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.

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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

17

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

18

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-

19

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,

21

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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