cell response to imaging contrast agents suggested for...
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Linköping University Medical Dissertations No. 1357
Cell response to imaging contrast agents suggested for atherosclerotic plaque imaging
Amit Laskar
Department of Clinical and Experimental Medicine,
Faculty of Health Sciences, Linköping University, Sweden
Linköping 2013
© Amit Laskar 2013
Published papers have been reprinted with the permission of the copyright holders
Printed in Sweden by LiU-Tryck, Linköping, Sweden 2013
ISBN: 978-91-7519-671-8
ISSN: 0345-0082
To my family
“Even if I am a minority of one, truth is still the truth”
Mohandas Karamchand Gandhi
ABSTRACT
Oxysterols are the major cytotoxic components of oxidized low-density lipoprotein
(OxLDL) that accumulate in atherosclerotic plaques. Their uptake by macrophages ensue
foam cell formation, atherogenesis and plaque progression. Magnetic resonance imaging
(MRI) has grown as a modality to track such intra-plaque developments by using
intracellular contrast agents. The focus of this study was to evaluate the effects of two
contrast agents; manganese based mangafodipir (TeslascanTM) and iron based super-
paramagnetic iron oxide nanoparticles (SPION, ResovistTM) on cell functions and examined
their interaction with oxysterol laden cells.
Mangafodipir has antioxidant property and provides protection against oxidative stress.
The chemical structure of mangafodipir comprises of organic ligand fodipir (Dipyridoxyl
diphosphate, Dp-dp) and Mn (manganese). Mangafodipir is readily metabolized within the
body to manganese dipyridoxyl ethyldiamine (MnPLED) after an intravenous injection.
MnPLED has superoxide dismutase (SOD) mimetic activity, and Dp-dp has iron chelating
effects. The second contrast agent tested in this study is ResovistTM. These SPION are
primarily ingested by macrophages and accumulated in lysosomes where they are
gradually degraded ensuing increased cellular iron.
In paper I, we examined whether the above-noted effects of mangafodipir could be utilized
to prevent 7β-hydroxycholesterol (7βOH) induced cell death. We found that mangafodipir
prevents 7βOH induced cell death by attenuating reactive oxygen species (ROS) and by
preserving lysosomal membrane integrity and mitochondrial membrane potential.
The second part of this study (paper II) was designed to identify the pharmacologically
active part of mangafodipir, which exerts the above-noted effects. We compared the
activity of parent compound (mangafodipir) with MnPLED and Dp-dp. We found that
mangafodipir; MnPLED and Dp-dp provide similar cyto-protection against 7βOH induced
cell death. These results suggest that MnPLED and Dp-dp both contribute to the
pharmacologically active part of mangafodipir.
In paper III, we aimed to examine the interaction of SPION with monocytes and
macrophages exposed or not to atheroma relevant oxysterols. We demonstrate that SPION
loading up-regulates cellular levels of cathepsin and ferritin and induces membranous
ferroportin expression. Additionally, SPION incites secretion of ferritin and both pro-
inflammatory and anti-inflammatory cytokines. Moreover, exposure to oxysterols resulted
in a reduced SPION uptake by cells, which may lead to inefficient targeting of such cells.
Although SPION uptake was reduced, the ingested amounts significantly up-regulated the
expression of 7βOH induced cathepsin B, cathepsin L and ferritin in cells, which may
further aggravate atherogenesis.
The fourth part of the study (paper IV) was designed to examine the interaction of SPION
with macrophage subtypes and compare the cellular effects of coated and un-coated iron-
oxide nanoparticles. We found that iron in SPION induces a phenotypic shift in THP1 M2
macrophages towards a macrophage subtype characterized by up-regulated intracellular
levels of CD86, ferritin and cathepsin L. Differential levels of these proteins among
macrophage subtypes might be important to sustain a functional plasticity. Additionally,
uncoated iron-oxide nanoparticles induced dose dependent cell death in macrophages,
which elucidates the potential cyto-toxicity of iron in iron-oxide nanoparticles.
In conclusion, evidence is provided in this study that intracellular MRI contrast agents have
the potential to modulate cell functions. The study reveals a therapeutic potential of
mangafodipir, which could be utilized for future development of contrast agents with both
diagnostic and curative potentials. Additionally, we found that surface coating in SPION
may provide cell tolerance to iron toxicity by modulation of cellular iron metabolism and
cell functions. Such alterations in cellular metabolism call for careful monitoring and also
highlight new concepts for development of iron containing nanoparticles. A reduced uptake
of SPION by atheroma relevant cells justifies development of functionalized SPION to target
such cells in atherosclerotic plaques.
POPULAR SUMMARY
Atherosclerosis is a common vascular disease and its clinical complications, (myocardial
infarction, stroke and angina pectoris), cause high morbidity and mortality in western
population. These clinical complications are all associated with the blockade in the blood
vessel by the fragments of the atherosclerotic plaques. Atherosclerotic plaque formation is
initiated by uptake of cytotoxic cholesterol oxidation products by white blood cells of lipid
rich foam cells. Thus, along with many other factors, white blood cells and foam cells
regulate the development of atherosclerosis.
Magnetic resonance imaging (MRI) is one of the most promising techniques for detecting
and tracking such cells in atherosclerotic plaques. For better detection of the plaques by
MRI, imaging contrast agents are used, which accumulate in the target cells to enhance the
accuracy of detection. Many such contrast agents are currently undergoing pre-clinical and
clinical trials. However, their effect on the cell functions is not fully examined. In this study,
we evaluated the effects of two contrast agents; manganese based mangafodipir
(TeslascanTM) and iron based super-paramagnetic iron oxide nanoparticles (SPION,
ResovistTM) on cell functions and examined their interaction with cells treated with lipid
oxidation products; 7βOH (7β-hydroxycholesterol) and 7keto (7 keto-cholesterol).
We found that mangafodipir was non-toxic to cells and pre-treating the cells with
mangafodipir prevented 7βOH induced cell death. In addition, a particular component in
the chemical structure of this contrast agent may be responsible for this protective effect.
However, when white blood cells were exposed to SPION, iron concentration in the cell was
increased, which lead to increases in iron regulatory protein levels in the cells, including
ferritin, ferroportin and cathepsins. Also, SPION elevates 7βOH and 7keto induced
cathepsin levels in the cells. All these proteins are associated with development of
atherosclerosis. Equally important, SPION affected immune functions of cells, which is a
very critical aspect determining atherosclerotic plaque stability and its progression. In
addition, we found that macrophages exposed to 7βOH have a reduced capacity to uptake
SPION. We also found that iron in SPION may itself induce toxicity in cells.
In conclusion, our study reveals that imaging contrast agents modulate cell functions which
could be an advantage or disadvantage. Mangafodipir provides protection against 7βOH
induced toxicity. This property of mangafodipir may be utilized in future development of
contrast agents with both detection and treatment abilities. However, we found that SPION
with surface modifications gives cell tolerance to iron toxicity by modulation of cellular
iron metabolism. Such alterations in cellular metabolism call for careful monitoring and
also highlight new ideas for development of iron containing nanoparticles.
Table of contents
LIST OF PAPERS ................................................................................................................................................. 12
LIST OF ABBREVIATIONS ............................................................................................................................... 13
INTRODUCTION .................................................................................................................................................. 14
Macrophages in atherosclerosis .............................................................................................................. 14
Macrophages and functional plasticity ................................................................................................. 15
OxLDL and related oxysterols in atherogenesis ................................................................................ 16
Uptake of OxLDL by macrophages .......................................................................................................... 16
Iron in atherogenesis ................................................................................................................................... 17
Macrophage iron and lysosomes ............................................................................................................. 17
Ferritin and cell functions .......................................................................................................................... 18
Immuno-modulatory iron and macrophage phenotype ................................................................ 18
Imaging atherosclerotic plaques ............................................................................................................. 19
Magnetic resonance imaging .................................................................................................................... 20
Biomarkers and atherosclerotic plaques ............................................................................................. 21
Mangafodipir ................................................................................................................................................... 22
Super-paramagnetic iron-oxide nanoparticles (SPION) ................................................................ 23
AIMS ........................................................................................................................................................................ 25
MATERIALS AND METHODS ......................................................................................................................... 26
Materials ........................................................................................................................................................... 26
Methods............................................................................................................................................................. 27
Cell culture .................................................................................................................................................. 27
Cell viability ................................................................................................................................................ 28
Reactive oxygen species ......................................................................................................................... 29
Lysosomal membrane integrity .......................................................................................................... 29
Mitochondrial membrane potential .................................................................................................. 29
Phagocytic activity ................................................................................................................................... 30
Cellular iron ................................................................................................................................................ 30
Immuno-cytochemistry .......................................................................................................................... 30
Secreted, nuclear and cytosolic protein ........................................................................................... 31
Statistics ............................................................................................................................................................ 31
RESULTS ................................................................................................................................................................ 32
Paper I and II ................................................................................................................................................... 32
Mangafodipir, MnPLED and Dp-dp prevents 7βOH-induced cell death .............................. 32
Paper III and IV .............................................................................................................................................. 33
Monocyte differentiation induces functional plasticity in macrophages and
simultaneous increases in ferritin and cathepsin L .................................................................... 33
SPION up-regulates ferroportin, ferritin and cathepsin L levels, incites cytokine and
ferritin secretion and modulates cell phenotype ......................................................................... 34
Oxysterols reduce phagocytic activity in macrophages thereby reduce uptake of SPION
......................................................................................................................................................................... 37
Surface coating promotes uptake of iron-oxide nanoparticles and potentially prevents
iron induced cytotoxicity ....................................................................................................................... 37
GENERAL DISCUSSION .................................................................................................................................... 39
Mangafodipir mediated cyto-protection involves modulation of lysosomal and
mitochondrial pathways ............................................................................................................................. 39
A conserved structure in mangafodipir may attribute to its cyto-protective effects ......... 39
Monocyte differentiation induces macrophage phenotype with elevated levels of ferritin
and cathepsin L .............................................................................................................................................. 40
Iron in SPION primes THP1 M2 macrophages towards a high CD86+ macrophage subtype
with elevated levels of ferritin and cathepsin L ................................................................................ 40
Lysosomal cathepsins have a functional role in ferritin degradation ...................................... 41
SPION loading incites secretion of both pro-inflammatory and anti-inflammatory
cytokines ........................................................................................................................................................... 41
Non-functionalized SPION may be inefficient to target intra-plaque macrophages ........... 42
Functional coating of iron-oxide nanoparticles gives cell tolerance to potential
cytotoxicity by regulation of cell functions ......................................................................................... 43
A need for better characterization of macrophage subtypes ....................................................... 46
CLINICAL AND FUTURE PERSPECTIVES .................................................................................................. 48
CONCLUSIONS ..................................................................................................................................................... 50
ACKNOWLEDGEMENT ..................................................................................................................................... 51
REFERENCES ....................................................................................................................................................... 53
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LIST OF PAPERS
This thesis is based on the following papers, which are referred to by their roman
numerals:
I. Laskar, A., Miah, S., Andersson, R.G.G., Li, W., 2010. Prevention of 7 beta-
hydroxycholesterol-induced cell death by mangafodipir is mediated through
lysosomal and mitochondrial pathways. Eur J Pharmacol 640, 124-128.
II. Laskar, A., Andersson, R.G.G., Li, W., 2013. Fodipir (Dp-dp) and its
dephosphorylated derivative PLED are involved in mangafodipir mediated cyto-
protection against 7β-hydroxycholesterol induced cell death. Submitted to The J of
Cardio Pharmcol.
III. Laskar, A., Ghosh, M., Khattak, S.I., Li, W., Yuan, X.M., 2012. Degradation of
superparamagnetic iron oxide nanoparticle-induced ferritin by lysosomal
cathepsins and related immune response. Nanomedicine-Uk 7, 705-717.
IV. Laskar, A., Eilertsen, J., Li, W., Yuan, XM., 2013. SPION primes M2 macrophages
towards a pro-inflammatory M1 subtype characterized by elevated levels of ferritin
and cathepsin L. Manuscript.
OTHER PUBLICATIONS NOT INCLUDED IN THIS THESIS
1. Laskar, A., Yuan, X.M., Li, W., 2010. Dimethyl sulfoxide prevents 7beta-
hydroxycholesterol-induced apoptosis by preserving lysosomes and mitochondria. J
Cardiovasc Pharmacol 56, 263-267.
2. Li, W., Laskar, A., Sultana, N., Osman, E., Ghosh, M., Li, Q.Q., Yuan, X.M., 2012. Cell
death induced by 7-oxysterols via lysosomal and mitochondrial pathways is p53-
dependent. Free Radical Bio Med 53, 2054-2061.
3. Ghosh, M., Carlsson, F., Laskar, A., Yuan, X.M., Li, W., 2011. Lysosomal membrane
permeabilization causes oxidative stress and ferritin induction in macrophages.
FEBS Lett 585, 623-629.
13
LIST OF ABBREVIATIONS
7keto 7keto cholesterol
7βOH 7β-hydroxycholesterol
AO Acridine orange
CLIK-148 (N-(trans-carbamoyloxyrane-2-carbonyl)-L-phenylalanine-dimethylamide)
DFO Desferrioxamine
DHE Dihydroethidium
Dp-dp Dipyridoxyl diphosphate
E64d Trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane
Fe2O3 Maghemite
Fe3O4 Magnetite
FeAC Ferric ammonium citrate
FBS Fetal bovine serum
FD40 FITC conjugated dextran
GM-CSF granulocyte macrophage colony-stimulating factor
hIgG Human immunoglobulin G
HMDMs human monocyte derived macrophages
IL-10 Interleukin-10
IL-13 Interleukin-13
INPs Iron-oxide nanoparticles
IFN-γ Interferon gamma
IL-4 Interleukin- 4
LDL Low-density lipoprpotein
LPS Lipopolysaccharide
LMP Lysosomal membrane permeability
MRI Magnetic resonance imaging
MnPLED Manganese dipyridoxyl ethyldiamine
NH4Cl Ammonium Chloride
OxLDL Oxidized low-density lipoprotein
PBS Phosphate buffer saline
PMA Phorbol 12-myristate 13-acetate
PLED Dipyridoxyl ethyldiamine
ROS Reactive oxygen species
SOD Superoxide dismutase
SPION Super-paramagnetic iron oxide nanoparticles
MMP Mitochondrial membrane permeability
TNF-α Tumour necrosis factor α
USPION Ultra-small iron-oxide nanoparticle
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INTRODUCTION
Macrophages in atherosclerosis
Atherosclerosis is a chronic inflammatory disease in which accumulation of inflammatory
cells contributes to atheroma plaque development and vulnerability [1]. Development of
atherosclerotic plaque is a complex process and involves activation of endothelium by
components of OxLDL found in the intima of the lesion prone areas [2]. OxLDL in the intima
is chemotactic for monocytes in the blood. Activated endothelium expresses cell adhesion
molecules, chemokine and cytokines and thereby promotes leukocyte recruitment [3-5].
This is a crucial step of atherogenesis in which monocytes infiltrate the sub-endothelial
space of larger arteries where they differentiate into intimal macrophages [2, 6-8]. These
monocyte derived macrophages that are recruited from the blood account for the majority
of the leukocytes in atherosclerotic plaques. These recruited macrophages play reparatory
roles in early plaques characterized by phagocytosis of oxidized lipids and apoptotic cells
[9]. However, during progression of atherosclerosis, these macrophages secrete
inflammatory factors and proteolytic enzymes and contribute to plaque rupture and
thrombosis [9]. High macrophage content, apoptosis and reduced clearance of apoptotic
macrophages are all associated to vulnerability of atherosclerotic plaques [6]. These intra-
plaque macrophages are rich in iron and lipids [10-12] and exhibit functional plasticity in
atherosclerotic plaques.
Among others, macrophages are considered the predominant cells which not only promote
atheroma plaque development and vulnerability [6, 13] but also play an important role in
cardiac damage after infarction [14]. A ruptured atherosclerotic plaque exposes their
thrombogenic interior to the luminal blood causing platelet aggregation and thrombus
formation [2, 15, 16]. Together these events lead to the major clinical manifestations of
atherosclerosis such as myocardial infarction and stroke, which are the most common
cause of death among the western society [17-19].
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Macrophages and functional plasticity
Macrophages are the highly plastic cell type, which display functional plasticity in
atherosclerotic plaques [6, 8, 9, 13, 20, 21]. Both pro-inflammatory (M1) and anti-
inflammatory (M2) macrophage subtypes are present in atherosclerotic lesions. In chronic
inflammatory diseases like atherosclerosis, macrophage plasticity is determined by
immunological microenvironment. Interferon gamma (IFN-γ) and lipopolysaccharide (LPS)
or granulocyte macrophage colony-stimulating factor (GM-CSF) or tumour necrosis factor α
(TNF-α) direct macrophages towards a classically activated macrophage subtype (M1)
which produces pro-inflammatory cytokines and high amounts of reactive oxygen and
nitrogen species [8, 21-24]. Macrophages are the main source of pro-inflammatory
mediators in the atherosclerotic plaques. In contrast, exposure to interleukin-4 (IL-4) or
macrophage colony stimulating factor (M-CSF) polarizes macrophages towards an
alternatively activated macrophage subtype (M2) which are associated with secretion of
anti-inflammatory cytokines and tissue repair [8]. However, based upon the stimulus to
generate these macrophages, the M2 macrophages are further subdivided into M2a (after
exposure to IL-4 or interleukin-13 (IL-13)), M2b (after exposure to immune complexes in
combination with interleukin-1beta (IL-1β) or LPS) and M2c (after exposure to IL-10, TGFβ
or glucocorticoids) [25]. Mox is an additional macrophage subtype that has been proposed
[23]. When exposed to 1-palmitoyl- 2arachidonoyl-sn-glycero-3-phosphorylcholine (ox-
PL), bone marrow derived macrophages differentiate to this unique population of Mox
macrophages with a unique profile of genes, including Heme-oxigenase-1 (OH-1) and
thioredoxin reductase 1 (Txnrd1) [23]. In addition, a fourth subtype of platelet chemokine
CXCL4 dependent macrophage (M4) has been proposed [26, 27] which is characterized by
a complete loss of CD163 [28] and higher levels of CD86 and mannose receptor [27]. CXCL4
promotes macrophage differentiation in-vitro. These M4 macrophages exhibit reduced
phagocytosis of acetylated or OxLDL [28].
As indicated above, diverse methodologies, including cytokine stimulation and growth
factor induced differentiation has been used for classical (M1) and alternative (M2)
macrophage polarization in-vitro. Growth factors including GM-CSF and M-CSF induces
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both macrophage differentiation and macrophage polarization into M1 and M2
macrophages respectively [28]. Overall, these findings suggest that induction of
differentiation induces functional polarization in macrophages. However, Human
immunoglobulin G (hIgG) or Phorbol 12-myristate 13-acetate (PMA) has been used for
induction of differentiation in monocytes [29, 30] but the obtained cell types are
insufficiently characterized in terms of functional polarization.
OxLDL and related oxysterols in atherogenesis
LDLs produced within the liver carry cholesterol to other cells and tissues of the body,
including the arterial wall. LDL undergoes oxidative modification in the intima by ROS
probably produced by macrophages [2, 31, 32]. This OxLDL present in the intima is
chemotactic to blood monocyte and aids in recruitment of other leukocuytes and induces
differentiation of monocytes to macrophage foam cells [33]. Furthermore, OxLDL has been
shown to induce cell death in smooth muscle cells, endothelial cells and in macrophages
[34-37]. Many cytotoxic components of OxLDL have been identified including: oxysterols
and oxidized fatty acids [38, 39].
Handling of lipids, especially cholesterol is one of the most important functions of
macrophages in the context of atherosclerosis. An imbalance in cholesterol influx and efflux
leads to an excessive accumulation of cholesterol in macrophages and subsequent foam cell
formation [40-42]. Oxysterols are cholesterol oxidation products resulting from
spontaneous or enzymatic oxidation of cholesterols [43]. 7βOH and 7keto-cholesterol
(7keto) are the major cytotoxic components of OxLDL, which contribute to development of
atherosclerosis [43, 44]. Increased levels of 7βOH and 7Keto are found in atherosclerotic
plaques and in plasma of patients with cardiovascular diseases [43]. Thus oxidized lipid
rich macrophages are considered an important target for intra-plaque macrophage imaging
[45].
Uptake of OxLDL by macrophages
Macrophages express a group of scavenger receptors, including SR-A, CD36 and lectin-like
oxidized LDL receptor-1 (LOX-1) to interact and uptake OxLDL [9, 46]. LDL and OxLDL are
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lysosomotropic and after endocytosis are transferred into lysosomes [47, 48]. Cholesterol
esters in LDL and OxLDL are hydrolysed in the lysosomes of these macrophages [9]. After
lysosomal processing, some of the un-esterified cholesterol integrates into the plasma
membrane, and the rest is stored as lipid droplets [9, 49]. Cholesterol accumulation down-
regulates native LDL receptors thus preventing further uptake and accumulation of
cholesterol [2, 50, 51]. However, the scavenger receptors in macrophages are unaffected by
cholesterol accumulation [51] and thus continue to uptake OxLDL and atherogenic
lipoproteins and form foam cells and eventually die by apoptosis or necrosis [2]. The lipid
remains of these foam cells form the content of the necrotic core in the intima which is
eventually covered with a fibrous cap of collagen rich matrix and vascular smooth muscle
cells [2].
Iron in atherogenesis
Determining the role of iron in atherogenesis has been an area with continuous focus over
decades [11, 52]. The primary reason for that is the presence of catalytic redox-active iron
in atherosclerotic vascular tissues which remain trapped in ferritin and hemosiderin within
the lesion. High iron concentrations are seen in human atherosclerotic lesions as compared
to healthy artery tissue [10, 11].
Macrophage iron and lysosomes
Most of the iron in atherosclerotic plaques has been associated with macrophages and
foam cells. In advanced plaques, iron derived from erythrophagocytosis within the
microhaemorrhagic areas remain the potential source of redox-active iron [11, 12].
Phagocytosis of erythrocytes and their lysosomal degradation increases the intracellular
redox active iron [12]. Lysosomes not only contain abundant hydrolytic enzymes but also
are rich in low mass iron [53, 54]. Since lysosomes are acidic and rich in reducing
equivalents such as cysteine and glutathione, such low mass iron in the lysosomes would
likely be Fe(II) with a potential to generate hydroxyl radicals, promote lipid oxidation and
induce lysosomal permeabilization [53]. An after-effect would be release of lysosomal
content, including cystein proteases (cathepsins), which play a key role in apoptosis and
cellular immune functions [55, 56]. Additionally, iron has been shown to increase cathepsin
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activity [57]. Together such developments alone shall be deleterious in atherosclerotic
plaques.
In addition, iron excess might incite expression of macrophage scavenger receptors and
promote uptake of lipids [58]. Increasing lipid load and increased availability of redox
active iron within the lysosomal compartment of the cells results in formation of highly
toxic iron laden-ceroid and lipidaceous materials [10, 59]. Release of such cytotoxic
materials from these cells may determine viability of nearby existing cellular elements and
destabilize plaques. In addition, iron excess in presence of OxLDL has been shown to
enhance secretion of ferritin and iron [10, 60], which may regulate immune function in
atherosclerotic microenvironment.
Ferritin and cell functions
The content of iron in the human body is well controlled by complex mechanisms to
maintain homeostasis. Iron in the body is bound to haemoglobin, myoglobin, iron
containing enzymes and iron storage proteins; ferritin and hemosiderin [61, 62]. Ferritin is
a ubiquitous and highly conserved iron-binding protein. Its heavy-chain also has enzymatic
properties, converting Fe(II) to Fe(III) as iron is sequestered in the ferritin mineral core
[53, 63]. In cells, ferritin mostly is cytosolic. However, it is known that ferritin also resides
in nucleus and mitochondria of mammalian cells [64]. Apart from being an iron storage
protein ferritin is immuno-modulatory [65], associated with inflammation, and regulates
ROS production and apoptosis [66]. In addition, apoptotic macrophage foam cells of human
atheroma are rich in iron and ferritin [67]. Both apoptotic and anti-apoptotic activities of
ferritin are documented. Cytosolic ferritin regulates ROS production and apoptosis while
nuclear ferritin may protect cells against oxidative stress and DNA damage [64]. In
addition, ferritin may slowly release iron when exposed to reducing agents such as cysteine
and reduced glutathione [53].
Immuno-modulatory iron and macrophage phenotype
Macrophages play an important role in iron homeostasis by phagocytosing erythrocytes
degrading them and rendering iron available for erythropoiesis. Iron has immuno-
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regulatory properties, and any alterations in cellular iron levels may affect the cellular
immune response and inflammatory signalling [68, 69]. In addition functional polarization
in macrophages has been associated with differential regulation of iron metabolism [22].
Iron handling by macrophages may determine their functional polarization [22, 24]. Iron
regulatory proteins; ferritin and ferroportin have been shown to be differentially expressed
in macrophage subtypes. M2 macrophages are distinctively characterized by an iron
release with high expression of ferroportin (only known non-heam iron exporter) and low
ferritin expression [22, 24, 70]. This iron export mode in M2 macrophages promotes matrix
remodelling and cell proliferation. In contrast, M1 macrophages hold intracellular iron and
are distinctively characterized by high ferritin expression and low ferroportin [22, 24, 70].
M1 macrophages release pro-inflammatory cytokines that promote atherogenesis [71].
Both M1 and M2 macrophages are demonstrated in atherosclerotic plaques. Thus,
differential iron status in intra-plaque macrophages seems to be important to sustain
functional polarization. Elevation in arterial iron as a consequence of increased
erytrophagocytosis by macrophages or by utilizing an imaging modality involving iron
nanoparticles may eventually determine functional plasticity of macrophages and stability
of atherosclerotic plaques.
Imaging atherosclerotic plaques
Identification and characterization of vulnerable atherosclerotic plaques in asymptomatic
individuals remains a major focus of research pertaining to diagnostic imaging. Currently,
imaging techniques used for atherosclerotic plaque imaging are broadly classified as
invasive imaging techniques and non-invasive imaging techniques respectively [72].
Presently employed invasive imaging techniques include intravascular ultrasound (IVUS),
optical coherence tomography (OCT) and near-infrared (NIR) spectroscopy [72]. However,
their usage is limited, barely 1% in daily clinical practise pertaining to their invasive nature
and growing evidence that plaque activity rather than plaque morphology determines
plaque stability [72, 73]. Evolution of non-invasive imaging for evaluation of
atherosclerotic plaques may enable improved detection of vulnerable plaques and allow
early therapeutic intervention. At present, the available non-invasive imaging techniques to
20
visualize vessel wall and atherosclerosis are; ultrasound, computed tomography, MRI and
nuclear medicine.
Magnetic resonance imaging
MRI is considered to be the safest and promising imaging technology which provides a high
degree of special resolution and excellent anatomic information [2]. Among others, MRI
emerged as a leading non-invasive imaging modality that provides direct assessment of
plaque burden and has been shown to provide a platform for interrogating biological
characteristics of atherosclerotic plaques [74]. Additionally, with the development of target
specific MRI contrast agents a new modality of molecular MRI has emerged with abilities to
track cells and molecular pathways of a disease [2, 75-77]. These developments within the
field of MRI have been achieved with the usage and unceasing development of imaging
contrast agents.
Protons are abundant in human body, and they possess magnetic properties. When
exposed to strong magnetic fields, protons in the exposed tissue orient themselves within
the magnetic field. The orientation and energy of the proton nuclei changes, when a pulse
of radio wave of certain frequency is applied for a specific duration. Eventually, as the
nuclei relax, it emits MRI signal. Longitudinal (T1) or transverse (T2) relaxation times of
proton and the magnetic susceptibility are the main contrast determinants of MRI. These
relaxation times are different in one tissue to another and influence signal intensity. Signal
intensity of a tissue is directly proportional to its proton density. A shorter T1 corresponds
to a high signal intensity, whereas a shorter T2 corresponds to low signal intensity of a
tissue [78, 79].
MRI provides high-resolution imaging of atherosclerotic plaques. However, low sensitivity
is the inherent limitation with this technique. Thus, MRI often requires use of contrast
agents which enhance the image contrast of the target tissues by increasing or decreasing
the signal intensity. MRI contrast agents modify the amplitude of the signals generated by
protons in the presence of a magnetic field. MRI contrast agents are broadly classified as
T1-shortening (positive) or T2- shortening (negative) contrast agents [79]. Additionally,
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the dose of such contrast agents may also determine their contrast effects. Gadolinium (Gd)
or manganese (Mn) containing paramagnetic chelates are typically positive contrast agents
[80, 81]. At a lower dose, their predominant effect is T1 shortening and thus the uptake of
such agents by the tissue makes them hyperintense and bright. However, these agents also
produce T2 shortening effects when their concentration rises in the tissue. On the other
hand, iron-oxide nanoparticles are typical negative contrast agents [80, 81]. These super-
paramagnetic particles generate local magnetic-field gradient disturbing the primary
magnetic field on the tissue causing T2 relaxation and darkening of the tissue.
Biomarkers and atherosclerotic plaques
Plaque vulnerability is dependent on plaque composition and most vulnerable plaques
exhibit high macrophage density, necrosis and lipid rich cores [18, 82-85]. These biological
characteristics of atherosclerotic plaques are considered the clinically relevant key feature
beyond size and degree of stenosis and may serve as imaging targets. Macrophages and
foam cells are excellent imaging targets as they play an important role in plaque formation
and its progression. Additionally, selectins, integrins, vascular cell adhesion molecule-1
(VCAM-1) are considered important imaging targets as they modulate the leukocyte
recruitment process and thereby favour atherogenesis [73]. Cytokine secretion and release
of proteases by the macrophages, including metalloproteinase and cathepsins, destabilize
the plaque by damaging extracellular matrix and thinning of the fibrous cap [86, 87]. Thus,
these inflammatory mediators are also attractive imaging biomarkers.
Phagocytic function of the macrophages has been exploited for imaging intra-plaque
macrophages by using a variety of intracellular imaging contrast agents, including iron-
oxide nanoparticles (INPs) and Mn-based chelates. Although, targeting of macrophages in
human atherosclerotic plaques by INPs has shown promise [88-91], functionalization of
these INPs by chemical attachment of an affinity ligand, such as a peptide or an antibody
has emerged as a molecular imaging platform. Most of these functionalized nanoparticles
have been mainly validated in animal models for cellular and molecular imaging of intra-
plaque macrophages based upon their scavenger function and receptors [2, 92, 93].
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The current focus in the field of cardiovascular imaging is to develop agents with both a
diagnostic and therapeutic potential. It has been soon realized that even currently existing
contrast agents might have this potential. Mangafodipir is such an example. It is a Mn-based
imaging contrast agent that exhibits pharmacological effects, including anti-oxidant and
cyto-protective activities [94-98]. As these intracellular contrast agents are phagocytosed
and remain within the cells for longer durations, they are bound to modulate cell functions.
Another group of contrast agents that have been shown to modulate cell functions are iron-
oxide nanoparticles. These INPs affect vital cell functions, including cellular immune
response [99, 100].
Mangafodipir
Mangafodipir was clinically used as an intravenous paramagnetic contrast media for liver
MRI (tumour and metastasis) and was marketed as TeslascanTM. It was supplied as a sterile
aqueous solution. The recommended adult dose of mangafodipir was 5 µmol/kg body
weight [80]. Mangafodipir trisodium is the active constituent of TeslascanTM, but it also
contains ascorbic acid, sodium chloride, sodium hydroxide and water for injection.
Mangafodipir trisodium [manganese (II)-N,N-dipyridoxylethylenediamine-N,N'-diacetate-
5,5'-bis(phosphate) sodium salt] consists of organic ligand fodipir (Dp-dp) and Mn
(Manganese) [101]. After intravenous administration, mangafodipir is dephosphorylated to
MnPLED followed by transmetallation by zinc [98, 101]. The released manganese is rapidly
cleared from the blood and is taken up by the hepatocytes and thereby increases the signal
intensity of the normal liver tissue. The excretion of manganese is through the biliary route
whereas the fodipir metabolites are renally excreted.
Besides its clinical application for imaging of liver conditions, mangafodipir has also been
shown to exert pharmacological effects. Mangafodipir has been used as a viability marker
in patients with myocardial infarction [102] and showed vascular relaxation effect.
Mangafodipir also conferred myocardial protection against oxidative stress [96, 103].
These effects may be attributed to the SOD-mimitic activity of mangafodipir and its
metabolite MnPLED and also high iron binding capability of Dp-dp and dipyridoxyl
ethyldiamine (PLED) [98].
23
Super-paramagnetic iron-oxide nanoparticles (SPION)
SPION possesses magnetic properties and has been extensively used in many bio-
applications, including magnetic resonance imaging, cell separation and magnetic drug and
gene delivery [100]. SPION has been widely used as a contrast agent to target
macrophages. Low toxicity profile and ease of functionalization have made them a perfect
selection for targeted imaging [100]. SPION composes of a crystalline iron core coated with
carboxydextran [104, 105]. Based on their size, SPION are broadly categorized as standard (60 -
150 nm), large (>200 nm), ultrasmall (USPIO; 10 – 40 nm), monocrystalline (MION; 10 – 30
nm) and very small (VSOP; 4 – 8 nm) [106, 107]. SPION used in this study is ResovistTM,
with a hydrodynamic diameter of 60 – 62 nm [106]. It was clinically used for diagnostic
imaging of cirrhosis and liver cancer. ResovistTM is composed of a Fe3O4 (magnetite) and
Fe2O3 (maghemite) core, and coated by carboxy-dextran [104]. Both Fe3O4 and Fe2O3 were
found to be cytotoxic to a variety of cells [108-112]. These findings indicate the presence of
potentially cytotoxic iron in SPION. The carboxy-dextran coating not only increases the
uptake of SPION by macrophages but also prevents aggregation of the iron core [106, 113,
114] and may be important to prevent iron induced cyto-toxicity by increasing
biocompatibility.
SPION are intracellular contrast agents with enhanced duration of cellular residence
following phagocytosis. Clathrin receptors contribute to internalization of a variety of
nanoparticles, and its potential role in SPION uptake has been suggested [115]. After
internalization SPION accumulates in lysosomes, where they are gradually degraded [113,
115-117], marked by co-localization with dextranases and resulting in iron accumulation
[118].
Increased cellular iron levels in macrophages following SPION degradation may pose
critical consequences in iron rich atherosclerotic plaques. Iron excess accounts for
observed cellular ferritin up-regulation [119], altered immune response in macrophages
[120] and iron-oxide induced delayed cytotoxicity [104, 118, 121]. However, these iron-
oxide nanoparticles have been shown to be phagocytosed by intra-plaque macrophages in
both animal models and in humans [122-124]. Thus these nanoparticles are unceasingly
24
developed and their potential to target intra-plaque macrophages is extensively examined
in pre-clinical trials in atherosclerotic settings. Recent studies mainly focus on cytotoxicity
induced by these nanoparticles, which may leave modulation of important cellular
processes by these nanoparticles go unnoticed.
25
AIMS Imaging contrast agents are unceasingly developed to target macrophages in
atherosclerotic plaques. However, little is known about their impact on normal cellular and
physiological or patho-physiological functions. The objective of our study was to evaluate
effects of two such magnetic resonance contrast agents; manganese based mangafodipir
(TeslascanTM) and iron based super-paramagnetic iron oxide nanoparticles (SPION,
ResovistTM) on cell functions and their interaction with oxysterol laden cells. The specific
aims were:
To determine whether mangafodipir could affect 7βOH induced cell death and
identify the underlying mechanisms that might be involved in the process.
To identify the active moiety in mangafodipir exerting pharmacological effects.
To examine the interaction of SPION with monocytes and macrophages exposed or
not to atheroma relevant oxysterols, focusing on cell function and cellular iron
handling.
To examine the interaction of SPION with functionally polarized macrophages and
compare the cellular effects of coated and un-coated iron-oxide nanoparticles.
26
MATERIALS AND METHODS
Materials
Cysteine protease inhibitor
Trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E64d) is an irreversible
cysteine protease inhibitor, including cathepsin B and cathepsin L and also inhibits
calpains. Upon pinocytosis, E64d localizes in lysosomal compartment. (N-(trans-
carbamoyloxyrane-2-carbonyl)-L-phenylalanine-dimethylamide) (CLIK-148) is a specific
cathepsin L inhibitor.
Iron chelators
In paper III and IV, Desferrioxamine (DFO) was used. It is a potent iron chelator, which
when endocytosed accumulates in acidic vacuolar compartment. It binds to low molecular
weight iron, thereby preventing iron mediated redox-reactions. In paper III, Ammonium
Chloride (NH4Cl) was used, which is a lysosomotropic alkalizing agent, and has been shown
to exhibit intra lysosomal iron chelation [125].
Oxysterols
Oxysterols are oxidation products of cholesterol. Oxysterols mimic negatively charged LDL
(LDL-), which is an oxidatively modified form of LDL and has been shown to induce
endothelial dysfunction and cholesterol accumulation in vascular wall cells. 7keto and
7βOH are the major cytotoxic components of OxLDL, and it has been shown that
macrophages exposed to 7keto exhibit cholesterol accumulation and eventually lead to
foam cell formation. In this project, 7βOH was used in papers I, II, III and IV, whereas 7Keto
was used in paper III and IV.
27
Methods
Cell culture
Human monocyte derived macrophages
Human blood monocytes were isolated from buffy coats density gradient centrifugation
using OptiprepTM. To induce differentiation, the monocytes were plated on 16 mg/mL hIgG
coated air-dried glass coverslips in petri dishes. The cells were maintained in RPMI
supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 u/mL penicillin
G and 100 µg/mL streptomycin. The cells were cultured at 95% air and 5% CO2 in a
humidified atmosphere at 37°C. Medium was replaced every alternate day, and the
macrophages were used on day 7 for experiments.
U937 and THP1 monocytes
U937 and THP1 cells were maintained in RPMI with 10% fetal bovine serum, 2 mM l-
glutamine,100 µg/ml penicillin G and 100 µg/ml streptomycin. The cells were cultured
at 95% air and 5% CO2 in a humidified atmosphere at 37°C. Subdivided cells at
approximately 3×105 cells/ml were used in the experiments [126].
THP1 macrophages
THP1 monocytes were treated with 300 nM PMA for 24 h, to generate THP1
macrophages [63]. After 24 h, PMA was withdrawn and the cells were washed with RPMI
culture media and kept in the same media for another 24 h before experiment.
Macrophage subtypes
PMA-treated THP1 monocytes were exposed to IFN-γ and LPS or IL-4 and IL-13 to
generate M1 or M2 macrophages by following a standardized protocol [30]. Briefly, THP1
cells were treated with 320 nM PMA for 6 hours and then either exposed to 100
ng/ml lipopolysaccharide (LPS) and 20 ng/ml interferon-gamma (IFN-γ) for 18 hours
to generate M1 macrophages or exposed to 20 ng/ml interleukin-4 (IL-4) and 20 ng/ml
interleukin-13 (IL-13) for 18 hours to generate M2 macrophages.
28
Figure 1: Generation of M1 and M2 macrophages. PMA induced THP1 macrophages (PMA THP1 M2); THP1
derived M1 macrophages (THP1 M1 MØ) and THP1 derived M2 macrophages (THP1 M2)
Cell viability
Plasma membrane integrity: Plasma membrane integrity can be assessed by adding a
cell permeable dye, which can only stain cells with damaged or porous plasma membrane
[37]. In paper I, III and IV cell morphology and plasma membrane integrity was assessed by
trypan blue dye exclusion test and observed by light microscopy.
Apoptosis: In paper I, apoptosis was assessed after Wright-Giemsa staining or following
Annexin V/propidium iodide staining [127]. For Wright-Giemsa staining, cells in
suspension were cyto-centrifuged, washed in PBS and spun down onto glass slides using
cyto-spin. The cells were then fixed in 4% formaldehyde for 10 minutes followed by
Wright-Geimsa for 2 minutes at room temperature. Cell morphology was examined by light
microscopy. Apoptotic cells were characterized as shrunken cells with fragmented nuclei.
In paper I, II, III and IV apoptosis was assayed by detection of phosphatidylserine exposure
using flow-cytometry following Annexin V/propidium iodide staining. Cells scored as early
apoptotic when they were positive to Annexin V, while when cells were positive to both
Annexin V and PI, they were scored as post-apoptotic or necrotic cell death. In brief, control
and treated cells were collected, washed once with phosphate-bufferered saline (PBS),
and stained for 10 min on ice with Annexin V and propidium iodide and analyzed by
29
flow-cytometry.
Reactive oxygen species
Intracellular reactive oxygen species was assayed by flow-cytometry following
dihydroethidium (DHE) staining. DHE enters the cells and interacts with superoxides to
form oxyethedium which interacts with nucleic acids and emits bright-red colour and is
detected by a fluorescence microscope [128]. The cultured cells were collected, washed
once with PBS, incubated for 15 min at 37°C with 10 µM DHE and analysed with flow-
cytometry. Percentages of cells with increased DHE red fluorescence were gated and
considered as increased reactive oxygen species.
Lysosomal membrane integrity
The integrity of the lysosomal membrane was assessed using the acridine orange (AO)
uptake technique as established previously [127]. AO is a lysosomotropic weak base which
accumulates in the acidic vacuolar compartment or lysosomes, and is protonated and
trapped in the vacuole, thereby imparting red granular fluorescence upon excitation with
blue light. An increase in lysosomal membrane permeability results in leakage of AO from
the lysosomes which shift emission fluorescence to green. In brief, the cells following
different treatments were collected, stained with 5 µg/ml AO (15 min, 37 °C), and analysed
with fluorescence microscopy or flow-cytometry. Percentages of cells with decreased
lysosomal AO red fluorescence were gated and considered as increased LMP.
Mitochondrial membrane potential
The mitochondrial membrane potential (Δψm) was measured using the fluorescent probe
JC-1 [129]. JC-1 enters mitochondria and reversibly changes colour from red to green with
decrease in membrane potential. In healthy cells with high membrane potential, JC-1 forms
complexes j-aggregates and emits red fluorescence [130]. While in apoptotic cells with low
mitochondrial potential, JC-1 remains in monomeric form and emits green fluorescence. In
brief, following different treatments control and treated cells were collected, incubated
with JC-1 (5 µg/ml for 10 min at 37 °C) and analysed with flow-cytometry. The ratio of JC-1
red and green 1uorescence intensities was used to calculate mitochondrial membrane
30
potential.
Phagocytic activity
Phagocytic activity was measured by assay of dextran up-take (FITC conjugated dextran,
FD40, molecular weight 40 000) and yeast up-take assay (FITC conjugated yeast).
Dextran uptake assay: Following treatment cells were exposed to FD40 (1 mg/mL) at
37°C or 4°C for 1 h. After washing with phosphate-buffered saline (PBS), the up-take of
FD40 was determined by flow-cytometry [131, 132].
Yeast uptake assay: For FITC-yeast assay, cells were exposed to FITC conjugated yeast
(1.4 x 106/ml) for two hours and then washed with PBS. The uptake of FITC-yeast was
determined by fluorescence microscopy [132].
Cellular iron
Intracellular iron oxide was localized by Perls’ Prussian blue staining. In brief, the cells
were incubated for 30 min with potassium hexacyanoferrat in 10% HCl, washed and
counter-stained with nuclear fast red. Ferric ions (Fe3+) in the cells react with potassium
ferrocyanide to form a blue pigment (Prussian blue).
Immuno-cytochemistry
Following treatment the cells were collected, immuno-stained for surface or intracellular
proteins and analysed by flow-cytometry or fluorescence microscopy [127].
Intracellular protein: In brief, the cells were fixed with 4% paraformaldehyde,
permeabalized with permeabalizing buffer (0.1% saponin and 0.5% serum in PBS), and
then incubated with primary anti-bodies to protein of interest at 4°C overnight. They were
then incubated with Alex 488 conjugated secondary antibodies at 22°C for 1h.
Surface protein: For determining surface proteins, cells were washed with PBS, and
then incubated with primary antibody for 30 min at 4°C followed by incubation with Alex
488 conjugated secondary antibodies at 4°C for 30 min. Then the cells were fixed with
4% paraformaldehyde. Fluorescence intensity of immuno-stained cells was determined by
31
flow-cytometry or fluorescence microscopy.
Secreted, nuclear and cytosolic protein
ELISA: Secreted protein in culture media was measured by an ELISA kit according to
the company’s protocol. The protein concentrations in the culture media were assayed by
bicinchoninic acid protein assay (BCA) according to manufactory’s instruction (R & D
Systems, MN, USA), and normal media was measured as background.
Western blot: Cytosolic and nuclear proteins were extracted according to manufactory’s
instruction by using an extraction kit (Cayman Chemical, Michigan, USA). The extracted
cytosol and nuclear fractions were saved at -80°C until usage. Secreted proteins were
precipitated using nine volumes of absolute ethanol at -70°C for 2 h. Precipitated proteins
were collected by centrifugation (12,300 relative centrifugal force) and were re-
suspended in the sample buffer with dithiothreitol and bromophenol blue and boiled for
4 minutes at 95°C. The protein concentrations from extracts and in culture media were
assayed by BCA protein assay and normal media was measured as background.
Secreted proteins in culture media, whole cells, cytosolic and nuclear extracts from
different groups of cells were loaded onto a 12% sodium dodecyl sulphate-
polyacrylamide gel and transferred onto a nitrocellulose membrane. Membranes were
then incubated with antibodies against protein of interest at 4°C; overnight after being
blocked with 5% nonfat milk in tris-buffered saline with 0.1% Tween® 20. Finally,
membranes were incubated with horseradish peroxidase-conjugated secondary antibodies
at 20°C for 1h and assayed by an enhanced chemiluminescence plus western detection kit.
Ponceau S staining or b-actin served as loading control.
Statistics
For statistical analysis, unpaired student’s t test for two groups or one-way ANOVA
followed by posthoc Newman-Kuels test for multiple groups were performed. Results are
given, as means ± SEM. To study correlation, Pearson correlation coefficient test or
Spearman correlation coefficient test and linear regression test was used. P ≤ 0.05 was
considered statistically significant.
32
RESULTS
Paper I and II
Mangafodipir, MnPLED and Dp-dp prevents 7βOH-induced cell death
In these studies, we investigated whether mangafodipir its metabolite MnPLED and its
constituent Dp-dp with anti-oxidant effects could affect 7βOH induced oxidative stress and
subsequent cell death. We found that mangafodipir was non-toxic to cells even at
concentrations as high as 800 µM. Furthermore, mangafodipir at concentrations between
100 and 400 μM inhibited 7βOH-induced cell death in a dose-dependent manner, while it
had no protective effect when the concentrations were less than 100 μM as assayed by
trypan blue staining followed by analysis with light microscopy or by annexine V
propidium iodide staining followed by analysis with flow-cytometry. Identical to the parent
compound, mangafodipir metabolite MnPLED (100 µM) and its structural constituent Dp-
dp (100 µM) also provide similar cyto-protection against 7βOH induced cell death.
Involvement of lysosomal pathways
Oxysterol induced apoptosis involves permeabilization of lysosomal membranes and
redistribution of their contents to cytosol [127, 133]. To determine whether regulation of
LMP was involved in mangafodipir mediated cyto-protection against 7βOH induced cell
death, we examined the effect of mangafodipir on LMP by using the AO uptake method.
7βOH induced destabilization of the lysosomal membrane was characterized by a
decreased red lysosomal AO red fluorescence with an increased cytosolic green
fluorescence as analysed by fluorescence microscopy or flow-cytometry. Pre-treatment
with mangafodipir and its metabolite MnPLED and its structural constituent Dp-dp
significantly prevented lysosomal membrane permeability induced by 7βOH. No
differences at the level of protection against 7βOH induced LMP was observed between
mangafodipir, MnPLED and Dp-dp. These results indicate that mangafodipir mediated
cyto-protection against 7βOH involves prevention of lysosomal membrane damage.
33
Involvement of mitochondrial pathways
Mitochondria are both the source and the target for ROS. It is known that ROS regulates the
release of cytchome C from mitochondria and thus regulates cell death [134, 135].
Moreover, increased intracellular ROS and mitochondrial membrane permeability (MMP)
are involved in 7-oxysterol mediated cell death [136]. To determine whether MMP and ROS
were also involved in mangafodipir mediated protection against 7βOH induced cell death,
the effect of mangafodipir on 7βOH induced MMP and ROS were evaluated by using JC-1
and DHE staining respectively. As analysed by flow-cytometry, in 7βOH exposure for 18h
resulted in decreases in JC-1 induced orange/red fluorescence and increases in JC-1 green
fluorescence, which indicates an increase in MMP in cells exposed to 7βOH. Additionally,
7βOH exposure for 18h resulted in significant increases in DHE induced red fluorescence
indicating increases in cellular ROS. However, pre-treatment with mangafodipir preserved
mitochondrial membrane potential. In addition, mangafodipir, Mn-PLED and Dp-dp
prevented 7βOH induced ROS production. No differences at the level of protection against
7βOH induced ROS was observed between mangafodipir, MnPLED and Dp-dp. These
results indicate that mangafodipir mediated cyto-protection against 7βOH involves
modulation of mitochondrial pathways, including suppression of ROS production and
preserving mitochondrial membrane potential.
Paper III and IV
Monocyte differentiation induces functional plasticity in macrophages
and simultaneous increases in ferritin and cathepsin L
We found that induction of differentiation of human monocytes induces functional
polarization in macrophages. Although, it is known that PMA induced differentiation in
THP1 monocytes induces a M2 macrophage subtype secreting anti-inflammatory cytokines,
we further validated this result in our study. We found that PMA stimulation induced M2
macrophages (THP1 PMA M2) characterized by an up-regulation of endogenous CD163
levels. Human monocyte derived macrophages (HMDMs) were also examined for their
functional polarization. We found that hIgG induced HMDMs exhibit CD68 and CD86
immuno-positivity and these macrophages did not express CD163 as confirmed by
34
fluorescence microscopy. In addition, we found up-regulated levels of endogenous ferritin
and cathepsin L in THP1 PMA M2 macrophages upon induction of differentiation. Further,
polarizing THP1 macrophages into distinct M1 and M2 subtypes raised intracellular ferritin
and cathepsin L levels. As expected, we did not observe significant differences in CD163
levels between PMA induced THP1 macrophages (THP1 PMA M2) and macrophages
further treated with IL-4 and IL-10 (THP1 M2), which indicate that the M2 macrophages
cannot be further influenced by combined cytokine treatment. However, significant
differences were observed in the levels of cellular ferritin and cathepsin L between THP1
M1 and THP1 M2 macrophages which indicates that cellular levels of these proteins may be
representative of important functional orientation in the macrophages.
SPION up-regulates ferroportin, ferritin and cathepsin L levels, incites
cytokine and ferritin secretion and modulates cell phenotype
SPION are unceasingly developed to target various biological characteristics of
atherosclerosis, including foam cells and macrophages, which exhibit functional plasticity
in atherosclerotic microenvironment [2, 23, 124]. However, their interactions with such
targets are completely unknown. In these studies, we investigated their interactions with
such targets in-vitro. We found that SPION loading affects cell functions without affecting
cell viability. In addition, we found that oxysterols reduce the phagocytic activity in
macrophages, thereby reducing the uptake of SPION.
Iron export
SPION are primarily ingested by monocytes and macrophages. They accumulate in the
lysosomes and are gradually degraded resulting in iron accumulation [118]. Cellular
viability was un-altered following SPION exposure for 6 - 48 h as assayed by trypan blue
staining or by flow-cytometry following Annexin V/PI staining. SPION loading had no effect
on cellular ROS as analysed by flow-cytometry following DHE staining. However, SPION
loading significantly enhanced intracellular iron concentrations as confirmed by Perls’
Prussian blue staining for iron. This increase in cellular iron levels could be potentially
detrimental. Perhaps to reduce iron load, cellular iron export pathways were activated in
the cells upon SPION loading. We found that iron loading induced membranous ferroportin
35
expression in the cells as analysed by florescence microscopy. Ferric ammonium citrate
(FeAC) induced ferroportin was used as a positive control. Ferroportin expression was not
the only measure taken by the cells to reduce cellular iron levels. A dose and time
dependent increase in ferritin secretion was also observed in the culture supernatants
following SPION loading. Ferritin is an ubiquitous and highly conserved iron-binding
protein, and its secretion by the cells would result in release of iron load. Interestingly,
ferritin secretion was considerably up-regulated in untreated controls of Thp1 monocytes
at 72 h. Similar ferritin secretion was also observed in THP1 monocytes following LPS
exposure.
Phenotypic shift
SPION loading increases cellular iron concentrations. Potentially iron regulates
macrophage plasticity and has immuno-modulatory properties. We found a significant
increase in TNF-α secretion following SPION loading, as measured by ELISA. A dose-
dependent IL-10 secretion was also observed following SPION loading, which declined over
72 h. In these experiments cells treated with LPS were positive controls. LPS stimulation
significantly augmented TNF-α secretion. Additionally, we found that iron in SPION induces
a phenotypic shift in THP1 M2 macrophages marked by elevated CD86 levels in these
macrophages which is a characteristic feature of M1 macrophages. This phenotypic shift
can be detrimental in various immunological disorders and may aggravate inflammation.
Ferritin and cathepsin L up-regulation in macrophage subtypes
M1 macrophages hold intracellular iron, whereas M2 macrophages are distinctively
characterized by an iron release. To examine the effect of iron in SPION on cellular iron
storage, ferritin levels were examined by flow-cytometry. Since SPION is degraded in the
lysosomes and thereby increases lysosomal iron levels, we evaluated the potential of iron
in SPION to modulate lysosomal functioning by focusing on lysosomal enzymes. We found
that iron in SPION simultaneously up-regulates ferritin and cathepsin L levels in monocytes
and in both M1 and M2 macrophages. Furthermore, the subcellular location of SPION
induced ferritin was both nuclear and cytoplasmic as confirmed by western blot of the
nuclear and cytosolic fractions of the monocytes after indicated treatments. FeAC was used
36
as a positive control in the model. DFO significantly diminished SPION as well as FeAC
induced ferritin and cathepsin L in the cells which confirms the role of iron in the process.
These results indicate that SPION elevates ferritin and cathepsin L levels in monocytes and
macrophages irrespective of their functional polarization.
Cathepsin in ferritin degradation
Lysosomes not only contain abundant hydrolytic enzymes but are also rich in iron.
Lysosomal enzymes are pivotal to ferritin proteolysis to maintain ferritin levels and
balance redox-active iron. We examined the possibility that the elevated ferritin level upon
SPION loading was the driving force for up-regulated cathepsin L. Cells were pre-treated
with or without E64 (cathepsin B/L inhibitor) or CLIK 148 specific cathepsin L inhibitor
followed by incubation with SPION. Both E64 and CLIK not only elevated SPION and FeAC
induced ferritin, but also elevated basal levels of ferritin in monocytes. These results
indicate a functional role of lysosomal cathepsins in ferritin degradation.
Ferritin, cathepsin L and macrophage plasticity
Iron regulates macrophage plasticity and thus levels of iron-related proteins among
macrophage subtypes might be important to sustain a polarized state. We found that
induction of differentiation in monocytes imparts functional polarization in resulting
macrophages and is associated with up-regulation of endogenous ferritin and cathepsin L
levels. Up-regulation in the endogenous levels of ferritin and cathepsin L were observed in
hIgG induced CD68+/CD86+/CD163- HMDMs and PMA induced THP1 macrophages.
Furthermore, polarizing THP1 macrophages into distinct M1 and M2 subtypes also raised
ferritin and cathepsin L levels in these macrophages. However, significant differences were
observed in the levels of cellular ferritin and cathepsin L between THP1 M1 and THP1 M2
macrophages, which indicates that cellular levels of these proteins may be representative
of macrophage phenotype.
37
Oxysterols reduce phagocytic activity in macrophages thereby reduce
uptake of SPION
Foam cells and lipid rich macrophages are biomarkers of atherosclerotic plaques. Such cells
are important imaging targets. SPION are unceasingly developed to target such
macrophage foam cells in atherosclerotic plaques. We found that oxysterol exposure
reduces phagocytic activity and thereby reduces SPION uptake by both THP1 monocytes
and THP1 macrophages and HMDMs. SPION uptake was confirmed by Perls’ Prussian blue
staining. In contrast to control cells, most cells exposed to SPION were Perls’ positive.
However, pre-treating cells with atheroma relevant 7βOH or 7keto and then exposure to
SPION resulted in decreased numbers of Perls’ -positive cells, indicating reduced
intracellular iron and reduced SPION uptake. This reduced uptake of SPION by cells
exposed to oxysterols resulted from a decline in phagocytic activity. Phagocytic activity
was measured by assaying uptake of FITC conjugated dextran (FD40) or FITC conjugated
yeast. Oxysterol exposure significantly declined FD40 uptake by monocytes and
macrophages. Moreover, in comparison to unexposed cells, a significantly low uptake of
FITC-yeast was observed in macrophages exposed to oxysterols. These results indicate a
decline in phagocytic activity in monocytes and in macrophages, which eventually lead to
reduced uptake of SPION by such macrophages. We also found an additive effect of SPION
on 7βOH induced cathepsin and ferritin. Although SPION uptake was reduced, the ingested
amounts significantly up-regulated the expression of cathepsin B, cathepsin L and ferritin
in both monocytes and macrophages, which may further aggravate atherogenesis.
Surface coating promotes uptake of iron-oxide nanoparticles and
potentially prevents iron induced cytotoxicity
Uptake INPs by macrophages was observed, irrespective to the presence or absence of a
surface coating, which resulted in increases of intracellular iron. However, the amounts of
INPs taken up by macrophages were dependent on the presence of a surface coating.
Carboxy-dextran coating in SPION enhances its solubility and uptake by macrophages
[100]. In the lysosomes, this outer dextran coating is degraded and the contents of SPION
38
are released, which are Fe2O3 and Fe3O4. Despite such dramatic increases in cellular iron
levels upon SPION loading, cell viability was unaltered. We compared the effects of SPION
with uncoated INPs (Fe2O3 and Fe3O4) on cell viability and functions and observed many
differences. We found that, intra-cellular iron levels in macrophages exposed to uncoated
INPs were considerably lower compared to macrophages exposed to SPION. SPION are
non-toxic to cells even at concentrations as high as 400 µg Fe/ml. In contrast, exposure to
uncoated INPs induced dose dependent cell death in macrophages. Additionally, uncoated
INPs affected phagocytic activity in macrophages. Cells exposed to sub-lethal
concentrations of Fe2O3 and Fe3O4 showed a reduced phagocytic activity compared to cells
exposed or unexposed to SPION as analysed by FITC-yeast assay. This reduction in
phagocytic activity may be early indications of subsequent loss of cell viability.
39
GENERAL DISCUSSION
Mangafodipir mediated cyto-protection involves modulation of
lysosomal and mitochondrial pathways
Our study substantiates the existing knowledge about underlying mechanisms behind cyto-
protection conferred by mangafodipir. The anti-oxidant activity of mangafodipir is well
documented [96, 98, 103] which ascertains its role in decreasing cellular ROS levels and
thereby protecting cells against oxidative stress. We found that mangafodipir not only
prevents 7βOH induced ROS production but also preserves the mitochondrial membrane
potential and thereby preventing cell death. By preserving mitochondrial membrane
potential, mangafodipir may stop the potential release of cytochrome C and subsequent cell
death. These results elucidate that the preservation of mitochondrial membrane potential
is an additional effect of mangafodipir on the mitochondrial compartment apart from
preventing ROS generation. The role of LMP and related cathepsins as apoptotic initiator is
now regarded as important apoptotic pathways [137, 138]. Lysosomal pathways are also
involved in cyto-protection conferred by mangafodipir. Our notion is based upon the
results where mangafodipir prevented LMP induced by oxysterols, which may prevent the
potential release of lysosomal cathepsins and subsequent activation of cell death pathways.
A conserved structure in mangafodipir may attribute to its
cyto-protective effects
We compared the parent compound, mangafodipir with its primary metabolite MnPLED
and its constituent Dp-dp. Mangafodipir, MnPLED and Dp-dp provide similar cyto-
protection against 7βOH induced cell death. These results suggest that the active part of
mangafodipir, which imparts the above-noted effects, is conserved among Dp-dp and
MnPLED. As Mn is rapidly released and cleared from the blood on intravenous
administration. The most likely conserved domain shall be Dp-dp and its dephosphorylated
derivative PLED, which have been shown to possess high iron binding capacity [98]. These
intriguing results lead us to question whether modulation of cellular iron is involved in
cyto-protection mediated by mangafodipir and its metabolites. We tested this hypothesis
40
by measuring the levels of ferritin in cells exposed to MnPLED and Dp-dp. We did not
observe any alterations in ferritin levels upon treatment with MnPLED and Dp-dp, which
does not exclude the possible involvement of other iron regulatory proteins or modulation
of labile iron pool in MnPLED and Dp-dp mediated cyto-protection.
Monocyte differentiation induces macrophage phenotype with
elevated levels of ferritin and cathepsin L
Induction of differentiation in monocytes induces functional polarization in acquired
macrophages. This notion is supported by our results where HMDMs induced by hIgG were
characterized as a distinct CD68+, CD86+ and CD163- macrophage subtype and PMA
induced THP1 macrophages were characterized as M2 macrophages which exhibit higher
levels of CD163. Additionally, cellular levels of ferritin and cathepsin L might be important
in processes like macrophage differentiation and their functional polarization. We found
that induction of differentiation in THP1 monocytes by PMA and in human blood
monocytes by hIgG up-regulated endogenous levels of ferritin and cathepsin L in resulting
macrophages. We further confirmed our observations by polarizing THP1 macrophages
into distinct M1 and M2 subtypes following an established protocol [30]. These treatments
raised endogenous ferritin and cathepsin L levels in both THP1 M1 and THP1 M2
macrophages. However, significant differences were observed in the levels of cellular
ferritin and cathepsin L between THP1 M1 and THP1 M2 macrophages.
Iron in SPION primes THP1 M2 macrophages towards a high
CD86+ macrophage subtype with elevated levels of ferritin and
cathepsin L
Iron in SPION governed the cellular levels ferritin and cathepsin L, as indicated by the
actions of DFO which significantly down-regulated SPION induced ferritin and cathepsin L
in our model. It is suggested that M1 macrophages hold intracellular iron whereas M2
macrophages release iron [24, 139]. However, we found similar handling of iron in SPION
by THP1 monocytes, THP1 M1 macrophages, THP1 M2 macrophages, THP1 PMA M2
macrophages and HMDMs. Iron in SPION simultaneously up-regulated ferritin and
41
cathepsin L in all cell types tested in this study. However, the levels of SPION induced
ferritin and cathepsin L was significantly higher in THP1 M1 macrophages in comparison to
THP1 PMA M2 macrophage and THP1 monocytes. Higher endogenous levels of ferritin [24,
139] and cathepsin L in M1 macrophages compared to M2 macrophages may reason the
highest levels of SPION induced ferritin and cathepsin L in THP1 M1 macrophages. In
addition iron in SPION induced a phenotypic shift in THP1 M2 macrophages towards a
macrophage subtype characterized by high levels of CD86, ferritin and cathepsin L, which
we found as a characteristic hallmark of M1 macrophages. This phenotypic shift can be
detrimental in atherosclerosis and in various immunological disorders and may aggravate
inflammation.
Lysosomal cathepsins have a functional role in ferritin
degradation
We examined the reasons behind simultaneous up-regulation of ferritin and cathepsin L in
monocytes and macrophages exposed to SPION. Lysosomal cathepsin is pivotal to ferritin
proteolysis to maintain ferritin levels and balance redox-active iron [140, 141]. Inhibition
of lysosomal cathepsins resulted in an increase in endogenous and SPION induced ferritin,
which is likely due to the prevention of ferritin degradation by lysosomal cathepsins. These
results indicate that lysosomal cathepsins have a functional role in cellular ferritin
metabolism and are pivotal to maintain ferritin levels following SPION degradation.
However, we cannot exclude the possibility that cathepsin inhibition may also interfere
with ferritin synthesis as demonstrated by Zhang et al [31].
SPION loading incites secretion of both pro-inflammatory and
anti-inflammatory cytokines
In atherosclerosis, immunological microenvironment determines the plaque progression
and its vulnerability [23]. Iron-oxide nanoparticles are mostly ingested by immune cells,
which increase their intra-cellular iron concentrations. Iron overload or its deficiency may
incite deleterious physiological effects [10, 142]. Iron overload has been also shown to
impair production of nitric oxide (NO) by macrophages [143]. Interestingly, iron-oxide
42
nanoparticles has been shown to induce immune response characterized by secretion of
both pro-inflammatory [144] and anti-inflammatory cytokines [145] by macrophages.
Similarly, in our study, we find that SPION loading induces secretion of both (TNF-α) pro-
inflammatory and (IL-10) anti-inflammatory cytokines. Additionally, at low concentration
of SPION (5µg Fe/ml) this response was noticed, which indicates the potency of these iron-
oxide nanoparticles. While a pro-inflammatory response may be the direct effect of iron
excess, an anti-inflammatory cytokine IL-10 secretion may correspond to a cellular defence
mechanism activated by cellular iron overload. Such immuno-modulatory effects of iron-
oxide nanoparticles must be strictly considered in future development of these particles
Non-functionalized SPION may be inefficient to target intra-
plaque macrophages
Macrophages are determinants of plaque progression and stability. Clearance of debris,
lipid and apoptotic and necrotic cells is one of the vital functions of macrophages in
atherosclerotic plaques [87, 146]. Attenuation of phagocytic functions in these
macrophages may enhance the rate of plaque progression. INPs are increasingly developed
to target such macrophages and foam cells in atherosclerotic plaques. Instead of
conventional targeting of such macrophages by INPs, molecular targeting by
functionalization of these INPs is the preferred approach today. Functionalization of INPs
includes modifying surface coating material and tagging them with specific antibodies and
peptides, which increases their target specificity [2, 100].
Reduced clearance indicates reduced phagocytic activity of macrophages in atherosclerotic
plaques, which may reason the development of such an approach. We observed reduced
uptake of INPs by macrophages exposed to atheroma relevant oxysterols, which can be
directly attributed to reduction in their phagocytic activity. In this study, we did not
examine whether oxysterols activated cell death pathways in macrophages. Thus we do not
exclude the possibility that initiation of apoptosis in macrophages by atheroma relevant
oxysterols may cause the reduction in phagocytic activity of these macrophages.
43
These results emphasize the necessity to develop functionalized INPs, which can increase
intracellular iron levels in such cells without affecting their viability and functions. And
such INPs should be able to distinctively identify macrophages and foam cells for effective
determination of plaque severity and vulnerability.
Although, macrophages exposed to oxysterols display a poor uptake of INPs, the uptake
does not entirely stop as indicated by up-regulation of oxysterol induced cathepsin L and B
by SPION in our cell model. 7-oxysterols induced cell death involves activation of lysosomal
pathways, including the release of lysosomal cathepsins. In chronic inflammatory disorders
like atherosclerosis lysosomal cathepsins may modulate apoptosis and cellular immune
functions. Our results for the first time show that iron in SPION up-regulates both
endogenous cathepsin L, and oxysterol induced cathepsin L and cathepsin B in monocytes
and macrophages. While up-regulation of oxysterol induced cathepsins by SPION is
indicative of an additive effect, the up-regulated cathepsins may further aggravate
atherogenesis.
Functional coating of iron-oxide nanoparticles gives cell
tolerance to potential cytotoxicity by regulation of cell
functions
Concentration of iron at atherosclerotic plaques exceeds that found in healthy arterial
tissue [12], and it is potentially redox active [11]. Erythrophagocytosis stands as an
important source of redox active iron in atherosclerotic plaques [11, 12]. In addition,
reduction and removal of stored iron by systemic iron chelation or dietary iron restrictions
has been shown to reduce lesion size and increased plaque stability in animal studies [7,
11]. These findings illustrate the importance of critical iron balance in atherosclerotic
plaques. Increasing cellular iron concentration by iron-oxide nanoparticles in macrophages
to image vulnerable and iron rich atherosclerotic plaques is certainly a major concern.
Iron catalyses the formation of ROS and OxLDL and potentiate OxLDL mediated
macrophage cell death [147], which may result in plaque formation and increase severity of
atherosclerosis [148, 149]. Although in our study, we found that carboxy-dextran coated
44
SPION is well tolerated by monocytes and macrophages, we also noticed the potential of
uncoated iron oxide nanoparticles to induce cell death. These results clearly indicate that
iron in iron oxide nanoparticles is potentially cytotoxic. However, coating their surface by
dextran makes them more compatible in biological compartments. Indeed, the biological
response generated by these nanoparticles has been attributed primarily to their size and
surface coating [2, 100]. The cytotoxic activity of uncoated iron-oxide nanoparticles,
including oxidative stress and DNA damage is well documented [2, 100, 104, 112]. In
contrast, high compatibility of dextran coated nanoparticles is also documented [100].
These iron-oxide nanoparticles have a long cellular residence time. Although, we could not
detect ROS production or cell death on exposure to SPION for as long as 72 h, there is
evidence that uncoated iron-oxide nanoparticles and ultrasmall iron-oxide nanoparticles
(USPION) induce ROS production and cell death [100, 150]. In addition, we and others have
shown that iron-oxide loading up-regulates ferritin in the cells [100, 151, 152]. Iron in iron-
oxide nanoparticles may be sustained in ferritin and hemosiderin and remain as a reservoir
of potentially reactive iron, which may be detrimental in plaque microenvironment.
However, releasing potentially reactive iron is not the only way by which iron-oxide
nanoparticle induced ferritin could determine the fate of plaque. Apart from being an iron
storage protein ferritin is immuno-modulatory [65, 153], associated with inflammation,
and has been shown to regulate ROS production and cell death [64] which may increase the
severity and progress of atherosclerosis. Additionally, iron released from iron-oxide
nanoparticle loaded cells could trigger apoptoic and necrotic cell death. We found that
SPION loading incites membranous ferroportin expression and ferritin secretion which
might be a cellular response to reduce elevated intracellular iron levels. However, this
action could be potentially unsafe to nearby phagocytes as it is suggested that endocytosis
of extracellular ferritin increases the level of free ferrous iron in the lysosomal
compartment inducing oxidative stress, LMP and trigger apoptosis and necrosis [142].
One major concern in the field of nano-toxicity is that SPION gradually increases cellular
iron concentrations in cells, which may ascertain the adverse effects of iron excess.
However, we observed differences in biological response incited by uncoated INPs and
carboxy dextran coated SPION. While, uncoated INPs induced dose dependent cell death,
45
carboxy dextran coated SPION used in our study affected vital cell functions, including cell
phenotype, immune response and altered the levels of iron dependent protein. No
alteration in cell viability was observed in cells exposed to SPION at doses as high as 400 µg
Fe/ml. Our results complied with previously published data, where SPION loading induced
minimal or no toxicity [100]. Toxicity induced by SPION, if any, is dependent on factors
such as surface coating, break-down products and oxidation state of iron in the SPION
[100]. In fact, the surface properties including surface coating of SPION determines the
efficiency of their cellular uptake, metabolism and potential toxicity. Masking the iron core
in SPION with carboxydextran, its lysosomal accumulation and degradation and gradual
release of iron may very well be the underlying reasons behind excellent cellular iron
tolerance. However, a recent study indicates significant cyto-toxicity induced by SPION
[104]. The authors in this study suggest that degradation of the carboxydextran shell and
exposure of the iron oxide core are delayed phenomena, and thus they employed a longer
period (five days) of exposure with SPION in their study. However, in our study, we find
that SPION loading significantly up-regulates cellular ferritin levels within 24 h, which
reaches its peak at 48 h and gradually declines at 72 h. These results are indicative of the
fact that degradation of the carboxy dextran shell, iron release and its entrapment in
ferritin is a much faster process than anticipated in the above study. We do not exclude the
possibility that incubating cells with SPION for as long as five days would have yielded us
different results. Although, we consider that this would be a good approach in animal
studies, but the results from in vitro study like in ours and from Lunov, et al., 2010 [104]
shall be governed by other additional factors. In our study, we observed an increased level
of ferritin and IL-10 in the culture medium of untreated control cells after 72 h of
incubation. This may indicate immune response by cells in nutrient deprived culture
media. The phenomenon was previously observed in melanoma cells, where the cells
secreted H-chain rich ferritin to stimulate IL-10 production [154]. This extracellular
protein accumulation might trigger or inhibit the observed differences between our
studies.
Overall, increases in cellular iron concentration have been attributed to the observed
alteration in cell functions and loss of cell viability. In our study, we highlighted several
46
pathways that the cell could employ to reduce iron burden and thus may escape iron
induced cyto-toxicity. SPION induces membranous ferroportin expression, which is by far
the only discovered iron export pathway [155]. Membranous ferroportin may reduce the
INPs induced cellular iron burden by exporting intracellular iron. Up-regulation of ferritin
could be employed as an initial step by the cells to accommodate iron released from
lysosomal degradation of SPION. It is suggested that cellular ferritin stores have the
capacity to bind to 2.6 folds of excess iron. By doing this cells entrap potentially active iron
in ferritin cage thereby reducing cellular free iron concentrations. Additionally, SPION
induced nuclear ferritin up-regulation in the cells which may protect cells against oxidative
stress and DNA damage [64]. Ferritin secretion by cells exposed to SPION might also
release the iron burden. Membranous ferroportin expression and ferritin secretion may
together help reducing the cellular iron load in cells exposed to SPION. However, the
impact of iron released by ferroportin and secreted ferritin on the adjacent cells has to be
evaluated as it has been suggested that released ferritin may also contribute to
immunological signalling events [10, 60]. Additionally, SPION loading also resulted in an
anti-inflammatory cytokine IL-10 secretion which is indicative of a cellular defence
mechanism activated by cellular iron overload and this may also protect the cells.
A need for better characterization of macrophage subtypes
Diverse methodologies, including cytokine stimulation and growth factor induced
differentiation have been used for classical (M1) and alternative (M2) macrophage
polarization from monocytes in-vitro. We characterized hIgG induced macrophages as a
distinct CD68+, CD86+ and CD163- macrophage subtype. This phenotype closely resembles
CXCL4 induced M4 macrophages characterized by a complete loss of CD163 [28] and
higher levels of CD86 [27]. However, we do not exclude the possibility that procedure used
in this study to generate HMDMs may also be utilized to generate DC (dendritic cells). Both
macrophages and dendritic cells may differentiate from monocytes depending on culture
conditions and use of additional growth factors. To differentiate them is an intriguing and
much discussed issue [156]. Thus, based on the fact that in our study we did not use
additional growth factors and the obtained HMDMs phenotype closely resemble M4
47
macrophages, we claim these cells as macrophages. However, these macrophages have to
be further characterized based upon their cytokine secretion response and a larger panel of
macrophage phenotypic markers [156-158] can be utilized to verify our results.
Additionally, we followed an established protocol from a well cited study [30] to generate
both M1 and M2 macrophages from THP1 cells. These macrophages have been functionally
characterized as M1 and M2 macrophages previously. However, we further validated the
phenotype of these macrophages by using widely employed macrophage phenotype
markers CD86 and CD163, which mark M1 and M2 macrophages respectively [139, 159-
161]. Although, it is known that DC are also CD86 positive cells, and may be differentiated
from THP1 monocytes by a similar protocol, it is accepted that THP1 cells have already
developed too far along the monocyte/macrophage differentiation pathway to allow
redirection toward DC [162]. Additionally, differentiating THP1 monocytes to dendritic
cells in-vitro requires additional supplements such as GM-CSF and serum free medium.
Such conditioning is lacking in our experimental model. In addition, it is also documented
that even after fulfilling most of the above requirements, the capacity of THP1 cells to
differentiate to DC is merely 5 % and they fail to acquire DC functions [163-165].
In addition, as indicated in the introduction section of this thesis, M2 macrophages are
further subdivided into M2a, M2b and M2c macrophages based upon their stimulus to
generate these macrophages [25]. In this study, we used IL-4 and IL-13 to generate M2
macrophages. Thus, our results from THP1 M2 macrophages may be confined to a specific
M2a phenotype of macrophages and may be verified in other M2 macrophage phenotypes
in future studies.
48
CLINICAL AND FUTURE PERSPECTIVES
Mangafodipir and SPION based particulate agents such as ResovistTM have been shown to
possess similar accuracy in detecting human liver lesions and hepatic metastasis [166-
169]. However, SPION based contrast agents have grabbed most attention pertaining to the
ease of their functionalization directed for targeted imaging in various imaging modalities,
including cardiovascular imaging. Nonetheless, these advancements and benefits of SPION
accompany the risk and concerns associated with their exposure. It is of particular concern
in the field of atherosclerotic plaque imaging where iron excess may eventually determine
the plaque vulnerability [170]. In contrast, Mangafodipir with proven pharmacological
effects, including our current findings that it prevents oxysterol induced cytotoxicity,
stands a better option in the field of atherosclerotic plaque imaging. In fact, new manganese
doped iron-oxide nanoparticles are currently been developed with the intensions to reduce
toxicity and intolerance of INPs [80]. Such developments in the future may produce a safe
contrast agent with both therapeutic and diagnostic potential.
The results from paper III and paper IV suggest that cellular immune functions can be
modulated by SPION pertaining to change in macrophage phenotype. Although, we
observed a SPION induced phenotypic shift in macrophages, the phenomenon was
examined by analysing the levels of macrophage phenotype marker protein in these
macrophages. It would be interesting to observe an altered cytokine secretion response by
M2 macrophages exposed to SPION. In addition, future studies should further examine the
influence of iron in SPION on the immune-functions of atheroma relevant macrophages. It
is known that exposure to OxLDL or oxysterols induces a pro-inflammatory macrophage
subset [171-173]. Thus it would be interesting to examine the effects of SPION induced
additional iron load on immune response of such macrophages. However, elevation in pro-
inflammatory response of such macrophages might be the most probable outcome.
Results from paper III and IV indicate that oxysterols reduce phagocytic activity in
macrophages and thereby reduce the uptake of iron-oxide nanoparticles. Cells uptake
nanoparticles by diverse mechanisms, including; phagocytosis, micropinocytosis, clathrin
49
mediated endocytosis and caveolae mediated endocytosis. Recently, clathrin mediated
uptake of SPION (ResovistTM) has been suggested [115]. Thus, it would be of interest to
examine the levels of clathrin in cells exposed to oxysterols. Perhaps, a decline in the level
of clathrin in macrophages exposed to oxysterols may reason the observed reduction in
SPION uptake by such cells. However, affecting clathrin mediated endocytosis might not be
the only way by which oxysterols may affect nanoparticle uptake. It has been also shown
that oxysterols down-regulate mRNA levels of caveolin [174], which may very well affect
endocytic function of cells. These findings suggest that oxysterols may affect endocytic
pathways in macrophages in multiple ways. These results also emphasize the necessity for
further investigation of the effects of oxysterol on endocytosis as it may directly correlate
to lipid accumulation in atherosclerotic plaques.
50
CONCLUSIONS
Mangafodipir mediated cyto-protection against 7βOH induced apoptosis involves
modulation of lysosomal and mitochondrial pathways.
This cyto-protective activity of mangafodipir is conserved within its primary
metabolite MnPLED and its structural constituent Dp-dp. These results reveal a
therapeutic potential of mangafodipir, which could be utilized for future
development of contrast agents with both therapeutic and diagnostic potential.
Iron in SPION, up-regulates cellular levels of ferritin and cathepsin L. Lysosomal
cathepsins have a functional role in cellular ferritin metabolism and are pivotal to
maintain ferritin levels following SPION degradation. Atheroma relevant oxysterols
reduce phagocytic activity in macrophage subtypes and thereby reduce uptake of
SPION. These results indicate that targeting by SPION may be inefficient to detect
lipid rich intra-plaque macrophages and foam cells and justifies the development of
functionalized iron-oxide nanoparticles to target such macrophages
Iron in SPION induces a phenotypic shift in M2 macrophages towards a CD86+
macrophage subtype with elevated levels of ferritin and cathepsin L. Cellular levels
of ferritin and cathepsin L among macrophages might be important to sustain a
functionally polarized state. Iron in SPION is potentially cytotoxic. In addition,
SPION up-regulate oxysterol induced cathepsins in cells, which may promote
atherogenesis. Thus, their clinical usage and future development should be strictly
monitored.
51
ACKNOWLEDGEMENT Interdependence is certainly more valuable than independence. This thesis is the result of
four years of work where I have been supported by many people. I now have the
opportunity to express my sincere gratitude and appreciation to all of them.
My main supervisor, Dr. Ximing Yuan for having offered me the project, sharing of
his scientific knowledge, developing enthusiasm and his invaluable support
throughout the project. I certainly owe him lots of gratitude for having me shown
this way of research.
My supervisor, Dr. Wei Li among others for the valuable support she tendered
throughout my project period. Her overly enthusiasm and integral view on research
has made a deep impression on me.
My supervisor, Professor Rolf GG Andersson, who kept an eye on the progress of my
work and was always available when I needed his advice.
The head of experimental pathology at Linköping University, Professor Karin
Öllinger, for providing a good scientific environment.
Moumita Ghosh; Sayem Miah; Sikander Iqbal Khattak and Jonas Eilertsen for
precious contributions to the papers.
Former and present colleagues at division of pathology: Aida Vahdat Shariatpanahi;
Anna Ansell; Ann-Charlotte Johansson; Bengt-Arne Fredriksson; Camilla Janefjord;
Fredrik Jerhammar; Hanna Appelqvist; Ida Eriksson; Jakob Domert; Katarina
Kågedal; Karin Roberg; Linda Vainikka; Linnea Sandin; Livia Civitelli; Lotta
Agholme; Moumita Ghosh; Nargis Sultana; Nabeel Siraj; Petra Wäster; Sangeeta
Nath; Sikander Iqbal Khattak and Uno Johansson. A special thanks to Lotta Agholme;
Hanna Appelqvist; Jakob Domert; Karin Öllinger; Katarina Kågedal and Linnea
Sandin for their advices throughout the process.
My friends among others, Sayem Miah, Nabeel Siraj, Mohammad Mirazul Islam,
Jacob Kuruvilla, Mayur Jain, Linnea Sandin and Nargis Sultana for giving me lots of
energy and great laughs.
52
My family, for their support, generosity and love: My parents Ashutosh Laskar and
Dipali Laskar. My sister Minakshi Laskar, her husband Ranajoy Laskar and her son
Anshuman Laskar. My brother Sumit Laskar. My father-in-law Dayal Krishna Ghosh,
mother-in-law Ila Ghosh and sister-in-law Paramita Ghosh.
And my wife, Moumita Ghosh. Mou, I am the luckiest because you chose me, and I
am the wealthiest because the bond we share is my most precious possession. You
have been my strength all these years, and so it shall be in the years to come.
Remember when I said that I shall be the change I want to see in you, I haven’t
changed since then.
This work was supported by the Swedish Heart-Lung Foundation, the Stroke Foundation,
and Research Funds of Linköping University Hospital.
53
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