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Page 1: effects of native and oxydized ldl on the endothelial cells
Page 2: effects of native and oxydized ldl on the endothelial cells

À la mémoire de ma grand-mère

Page 3: effects of native and oxydized ldl on the endothelial cells

Faculté de génie

Département de génie chimique et biotechnologique

EFFETS DES LDL NATIVES ET OXYDÉES SUR L’ÉVOLUTION DES PROPRIÉTÉS BIOMÉCANIQUES DES CELLULES ENDOTHÉLIALES ET IMAGERIE DES LDL PAR MICROSCOPE À FORCE ATOMIQUE.

Mémoire de maîtrise es sciences appliquées Spécialité : génie chimique et biotechnologique

_________________________ Julie CHOUINARD____

Sherbrooke (Québec), Canada Octobre 2006

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I

RÉSUMÉ

EFFETS DES LDL NATIVES ET OXYDÉES SUR L’ÉVOLUTION DES PROPRIÉTÉS

BIOMÉCANIQUES DES CELLULES ENDOTHÉLIALES ET IMAGERIE DES LDL PAR MICROSCOPE À FORCE ATOMIQUE.

par

Julie Chouinard

Département de génie chimique

Université de Sherbrooke

Le but principal de cette étude était de définir l’effet des lipoprotéines de basses densité natives (LDL) et oxydées (ox-LDL) sur les fonctions des cellules endothéliales en relation avec les processus physiopathologiques de l’athérosclérose. Le microscope à force atomique (AFM) fut utilisé en combinaison avec les méthodes biochimiques traditionnelles afin d’acquérir de l’information sur les propriétés biomécaniques des cellules endothéliales. L’AFM est un outil permettant l’acquisition d’images et de mesures de forces quantitatives concernant les propriétés viscoélastiques des cellules vivantes selon leur exposition aux LDL ou ox-LDL. L’AFM rassemble localement des informations sur la membrane cellulaire et le cytosquelette des cellules et ce, de manière non invasive. Il est ensuite possible de corréler les résultats obtenus avec les marquages immunohistochimiques afin d’évaluer la réponse cellulaire suite à une exposition à des LDL ou ox-LDL. Ces données recueillies, les protocoles étant au point, il ne restera plus qu’à effectuer les tests avec les antioxydants afin de déterminer les agents et les dosages appropriés permettant une protection de l’endothélium. Ce travail amène donc de nouvelles connaissances sur les mécanismes moléculaires fondamentaux de la dysfonction endothéliale en vue éventuellement de développer de nouvelles thérapies cytoprotectrices efficaces. Une méthode d’imagerie des LDL a également été mise au point en utilisant l’AFM. Il est maintenant possible d’obtenir des images de bonne qualité permettant aussi de mesurer les dimensions de LDL individuelles. Cette technique pourrait entre autre servir à évaluer des pathologies touchant les LDL comme le diabète.

Mots clés: AFM, HUVEC, LDL, ox-LDL, cellules vivantes, dysfonction endothéliale, athérosclérose, mécanique cellulaire, rigidité cellulaire, actine, vimentine.

Page 5: effects of native and oxydized ldl on the endothelial cells

II

REMERCIEMENTS

Ce travail fut réalisé dans le Laboratoire de Bioingénierie et Biophysique de

l’Université de Sherbrooke en collaboration avec le laboratoire du stress oxydatif,

athérosclérose et système immunitaire du Centre de recherche sur le vieillissement.

C’est sous la direction du Docteur Patrick Vermette, professeur au département de

génie chimique et biotechnologique, ainsi que sous la co-supervision du Docteur

Abdelouahed Khalil, professeur au département de physiologie et biophysique, que ce

travail a été effectué. Je tiens à leur exprimer ma plus profonde gratitude pour m’avoir

accueilli chez eux, me permettant ainsi de poursuivre des études graduées. Je les remercie

pour leur guidance et les nombreux conseils qui ont grandement contribué à ma

formation en sciences. Je leur suis également très reconnaissante de m’avoir permis de

contribuer à la littérature scientifique et de participer à de nombreux congrès tout au long

de ma maîtrise.

J’adresse mes plus sincères remerciements au Dr Guillaume Grenier et au Dr Pierre

Proulx, professeurs à l’Université de Sherbrooke, pour avoir bien gentiment accepté

d’évaluer ce travail de recherche.

Je tiens particulièrement à remercier le Dr Guillaume Grenier pour son temps, les

nombreuses discussions constructives et son intérêt face à mon projet.

Je voudrais remercier le Dr Charles Doillon, du Département d’endocrinologie

moléculaire et oncologie du Centre Hospitalier Universitaire de l’Université Laval, pour

son aide précieuse lors de l’élaboration du protocole d’extraction des cellules

endothéliales et ses conseils sur la culture cellulaire. Je suis également reconnaissante à

Anis Larbi, ancien étudiant au doctorat, pour m’avoir initié aux techniques de culture et

d’extraction de cellules, ainsi qu’au Dr Nathalie Faucheux pour ses avis éclairés

concernant les marquages et la culture cellulaire en général.

Je remercie le Dr Hicham Berrougui, Martin Cloutier et Maxim Isabelle du Centre

de Recherche sur le Vieillissement pour leur collaboration et soutien durant les

Page 6: effects of native and oxydized ldl on the endothelial cells

III

séparations de lipoprotéines. Merci également aux gens du 5e étage du Centre de

Recherche sur le Vieillissement pour votre accueil.

J’aimerais remercier tous mes collègues de laboratoire et les techniciens du

Département de génie chimique pour l’ambiance sympathique qui a contribué au bon

déroulement de ce travail. J’adresse un merci particulier à Emmanuelle Monchaux, Heïdi

Brochu et Anne Danion pour leur support, leur aide et surtout leur amitié; ce fut un

privilège de vous connaître et de travailler avec vous.

J’exprime mes sincères remerciements aux infirmières et médecins du Centre

Hospitalier Universitaire de Sherbrooke pour leur précieuse collaboration dans la collecte

des cordons ombilicaux. Je suis particulièrement reconnaissante envers les infirmières

Micheline Gagné, Michelle Lafleur, Pamela Keenan et Johanne Breton pour tous leurs

efforts concernant le recrutement des patientes et le suivi des échantillons.

Je souhaite exprimer ma plus profonde reconnaissance à Simon Jubinville pour son

aide dans l’élaboration d’un programme me permettant de calculer rapidement

l’indentation d’un cantilevier dans un substrat. Je le remercie également pour son soutien

moral constant très apprécié tout au long de ma maîtrise; sans compter ses précieux

commentaires dans la correction de presque tous mes manuscrits.

J’aimerais souligner la participation financière des Instituts de recherche en santé

du Canada (IRSC), du Centre de recherche sur le vieillissement et du réseau de

Formation interdisciplinaire en recherche sur la santé et le vieillissement (FORMAV)

dans ce projet. Enfin, je n’oublierai pas de remercier sincèrement les membres de ma

famille qui ont toujours été là pour moi : mon père qui s’est toujours intéressé à ce que je

fais, ma mère qui m’a continuellement encouragé à poursuivre mes études dans un

domaine que j’aimais et qui m’a donné de nombreux contacts au CHUS, mon frère

François et finalement ma grand-mère Jeannine, qui ne verra malheureusement pas

l’accomplissement de ce travail, mais qui n’a cessé de croire en moi. Un gros merci à ma

coloc Amanda Larose pour m’avoir soutenue au quotidien dans les hauts comme dans les

bas. Enfin, merci à tous mes amis.

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IV

TABLE DES MATIÈRES

LISTE DES TABLEAUX ET DES FIGURES ................................................................ VI

LISTE DES ABRÉVIATIONS........................................................................................ VII

1. INTRODUCTION .........................................................................................................1

2. BIBLIOGRAPHIC STUDY ..........................................................................................3

2.1 ATHEROSCLEROSIS .........................................................................................3

2.2 PLASMA LIPOPROTEINS .................................................................................4

2.2.1 Low Density Lipoproteins ........................................................................5

2.2.2 High Density Lipoproteins ........................................................................6

2.3 ANTIOXYDANTS ...............................................................................................8

2.4 HUMAN ENDOTHELIAL CELLS ...................................................................10

2.5 CELLULAR SENESCENCE .............................................................................10

2.6 ANIMAL MODELS ...........................................................................................12

2.7 ATOMIC FORCE MICROSCOPE ....................................................................13

2.7.1 Contact Mode AFM ................................................................................14

2.7.2 Tapping ModeTM AFM ...........................................................................15

2.7.3 AFM Force Measurements .....................................................................16

REFERENCES ............................................................................................................17

3. PROJECT DESCRIPTION AND OBJECTIVES .......................................................28

4. RESULTS ....................................................................................................................29

4.1 Effect of native and oxidized LDL on the biomechanical properties of endothelial cells ........................................................................................................................29

4.2 Method of imaging low density lipoproteins by atomic force microscopy ...........51

CONCLUSION ET PERSPECTIVES ...............................................................................64

RÉFÉRENCES ..................................................................................................................65

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V

ANNEXE 1 EXTRACTION CELLULAIRE ET CARACTÉRISATION... .....................67

A.1 HUVEC……….. .................................................................................................67

A.1.1 Extraction de cellules endothéliales à partir de cordons ombilicaux ......67

A.1.2 Marquage vWF .......................................................................................73

A.1.3 Marquage Dil-Acétyl-LDL .....................................................................75

A.1.4 Marquage Live/Dead ..............................................................................76

A.1.5 Marquage F-Actine et Vimentine ...........................................................77

A.1.6 Marquage sénescence ..............................................................................79

RÉFÉRENCES ...................................................................................................80

ANNEXE 2..... ...................................................................................................................81

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VI

LISTE DES TABLEAUX ET FIGURES

FIGURE 2.1 SA-β-gal staining ....................................................................................11

FIGURE 2.2 SEM picture of an AFM cantilever with sphere .....................................13

FIGURE 2.3 AFM imaging of confluent HUVEC in contact mode ............................14

FIGURE 2.4 AFM imaging of a HUVEC in Tapping modeTM ....................................15

FIGURE 2.5 Typical AFM force curves obtained on a hard surface and on a cell .....16

ANNEXE 1.1 Technique d’attache de la canule au cordon. ..........................................69

ANNEXE 1.2 Montage d’un cordon avec canules et forceps ........................................70

ANNEXE 1.3 Photo d’une extraction de cellules HUVEC ...........................................71

ANNEXE 1.4 HUVEC en culture depuis environ 2 jours .............................................72

ANNEXE 1.5 Répartition des produits pour le marquage vWF. ...................................73

ANNEXE 1.6 Exemple d’un marquage vWF sur des HUVEC .....................................74

ANNEXE 1.7 Marquage Dil-Ac-LDL sur des extraits de cordons ombilicaux .............75

ANNEXE 1.8 Photo combinée d’un marquage Live/Dead ............................................76

ANNEXE 1.9 Marquage des filaments d’actine en rouge et des noyaux en bleu. .........78

ANNEXE 1.10 Marquage des filaments intermédiaires de vimentine ............................78

ANNEXE 1. 11 Marquage des cellules sénescentes par la méthode du SA-β-gal ...........79

ANNEXE 2.0 Imagerie AFM d’un substrat de HOPG seul ...........................................81

Page 10: effects of native and oxydized ldl on the endothelial cells

VII

LISTE DES ABRÉVIATIONS AFM Microscope à force atomique

Apo Apolipoprotéine

BSA Albumine de sérum bovin

CACE Cellule endothéliale de l’artère coronaire

CE Cholestérol estérifié

CHD Cardiopathie coronarienne

CM Chylomicron

Dil-LDL 1,1’-dioctadecyl-3,3,3’,3’-tetramethyl-indocarbocyanine lipoprotéine

de basse densité acétylée

Dil-ox-LDL 1,1’-dioctadecyl-3,3,3’,3’-tetramethyl-indocarbocyanine lipoprotéine

de basse densité oxydée acétylée

ECGS Supplément de croissance pour cellules endothéliales

EDTA Acide éthylène-diamine-tétraacétique

EM Microscopie électronique

eNOS Oxyde nitrique synthase endothéliale

F-actine Filaments d’actine

FBS Sérum de veau foetal

FE Cholestérol libre

HBSS Solution de sels balancés de Hanks

HDL Lipoprotéine de haute densité

HEPES 4-(2-hydroxyethyl)poperazine-1-ethanesulfonic acid

HUVEC Cellule endothéliale humaine de la veine du cordon ombilical

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VIII

HOPG Graphite pyrolitique hautement organisé

IDL Lipoprotéine de densité intermédiaire

LCAT Lécithine-cholestérol-acyl-transférase

LDL Lipoprotéine de faible densité

M199 Medium 199

NO Oxyde nitrique

O2˙ Anion superoxyde

OH˙ Hydroxyle

Ox-LDL Lipoprotéine de faible densité oxydée

PBS Solution de tampon phosphate

Pl Phospholipide

PNB Alpha-phenyl N-tert-butynitrone

PON Paraoxonase

rHDL Lipoprotéine de haute densité recombinante

SA-β-gal β-galactosidase associée à la sénescence

SEM Microscopie électronique

TEM Microscopie électronique à transmission

Tg Triglycéride

VLDL Lipoprotéine de très basse densité

Page 12: effects of native and oxydized ldl on the endothelial cells

INTRODUCTION

The last half of the 20th century saw the emergence of a great increase in human life

expectancy, which brought a new perspective of the universally observed process that is

aging. What is now left to define is: when can we possibly consider someone old? Where

does the turning point stand? In 1884, a German chancellor once fixed the retirement age at

65 years old, which was almost unthinkable considering that the average life expectancy

was around 37 years at that time86. Today, industrialized countries are still using 65 as the

reference age to mark the passage to old age, but again, are not we confusing aging and

retirement here86? Aging could be described as a result of several progressive, noxious and

irreversible processes that decrease the capacity of the organism to adapt to the changing

conditions of its environment105. There is no established universal definition of aging in the

litterature. However, we know that aging has many consequences. Organs perform less

efficiently and so does the immune system. These changes create the right conditions for

certain diseases to develop54. Cardiovascular diseases, the number one killers in America,

claim more lives each year than accidents, cancer and AIDS, all together7. One major

chronic ailment among the cardiovascular troubles family is atherosclerosis, which clogs

arteries, damages the endothelium and leads to heart attacks and stroke21. An interesting

factor that appears to contribute to the impairment of the endothelial cell monolayer in

atherosclerosis cases is the decrease of nitric oxide (NO) synthesis in aged endothelial

cells57. Also in vitro studies have demonstrated that, compared to native low-density

lipoproteins (LDL), oxidized low-density lipoproteins (ox-LDL) are associated with an

increased atherogenecity90. In vivo and in vitro studies have highlighted the anti-

atherogenic action of high-density lipoproteins (HDL)121. Indeed, results demonstrate that

HDL protect the endothelial cells by acting as an antioxidant against LDL oxidation104. The

protective effect of HDL comes from an associated enzyme called paraoxonase 1

(PON1)129. However, the mechanism by which PON1 is implicated against the endothelial

cells dysfunction is not yet elucidated, but an age-related decrease in PON activity has been

documented87.

The main objectives of this project were to further investigate the effects of native

LDL and ox-LDL on the endothelial cell biomechanical properties. To achieve this, the

1

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project required gaining skills in endothelial cell extraction techniques along with a

sufficient knowledge of the Atomic Force Microscope (AFM) to operate it efficiently.

The second chapter encloses a bibliographic study which reviews the published data

in the various fields concerned by this project. Chapter 2 will thus present:

• The atherosclerosis pathology.

• Low and high density lipoproteins and their role in cholesterol transport.

• The anti-atherogenic role of antioxidants.

• The endothelial cell models used in research.

• The senescent state of primary cells.

• The existing animal models for atherosclerosis research.

• The AFM instrument with its biological applications.

In Chapter 3 a description of the project is presented followed in Chapter 4 by two

articles. The first article details the effects of native and oxidized LDL on the

biomechanical properties of endothelial cells over time. Cytoskeleton and general

morphology changes had been evaluated by standard immunohistochemistry methods while

cell rigidity was measured by AFM. The second article is a short paper on a method to

image and measure LDL by AFM. Finally, detailed protocols used in this study can be

found in the Annex section.

2

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RÉSUMÉ

Les maladies cardiovasculaires sont la principale cause de mortalité dans les pays

industrialisés présentant une population de plus en plus vieillissante. Parmi ces maladies,

l’athérosclérose, un état chronique de dégénération de l’artère ayant pour origine la

formation d’une plaque d’athérome (dépôt lipidique) sur sa paroi, est responsable à elle

seule de plus d’un tiers des décès. Les lipoprotéines de basse densité (LDL) sont des

transporteurs dont la fonction est d’amener le cholestérol dans la circulation jusqu’aux

cellules. Dans le processus d’athérosclérose, les LDL sont oxydées et s’accumulent dans les

artères. Des études in vitro et in vivo ont démontré l’action anti-athérogénique des

lipoprotéines de haute densité (HDL). En effet, il a été démontré que les HDL protégeaient

l’endothélium en agissant en tant qu’antioxydant envers les LDL, mais que cet effet

protecteur diminuait avec l’âge. De là vient l’intérêt d’évaluer l’effet cytoprotecteur de

différents antioxydants afin de contrer la dysfonction endothéliale. Afin de développer des

thérapies efficaces, il est nécessaire de trouver de bons modèles d’athérosclérose ainsi

qu’un appareil permettant d’évaluer de façon précise la réponse cellulaire à divers

traitements de LDL oxydées et antioxydants.

2. BIBLIOGRAPHIC STUDY

2.1 Atherosclerosis

Not so long ago, doctors could still claim that atherosclerosis consisted of a simple

“pipes trouble”: fatty deposits on the arterial wall luminal surface. However, we now know

that arteries are much more complex than this. In fact, vascular endothelial cells play a

major role in the development and grow of fatty sediments inside the vascular intima76,

which provokes atheromatous plaque formation and an increase of wall stiffening and

thickness106. The increasing popularity of fast food and sugar-containing beverages in the

past years brought a high frequency of lipid plaque formation even in young children12.

Epidemiologic studies revealed that those plaques, over the decades, are the source of

fibrotic and calcified lesions observed in adults and elderly people60. A long-term lipid-rich

diet is a key initiator of atherosclerosis, but its combination with diabetes has demonstrated

a marked acceleration of atherosclerosis in pig models45, 91. In a normal body, low density

lipoproteins (LDL) are in charge of cholesterol transport from their formation point in the

3

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liver to the whole organism. As much as LDL and cholesterol are good, an excess rapidly

becomes a problem since they start to accumulate in the vascular wall where lipids are

subject to oxidation and proteins to glycosylation. Endothelial cells appear to mistake those

modifications as a potential threat and start secreting chemokines, thus initiating an

immune response. Monocytes and lymphocytes therefore secrete cytokines, enhancing the

inflammatory process in arteries. Macrophage cells capture modified LDL and take a foam-

like morphology once saturated. Instead of healing the arteries, those events reshape them

by changing their characteristics producing a thick plaque. The inflamed intima produces

factors that urge the sequential division of smooth muscles cells (SMC) which finally leads

to a phenotype change. SMC start an increased and disorganized production of cellular

matrix substrates that are crucial for the fibrous plaque formation22, 76. Arteries become

stiffer and lose elasticity. A growing plaque eventually reaches a rupture point and when

that happens, tissue components are released into the bloodstream. These components are

highly reactive with plasma factors and when mixed together, a clot is formed. It can

eventually block the whole vessel creating either a cerebral vascular accident or a coronary

thrombosis. In some rare cases, atherosclerosis is not activated by inflammation, but rather

by micro-organisms such as the herpes virus or Chlamydia pneumonia. In any case, there is

no known efficient treatment for atherosclerosis since researchers have been unable to

reverse the process, so far. The only available therapies consist in using anti-inflammatory

molecules, but a permanent inhibition of the immune system greatly exposes the patient to

infections76.

2.2 Plasma Lipoproteins

The most common lipids in the human body are triglycerides (Tg), cholesterol (free

cholesterol (FC) and cholesterol esters (CE)) and phospholipids (Pl)46, 48. Triglycerides are

stored in adipose tissues and are a source of energy. Cholesterols are precursor of hormones

and bile acid as well as being a component of the cellular membrane. Thanks to their

amphipatic properties, phospholipids are the main component of cell walls and of lipid-

carrying liproproteins. Cholesterol and triglycerides, being hydrophobic compounds, are

thus using phospholipids to be transported in the circulation.

4

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All lipoproteins present the same organisation: a hydrophobic core containing

neutral lipids (Tg and CE) and an anhydrophilic surface exposing apolipoproteins and polar

lipids such as FC and Pl. Lipoproteins have been traditionally classified into five major

categories based on their density: chylomicrons (CM), very low density proteins (VLDL),

intermediate density protein (IDL), low density lipoprotein (LDL) and finally high density

lipoproteins (HDL)46, 48.

It is possible to divide the endogenous lipid transport system into two subsystems:

the apoB-100 lipoprotein system (VLDL, IDL and LDL) and the ApoA-1 lipoprotein

system (HDL)46. Most VLDL and IDL are either cleared from the circulation by hepatic

receptors or put through the VLDL-IDL-LDL cascade transforming them in IDL and LDL

particles, which will later be removed mainly by the liver either by LDL receptors or other

receptors19, 46. HDL particles are complex precursor derivatives secreted by the liver and

the intestine and are the major mediators of the reverse cholesterol transport where the

cholesterol deposits on peripheral cells are returned to the liver46,71. There exists a

correlation between coronary artery disease and high serum levels of total cholesterol,

which includes LDL, apolipoprotein B (apoB) and triglycerides, as well as HDL18, 25, 49, 71,

103. It is believed that variability of these lipids and lipoproteins in the serum is mainly of

genetic origin; 17 one of the possible responsible being the apoB gene. Several other non-

genetic factors influence the plasma-level lipid metabolism including both dietary and other

lifestyle factors such as age, gender and body fat distribution59. Besides nutrition, lipid and

lipoprotein levels are also linked to tobacco smoking41, physical activity13 and

psychological stress83.

2.2.1 Low Density Lipoproteins

LDL particles are the key players in cholesterol transfer and metabolism in the

human circulation. LDL form an heterogeneous family whose members vary greatly in size,

composition and structure. They present an average density of 1,019-1,063 g/ml56, 70. Men

have smaller LDL particles than women40, 84 and there is a little change in size with age40,

51, but generally the average LDL diameter is 22 nm40, 56, but LDL size can vary between

18-25 nm10, 37, 85, 112, 116. The particle core consist of about 170 TG and 1600 CE molecules

while the surface monolayer shows about 700 Pl and a single copy of apoB-10034, which is

5

Page 17: effects of native and oxydized ldl on the endothelial cells

the largest known monomeric protein consisting of 4563 amino acid residues (4536 amino

acid mature peptides and 27 amino acid signal peptides)29, 112. LDL are subject to oxidation

in the arterial wall considering the wide range of biologically active hydrolytic enzymes

and pro-oxidative agents present in the arterial intima. In fact, LDL oxidation is unlikely to

occur in plasma since it contains a high concentration of antioxidants and metal ions

chelating proteins14. LDL oxidation leads to a loss of endogenous antioxidant molecules

and polyunsaturated lipid fatty acids. An extensive oxidation has been demonstrated to be

associated to a loss of LDL particles integrity56. There are increasing evidences suggesting

that oxidized LDL (ox-LDL) play a critical role in endothelial injury and atherosclerosis14,

75 As explained earlier, ox-LDL are fully implicated in the atherogenic process and

atherosclerotic plaque rupture by promoting lipid accumulation, pro-inflammatory signals

as well as apoptotic cell death. Studies have demonstrated that the LOX-1 vascular cell

receptor mediates ox-LDL uptake by endothelial cells inducing apoptosis28, 31.

LDL oxidation in vitro can be induced by γ-radiolysis in oxygenated aqueous

solution containing 10-2 M sodium phosphate buffer at pH 7.068 or copper-mediated

oxidation. In the later technique, it has been reported that the used copper concentration is a

major factor to consider to evaluate the formation of various ox-LDL forms with specific

physico-chemical characteristics and probably biological functions131.

2.2.2 High Density Lipoproteins

HDL are implicated in the reverse cholesterol transport where their role consist in

removing the excess of cholesterol from peripheral tissues or other lipoproteins and

delivering it to the liver for disposal in the bile. HDL particles are believed to exist in two

different forms: a nascent discoidal one, which is abundant in the plasma of people with

lecithin cholesterol acyltransferase (LCAT) deficiency and a mature spherical form,

resulting from discoidal HDL transformation under LCAT activity and plasma lipid transfer

proteins, predominant in normal people. The main challenge with functional study of HDL

was related to the inability to obtain clear structural information. Production of lipoproteins

and apolipoproteins crystals being extremely difficult to achieve, high-resolution X-ray

crystallography was not considered24. Transmission electron microscopy (TEM) of

negatively stained samples38 showed cylindrical stacks and circular objects, but the tube

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structure is believed to be an artefact from the negative staining process since they change

in height according to dilution39. The presently accepted nascent HDL structure model is

that of a phospholipid bilayer, which is stabilized at the edges by amphipathic

apolipoprotein helixes110. The first discoidal structure of reconstituted HDL (rHDL) images

under native conditions were obtained with AFM showing that the particles adsorb readily

onto mica and would not adsorb on top of each other. Given that the rHDL disks height is

consistent with the thickness of a phospholipid bilayer, it suggests that the disks rest with

the acyl chains of the phospholipid perpendicular to the mica surface24. Normal HDL

particle size range between 7 to 10 nm in diameter and typically form hexagonal arrays in

areas of high particle concentration38. Apolipoprotein A-1 (apoA-1) is the HDL primary

protein component, which is the most potent activator for LCAT36. ApoA-1 is believed to

fold into amphipathic helixes that stabilize the lipid particles and bind to the HDL

surfaces111. The various apolipoproteins, including apoA-1, as well as Pl, CE, FC, Tg and

lysolecithins composing the native HDL make it a highly heterogeneous entity8. This

heterogeneity easily explains why people often favour rHDL since they can be assembled

with specific components so it is possible to control their size, phospholipid and protein

composition while retaining the major characteristics of native HDL63, 128. A strong inverse

correlation exists between plasma HDL levels and coronary heart disease incidence (CHD).

In fact, low levels of HDL cholesterol are common findings in patients with premature

CHD44 and genetic syndromes of high HDL cholesterol are more than often associated with

longevity and decreased atherosclerotic cardiovascular disease (ASCVD)47. HDL have been

found to prevent ox-LDL-induced cytotoxicity on cultured endothelial cells and smooth

muscle cells55. Mild oxidative modification of LDL in co-cultures of human aortic wall

cells was completely inhibited by HDL92 and so was the copper-catalyzed oxidation of

LDL. This HDL inhibition represents an effect that might reflect the exchange lipid

peroxidation products between high and low density lipoproteins100. HDL have also been

shown to block the induction of monocyte adhesion to the endothelium even if the exact

mechanisms are still unclear82. HDL and apoA seem to act at the cellular level by

increasing the resistance of endothelial cells against the cytotoxicity of ox-LDL during a

relatively long period (protective activity can last several days) and HDL inhibit the

pathogenetic intracellular signalling triggered by ox-LDL that induces the sustained Ca2+

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Page 19: effects of native and oxydized ldl on the endothelial cells

rise leading to cell death122. Considering the impact of HDL in vascular diseases, studies

have even proposed their use as a therapeutic strategy in managing and preventing

atherosclerosis95, 101, but current pharmacological and non-pharmacological interventions

have limitations because of the functional heterogeneity of HDL themselves114. A proposed

alternative consists of a direct administration of functionally competent HDL, its associated

protein ApoA-1, or synthetic mimetics. For example, repeated recombinant Apo A-1milano-

phospholipid complex parenteral administration prevented aortic atherosclerosis

progression in ApoE-deficient mice despite severe hypercholesterolemia6, 115. These studies

showed that it is possible to stimulate reverse cholesterol transport with ApoA-1, but little

is said about the LDL oxidation issue.

2.3 Antioxidants

The anti-atherogenic effect of HDL originates partly from the ApoA-1, but the

antioxidant role of the molecules comes more from the human serum paraoxonase (PON1),

which is a 44-kDa glycoprotein42 almost exclusively found on ApoA-1 and ApoJ80. This

HDL-associated enzyme is one of the three known members of the paraoxonase family102,

the others being called PON2 and PON3. PON3 appeared not only to prevent the formation

of mildly ox-LDL, but also to inhibit ox-LDL-induced monocyte chemotactic activity thus

suggesting a resemblance to PON1. While PON1 mRNA expression in the liver is

repressed by oxidized lipids, PON3 message is constantly expressed and remains

unaffected. Data suggest that PON1 and PON3 may play distinct roles in the prevention of

atherosclerosis. PON3 may provide a basal constitutive atheroprotective function, whereas

the PON1 protective effect is more variable in as much as PON1 expression is repressed by

proatherogenic stimuli104. Studies on PON2 showed significantly less intracellular

oxidative stress following treatment with hydrogen peroxide or oxidized phospholipids in

cells. PON2 possesses antioxidant properties similar to those of PON1 and PON3. Activity

of PON1 in vitro is routinely evaluated by its ability to hydrolyze paraoxon (PON activity)

and phenylacetate (arylesterase activity). PON3 seems to lack detectable arylesterase and

PON activities, which still does not preclude it from possessing the ability to protect LDL

from oxidation since those activities appears to be distinct in vivo104. However, in contrast

to PON1 and PON3, PON2 may instead exert its antioxidant functions at the cellular level,

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joining the host of intracellular antioxidant enzymes that protect cells from oxidative

stress96.

Like HDL, isolated PON inhibits copper-catalyzed oxidative modification of LDL79.

PON activity in serum had been shown to be significantly lower in both familial

hypercholesterolemia and insulin-dependent diabetes mellitus, which are greatly associated

in humans with increased risk for vascular diseases78. Is has also been suggested that the

mechanism by which smoking increases CHD risks may be directly on reducing PON

activity thus promoting lipid oxidation [reviewed in 15]. The most investigated PON family

members are by far the two PON1 isoenzymes Q and R, that are calcium-dependent

hydrolases that catalyze the hydrolysis of a large spectrum of carboxylic acid esters and

organophosphates72. Is has also been reported that serum PON1 can hydrolyze a variety of

lactones and cyclic carbonate esters, including naturally occurring lactones and

pharmacological agents15. However, despite many efforts, the structure and mechanism of

action of PONs are still enigmatic1 and are widely subject to speculation. PON1 is

represented in the population in three possible phenotypes as determined by a dual substrate

method requiring the use of 1M NaCl to stimulate PON and monitoring phenylacetate

hydrolysis. Cut-off values between phenotypes can be described as follows: ratio < 3.0 for

AA, ratio between 3.0 and 7.0 for AB and > 7.0 for BB phenotype119. PON1 activity had

been found to decrease with age while its arylesterase activity as well as its concentration in

the serum did not change significantly. It is suggested that the decreased PON1 activity

may be related to the development of oxidative stress conditions with aging thus increasing

the HDL susceptibility to oxidation in elderly subjects113.

Many other molecules can be used to protect endothelial cells against oxidative

damage. Vitamin E (α-tocopherol) presented an LDL protective effect towards endothelial

cell functions97, when water-soluble vitamin E derivated anti-oxidant Trolox® was used to

pretreat human coronary artery endothelial cells. Trolox® succeeded to inhibit the

formation of superoxide anions as well as the down-regulation of LDL-receptors in

response to ox-LDL58. The lipophilic spin trap, alpha-phenyl N-tert-butynitrone (PNB) also

possesses an inhibition effect on cell and LDL oxidation from cupric ions64. It is likely that

PNB inhibits the oxidative and biological modification of LDL by scavenging the LDL-

lipid-derived radical. Results showed that LDL incubated in the presence of PBN with

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either endothelial cells or cupric ions were less readily degraded by macrophages than LDL

incubated in the absence of PBN64. Vitamin C and lipoic acid had been shown to potentiate

NO systhesis and bioactivity in endothelial cells. Alpha lipoic acid has the ability to quench

oxygen singlet, hydroxyl and syperoxide species, while its reduced form, dihydrolipoic

acid, stabilizes the peroxyland peroxynitrate radicals. Initial and reduced forms are both

regenerated through redox cycling of other antioxidants like vitamins C and E99.

2.4 Human Endothelial Cells

The human umbilical vein endothelial cells (HUVEC) had widely been used in

research to study in vitro endothelial functions and pathologies30, 125. However, it is

reported that these cells, being of the primary type, undergo senescence as soon as the

fourth passage and lose their endothelial characteristics33. Also, these cells are often close

to senescence and are taken from hypoxic and surely activated blood vessels43. Although

HUVEC are now a common well-known system in human vascular studies in vitro, it still

represents an imperfect model to evaluate the anti-atherogenic properties of HDL, since it

originates from an endothelium that is not susceptible to coronary atherosclerosis120. The

coronary artery endothelial cells (CAEC) represent a much more relevant model of

endothelial dysfunction since the cells come directly from vessels affected by

atherosclerosis3, 4 as is the model using human aortic endothelial cells from macrovessels

developped by Donnini et al.33 However, given the abundance of literature on HUVEC and

their easy access, HUVEC were selected as model cells to carry out cell testing in the

present studies.

2.5 Cell Senescence

Cell senescence is the limited ability of primary human cells to divide when

cultured in vitro and is accompanied by a specific set of phenotypic changes in

morphology, gene expression and cell functions65, 89. These phenotypic changes had notably

been implicated in human aging35. The hypothesis of cellular aging was first described by

Hayflick53 and later supported by the evidence that the growth potential of cultures

correlates well with mean maximum lifespan of the species from which the cultures are

derived107. Human primary cultures derived from patients with premature aging syndromes,

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such as Bloom syndrome and Werner syndrome, are known to have a shorter lifespan than

cultures from age-matched healthy populations, thus supporting the hypothesis of aging123.

Senescent cells show impaired functions such as a decreased expression of endothelial

nitric oxide synthase (eNOS)27, 118 and an increased expression of pro-inflammatory

molecules88. It has been reported that replicative cell senescence can be associated with the

attrition of telomeres, although no direct correlation between the two has been found16.

Telomeres are believed to act as a mitotic clock, counting the number of possible divisions

a cell has gone through and eventually activate replicative senescence26, 69. Expression of

negative regulators such as p53 and p16 in the cell cycle is increased with cell division and

thereby promotes growth arrest27. Most senescent cells, including endothelial cells, remain

metabolically active in cell culture and probably in vivo as well61. Flattened and enlarged

cell morphology is reported as a known characteristic of cell senescence23. Beside

morphology, which is not always easy to evaluate, some techniques exist to verify the

senescent states of cells. Primary cultured cells undergoing cell senescence in vitro express

an increased activity of β-galactosidase (β-gal) when assayed at pH 6, which can be

differentiated from the endogenous lysosomal β-gal activity detected at pH 4. In situ

staining for SA-β-gal showed a few SA-β-gal-positive HUVEC (11%) at passage 5, but

positive cells increased at passage 45 (over 90% labelled)32, 65. See Figure 2.1 for SA-β-gal

staining results on HUVEC. Proteomics-based approach had also been used to identify

three up-regulated and five down-regulated proteins during replicative senescence65.

A B

Figure 2.1 SA-β-gal staining of HUVEC using SA-β-gal. On Fig. 2.1A: HUVEC at passage 4, two weeks following confluence. Senescent cells appear in blue. It can beobserved that senescent cells are much bigger than young cells. Fig. 2.1B presents a magnified senescent cell (enlarge cell with the blue stain in the nucleus’ region).

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Repeated cell passages would be an interesting model of cell aging only if vascular

cells in vivo were all senescent when the subject reaches a certain age. Even if senescence

had widely been investigated in vitro, its presence in vivo has not yet been clarified88.

Moreover, cells in culture grow in a controlled environment without many cell interactions,

almost free of influence from hormones and the humorous system. It is possible to mimic

aging in vitro by increasing the number of passages, which leads to replicative senescent

cells having enhanced apoptosis when cultured for a prolonged time period without

passaging132. It is reported that HUVEC can undergo senescence as soon as the fourth

passage by losing their endothelial characteristics33 and that the prostacyclin/thromboxane

A2 formation ratio decreases in each subsequent passage94. HUVEC at passage 22 showed a

low to undetectable level of telomerase activity as compared to passage 5 cells27. HUVEC

are a well-known model, but these cells are not a perfect mirror of the real in vivo process.

This is why an animal model is essential to validate the implications of atherogenesis50.

2.6 Animal Models

There are not many animal models for atherosclerosis research presenting the major

human pathology characteristics such as:

• Naturally developing lesions under a reasonable feed diet.

• Lesions more frequent in males than in females and first appearing in the aorta.

• Atheromatous plaques with complications like mineralization, ulceration and

hemorrhage.

• The right cholesterol profile with an LDL level concentration higher than HDL50, 62.

Of all the various atherosensitive animals found in the literature (e.g., pigs,

monkeys, pigeons, chickens, rabbits), only two appeared to be of interest to study

atherosclerosis. Indeed, the monkey and the pig are the sole species phylogenetically close

to the human being, developing a spontaneous atherosclerosis favored by dietary

cholesterol, hormones or psychological stress. Also, they are the only two animals with

convenient artery size that are presenting the natural lipoprotein profile i.e., [LDL] >

[HDL] as observed in humans50, 62. Since the importation of monkeys is generally expensive

if not banned, the pig model would be the best choice, as it is a largely used vascular model

in science67, 130.

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2.7 The Atomic Force Microscope (AFM)

The AFM has considerably evolved during the past decade, providing nanometer

scale resolution in the imaging of biological samples ranging from single molecules, like

DNA77, to intact cells adsorbed on biomaterials20. AFM images of bull sperm have even

been able to rival with electron microscopy (EM)2, but unlike EM, AFM imaging presents

the advantage to require little to no sample chemical treatments and can be performed

directly in fluid on living cells. The AFM does not only deal with topographical

measurements, it can also provide additional information about other surface properties

such as stiffness, hardness, friction or elasticity5. The AFM instrument provides unique

information for cell analysis. For example, AFM was used to image in real-time plasma

membrane in migrating cells98. The cell membrane immunogold labelling allows high-

resolution mapping of cell surface antigens93. AFM cell indentation analyses can be used to

create viscoelastic maps of different cell types109. Monocytes adhesion on HUVEC showed

a decreasing elastic modulus of the cells, which was correlated with the distribution of F-

actin filaments66. Local mechanical properties and cytoskeletal structure changes of

cultured bovine endothelial cells exposed to shear stress were also evaluated by AFM108. It

is also possible to widen the AFM analysis capacity by functionalizing the cantilever to

study molecular properties such as a ligand-receptor or antigen-antibody attraction and

pull-off forces in near physiological conditions74.

However, standard coated tips can only perform a limited number of measurements,

since it damages the cells. Also, the non-covalent fixation methods often used to attach

ligands to AFM probes limit the repeatability of these measurements. These problems can

solved by using glass spheres instead of the tips (see Figure 2.2)11, 52, 73 and by covalently

attaching ligands to these colloidal probes. Figure 2.2 Scanning ElectronicMicroscopy (SEM) photograph of acantilever tip on which a silica spherewas glued. It can be observed that thesphere surface is relatively clean andfree of residues.

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Page 25: effects of native and oxydized ldl on the endothelial cells

2.7.1 Contact Mode AFM

The contact mode is the original AFM imaging method where the tip is in constant

contact with the sample. A piezoelectric scanner gently moves the tip across the sample

while changes in the cantilever deflection are sensed and passed to a feedback amplifier.

The amplifier applies a voltage to the piezoelectric ceramic thus raising or lowering the

cantilever from the surface to restore the deflection to the setpoint value to keep a constant

height124. Images are generated by mapping the z-position variations of the sample during

scanning to create a height image where color contrast is used to show the topography5

(Figure 2.3, left part). Unfortunately, the combination of lateral forces and high forces

normal to the surfaces may damage soft samples like cells and can also result in lowered

spatial resolution124. The deflection error signal can also be recorded and mapped while

performing contact mode imaging which gives a better detailed appreciation of the sample

general features that are more difficult to observe in height topography images (Figure 2.3,

right part).

Figure 2.3 AFM imaging of confluent HUVEC in contact mode. The image on the left represents color-coded topography where brighter colors represent highest features. Theimage on the right is the corresponding deflection signal.

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2.7.2 Tapping ModeTM AFM

Tapping mode topography images (Figure 2.4, left part) are produced by mapping

the vertical distance travelled by a cantilever to maintain constant cantilever oscillation

amplitude while scanning. Phase imaging (Figure 2.4, right part), can be performed at the

same time as topographic images, both being recorded in a single scan. Phase imaging

consists of measuring the sinusoidal oscillations of the cantilever compared to the applied

driving signal causing the oscillations. The phase shifts obtained in Tapping modeTM are

recorded to produce an image, which allows the detection of sample stiffness where lighter

areas correspond to regions of higher stiffness9, 81 (Figure 2.4, right part). Tapping modeTM

also presents the advantage of reducing the lateral shear force existing in contact mode

which damages soft samples5.

Figure 2.4 AFM imaging of a HUVEC in Tapping modeTM. The left image represents theheight with higher features in bright colors while the right image is the correspondingphase image where the brighter color is associated to a harder surface.

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Page 27: effects of native and oxydized ldl on the endothelial cells

2.7.3 AFM Force Measurements

The AFM is able to record the amount of force felt by a cantilever as the tip is

brought close to a sample surface and then pulled away. This technique is used to probe

local mechanical properties like adhesion and elasticity5. To calculate the force, Hooke’s

law comes handy: F = -kx where F is the total applied force, k represents the cantilever

spring constant and x is the spring displacement, calculated from the laser deflection

recorded by the photodetector124. Force measurements done in liquid present the advantage

of cancelling attractive interaction that gives the adhesion force117. To acquire good force

measurement data on cells, the cantilever needs to be calibrated on a hard surface in the

same liquid environment, without changing the tip or the position of the laser beam on the

cantilever11, 127. The difference between the deflection of the cantilever on a hard substrate

and a soft one can be associated with the level of sample indentation (Figure 2.5). Force

plots on soft surfaces show a lower cantilever deflection slope since the tip indents the

sample. It is crucial to use the approaching part of the force curves for the calculation of

indentation since the retracting curves lead to wrong measurements like those due to

adhesive forces126.

Figure 2.5 Typical AFM force curves obtained on a hard surface (glass slide) with a slope (S) of 1 and on a cell with a slope smaller than 1. The distance in deflection between the two curves is a measure of the indentation.

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3.0 PROJECT DESCRIPTION AND OBJECTIVES

The membrane of endothelial cells is known to undergo numerous alterations

during the aging process, which often leads to endothelial dysfunctions. The aim of this

study was to define the effect of ox-LDL on endothelial cells function in relation to the

age-related physio-pathological process of atherosclerosis. To achieve this, a first study

focused on the comparison of endothelial cells’ biomechanical properties following

exposition to LDL and ox-LDL. A second one concentrated on the LDL themselves by

developing a method to image the LDL and obtain more information about their general

structure and dimensions.

The first article describes how, in combination with standard biochemical assays,

the AFM was used to gain new knowledge on the biomechanical properties of individual

endothelial cells. This instrument provides images and quantitative force measurements

in the nanoscopic range. AFM measurements gather localized information, in a non-

invasive way, on the cell membrane and on the cytoskeletal properties of endothelial

cells. These data were then correlated with biological functions in relationship to the

endothelial cells responses to LDL and ox-LDL. This work provides new data on the

molecular mechanisms underlying endothelial cells dysfunction on the way to develop

efficient cytoprotective therapies.

The objective of the second article was to elaborate a method to image individual

LDL particles. In this short paper, LDL were imaged on highly ordered pyrolytic graphite

(HOPG), which is a very plane surface (See Annex 2), to gather some more information

on lipoproteins.

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4.0 RESULTS

4.1 Effect of native and oxidized low density lipoproteins (LDL) on the

biomechanical properties of endothelial cells

Résumé

Il existe de plus en plus d’études qui suggèrent que les lipoproteins de basse

densité oxidées (ox-LDL) jouent un rôle primordial dans la dysfonction endothéliale et le

processus d’athérosclérose. Le but de cette étude était d’étudier les effets des LDL

natives et oxydées sur les propriétés biomécaniques des cellules endothéliales de la veine

de cordons ombilicaux humains (HUVEC) par microscopie à force atomique (AFM). De

plus, la contribution des filaments d’actine (F-actine) et du réseau de vimentine fut

examinée par microscopie à fluorescence. Nous avons trouvé que les ox-LDL ont des

effets majeurs sur le cytosquelette des HUVEC. Ceux-ci changent la morphologie

cellulaire ainsi que l’organisation de la F-actine et la vimentine tandis que les résultats

obtenus avec les LDL natives étaient très comparables à ceux des cellules non traitées.

Les Dil-ox-LDL furent absorbés par les cellules très rapidement alors que les Dil-LDL

furent plus lents. L’AFM nous a permis de mesurer les changements de rigidité

directement sur de jeunes HUVEC vivantes exposées aux LDL natives ou oxydées in

vitro en fonction du temps. Les résultats démontrent que le cytoplasme des cellules

devient significativement plus rigide suivant une longue période d’incubation en présence

de ox-LDL tandis que les cellules exposées aux LDL ont démontré une rigidité similaire

aux échantillons de contrôle après 24 heures. Ce travail amène de nouvelles données

quantitatives sur les mécanismes biomécaniques liés à la dysfonction endothéliale et

donne une méthode d’évaluation des effets de potentielles thérapies cytoprotectrices dans

le futur.

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Abstract

There is increasing evidence suggesting that oxidized low density lipoproteins

(ox-LDL) play a critical role in endothelial injury and atherosclerosis process. The aim of

this study was to investigate the effects of native and ox-LDL on the mechanical

properties of human umbilical vein endothelial cells (HUVEC) by atomic force

microscopy (AFM). In addition, the contribution of filamentous actin (F-actin) and

vimentin networks were examined by fluorescence microscopy. We found that ox-LDL

had major effects on the cytoskeleton of HUVEC. They changed cells’ shape as well as

F-actin and vimentin organisation while native LDL results were very close to untreated

cells. Dil-ox-LDL were quickly absorbed by the cells while Dil-LDL took longer. AFM

allowed us to directly measure the changes in rigidity of living young individual HUVEC

in vitro exposed to native and oxidized LDL through time. Results demonstrated that the

cell body became significantly stiffer after a long period of incubation in the presence of

ox-LDL while cells exposed to LDL showed similar rigidity to our control sample after

24 hours. This work thus brings new quantitative data on the biomechanical mechanisms

related to endothelial cells dysfunction and provides a way to evaluate the effects of

potential cyto-protective therapies in the future.

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Introduction

Atheroslerosis is a chronic ailment which clogs arteries, damages the endothelium,

and leads to heart attacks and stroke9. It is now admitted that vascular endothelial cells

play a major role in the development and growth of fatty sediments inside the vascular

intima28, which participate to the atheromatous plaque formation and to the increase of

wall stiffeness and thickness37. In normal conditions, low-density lipoproteins (LDL)

provide cholesterol transport from the liver to the whole organism. As much as LDL and

cholesterol are essential to maintain the homeostasis, an excess quickly becomes a

problem since they start to accumulate in the vascular wall where lipids and proteins are

subject to oxidation and to glycosylation, respectively. LDL oxidation leads to a loss of

endogenous antioxidant molecules and polyunsaturated fatty acids21. Extensive LDL

oxidation has been demonstrated to lead to a loss of LDL particles’ integrity21. There is

increasing evidence suggesting that oxidized LDL (ox-LDL) play a critical role in

endothelial injury and atherosclerosis4,26. Ox-LDL are implicated in the atherogenic

process and in the atherosclerotic plaque rupture by promoting lipid accumulation, pro-

inflammatory signals as well as apoptotic cell death28. Some studies have demonstrated

that the LOX-1 vascular cell receptor that mediates ox-LDL uptake by endothelial cells,

induces apoptosis10,15. It has also been reported that circulating ox-LDL may significantly

delay endothelial wound healing5. Vascular endothelium exposed to ox-LDL in vitro and

in vivo shows an increased permeability27,35, a telltale sign of endothelial dysfunction. In

addition, it has been demonstrated that ox-LDL induce cytoskeletal rearrangements like

F-actin distribution, cell contraction and formation of intercellular gaps16, 47, 48, all

affecting the endothelial barrier function35. Many studies5, 16, 27, 35, 47, 48 have been done on

the effects of ox-LDL in endothelial dysfunction, but almost none had been able to assess

the biophysical alterations effects. One study reports an increasing cell stiffness after ox-

LDL exposition using micropipette aspirations8. We were interested in investigating

cells’ rigidity with atomic force microscopy; an other more sensitive and less cell

disturbing technique that allows repeatable data collection in the picoNewton range on

different cell parts like the nucleus and the cytoplasm.

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The Atomic Force Microscope (AFM) has considerably evolved during the past

decade, providing nanometer scale resolution in the imaging of biological samples

ranging from single molecules such as DNA30 to intact cells adsorbed on biomaterials7.

Also, unlike electron microscopy, AFM imaging presents the advantage to require little

sample preparation and can be performed in fluid directly on living cells3, 19, 29, 36, 41, 46.

The AFM instrument provides unique information for cell analysis. The AFM does not

only make topographical measurements, it can also provide additional information about

other surface properties such as stiffness, hardness, friction or elasticity1, which are

properties of interest to study cell mechanics and its cytoskeleton. For example, AFM

studies on transformed mouse fibroblasts45 showed that actin fibers have a great influence

on cell rigidity. Living cells represent complex and heterogenous viscoelastic structures39

which can be correlated with the cytoskeleton. The cytoskeleton is in constant

reorganization during cell motility and during cellular response to environmental factors,

making it a highly dynamic structure. Since viscous and elastic properties of the cells

have an influence on their response to an applied stress, it is considered crucial to

comprehend the cell mechanical behaviour in response to applied mechanical forces.

Unfortunately, the standard pyramidal tips normally used in AFM imaging can only

perform a limited number of measurements, since they damage the cells3, 46. That issue

can be reduced by using colloidal particles glued on AFM tips instead of the bare

pyramidal tips3, 18, 25. In the force measurement mode, the larger area between the sphere

and the cell provides average force profiles leading to less variations in the Young’s

modulus measurements, for instance, thus requiring less measurements to carry out good

statistical analyses3.

Human umbilical vein endothelial cells (HUVECs) had been widely used in research

to study endothelial functions and pathologies in vitro13, 43. However, it is reported that

those cells may undergo senescence as soon as the fourth passage and lose their

endothelial functional characteristics in culture undergoing senescence31 so young cells

must be used.

The aim of this study was to observe the effects of native and oxidized LDL on the

HUVEC mechanical properties by AFM. In addition, the contribution of filamentous

actin (F-actin) and vimentin networks were examined by fluorescence microscopy.

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The results obtained from this work will bring considerable quantitative

knowledge on ox-LDL caused endothelial dysfunction providing a way to evaluate the

effects of potential cyto-protective therapies using antioxidants in the future.

Experimental methods

3.1 Materials

4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES, 99.5%, H-3375), t-

octylphenoxypolyethoxyethanol (Triton X-100, T9284), fetal bovine serum (FBS, F-

1051), Hanks buffered salt solution (HBSS, H6136), bovine serum albumin (BSA,

A7906), Medium 199 (M199, M5017), endothelial cell growth supplement (ECGS

E2759), heparin (H1027), gelatin type B (G9391), phalloidin-TRITC (P1951), and

Hoechst No.33258 (B1155) were purchased from Sigma-Aldrich (Oakville, ON,

Canada).Sodium chloride (NaCl, ACS grade), formaldehyde (F79-1), PBS (BP665-1),

and disposable plastic wares came from Fisher Scientific (Ottawa, ON, Canada).Alexa

Fluor 488 goat anti-mouse (A11001), Live/Dead assay (L-3224), trypsin EDTA (25200-

056), and penicillin G + streptomycin sulphate (15140-122) were obtained from

Invitrogen (Burlington, ON, Canada). Antibodies directed against human vimentin and

vWf were purchased from BD Biosciences (550513, Mississauga, On, Ca) and Cedarlane

(PAHVWF-G-1MG) respectively.1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine

acetylated low-density lipoprotein (Dil-Ac-LDL, BT-902), non acetylated Dil-LDL (BT-

904) and Dil-ox-LDL (BT-920) were purchased from Biomedical Technologies Inc.,

MA, USA.Collagenase type 3 from Clostridium histolyticum (4180 CLS3, 149U/mg) was

bought from Wortington, NJ, USA.Dialysis bags were purchased from Spectrum Medical

Industries Inc. (Texas, USA).

3.2 Subjects

Human umbilical cords were obtained from healthy mothers at the local hospital

maternity ward. All procedures were approved by the Ethic Committee on Human

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Research of the Centre Hospitalier Universitaire de Sherbrooke (CHUS) protocol

number CRC 04-037. After the subjects were thoroughly informed about the nature and

goal of the study, they provided written consent.

Blood plasma was obtained from healthy normo-lipidemic subjects from 18 to 25

years of age after overnight fasting. No study subject had kidney, liver or thyroid disease.

Blood pressure profile was in the normal range and all were non-smokers. Glycemia,

fibrinogen level, lipid profile and coagulation profile were also within the normal ranges.

3.3 Methods

3.3.1 HUVEC extraction and culture

Endothelial cells were harvested from human umbilical cord veins by an adaptation

of the method described by Jaffe et al22. The cords were placed in a sterile container

containing M199 and heparin (90 μg/l) soon after birth. Cords were then inspected, to

insure that they contained no clamps or puncture marks, and rinsed right away, vein

included, with HBSS containing penicillin G (250 U/l), streptomycin sulphate (250 μg/l)

to remove as much blood as possible. Collagenase (2mg/ml) in a serum-free M199 was

used to remove the endothelial cell layer inside the vein.

Near confluence cells were harvested by a short trypsin-EDTA treatment and

cultured on plastic flasks that were previously coated with 100 µg/ml gelatin to help cell

attachment and to limit batch-to-batch variation in the plastic ware response towards cell

attachment. HUVEC were grown in medium 199 containing 20% foetal bovine serum,

heparin (90 μg/ml), L-glutamine (2mM), penicillin G (50 U/ml), streptomycin sulphate

(50 μg/ml), and ECGS (20 μg/ml). The cells were cultured in an incubator (5% CO2 in

humid atmosphere). After the first passage, FBS level was lowered to 10%. Endothelial

cells’ phenotype was confirmed by the specific labelling Dil-Ac-LDL uptake and by the

presence of factor VIII related antigen (von Willebrand factor) using standard

immunocytochemistry methods. Cells were used until passage 4.

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3.3.2 Morphological studies

Cells were fixed with 3.7% (v/v) formaldehyde for 30 minutes at room

temperature. After three 5-minute washes with PBS, the cells were permeabilized by

0.3% (v/v) Triton X-100 made in PBS for 5 minutes at room temperature and washed

twice with PBS and then stored in 0.01% (wt/v) sodium azide in PBS. Filamentous actin

(F-actin) and nucleus were stained by addition of the phalloidin TRITC 1/100 (once

diluted according to manufacturer’s note) and of the specific DNA dye Hoechst at a

concentration of 1/10 000, respectively, in blocking solution (PBS containing 15% FBS

and 5% BSA) for 30 minutes at room temperature, followed by three final washes in

PBS. For vimentin staining, cells were fixed in 90% cold acetone solution for 20 minutes

at -20ºC. Anti-vimentin antibodies 1/200 in blocking solution was added to the cells for

one hour at room temperature and rinsed 3 times (5 minutes each) with PBS. Blocking

solution containing Hoescht 1/10 000 and Alexa Fluor 1/1000 was then added to the cells

and incubated for one hour in the dark at the same temperature. After a final 3 washes

with PBS, cells were stored in PBS with 0.01% sodium azide. The double-stain allowed

the simultaneous visualization of the cell cytoskeleton and nucleus. Pictures were

randomly taken in the plate with the same objective (400X) using a Nikon Eclipse

TE2000-S inverted optical microscope combined to a Retiga 1300R camera. Each

experiment was repeated at least twice.

3.3.3 Cellular uptake of LDL and ox-LDL

Dil-LDL or Dil-ox-LDL at a concentration of 10μg/ml were added to HUVEC at

passage 4 in standard culture media containing 10% FBS. After 30, 60, 120, 300, 720 and

1440 minutes, samples were rinsed 3 times in HBSS, and then observed under

fluorescence microscopy.

3.3.4 Isolation of LDL

Isolation of LDL (1.019 < d < 1.063) and HDL (1.063 < d < 1.21) was performed

according to the method of Sattler et al.40. Briefly, blood samples harvested with EDTA

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anticoagulant were centrifuged using a Beckman Optima TLX ultracentrifuge equipped

with TLA 100.4 rotor, in the presence of EDTA (0.4 mg/ml) as already described23, 24.

After separation, LDL and HDL were dialyzed overnight at 4°C with 10–2M sodium

phosphate buffer (pH 7.4). Protein concentrations were measured by commercial assay

(Pierce method, Rockford, IL, USA).

3.3.5 LDL oxidation by gamma radiolysis of water

Oxygen free radical species were generated by irradiation of aqueous solutions of

LDL using a 60Co Gamma cell 220 (Atomic Energy of Canada Ltd.) at a dose rate of 0.13

Gy/s as determined with the Fricke (ferrous sulfate) dosimeter17, 42. Irradiations were

performed at room temperature as previously described6. In brief, solutions (2 ml) of

LDL containing 10−2M sodium phosphate buffer (pH 7.4), saturated with oxygen, were

exposed to γ-radiation. Under these conditions, the main free radical species produced

selectively and simultaneously were hydroxyl (·OH) and superoxide anion (O2-·) radicals

with yields of 2.8×10−7 and 3.4×10−7 mol/J, respectively42. The total radiation dose used

in this study was 390 Gy.

3.3.6 AFM imaging of living cells

Atomic Force Microscopy (AFM) imaging was performed using a Digital

Nanoscope IIIa Bioscope (Veeco Instruments, Santa Barbara, CA, USA) mounted on an

inverted microscope (Zeiss Axiovert 200, Carl Zeiss, Thornwood, NY). TappingTM mode

imaging greatly reduces the magnitude of lateral forces applied to samples and was

therefore used in the present study to image living cells. Another advantage is that

Tapping modeTM is less sensitive to drift of the cantilever34. The drive frequencies were

chosen between 7.8 and 8.1 kHz. The RMS amplitude was fixed at 0.3 V. Oxide

sharpened silicon nitride (Si3N4) cantilevers (model DNP-20, Veeco NanoProbe Tips)

with a spring constant of 0.32 N/m were used in this study. Cantilevers were cleaned in

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Page 48: effects of native and oxydized ldl on the endothelial cells

Liquinox (#1232, Alconox, NY, USA) for one hour, rinsed in Milli-Q water and then

passed under a UV lamp (PSD-UV, ozone cleaner 185 & 254 nm, Novascan

Technologies).

3.3.7 AFM force measurements on living cells

Atomic Force Microscopy (AFM) force measurements were performed using the

same Digital Nanoscope IIIa Bioscope described above. Oxide sharpened silicon nitride

(Si3N4) cantilevers with integrated pyramidal tips (Model DNP-S, Veeco NanoProbe

Tips) were used in force measurements. Cantilevers were cleaned as described above.

One 4.12-μm diameter silica particle (SS05N, Bangs Laboratories, Inc.) was glued

to each DNP-S cantilever by UV adhesive (Electro-lite, CT, USA, ELC-4481) under a

375 nm UV light. Cantilever deflection and z-piezo position were transformed into force-

versus-indentation curves using a Matlab routine. Young’s modulus was obtained with a

homemade software, which is made freely available upon request to P. Vermette. The

spring constant of the modified cantilevers was determined to be 0.1399 N/m using the

resonance method proposed by Cleveland et al12.

HUVEC were seeded at passage 4 in gelatin-coated petri dishes and cultured at

least 24h prior to AFM experiments. Culture media was supplemented with phosphate

buffer (10mM NaH2PO4 with 0.9% NaCl at pH 7.4) containing LDL or ox-LDL to obtain

a final protein concentration of 0.08 mg/ml. For control, buffer without LDL or ox-LDL

was added to the media. Samples were kept in the incubator until required for AFM

analysis. Native LDL from fresh plasma were isolated, dialysed and used in the following

3 days to limit oxidation over time. At pre-selected times, just before analysis, cells were

rinsed twice with HBSS containing penicillin G (250 U/l), streptomycin sulphate (250

μg/l) and stored in the same HBSS to which 1% HEPES buffer was added to keep pH

stable during the AFM experiments. We waited 15 minutes before beginning data

collection to give the cells a chance to get used of the new media and temperature

conditions.

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Page 49: effects of native and oxydized ldl on the endothelial cells

The cantilever was placed on the fluid holder and connected to the AFM before

each experiment. A drop of HBSS media from the sample was added on the cantilever

before inverting the whole scanner on top of the petri dish containing the cells. The step

motor was used to move the piezoelectric ceramic toward the sample so that the

cantilever appeared very close to the surface. The probe was engaged to the surface at a

scan rate of 0.1 Hz with a scan size set at 0 nm to minimize the initial contact area

between the probe and the cells. Data were obtained with the AFM software (version

6.13R1). At least seven different spots (10 curves each) were collected on each sample (3

spots on the nucleus and 4 spots on the cytoplasm were analysed) at a z-scan velocity of

150 nm/s where hysteresis was minimal32. At least 3 samples per conditions with over 70

force curves each were tested. Chosen cells (under 400x microscope magnification) were

around the same size, had a central nucleus, were well spread in all directions and looked

healthy. In each experiment, the AFM cantilever sensitivity was calibrated against a small

piece of coverslip immersed directly in the HBSS in the Petri dish containing the cells

and the approach force curves were used for calculation of the rigidity.

Nucleolus were chosen as nucleus points because it was easy to align the cantilever

sphere over it, allowing to collect data on the very same spot over the experiment and

avoiding drifting or changes due to cell movements, like contraction. Since no point of

reference was available in the cytoplasm, locations between the nucleus and the cell edge

were selected to avoid the nucleus’ immediate region.

All AFM experiments (imaging and force measurements) were carried out in less

than 4 days following cell seeding, collecting as much data as possible in the first 2 days

considering that Sato et al.38 reported that the length of culture period remarkably affects

the elasticity of HUVEC, especially when the culture time was carried out over 4 days.

Results and Discussion

The cytoskeletal morphology of HUVEC at passage 4 exposed to physiological

concentration (80 µg of protein/ml) of native or ox-LDL was compared under the same

culture conditions. Vimentin staining of cells was done following 2- and 12-hours

38

Page 50: effects of native and oxydized ldl on the endothelial cells

exposure to either LDL or ox-LDL. Cells exposed during 2 hours to LDL shown a similar

morphology than control cells (cells incubated alone) while cells exposed to ox-LDL

((Fig, 1A and 1B respectively)) showed a fiber-like vimentin dispersion instead of a

spread network covering the whole cytoplasm. Cells exposition during 12 hours to LDL

(Fig. 1C) induces a formation of a denser vimentin pattern when compared to 2-hours

exposition to LDL. Figure 1 also shows vimentin structure of HUVEC exposed 12 hours

to ox-LDL (Fig. 1D), revealing a dense network with larger fibers and even uncovered

regions giving the appearance of holes in the vimentin pattern.

A

Figure 1. Immuno-staining of vimentin on HUVEC at passage 4.A, B: Cells exposed 2 hours and 12 hours to native LDLs,respectively. C, D: Cells exposed 2 hours and 12 hours to ox-LDLs, in that order.

39

Page 51: effects of native and oxydized ldl on the endothelial cells

Phalloidin-TRITC staining of control HUVEC and those exposed to LDL or

ox-LDL following 12-hour incubation still showed well spread cells in a cobblestone

pattern, in contact with each other with some stress fibers visible from one side of the

cells to the other one (Fig. 2A). Phalloidin-TRITC staining was complemented with

Hoescht to ascertain that cells had a central unique nucleus of normal appearance (results

not shown). In petri dishes in which HUVEC were exposed to ox-LDL for 12 hours (Fig.

2B), peripheral bands of F-actin were observed and intercellular gaps appeared between

adjacent cells. Individual stress fibers disappeared partly and F-actin clustered in

peripheral region indicating of an F-actin redistribution. At some points, shaky outline

with membrane ruffles (Fig 2B, arrows) could be observed. Those membrane ruffles seen

in Figure 2B had also been observed by Chow et al.11 following cell exposure to ox-LDL.

This finding was shown to be a consequence of pinocytosis, which was also observed

with LDL, but was not associated with an apparent membrane ruffling suggesting that the

actin mechanism might be different11. Essler et al.16, and Zhao et al.47, 48 found that ox-

LDL induce cell contraction, formation of actin stress fibers and intercellular gaps

leading to an increase in endothelial cell permeability, which is in agreement with the

present study.

Figure 2. Immuno-staining of actin filaments with phalloidin-TRITC on HUVEC at passage 4. A: Cells exposed 12 hours to LDLs. B: Cells exposed 12 hours to ox-LDLs.

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Figure 3. Follow up of Dil-LDL and Dil-ox-LDL cell uptake following1-hour and 24-hour incubation with HUVEC.

The distribution of Dil-LDL and Dil-ox-LDL at pre-selected times was followed

and it was found that in the first hour, Dil-ox-LDL were taken and distributed quicker

than Dil-LDL by the cells, thus giving a clearer and better localized signal (Fig. 3). At 24

hours after injection though, we noticed that Dil-LDL’s signal was much stronger (Fig. 3)

than that observed with Dil-ox-LDL. In all cases, it could be clearly seen that Dil-LDL

and Dil-ox-LDL accumulated around the nucleus over time.

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Page 53: effects of native and oxydized ldl on the endothelial cells

Figure 4. AFM imaging in Taping™ mode of HUVEC before treatment (A,C) and 2 hours following LDL injection (B,D). Images A and B show sample height, while images C and D show the phase imaging (corresponding to the surface hardness - the brighter colour is associated to a harder surface). Scan size: 70 μm.

Phase imaging (Fig. 4 C,D), is able to be performed, in tapping modeTM, in the

same time as topographic image (Fig. 4 A,B), both being recorded in a single scan. Phase

imaging allows the detection of different components in a sample related to their stiffness

where lighter areas correspond to regions with higher stiffness2. Nucleoluses seem to

relatively keep the same shape, height and hardness (according to the color scale) 2 hours

following LDL injection. It can be seen that the fibers located over the nucleus look

harder (Fig. 4C) than the cytoplasm prior to the LDL injection to become softer than the

cytoplasm 2 hours following LDL injection (Fig. 4D). Images obtained with ox-LDL

were similar (not shown). In AFM height images, fibers are easily visible before any

treatment (Fig. 4A), while they are more difficult to distinguish 2 hours following LDL

42

Page 54: effects of native and oxydized ldl on the endothelial cells

injection (Fig. 4B). Figure 4D shows that the fibers are still present after two hours, only

hidden from topographic image, so we can only conclude that the LDL came between the

cytoskeleton and the cell’s surface. Figure 4C shows that fibers are packed very close

against each other, while they are well apart in Figure 4D, almost perfectly parallel.

Changes were noticed in the cytoplasm, revealing a foam-like appearance around the

nucleus (Fig. 4B). According to Figure 4D, this region of the cytoplasm became softer

following LDL exposition. Considering that the LDL accumulate around the nucleus

(Figure 3), it can be hypothesized that these changes observed in Figure 4 were due to the

presence of the LDL. This phenomenon was not seen on all our samples, but it can be

postulated that this would be transient phenomena and that it could be affected by the

scanning parameters such as quality of cantilever, frequency peak selection, tip-sample

distance, RMS amplitude, scanning speed, gains and setpoints44. Figure 4 reveals that

nucleoluses are one of the highest components (A-B) in the cell, but also one of the

hardest parts (C-D). Henderson and Sakaguchi demonstrated with fluorescent staining

that the fibers seen in AFM imaging of glial cells were actin filaments20. We thus

conclude that the fibers seen in Figure 4 represent actin filaments since they also

correspond to the pattern observed in Figure 2. Figure 4B does show that the region over

the nucleus is more rigid (light areas) than the cell body. Researchers usually obtained a

harder nucleus than cytoplasm on rabbit aortic endothelial cells33 and HUVEC32 which is

in accordance with our observation. AFM imaging in TappingTM mode did not seem to

disturb the cells since samples were scanned over and over again for more than 3 hours

without seeing any changes. Also, following AFM imaging, we were able to re-culture as

if nothing happened. Cells were also stained using the Live/Dead assay to make sure that

the concentration of ox-LDL used in our study did not affect cell viability or cell

integrity.

AFM force measurements were done on HUVEC (passage 4) exposed to LDL

(Fig. 5A and 5B) and ox-LDL (Fig. 5C and 5D) following different exposure times.

Force measurements were expressed as force-vs-indentation curves. Figures 5A and 5B

show AFM surface force measurements performed between a silica colloidal probe and

nucleolus and cytoplasm areas, respectively, of cells exposed to LDL. In Figure 5A,

comparison of the force-vs-indentation curves of the nucleolus area of cells exposed to

43

Page 55: effects of native and oxydized ldl on the endothelial cells

LDL at different exposure times to those of the same area of cells that were not exposed

to LDL revealed no major difference. From Figure 5A, it may be hypothesized that the

nucleolus area of the cells following 24-hour exposition to LDL became softer than that

of control cells that were not exposed to LDL. In Figure 5B, comparison of the force-vs-

indentation curves on the cytoplasm area of cells exposed to LDL at different exposure

times to those of the same area of cells that were not exposed to LDL revealed no major

difference. From Figure 5B, it could be hypothesized that the cytoplasm area of cells

following 12-hour exposition to LDL became harder than that of control cells that were

not exposed to LDL. However, this phenomenon seems transient as the force analysis of

the same cytoplasm area of the cells following 24-hour exposition to LDL was no

different than that of control cells that were not exposed to LDL. This could possibly

represent an adaptative response from the cells after 24 hours.

Figures 5C and 5D show AFM surface force measurements performed between a

silica colloidal probe and nucleolus and cytoplasm areas, respectively, of cells exposed to

ox-LDL. On one hand, in Figure 5C, comparison of the force-vs-indentation curves of the

nucleus area of cells exposed to ox-LDL at different exposure times to those of the same

area of cells that were not exposed to ox-LDL revealed no major. On the other hand, in

Figure 5D, comparison of the force-vs-indentation curves of the cytoplasm area of cells

exposed to ox-LDL at different exposure times to those of the same area of cells that

were not exposed to ox-LDL revealed major differences over time which is in agreement

with what has been reported by Byfield et al using micropipette aspiration8. From Figure

5D, the cytoplasm area of cells exposed 12 and 24 hours to ox-LDL became harder than

the same area of control cells that were not exposed to ox-LDL. As for the cytoplasm

area of cells exposed to LDL (Fig. 5B), this phenomenon, however, seems transient as the

analysis of the force-indentation profiles indicates that the cytoplasm area of the cells

became softer from 12-hour to 24-hour exposition. Nevertheless, analysis of the force-

indentation profiles of the cytoplasm area of cells exposed 12 and 24 hours to ox-LDL

revealed that ox-LDL had a stiffening effect on the this area of the cells. Figure 1D

______

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Page 56: effects of native and oxydized ldl on the endothelial cells

0,00

10,00

20,00

30,00

40,00

50,00

60,00

70,00

80,00

90,00

0,00 30,00 60,00 90,00 120,00

Indentation (nm)

Forc

e (p

N)

A B

C D

0,00

10,00

20,00

30,00

40,00

50,00

60,00

70,00

80,00

90,00

0,00 30,00 60,00 90,00 120,00

Indentation (nm)

Forc

e (p

N)

0,00

10,00

20,00

30,00

40,00

50,00

60,00

70,00

80,00

90,00

0,00 30,00 60,00 90,00 120,00

Indentation (nm)Fo

rce

(pN

)

0,00

10,00

20,00

30,00

40,00

50,00

60,00

70,00

80,00

90,00

0,00 30,00 60,00 90,00 120,00

Indentation (nm)

Forc

e (p

N)

Figure 5. AFM force measurements expressed as force-vs-indentation curves on HUVEC at passage 4 following LDL (A,B) and ox-LDL treatment (C,D). Force profiles on the nucleus area (A,C) on cytoplasm area (B,D).

45

Page 57: effects of native and oxydized ldl on the endothelial cells

reveals a higher vimentin network density for cells incubated 24 hours with ox-LDL,

which could be partly related to the increasing stiffness showed by the AFM force

measurements.

Force curves were obtained for indentation depths (135 nm) smaller than 10 % of

the cell thickness (2-2.7μm), therefore they were not influenced by the substrate32.

Analysis of the AFM force measurement profiles obtained on cells did not show a

significant difference in term of rigidity between the nucleolus and cell body areas. This

was also observed by Berdyyeva et al. on epithelial cells, who suggested that the broader

distribution of rigidity for young cells might be explained by a higher variation in

cytoskeleton density3, while comparing nucleus and cell body regions. Even though the

approach/retraction speed of the AFM tip was carefully selected to minimize the

dissipated energy (by viscous effects), minimal hysteresis was still observed. Therefore,

the AFM force measurement profiles do not reflect a purely elastic modulus, but rather an

apparent elastic modulus14, 29._______

5. Conclusions

The objective of this study was to evaluate the effects of native and oxidized LDL

on the cells mechanical properties. Ox-LDL were shown to change cell shape as well as

F-actin and vimentin organisation while results obtained with native LDL revealed

almost no noticeable changes. AFM was successfully used to measure in vitro the

changes in rigidity directly on living young individual HUVEC exposed to native and ox-

LDL in function of exposition time. Analysis of the AFM force measurement profiles

revealed that the cytoplasm became significantly stiffer following 12-hour and 24 hour

incubation with ox-LDL while cells exposed 24 hours to LDL showed no major changes

in term of their rigidity when compared to control cells that were not exposed to LDL. To

our knowledge, this is the first study reporting biomechanical analyses of the effects of

LDL and ox-LDL on young living cells rigidity using AFM.

46

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39. Sato M. Theret DP. Wheeler LT. Ohshima N. & Nerem RM. Application of the micropipette technique to the measurement of cultured porcine aortic endothelial cell viscoelastic properties. J. Biomech. Eng 112, 263-268 (1990).

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40. Sattler W. Mohr D. & Stocker R. Rapid isolation of lipoproteins and assessment of their peroxidation by high-performance liquid chromatography postcolumn chemiluminescence. Methods Enzymol. 233, 469-489 (1994).

41. Simon A. et al. Heterogeneous cell mechanical properties: an atomic force microscopy study. Cell Mol. Biol. (Noisy. -le-grand) 50, 255-266 (2004).

42. Spinks JWT. Woods RJ. An Introduction to Radiation Chemistry New York, 1990).

43. Unterluggauer H. Hampel B. Zwerschke W. & Jansen-Durr P. Senescence-associated cell death of human endothelial cells: the role of oxidative stress. Exp. Gerontol. 38, 1149-1160 (2003).

44. Wright M. & Revenko I. TappingMode Atomic Force Microscopy Operation in Fluid. Veeco Instruments Inc. 2004.

45. Wu HW. Kuhn T. & Moy VT. Mechanical properties of L929 cells measured by atomic force microscopy: effects of anticytoskeletal drugs and membrane crosslinking. Scanning 20, 389-397 (1998).

46. You HX. Lau JM. Zhang S. & Yu L. Atomic force microscopy imaging of living cells: a preliminary study of the disruptive effect of the cantilever tip on cell morphology. Ultramicroscopy 82, 297-305 (2000).

47. Zhao B. Ehringer WD. Dierichs R. & Miller FN. Oxidized low-density lipoprotein increases endothelial intracellular calcium and alters cytoskeletal f-actin distribution. Eur. J. Clin. Invest 27, 48-54 (1997).

48. Zhao B. Zhang Y. Liu B. Nawroth P. & Dierichs R. Endothelial cells injured by oxidized low density lipoprotein. Am. J. Hematol. 49, 250-252 (1995).

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4.2 Method of imaging low density lipoproteins (LDL) by atomic

force microscopy

Julie A. Chouinard1,2, Abdelouahed Khalil2,3, Patrick Vermette1,2*

1Laboratoire de Bioingénierie et de Biophysique de l’Université de Sherbrooke,

Department of Chemical Engineering, 2Centre de Recherche sur le Vieillissement, Institut

universitaire de gériatrie de Sherbrooke and 3Department of Medicine, Faculty of

Medicine, Université de Sherbrooke, Sherbrooke, (Qué), Canada.

Article soumis au Microscopy Research and Technique, référence 1027IB.

Inclus dans le mémoire avec la permission des auteurs en octobre 2006.

Keywords: LDL, AFM imaging.

Running title: AFM imaging of LDL.

*Corresponding author: Department of Chemical Engineering, Université de Sherbrooke, 2500, boul. de l’Université, Sherbrooke, Québec, Canada, J1K 2R1. Phone: +1 819 821-8000 ext. 62826; Fax: +1 819 821 7955. E-mail: [email protected]

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Résumé

Ce court article décrit une méthode permettant d’imager des lipoprotéines de basse

densité (LDL) en utilisant le microscope à force atomique (AFM). Cet instrument permet

l’imagerie d’échantillons biologiques en milieu liquide et présente l’avantage de ne

nécessiter aucune préparation d’échantillon telle que les colorations ou les fixations

pouvant affecter leur structure générale. Les dimensions (hauteur et diamètre) de LDL

individuelles furent mesurées avec succès. Les images AFM démontrent que les LDL ont

une structure quasi-sphérique sur l’axe des x et y et une structure sphéroïde oblate

considérant l’axe des z (hauteur). Les LDL observées présentent un diamètre moyen de

23 ± 3 nm. La moyenne obtenue pour la hauteur est de 10 ± 2 nm.

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1. Abstract

This short paper reports a method to image low density lipoproteins (LDL) using

Atomic Force Microscopy (AFM). This instrument allows imaging of biological samples

in liquid and presents the advantage of needing no sample preparation such as staining or

fixation that may affect their general structure. Dimensions (diameter and height) of

individual LDL particles were successfully measured. AFM imaging revealed that LDL

have a quasi-spherical structure on the x and y axis with an oblate spheroid structure in

the z axis (i.e., height). LDL were found to have an average diameter of 23 ± 3 nm. The

obtained mean height was 10 ± 2 nm.

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2. Introduction

Lipoproteins have been traditionally classified into five major categories based on

their density: chylomicrons (CM), very low density lipoproteins (VLDL), intermediate

density lipoproteins (IDL), low density lipoproteins (LDL), and high density lipoproteins

(HDL) (Ginsberg, 1998; Gotto et al., 1986). LDL particles are playing key roles in the

transfer of cholesterol to peripheral cells and its metabolism in the human blood

circulation. LDL form a heterogeneous family of molecules, which vary greatly in size,

composition and structure. They have an average density ranging between 1,019-1,063

g/ml (Hevonoja et al., 2000; Krauss and Burke, 1982). LDL particles found in men are

smaller than those found in women (Freedman et al., 2004; McNamara et al., 1987).

Also, LDL size is known to change in the presence of cardiovascular risk factors

including aging, diabetes and hypercholesterolemia (Freedman et al., 2004; Haffner et al.,

1994). LDL diameter is reported to range from 18 to 25 nm with an average diameter of

22 nm (Belo et al., 2004; Freedman et al., 2004; Hevonoja et al., 2000; Segrest et al.,

2001). The LDL particle core consists of approximately 170 triglycerides and 1600

cholesteryl ester molecules, while the surface monolayer is composed of 700

phospholipids and a single copy of ApoB-100 (Esterbauer et al., 1992), which is the

largest known monomeric protein consisting of 4563 amino acid residues (4536 amino

acid mature peptides and 27 amino acid signal peptides) (Chen et al., 1986; Segrest et al.,

2001).

The Atomic Force Microscope (AFM) is an instrument able to provide nanometer

scale resolution images of native biological samples in liquid (Allen et al., 1995; Hassan

et al., 1998; Lehenkari and Horton, 1999). The aim of this study was therefore to obtain

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good quality images of fresh LDL in a near physiological environment that does not alter

the sample.

3. Materials and Methods

3.1 Materials

Highly ordered pyrolytic graphite (HOPG) was obtained from SPI (SPI-2 Toronto,

ON, Canada). Dialysis bags were purchased from Spectrum Medical Industries Inc.

(Texas, USA).

3.2 Subjects

Blood plasma was obtained from healthy normo-lipidemic subjects from 18 to 25

years of age after overnight fasting. All procedures were approved by the Ethics

Committee of the Research Centre on Aging (Sherbrooke). After the subjects were

thoroughly informed about the nature and goal of the study, they provided written

consent.

3.3 Methods

3.3.1 Isolation of LDL

Isolation of LDL (1.019 < d < 1.063) and HDL (1.063 < d < 1.21) was performed

according to the method of Sattler et al. (Sattler et al., 1994), using a Beckman Optima

TLX ultracentrifuge equipped with a TLA 100.4 rotor, in the presence of

ethylenediaminetetraacetic acid (EDTA, 0.4 mg/ml) as already described (Khalil et al.,

1996; Khalil, 1998). After separation, LDL and HDL were dialyzed overnight at 4°C

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with 10–2M sodium phosphate buffer containing 0.15M NaCl (pH 7.4). Protein

concentrations were measured by a commercial assay (Pierce method, Rockford, IL,

USA).

3.3.2 Atomic Force Microscopy (AFM) imaging

AFM imaging was performed using a Digital Nanoscope IIIa Bioscope (Veeco

Instruments, Santa Barbara, CA, USA) mounted on an inverted microscope (Zeiss

Axiovert 200, Carl Zeiss, Thornwood, NY). All imaging were carried out using Tapping

modeTM with oxide sharpened silicon nitride (Si3N4) cantilevers with integrated

pyramidal tips (Model DNPS, Veeco NanoProbe Tips). Cantilevers were cleaned in

Liquinox (Alconox, NY, USA) for one hour, rinsed in Milli-Q water and then passed

under a UV lamp (PSD-UV, ozone cleaner 185 & 254 nm, Novascan Technologies).

Tapping modeTM imaging greatly reduces the magnitude of lateral forces applied to

samples and thus appears more appropriate for imaging LDL. Another advantage is that

Tapping modeTM is less sensitive to drift of the cantilever (Radmacher et al., 1995). The

drive frequencies were chosen between 7.8 and 8.1 kHz. The RMS amplitude was fixed

at 0.3 V. Cantilevers used in this study have a spring constant of 0.32 N/m.

LDL were added to 10-2 M phosphate buffer with 0.15M NaCl pH 7.4 to obtain a

final protein concentration of 50 µg/ml. The sample was then deposited on a freshly

cleaved HOPG surface and left 15 minutes before beginning the AFM imaging. The

cantilever was placed on the fluid holder and connected to the AFM before each

experiment. A drop of buffer was added to the cantilever holder before inverting the

whole scanner on top of the sample. The step motor was used to move the piezoelectric

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ceramic toward the sample so that the cantilever appeared very near to the surface. The

probe was engaged to the surface at a scan rate of 1.00 Hz. Data were obtained with the

AFM software version 6.13R1 using the Tapping modeTM scanning. Integral and

proportional gains were set to 0.2 and 0.3, respectively. Drive amplitude of 60 mV was

used, with an amplitude setpoint of 0.26, scanning at a resolution of 512 by 512 points.

4 Results and Discussion

AFM images of LDL were obtained under 10-2 M phosphate buffer with 0.15M

NaCl at pH 7.4 and with no sample treatment. Individual LDL can be easily identified on

the AFM images (Figs. 1 and 2), appearing as spherical particles on the HOPG substrates.

Such spherical particles were not seen on bare HOPG substrates (see Annex 2). Although

all LDL look alike, each particle was different in size (Fig. 1A). This could be explained

considering that there are at least 8 known distinct subclasses of LDL presenting different

lipid composition and ApoB-100 conformational changes (Krauss and Burke, 1982;

McNamara et al., 1996). ApoB-100 protein is believed to unfold to adapt to LDL particle

sizes, which vary in function of the LDL subclasses (McNamara et al., 1996). These

authors also calculated that ApoB’s thickness at the interface decreases from

approximately 2,5 nm to 1,6 nm (McNamara et al., 1996).

LDL show an average diameter of 23 ± 3 nm (n = 27). The measured mean height

was 10 ± 2 nm. Therefore, the measured height of the LDL molecules did not correspond

to the measured diameter. On one hand, this finding could be a result of the LDL

compression by the AFM tips, showing an oblate spheroid shape. The volume of a

spheroid particle can be calculated by V = (4/3)πa2b where a is the half-length of the

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Page 69: effects of native and oxydized ldl on the endothelial cells

principal axis (diameter) and b the half-length of the secondary axis (height). If it is

hypothesized that the volume did not change upon compression, the volume of a sphere,

given by V = (4πr3)/3, can be used to calculate the unaltered LDL spherical diameter,

which in our case would have been approximately 17 nm. This calculated diameter is in

good agreement with the one obtained by Legleiter et al.(2004), using AFM also. On the

other hand, it can be questioned whether or not LDL are spherical. In fact, Orlova et al.

(1999) reported an ellipsoid LDL structure of 25 x 21 x 17,5 nm using electron cryo-

microscopy. Van Antwerpen et al.(1997, 1994) also reported images of LDL taken in

vitrified frozen-hydrated conditions without any use of staining or chemical fixation.

They concluded that human LDL had a discoidal shape with a diameter of 21,4 ± 1,3 nm

and a height of 12,1 ± 1,1 nm (Van Antwerpen et al., 1997; Van Antwerpen and Gilkey,

1994). The same conclusion was reached by Spin and Atkinson (1995), also using

electron cryo-microscopy, although these authors considered the observed discoidal

shape as an artefact (Spin and Atkinson, 1995). Their study presented LDL as almost

spherical structure with diameters ranging from 22 to 24 nm where, in some images,

particles were found to have an egg shape with a pointy end, believed to represent the N-

terminal globular region of the ApoB (Spin and Atkinson, 1995).

AFM phase imaging (Fig. 1B) was recorded in parallel to AFM topographic

imaging (Fig. 1A). Phase imaging allows the characterization of the sample stiffness over

the scanned area. Brighter areas correspond to stiffer regions (Babcock, 2004). Figure 1B

shows that LDL were softer than the underneath HOPG surface. This finding is not

surprising as LDL are mainly composed of lipids while HOPG is a rigid carbon substrate.

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5 Conclusions

Using the atomic force microscopy (AFM), good quality images were obtained

allowing identification of individual LDL molecules. LDL particles were found to have

an oblate spheroid structure with an average diameter of 23 ± 3 nm and a height of 10 ± 2

nm.

6 Acknowledgements

This work was supported by the Canadian Institute of Health Research (CIHR), the

Research Centre on Aging and by the Université de Sherbrooke. The authors wish to

thank Maxim Isabelle, Martin Cloutier and Hicham Berrougui for their technical

assistance with the LDL extraction. The authors are also grateful to Félix Dupont for his

technical assistance with the AFM.

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References

Allen M J, Bradbury EM, and Balhorn R. 1995. The natural subcellular surface structure of the bovine sperm cell: J.Struct.Biol., 114(3):197-208.

Babcock KL, Prater CB. 2004. Phase Imaging: Beyond Topography. Veeco Instruments Inc.

Belo L, Caslake M, Santos-Silva A, Castro EM, Pereira-Leite L, Quintanilha A, Rebelo I. 2004. LDL size, total antioxidant status and oxidised LDL in normal human pregnancy: a longitudinal study: Atherosclerosis, 177(2):391-399.

Chen SH, Yang CY, Chen PF, Setzer D, Tanimura M, Li WH, Gotto AMJr, Chan L. 1986. The complete cDNA and amino acid sequence of human apolipoprotein B-100: J.Biol.Chem., 261(28):12918-12921.

Esterbauer H, Gebicki J, Puhl H, Jurgens G. 1992. The role of lipid peroxidation and antioxidants in oxidative modification of LDL: Free Radic.Biol.Med., 13 (4):341-390.

Freedman DS, Otvos JD, Jeyarajah EJ, Shalaurova I, Cupples LA, Parise H, D'Agostino RB, Wilson PW, Schaefer EJ. 2004. Sex and age differences in lipoprotein subclasses measured by nuclear magnetic resonance spectroscopy: the Framingham Study: Clin.Chem., 50(7):1189-1200.

Ginsberg HN. 1998. Lipoprotein physiology: Endocrinol.Metab Clin.North Am., 27(3):503-519.

Gotto AMJr, Pownall HJ, Havel RJ. 1986. Introduction to the plasma lipoproteins: Methods Enzymol., 128:3-41.

Haffner SM, Mykkanen L, Stern MP, Paidi M, Howard BV. 1994. Greater effect of diabetes on LDL size in women than in men: Diabetes Care, 17(10):1164-1171.

Hassan E, Heinz WF, Antonik MD, D'Costa NP, Nageswaran S, Schoenenberger CA, Hoh JH. 1998. Relative microelastic mapping of living cells by atomic force microscopy: Biophys.J., 74(3):1564-1578.

Hevonoja TM, Pentikainen O, Hyvonen MT, Kovanen PT, La-Korpela M. 2000. Structure of low density lipoprotein (LDL) particles: basis for understanding molecular changes in modified LDL: Biochim.Biophys.Acta, 1488(3):189-210.

Khalil A, Wagner JR, Lacombe G, Dangoisse V, Fulop TJr. 1996. Increased susceptibility of low-density lipoprotein (LDL) to oxidation by gamma-radiolysis with age: FEBS Lett., 392(1):45-48.

Khalil A, Jay-Gerin J-P, Fulop TJr. 1998. Effect of aging on high density lipoproteins susceptibility to oxidation induced by radiolysis of water. FEBS Lett. 435:153-158.

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Krauss RM, Burke DJ. 1982. Identification of multiple subclasses of plasma low density lipoproteins in normal humans: J.Lipid Res., 23(1):97-104.

Legleiter J, DeMattos RB, Holtzman DM, Kowalewski T. 2004. In situ AFM studies of astrocyte-secreted apolipoprotein E- and J-containing lipoproteins: J.Colloid Interface Sci., 278(1):96-106.

Lehenkari PP, Horton MA. 1999. Single integrin molecule adhesion forces in intact cells measured by atomic force microscopy: Biochem.Biophys.Res.Commun., 259(3):645-650.

McNamara JR, Campos H, Ordovas JM, Peterson J, Wilson PW, Schaefer EJ. 1987. Effect of gender, age, and lipid status on low density lipoprotein subfraction distribution. Results from the Framingham Offspring Study: Arteriosclerosis, 7(5):483-490.

McNamara JR, Small DM, Li Z., Schaefer EJ. 1996. Differences in LDL subspecies involve alterations in lipid composition and conformational changes in apolipoprotein B: J.Lipid Res., 37(9):1924-1935.

Radmacher M, Fritz M, Hansma PK. 1995. Imaging soft samples with the atomic force microscope: gelatin in water and propanol: Biophys.J., 69(1):264-270.

Sattler W, Mohr D, Stocker R. 1994. Rapid isolation of lipoproteins and assessment of their peroxidation by high-performance liquid chromatography postcolumn chemiluminescence: Methods Enzymol., 233:469-489.

Segrest JP, Jones MK, De LH, Dashti N. 2001. Structure of apolipoprotein B-100 in low density lipoproteins: J.Lipid Res., 42(9):1346-1367.

Spin JM, Atkinson D. 1995. Cryoelectron microscopy of low density lipoprotein in vitreous ice: Biophys.J., 68(5):2115-2123.

Van Antwerpen R, Chen GC, Pullinger CR, Kane JP, LaBelle M, Krauss RM, Luna-Chavez C, Forte TM, Gilkey JC. 1997. Cryo-electron microscopy of low density lipoprotein and reconstituted discoidal high density lipoprotein: imaging of the apolipoprotein moiety: J.Lipid Res., 38(4):659-669.

Van Antwerpen R, Gilkey JC. 1994. Cryo-electron microscopy reveals human low density lipoprotein substructure: J.Lipid Res., 35(12):2223-2231.

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Figure legends

Figure 1: AFM images of freshly extracted LDL from a young healthy male. A) Height

color coded image, in which brighter areas correspond to higher features. B)

Corresponding AFM phase image, in which darker tones represent a softer material. Scan

size = 110 nm.

Figure 2: AFM images of freshly extracted LDLs from a young healthy male. A) Height

color coded image, in which brighter areas correspond to higher features. B)

Corresponding AFM phase image, in which darker tones represent a softer material. Scan

size = 50 nm.

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Figure 1

Figure 2

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Conclusion et perspectives générales

En conclusion, ce projet a permis d’établir les effets des LDL et ox-LDL sur les

propriétés biomécaniques des cellules endothéliales. Les résultats des marquages

immunohistochimiques démontrent que les LDL ne semblent pas avoir d’influence sur le

cytosquelette des cellules alors que les ox-LDL induisent de grands changements de

forme et de conformation, modifiant ainsi la morphologie cellulaire générale. Ces

modifications sont également rapportées dans la littérature scientifique comme étant des

facteurs augmentant la perméabilité de l’endothélium2, 3 et retardant la guérison des

blessures1, signes révélateurs de dysfonctionnement endothélial. L’utilisation de l’AFM a

apporté de nouvelles données sur les propriétés biomécaniques des cellules HUVEC

exposées aux LDL et ox-LDL. Cette fois-ci encore, les LDL n’ont pas semblé avoir

d’impact majeur sur les cellules alors que les ox-LDL ont largement augmenté la rigidité

de ces dernières, plus particulièrement au niveau du cytoplasme. À notre connaissance, il

s’agit de la première démonstration de l’étude par AFM de la rigidité cellulaire de jeunes

cellules endothéliales vivantes exposées à des LDL ou ox-LDL. Nos résultats sont

originaux du fait qu’ils démontrent une nouvelle méthode de quantifier de façon précise

la dysfonction endothéliale permettant dans un futur proche d’évaluer l’efficacité

cytoprotectrice de traitements aux antioxydants dans la thérapie et la prévention de

l’athérosclérose.

Une méthode d’imagerie des LDL a aussi été mise au point en utilisant l’AFM. Il

est maintenant possible d’obtenir des images de bonne qualité permettant aussi de

mesurer les dimensions des LDL individuelles. Le débat quant à la forme réelle des LDL,

sphérique ou sphéroïde oblate, n’est cependant toujours pas résolu. La technique

développée dans ce projet permettra dans le futur d’imager, par exemple, les LDL de

personnes atteintes de pathologies telles que le diabète ou autres maladies affectant le

métabolisme des lipides.

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65

References

1. Boissonneault,G.A., Wang,Y., & Chung,B.H. Oxidized low-density lipoproteins delay endothelial wound healing: lack of effect of vitamin E. Ann. Nutr. Metab 39, 1-8 (1995).

2. Liao,L., Aw,T.Y., Kvietys,P.R., & Granger,D.N. Oxidized LDL-induced microvascular dysfunction. Dependence on oxidation procedure. Arterioscler. Thromb. Vasc. Biol. 15, 2305-2311 (1995).

3. Rangaswamy,S., Penn,M.S., Saidel,G.M., & Chisolm,G.M. Exogenous oxidized low-density lipoprotein injures and alters the barrier function of endothelium in rats in vivo. Circ. Res. 80, 37-44 (1997).

Page 77: effects of native and oxydized ldl on the endothelial cells

ANNEXES

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ANNEXE 1: EXTRACTION CELLULAIRE ET CARACTÉRISATION

1.1 Human Umbilical Vein Endothelial Cells (HUVEC)

Les cellules utilisées dans ce projet furent les HUVEC. Nous étions

principalement intéressés au type natif (sans modifications génétiques). Pour ce faire, une

technique d’extraction capable de donner des cellules très jeunes en grande quantité fut

mise au point pour les besoins de ce projet. En effet, les HUVEC, étant des cellules

primaires, sont connues pour devenir rapidement sénescentes et ne peuvent par

conséquent pas être conservées très longtemps. L’un des premiers protocoles d’extraction

de ce genre de cellule endothéliale fut publié par Jaffe et al1 en 1973. et constitue encore

aujourd’hui la référence principale2-5. C’est grâce à la littérature et aux conseils du

professeur Charles Doillon du Centre Hospitalier Universitaire de Laval (CHUL) à

Québec que le protocole suivant a finalement été mis au point.

1.1.1 Extraction de cellules endothéliales à partir de cordons ombilicaux

Matériels et produits : Avant de commencer, s’assurer d’avoir (pour 1 cordon) : Au moins 200 ml de HBSS 1X et de HBSS 5X à 37oC; Au moins 100 ml de milieu M199 à 37oC; Au moins 500 ml de PBS à 37oC; De la collagénase; Un aliquot d’Endothelial Cell Growth Supplement (ECGS); Un aliquot de L-glutamine; Une lame de scalpel; Deux tubes stériles de 50 ml; Un tube stérile de 15 ml; Un T-flask de 25 cm2; Une solution de gélatine à 100µg/ml; Une trousse de cordon contenant 1 grand papier d’aluminium stérile, 2 pinces à

forceps (ciseaux bleus), 2 pinces, 2 tubes de verres avec bouts de plastique (canules) et 3 triples fils;

Deux béchers de plastique stériles de 250ml; Une seringue stérile de 60 ml, une de 20 ml et un petit filtre 0,45μm; Un pied de métal avec pince pour y accrocher la seringue de 60 ml; Des pipettes stériles de 10 ml en plastique; Beaucoup d’éthanol 70% en vaporisateur.

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EN TOUT TEMPS LE MATÉRIEL DOIT ÊTRE STÉRILE, LES LIQUIDES MAINTENUS À 37oC ET LES OPÉRATIONS SE FONT SOUS UNE HOTTE LAMINAIRE. Récupération du cordon :

• Dans un pot stérile contenant 60 ml de milieu + antibio 1X et héparine sans sérum. • Bien inspecter pour s’assurer de l’absence de ponctions ou de marques de pinces. • Couper les bouts abîmés. • Laver le cordon autant que possible avec le tampon dans un bécher de 250 ml. • Effectuer 2 rinçages de la veine au HBSS 5X lorsque le cordon est très frais (dès

réception par les infirmières) pour éviter la formation de caillots. • Mettre le cordon dans un pot neuf avec milieu et antibiotiques pour le transport. • Conserver les pots à cordon au réfrigérateur jusqu’au moment du transport. • Extraire les cellules rapidement (< 4 heures). • Température de transport : 4-12°C (présence de glace)

Note : Si besoin, ajuster le pH des solutions avec 1 % de tampon HEPES 1M. Collagénase :

• Mettre à température de la pièce 30 minutes avant d’ouvrir, car la collagénase est hygroscopique.

• Préparer la collagénase (Wortington, 10000U à ~ 140U/mg) dans une petite bouteille de verre : diluer l’enzyme à 2mg/ml dans du milieu sans sérum.

• Incuber à 37°C 15 minutes. • Filtrer avec la seringue de 20ml et le filtre de 0.45 μm dans un tube de 15ml

stérile (ne pas trop agiter la solution pour ne pas abîmer l’enzyme !). Manipulations :

Tremper le cordon dans du HBSS contenant des antibiotiques (5X) à 37oC et le laver doucement.

Travailler sur une feuille de papier d’aluminium stérile.

Déballer les pièces de la trousse à cordon en prenant soin de conserver les grands

papiers d’aluminium stérile, ils serviront à emballer le bécher à cordon pour la période d’incubation avec la collagénase.

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Installer la première canule, la solidifier avec un triple fil à une distance d’environ 1cm avant le début du plastique (Annexe1.1A), faire quelques tours, puis monter attacher plus haut sur la canule environ 2cm plus haut que la jonction cordon-canule (Annexe1.1B). Redescendre solidifier plus bas (Annexe1.1C). Ajouter le forcep sur le nœud final pour tenir le tout. Il est important de bien serrer le cordon avec le fil et ce sans avoir peur. Il est plus probable d’ailleurs que le fil casse, plutôt que le cordon.

A B

A c a t c R

C

nnexe 1.1 Technique d’attache de la anule au cordon. A) Commencer par ttacher le bas sur le cordon où passe la ige de verre. B) Remonter les fils en roisant pour solidifier sur le tube. C) edescendre en croisant de nouveau

pour attacher solidement le bas.

Accrocher la seringue de 60 ml sur le pied de métal, retirer le piston et le remettre dans son étui en attendant, puis enfoncer le cordon sur la seringue par la canule.

Rincer le cordon avec du HBSS + 1x pour nettoyer la veine (2 x 50 ml).

de petits caillots (ne pas forcer, ne pas masser !). (Voir montage à l’Annexe 1.2)

Mettre la deuxième canule, fermer celle du bas avec une pince, faire passer du

tampon doucement, la veine doit gonfler mais pas trop, refermer le haut du cordon avec une autre pince, laisser reposer 5 min puis ouvrir et laisser couler le tampon. Rincer une deuxième fois en laissant passer du tampon (~ 40 ml). Mieux vaut gonfler légèrement la veine si jamais il y a

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Annexe 1.2 Montage d’un cordon avec canules et forceps pour les étapes de rinçage ou de collecte de cellules suite à la digestion par collagénase. Les pinces sont ici absentes, mais elles vont normalement sur les tubes des canules (flèches) lorsque l’on désire couper le flot.

Avant de faire passer la collagénase, coincer l’extrémité du bas avec l’une des 2 pinces. Faire passer la collagénase en relâchant la pince du bas (pour permettre à la collagénase de remplacer l’air) et dès que des gouttes commencent à tomber du tube, refermer la pince. Quand toute la collagénase est dans le cordon (attention à ne pas laisser entrer d’air), refermer le cordon en haut avec la pince.

Le cordon doit ensuite être placé dans un bécher de 250 ml contenant du PBS à

37oC et bien emballé (car sortie à l’extérieur de la hotte) pour un séjour de 15 minutes au bain-marie à 37oC. Bien s’assurer que les extrémités ne soient pas immergées.

Récupérer rapidement les cellules endothéliales libres qui baignent dans le

mélange milieu + collagénase. Placer un tube de 50 ml, contenant 0.5 ml de sérum, sous le cordon, et ouvrir la pince de la canule du bas, puis du haut.

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Faire passer ± 10ml de milieu. Normalement, le liquide devrait s’écouler tout seul, mais il faut souvent l’aider avec le piston de la seringue (Attention, car la veine est très fragile à ce stade et il ne faut pas la faire éclater sous la pression). Enfin, faire passer doucement ± 30ml de HBSS pour récupérer un maximum de cellules.

Sans perdre de temps, faire centrifuger à 1200 rpm pendant 10 min.

Enlever le surnageant. Pour cette opération et les subséquentes, utiliser les

pipettes stériles de 10 ml.

Re-suspendre les cellules dans 5ml de milieu + 20% sérum + antibiotiques.

Placer ces 5 ml dans un T-flask de 25 cm2 (T25) et ajouter l’ECGS + L-glutamine pour démarrer la culture. Les cellules à ce stade ressemblent à l’Annexe 1.3.

Annexe 1.3 Photo au microscope d’une extraction de cellules

HUVEC prise au moment de la mise en culture. Les cellules sont en amas et le plus souvent rattachées à des débris de tissus.

Changer le milieu au bout de 2 heures pour éliminer les cellules musculaires lisses

et les hématies, puis laisser le T-flask dans l’incubateur à 37oC pour la nuit.

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Le lendemain, changer le milieu pour éliminer les globules rouges avec encore 10ml de milieu + 10% sérum + antibiotiques + ECGS + L-glutamine. Les cellules commencent à faire des îlots ressemblant à l’Annexe 1.4.

Changer le milieu tout les deux jours et ajouter l’ECGS jusqu’à confluence de

80%. Les cellules ne doivent pas tapisser complètement la surface, car elles risquent de perdre leurs propriétés si elles subissent trop d’inhibition de contact.

Le matériel ayant servi pour la procédure et qui sera réutilisé (bécher, clamp,

forceps, canules…) doit tremper au moins 24h dans l’eau de javel avant d’être nettoyé à l’exception du matériel métallique.

Lors du premier passage, il faut faire très attention de changer le milieu du T-flask

et de bien rincer au moins 3 fois un peu moins de une heure après le passage lorsque les cellules commencent à adhérer. Ceci afin de se débarrasser des quelques fibroblastes en culture sachant que les HUVEC adhèrent très rapidement et les fibroblastes en environ 2 heures.

Annexe 1.4 HUVEC en culture depuis environ 2 jours. La vitesse derecouvrement varie d’un cordon à l’autre selon la quantité initiale decellules extraites.

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1.1.2 MARQUAGE DU FACTEUR VON WILLEBRAND (VWF) Ce marquage sert à identifier les cellules endothéliales.

Matériel :

• PBS (température ambiante). • Formaldéhyde 3.7% v/v dans PBS (4°C température ambiante). • Triton X100 0.5% v/v dans PBS (4°C température ambiante). • 2% de BSA dans du PBS (4°C 4°C). • Rabbit serum (4°C 4°C, obscurité). • AcI anti-vWF (-20°C 4°C). • AcII rabbit anti-mouse, FITC conjugate (4°C 4°C, obscurité). • Hoechst (4°C 4°C, obscurité).

Note : (Condition d’entreposage condition(s) à conserver durant le marquage) Protocole :

• Rincer 3 fois les cellules avec du PBS. • Fixer avec formaldéhyde à 3.7 % w/v dans le PBS, 15 min à température ambiante. • Lavages PBS x3. • Perméabiliser les cellules avec une solution de Triton X-100 0.5% (PBS) pendant 5

min à température pièce. • Rinçage au PBS x3. • Bloquer avec du sérum du second animal (rabbit) (1/20 dans le PBS avec 2% de

BSA) pendant 20 min (chambre humide, température ambiante). • Rinçage au PBS x3. • Ajouter AcI anti-vWF, 1/500 dans PBS/BSA pendant 1 heure (chambre humide,

température pièce). • Rinçage au PBS x3. • Incuber le 2ème anticorps, 1/100 dans PBS/BSA, 1 heure dans le noir (chambre

humide, température ambiante). • Rinçage au PBS x3. • Montage entre lame et lamelle en utilisant soit le PBS-glycérol (50/50 v/v).

Pour 1 ml Serum AcI Hoechst 1/100 Mix AcII

AcII Hoechst 1/100

produit 50 μl 2 μl 10 μl 10 μl 10 μl

PBS/BSA 950 μl 998 μl 990 μl 980 μl

Annexe 1.5 Répartition des produits pour le marquage vWF.

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Les cellules endothéliales possèdent des réserves de facteurs von Willebrand qui

au marquage donnent l’aspect de petites granules concentrées à l’intérieur du cytoplasme.

Malheureusement, nous avions un problème avec notre lot d’anticorps couplé à la FITC

qui faisait que la fluorescence disparaissait à vue d’œil. Le temps était suffisant pour

confirmer un marquage positif, mais il fut impossible d’obtenir des photos nettes étant

donné l’impossibilité d’ajuster les paramètres de la caméra. Voilà pourquoi l’Annexe 1.6

n’est pas de la plus grande qualité. L’Alexa Fluor 488 est plus stable que la FITC et sera

désormais utilisée pour ce type de marquage.

Annexe 1.6 Exemple d’un marquage vWF sur des HUVEC.

L’aspect de granules à l’intérieur des cellules est typique aux cellules endothéliales.

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1.1.3 MARQUAGE DIL-ACÉTYL-LDL Les LDL acétylées marquées au 1,1'-dioctadecyl – 3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (Dil) marquent seulement les cellules endothéliales et les macrophages. Matériel :

• Dil-Ac-LDL (Biomedical Technologies Inc., stock à 200 μg/mL). • Milieu de culture standard complet stérile.

Protocole :

• Diluer de façon stérile le Dil-Ac-LDL dans du milieu de culture pour obtenir [10μg/ml] au final. Ex. : pour 3 ml : 150μL de Dil-Ac-LDL dans 2.85 ml de M199).

• Ajouter le mélange aux cellules (au moins 36 heures post-trypsine/collagénase) de manière à couvrir le fond du plat de culture.

• Incuber le tout 4 heures à 37°C. • Retirer le milieu contenant le Dil-Ac-LDL. • Rincer plusieurs fois avec du milieu simple. • Visualiser au microscope avec le filtre standard rhodamine. (Voir Annexe 1.7) • Il est possible de fixer les cellules avec 3,7 % formaldéhyde dans du PBS (ne jamais

fixer au méthanol ou à l’acétone, le Dil étant soluble dans les solvants organiques).

75

Annexe 1.7 Marquage Dil-Ac-LDL sur des extraits de cordons ombilicaux. Il est ànoter que même les cellules sénescentes prennent ce marquage (flèche). Seules lesHUVEC et les macrophages ont un marquage positif. Photo de droite : différenceentre les HUVEC qui prennent le Dil-Ac-LDL et un autre type cellulaire,possiblement des fibroblastes, qui ne le prend pas.

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1.1.4 MARQUAGE LIVE / DEAD Ce marquage indique en rouge le noyau des cellules mortes ou dont l’intégrité membranaire est compromise et en vert les cellules vivantes. Matériel :

• HBSS 1X incolore (4°C 37°C). • Éthidium homodimère (rouge) (- 20°C, obscurité). • Calcéine (vert) (- 20°C, obscurité).

Note : (Condition d’entreposage condition à conserver durant le marquage)

ATTENTION : travailler dans le noir !

Protocole :

• Préparer la solution de marqueurs (on utilise des concentrations diluées de moitié de celles recommandées par le fabricant, car le marquage marche aussi bien) :

o HBSS 1X incolore : 2.5 ml. o Éthidium homodimère : 2.5 μl. o Calcéine : 0.625 μl.

• Vortexer le tout. • Rincer les cellules au HBSS chaud. • Mettre la solution de marqueurs (s’assurer de couvrir le fond). • Emballer le contenant dans du papier d’aluminium pour protéger de la lumière. • Incuber 30 minutes à 37°C. • Rincer au HBSS chaud et prendre les photos en fluorescence.

Annexe 1.8 Photo combinée d’un marquage Live/Dead où les cellules vivantes apparaissent en vert et le noyau des mortes, en rouge.

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1.1.5 MARQUAGE F-ACTINE ET VIMENTINE

Les filaments d’actine sont les principaux composants du cytoskelette avec les filaments de vimentine aussi appelés filaments intermédiaires.

Matériel :

• PBS (température ambiante). • Formaldéhyde 3.7% v/v dans PBS (4°C température ambiante). • Triton X100 0.5% v/v dans PBS (4°C température ambiante). • Acétone froid 90% (-20°C). • PBS contenant BSA 5% et 15% FBS (4°C). • AcI anti-vimentine (-20:C 4°C). • AcII goat anti-mouse, Alexa Fluor 488 (-20°C 4°C, obscurité). • Phalloidin-TRITC (-20°C 4°C, obscurité). • Hoechst (4°C, obscurité).

Note : (Condition d’entreposage condition(s) à conserver durant le marquage) Protocole :

• Rincer 3x les cellules avec du PBS. • Pour phalloïdine seulement. Fixer les cellules au formaldéhyde 3.7% w/v dans PBS

30 min à température pièce. Rincer 3x 5min PBS. • Pour phalloïdine seulement. Perméabiliser les cellules avec une solution de Triton

X100 0.3% / PBS pendant 5 min à température pièce. • Pour vimentine seulement. Fixer les cellules avec de l’acétone froid 90 % pendant 20

minutes à -20°C. (Cette étape a également l’avantage de perméabiliser). • Rincer 3x 5min PBS. • Saturer avec PBS/BSA 5% + FBS 15% 30 min à température pièce. • Rincer 3x PBS. • Pour vimentine seulement. Incuber avec AcI anti-vimentine (souris) dilué au 1/200

dans du PBS/BSA+FBS 1h à température pièce. Rincer 3x 5min PBS. • Pour vimentine seulement. Incuber avec AcII chèvre anti-souris alexa fluor 488 dilué

au 1/1000, contenant aussi du Hoechst au 1/10 000. 1h à température pièce / noir. • Pour actine seulement. Incuber avec phalloïdine-TRITC diluée à 1/100, contenant

également du Hoescht à 1/10 000, pendant 1 h dans le noir à température pièce. • Rincer 3x 5 min PBS. • Montage entre lame et lamelle en utilisant soit le PBS-glycérol (50/50 v/v) soit le

Prolong gold antifade ou le DakoCytomation Mounting Medium S3023.

Note : Si le marquage et la fixation n’ont pas lieu le même jour, ajouter 0.01% d’azide sodique au PBS final pour la conservation.

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Annexe 1.9 Marquage des filaments d’actine en rouge et des noyaux

en bleu grâce au double marquage phalloïdine-TRITC (actine) etHoescht (ADN).

Annexe 1.10 Marquage des filaments intermédiaires de vimentine.

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1.1.6 MARQUAGE SÉNESCENCE

Les cellules sénescentes démontrent une activité accrue de β-galactosidase à pH 6.0.

Matériel :

• Solution de fixation (- 20°C Température pièce) • X Gal (- 20°C Température pièce) • Solution de marquage (- 20°C Température pièce) • Supplément de marquage (- 20°C Température pièce) • PBS 1X • DMF (N-N-diméthylformamide)

Note : (Condition d’entreposage condition à conserver durant le marquage)

Préparation :

• Dissoudre 20 mg de X Gal dans 1 ml de DMF afin de préparer une solution stock 20X. Cette solution peut être conservée un mois à l’obscurité à - 20°C. Toujours stocker le X Gal dans du verre ou du polypropylène, ne jamais utiliser de polystyrène.

Protocole (pour un puit d’une plaque 12 puits):

• Enlever le milieu de culture et rincer les cellules une fois avec 1 ml de PBS 1X. • Fixer les cellules avec 0.5 ml de solution de fixation à température ambiante pendant

10 à 15 minutes. • Pendant ce temps préparer le mélange dans un tube de polypropylène. Pour chaque

puit, ajouter 470 μl de la solution de marquage, 5 μL du supplément de marquage et 25 μl de la solution de X Gal 20X dans le DMF.

• Rincer les cellules deux fois avec 1 ml de PBS 1X. • Ajouter 0.5 ml du mélange à chaque puit. • Incuber toute la nuit à 37°C et observer au microscope les cellules en bleu. Note : Pour conserver les cellules, retirer le mélange, couvrir de glycérol 70% et mettre à 4°C.

Annexe 1.11 Marquage des cellules sénescentes en bleu par la méthode du SA- β-gal.

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Reference List

1. Jaffe,E.A., Nachman,R.L., Becker,C.G., & Minick,C.R. Culture of human

endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J. Clin. Invest 52, 2745-2756 (1973).

2. Neubert,K., Haberland,A., Kruse,I., Wirth,M., & Schimke,I. The ratio of formation of prostacyclin/thromboxane A2 in HUVEC decreased in each subsequent passage. Prostaglandins 54, 447-462 (1997).

3. Sato,H. et al. Kinetic study on the elastic change of vascular endothelial cells on collagen matrices by atomic force microscopy. Colloids Surf. B Biointerfaces. 34, 141-146 (2004).

4. Varani,J. et al. Age-dependent injury in human umbilical vein endothelial cells: relationship to apoptosis and correlation with a lack of A20 expression. Lab Invest 73, 851-858 (1995).

5. Wagner,M. et al. Replicative senescence of human endothelial cells in vitro involves G1 arrest, polyploidization and senescence-associated apoptosis. Exp. Gerontol. 36, 1327-1347 (2001).

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ANNEXE 2

Annexe 2.0 Imagerie AFM d’un substrat de HOPG. Scan size = 500 nm.

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