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À la mémoire de ma grand-mère
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
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.
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
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.
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
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
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
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
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
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
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
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
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
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
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
6
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+
7
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,
8
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
9
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,
10
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).
11
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.
12
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.
13
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.
14
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.
15
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.
16
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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.
28
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.
29
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.
30
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.
31
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.
32
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
33
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.
34
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
35
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
36
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.
37
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
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
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.
40
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.
41
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
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
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
______
44
0,00
10,00
20,00
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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
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0,00 30,00 60,00 90,00 120,00
Indentation (nm)
Forc
e (p
N)
0,00
10,00
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30,00
40,00
50,00
60,00
70,00
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90,00
0,00 30,00 60,00 90,00 120,00
Indentation (nm)Fo
rce
(pN
)
0,00
10,00
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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
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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17. Fricke HMS. Am.J.Roentgenol.Radiat.Ther. 18, 430-432. 1927.
18. Hansma HG. & Hoh JH. Biomolecular imaging with the atomic force microscope. Annu. Rev. Biophys. Biomol. Struct. 23, 115-139 (1994).
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50
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]
51
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.
52
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.
53
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
54
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
55
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
56
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
57
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.
58
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.
59
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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.
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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.
62
Figure 1
Figure 2
63
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.
64
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).
ANNEXES
66
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
69
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.
70
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).
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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.
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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