Panels A, B, C, D, P<0.05).
50
Although not quantitated, picrosirius red staining suggested that the degree of MCT-
induced collagen deposition was less in the CeO2 treated animals when compared to
that in the MCT only animals (Figure 3-7, Panel G).
In an effort to extend these findings, we next performed immunoblotting to
examine the effect of CeO2 nanoparticle treatment on myosin heavy chain expression.
Compared to control animals, MHC-β expression was 91% higher in the MCT animals
but only 3% higher in MCT + CeO2 group, respectively (Figure 3-8, Panel B, P<0.05).
Similarly, MCT-induced fibronectin expression was 113% higher in the MCT animals but
only 4% more in the MCT + CeO2 animals (Figure 3-8, Panel A, P<0.05).
CeO2 nanoparticles treatment decreases MCT-induced increases in oxidative
stress signaling
As an index of oxidative stress, we determined superoxide anion levels in tissue
sections obtained from the RV by quantifying dihyroethidium (DHE) fluorescence levels.
Compared to the control rats, the DHE fluorescence intensity was 71% and 12% higher
in the MCT and MCT + CeO2 nanoparticle treatment rats, respectively (Figure 3-9,
P<0.05). In an effort to extend these findings, we next examined the effects of PAH and
treatment on the amount of protein exhibiting 3-nitrotyrosine (3-NT) modification.
Compared to the control rats, 3-NT levels were 15% higher in the MCT animals (Figure
3-10, Panel A, P<0.05). Interestingly, CeO2 nanoparticle treatment actually reduced 3-
NT levels by 17% compared to that observed in the control animals (Figure 3-10, Panel
51
A, P<0.05). Likewise, compared to control rats, the amount of protein carbonylation was
14% and 4% higher in the MCT and MCT + CeO2 groups, respectively (Figure 3-10,
Panel B, P<0.05).
CeO2 nanoparticles treatment attenuates MCT-induced increases in AMPK alpha,
ERK1/2 and JNK phosphorylation
Compared to the control rats, the amount of p-AMPK alpha was 9% higher and
MCT animals and 11% lower in the MCT + CeO2 group (Figure 3-11, Panel A, P<0.05).
Total AMPK alpha levels were 1% and 9% lower in the MCT and MCT + CeO2
nanoparticle treated rats, respectively (Figure 3-11, Panel A, P<0.05). Compared to the
control rats, the amount of p-ERK 1/2 protein was 17% higher and 7% lower in MCT
and MCT + CeO2 animals, respectively (Figure 3-11, Panel B, P<0.05). Total ERK 1/2
was 12% and 2% higher in the MCT and MCT + CeO2 nanoparticle treatment rats,
respectively (Figure 3-11, Panel B).
Compared to the controls, p-JNK levels were 19% and 11% higher in the MCT
and MCT + CeO2 rats, respectively (Figure 3-11, Panel C, P<0.05). Total JNK protein
was unchanged and 6% higher in the MCT and MCT + CeO2 nanoparticle treatment
rats, respectively (Figure 3-11, Panel C).
In an effort to extend these findings, we next examined the effect of CeO2
nanoparticle treatment on the regulation of HSP-27 following MCT insult. Compared to
52
the control rats, the amount of p-HSP-27 was 88% and 9% higher in the MCT and MCT
+ CeO2 groups, respectively (Figure 3-12, Panel A, P<0.05). Similarly, total HSP-27
levels were 55% and 15% higher in the MCT and MCT + CeO2 nanoparticle treatment
rats, respectively (Figure 3-12, Panel B). No changes in the expression and
phosphorylation levels of HSP-70 and HSP-90 were observed (Data not shown).
CeO2 nanoparticles treatment attenuates MCT-induced increases in the
expression of NF-kB-p-50
Compared to the control rats, the amount of p-NF-kB-p-50 was 56% and 12%
higher in the MCT and MCT + CeO2 nanoparticles rats, respectively (Figure 3-12, Panel
B, P<0.05). Total NF-kB-p-50 was 10% lower in MCT group but only 3% lower with
CeO2 nanoparticle treatment (Figure 3-12, Panel B, P<0.05).
Effects of CeO2 nanoparticles treatment on serum protein biomarkers
Compared to control rats, MCT insult appeared to increase the amount of serum
C-reactive protein (CRP), TNF-α, CD40 Ligand, serum amyloid protein, von Willebrand
factor, vascular endothelial growth factor A, macrophage inflammatory protein-1 beta,
macrophage colony-stimulating factor-1, and tissue inhibitor of metalloproteinase-1,
which appeared to be attenuated with CeO2 nanoparticle treatment (Figure 3-13).
53
CeO2 nanoparticles treatment decreases MCT-induced cardiomyocytes apoptotic
signaling
The TdT-mediated dUTP nick end labeling (TUNEL) procedure was used to
determine the number of cardiomyocytes in the right ventricle that exhibited DNA
fragmentation. Compared to control rats, MCT insult was associated with an increase in
the number of TUNEL positive nuclei (Figure 3-14). To extend upon these findings, we
next examined the effects of treatment on Bax and Bcl-2 expression. Compared to
control rats, the ratio of Bax/Bcl-2 was 84% higher in the MCT animals but only 29%
higher in the MCT + CeO2 group (Figure 3-15, Panel A, P<0.05). Similarly, the amount
of cleaved caspase-3 (19 kDa) was 19% higher in the MCT animals but only 1% higher
in the MCT + CeO2 rats, respectively (Figure 3-15, Panel B, P<0.05).
54
Discussion
In spite of recent advances in the early diagnosis and treatment of pulmonary
arterial hypertension (PAH), right ventricular hypertrophy and failure remain the leading
cause of death seen with this disease [158]. The monocrotaline model of PAH is
characterized by elevated pulmonary pressure and increased tissue superoxide levels
that are associated with the development of right ventricular hypertrophy and failure
[46]. Previous data has suggested that antioxidant treatment in the rat monocrotaline
model of PAH can reduce oxidative stress levels and diminish the extent of right
ventricular remodeling [98, 159]. Supporting this contention, and consistent with
previous data demonstrating that CeO2 nanoparticles exhibit the ability to function as an
antioxidant both in vitro and in vivo [15, 110] we demonstrate that CeO2 nanoparticle
administration diminishes the development of right ventricular (RV) hypertrophy and
evidence of reduced MAPK, and pro-apoptotic signaling (Table 3-1, Figures 3-2, 3-3, 3-
4, 3-9,3-10,3-11, 3-12 and 3-15).
Effect of CeO2 nanoparticle administration on monocrotaline-induced pulmonary
hypertension and cardiac remodeling
Consistent with previous work [60], we observed that monocrotaline insult was
associated with increased pulmonary arterial pressure and right ventricular hypertrophy
(Table 3-1, Figures 3-2, 3-3 and 3-4). Specifically, our echocardiography data
demonstrated that MCT treated rats exhibited an 18% increase in pulmonary arterial
pressure (Figure 3-3 A), a 27% increase in mean pulmonary arterial diameter (Figure 3-
55
3 B), a 46% higher mean pulmonary arterial area (Figure 3-3 C), and a 14% increase in
right ventricular out flow tract diameter (Figure 3-3 D). More importantly, however, we
found that CeO2 nanoparticle treatment appeared to attenuate monocrotaline-induced
changes in pulmonary flow and the development of PAH and PAH associated RV
hypertrophy (Table 3-1, Figures 3-2, 3-3 and 3-4). These data are consistent with
previous work showing that CeO2 nanoparticles can protect cultured cardiac myocytes
from cigarette smoke extract [148] and hydrogen peroxide-induced oxidative damage in
culture [160].
In the present study, we noted monocrotaline-associated increases in RV tissue
weight, right ventricular anterior free wall thickness, and ventricular septal diameter was
diminished with CeO2 nanoparticle treatment (Table 3-1, Figures 3-4, 3-5). In addition,
our echocardiography analysis suggested the presence of altered atrial and ventricular
geometry including the bowing of the right atria towards left atria, septal flattening during
diastole, septal bulging into the LV cavity during systole, and tricuspid regurgitation in
the monocrotaline treated animals (Figure 3-6) but not in the CeO2 nanoparticle treated
animals.
In an effort to better characterize the hypertrophic response, we next investigated
whether monocrotaline induced increases in right ventricular mass were associated with
cardiomyocyte hypertrophy and fibrosis. To this end, we first measured cardiomyocyte
cross sectional area (CSA) in each of the three animal groups. Similar to our findings at
the whole tissue level, our histological studies and quantification of the data
56
demonstrated that CeO2 nanoparticle treatment diminished monocrotaline-induced
increases in right ventricular cardiomyocyte CSA (Figure 3-7), cardiac fibrosis (Figure 3-
7) and fibronectin protein levels (Figure 3-8 A).
In addition to changes in tissue morphology, cardiac hypertrophy is also
associated with changes in contractile protein expression. During the hypertrophic
process, the heart undergoes an up-regulation in the amount of β-MHC and a down-
regulation in the amount of α-MHC [161], which results in an increased efficiency of
cardiac contraction [162]. In the present study, and similar to previous work [163], we
found that MCT-induced increases in cardiac mass were associated with increases in β-
MHC expression (Figure 3-8 B) and importantly, that this up-regulation was attenuated
following CeO2 nanoparticle administration (Figure 3-8 B).
CeO2 nanoparticle administration diminishes indices of oxidative stress during
pulmonary arterial hypertension
It has been postulated that increases in oxidative stress may be involved in the
initiation and progression of cardiac hypertrophy in the MCT-PAH rat model [164, 165].
Consistent with previous work demonstrating the antioxidant activity of CeO2
nanoparticles [106], we found that CeO2 nanoparticle treatment was associated with
decreased tissue superoxide anion levels (Figure 3-9), diminished tyrosine nitration
(Figure 3-10 A) and an attenuation in the amount of MCT-induced protein carbonylation
(Figure 3-10 B). Whether the changes we observe in ROS stress are due entirely to the
57
antioxidant activity of CeO2 nanoparticles or if they may be related to increases in
antioxidant activity is currently unclear.
Recent work has suggested that AMPK, MAPK, HSP and NF-kB signaling are
regulated, at least in part, by oxidative stress [166-169] and that antioxidants can
function to block the activation of these molecules both in vitro and in vivo [170, 171].
Other data has demonstrated that these signaling pathways are important regulators of
ventricular remodeling following pressure overload [170, 172]. Supporting these
findings, we found that MCT-induced right ventricular hypertrophy was associated with
increased phosphorylation (activation) of AMPK-alpha, ERK 1/2, JNK-MAPK (Figure 3-
11), HSP-27 and NF-kB p50 (Figure 3-12). The effect of activation was diminished with
CeO2 nanoparticle treatment (Figure 3-11, 3-12). Whether these alterations in these
signaling pathways are responsible for the diminished cardiac remodeling we observed
with CeO2 nanoparticle treatment is currently unclear.
In humans, the involvement of leukocytes and macrophages in the inflammatory
process leading to the development of PAH associated vascular lesions is well
documented [173, 174]. It is thought that inflammation plays a significant role in MCT-
induced PAH [175]. We found that MCT administration was associated with the up-
regulation of several serum protein markers associated with inflammation (e.g. C-
reactive protein (CRP), tissue necrosis factor alpha (TNF-α)) and that this up-regulation
was attenuated following CeO2 nanoparticle administration (Figure 3-13).
58
CeO2 nanoparticle administration diminishes monocrotaline-induced increases in
apoptosis
Recent data has suggested that cardiomyocyte apoptosis may be involved in the
ventricular remodeling seen during MCT-induced PAH [176]. To examine this we
determined the number of TUNEL positive nuclei in right ventricular tissue sections
obtained from the control, MCT, and MCT + CeO2 nanoparticle groups. Consistent with
our present data we found that CeO2 nanoparticle treatment was associated with a
reduced number of TUNEL positive nuclei (Figure 3-14), diminished Bax/Bcl2 ratio
(Figure 3-15, Panel A), a decreased caspase-3 cleavage (activation) (Figure 3-15,
Panel B) which are suggestive of diminished cellular apoptosis.
Taken together, the data of the present study demonstrated that CeO2
nanoparticle administration attenuates MCT-induced increases in ventricular
hypertrophy and that this finding is associated with diminished levels of tissue oxidative
stress and apoptosis. Future experiments designed to directly uncover the
mechanism(s) of this finding may be warranted.
59
Table 3-1. Cerium oxide nanoparticle treatment attenuates monocrotaline-induced increases in right ventricular remodeling. * indicates significant difference from control animals, † indicates significant difference from the MCT only group.
60
Figure 3-1.Characterization of CeO2 nanoparticles size. Nanoparticle size was determined by DLS (Panel A) and AFM (Panel B).
61
Figure 3-2.CeO2 nanoparticle treatment improves pulmonary flow. Pulsed-wave Doppler echocardiography demonstrating normal, round shaped flow profile on day 1 (base line) of MCT rats (Panel A), triangular flow profile with mid systolic notching on day 28 of MCT rats progression to pulmonary arterial hypertension (Panel B) (arrow), - normal, round shaped flow profile on day 1 (base line) of MCT + CeO2 nanoparticle treated rats (Panel C), - attenuation of pulmonary arterial hypertension and restoring the normal, round shaped flow profile as of baseline observed with CeO2 nanoparticle treatment (Panel D).
62
Figure 3-3.CeO2 nanoparticle administration diminishes pulmonary artery remodeling. Pulsed-wave Doppler echocardiography quantified data form Pulmonary arterial pressure (Panel A), mean pulmonary arterial diameter (Panel B), mean pulmonary arterial area (Panel C), right ventricular outflow tract diameter (Panel D). Data are mean ± SEM (n= 6 rats/group). In the graphs, Grey bars represent base line and black bars represent final echocardiographic values. * indicates significant difference from base line values of MCT only group and † indicates significant difference from final values of MCT only group (p<0.05).
63
Figure 3-4.CeO2 nanoparticle treatment attenuates MCT-induced increases in right ventricle wall thickness. M-mode echocardiography images representing right ventricle wall thickness of the MCT only rats on day 1 (Panel A) and at day 28 (Panel B) or the MCT + CeO2 nanoparticle treated rats on day 1 (Panel C) and at day 28 (Panel D). The larger arrow in Panel B represents the increases in RV anterior wall thickness.
64
Figure 3-5.CeO2 nanoparticle treatment attenuates MCT-induced cardiac remodeling. Ventricle wall thickness (Panel A) and intra-ventricular septum diameter (Panel B) in the MCT only and MCT treated animals. Data are mean ± SEM, (n= 6 rats/group). In the graphs, grey bars represent the base line and black bars represent the final echocardiography values. * indicates significant difference from base line values of MCT only group and † indicates significant difference from final values of MCT only group (p<0.05).
65
Figure 3-6.PAH associated changes in cardiac function in the MCT only animals. Short-axis view of transthorasic echocardiogram showing right atrium bowing towards the left atrium (Panel A). Dilatation of the RV with septal flattening of the interventricular septum causing the left ventricle to conform into a “D” shape (Panel B). Tricuspid regurgitation (Panel C).
66
Figure 3-7.CeO2 nanoparticle administration attenuates monocrotaline-induced increases in cardiomyocyte cross sectional area and cardiac fibrosis. Dystrophin stained right ventricular sections from control (Panel A), MCT only (Panel B), and MCT + CeO2 nanoparticle treatment animals (Panel C). Quantification of cardiomyocyte cross-sectional area (Panel D). Picrosirius red staining was used to evaluate cardiac fibrosis in the right ventricles of control (Panel E), MCT only (Panel F) and MCT + CeO2 nanoparticle treatment animals (Panel G). Scale bar 50μm. Data are mean ± SEM (n = 3 rats/group). For each group 9 sections were analyzed. * indicates significant difference from the control and † indicates significantly different from the MCT only group (p<0.05).
67
Figure 3-8.MCT induced increases in RV fibronectin and MHC-beta are attenuated with cerium oxide nanoparticle treatment. Protein isolates from the right ventricle of control, MCT only, MCT + CeO2 nanoparticle treated rats were analyzed by immunoblotting for changes in fibronectin (Panel A) and myosin heavy chain-beta (Panel B) protein levels. Protein abundance was normalized to the expression of GAPDH. n=6 for each group. * indicates significant difference from the control and † indicates significantly different from the MCT only group (p<0.05).
68
Figure 3-9.CeO2 nanoparticle treatment decreases the incidence of superoxide levels in MCT-induced RV hypertrophy. Cardiac ROS determined by fluorescence intensity of ethidium bromide – stained nuclei. * Significantly different from control rats, † Significantly different from the MCT only rats (P<0.05).
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Figure 3-10.Cerium oxide nanoparticle treatment decreases MCT-induced increases in protein nitration and carbonylation. Protein isolates obtained from the right ventricles of control, MCT only and MCT + CeO2 nanoparticle treated rats were analyzed by immunoblotting for alterations in protein nitrosylation (Panel A) and carbonylation (Panel B). * Significantly different from control rats, † Significantly different from the MCT only rats (P<0.05). n= 6 animals / group
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Figure 3-11.Cerium oxide nanoparticle treatment decreases the MCT-induced increases in AMPK-alpha, ERK1/2 and JNK phosphorylation. Protein isolates obtained from the right ventricles of control, MCT only and MCT + CeO2 nanoparticle treated rats were analyzed by immunoblotting to determine the phosphorylation of AMPK-alpha (Panel A), ERK1/2 (Panel B) and JNK (Panel C). * Significantly different from control rats, † Significantly different from the MCT only rats (P<0.05). n= 6 animals / group.
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Figure 3-12. Cerium oxide nanoparticle treatment decreases the MCT induced oxidative stress associated phosphorylation of HSP-27 and NF-kB p50. Protein isolates of RV from control, MCT only and MCT + CeO2 nanoparticle treated rats were analyzed by immunoblotting to determine alterations in HSP-27(Panel A) and NF-kB regulation (Panel B). * Significantly different from control rats, † Significantly different from the MCT only rats (P<0.05).n = 6 animals / groups.
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Figure 3-13. Alterations in serum protein biomarkers with MCT and MCT + CeO2
nanoparticle treatment.
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Figure 3-14.CeO2 nanoparticles treatment decreases the incidence of TUNEL positive nuclei in MCT induced RV hypertrophy. Immunofluoresence labeling of TUNEL positive nuclei (FITC) in control (Panel A), MCT only (Panel B), MCT + CeO2 nanoparticle treated animals (Panel C). Bar 50 μm. n = 3 animals / group.
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Figure 3-15.Cerium oxide nanoparticle treatment is associated with diminished pro-apoptotic signaling. Protein isolates obtained from the right ventricles of control, MCT only and MCT + CeO2 nanoparticle treated rats were analyzed by immunoblotting to determine the Bax/Bcl-2 protein levels (Panel A) and caspase-3 cleavage (Panel B).*Significantly different from control rats, †Significantly different from the MCT only rats (P<0.05). n = 6 animals / group.
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PAPER 3
The following paper corresponds to the specific aim II
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Curcumin nanoparticles attenuate cardiac remodeling due to pulmonary arterial
hypertension
Key words:
pulmonary arterial hypertension, cardiac hypertrophy, curcumin, nanoparticles
Running title:
Effect of curcumin nanoparticles on cardiac hypertrophy following pulmonary arterial
hypertension.
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Abstract
Introduction: Pulmonary arterial hypertension (PAH) is a progressive disorder that can
cause right ventricular failure and death. Curcumin is a polyphenol compound that
exhibits anti-inflammatory and antioxidant properties whose clinical application has
been hindered by poor solubility, short biological half-life, and low bioavailability after
oral administration. Herein, we investigate whether curcumin nanoparticles (Cur NP) are
effective for the treatment of monocrotaline (MCT)-induced PAH in the laboratory rat.
Methods: Cur NP was prepared using an emulsion-diffusion-evaporation technique.
Male Sprague Dawley rats (175-200 gm) were randomized into control, MCT only or
MCT + Cur NP groups (n=6 each / group). PAH was induced by a single subcutaneous
injection of MCT (60 mg/kg). MCT + Cur NP treated rats received 50 mg/kg of Cur NP in
0.5 ml of distilled water i.p. daily for seven days while the control and MCT groups
received only distilled water. Echocardiography was done at the start of the study and
twenty eight days after MCT injection. Results: Compared to MCT only animals, Cur
NP administration was associated with diminished pulmonary arterial pressure (71 ± 2
vs. 67 ± 2 mmHg, P<0.05), reduced right ventricular wall thickness (0.27 ± 0.02 cm vs.
0.23 ± 0.01 cm,) and a decreased right ventricle weight/body weight ratio (0.74 ± 0.06
vs. 0.57 ± 0.02 mg/gm, P<0.05). MCT insult elevated the serum TNF-α levels (19%)
which were reduced with Cur NP treatment. Cur NP treatment attenuated cardiac
fibrosis and the development of RV hypertrophy. Conclusions: Taken together, these
data suggest that Cur NP may attenuate MCT-induced pulmonary arterial hypertension
and right ventricle hypertrophy. WC: (261)
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Introduction
Pulmonary arterial hypertension (PAH) is a debilitating disease characterized by
progressive obstruction of the pulmonary arteries which can cause a rise in pulmonary
vascular resistance (PVR) and right ventricular (RV) hypertrophy [177]. The median
survival rate in PAH patients without treatment is 2.8 years after diagnosis and the
estimated survival rates at 1-, 3- and,5-years is 68%, 48% and 34%, respectively [178].
Not unexpectedly, RV failure is the most common cause of death in PAH patients (~ 30-
50% of deaths) [179]. Current therapeutic treatments, although effective in improving
symptomology, only delay disease progression. As of yet, a cure for PAH has not been
found and long term survival remains poor [180].
Curcumin (diferuloylmethane) is a polyphenolic compound in the curry spice
turmeric that has been used in Indian Ayurvedic medicine for the treatment of
inflammatory diseases [181]. Recent data has shown that curcumin exhibits anti-
inflammatory, anti-oxidant and anti-proliferative activity [28], most likely through its
ability to scavenge free radicals, diminish cytokine expression (e.g, tumor necrosis
factor (TNF)- α, interleukin (IL)-1, and IL-6) and inhibit the activity of nuclear factor
kappa-light-chain-enhancer of activated B cells (NF-kB) [182, 183]. Curcumin has been
used to improve left ventricular function in rabbits subjected to pressure-overload [184]
and inhibit cardiac inflammation and fibrosis in rats [35].
Thus far, the biomedical application of curcumin has been hampered due to its
poor water solubility and short biological half-life [38]. Recent studies, using
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nanoparticle-based systems, have shown increased curcumin bioavailability [39, 137].
Here, we examine whether the systemic administration of curcumin nanoparticles (Cur
NP) can diminish PAH-induced cardiac hypertrophy in the rat. To induce PAH, rats were
subjected to a single subcutaneous injection of monocrotaline (MCT; 60 mg/kg). MCT is
a macrocylic pyrrolizidine alkaloid that when metabolized by cytochrome P450
(CYP3A4) is converted to dehydromonocrotaline, which selectively injuries the vascular
endothelium of lung causing an increase in pulmonary arterial pressure [185]. Given the
pivotal role that inflammation plays in the development of PAH [10], we hypothesized
that Cur NP treatment would diminish the expression of inflammatory mediators and
that this reduction in inflammation would be associated with decreased right ventricular
hypertrophy.
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Materials and Methods
PAH model and experimental design
All procedures were approved by the Institutional Animal Care Committee of
Marshall University using the criteria outlined by the American Association of Laboratory
Animal Care (AALAC). Male Sprague Dawley rats (7 weeks old, 175-200 gm) were
obtained from Hilltop laboratories (Scottsdale, PA) and were housed two per cage in an
AALAC approved vivarium at Marshall University. The animal housing facility was
operated with a 12 hour: 12 hour dark – light cycle and maintained at 22 ± 2°C. Rats
were provided with food and water ad libitum and allowed to acclimate to the housing
facilities for at least two weeks before experimentation. Animals were randomly
assigned to one of three experimental groups 1.) Control (n=6), 2.) MCT only (n=6), or
3.) MCT + Cur NP treatment (n=6). PAH was induced by a single injection of MCT (60
mg/kg s.c.) (Sigma-Aldrich, St.Louis, MO) as described previously [149]. Animals
injected with MCT were given either Cur NP (50 mg/kg i.p.) or vehicle (DI water) once
daily for seven days following the MCT injection.
Preparation of curcumin nanoparticles
Curcumin (100 mg) was solubilized in dichloromethane (10 ml), and 1 ml of this
solution was sprayed into deionized water (25 ml) drop wise at a flow rate of 0.2 ml/min
in 5 min under ultrasonic conditions, with an ultrasonic power of 100 W and a frequency
of 30 kHz. After sonication for 10 min, the contents were stirred at 400-800 rpm at room
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temperature for about 30 min until a clear orange-colored solution was obtained. The
solution was concentrated under reduced pressure at 50°C and then dried at 40°C to
obtain an orange powder.
Scanning Electron Microscopy
Curcumin nanoparticles’ size and morphology were characterized using scanning
electron microscope (SEM) (JEOL’s JSM 7400 SEM). The SEM was operated at 5 kv.
The samples were prepared by fixing the curcumin nanoparticles to a microscope
holder using a conducting carbon strip.
Atomic Force Microscopy
Curcumin nanoparticles (1µg/µl) were deposited on freshly cleaved mica (V1
mica, SPI Inc, West Chester, PA). After a 10 min incubation period, the surface was
gently rinsed with deionized (DI) water to remove any unbound nanoparticles and the
sample was dried under nitrogen. Particles were imaged in noncontact mode at a 319
kHz with a scan speed 0.5 Hz using a Nano-R microscope (Pacific Nanotechnology,
Santa Clara, CA) equipped with a TM300-A probe (Sensa Probes Inc, Santa Clara,
CA.).
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Echocardiography
Echocardiography was performed using a Philips 5500 ultrasound machine with
an S-12 transducer frequency at the beginning of the study and at 4 weeks to assess
pulmonary and right ventricular structure and function as outlined previously [186].
Briefly, rats were anesthetized with an intraperitoneal injection of ketamine and xylazine
(4:1, 50 mg/kg). Once anesthetized, the ventral thorax area was shaved and covered
with ultrasonic transmission gel. Electrocardiograms were recorded simultaneously to
monitor heart rate. Echocardiography images were obtained in the 2-dimensional
guided M-mode and Doppler M-mode from parasternal long-axis (PLAX) and
parasternal short-axis (PSAX) views. M-mode measurements of the RV, LV and wall
thickness were performed from PSAX image plane. Pulmonary artery flow was
measured at the pulmonary valve level and pulmonary artery diameter was measured at
the level of pulmonary out flow tract during systole using the PSAX view. An apical four-
chamber view was employed to measure end-diastolic right ventricular area. Pulsed-
wave Doppler was used to measure pulmonary artery acceleration time and flow.
Measurements from six rats in each group were obtained for the assessment of cardiac
structure and function.
Tissue processing and immunoblotting
RV cardiac muscles were pulverized in liquid nitrogen using a mortar and pestle
until a fine powder was obtained and homogenized in TPER lysis buffer (Pierce,
Rockford, IL) containing protease (P8340, Sigma-Aldrich, Inc., St. Louis, MO) and
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phosphatase inhibitors (P5726, Sigma-Aldrich, Inc., St. Louis, MO). After incubation on
ice for 30 min, the homogenate was collected by centrifuging at 12,000 g for 5 min at
4°C. The protein concentration of homogenates was determined in triplicate via the
Bradford method (Fisher Scientific, Rockford, IL). Homogenate samples were boiled in a
Laemmli 2X sample buffer (Sigma-Aldrich, Inc., St. Louis, MO) for 5 min before loading
forty micrograms of total protein from each sample on 10% PAGEr Gold Precast gels
(Lonza, Rockland, ME). Proteins were transferred onto nitrocellulose membranes using
standard conditions [187]. For immunodetection, membranes were blocked for 1 h at
room temperature in blocking buffer (5% non-fat dry milk in TBS-T (20mM Tris-base,
150mM NaCl, 0.05% Tween-20), pH 7.6), serially washed in TBS-T at room
temperature, then probed with primary antibodies for the detection of β-myosin heavy
chain (M 8421, Sigma Aldrich, St. Louis, MO), fibronectin (AB23750, Abcam,
Cambridge, MA), p-AMPK-α (#2535), AMPK-α (#2532), HSP-27 (#2442s), HSP-70
(#4872), Bax (#2772), Bcl2 (#2870s), Caspase-3 (#9662), cleaved caspase-3 (#9661s),
caspase-9 (#9506), ERK1/2 (#9106s), p-ERK1/2 (#4377s), p-p-38 (#9216L), p-38
(#9212), p-JNK (#9251s), JNK (#9252), glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) (#2118), (from Cell Signaling Technology, Inc., Beverly, MA) and 3-
nitrotyrosine (SC-55256) (from Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
Membranes were incubated overnight at 4°C in primary antibody buffer (5% BSA in
TBS-T, pH 7.6, primary antibody diluted 1:1,000), followed by washing in TBS-T (3 x 5
min), and incubation with HRP-conjugated secondary antibody (anti-rabbit ((#7074)) or
anti-mouse ((#7076)), Cell Signaling Technology, Inc., Danvers, MA) in blocking buffer
for 1 h. After removal of the secondary antibody, membranes were washed (3 x 5 min)
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in TBS-T and protein bands were visualized following reaction with ECL reagent
(Amersham ECL Western Blotting reagent RPN 2106, GE Healthcare Bio-Sciences
Corp., NJ). Target protein levels were quantified by AlphaView SA image analysis
software (Alpha InfoTech, San Leandro, CA) and normalized to GAPDH.
Histology
Picrosirius red staining was used to assess the collagen content deposition in the
right ventricle as detailed elsewhere [188]. Briefly, frozen tissue sections were fixed
with 95% alcohol for 1 min, washed three times with running tap water, and then stained
with hematoxylin for 8 min and picrosirius red for 20 min. After dehydration, sections
were mounted and visualized at 20x or 40x magnification using an Olympus BX51
microscope (Olympus, Center Valley, PA).
Determination of cytokine levels in serum by ELISA method
Serum concentrations of tumor necrosis factor (TNF)-α and interleukin-1β (IL-
1β) were measured using commercially available enzyme-linked immunosorbent assay
(ELISA) kits (BD Bioscience, San Diego, CA) according to the manufacturer’s
instructions [24]. Absorbance measurements for each plate were taken at 450 nm using
ELISA plate reader (BioTek, Winooski, Vermont).
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Determination of cardiac TNF-α and IL-1βmRNA levels
RV tissue was homogenized in TRI reagent (Ambion, Austin, TX) at a
concentration of 1ml for every 100mg of tissue as detailed elsewhere [189]. Briefly, after
5 minutes of incubation with TRI reagent, the homogenates were centrifuged (12,000 g
for 15 min at 4 °C). The supernatant was collected and mixed with 200 μl of chloroform
(Sigma-Aldrich, Inc., St. Louis, MO). After a 10 min incubation at room temperature, the
aqueous phase of the mixed solution was collected via centrifugation (12,000 g for 15
min at 4 °C) and then mixed with 500 μl of isopropanol (Sigma-Aldrich, Inc., St. Louis,
MO). RNA pellets were precipitated (12,000 x g for 10 min at 4 °C), washed with 75%
ethanol, and then dried under vacuum to remove residual ethanol. RNA pellets were
dissolved in 50 μl of 10mM Tris, pH8.0 and 1mM EDTA. RNA was quantified at 260 nm
using a NanoVue UV-Vis Spectrophotometer (GE Healthcare, Piscataway, NJ), and
RNA integrity was confirmed using a RNA Nanokit and Agilent 2100 Bio-analyzer
(Agilent Technologies, Santa Clara, CA). RNA samples were stored at -80°C until
further use.
Reverse transcription coupled with RT-PCR was used to quantify the relative
abundance of TNF-α and IL-1β mRNA levels in control, MCT and MCT + Cur NP
animals. Complementary DNA (cDNA) libraries were synthesized using a High Capacity
cDNA Reverse Transcription Kit (Applied Biosystems Inc., Foster City, CA) as detailed
elsewhere [189].
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RT-PCR was performed using the Power SYBER Green PCR Master Mix
(Applied Biosystems Inc., Foster City, CA) as outlined previously [190]. PCR reactions
were run on an ABI 7000 Real-Time PCR system (Applied Biosystems Inc., Foster City,
CA) using the following cycle conditions: 50°C for 2min, 95°C for 10min, followed by 40
cycles at 95°C for 15s and 60°C for 1min as detailed elsewhere [191]. All experiments
were repeated in triplicate. β-actin or glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) were used as internal controls. The forward and reverse primers were
purchased from Integrated DNA technologies (San Diego, CA). TNF-α Gene bank
accession number: NM_012675.3 with forward primer (5'-3') -
CCAGAACTCCAGGCGGTGTC, reverse primer (5'-3')-GGCTACGGGCTTGTCACTCG,
IL-1β Gene bank accession number: NM_031512.2 forward primer (5'-3') -
TGCCCGTGGAGCTTCCAGGA, reverse primer (5'-3')-TCCAGCTGCAGGGTGGGTGT.
The expression of TNF-α and IL-1β mRNA levels were normalized to the geometric
means of β-actin and GAPDH mRNA levels, and the 2-Δct method was used to calculate
the relative mRNA expression levels.
Data analysis
Results are presented as mean ± SEM. Data were analyzed using SigmaPlot
11.0 software. One-way analysis of variance (ANOVA) was used for overall
comparisons. The Student Newman Keuls post hoc test was used where appropriate to
determine differences between groups. The level of significance accepted a priori was P
≤ 0.05.
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Results
Curcumin NP characterization
The particle size analysis of the nanoparticles was performed by scanning
electron microscope (SEM) and atomic force microscope (AFM). The SEM of powdered
sample showed the particle size to be in the range of 300 ± 50 nm (Figure 3-16 A, B)
and AFM of aqueous dispersion showed the particles to be approximately 50 nm in size
(Figure 3-16 C).
Curcumin NP treatment decreases PAH induced right ventricular tissue weight
Compared to control animals, body weight was 19% less in the MCT and MCT +
Cur NP rats, respectively (P<0.05) (Table 3-3). Conversely, the ratio of right ventricle
(RV) weight to body weight ratio was 27% and 10% higher in the MCT and MCT + Cur
NP animals (P<0.05). Similarly, the lung weight to body weight ratio was 356% and
214% higher in the MCT and MCT + Cur NP groups, respectively (P<0.05).
Curcumin NP treatment decreases MCT-induced changes in pulmonary arterial
pressure and cardiac remodeling
Compared to the MCT + Cur NP animals, echocardiographic analysis of
pulmonary arterial flow demonstrated a mid-systolic notch in the MCT animals (Figure
3-17, Panels A-D). Consistent with this finding, pulmonary arterial pressure (PA Pr) was
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18% and 10% higher in the MCT and MCT + Cur NP animals, respectively (Figure 3-17
E, P<0.05). Likewise, the slope of the pulmonary artery acceleration vector was 200%
and 52% in higher in the MCT and MCT+ Cur NP animals, respectively (Figure 3-17 F,
P<0.05). Similar to our findings at the whole tissue level, echocardiography analysis
demonstrated that Cur NP treatment attenuated MCT-induced increases in RV anterior
free wall thickness (Figure 3-18).
To examine the molecular changes that might accompany PAH-induced changes
in RV thickness we next performed immunoblotting to examine myosin heavy chain
expression. Compared to the control animals, MHC-β expression was 278% and 106%
higher in the MCT and MCT + Cur NP treated animals, respectively (Figure 3-19 A,
P<0.05). Similarly, picrosirius red staining of the right ventricle suggested that the
degree of MCT-induced collagen deposition was less in the Cur NP treated animals
when compared to that in the MCT only treated animals (Figure 3-19, Panel B, C and
D). In an effort to extend these findings, we next examined the effect of Cur NP
treatment on fibronectin expression with MCT treatment. Compared to that observed in
the control animals, fibronectin protein levels were 95% and 57% higher in the MCT and
Cur NP MCT animals, respectively (Figure 3-19, Panel E, P<0.05).
Curcumin nanoparticle treatment decreases serum TNF-α levels
Compared to the control animals, serum TNF-α levels were 19% and 5% higher
in the MCT and MCT + Cur NP animals, respectively (Figure 3-20, Panel A, P<0.05).
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The amount of serum IL-1βwas not changed in the MCT or MCT + Cur NP animals
(data not shown). At the tissue level, the abundance of TNF-α level was 124% and 2%
higher in the MCT and MCT + Cur NP animals, respectively (Figure 3-20, Panel B,
P<0.05) and IL-1β message level was 227% higher and 7% lower in the MCT and MCT
+ Cur NP animals, respectively (Figure 3-20, Panel C, P<0.05).
Curcumin nanoparticle treatment decreases AMPK alpha phosphorylation but not
MAPK or apoptotic signaling
Compared to the control animals, the amount of p-AMPK alpha was decreased
by 36% and 22% in the MCT and MCT + Cur NP animals, respectively (P<0.05) (Figure
3-21). No differences in the ratio of Bax/Bcl-2, the amount of cleaved caspase-3, p-
ERK1/2, p-p38, or p-JNK1/2 was observed with nanoparticle treatment.
Curcumin nanoparticle treatment decreases MCT-induced increases in oxidative
stress signaling
Compared to the control animals, the amount of HSP 27was 25% and 15%
higher in the MCT and MCT + Cur NP animals (Figure 7, Panel A, P<0.05). Like that
observed for HSP 27, HSP70 protein levels were 42% and 22% higher in the MCT and
MCT + Cur NP animals (Figure 3-22, Panel B, P<0.05). Likewise, 3-nitrotyrosine protein
was 23% higher in the MCT animals and 11% higher in the MCT + Cur NP animals
(Figure 3-22, Panel C, P<0.05).
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Discussion
Recent studies have suggested that curcumin may be effective for the treatment
of neurodegenerative, pulmonary, autoimmune, and neoplastic diseases [28]. In
addition, other data have demonstrated that curcumin is able to prevent cardiac
hypertrophy and heart failure in a murine model of salt-induced hypertension [35]. In an
effort to extend these findings, the present study investigated whether treatment with
curcumin nanoparticles was able to attenuate the development of monocrotaline-
induced RV hypertrophy following pulmonary arterial hypertension. Herein, we
demonstrate that the administration of curcumin nanoparticles (50 mg/kg /i.p.) for seven
days is capable of diminishing the development of pulmonary arterial pressure and right
ventricular remodeling in the rat MCT model of pulmonary arterial hypertension (Figures
3-17, 3-18, and 3-19). These changes appear to be associated with decreased serum
TNF-α levels and evidence of diminished hypertrophic signaling and oxidative stress in
the heart (Figures 3-20, 3-21, and 3-22). Taken together, these results are consistent
with the possibility that curcumin nanoparticle treatment may be efficacious for the
treatment of pulmonary arterial hypertension.
The most commonly used animal model of PAH utilizes monocrotaline to
selectively injure the pulmonary vasculature, which causes an increase in pulmonary
vascular resistance [70, 192]. Similar to the previous reports [186, 193], our
echocardiography data demonstrated that MCT insult resulted in the obstruction to
pulmonary arterial flow and increases in pulmonary artery pressure (Figure 3-17). These
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changes in vascular function pressure, in turn, are associated with increased right
ventricular mass and free wall thickness (Table 3-3, Figure 3-18).
In an effort to better understand the nature of RV hypertrophy we next examined
if MCT insult was associated with changes in contractile protein expression and an
increase in non-contractile tissue. Consistent with previous data [161, 194], we found
that MCT-insult was associated with an increase in the amount of slow β-myosin heavy
chain (MHC) isoform, tissue fibrosis and fibronectin protein expression in the pressure
overloaded right ventricle (Figure 3-19, Panels A, B, C). Importantly, we found that the
administration of Cur NP was associated with evidence of diminished cardiac
hypertrophy and tissue fibrosis (Table 3-3, Figures 3-18 and 3-19).
How Cur NP administration may function to diminish cardiac hypertrophy and the
development of fibrosis is currently unclear. Previous reports have suggested that
curcumin can act as an anti-fibrotic agent and that the chronic administration of
curcumin can attenuate the development of pulmonary fibrosis in the rat. It has been
hypothesized that this effect may be related to the ability of curcumin to reduce
inflammation [33]. Consistent with this possibility, and the work of others using bulk
curcumin preparations [195], we found that Cur NP associated decreases in cardiac
fibrosis were associated with diminished serum TNF-α protein and decreased
ventricular TNF-α mRNA expression (Figure 3-20, Panels A, B). Whether these
changes in TNF-α regulation are directly responsible for the changes we see in cardiac
remodeling is unknown and will require further experimentation.
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The molecular events that govern the development of cardiac hypertrophy are
not entirely clear. Recent data has suggested that the phosphorylation (activation) of α-
AMPK functions to inhibit cardiac hypertrophy following chronic pressure overload [196].
Supporting this contention, we observed that MCT-induced cardiac hypertrophy was
associated with decreases in α-AMPK phosphorylation (Figure 3-21). As expected from
our analysis of cardiac hypertrophy at the tissue level, we noted that Cur NP
administration appeared to up-regulate α-AMPK phosphorylation (Figure 3-21 Panel A).
Whether this up-regulation of α-AMPK phosphorylation is related to decreases in
cellular inflammation or changes in TNF-α expression is unknown and will require
further experimentation.
It has been reported that the increases in tissue inflammation/oxidative stress
seen during pressure-overload induced RV hypertrophy can cause an up-regulation in
heat shock protein expression [197, 198]. Supporting this contention, we observed that
PAH induced cardiac hypertrophy was associated with increased expression of HSP-27
and HSP-70 in the right ventricle (Figure 3-22). Consistent with our hypertrophy data,
we also noted that Cur NP administration was associated with a down-regulation of the
expression of HSP-27 (Figure 3-22 Panel A) and HSP-70 (Figure 3-22 Panel B) and
decreased levels of oxidative stress. In addition to increases in HSP expression,
pressure overload has also been shown to increase tissue oxidative stress [199].
Similar to our HSP data, we found that MCT-insult caused an elevation in 3-nitrotyrosine
levels and that 3-nitrotyrosine levels were diminished with Cur NP administration (Figure
3-22 Panel C). These data are consistent with the previous work of others showing that
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curcumin treatment prevented diabetes-induced increases in retinal nitro tyrosine levels
[200].
In conclusion, the data of the present study demonstrate that Cur NP treatment
can attenuate the development of RV hypertrophy in the rat MCT model of PAH.
Although the exact mechanism of action is not clear, our data suggest that the
attenuated hypertrophy we observe following Cur NP administration may be related to
decreases in TNF-α levels, the activation of α-AMPK, and diminished oxidative stress.
Although prior work studies have reported that curcumin administration can prevent
cardiac remodeling it should be noted that the curcumin levels used in this study were
much higher (150mg/kg/day for 7 weeks) than those used in the present study [201].
Whether Cur NP treatment is effective in preventing other types of cardiac hypertrophy
will require additional study.
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Table 3-2. Body and organ weight for control, MCT only and MCT + Cur NP rats collected 4 weeks after injection of MCT (60mg/kg). Values are expressed as mean ± SEM; n=6 for each group. * P<0.05 significantly different from control rats and † P<0.05 significantly different from MCT only rats.
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Figure 3-16.Optical characterization of curcumin nanoparticles. Curcumin nanoparticles were observed under SEM (Panels A, B) and AFM imaging (Panel C).
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Figure 3-17.Curcumin nanoparticle treatment improves MCT-induced decreases in pulmonary artery flow. Representative image from a MCT only animal at day 1 (Panel A) and at day 28 (Panel B). Note mid-systolic notch at day 28 (arrow). Images from a MCT + Cur NP treated animal at day 1 (Panel C) and at day 28 (Panel D). Curcumin nanoparticle treatment decreases MCT-induced increases in pulmonary artery flow acceleration (Panel E) and increases the slope of the pulmonary artery acceleration vector (Panel F). Values are expressed as mean ± SEM; n=6 for each group. * P<0.05 vs. the control group; † P<0.05 vs. the MCT + Cur NP group.
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Figure 3-18.Curcumin nanoparticle treatment attenuates PAH induced increases in RV anterior free wall thickness. M-mode echocardiography images of a MCT only animal at day 1 (Panel A), and at day 28 (Panel B). Images from a MCT + Cur NP treated animal at day 1 (Panel C) and at day 28 (Panel D).Quantification of Doppler echocardiography (Panel E). Values are expressed as mean ± SEM; n=6 for each group. * P<0.05 vs. the base line of MCT only group;
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Figure 3-19.Curcumin nanoparticle treatment decreases PAH-induced cardiac remodeling. Myosin heavy chain-β abundance in the RV as determined by immunoblotting and normalized to the amount of GAPDH (Panel A). Picrosirius red staining of right ventricular tissue from control (Panel B), MCT only (Panel C) and MCT + Cur NP treated animals (Panel D). Fibronectin protein abundance normalized to the expression of GAPDH (Panel E). Scale bar 50 μm. Graphical data are expressed as mean ± SEM; n=6 for each group. * P<0.05 vs. the control group; † P<0.05 vs. the MCT + Cur NP group.
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Figure 3-20. Curcumin nanoparticle treatment diminished MCT-induced increases in inflammatory cytokines. Serum protein TNF-α levels (Panel A).Right ventricular mRNA expression levels of TNF-α (Panel B) and IL-1β (Panel C). Values are expressed as mean ± SEM; n=5 for each group. * P<0.05 vs. the control group; † P<0.05 vs. the MCT + Cur NP group.
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Figure 3-21. Curcumin nanoparticle treatment alters AMPK-α activation but does not change MAPK or apoptotic signaling. Protein expression was determined in RV tissue by immunoblotting and normalized to the expression of GAPDH. Values are expressed as mean ± SE; n=6 for each group. * P<0.05 vs. the control group; † P<0.05 vs. the MCT + Cur NP group.
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Figure 3-22.Curcumin nanoparticle treatment attenuates PAH-associated increases in heat shock protein and protein nitrosylation. The abundance expression of Hsp-27 (Panel A), Hsp-70 (Panel B) and nitro tyrosine (Panel C) were determined by immunoblotting and normalized to the expression of GAPDH. Values are expressed as mean ± SEM; n=6 for each group. * P<0.05 vs. the control group; † P<0.05 vs. the MCT + Cur NP group.
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CHAPTER IV
GENERAL DISCUSSION
Pulmonary arterial hypertension (PAH) affects almost 15-52 people per million
people [202]. As of yet, no cure for PAH has been found and the long term survival
remains poor. Even with modern medical therapy, most of the patients experience
progression of their disease and many are referred for lung transplantation [5]. Right
ventricular failure is the leading cause of death in patients with PAH. The exact
molecular mechanisms underlying the structural and functional alterations that lead to
the RV dysfunction are not well understood. Although animal models lack the
development of the classical plexiform lesions seen in human PAH, it is thought that the
rat monocrotaline injury is useful for studying the pathological processes seen in human
PAH.
Recent data suggests that increases in pulmonary artery resistance are the main
causative agent in the development of PAH-induced RV hypertrophy and RV
dysfunction [203]. In addition to increased mechanical loading, other data has
demonstrated that the development of right heart failure may also be mediated, at least
in part, by increases in reactive oxygen species and inflammatory mediators [9].
The primary purpose of this study was to determine the efficacy of CeO2 and
curcumin nanoparticles to attenuate monocrotaline-induced PAH and RV hypertrophy.
To accomplish this goal, we examined the effects of nanoparticle administration on
pulmonary artery pressure/flow, RV thickness as determined by echocardiography, RV
103
mass, RV fibrosis, myocyte cross sectional area, and indices of oxidative stress and
myocardial apoptosis.
Effects of CeO2 nanoparticle administration on monocrotaline-induced RV
hypertrophy in Sprague-Dawley rats
Previous study has shown that CeO2nanoparticles exhibit antioxidant activity
both in vitro and in vivo [106]. Other data has shown that CeO2 nanoparticles may also
exhibit catalytic activities similar to superoxide dismutase and catalase [109]. Recent
work has shown that the administration of CeO2 nanoparticles can inhibit left ventricular
dysfunction and myocardial oxidative stress in the MCP-1 transgenic mouse model of
cardiomyopathy [23]. Similarly, Kong and coworkers demonstrated that CeO2
nanoparticles could inhibit retinal degeneration in tubby mice and that this finding was
associated with diminished oxidative stress and a down-regulation of apoptotic signaling
[109].
Here, we observed that that CeO2 nanoparticle administration is capable of
preventing monocrotaline-induced PAH and RV hypertrophy in male Sprague Dawley
rats (Chapter 3, Paper #1). Specifically, our echocardiography and histological data
demonstrate that CeO2 nanoparticle administration was associated with a lower RV
weight to body weight ratio, decreased cardiomyocyte fiber cross sectional area, and
diminished RV myocardial fibrosis.
104
Other data from this study suggest that CeO2 nanoparticle treatment is able to
diminish monocrotaline-induced increases in cellular ROS and cardiomyocyte apoptosis
(Chapter 3, Paper #1). Specifically, we observed that CeO2 nanoparticle administration
was associated with decreases in superoxide anion, diminished protein nitrosylation, an
attenuation in apoptotic signaling ( Bax/Bcl2 ratio and caspase-3 cleavage), reduced
activation of stress-associated signaling ( p-ERK1/2, p-JNK, HSP-27, and NF-kB p-50)
and the down-regulation of inflammatory cytokines. Whether these changes in cellular
ROS and signaling are directly responsible for the attenuation of monocrotaline-induced
RV hypertrophy will require further investigation.
Effects of curcumin nanoparticle administration on monocrotaline-induced right
ventricular hypertrophy in Sprague Dawley rats
Curcumin (diferuloylmethane) is a polyphenolic compound found in turmeric that
has been used in the ancient Indian Ayurvedic medicine for centuries to treat biliary
disorders, anorexia, cough, diabetic wounds, liver disorders, rheumatism, and
respiratory conditions [116]. Curcumin is a phenolic compound that has been shown to
exhibit anti-oxidant, anti-inflammatory, anti-microbial, and anti-carcinogenic activities
[26, 27, 177]. Recent studies have shown that curcumin administration can prevent
cardiac hypertrophy and heart failure in a murine model of salt-induced hypertension
[201]. In addition, curcumin has been shown to diminish the deleterious effects of
inflammatory cytokines [204], and to inhibit pulmonary fibrosis in rats [33].
105
Similarly, Yao and coworkers demonstrated that curcumin could inhibit the
pressure overload left ventricular hypertrophy in rabbits and that this finding was
associated with diminished tumor necrosis factor-alpha levels, decreased matrix
metalloproteinase-2 expression and the inhibition of collagen remodeling [184].
Here for the first time we observed that Cur NP administration is capable of
preventing monocrotaline-induced PAH and RV hypertrophy in male Sprague Dawley
rats (Chapter 3, Paper #2). Our echocardiography and histological data demonstrate
that Cur NP administration was associated with diminished RV weight to body weight
ratio, decreased RV anterior free wall thickness and diminished RV myocardial fibrosis.
We also observed that Cur NP administration was associated with decreases in protein
expression of cardiac remodeling ( β-MHC and fibronectin), decreases in oxidative
stress associated signaling ( HSP-27, HSP-70, and nitro-tyrosine) and the down-
regulation of inflammatory cytokines. Whether these changes in molecular signaling are
directly responsible for the amelioration of monocrotaline-induced RV hypertrophy will
require further investigation.
SUMMARY
1. Monocrotaline insult is associated with significant increases in pulmonary arterial
pressure and right ventricular hypertrophy in the male Sprague Dawley rats.
106
2. Monocrotaline-induced right ventricular hypertrophy is associated with increases
in indices of oxidative stress (superoxide anion, protein carbonylation) and
protein nitrosylation.
3. The administration of CeO2 nanoparticles appeared to attenuate monocrotaline-
induced increases in pulmonary arterial pressure along with right ventricular
cardiomyocyte cross sectional area and fibrosis.
4. The administration of CeO2 nanoparticles attenuated monocrotaline-induced
increases in oxidative stress.
5. The administration of CeO2 nanoparticles reduced monocrotaline-induced
increases in ERK1/2, JNK, HSP-27 and NF-kB activation.
6. CeO2 nanoparticles treated animals exhibited diminished monocrotaline
associated apoptotic signaling.
7. Curcumin nanoparticle administration attenuated monocrotaline-induced
increases in right ventricular hypertrophy and fibrosis.
8. Curcumin nanoparticle administration was associated with diminished
monocrotaline associated increases in serum TNF-α and decreased cardiac heat
shock protein expression.
107
Figure 4-1. Possible role of CeO2 nanoparticles in attenuation of MCT-induced RV
hypertrophy following pulmonary arterial hypertension.
108
Figure 4-2. Possible role of Cur NP in cardiac remodeling following pulmonary
arterial hypertension.
109
FUTURE DIRECTIONS
The present study investigated the efficacy of using nanoparticles to attenuate
the development of the cardiac hypertrophy seen following pulmonary arterial
hypertension in the male Sprague Dawley rats. Our data suggest that the administration
of CeO2 and curcumin nanoparticles is capable of diminishing MCT-induced increases
in pulmonary arterial pressure and indices of right ventricular hypertrophy, myocardial
oxidative stress, and apoptosis in male Sprague Dawley rats. Although these results are
promising, additional studies are required to determine the exact mechanism(s)
responsible for these findings. Future areas of study could include the following areas of
inquiry.
It is possible that the distribution and accumulation of nanoparticles in the body
may pose deleterious effects on the health of individuals. Determination of
CeO2/curcumin nanoparticles levels in the blood and tissues (pulmonary artery, lung,
heart, liver and kidney) is of interest to determine whether treatment with these
materials is potentially hazardous. Findings from the present study suggest that
treatment with CeO2/curcumin nanoparticles is able to diminish MCT-induced increases
in cardiac oxidative stress in male Sprague Dawley rats. Little is currently known about
how the administration of CeO2/curcumin nanoparticles may provide cardioprotective
effects. Previous reports suggested that CeO2 nanoparticles may act as antioxidant
[107]. Similarly, curcumin has been suggested to exhibit antioxidant and anti-
inflammatory effects [205]. Future studies could be directed at investigating whether the
110
effect of CeO2 or curcumin nanoparticle treatment is due to increased free radical
scavenging or due to the increased expression of antioxidant enzymes.
Previous studies have shown that CeO2 nanoparticles can attenuate the effects
of chronic inflammation by acting as free radical scavengers [15]. To directly assess this
possibility in endothelial cells, bovine pulmonary artery endothelial cells (BPAE) cells
could be exposed to various concentrations of H2O2 with and without CeO2
nanoparticles in the media. Treatment effectiveness could be established with the
measurement of 2’,7’-dichlorfluorescein-diacetate (DCFH-DA) fluorescence intensity or
by assessing cell viability with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay.
Findings from the present study suggest that CeO2/curcumin nanoparticle
treatment may attenuate MCT-induced RV hypertrophy following pulmonary arterial
hypertension. How CeO2/curcumin nanoparticle administration attenuated pulmonary
arterial pressure is not clear. Future studies could explore whether nanoparticle
administration has an effect on MCT-induced pulmonary artery fibrosis and stiffness.
Similarly, other experiments could examine the lungs for changes in inflammatory
markers, oxidative stress levels and cellular apoptosis. Results from such studies will no
doubt help us to better understand the disease process and therapeutic efficiency.
According to the reports from the Center for Disease Control and Prevention the
incidence of PAH is ~4:1 higher in women than men [206]. Why more women suffer
111
from PAH than men is currently not clear. Future studies using female rats could further
our understanding of the influence of sex on the pathophysilogy of monocrotaline-
induced pulmonary arterial hypertension and could be invaluable for helping to
determine whether treatment with CeO2/curcumin nanoparticles is safe.
112
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Appendix
Letter from Institutional Research Board
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Curriculum Vitae
Madhukar B. Kolli 6133, Country Club Dr, Huntington,WV-25705
E-mail:[email protected] Phone :( 304) 942-5920
CAREER OBJECTIVE: To obtain a position where my scientific and academic knowledge will contribute for the development field of science grow along with the institute. EDUCATION Marshall University, Huntington, WV John C Edward School of Medicine Ph.D in Biomedical sciences Graduated – Aug 2012
GPA-3.47/4. Marshall University, Huntington, WV MS in Biological Sciences Graduated -Aug 2008 GPA-3.9/4.0 Acharya N G Ranga Agricultural University, Hyderabad, India. DVM Graduated - Feb 2004 GPA-4.0/4.0
LICENSURE West Virginia Board of Veterinary Medicine # 22-2012. RESEARCH EXPERIENCE Graduate research Assistant, Center for diagnostic nanosystems, Marshall University, Huntington, WV-25705 (2007- Present) Ph.D Dissertation: The use of nanoparticles as therapeutic agents for the treatment of ventricular hypertrophy following pulmonary arterial hypertension M.S Thesis: Assembly and Function of Myosin II on Ultraviolet/Ozone Patterned Trimethyl-chlorosilane Substrates.
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RESEARCH SKILLS
Immunohistochemistry
Flourescent and Confocal Microscopy
Differential staining procedures
Sample preparation from Laboratory animals
Protein Quantitation using Bradford assay
SDS gel electrophoresis
Immunoblotting
Immunoprecipitation and Westerns
Quantitation using Densitometry
DNA, RNA and microRNA isolation
DNA and Protein gel electrophoresis
PCR, RT-PCR
Cell culture
Small animal surgery
Handling of Laboratory animals
Protein Identification by MALDI- TOF MS
Cardiac Ischemia-Reperfusion Experiments in Rats and Mice PUBLICATIONS:
1. Intra tracheal instillation of cerium oxide nanoparticles induces hepatic toxicity in male Sprague-Dawley rats. Nalabotu SK, Kolli MB*, Triest WE, Ma JY, Manne ND, Katta A, Addagarla, Rice KM and Blough ER. Int J Nanomedicine. 2011;6:2327-35. Epub 2011 Oct 14.
2. Transport of single cells using an actin bundle-myosin bionanomotor transport system. Takatsuki H, Tanaka H, Rice KM, Kolli MB*, Nalabotu SK, Kohama K, Famouri P and Blough ER. Nanotechnology. 2011 Jun 17;22(24):245101. Epub 2011 Apr 20.
3. Application of poly(amidoamine) dendrimers for use in bionanomotorsystems.Kolli MB*, Day BS, Takatsuki H, Nalabotu SK, Rice KM, Kohama K, Gadde MK, Kakarla SK, Katta A and Blough ER. Langmuir. 2010 May 4;26(9):6079-82.
4. Possible molecular mechanisms underlying age-related cardiomyocyte apoptosis in the F344XBN rat heart.Kakarla SK, Rice KM, Katta A, Paturi S, Wu M, Kolli MB*, Keshavarzian S, Manzoor K, Wehner PS and Blough ER. J Gerontol A BiolSci Med Sci. 2010 Feb;65(2):147-55. Epub 2010 Jan 7.
5. Altered regulation of contraction-induced Akt/mTOR/p70S6k pathway signaling in skeletal muscle of the obese Zucker rat.Katta A, Kakarla S, Wu M, Paturi S,
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Gadde MK, Arvapalli R, Kolli MB*, Rice KM and Blough ER. Exp Diabetes Res. 2009; Epub 2010 Mar 30.
6. Assembly and Function of Myosin II on Ultraviolet/Ozone Patterned Trimethylchlorosilane Substrates.Takatsuki H, Kolli MB*, Rice KM, Day BS Asano, Shinichi; Rahman, Mashiur; Zhang, Yue; Ishikawa, Ryoki; Kohama K and Blough, ER. Journal of Bionanoscience, Volume 2, Number 1, June 2008 , pp. 35-41(7)
7. Diminished muscle growth in the obese Zucker rat following overload is associated with hyperphosphorylation of AMPK and dsRNA-dependent protein kinase kinase. Katta A, Wu M, Manne N, Kolli MB*, Rice KM and Blough ER J Appl Physiol. 2012 May 31
8. Control of myosin motor activity by the reversible alteration of protein structure for applications in the development of a bio nano device. Nalabotu SK, Takatsuki H, Kolli MB* and Blough, ER (Accepted for publication in Advanced Science Letters. Jan-2012)
MANUSCRIPTS IN PREPARATION:
1. Cerium oxide nanoparticles attenuate Monocrotaline induced pulmonary arterial hypertension and associated right ventricular hypertrophy Kolli MB*, Nalabotu SK, Para RK and Blough ER
2. Antioxidant cerium oxide nanoparticles ameliorates monocrotaline induced right ventricular hypertrophy Kolli MB*, Nalabotu SK, Para RK and Blough ER
3. Curcumin nanoparticles attenuate cardiac remodeling due to pulmonary arterial hypertensionKolli MB*, Para RK, Nalabotu SK and Blough ER
4. Role of Oxidative Stress and Apoptosis in the hepatic Toxicity induced by Cerium Oxide Nanoparticles Following Intratracheal Instillation in Male Sprague-Dawley Rats. Nalabotu SK , Manne DPK, Kolli MB*, Para RK and Blough ER
5. Exposure to cerium oxide nanoparticles is associated with activation of MAPK signaling and apoptosis in the rat lungs. Nalabotu SK , Manne DPK, Kolli MB*, Para RK and Blough ER
ABSTRACTS AND POSTER PRESENTATIONS:
1. Madhukar B. Kolli, Arun Kumar, FerasElbash, Radha Para, Siva K. Nalabotu, Nandini D Manne, GeetaNandyala, Paulette Wehner and Eric R. Blough. Efficacy Of Curcumin Nanoparticles On Monocrotaline Induced Pulmonary Arterial
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Hypertension And Right Ventricular Hypertrophy. (American Heart Association HBPR, 2011).
2. MadhukarB. Kolli, RadhakirshnaPara,SivaNalabotu, Salah M El Bash, Lucy Dornon, Paulette Wehner, and Eric R. Blough. Cerium oxide nanoparticles ameliorate monocrotaline induced pulmonary arterialhypertension and associated right ventricular hypertrophy in Sprague Dawley rats.CDDC- Research Symposium, Marshall univeristy, March, 2012
3. Siva K. Nalabotu, NandiniManne, MadhukarKolli, GeetaNandyala, Radha K Para, Valentovic Monica, Jane Ma and Eric R. Blough. Evaluation of oxidative stress and apoptosis in the liver following a single intratracheal instillation of cerium oxide nanoparticles in male Sprague dawley rats. (Society of Toxicology Annual Meeting, San Francisco 2012)
4. Siva K. Nalabotu, AshuDhanjal, Lucy Dornon, NandiniManne, Madhukar B. Kolli, Paulette Wehner, and Eric R. Blough. Intratracheal instillation of the cerium oxide nanoparticles may induce cardiac alterations in the male Sprague-Dawley rats. (West Virginia-ACC Annual Meeting 2011)
5. Katta, A, Kundla S, Kakarla S, Wu M, Paturi S, Gadde M.K., Arvapalli R, Madhukar B. Kolli, Siva K. Nalabotu. Rice, Kevin M. and Eric R. Blough. Impaired Overload-induced Hypertrophy Is Associated With Diminished mTOR Signaling In Insulin Resistant Obese Zucker Rat (American College of Sports & Medicine, Baltimore, 2010)
6. Madhukar B. Kolli, Hideyo Takatsuki, Devashish Desai, Kevin M. Rice, Sunil Kakarla, AnjaiahKatta, Sriram P. Mupparaju, SarathMeduru, Anil K. Gutta, SatyanarayanaPaturi and Eric R.Blough. Confinement of myosin motor activity on Trimethylchlorosilane surface by Ultra Violet lightexposure for nano-technology applications.ACSM Conference – May 2008, Indianapolis,IN,USA.
7. Madhukar B. Kolli, Hideyo Takatsuki, Devashish Desai, Kevin M. Rice, Sunil Kakarla, AnjaiahKatta, Sriram P. Mupparaju, SarathMeduru, Anil K. Gutta, SatyanarayanaPaturi and Eric R.Blough. Surface modification of TMCS by Ultra Violet Light exposure for confinement of Myosin motor activity. "Multifunctional Nanomaterials" International Symposium, April 2008,WV.
8. Murali K. Gadde, Hideyo Takatsuki, Madhukar B. Kolli, Kevin M. Rice, Siva K Nalabotu, Kazuhiro Kohama and Eric Blough. Disassembly of fascin bundled actin filaments induced by myosin II motors in an in-vitro motility assay and its applications to nanotechnology. (Sigma Xi Research day, April 2008)
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PROFESSIONAL MEMBERSHIPS Member- West Virginia Board of Veterinary Medicine Member- Society of Toxicology Member - Veterinary council of INDIA Member - Educational Commission for Foreign Veterinary Graduates(ECFVG) of
American Veterinary Medical Association (AVMA), 2007-present Member - Cell Differentiation and Development Center-2007, Marshall University.
AWARDS AND ACCOMPLISHMENTS
BMS Graduate Student travel award (2010), Marshall University, Huntington, WV-25705
Runner up for the graduate studentsposter presentation at regional CDDC symposium March 24, 2012
Dean's Award for Academic Excellence, ANGRA University, Hyderabad, India (2003)
Acharya NG Ranga merit scholarship, College of Veterinary Science, Tirupati, India (2001)
REFERNCES Dr.EricR.Blough, Ph.D Associate professor Dept. of Pharmaceutical Science& Research School of Pharmacy, Marshall University, Huntington, WV E-mail: [email protected], Phone: (304)696-2708 Dr. Monica Valentovic, Ph.D Professor Dept.Pharmacology, Physiology and Toxicology Joan C. Edwards School of Medicine, Marshall University,Huntington, WV E-mail: [email protected] Phone: (304) 696-7332 Dr. Nalini Santanam, Ph.D Professor, Dept. Pharmacology, Physiology and Toxicology Joan C. Edwards School of Medicine, Marshall University, Huntington, WV E-mail: [email protected] Phone: (304) 696-7321