Marshall University Marshall Digital Scholar eses, Dissertations and Capstones 2012 e Use of Cerium Oxide and Curcumin Nanoparticles as erapeutic Agents for the Treatment of Ventricular Hypertrophy Following Pulmonary Arterial Hypertension Madhukar Babu Kolli [email protected]Follow this and additional works at: hp://mds.marshall.edu/etd Part of the Medical Biochemistry Commons , Medical Biotechnology Commons , Medical Molecular Biology Commons , Medical Pharmacology Commons , and the Medical Toxicology Commons is Dissertation is brought to you for free and open access by Marshall Digital Scholar. It has been accepted for inclusion in eses, Dissertations and Capstones by an authorized administrator of Marshall Digital Scholar. For more information, please contact [email protected]. Recommended Citation Kolli, Madhukar Babu, "e Use of Cerium Oxide and Curcumin Nanoparticles as erapeutic Agents for the Treatment of Ventricular Hypertrophy Following Pulmonary Arterial Hypertension" (2012). eses, Dissertations and Capstones. Paper 353.
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Marshall UniversityMarshall Digital Scholar
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]
Follow this and additional works at: http://mds.marshall.edu/etdPart of the Medical Biochemistry Commons, Medical Biotechnology Commons, Medical
Molecular Biology Commons, Medical Pharmacology Commons, and the Medical ToxicologyCommons
This Dissertation is brought to you for free and open access by Marshall Digital Scholar. It has been accepted for inclusion in Theses, Dissertations andCapstones by an authorized administrator of Marshall Digital Scholar. For more information, please contact [email protected].
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.
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].
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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
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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.
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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
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).
69
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
70
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.
71
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.
72
Figure 3-13. Alterations in serum protein biomarkers with MCT and MCT + CeO2
nanoparticle treatment.
73
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.
74
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.
75
PAPER 3
The following paper corresponds to the specific aim II
76
Curcumin nanoparticles attenuate cardiac remodeling due to pulmonary arterial
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).
89
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).
90
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
91
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.
92
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
93
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.
94
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.
95
Figure 3-16.Optical characterization of curcumin nanoparticles. Curcumin nanoparticles were observed under SEM (Panels A, B) and AFM imaging (Panel C).
96
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.
97
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;
98
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.
99
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.
100
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.
101
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.
102
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
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
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