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Fluid shear stress directly stimulates synthesis of the endothelial glycocalyx : peturbations byhyperglycemia
Gouverneur, M.C.L.G.
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Citation for published version (APA):Gouverneur, M. C. L. G. (2006). Fluid shear stress directly stimulates synthesis of the endothelial glycocalyx :peturbations by hyperglycemia.
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Download date: 31 Dec 2019
Fluid shear stress directly stimulates synthesis
of the endothelial glycocalyx
Perturbations by hyperglycemia
© 2006 by Mirella Gouverneur
Fluid shear stress directly stimulates synthesis of the endothelial glycocalyx: perturbations by
hyperglycemia; PhD thesis, University of Amsterdam
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ISBN10: 90-9020855-0
ISBN13: 978-90-9020855-8
Fluid shear stress directly stimulates synthesis
of the endothelial glycocalyx
Perturbations by hyperglycemia
ACADEMISCH PROEFSCHRIFT
ter verkrijging van de graad van doctor
aan de Universiteit van Amsterdam
op gezag van de Rector Magnificus
prof. mr. P.F. van der Heijden
ten overstaan van een door het college voor promoties ingestelde
commissie, in het openbaar te verdedigen in de Aula der Universiteit
op vrijdag 8 september 2006, te 10.00 uur
door
Maria Cornelia Lucia Gerdina Gouverneur
geboren te Waalwijk
Promotiecommissie Promotor Prof.dr. Hans Pannekoek Prof.dr. ir. Jos A.E. Spaan Co-promotor Dr. Hans Vink Overige leden Prof.dr. M. Daemen
Prof.dr. V.W. van Hinsbergh Dr. A.J.G. Horrevoets
Prof. dr. V.H. Huxley Prof.dr. C.Ince
Faculteit der Geneeskunde
Financial support by the Netherlands Heart Foundation and Dutch Diabetes Research
Foundation for the publication of this thesis is gratefully acknowledged.
If a person was to ask my advice, before undertaking a long voyage, my answer would
depend upon his possessing a decided taste for some branch of knowledge, which could by
this means be advanced. No doubt it is a high satisfaction to behold various countries and the
many races of mankind, but the pleasures gained at the time do not counterbalance the evils.
It is necessary to look forward to a harvest, however distant that may be, when some fruit will
be reaped, some good effected.
Charles Darwin, A Naturalist’s voyage (1845)
7
Table of Contents Chapter 1 General Introduction
1 The Cardiovascular System ………………………………… 13 2 The Endothelial glycocalyx ………………………………… 16 3 Clinical relevance ………………………………… 29
References ………………………………… 32
Outline of thesis ………………………………… 37
Chapter 2 Fluid shear stress stimulates incorporation of hyaluronan into the endothelial cell glycocalyx Am J Physiol Heart Circ Physiol. 290: H458-H452, 2006 Abstract ………………………………… 41 2.1 Introduction ………………………………… 43 2.2 Materials and methods ………………………………… 43 2.3 Results ………………………………… 47 2.4 Discussion ………………………………… 53 References ………………………………… 56
Chapter 3 Hyperglycemia attenuates flow-induced hyaluronan production by cultured RF24 endothelial cells Submitted for publication Abstract ………………………………… 61 3.1 Introduction ………………………………… 63 3.2 Materials and methods ………………………………… 63 3.3 Results ………………………………… 65 3.4 Discussion ………………………………… 68 References ………………………………… 70
Chapter 4 Acute hyperglycemia stimulates shedding of the glycocalyx from cultured endothelial cells. Submitted for publication Abstract ………………………………… 75 4.1 Introduction ………………………………… 77 4.2 Materials and methods ………………………………… 77 4.3 Results ………………………………… 79 4.4 Discussion ………………………………… 85 References ………………………………… 87
8
Chapter 5 Loss of Endothelial Glycocalyx during Acute Hyperglycemia Coincides with Endothelial Dysfunction and Coagulation Activation in vivo Diabetes 2006 Feb; 55(2): 480-6 Abstract ………………………………… 91 5.1 Introduction ………………………………… 93 5.2 Materials and methods ………………………………… 94 5.3 Results ………………………………… 97 5.4 Discussion ………………………………… 103 References ………………………………… 107
Chapter 6 Vasculoprotective Properties of the Endothelial Glycocalyx: Effects of Fluid Shear Stress (review) J. Intern. Med. 2006 Apr;259(4):393-400 6.1 Introduction ………… 115 6.2 Structural properties of the endothelial glycocalyx ………… 116 6.3 Glycocalyx at arterial bifurcations ………… 118 6.4 Mechanism of glycocalyx reduction at high regions ………… 121 6.5 Glycocalyx and systematic aherogenic stimuli ………… 122 6.6 Human glycocalyx measurements ………… 123 6.7 Summary ………… 125 References ………… 126
Summary ………………………………… 131
Nederlandse Samenvatting ………………………………… 133
Dankwoord ………………………………… 137
Curriculum Vitae ………………………………… 141
Publication list ………………………………… 143
Conference abstracts ………………………………… 145
9
Prologue
‘By now we have established the point that many plant and animal cells possess an
extra cellular coating rich in polysaccharides ... I deem it desirable to assign a single, general,
inclusive term to this extra cellular, sugary coating, wherever it may be found. The ancient
Greeks had no word for sugar, but they had one for sweet-a taste, which we often associate
with sugars. I propose that we choose to speak generally of this polysaccharide-rich coating
of cells as the “glycocalyx”. This word means “sweet husk”... We know of very many kinds
of plant, animal, bacterial and other cells, which possess a glycocalyx. Do all cells possess
one? Is a glycocalyx a general feature of cells, as is the plasma membrane? We do not know.’
These words from Bennet HS in 1963 (1) introduced the term glycocalyx for the sugar
coating of cells to the scientific community. Much research was to follow and our interest is
the ‘sugar coat’ of the cells that line the vascular system, the endothelial glycocalyx. The
endothelial glycocalyx protects the endothelial cells from the shear forces of flowing blood
and is highly hydrated, giving it gel-like properties. Since the use of intravital microscopy, it
has been possible to visualize and study the in vivo endothelial glycocalyx in the smallest
blood vessels during blood flow. The endothelial glycocalyx has been studied by a select
number of research groups in the world and is gaining more and more attention for its key
role in the protection of the vasculature in clinical settings such as atherosclerosis and
diabetes. Furthermore, the relatively young research field of glycobiology is opening doors
even further to study the highly complex nature of glycocalyx structures and the organization
of its proteins and saccharides to increase our understanding of its role in health and disease.
In this thesis, I present to you my studies on the biochemistry and dynamics of the
glycocalyx of cultured endothelial cells exposed to stimuli designed to obtain insight into its
possible role in the development of atherosclerotic and diabetic disease. An attempt is made
to translate these findings into the clinical settings we are encountering at this time. I hope I
will find you enjoyed and intrigued by the endothelial glycocalyx, as I have been.
10
Chapter 1
General Introduction
Chapter 1 General Introduction
12
Chapter 1 General Introduction
13
1 The Cardiovascular System
1.1 A historical perspective
In the early days of Hippocrates (460-377 BC), the founder of modern medicine, the heart
was not seen as a propulsive organ and neither did anyone realize that the heart recirculated
blood. Several decades later, another Greek physician by the name of Galen (130-200) was
known to be an extremely prolific investigator. Galen did not recognize the fact that blood
recirculates, he stated blood to be produced in the liver and is distributed through veins to
organs where it is consumed. He hypothesized that arteries carry a vital spirit from lung to
peripheral tissues. For those times, these erroneous conclusions were as accurate as possible
(2). It was after nearly 1500 years, in 1629, that William Harvey (1578-1657) discovered and
reported that blood recirculates and surmised the existence of capillaries, even though he
could not see them, using a theoretical model approach. Marcello Malphighi (1628-1694)
first visualized blood capillaries in the lung of a frog in 1661 and shortly after, Antoni van
Leeuwenhoek (1632-1723) established the existence of capillaries in mammalian tissues and
organs (3).
1.2 Structure and function
1.2.1 Anatomy
The vascular system consists of the macrovascular conduit vessels, the large arteries and
veins, and the microcirculation, consisting of vessels with a diameter smaller than 300 µm,
arterioles, capillaries and venules, of which the capillaries have the smallest diameters
between 3-8 µm, which distribute the blood volume in the body (Figure 1.1).
Chapter 1 General Introduction
14
Blood vessels, except capillaries, contain three subsequent layers. The intima is the innermost
layer closest to the blood perfused lumen, and consists of an endothelial cell monolayer,
basale membrane, and subendothelial space. The media consists of several layers of smooth
muscle cells. The adventitia, the outermost layer, contains connective tissue. In addition, the
large arteries and veins contain large amounts of elastic tissue in the internal elastic lamina
arranged between intima and media, and in the external elastic lamina between media and
adventitia. The capillary wall only consists of endothelium, the basal membrane and pericytes
(4).
1.2.2 Pressure control
The vascular system consists of arteries, arterioles, capillaries, venules and veins, in which
the pulsatile pressure drops subsequently from 100mmHg to central venous pressure between
0 and 5 mmHg. The major pressure drop takes place after arteries become smaller than 300
µm in diameter, which are therefore also known as resistance vessels (5). The arteries and
arterioles consist of highly elastic components and collagen giving them elasticity as well as
mechanical strength to deal with the pulsatile nature of pressure and flow. Pressure in venules
Figure 1.1
Distribution of blood volume in the
different components of the circulatory
system. Reprinted from textbook of
Medical Physiology, Guyton & Hall 9th
edition, page162 with permission from
Elsevier.
Chapter 1 General Introduction
15
and veins is low and both pressure and flow are non-pulsatile. Consequently, venules differ
mostly from arteries in respect to their elastic requirements.
1.2.3 Transport and exchange
The cardiovascular system keeps the other bodily systems working. The heart pumps blood
rich in oxygen and nutrients through the arteries to the organs where the oxygen is consumed
and via the venous piping system the oxygen poor blood is returned back to the heart from
where it is pumped to the lungs to be oxygenated again. The exchange of oxygen and
nutrious components takes place in the capillaries. Capillaries are the smallest vessels with
diameters of only a few microns, consisting of a single layer of endothelial cells. However,
due to their great number, capillary endothelial surface area accounts for over 99% of the
total surface area of the vascular system, hence, the endothelial lining of the microcirculation
is of extreme importance for control of transvascular exchange (Figure 1.2) (2; 6; 7).
Figure 1.2
Surface area of the human vasculature.
Shown is the estimated total cross-
sectional area of the vascular tree in
humans (top) and the linear velocity
(bottom). The cross-sectional area of the
more clinically recognized blood vessels
(coronary, carotid and peripheral
arteries) represents a tiny fraction of the
total. Reprinted with permission from
Aird et al.
Chapter 1 General Introduction
16
2 The Endothelial glycocalyx
2.1 A historical perspective We have seen that endothelial cells line the vascular system and therefore are cells closest to
flowing blood. At the end of the 17TH century, when it was possible to visualize blood flow in
capillaries, researchers were able to study the vascular system more closely. Malpighi (1628-
1694), who first visualized capillaries in frog, reported a plasmatic zone in circulating blood
(3). Poiseuille (1880) postulated an immobile part portion of the plasmatic zone in close
proximity to the endothelium. He investigated the quantitative nature of hemodynamics and
his studies lead to the formulation of the Poiseuille equation:
∆Pd4
Q = 128ηl
in which flow (Q) is described in terms of a pressure gradient (∆P), viscosity (η) and diameter
(d) and length (l) of the vessel (8). Fåhraeus (1929) showed a reduction in tube hematocrit,
which is defined as the percentage of red blood cell volume in total blood volume, as blood
flows through artificial small-bore tubes. Subsequent studies were done on the corresponding
reduction in blood viscosity (8). He noted that the radial distribution of red blood cells is not
uniform in microvessels < 300 µm, but that the radial erythrocyte concentration is largest in
the middle of the vessel (9). Studies using small artificial bore tubes showed that microvessel
hematocrit may be an important determinant of microvessel perfusion by virtue of the strong
dependence of the apparent viscosity of blood on hematocrit (10). The introduction of
intravital microscopy enabled the study of blood flow in tissue capillaries under high
magnification and provided researchers with even more detail of the vasculature with respect
to the behavior of blood near the endothelial surface.
Chapter 1 General Introduction
17
2.2 Functional implications
2.2.1 Hematocrit
Duling and coworkers intensively studied capillary hematocrit and the Fåhraeus effect using
intravital microscopy and postulated a slow moving plasma layer or glycocalyx of 1µm
thickness on the luminal surface of capillary endothelium (11). Experiments by Duling and
Desjardins, who infused capillaries with the enzyme heparinase, showed a reduction in the
functional volume of this plasma layer as reflected by an increase of capillary tube hematocrit
of 2 to 4 fold, in the absence of major changes in red blood cell velocity. Mathematically
simulated blood flow in microvascular networks confirmed that such changes in
microvascular hemodynamics are consistent with a 50% reduction of the dimension of the
endothelial surface layer, in agreement with the experimental findings (12). Vink and Duling
showed that light dye damage to the endothelial glycocalyx increased capillary hematocrit by
60% in 5 µm wide capillaries compared to untreated sites in the same vessels (Figure 3). This
finding resulted in an important note of caution in the use of (fluorescence) light microscopy
to study the endothelial glycocalyx: be aware not to destroy the object you study with the
method selected to study it (13). Constantinescu et al. showed increased hematocrit and a
reduction of the effective thickness of the glycocalyx when capillaries were treated with
oxidized low density lipoprotein (oxLDL) (14). These results clearly state the overwhelming
impact of the endothelial surface layer on the vasculature in relation to microvascular tube
hematocrit values. The question arises how this relation between the endothelial glycocalyx
and blood hematocrit values can be used in obtaining a better understanding of
pathophysiological clinical situations.
Chapter 1 General Introduction
18
2.2.2 Permeability
The large surface area of vascular endothelium provides our vascular system with an efficient
compartment for regulation of transvascular exchange. It is estimated that the endothelial
surface area can amount up to several thousand square meters in a 70 kg adult man, which is
predominantly situated in the microvascular system. Starling (1896) stated that ‘the net force
determining capillary fluid exchange is the balance between capillary hydrostatic pressure
and the effective colloid osmotic pressure of the plasma proteins’ and provided the most
fundamental insight on the driving forces that regulate exchange of fluid and solute between
the circulation and tissues (15). Landis (1926) provided direct experimental support for
Starling’s hypothesis. Using a microinjection technique to measure capillary pressure and
observation of the movement of red blood cells to monitor transcapillary fluid exchange, he
was able to estimate fluid flux through capillary walls as a function of capillary hydrostatic
Bright field Fluorescence
4.7 µm5.4 µm
C apillaryd iam eter
Exclusion zone(Endothelial surface layer)
R ed b lood cell(R B C )
F luorescent dye(F ITC -dextran 70)
W hite b lood cell(W B C )
Bright field Fluorescence
4.7 µm5.4 µm
Bright field Fluorescence
4.7 µm5.4 µm5.4 µm5.4 µm
C apillaryd iam eter
Exclusion zone(Endothelial surface layer)
R ed b lood cell(R B C )
F luorescent dye(F ITC -dextran 70)
W hite b lood cell(W B C )
C apillaryd iam eter
Exclusion zone(Endothelial surface layer)
R ed b lood cell(R B C )
F luorescent dye(F ITC -dextran 70)
W hite b lood cell(W B C )
Figure 1.3
Left: Digitized images of a capillary segment. RBC width (A) using bright field microscopy and the width of
the fluorescent FITC-dextran column (B) were significantly smaller than the anatomic capillary diameter. The
scale bar represents 5 µm (Left). Right: Schematic illustration of method for measuring permeation of
fluorescently labeled plasma solutes into endothelial surface layer (glycocalyx). Method is based on exclusion
of fluorescent tracers and red blood cells (RBC) from a space adjacent to capillary endothelial cell surface.
Measurements of anatomical capillary diameter, dye column width, and RBC width were compared to obtain
estimates of the apparent endothelial exclusion zone. Reprinted with permission from Vink et al.
A B
Chapter 1 General Introduction
19
pressure. He observed that when he occluded a capillary with a glass rod, the column of red
cells in the capillaries tended to move forward, indicating filtration of fluid from blood to
tissue, whereas capillaries with the lowest pressure tended to reabsorb fluid from the
surrounding tissues (16). Distinctions can be made between continuous and fenestered
endothelium. Experimental evidence has shown a pathway that runs through the breaks or
openings in the junctional strands, which represent a pathway that is not narrower than the
wide regions of the intercellular clefts (i.e., 15–20 nm), within intercellular clefts, when
investigating permeability and ultrastructure of continuous endothelium in microvessels.
From these findings it was concluded that the most important feature of these realistic models
is that the tight junctions are not the site of the endothelial barrier to macromolecules (17).
The permeability barrier was supposedly provided by a fiber matrix filter close to the
entrance of the clefts from the luminal surface, and it was proposed that this ultrafilter was
formed by the luminal glycocalyx of the endothelium (18).
The initial role of the glycocalyx in relation to control of endothelial permeability came in
focus in the 1940s when a concept was proposed by Danielli (19; 20) that there was a non
cellular layer on the endothelial surface, called endo capillary layer, which was thought to
include absorbed plasma proteins. In order to study the permeation and the magnitude of the
endothelial glycocalyx in relation to microvascular permeability, Vink and Duling, used
various plasma tracers of different sizes and charges. It was demonstrated that the endothelial
glycocalyx limits access of plasma macromolecules into a domain of 0.4 to 0.5 µm thick on
the luminal endothelial surface of hamster cremaster capillaries (21), while red blood cells
under normal flow conditions are excluded from a region extending even farther into the
lumen (22). In vivo studies using FITC-labeled tracers and TNFα exposure show that pro
inflammatory cytokines can cause disruption of the endothelial apical glycocalyx, leading to
an increased macromolecular permeation (23). The effect of adenosine on the endothelial
glycocalyx indicates that adenosine causes a rapid and profound decrease in the ability of the
glycocalyx to exclude dextran 70kDa, but only affects red blood cell exclusion at
pharmacological levels (24).
Subsequent studies using specific enzymes to degrade the endothelial glycocalyx, further
studied its specific contribution to control of vascular permeability. Huxley (25) evaluated the
hypothesis that a glycocalyx contributes to the resistance to transvascular protein flux
Chapter 1 General Introduction
20
measured in coronary arterioles. Apparent solute permeability (P-s) to two proteins of
different size and similar charge, alpha-lactalbumin (alpha-lactalb) and porcine serum
albumin (PSA), was determined. Frog mesenteric microvessels were treated with glycocalyx
degrading enzymes, pronase and heparitinase. Data demonstrate that in intact coronary
arterioles an enzyme-sensitive layer, most likely at the endothelial cell surface, contributes
significantly to the net barrier resistance to solute flux. The effect of hyaluronidase on the
endothelial glycocalyx was studied by Henry and Duling. After infusion of one of several
FITC-dextrans (70, 145, 580, and 2,000 kDa) via a femoral cannula, microvessels were
observed with bright-field and fluorescence microscopy to obtain estimates of the anatomic
diameters and the widths of fluorescent dextran columns and of red blood cell columns. After
1 hour of treatment with active Streptomyces hyaluronidase, there was a significant increase
in access of 70- and 145-kDa FITC-dextrans to the space bounded by the apical glycocalyx,
but no increase in access of the red blood cells into the glycocalyx and in the absence of
changes in the anatomic diameter of capillaries, arterioles, and venules. Hyaluronidase did
not affect access of FITC-Dextrans 580 and 2,000 to the endothelial glycocalyx. Infusion of a
mixture of hyaluronan and chondroitin sulfate after enzyme treatment reconstituted the
glycocalyx, although treatment with either molecule separately had no effect (22).
Extensive studies on renal filtration have also tuned in on the role of the endothelial
glycocalyx in glomerular sieving process. Extensive studies by Haraldsson reveal the
important contribution of endothelial barrier capability, the endothelial surface layer and
development of proteinuria (26-29).
These studies show the importance of the endothelial glycocalyx as the first ‘sieve’ of the
vascular permeability barrier and establish its role as an initial protective barrier against
clinical disturbances such as vascular leakage.
2.2.3 anti-adhesive barrier
In a healthy state, the endothelium provides an anti-adhesive lining in the vasculature. Initial
stages of vascular injury show disruption of this anti-adhesive property where the, normally
in blood circulating, white blood cells start rolling along the endothelium. The initial
endothelial-leukocyte interactions are mediated by E-selectin and P-selectin molecules on the
Chapter 1 General Introduction
21
endothelium and L-selectin on leukocytes. When the leukocyte is slowed down, it is able to
attach to the vascular wall using adhesion molecules such as ICAM-1 on endothelial cells and
integrins CD11/18 on leukocytes (30) and, subsequently invade the vascular wall (31). The
receptors for this attachment to the endothelium can become available through different
stimuli such as inflammatory factors and oxidative stress (32). Several studies have shown
that in addition to the expression of the adhesion molecules on the endothelial surface,
leukocyte-endothelial adhesion also requires modulation of glycocalyx components (33-35).
Stimulation of endothelial cells with platelet-activating factor (PAF) induced expression of
adhesion molecules in parallel with loss of sulfated proteoglycans of the endothelial
glycocalyx, which resulted in leukocyte-endothelial adhesion (33) Cultured endothelial cell
surfaces loose their anti-adhesiveness to leukocytes after enzymatic removal of sulfated
proteoglycans (36). Binding of CD44 to hyaluronic acid can mediate rolling, as demonstrated
by the BW5147 cell line that rolls either on purified hyaluronate or cultured endothelial cells
(31). In addition, heparan sulfate proteoglycans have binding sites for L-selectin (37),
implicating a direct role of the glycocalyx as an endothelial surface ligand for rolling
leukocytes. Unfractionated heparin also binds selectins, and this may account in part for the
anti-inflammatory role of heparin (38). Loss of sulfated proteoglycans could also occur
through cleavage by neutrophil derived proteases, which may therefore play a critical role in
the vascular injury associated with inflammation (39).
2.2.4 anti-coagulatory coating
The coagulation of blood is mediated by cellular components and soluble plasma
proteins. In response to vascular injury, circulating platelets adhere, aggregate, and provide
cell-surface phospholipid for the assembly of blood-clotting enzyme complexes. The
extrinsic pathway of blood coagulation is initiated when blood is exposed to non-vascular-
cell–bound tissue factor in the subendothelial space. Tissue factor binds to activated factor
VII, and the resulting enzyme complex activates factors IX and X of the intrinsic and
common coagulation pathways, respectively. Factor IX activated by the tissue-factor pathway
in turn activates additional factor X, in a reaction that is greatly accelerated by a cofactor,
factor VIII. Once activated, factor X converts prothrombin to thrombin (factor IIa) in a
reaction that is accelerated by factor V. In the final step of the coagulation pathway, thrombin
cleaves fibrinogen to generate fibrin monomers, which then polymerize and link to one
another to form a chemically stable clot. Thrombin also feeds back to activate cofactors VIII
Chapter 1 General Introduction
22
and V, thereby amplifying the coagulation mechanism. The blood-coagulation cascade has
the ability to transduce a small initiating stimulus into a large fibrin clot. The potentially
explosive nature of this cascade is offset by natural anticoagulant mechanisms. The
maintenance of adequate blood flow and the regulation of cell-surface activity limit the local
accumulation of activated blood-clotting enzymes and complexes (40).
One of the predominant sulfated proteoglycans in the vascular system is heparan sulfate
proteoglycan (41). The physiologic role of endothelial cell anticoagulant HSPGs has been
defined by perfusing rat hindlimbs with Thrombin and AntiThrombin, and showing that T–
AT complex generation is dramatically accelerated by a heparan sulfate vessel wall
component. The in vivo location of anticoagulant Heparan Sulfate Proteoglycans (HSPGs)
was ascertained by light and EM level autoradiography, which revealed small amounts of
anticoagulant HSPGs on the luminal surface of endothelial cells with much larger quantities
deposited in the subendothelial space (42).
Using oxidative stress, disruption of the endothelial surface layer by oxidized low-density
lipoproteins (Ox-LDL) contributes to atherogenic increases in vascular wall adhesiveness for
blood platelets. The dimension of the EC surface layer is diminished by 60% within 25
minutes, which correlated with a transient increase in the number of platelet-EC adhesions.
Combined administration of superoxide dismutase and catalase completely blocked the effect
of Ox-LDL (43).
Chapter 1 General Introduction
23
2.3 Structural properties
2.3.1 Electron microscopic visualization
In order to visualize the endothelial surface layer (ESL) or glycocalyx, experiments using
Alcian blue by Chambers & Zweifach (44) showed ‘thin strands and sheets of a faintly
colored blue translucent material sloughing of the inner surface of capillaries in frog
mesentery’. Copley (45; 46) injected pontamine sky blue and observed an unstained
plasmatic zone adjacent to the endothelial cells, proposing an endothelial surface covered by
a thin molecular layer and immobile sheet of plasma. Electron microscopic studies using
ruthenium red, which generates detectable election density in the presence of osmium
tetroxide, showed a layer with puffy appearance and a thickness of 20nm (47). Studies by
Haldenby using Alcian blue, which gives information on charge density of the endothelial
layer, showed a staining up to 60nm in the capillary lumen. Sims and Horne (48) stated that
used aquous solutions for the preservation of tissue for electron microscopic observation
likely dissolves all but the protein cores of glycoproteins more quickly than they can be
preserved.
EM studies using fluorocarbons-osmiumtetraoxide showed a stained layer of 60-110nm (49);
gold colloids and immunoperoxidase labeling showed extended staining ranging up to
100nm. (50). Van den Berg preserved and stained the endothelial glycocalyx using Alcian
blue and the staining even ranged up to 500nm into the capillary lumen of rat heart capillaries
(51) (Figure 1.4).
Note: these days the term ‘glycocalyx’ is often used as a synonym for ‘endothelial surface
layer’, the endothelial layer with its glycoproteins, proteoglycans, glycosaminoglycans and
absorbed plasma proteins.
Chapter 1 General Introduction
24
2.3.2 Proteoglycans and glycosaminoglycans
Proteoglycans consist of a family of core proteins, which are modified by extensive
glycosylation. The main proteoglycans present in the cardiovascular system consist of three
families of core proteins, the membranous core protein glypican, the transmembrane core
protein syndecan and the secreted core protein perlecan (41).
The negatively charged repeated disaccharide units, the glycosaminoglycans (GAGs), can be
even more negatively charged when sulfated. These glycosaminoglycans can range up to 200
disaccharide units, displaying highly specific sulfation patterns. These extensive core protein
modifications give the endothelial glycocalyx its highly negative charge and can extend far
into the blood vessel lumen (Figure 1.5).
Figure 1.4
Electron microscopic overview of an
Alcian blue 8GX–stained rat left
ventricular myocardial capillary (bar: 1
µm). Reprinted with permission from van
den Berg et al.
GAG ( ) attachment sites
Syndecan Glypican
Cell membrane
GAG ( ) attachment sites
Syndecan Glypican
Cell membrane
GAG ( ) attachment sites
Syndecan Glypican
Cell membrane
Figure 1.5
One of the main components of the
endothelial glycocalyx (left panel stained
with Alcian blue 8GX. Van den Berg) are
the proteoglycans. The proteoglycan core
proteins can be extensively modified by
addition of disaccharide repeat units, the
glycosaminoglycans, which can be sulfated
at specific sites in order to establish a
highly specific and diverse endothelial
surface layer, reaching far into the lumen.
Bar: 1 µm.
Chapter 1 General Introduction
25
There are five known glycosaminoglycan families, namely, heparan sulfate, chondroitin
sulfate, keratin sulfate, dermatan sulfate and hyaluronan. Of these families, three are
predominantly present in the vascular system, heparan sulfate, chondroitin sulfate and
hyaluronan. They differ in disaccharide component and degree of sulfation (Figure 1.6).
This posttranslational system is highly organized, with multiple enzymes linking the
disaccharides and a battery of sulfotransferases attaching a sulfate group where needed, each
sulfotransferase attaching a sulfate group at a specific site of the disaccharide. Whether a site
needs sulfation is dependent on the downstream disaccharide-sulfation sequence, making the
whole system highly organized and very controlled (41) (Figure 1.7). Hyaluronan differs
from other glycosaminoglycans for it is unsulfated and not attached to a core protein (52).
Figure 1.7
HS proteoglycans turn over both by shedding
from the cell surface and by endocytosis and
step-wise degradation inside lysosomes.
Reprinted with permission from Varki et al.
Figure 1.6
Structure of heparan sulfate and hyaluronan
dissacharide units. The glycosaminoglycan
building blocks of heparan sulfate and
hyaluronan both consist of repeated units of
glucuronic acid and N-acetyl glucosamine.
The main difference between heparan sulfate
and hyaluronan is the degree of sulfation.
While hyaluronan is unsulfated, heparan
sulfate can be sulfated at specific sites in the
structure, depicted by bold arrows.
nNHCOCH2
OOO
COOH
OHOH
CH2-OH
O O
nNH-SO3
-
OO
O
COOH
O-S O3-
OSO3-
CH2-O-SO3-
OO
OH
Hyaluronan (HA) Heparan sulfate (HS)
GlcA Glucosamine GlcA Glucosamine
OH
GlcA GlcNAc GlcA GlcNAc
nNHCOCH2
OOO
COOH
OHOH
CH2-OH
O O
nNH-SO3
-
OO
O
COOH
O-S O3-
OSO3-
CH2-O-SO3-
OO
OH
Hyaluronan (HA) Heparan sulfate (HS)
GlcA Glucosamine GlcA Glucosamine
OH
GlcA GlcNAc GlcA GlcNAc
Chapter 1 General Introduction
26
2.3.2.1 Interactions between plasma proteins and sulfated glycosaminoglycans of the
endothelial glycocalyx.
Glycosaminoglycans, such as heparan sulfate and chondroitin sulfate, consist of different
sulfation sequences that give the glycocalyx its diversity and specificity for the binding of
numerous factors (Table 1.1). These highly organized, dynamic properties of the endothelial
cell glycocalyx post-translational modifications contribute to the endothelial heterogeneity
supposedly present in the vascular system.
In contrast, hyaluronan lacks specific protein binding sites and could best be seen as an
important space filling gel-like component of the glycocalyx that plays an important role in
maintaining the endothelial permeability barrier (22), and possibly in the
mechanotransduction of fluid shear stress into activation of intracellular biochemical
pathways (59; 61).
2.3.2.2 Hyaluronan
Hyaluronan is a glycosaminoglycan, often overlooked for its unsulfated, ‘inert’ state, being
therefore a less desirable candidate for binding e.g. growth factors and coagulation factors
present in the blood. It is however a major constituent of the endothelial glycocalyx, crucial
for maintaining endothelial barrier properties for plasma macromolecules (22). Hyaluronan is
a glycosaminoglycan, which is not sulfated, not attached to a core protein and is synthesized
in the cell membrane by hyaluronan synthase (52; 53). Overall, post- translational
Table 1.1
Examples of proteins that bind to glycosaminoglycans (GAGs).
Reprinted with permission from Varki et al.
Chapter 1 General Introduction
27
modifications of proteins take place in the ER and Golgi apparatus, where the disaccharides
are sulfated and attached to core protein as is the case for glycosaminoglycans attached to
their specific core proteins, except for the synthesis of hyaluronan. Hyaluronan consists of
repeated disaccharide units of glucuronic acid and glucosamine, which are present in the
cell’s cytoplasm. Hyaluronan is produced directly in the cell membrane layer, where
activated precursor molecules, glucuronic acid and glucosamine, are linked by
transmembrane enzyme hyaluronan synthases and pushed to the outside of the cell. So far,
three distinct hyaluronan synthasis have been identified and differ in their enzymatic activity
and specificity of hyaluronan product size (54). The catabolism of hyaluronan involves the
cell surface hyaluronan receptor CD44, two hyaluronidases, Hyal-1 and Hyal-2, and two
lysosomal enzymes, beta-glucuronidase and beta-N-acetylglucosaminidase. This metabolic
cascade begins in lipid raft invaginations at the cell membrane surface. Degradation of the
high-molecular-weight extracellular hyaluronan occurs in a series of discrete steps generating
hyaluronan chains of decreasing sizes. The biological functions of the oligomers at each
quantum step differ widely, from the spacefilling, hydrating, anti-angiogenic,
immunosuppressive 10000-kDa extracellular polymer, to 20-kDa intermediate polymers that
are highly angiogenic, immuno-stimulatory, and inflammatory. This is followed by
degradation to small oligomers that can induce heat shock proteins and that are anti-
apoptotic. The single sugar products, glucuronic acid and a glucosamine derivative are
released from lysosomes to the cytoplasm, where they become available for other metabolic
cycles. There are 15 grams of hyaluronan in a 70-kg individual, of which 5 grams are cycled
daily through this pathway (53).
Figure 1.8
Regulation of hyaluronan amount and chain length by
expression of a specific HAS protein. Biochemical
characterizations of the vertebrate HAS enzymes
expressed in mammalian cell culture have revealed
similarities and differences between the respective
mammalian hyaluronan synthase enzymes. The
physiological significance of these differences in
enzymatic activity is not yet known.
Reprinted with permission from Spicer AP and
McDonald.
Chapter 1 General Introduction
28
Because of its direct cell membrane synthesis site, sudden environmental changes are
proposed to have immediate effect on hyaluronan metabolism (53) and make it a very
desirable candidate to study acute changes in endothelial glycocalyx structure and function
(Figure 1.8).
2.4 Hemodynamics
The role of the endothelial glycocalyx in mechanosensing has been shown by the results of
studies of Mochizuki (55), exposing isolated canine femoral arteries to hyaluronidase They
show that hyaluronic acid glycosaminoglycans within the glycocalyx play a pivotal role in
detecting and amplifying the shear force of flowing blood that triggers endothelium-derived
nitric oxide production. Florian et al (56) exposed endothelial cells to heparitinase, in order to
specifically remove heparan sulfate from the endothelial glycocalyx. They subsequently
exposed the endothelium to fluid shear stress after which endothelial nitric oxide (NO) was
measured in the cell culture system. Results show that the heparan sulfate component of the
endothelial cell glycocalyx participates in mechanosensing that mediates NO production in
response to shear stress. Though it is demonstrated that heparan sulfates are involved in flow-
mediated mechano-transduction, the possibility of the involvement of other components of
the glycocalyx to participate in this process is not excluded. Constituents of the glycocalyx
form an entangled matrix covering the endothelial surface, and it is highly likely that
mechanical properties of this surface layer are given not only by the scaffolding components,
such as heparan sulfates, but also by associated macromolecules including sialic acid-
containing glycoproteins, proteoglycans, and oligosaccharides that are degraded by e.g.
neuraminidase. Pohl (57) showed in experiments using small saline-perfused rabbit
mesentery arteries that increasing perfusate viscosity with dextran solutions induced shear
stress related increases in vascular dimension. After preincubation with neuraminidase (0.2
U/ml, 30 min), which removes part of the membrane glycocalyx, the flow-dependent dilation
was abolished, again showing a role for the endothelial glycocalyx in shear force sensing.
Chapter 1 General Introduction
29
3 Clinical relevance
3.1 Cardiovascular disease and Diabetes Mellitus; a global view
Most deaths in the world are attributable to non-communicable diseases (32 million per year)
and just over half of these (16.7 million) are due to cardiovascular diseases. The term
cardiovascular disease (CVD) represents a group of disorders of heart and blood vessels, such
as hypertension, coronary heart disease, cerebrovascular disease, peripheral vascular disease,
heart failure, rheumatic heart disease, congenital heart disease and cardiomyopathies. Heart
disease and stroke are first and second leading cause of death for male and female adults in
developed countries, and are most common viewed as life style diseases. The main CVD risk
factors are raised blood pressure, alcohol consumption, high plasma cholesterol, obesity, and
diabetes.
Diabetes is a chronic disease presented by pathogenic high plasma levels of glucose
(hyperglycemia), which is caused by a deficiency in insulin production (diabetes type 1) or,
more commonly, by an ineffectiveness of insulin’s action to control blood glucose levels
(diabetes type 2). Data shows that approximately 150 million people have diabetes mellitus
worldwide and that this number may well double by the year 2025, which will mostly occur
in developing countries due to population growth, aging, unhealthy diets, obesity and
sedentary lifestyles. The high incidence of cardiovascular disease worldwide and the
upcoming diabetic epidemic, of which 50% will result in mortal cardiovascular
complications, provokes the question what the relationship between these two pathological
schemes is (58).
Despite the prevelance of atherogenic vascular disease and the contribution of diabetic
pathologies, the mechanistic causes of pathogenic vascular damage is still unclear. Currently,
it is generally recognized that the earliest measurable pathogenic functional abnormality of
the vessel wall is endothelial dysfunction (ED), which is characterized by an imbalance
between relaxing and contracting factors, procoagulant and anticoagulant substances, and
between proinflammatory and inflammatory mediators. Although abundant experimental
evidence is now available that the protective properties of the endothelial cell glycocalyx are
Chapter 1 General Introduction
30
essential to maintain optimal endothelial function, studies to unravel the contribution of
glycocalyx perturbation in the development of vascular dysfunction are lacking.
3.2 Focal nature of atherosclerotic lesions and glycocalyx
Atherosclerosis is known to be a local vascular phenomenon that is confined to specific sites
in the vasculature that are exposed to complex and disturbed flow profiles. These sites
demonstrate endothelial dysfunction, as reflected by impaired local production of nitric
oxide, increased leakage of lipids into the vascular wall, recruitment of adhering platelets and
thrombus formation, leukocyte-endothelial adhesion and finally plaque formation.
The glycocalyx has been shown to serve as a mechanosensor of shear stress, mediating shear-
induced release of NO by endothelial cells (55; 56; 59). In fact, selective perturbation of the
glycocalyx leads to increased vascular permeability, attenuated NO availability, and
increased adhesion of leukocytes and platelets. Reconstitution of the glycocalyx results in the
restoration of its barrier and anti adhesive properties (22; 60). In view of the intricate relation
between glycocalyx integrity and vascular homeostasis in experimental models, it has been
postulated that glycocalyx derangement could contribute to increased focal atherogenic
vascular vulnerability in humans (61).
In a recent study, it was hypothesized that endothelial glycocalyx perturbation contributes to
increased vulnerability of the arterial wall exposed to atherogenic risk factors. Glycocalyx
and intima-to-media ratios (IMR) were studied at a low- and a high-risk region within the
murine carotid artery (common region) and internal carotid branch (sinus region) in control
C57BL/6J (C57BL6) and age-matched C57BL/6J/apoE*3-Leiden (apoE*3; on an atherogenic
diet) mice. Electron micrographs revealed significantly thinner glycocalyces [73 vs. 399
nm] and greater IMR [0.096 vs. 0.044] at the sinus region of C57BL6 mice than in the
common region. Thinner glycocalyces [100 vs. 399 nm] and greater IMR [0.071 vs. 0.044]
were also observed in the common region of age-matched apoE*3 mice on an atherogenic
diet for 6 wk vs. C57BL6 mice on a normal diet. Greater IMR were due to greater intima
layers, without significant changes in media layer dimension. In addition, atherogenic diet
resulted in increased endothelial cell thickness at the sinus region [0.85 vs. 0.53 µm] but not
at the common region [0.66 vs. 0.62 µm]. It was therefore concluded that both regional and
Chapter 1 General Introduction
31
diet-induced increases in atherogenic risk are associated with smaller glycocalyx dimensions
and greater IMR and that vascular sites with diminished glycocalyx are more vulnerable to
proinflammatory and atherosclerotic sequelae (62).
3.3 Glycocalyx in diabetes
The cardiovascular complications due to poor hyperglycemic control appear to be a systemic
phenomenon since they are at different sites of the vasculature, ranging from the heart and
eye to the foot (58). Hyperglycemia itself has been shown to induce a wide array of
downstream effects, which adversely affect the protective capacity of the vessel wall (63).
Hyperglycemia has been associated with enhanced endothelial permeability, increased
leukocyte-endothelium adhesion and impaired nitric oxide (NO) bioavailability (64-66),
which are all phenomena that are also associated with changes in glycocalyx composition.
Furthermore, because increased degradation of proteoglycans has previously been
demonstrated in hyperglycemic conditions (67; 68), we felt that the impact of hyperglycemia
on the glycocalyx merits special interest.
These findings let us to the first hypothesis of this thesis that exposure of
vascular endothelium to physiological levels of fluid shear stress is essential
for the synthesis of the endothelial glycocalyx, and that lack of shear stress
induced glycocalyx synthesis contributes to the increased vascular
vulnerability of endothelium at regions exposed to complex flow profiles.
This led us to formulate the second hypothesis of this thesis that
hyperglycemic perturbation of (shear stress induced) glycocalyx synthesis
contributes to increased vascular vulnerability and elevated atherogenic risk
in diabetes.
Chapter 1 General Introduction
32
References
1. Bennett HS. Morphological aspects of extracellular polysaccharides. Journal of histochemistry & cytochemistry 11: 14-23, 1963.
2. Aird WC. Endothelial cells in health and disease. Boca Raton, FLA: Taylor and Francis Group, 2005.
3. Fulton JF. Selected readings in the History of Physiology. Springfield Illinois U.S.A.: 1966.
4. Simionescu N. and Simionescu M. The cardiovascular system. In: Histology, Cell and Tissue Biology, edited by Weiss L. New York: Elsevier Biomedical, 1983, p. 355-398.
5. Chilian WM, Layne SM, Klausner EC, Eastham CL and Marcus ML. Redistribution of Coronary Microvascular Resistance Produced by Dipyridamole. American Journal of Physiology 256: H383-H390, 1989.
6. Burton AC. Physiology and biophysics of the circulation. Chicago: Medical Publishers Inc., 1972.
7. Wolinsky H. A proposal linking clearance of circulating lipoproteins to tissue metabolic activity as a basis for understanding atherogenesis. Circ Res 47: 301-311, 1983.
8. Leake C.D. The historical development of cardiovascular physiology. In: Handbook of Physiology. Section 2. Circulation. Vol. 1, edited by Renkin E.M. MCC. Washington DC: American Physiological Society, 1962, p. 11-22.
9. Fahraeus R. and Lindqvist T. The viscosity of the blood in narrow capillary tubes. Am J Physiol 562-568, 1931.
10. Pappenheimer J.R., Renkin E.M. and Borrerro J.M. Filtration, diffusion and molecular sieving through the peripheral capillary membranes: a contribution to the pore theory of capillary permeability. Am J Physiol 13-46, 1951.
11. Klitzman B and Duling BR. Microvascular hematocrit and red cell flow in resting and contracting striated muscle. Am J Physiol 237: H481-H490, 1979.
12. Pries AR and Secomb TW. Microvascular blood viscosity in vivo and the endothelial surface layer. American Journal of Physiology-Heart and Circulatory Physiology 289: H2657-H2664, 2005.
13. Vink H and Duling BR. Identification of distinct luminal domains for macromolecules, erythrocytes, and leukocytes within mammalian capillaries. Circulation Research 79: 581-589, 1996.
14. Constantinescu AA, Vink H and Spaan JA. Elevated capillary tube hematocrit reflects degradation of endothelial cell glycocalyx by oxidized LDL. Am J Physiol Heart Circ Physiol 280: H1051-H1057, 2001.
15. Starling E.H. On the absorption of fluids from the connective tissue spaces. J Physiol 312-326, 1896.
16. Landis E.M. The capillary pressure in frog mesentary as determined by micro-injection methods. Am J Physiol 548-570, 1926.
17. Adamson RH, Michel CC, Parker KH, Phillips CG and Wang W. Pathways Through the Intercellular Clefts of Frog Mesenteric Capillaries. Journal of Physiology-London 466: 303-327, 1993.
Chapter 1 General Introduction
33
18. Michel CC and Curry FE. Microvascular permeability. Physiological Reviews 79: 703-761, 1999.
19. Danielli JF. Capillary permeability and oedema in the perfused frog. The journal of physiology 98: 109-129, 1940.
20. Danielli JF and Stock A. The structure and permeability of blood capillaries. Biological reviews 19: 81-94, 1944.
21. Vink H and Duling BR. Capillary endothelial surface layer selectively reduces plasma solute distribution volume. American Journal of Physiology - Heart & Circulatory Physiology 278: H285-H289, 2000.
22. Henry CB and Duling BR. Permeation of the luminal capillary glycocalyx is determined by hyaluronan. American Journal of Physiology 277: H508-H514, 1999.
23. Henry CB and Duling BR. TNF-alpha increases entry of macromolecules into luminal endothelial cell glycocalyx. American Journal of Physiology - Heart & Circulatory Physiology 279: H2815-H2823, 2000.
24. Platts SH and Duling BR. Adenosine A3 Receptor Activation Modulates the Capillary Endothelial Glycocalyx. Circ Res 2003.
25. Huxley VH and Williams DA. Role of a glycocalyx on coronary arteriole permeability to proteins: evidence from enzyme treatments. American Journal of Physiology - Heart & Circulatory Physiology 278: H1177-H1185, 2000.
26. Olsson U, Ostergren-Lunden G and Moses J. Glycosaminoglycan-lipoprotein interaction. Glycoconj J 18: 789-797, 2001.
27. Jeansson M and Haraldsson B. Glomerular size and charge selectivity in the mouse after exposure to glucosaminoglycan-degrading enzymes. Journal of the American Society of Nephrology 14: 1756-1765, 2003.
28. Ciarimboli G, Hjalmarsson C, Bokenkamp A, Schurek HJ and Haraldsson B. Dynamic alterations of glomerular charge density in fixed rat kidneys suggest involvement of endothelial cell coat. American Journal of Physiology-Renal Physiology 285: F722-F730, 2003.
29. Hjalmarsson C, Johansson BR and Haraldsson B. Electron microscopic evaluation of the endothelial surface layer of glomerular capillaries. Microvasc Res 67: 9-17, 2004.
30. Perry MA and Granger DN. Role of Cd11/Cd18 in Shear Rate-Dependent Leukocyte-Endothelial Cell-Interactions in Cat Mesenteric Venules. Journal of Clinical Investigation 87: 1798-1804, 1991.
31. DeGrendele HC, Estess P, Picker LJ and Siegelman MH. CD44 and its ligand hyaluronate mediate rolling under physiologic flow: a novel lymphocyte-endothelial cell primary adhesion pathway. J Exp Med 183: 1119-1130, 1996.
32. Ross R. Atherosclerosis is an inflammatory disease. Am Heart J 138: S419-S420, 1999.
33. Silvestro L, Ruikun C, Sommer F, Duc TM, Biancone L, Montrucchio G and Camussi G. Platelet-Activating Factor-Induced Endothelial-Cell Expression of Adhesion Molecules and Modulation of Surface Glycocalyx, Evaluated by Electron-Spectroscopy Chemical-Analysis. Seminars in Thrombosis and Hemostasis 20: 214-222, 1994.
34. Sabri S, Soler M, Foa C, Pierres A, Benoliel A and Bongrand P. Glycocalyx modulation is a physiological means of regulating cell adhesion. Journal of Cell Science 113: 1589-1600, 2000.
Chapter 1 General Introduction
34
35. Soler M, Desplat-Jego S, Vacher B, Ponsonnet L, Fraterno M, Bongrand P, Martin JM and Foa C. Adhesion-related glycocalyx study: quantitative approach with imaging-spectrum in the energy filtering transmission electron microscope (EFTEM). FEBS Letters 429: 89-94, 1998.
36. Fibbi G, Vannucchi S, cavallini P, Del Rosso M, Pasquali F, Cappelletti R and Chiarugi V. Involvement of chondroitin sulfate in preventing adhesive cellular interactions. Biochim Biophys Acta 762: 512-518, 1983.
37. Norgardsumnicht K and Varki A. Endothelial Heparan-Sulfate Proteoglycans That Bind to L-Selectin Have Glucosamine Residues with Unsubstituted Amino-Groups. Journal of Biological Chemistry 270: 12012-12024, 1995.
38. Koenig A, Norgard-Sumnicht K, Linhardt R and Varki A. Differential interactions of heparin and heparan sulfate glycosaminoglycans with the selectins. Implications for the use of unfractionated and low molecular weight heparins as therapeutic agents. Journal of Clinical Investigation 101: 877-889, 1998.
39. Key NS, Platt JL and Vercellotti GM. Vascular Endothelial-Cell Proteoglycans Are Susceptible to Cleavage by Neutrophils. Arteriosclerosis and Thrombosis 12: 836-842, 1992.
40. Rosenberg RD and Aird WC. Vascular-bed--specific hemostasis and hypercoagulable states. N Engl J Med 340: 1555-1564, 1999.
41. Rosenberg RD, Shworak NW, Liu J, Schwartz JJ and Zhang L. Heparan sulfate proteoglycans of the cardiovascular system. Specific structures emerge but how is synthesis regulated?. [Review] [88 refs]. Journal of Clinical Investigation 100: S67-S75, 1997.
42. Marcum JA and Rosenberg RD. The biochemistry, cell biology and pathophysiology of anticoagulantly active heparin-like molecules of the vessel wall. In: Heparin, Chemical and Biological Properties: Clinical Applications., edited by D.A.Lane and U.Lindahl. London: Edward Arnold, 1989, p. 275-294.
43. Vink H, Constantinescu AA and Spaan JA. Oxidized lipoproteins degrade the endothelial surface layer : implications for platelet-endothelial cell adhesion. Circulation 101: 1500-1502, 2000.
44. Chambers R and Zweifach BW. Intercellular cement and capillary permeability. Physiol Rev 27: 436-463, 1947.
45. Copley AL. Hemorheological aspects of the endothelium-plasma interface. Microvasc Res 8: 192-212, 1974.
46. Copley AL. The endoendothelial fibrin lining, fibrinogen gel clotting, and the endothelium-blood interface. Ann N Y Acad Sci 416: 377-396, 1983.
47. Luft JH. Fine structure of capillary and endocapillary layer as revealed by ruthenium red. Microcirc Symp Fed Proc 25: 1773-1783, 1966.
48. Sims DE and Horne MM. Non-aqueous fixative preserves macromolecules on the endothelial cell surface: an in situ study. Eur J Morphol : 32: 59-64, 1993.
49. Sims DE, Westfall JA, Kiorpus AL and Horne MM. Preservation of tracheal mucus by nonaqueous fixative. Biotech Histochem 66: 173-180, 1991.
50. Pries AR, Secomb TW and Gaehtgens P. The endothelial surface layer. Pflugers Arch 440: 653-666, 2000.
51. van Den Berg BM, Vink H and Spaan JA. The endothelial glycocalyx protects against myocardial edema. Circ Res 92: 592-594, 2003.
Chapter 1 General Introduction
35
52. Hascall VC. Hyaluronan, a common thread. Glycoconj J 17: 607-616, 2000.
53. Stern R. Hyaluronan catabolism: a new metabolic pathway. European Journal of Cell Biology 83: 317-325, 2004.
54. Itano N, Sawai T, Yoshida M, Lenas P, Yamada Y, Imagawa M, Shinomura T, Hamaguchi M, Yoshida Y, Ohnuki Y, Miyauchi S, Spicer AP, McDonald JA and Kimata K. Three isoforms of mammalian hyaluronan synthases have distinct enzymatic properties. J Biol Chem 274: 25085-25092, 1999.
55. Mochizuki S, Vink H, Hiramatsu O, Kajita T, Shigeto F, Spaan JA and Kajiya F. Role of hyaluronic acid glycosaminoglycans in shear-induced endothelium-derived nitric oxide release. Am J Physiol Heart Circ Physiol 285: H722-H726, 2003.
56. Florian JA, Kosky JR, Ainslie K, Pang Z, Dull RO and Tarbell JM. Heparan Sulfate Proteoglycan Is a Mechanosensor on Endothelial Cells. Circ Res 2003.
57. Pohl U, Herlan K, Huang A and Bassenge E. Edrf-Mediated Shear-Induced Dilation Opposes Myogenic Vasoconstriction in Small Rabbit Arteries. American Journal of Physiology 261: H2016-H2023, 1991.
58. World Health Organization (WHO). World health report. 2003.
59. Weinbaum S, Zhang X, Han Y, Vink H and Cowin SC. Mechanotransduction and flow across the endothelial glycocalyx. Proc Natl Acad Sci U S A 100: 7988-7995, 2003.
60. Constantinescu AA, Vink H and Spaan JA. Endothelial Cell Glycocalyx Modulates Immobilization of Leukocytes at the Endothelial Surface. Arterioscler Thromb Vasc Biol 23: 1541-1547, 2003.
61. Thi MM, Tarbell JM, Weinbaum SS and Spray DD. The role of the glycocalyx in reorganization of the actin cytoskeleton under fluid shear stress: A "bumper-car" model. PNAS 101: 16483-16488, 2004.
62. van den Berg BM, Spaan JAE, Rolf TM and Vink H. Atherogenic region and diet diminish glycocalyx dimension and increase intima-to-media ratios at murine carotid artery bifurcation. American Journal of Physiology-Heart and Circulatory Physiology 290: H915-H920, 2006.
63. Nathan DM, Lachin J, Cleary P, Orchard T, Brillon DJ, Backlund JY, O'Leary DH and Genuth S. Intensive diabetes therapy and carotid intima-media thickness in type 1 diabetes mellitus. New England Journal of Medicine 348: 2294-2303, 2003.
64. Algenstaedt P, Schaefer C, Biermann T, Hamann A, Schwarzloh B, Greten H, Ruther W and Hansen-Algenstaedt N. Microvascular alterations in diabetic mice correlate with level of hyperglycemia. Diabetes 52: 542-549, 2003.
65. Morigi M, Angioletti S, Imberti B, Donadelli R, Micheletti G, Figliuzzi M, Remuzzi A, Zoja C and Remuzzi G. Leukocyte-endothelial interaction is augmented by high glucose concentrations and hyperglycemia in a NF-kB-dependent fashion. J Clin Invest 101: 1905-1915, 1998.
66. Du XL, Edelstein D, Dimmeler S, Ju QD, Sui C and Brownlee M. Hyperglycemia inhibits endothelial nitric oxide synthase activity by posttranslational modification at the Akt site. Journal of Clinical Investigation 108: 1341-1348, 2001.
67. Ceriello A, Giugliano D, Dello Russo P, Passariello N, Saccomanno F and Sgambato S. Glycosaminoglycans in human diabetes. Diabete Metab 9: 32-34, 1983.
68. Ikegami-Kawai M, Okuda R, Nemoto T, Inada N and Takahashi T. Enhanced activity of serum and urinary hyaluronidases in streptozotocin-induced diabetic Wistar and GK rats. Glycobiology 14: 65-72, 2004.
36
37
Outline of the Thesis
The hypotheses we want to test in this thesis as stated before are:
(I) The exposure of vascular endothelium to physiological levels of fluid shear stress is
essential for the synthesis of the endothelial glycocalyx, and that the lack of shear stress
induced glycocalyx synthesis contributes to the increased vascular vulnerability of
endothelium at regions exposed to complex flow profiles.
(II) Hyperglycemic perturbation of (shear stress induced) glycocalyx synthesis contributes to
increased vascular vulnerability and elevated atherogenic risk in diabetes.
Chapter 1 gives a general introduction of the endothelial glycocalyx and its role in the
vascular system.
Chapter 2 studies the effect of shear stress on glycocalyx production in cultured endothelial
cells. We show that shear stress increases the incorporation of hyaluronan component of the
endothelial glycocalyx in an in vitro cell culture system.
The effect of hyperglycemia on the hyaluronan production in endothelial cells under flow
conditions described in Chapter 3 shows an attenuated hyaluronan production by
hyperglycemia on endothelial cells exposed to shear stress.
The study in Chapter 4 where we looked at the effect of hyperglycemia on glycocalyx
synthesis shows an initial increase of glycocalyx production followed by a decreased
endothelial glycocalyx content after 6 hours of hyperglycemic exposure, in accordance with
the hyaluronan plasma content profile after 6 hrs of hyperglycemic clamping.
From the clinical study in Chapter 5 we learn that hyperglycemia causes a loss of glycocalyx
volume in healthy human subjects after 6 hours of hyperglycemic clamping.
Chapter 6 gives an overview of the effects of fluid shear stress on the vasculoprotective
properties of the endothelial glycocalyx and discusses the role of the endothelial glycocalyx
in various experimental models, cell culture, animal and human studies and its role in disease
states.
38
Chapter 2
Fluid shear stress stimulates incorporation of hyaluronan
into the endothelial cell glycocalyx
Mirella Gouverneur,1 Jos A. E. Spaan,1 Hans Pannekoek,2
Ruud D. Fontijn2 and Hans Vink1
Departments of 1Medical Physics and 2Medical Biochemistry, Academic Medical Center,
University of Amsterdam, Amsterdam, The Netherlands
Am J Physiol Heart Circ Physiol 2006 290(1):H458-62
Chapter 2 Fluid shear stress stimulates hyaluronan incorporation
40
Chapter 2 Fluid shear stress stimulates hyaluronan incorporation
41
Abstract
Vascular endothelial cells are shielded from direct exposure to flowing blood by the
endothelial glycocalyx, a highly hydrated mesh of glycoproteins, sulfated proteoglycans and
associated glycosaminoglycans (GAGs). Recent data indicate that incorporation of the
unsulfated GAG hyaluronan (HA) into the endothelial glycocalyx is essential to maintain its
permeability barrier properties and we hypothesized that fluid shear stress is an important
stimulus for endothelial HA synthesis. In order to evaluate the effect of shear stress on
glycocalyx synthesis and shedding of its GAGs into the supernatant, cultured human
umbilical vein endothelial cells (i.e. the stable cell line EC-RF24) were exposed to 10
dynes/cm2 non-pulsatile shear stress for 24 h and incorporation of [3H]glucosamine and
Na2[35S]O4 into GAGs was determined. Furthermore, the amount of HA in the glycocalyx
and in the supernatant was determined by ELISA. Shear stress did not affect the
incorporation of 35S, but significantly increased the amount of glucosamine-containing GAGs
incorporated in the endothelial glycocalyx (168 ± 17% of static levels, P < 0.01) and shedded
into the supernatant (231 ± 41% of static levels, P < 0.01). Correspondingly with this finding,
shear stress increased the amount of HA in the glycocalyx (from 26 ± 24 · 10-4 ng/cell to 46 ±
29 · 10-4 ng/cell, static vs. shear stress, P < 0.05) and in the supernatant (from 28 ± 11 · 10-4
ng/cell/h to 55 ± 16 · 10-4 ng/cell/h, static vs. shear stress, P < 0.05). The increase in the
amount of hyaluronan incorporated in the glycocalyx was confirmed by a 3-fold higher level
of hyaluronan binding protein within the glycocalyx of shear stress-stimulated endothelial
cells. In conclusion, fluid shear stress stimulates incorporation of hyaluronan in the
glycocalyx, which may contribute to its vasculo-protective effects against pro-inflammatory
and pro-atherosclerotic stimuli.
Chapter 2 Fluid shear stress stimulates hyaluronan incorporation
42
Chapter 2 Fluid shear stress stimulates hyaluronan incorporation
43
2.1 Introduction
The endothelial glycocalyx is a highly negatively charged, organized mesh on the endothelial
cell surface, consisting of membranous glycoproteins, proteoglycans, glycosaminoglycans
and associated plasma proteins, and is situated at the luminal side of blood vessels(20). This
endothelial layer functions as a protective barrier between endothelial cells and flowing blood
by contributing to the endothelial permeability barrier (19), binding anticoagulation factors
(13) modulating leukocyte interactions with the endothelium (3; 12), limiting myocardial
edema (16), and has become in focus for its role as a mechano-shear sensor (22). Recently,
we demonstrated that atherogenic stimuli, like oxidative stress (18) and oxidized low-density
lipoproteins (ox-LDL) (2; 3; 17), perturb the endothelial glycocalyx, resulting in increased
glycocalyx permeability and adhesiveness of platelets and leukocytes to the endothelial
membrane. Earlier studies, using sialic acid binding lectins (8) and alcian blue (9), showed
that reduced dimensions of the endothelial glycocalyx at arterial sites exposed to disturbed
flow patterns associate with increases in endothelial permeability and susceptibility to
atherosclerotic lesion formation. Additionally, studies by Woolf (23) and Wang et al. (21)
revealed thicker glycocalyces at high shear regions compared to low shear regions and
demonstrated that glycocalyx dimension is reduced when rabbits are fed an atherogenic diet.
Steady state glycocalyx dimension is the result of local synthesis and degradation of its
constituents and it is important to know the factors that determine this balance. We
hypothesized that fluid shear stress is an important stimulus for glycocalyx synthesis.
2.2 Materials and Methods
Chemicals
M199 media, L-glutamine, antibiotic-antimycotic and trypsin were obtained from Gibco-
BRL, PBS (pH 7.4) from Fresenius Kabi and Fetal Bovine Serum (FBS) from Biowhittaker.
The following chemicals were obtained from Sigma; heparin, endothelial cell growth
supplement (ECGS). The radiochemicals 6-[3H]glucosamine (specific activity: ~25-
40Ci/mmol) and carrier-free Na[35S]O4 (specific activity: ~43 Ci/mgS) were purchased from
ICN and Molecular Probes provided anti-heparan sulfate, conjugated anti-mouse IgM,
Chapter 2 Fluid shear stress stimulates hyaluronan incorporation
44
custom-made fluorescent (Alexa-555) hyaluronan binding protein (cat # 400762-1, Seikagaku
America) and nuclear stain Syto. The Hyaluronan Enzyme-linked Immunosorbent Assay kit
was obtained form Echelon biosciences incorporated, Salt Lake City, USA. Fibronectin was a
kind gift from Sanquin Research Foundation, Amsterdam, The Netherlands.
Cell culture
The human EC-RF24 cell line, representing human vascular umbilical endothelial cells that
have been immortalized with an amphotrophic replication-deficient retrovirus, containing
human papilloma virus 16 E6/E7 DNA (4). EC-RF24 cells have been shown to have retained
a diploid karyotype, display no abnormalities and are able to grow in a polar fashion when
compared to primary HUVEC cells after culture for up to one year. Furthermore, the presence
of von Willebrand factor, endoglin, PCAM-1 was established as well as expression of surface
adhesion molecules E-selectin, VCAM-1 and ICAM-1, and the results were comparable to
those obtained from primary endothelial cells. The cells were grown on 10 µg/ml fibronectin-
coated cell culture flasks in M199 media, supplemented with 20 % (v/v) heat-inactivated fetal
bovine serum, 50 µg/ml heparin and 12.5 µg/ml endothelial-cell growth supplement, 0.2
mmol/l L-glutamine and 100 U/ml penicillin-G, 100 U/ml streptomycin sulfate, 25µg/ml
amphotericin-B at 37oC in 5% CO2.
Parallel flow chamber
The parallel flow perfusion chamber used is described in detail by Sakariassen et al. (14). In
short, the chamber consists of two 1 cm thick polymethyl methacrylate rectangulars. The top
part has a 1 cm wide depression of 0.6 mm, which represents the chamber width and height,
when the parts are joined together. Two corks can be placed at the bottom part of the
chamber, which each fit the endothelial cell containing thermanox cover slips, matching the
flow path of the top part. Watson-Marlow pump model 323S/D was used to obtain flow and
in order to eliminate the pulse generated by the pump, two windkessels and resistance tubing
were placed in the flow set-up, the windkessels were placed before and after the flow
chamber and resistance tubing between first windkessel and flow chamber. The final flow
exposed to cells was 49.9 ± 2.73 ml/min, which translates into a shear stress of 9.7± 0.5
dynes/cm2. The parallel flow perfusion chamber was generously provided by dr. Ph.G. de
Groot, Department of Hematology, University Medical Center, University of Utrecht, The
Netherlands.
Chapter 2 Fluid shear stress stimulates hyaluronan incorporation
45
Radioactive incorporation studies
The cells were seeded on fibronectin-coated thermanox cover slips and attached to
confluency for 2 h in M199 media without antibiotics (approximately 2 x 105 cells per cover
slip). The cells were placed in parallel flow chamber and non-pulsatile flow at shear stress of
9.7 ± 0.5 dynes/cm2 was applied for 24 h in complete media M199 without antibiotics,
supplemented with 40 µCi/ml 6-[3H] glucosamine and 50 µCi/ml Na2[35S]O4. After 24 hours
of exposure either under static condition or flow condition, the cells were placed under static
condition and after 1 hour the supernatant was collected. After 1 hour, the cells were treated
for 30 min at room temperature with 0.05% (w/v) trypsin and for an additional 10 min at
37oC in order to separate membranous glycocalyx cell fraction from the cellular fraction.
Validation of trypsin fraction purity.
To test whether elevated levels of glucosamine to sulfate ratio in the glycocalyx were due to
specific incorporation of substrate into glycosaminoglycans (GAGs), four additional
experiments were performed from which the trypsin-derived glycocalyx fraction was further
purified by DEAE chromatography.(15) Briefly, the cells were incubated with for 30min at
room temperature 0.05% (w/v) trypsin and for an additional 10 min at 37o C and then
centrifuged at 1500 g for 5 min to separate cells from the trypsinated glycocalyx proteoglycan
fraction. The columns were prepared as described previously (15). The glycocalyx fraction
was applied to the column and the column was washed to remove non-proteoglycan proteins.
Column-retained proteoglycans were eluted with high salt (1M NaCl) and counted in a
scintillation counter. Additionally, the trypsin fraction of GAG-deficient cells (CHO-
pgsA745) (6) were also purified by column chromatography and compared with glucosamine
and sulfate incorporation of wild-type GAG-containing CHO cells (CHO-K1).
Fluorescent Imaging
Cells were cultured as stated above and exposed for 24 h to non-pulsatile flow. Afterwards,
cells were rinsed with 0.1% BSA/HEPES and subsequently incubated for 30min at RT with
10µg/ml anti-Heparan Sulfate antibody, 30min 10µg/ml Alexa fluor 488 conjugated anti-
mouse IgM, 30min 50µg/ml hyaluronan binding protein (HBP) labeled with Alexa fluor 555.
The thermanox coverslips were placed between microscopic object glass and glass coverslip,
immersed in 2x10-3 mmol/l nucleus stain 44 blue fluorescent nucleic acid (Syto). The cells
Chapter 2 Fluid shear stress stimulates hyaluronan incorporation
46
were imaged using a fluorescence microscope (Olympus BI51, objective: UplanApo 60x/1.2
WI) and 16 bit image stacks of 50 x 50 x 20 µm (XYZ) with pixel sizes of 144 x 144 x 200
nm were recorded using a cascade 650 digital camera (photometrics). The recorded image
stacks were deconvolved using Huygens2 software (Huygens Professional version 2.4.1,
Scientific Volume Imaging B.V., Hilversum, The Netherlands) and the intensity per plane
was calculated, from which cytoplasmic and glycocalyx intensities were determined.
Hyaluronan content determination
Hyaluronan mass was determined using enzyme linked immunosorbent assay kit,
commercially available from Echelon biosciences incorporated. The principle is based on
competitive ELISA assay in which the colorimetric signal is inversely proportional to the
amount of hyaluronan present in the sample.
Statistical analysis
For statistical analysis, two-way paired ([3H]glucosamine and Na2[35S]O4 incorporation data)
or unpaired (HABP intensity data), (Hyaluronan ELISA data) t-tests were used when
appropriate. A value of P < 0.05 was considered statistically significant. Values are means ±
SD.
Chapter 2 Fluid shear stress stimulates hyaluronan incorporation
47
2.3 Results
Effect of flow on [3H] glucosamine and Na2[35S]O4 incorporation.
EC-RF24 cells were seeded on fibronectin-coated cover slips and grown to confluency (105
cells/cover slip). After replacing the supernatant with medium without radiolabeled tracers,
radiolabeled glucosamine and sulfate-containing GAGs shedded into the supernatant were
measured after 1 h. Subsequently, the cells were counted and all radioactive counts per
minute (cpm) were normalized to cell number. Exposure to shear stress reduced the number
of cells per cover slip by a relatively large amount (from 0.7 ± 0.3 x 105 to 0.4 ± 0.3 x 105
cells/cover slip, static vs. flow respectively, N=7, P < 0.05), but shear stress did not affect
sulfate incorporation (sulfate cpm/cell of flow cells = 104 ± 60 % of static cells, N=7, NS). In
contrast, exposure of the cells to shear stress tended to increase total glucosamine
incorporation (glucosamine cpm/cell in flow cells = 181 ± 161% of static cells, N=7, NS).
Absolute counts of glucosamine and sulfate incorporation per 104 cells in the various
compartments are depicted in table 2.1.
Table 2.1. Absolute counts of [3H]glucosamine and [35S]sulfate incorporation per 104 cells in various
compartments.
Chapter 2 Fluid shear stress stimulates hyaluronan incorporation
48
To eliminate the confounding inter-experimental variation of substrate label intensity,
glucosamine over sulfate (G/S) ratios were determined for each individual experiment,
revealing a highly significantly increased level of glucosamine over sulfate incorporation
(160 ± 42% of static cells, N=7, P<0.01). This increase in G/S ratio was due to increased
incorporation of glucosamine containing glycosaminoglycans in the glycocalyx of flow cells
(N = 4, 168 ± 17% of static cells, P<0.01, Fig. 2.1 center), but no changes were detected in
the intracellular pool of incorporated substrate (N = 4, 117 ± 23% of static cells, Fig. 2.1 left).
Furthermore, the increased levels of glucosamine substrate in the glycocalyx were also
reflected by elevated levels of glucosamine containing glycosaminoglycans shedded into the
supernatant (N = 4, 231 ± 41% of static cells, P<0.01, Fig.2.1 right).
Figure 1. Effect of flow on cellular glucosamine / sulfate distribution.
Flow stimulates the cellular incorporation of glucosamine relative to sulfate substrate (G/S ratio), as reflected
by increases in the supernatant G/S ratio (P < 0.01, left panel) and in glycocalyx G/S ratios (P < 0.01, middle
panel) but no changes occurred in the intracellular G/S ratios (NS, right panel).
Chapter 2 Fluid shear stress stimulates hyaluronan incorporation
49
Specificity of substrate incorporation in glycocalyx glycosaminoglycans
To test whether the glucosamine and sulfate moieties, present in the glycocalyx, were
specifically incorporated into GAGs, glycocalyx fractions of shear stress-exposed and static
cells were passed through DEAE columns, and retained glycocalyx proteoglycan-rich
fractions were analyzed for relative glucosamine and sulfate content. The shear stress-
induced increase in glucosamine over sulfate ratio was not affected by DEAE
chromatography, being 168 ± 17% (N = 4, P<0.01) and 149±13% (N=4, P<0.01) before and
after this purification procedure chromatography, respectively.
The specificity of the column purification method for GAG-incorporated substrate was
confirmed by comparing glucosamine and sulfate incorporation levels in the glycocalyx
fractions obtained from GAG-containing CHO cells (CHO-K1), and GAG-deficient CHO-
cells (CHO-pgsA745). DEAE chromatography lowered CHO-pgsA745 substrate
incorporation levels to 6 ± 1% and 2 ± 1% of wild type controls, indicating that less than 10%
of glycocalyx incorporated substrate levels can be accounted for by non-specific cell surface
binding of substrate (data not shown).
Hyaluronan binding protein (HABP) and anti heparan sulfate (anti-HS) binding to static and
flow exposed ECRF24 cells
Shear stress did not significantly affect intracellular uptake of hyaluronan binding protein
(HAPB) compared to static cells (65 ± 65 vs. 53 ± 25 a.u. per µm2 endothelial surface area, in
flow (N=6) and static (N=4) cells, respectively, NS). In contrast, a significant 3-fold increase
was found in glycocalyx HABP levels in flow exposed cells compared to static cells (104 ±
6% vs. 32 ± 8% of cytoplasmic HABP intensity levels, P<0.0001, figure 2.2, left panel). The
anti-heparan sulfate binding showed no differences in glycocalyx heparan sulfate levels of
flow exposed cells compared to static cells (70 ± 30% vs. 110 ± 18% of cytoplasmic anti-
HABP intensity levels, NS, fig 2.2, right panel)
Chapter 2 Fluid shear stress stimulates hyaluronan incorporation
50
Effect of flow on hyaluronan content of glycocalyx and supernatant
The hyaluronan content of the glycocalyx of cells cultured under static condition and flow-
exposed cells was determined with hyaluronan ELISA kit. The glycocalyx of flow exposed
cells contained more hyaluronan, 46.3 ±28.7ng/cell*104 than the glycocalyx of static cultured
cells, 26.3 ±24.1 ng/cell*104 (P<0.05) (N=6, Figure 2.3, left panel). The hyaluronan content
in the supernatant collected under static condition 1 hour after 24 hours static condition or
flow exposure was 27.8±10.7 vs. 55.3±15.7 ng/cell*104 (P<0.05). (N=7, Figure 2.3, right
panel).
Figure 2.2. Glycocalyx HABP and anti-HS staining on flow and static cells.
Shear stress induced a 3-fold increase in glycocalyx HABP label intensity (P < 0.0001, left panel) compared to
glycocalyx of static cultured cells. The anti-heparan sulfate label binding showed no differences in glycocalyx
heparan sulfate levels of flow-exposed cells compared to static cells (NS, right panel).
Chapter 2 Fluid shear stress stimulates hyaluronan incorporation
51
Time-dependent effect of endothelial cell flow exposure on hyaluronan in culture media
Measuring the hyaluronan content in the media of shear stress stimulated endothelial cells
during different times of flow exposure revealed that the hyaluronan content in the media was
already significantly increased after 1 hour of flow stimulus, static versus flow exposed cells
23.5±10.9 vs. 58.6±36.5ng/cell*104 (P<0.05) (N=6, Figure 2.4, left bar). The amount of
hyaluronan produced by shear stress stimulated cells increased further to
83.3±74.7ng/cell*104 and 540.7±327.7 after 6 and 24 hours of shear stress stimulation (N=6,
P < 0.05, Figure 2.4 middle and right bars). No significant changes in the amount of
hyaluronan produced by static cells was observed over time.
Figure 2.3: Effect of flow on hyaluronan content of glycocalyx and supernatant.
Flow exposed cells contained more hyaluronan in glycocalyx compared to glycocalyx of cells cultured under
static condition (left panel). Similar differences were observed for the hyaluronan content in the supernatant
collected 1 hour after 24 hours static versus flow exposure (P<0.05) (right panel).
Chapter 2 Fluid shear stress stimulates hyaluronan incorporation
52
Figure 2.4 Time-dependent effect of endothelial cell flow exposure on hyaluronan content in cell culture media.
Measuring the hyaluronan content of endothelial cells in the collected during different times of flow exposure the
hyaluronan content in the media was already significantly increased after 1 hour of flow stimulus (P<0.05) (left panel).
After the cells were exposed for 24 hours of flow the hyaluronan content in the 24 hour collected media was about 20
times higher in flow exposed cells compared to 24 hours static cultured cells (P<0.05) (right panel).
Chapter 2 Fluid shear stress stimulates hyaluronan incorporation
53
2.4 Discussion
The present study demonstrates that exposure of cultured endothelial cells for 24 h to a shear
stress of 10 dynes/cm2 stimulates incorporation of glucosamine-containing GAGs in the
glycocalyx, which is accompanied by elevated levels of glucosamine-containing GAGs in the
supernatant. These increases were confirmed by direct demonstration of increased hyaluronan
concentrations in the glycocalyx and in the supernatant, as well as by a 3-fold increase in the
incorporation of hyaluronan binding protein in the glycocalyx. The fact that shear stress did
not affect net sulfate levels in the glycocalyx does not rule out an effect of shear stress on
incorporation of sulfated GAGs in the glycocalyx. In addition to its incorporation in
hyaluronan, glucosamine is also incorporated in sulfated sugars like heparan sulfate and
chondroitin sulfate. The lack of changes in sulfate incorporation could be due to altered
activities of sulfotransferase enzymes or modification of sulfated GAG chain length.
Therefore, more detailed studies are required to determine whether shear stress affects also
sulfated GAGs in addition to the clear increases in hyaluronan incorporated in the glycocalyx.
Previous studies by others have focused predominantly on the effects of shear stress on
endothelial metabolism of sulfated glycosaminoglycans. Arisaka et al.(1) used pig aortic
endothelial cells exposed to shear stress levels of 15 and 40 dynes/cm2 in a parallel flow
chamber for periods of 3, 6, 12 and 24 h. The cells were incubated for 12 h with Na[35S]O4
after shear-stress exposure. These authors demonstrated increased synthesis of sulfated GAGs
after high shear stress of 40 dynes/cm2, and also a small, but significant increase at 15
dynes/cm2. Our lack of shear stress induced increases in incorporation of sulfate at a
relatively low shear stress level of 10 dynes/cm2 is therefore consistent with their conclusion
that moderate to high shear stress levels are required to stimulated synthesis of sulfated
GAGs. Elhadj et al (5) exposed bovine aortic endothelial cells for 7 days to < 0.5 dynes/cm2
prior to increasing shear rates for 3 days to 5 and 23 dynes/cm2. Na[35S]O4 was added to the
sheared cells during the final 24 h. The focus of that study was on soluble released material,
which was purified by Sepharose CL-6B and DEAE chromatography. No significant
increase in the net sulfated GAG synthesis was detected, but a shift in its size distribution was
reported, indicating that modulation of specific sulfation patterns may occur despite limited
effects of low shear stress levels on sulfated GAG synthesis. Florian et al (7) exposed bovine
Chapter 2 Fluid shear stress stimulates hyaluronan incorporation
54
aortic endothelial cells to a short term (3 h) oscillatory shear stress of 10 ± 15 dynes/cm2 as
well as steady shear stress of 10 dynes/cm2. Shear stress-induced endothelial NO production
was found to be abolished when cells were treated with heparitinase, indicating that
endothelial sulfated GAGs may contribute to the mechano-transduction of shear forces from
the extracellular to the intracellular compartment.
In summary, these experiments demonstrate 1) that sulfated GAGs in the endothelial
glycocalyx may function as a mechano-transducers as reported before for hyaluronic acid
(11), 2) that shear stress exposure alters the size distribution of endothelial sulfated GAGs,
and 3) that high levels of shear stress may also increase sulfated GAG synthesis. The current
study, however, demonstrates for the first time that shear stress also increases hyaluronan
content in the endothelial glycocalyx. However, it must be noted that loss of a significant
number of cells in the shear stress experiments may have selected for an endothelial
phenotype that is most responsive to shear stress in terms of extracellular hyaluronan
incorporation and attachment. The current experiments where performed on an immortalized
HUVEC cell-line exposed to a laminar, non-pulsatile flow profile. Future studies are required
using different flow profiles on arterial as well as microvascular types of endothelium to test
whether shear stress stimulation of endothelial hyaluronan incorporation is a general
response, or possibly limited to specific conditions and certain vascular sites.
Functions of hyaluronan in the endothelial glycocalyx
The contribution of hyaluronan to the endothelial glycocalyx and its functional implications
are only recently recognized. Mochizuki et al. demonstrated that hyaluronidase treatment of
the endothelial glycocalyx in isolated canine femoral arteries (11) attenuates flow-induced
production of nitric oxide to 19 ± 9% of control. This reduced ability of the vascular
endothelium to transduce fluid shear stresses into biochemical synthesis of nitric oxide did
not affect agonist-stimulated endothelial nitric oxide production, indicating that hyaluronan
GAGs play a specific role in the mechano-stimulation of the endothelium. Electron
microscopic observation of discrete glycocalyx structures in rat myocardial capillaries
confirmed that hyaluronidase treatment eliminates most of these structures from the luminal
endothelial surface (16). Loss of glycocalyx hyaluronan subsequently resulted in significant
swelling of the pericapillary space and anatomic compression of myocardial capillaries,
indicating that removal of hyaluronan GAGs from the endothelial glycocalyx increases
transcapillary fluid loss and probably leakage of plasma macromolecules. This concept is
Chapter 2 Fluid shear stress stimulates hyaluronan incorporation
55
supported by previous in vivo studies (10) revealing that elevating plasma levels of
hyaluronidase increases the permeation of dextrans of MW ≤ 150 kDa into the endothelial
glycocalyx of hamster cremaster muscle capillaries. In the latter study, it was elegantly
demonstrated that glycocalyx permeability barrier properties could be restored by
reconstitution of the hyaluronidase-degraded hyaluronan GAGs by a mixture of chondroitin
sulfate and hyaluronan, demonstrating that association of hyaluronan with endothelial
proteoglycans is essential to exert its permeability barrier properties.
In line with these recent reports on the contribution of hyaluronan glycosaminoglycans to
glycocalyx vasculo-protective properties, our current finding that shear stress is an important
stimulus for incorporation of hyaluronan into the endothelial glycocalyx might increase our
understanding of the relation between spatial shear stress distributions and the localization of
vulnerable vascular sites.
In conclusion, we demonstrate in the current study for the first time that fluid shear stress
stimulates incorporation of hyaluronan in the endothelial glycocalyx. Enhanced incorporation
of hyaluronan in the endothelial glycocalyx might contribute to optimal endothelial function
and protect against pathogenic vascular perturbation, and warrants future studies on the
relation between hyaluronan synthesis and endothelial function at lesion prone vascular sites
exposed to complex flow patterns.
Chapter 2 Fluid shear stress stimulates hyaluronan incorporation
56
References
1. Arisaka T, Mitsumata M, Kawasumi M, Tohjima T, Hirose S and Yoshida Y. Effects of shear stress on glycosaminoglycan synthesis in vascular endothelial cells. Ann N Y Acad Sci 748: 543-554, 1995.
2. Constantinescu AA, Vink H and Spaan JA. Elevated capillary tube hematocrit reflects degradation of endothelial cell glycocalyx by oxidized LDL. Am J Physiol Heart Circ Physiol 280: H1051-H1057, 2001.
3. Constantinescu AA, Vink H and Spaan JA. Endothelial Cell Glycocalyx Modulates Immobilization of Leukocytes at the Endothelial Surface. Arterioscler Thromb Vasc Biol 23: 1541-1547, 2003.
4. Dekker RJ, van SS, Fontijn RD, Salamanca S, de GP, VanBavel E, Pannekoek H and Horrevoets AJ. Prolonged fluid shear stress induces a distinct set of endothelial cell genes, most specifically lung Kruppel-like factor (KLF2). Blood 100: 1689-1698, 2002.
5. Elhadj S, Mousa SA and Forsten-Williams K. Chronic pulsatile shear stress impacts synthesis of proteoglycans by endothelial cells: Effect on platelet aggregation and coagulation. J Cell Biochem 86: 239-250, 2002.
6. Esko JD, Stewart TE and Taylor WH. Animal cell mutants defective in glycosaminoglycan biosynthesis. Proceedings of the National Academy of Sciences of the United States of America 82: 3197-3201, 1985.
7. Florian JA, Kosky JR, Ainslie K, Pang Z, Dull RO and Tarbell JM. Heparan Sulfate Proteoglycan Is a Mechanosensor on Endothelial Cells. Circ Res 2003.
8. Gorog P and Born GV. Uneven distribution of sialic acids on the luminal surface of aortic endothelium. Br J Exp Pathol 64: 418-424, 1983.
9. Haldenby KA, Chappell DC, Winlove CP, Parker KH and Firth JA. Focal and regional variations in the composition of the glycocalyx of large vessel endothelium. J Vasc Res 31: 2-9, 1994.
10. Henry CB and Duling BR. Permeation of the luminal capillary glycocalyx is determined by hyaluronan. American Journal of Physiology 277: H508-H514, 1999.
11. Mochizuki S, Vink H, Hiramatsu O, Kajita T, Shigeto F, Spaan JA and Kajiya F. Role of hyaluronic acid glycosaminoglycans in shear-induced endothelium-derived nitric oxide release. Am J Physiol Heart Circ Physiol 285: H722-H726, 2003.
12. Mulivor AW and Lipowsky HH. Role of glycocalyx in leukocyte-endothelial cell adhesion. Am J Physiol Heart Circ Physiol 283: H1282-H1291, 2002.
13. Rosenberg RD. Redesigning heparin. N Engl J Med 344: 673-675, 2001.
Chapter 2 Fluid shear stress stimulates hyaluronan incorporation
57
14. Sakariassen KS, Aarts PA, de GP, Houdijk WP and Sixma JJ. A perfusion chamber developed to investigate platelet interaction in flowing blood with human vessel wall cells, their extracellular matrix, and purified components. J Lab Clin Med 102: 522-535, 1983.
15. Shworak NW. High-specific-activity 35S-labeled heparan sulfate prepared from cultured cells. Methods Mol Biol 171: 79-89, 1901.
16. van den Berg BM, Vink H and Spaan JA. The endothelial glycocalyx protects against myocardial edema. Circ Res 92: 592-594, 2003.
17. Vink H, Constantinescu AA and Spaan JA. Oxidized lipoproteins degrade the endothelial surface layer : implications for platelet-endothelial cell adhesion. Circulation 101: 1500-1502, 2000.
18. Vink H and Duling BR. Identification of distinct luminal domains for macromolecules, erythrocytes, and leukocytes within mammalian capillaries. Circulation Research 79: 581-589, 1996.
19. Vink H, Duling BR. Capillary endothelial surface layer selectively reduces plasma solute distribution volume. American Journal of Physiology - Heart & Circulatory Physiology 278: H285-H289, 2000.
20. Vink H, Wieringa PA and Spaan JA. Evidence that cell surface charge reduction modifes capillary red cell velocity-flux relationships in hamster cremaster muscle. Journal of Physiology 489: 193-201, 1995.
21. Wang S, Okano M, and Yoshida. Ultrastructure of endothelial cells and lipid deposition on the flow dividers of branchiocephalic and left subclavian arterial bifurcations of the rabbit aorta. J.Jpn.Atheroscler.Soc. 19, 1089-1100. 1991.
22. Weinbaum S, Zhang X, Han Y, Vink H and Cowin SC. Mechanotransduction and flow across the endothelial glycocalyx. Proc Natl Acad Sci U S A 100: 7988-7995, 2003.
23. Woolf N. The arterial endothelium. In: Pathology of Atherosclerosis, edited by Crawford ST. Butterworths & Co Ltd. London, England., 1982, p. 25-45.
58
Chapter 3
Hyperglycemia attenuates flow induced hyaluronan
production by cultured EC-RF24 endothelial cells
Mirella Gouverneur, Jos A. E. Spaan and Hans Vink
Department of Medical Physics, Academic Medical Center, University of Amsterdam,
Amsterdam, The Netherlands
Submitted for publication
Chapter 3 Hyperglycemia attenuates flow induced hyaluronan production
60
Chapter 3 Hyperglycemia attenuates flow induced hyaluronan production
61
Abstract The endothelial glycocalyx protects the vascular wall against atherogenic stimuli, and it is suggested that hyperglycemic perturbations of the endothelial glycocalyx contribute to increased vascular vulnerability of diabetic patients. We recently demonstrated that fluid shear stress is an important stimulus of endothelial hyaluronan production, an essential component of the endothelial glycocalyx. In the present study we tested the hypothesis that hyperglycemia impairs shear stress induced endothelial hyaluronan synthesis. Endothelial cells (EC-RF24) were incubated for 18 hours with or without hyperglycemic media (5 or 25mM glucose). Subsequently, the cells were exposed to fluid shear stresses of 1, 5 and 10 dynes/cm2 under normo- or hyperglycemic conditions. Samples were taken from the media solutions at t = 2 hours to determine the initial amount of hyaluronan released from the endothelium upon initiation of fluid shear stress. Measurements of hyaluronan levels at later time points up to 9 hours during exposure to fluid shear stress were performed to determine the amount of shear stress induced endothelial hyaluronan release. Hyaluronan levels were determined using a commercial hyaluronan ELISA kit. Initiation of fluid shear stress resulted in a rapid, initial endothelial release of hyaluronan, independent of the level of fluid shear stress. Initial release of hyaluronan from normoglycemic cells averaged 2.7 ± 0.9 ng / 10000 cells (pooled data). Exposure of endothelial cells to hyperglycemic conditions for 18 hours increased the initial shear stress induced hyaluronan release to 6.3 ± 1.5 ng / 10000 cells (P < 0.05). Continued exposure of normoglycemic endothelial cells to fluid shear stress of 10 dynes/cm2 induced endothelial hyaluronan release at a rate of 18.3 ± 1.4 ng / 10000 cells/6h, which is significantly greater than hyaluronan release rates of 5.5 ± 5.1 and 0.2 ± 2.3 ng / 10000 cells/6h at fluid shear stress levels of 5 and 1 dynes/cm2, respectively. Hyperglycemia significantly attenuated endothelial hyaluronan release rate at 10 dynes/cm2 to 7.6 ± 6.7 ng / 10000 cells/6h. Exposure of cultured endothelial cells to hyperglycemic conditions for 18 hours doubles the initial shedding of hyaluronan from the endothelial surface upon the start of fluid shear stress exposure. Hyperglycemic hyaluronan shedding is accompanied by impaired shear stress induced endothelial hyaluronan synthesis at 10 dynes/cm2, which is consistent with the hypothesis that hyperglycemic loss of hyaluronan from the endothelial glycocalyx impairs flow dependent endothelial hyaluronan synthesis by impaired mechano-transduction of fluid shear stress.
Chapter 3 Hyperglycemia attenuates flow induced hyaluronan production
62
Chapter 3 Hyperglycemia attenuates flow induced hyaluronan production
63
3.1 Introduction
Patients with diabetes mellitus are characterized by increased vascular vulnerability, leading
to accelerated macrovascular disease such as atherogenesis as well as microvascular
complications (10). Hyperglycemia and poor hyperglycemic control are considered as
important contributors to higher cardiovascular complication incidence in the diabetic state
(1). The in vivo endothelial glycocalyx is a highly negatively charged protective barrier
between endothelial cells and flowing blood consisting of glycoproteins, proteoglycans and
associated glycosaminoglycans and plasma proteins (27). Hyaluronan glycosaminoglycans,
one of the major constituents of the glycocalyx, are of crucial importance for maintaining
endothelial barrier properties and mechanotransduction of fluid shear stress (11).
Furthermore, it has recently been shown that fluid shear stress stimulates incorporation of
hyaluronan into the endothelial cell glycocalyx (8). The dimension of the endothelial
glycocalyx exceeds those of endothelial adhesive molecules (25), which may explain its
potent anti-adhesive effects towards both leukocytes and platelets (3; 4; 18; 20; 21).
Perturbations of the endothelial glycocalyx, such as hyperglycemia (26) are coming more into
focus as playing a major role in the initiation of vascular complications (15). The hypothesis
of this study therefore is whether hyperglycemia impairs shear stress induced hyaluronan
synthesis.
3.2 Materials and Methods
Chemicals
M199 media, L-glutamine, antibiotic-antimycotic and trypsin were obtained from Gibco-
BRL, PBS pH: 7.4 from Fresenius Kabi and Fetal Bovine Serum (FBS) from Biowhittaker.
The following chemicals were obtained from Sigma; heparin, endothelial cell growth
supplement (ECGS). D(+)glucose was obtained from Merck. The Hyaluronan Enzyme-linked
Immunosorbent Assay kit was obtained form Echelon biosciences incorporated, Salt Lake
City, USA. Fibronectin was a kind gift from Central Laboratory for Blood transfusion (CLB),
Amsterdam, The Netherlands.
Chapter 3 Hyperglycemia attenuates flow induced hyaluronan production
64
Cell culture
EC-RF24 cell line, human umbilical vein endothelial cells immortalized with an
amphotrophic replication-deficient retrovirus containing human papilloma virus 16 E6/E7
DNA (6). The cells were grown on 10µg/ml fibronectin-coated cell culture flasks in M199
media supplemented with 20% heat-inactivated fetal bovine serum, 50µg/ml heparin and
12.5µg/ml endothelial cell growth supplement, 0.2mmol/l L-glutamine and 100U/ml
Penicillin-G, 100U/ml Streptomycin sulfate, 25µµg/ml Amphotericin-B at 37oC in 5% CO2.
Parallel flow chamber
The parallel flow perfusion chamber system used is described in detail by Sakariassen KS et
al (19). The parallel flow perfusion chamber was generously provided by Prof. P.G. de Groot,
Dept. Hematology, University Medical Center, The Netherlands(9).
Short-term shear stress exposure under normo (5mM) and hyperglycemic (25mM) conditions
The cells were seeded on fibronectin coated thermanox cover slips and attached to
confluency for 18 hours in complete M199 media (approximately 2x105cells/thermanox)
under normo (5mM) and hyperglycemic (25mM) conditions, subsequently, the cells were
placed in parallel flow chamber and exposed to steady laminar flow at shear stresses of 1, 5
and 10dynes/cm2. At times 0, 2, 4, 6 and 9 hours an aliquot of 250µl (of total 20ml) was
taken from the media and stored at 4oC.
Hyaluronan content
Hyaluronan mass was determined using enzyme linked immunosorbent assay kit,
commercially available from Echelon biosciences incorporated. The principle is based on
competitive ELISA assay in which the colorimetric signal is inversely proportional to the
amount of hyaluronan present in the sample.
Statistical analysis
For statistical analysis, two-way unpaired t-tests were used. A value of P < 0.05 was
considered statistically significant. Values are means ± SE.
Chapter 3 Hyperglycemia attenuates flow induced hyaluronan production
65
3.3 Results
Effect of hyperglycemia on initial hyaluronan shedding from endothelial cells
EC-RF24 cells were seeded on fibronectin-coated cover slips and grown to confluency (105
cells/cover slip). The cells were exposed to normoglycemic (5mM glucose) or hyperglycemic
(25mM glucose) media for 18 hours. Upon initiation of endothelial fluid shear stress
exposure, hyaluronan content in media collected at t = 2 hours from hyperglycemic cells was
significantly elevated compared to the level of initial hyaluronan shedding from
normoglycemic endothelial cells, being 6.3 ± 1.5 and 2.7 ± 0.9 ng / 10000 cells (P < 0.05),
respectively (figure 3.1).
Effect of shear stress levels on endothelial hyaluronan synthesis under normo- and
hyperglycemic conditions
Following the initial hyaluronan shedding, exposure of normo- and hyperglycemic
endothelial cells to fluid shear stresses of 1, 5 and 10dynes/cm2 was continued up to 7 more
hours under normoglycemic (5mM) and hyperglycemic (25mM) media conditions. At low
Figure 3.1: Initial hyaluronan shedding under shear conditions: Effect of hyperglycemia. The initial
hyaluronan shedding is significantly higher under the hyperglycemic condition compared to the
normoglycemic condition at all shear levels (6.3±1.5 versus 2.7±0.9 ng hyaluronan/10000cells) (P<0.05).
Initial endothelial hyaluronan shedding from flow start till t = 2h
0
5
10
15
Initi
al e
ndot
helia
l HA
she
ddin
g (n
g/ 1
0000
cel
ls)
Normoglycemia
Hyperglycemia
Initial endothelial hyaluronan shedding from flow start till t = 2h
0
5
10
15
Initi
al e
ndot
helia
l HA
she
ddin
g (n
g/ 1
0000
cel
ls)
Normoglycemia
Hyperglycemia
Chapter 3 Hyperglycemia attenuates flow induced hyaluronan production
66
shear stress levels of 1 and 5 dynes/cm2, both normo- and hyperglycemic conditions do not
significantly stimulate endothelial hyaluronan shedding into the media over time (Figure 3.2
and 3.4).
At a shear level of 10dynes/cm2, normoglycemic endothelial cells are stimulated to release
hyaluronan into the media at a rate of 18.3 ± 1.4 ng / 10000 cells/6h, which is significantly
greater than hyaluronan synthesis at 10 dynes/cm2 by hyperglycemic endothelial cells, being
7.6 ± 6.7 ng / 10000 cells/6h (Figure 3.3 and 3.4).
Figure 3.2: Hyaluronan release under 1 dyne/cm2 shear stress: Effect of hyperglycemia. Endothelial
cell exposed to a low shear stress of 1 dyne/cm2 is not significantly changed after 6 hours and
unaltered by hyperglycemic condition.
Effect of hyperglycemia on 1 dynes/cm2 shear stress induced endothelial hyaluronan release
y = -0.4x + 7.2
y = 0.1x + 3.2
0
5
10
15
0 2 4 6 8
Duration of 1 dynes/cm2 shear stress exposure (hours)
Endo
thel
ial h
yalu
rona
n(n
g/ 1
0000
cel
ls)
NormoGlycemia HyperGlycemia
Initial hyaluronan shedding upon flow start
Effect of hyperglycemia on 1 dynes/cm2 shear stress induced endothelial hyaluronan release
y = -0.4x + 7.2
y = 0.1x + 3.2
0
5
10
15
0 2 4 6 8
Duration of 1 dynes/cm2 shear stress exposure (hours)
Endo
thel
ial h
yalu
rona
n(n
g/ 1
0000
cel
ls)
NormoGlycemia HyperGlycemia
Initial hyaluronan shedding upon flow start
Chapter 3 Hyperglycemia attenuates flow induced hyaluronan production
67
Figure 3.3: Hyaluronan release under 10 dynes/cm2 shear stress: Effect of hyperglycemia. Endothelial cell
exposed to a shear stress of 10 dyne/cm2 is significantly increased over time (21.4±1.4 ng
hyaluronan/10000cells) (P<0.05) and attenuated by the hyperglycemic condition (8.9±5.3) (P<0.05).
Figure 3.4: Hyaluronan shedding under hyperglycemia: Effect of shear stress level. At shear stress levels of 1
and 5 dynes/cm2, the hyaluronan shedding is not significantly increased over time and the hyperglycemic
condition shows no effect on hyaluronan shedding. At shear level of 10 dynes/cm2, the hyaluronan shedding is
significantly increased over time and under hyperglycemic condition the hyaluronan shedding is depressed (18.3
± 1.4 versus 7.6 ± 6.7 ng / 10000 cells) (P<0.05).
Effect of hyperglycemia on 10 dynes/cm2 shear stress induced endothelial hyaluronan release
y = 3.1x - 0.4
y = 1.3x - 0.5
0
5
10
15
20
25
0 2 4 6 8Time - T2 (hours)
Endo
thel
ial h
yalu
rona
n -i
nitia
l HA
(n
g/ 1
0000
cel
ls)
NormoglycemiaHyperglycemia
Effect of hyperglycemia on 10 dynes/cm2 shear stress induced endothelial hyaluronan release
y = 3.1x - 0.4
y = 1.3x - 0.5
0
5
10
15
20
25
0 2 4 6 8Time - T2 (hours)
Endo
thel
ial h
yalu
rona
n -i
nitia
l HA
(n
g/ 1
0000
cel
ls)
NormoglycemiaHyperglycemia
Shear stress induced hyaluronan release from normo-and hyperglycemic endothelial cells
-5
0
5
10
15
20
25
1 dynes/cm2 5 dynes/cm2 10 dynes/cm2
Endo
thel
ial h
yalu
rona
n re
leas
e (n
g/ 1
0000
cel
ls /
6hou
rs)
NormoglycemiaHyperglycemia
*
Shear stress induced hyaluronan release from normo-and hyperglycemic endothelial cells
-5
0
5
10
15
20
25
1 dynes/cm2 5 dynes/cm2 10 dynes/cm2
Endo
thel
ial h
yalu
rona
n re
leas
e (n
g/ 1
0000
cel
ls /
6hou
rs)
NormoglycemiaHyperglycemia
*
Chapter 3 Hyperglycemia attenuates flow induced hyaluronan production
68
3.4 Discussion
The most important finding of the current study is that hyperglycemia impairs flow mediated
endothelial cell hyaluronan synthesis at physiological shear stress of 10dynes/cm2. In
addition, it is demonstrated that low shear stress levels of 1 dyne/cm2 are not sufficient to
stimulate endothelial hyaluronan synthesis, which is consistent with previous findings of very
little endothelial cell hyaluronan synthesis under static conditions (8). Furthermore, we report
that overnight incubation of static endothelial cells with hyperglycemic medium is
accompanied by greater amounts of hyaluronan that are initially released from the endothelial
surface upon initiation of fluid shear stress exposure.
Acute effect of hyperglycemia on hyaluronan synthesis
Our findings are consistent with reports by others using cell, animal and human models on
the effects of acute hyperglycemia on endothelial cell hyaluronan in relation to vascular
complications. Studies show that hyaluronan is shedded by oxygen radicals induced by
hyperglycemia in cultured endothelial cells (7), and the effect of advanced glycation end
products (AGEs) on vitreous gel of the eye showed that in addition to light exposure, AGEs
promote the decrease of MW hyaluronan in vitreous of diabetic patients (13). Type 2 diabetic
rats show 2-fold increase in hyaluronidase (12) and increased shedding of hyaluronan in
plasma compared to healthy controls (2). Furthermore, short-term hyperglycemia increases
endothelial glycocalyx permeability with acute capillary shutdown (30). Additionally, Wang
and Hascall show that hyaluronic structures synthesized by rat mesengial cells in response to
hyperglycemia induce monocyte adhesion (28). Recently, human studies show that
hyperglycemic clamping elicits a profound reduction in glycocalyx volume coinciding with
increased circulating plasma levels of glycocalyx constituents like hyaluronan, which is
consistent with release of glycocalyx constituents into the circulation (15).
Mechano-transduction of shear stress and the glycocalyx.
Presently, the mechanism by which endothelial cells sense shear stress is incompletely
understood. In vivo experiments have shown a decreased glycocalyx in of the vasculature
exposed to disturbed shear stress regions, which are considered at high- atherogenic risk (24).
In addition, static endothelial cell shown decreased hyaluronan staining compared to flow
Chapter 3 Hyperglycemia attenuates flow induced hyaluronan production
69
exposed endothelial cells (8). Shear stress may upregulate hyaluronan synthase (HAS)
synthesis, but recent studies have failed to demonstrate significant differences in HAS gene
activation at undisturbed versus disturbed shear stress regions (16). Alternatively, the
hyaluronan synthase enzyme HAS is located at the plasma membrane of endothelial cells. It
is therefore possible that mechanotransduction of fluid shear stress can directly stimulate
HAS activity.
We and others have demonstrated that an intact glycocalyx is required for endothelial cell
release of nitric oxide (NO) in response to shear stress. Studies using glycocalyx component
removing enzymes, hyaluronidase (14) and heparitinase (5) neuraminidase (17) to disturb the
endothelial glycocalyx, show these components to be participating in mechanosensing that
mediates NO production in response to shear stress. In addition, compromising the
endothelial glycocalyx or endothelial surface layer by enzyme treatment and/or decreasing
environmental protein concentration show a decreased cytoskeletal reorganization of the
endothelial cells in response to shear stress (23), elaborating on the findings that the core
proteins in the bush-like glycocalyx structures have a flexural rigidity that is sufficiently stiff
to serve as a molecular filter for plasma proteins and shows to be an exquisitely designed
transducer of fluid shearing stresses (22; 29). Therefore, our finding of hyperglycemic
induced hyaluronan shedding prior to the shear stress exposure is consistent with the
possibility with impaired mechanotransduction of shear stress due to hyperglycemic
glycocalyx perturbations. In support, in a recent study we demonstrated that hyperglycemic
shedding of glycocalyx hyaluronan in humans is associated with impaired flow mediated
arterial dilatation (15).
Chapter 3 Hyperglycemia attenuates flow induced hyaluronan production
70
References
1. Ceriello A. Impaired glucose tolerance and cardiovascular disease: The possible role of post-prandial hyperglycemia. American Heart Journal 147: 803-807, 2004.
2. Chajara A, Raoudi M, Delpech B, Leroy M, Basuyau JP and Levesque H. Circulating hyaluronan and hyaluronidase are increased in diabetic rats. Diabetologia 43: 387-388, 2000.
3. Constantinescu AA, Vink H and Spaan JA. Endothelial Cell Glycocalyx Modulates Immobilization of Leukocytes at the Endothelial Surface. Arterioscler Thromb Vasc Biol 23: 1541-1547, 2003.
4. Fibbi G, Vannucchi S, cavallini P, Del Rosso M, Pasquali F, Cappelletti R and Chiarugi V. Involvement of chondroitin sulfate in preventing adhesive cellular interactions. Biochim Biophys Acta 762: 512-518, 1983.
5. Florian JA, Kosky JR, Ainslie K, Pang Z, Dull RO and Tarbell JM. Heparan Sulfate Proteoglycan Is a Mechanosensor on Endothelial Cells. Circ Res 2003.
6. Fontijn R, Hop C, Brinkman HJ, Slater R, Westerveld A, van MJ and Pannekoek H. Maintenance of vascular endothelial cell-specific properties after immortalization with an amphotrophic replication-deficient retrovirus containing human papilloma virus 16 E6/E7 DNA. Exp Cell Res 216: 199-207, 1995.
7. Giardino I, Edelstein D and Brownlee M. BCL-2 expression or antioxidants prevent hyperglycemia-induced formation of intracellular advanced glycation endproducts in bovine endothelial cells. J Clin Invest 97: 1422-1428, 1996.
8. Gouverneur M, Spaan JAE, Pannekoek H, Fontijn RD and Vink H. Fluid shear stress stimulates incorporation of hyaluronan into endothelial cell glycocalyx. American Journal of Physiology-Heart and Circulatory Physiology 290: H458-H462, 2006.
9. Grimm J, Keller R and de Groot PG. Laminar flow induces cell polarity and leads to rearrangement of proteoglycan metabolism in endothelial cells. Thromb Haemost 60: 437-441, 1988.
10. Haffner SM, Lehto S, Ronnemaa T, Pyorala K and Laakso M. Mortality from coronary heart disease in subjects with type 2 diabetes and in nondiabetic subjects with and without prior myocardial infarction. New England Journal of Medicine 339: 229-234, 1998.
11. Henry CB and Duling BR. TNF-alpha increases entry of macromolecules into luminal endothelial cell glycocalyx. American Journal of Physiology - Heart & Circulatory Physiology 279: H2815-H2823, 2000.
Chapter 3 Hyperglycemia attenuates flow induced hyaluronan production
71
12. Ikegami-Kawai M, Okuda R, Nemoto T, Inada N and Takahashi T. Enhanced activity of serum and urinary hyaluronidases in streptozotocin-induced diabetic Wistar and GK rats. Glycobiology 14: 65-72, 2004.
13. Katsumura C, Sugiyama T, Nakamura K, Obayashi H, Hasegawa G and Ikeda T. Effects of advanced glycation endo products on hyaluronan proteolysis: A new mechanism of diabetic vitreopathy. Ophthalmic research 36: 327-331, 2004.
14. Mochizuki S, Vink H, Hiramatsu O, Kajita T, Shigeto F, Spaan JA and Kajiya F. Role of hyaluronic acid glycosaminoglycans in shear-induced endothelium-derived nitric oxide release. Am J Physiol Heart Circ Physiol 285: H722-H726, 2003.
15. Nieuwdorp M, van Haeften TM, Gouverneur MCLG, Mooij HL, van Lieshout MHP, Levi M, Meijers JCM, Holleman F, Hoekstra JBL, Vink H, Kastelein JJP and Stroes ESG. Loss of endothelial glycocalyx during acute hyperglycemia coincides with endothelial dysfunction and coagulation activation in vivo. Diabetes 55: 480-486, 2006.
16. Passerini AG, Polacek DC, Shi C, Francesco NM, Manduchi E, Grant GR, Pritchard WF, Powell S, Chang GY, Stoeckert CJJ and Davies PF. Coexisting proinflammatory and antioxidative endothelial transcription profiles in a disturbed flow region of the adult porcine aorta. Proc Natl Acad Sci U S A 101: 2482-2487, 2004.
17. Pohl U, Herlan K, Huang A and Bassenge E. Edrf-Mediated Shear-Induced Dilation Opposes Myogenic Vasoconstriction in Small Rabbit Arteries. American Journal of Physiology 261: H2016-H2023, 1991.
18. Sabri S, Soler M, Foa C, Pierres A, Benoliel AM and Bongrand P. Glycocalyx modulation is a physiological means of regulating cell adhesion. Journal of Cell Science 113: 1589-1600, 2000.
19. Sakariassen KS, Aarts PA, de GP, Houdijk WP and Sixma JJ. A perfusion chamber developed to investigate platelet interaction in flowing blood with human vessel wall cells, their extracellular matrix, and purified components. J Lab Clin Med 102: 522-535, 1983.
20. Silvestro L, Ruikun C, Sommer F, Duc TM, Biancone L, Montrucchio G and Camussi G. Platelet-Activating Factor-Induced Endothelial-Cell Expression of Adhesion Molecules and Modulation of Surface Glycocalyx, Evaluated by Electron-Spectroscopy Chemical-Analysis. Seminars in Thrombosis and Hemostasis 20: 214-222, 1994.
21. Soler M, Desplat-Jego S, Vacher B, Ponsonnet L, Fraterno M, Bongrand P, Martin JM and Foa C. Adhesion-related glycocalyx study: quantitative approach with imaging-spectrum in the energy filtering transmission electron microscope (EFTEM). FEBS Letters 429: 89-94, 1998.
22. Squire JM, Chew M, Nneji G, Neal C, Barry J and Michel C. Quasi-periodic substructure in the microvessel endothelial glycocalyx: a possible explanation for molecular filtering? J Struct Biol 136: 239-255, 2001.
Chapter 3 Hyperglycemia attenuates flow induced hyaluronan production
72
23. Thi MM, Tarbell JM, Weinbaum S and Spray DC. The role of the glycocalyx in reorganization of the actin cytoskeleton under fluid shear stress: A "bumper-car" model. Proceedings of the National Academy of Sciences of the United States of America 101: 16483-16488, 2004.
24. van den Berg BM, Spaan JAE, Rolf TM and Vink H. Atherogenic region and diet diminish glycocalyx dimension and increase intima-to-media ratios at murine carotid artery bifurcation. American Journal of Physiology-Heart and Circulatory Physiology 290: H915-H920, 2006.
25. van Haaren PMA, VanBavel E, Vink H and Spaan JAE. Localization of the permeability barrier to solutes in isolated arteries by confocal microscopy. American Journal of Physiology-Heart and Circulatory Physiology 285: H2848-H2856, 2003.
26. Vink H, Constantinescu AA and Spaan JA. Oxidized lipoproteins degrade the endothelial surface layer : implications for platelet-endothelial cell adhesion. Circulation 101: 1500-1502, 2000.
27. Vink H, Wieringa PA and Spaan JA. Evidence that cell surface charge reduction modifes capillary red cell velocity-flux relationships in hamster cremaster muscle. Journal of Physiology 489: 193-201, 1995.
28. Wang A and Hascall VC. Hyaluronan structures synthesized by rat mesangial cells in response to hyperglycemia induce monocyte adhesion. J Biol Chem 279: 10279-10285, 2004.
29. Weinbaum S, Zhang X, Han Y, Vink H and Cowin SC. Mechanotransduction and flow across the endothelial glycocalyx. Proc Natl Acad Sci U S A 100: 7988-7995, 2003.
30. Zuurbier CJ, Demirci C, Koeman A, Vink H and Ince C. Short-term hyperglycemia increases endothelial glycocalyx permeability and acutely decreases lineal density of capillaries with flowing red blood cells. Journal of Applied Physiology 99: 1471-1476, 2005.
Chapter 4
Hyperglycemic glycocalyx loss is secondary to an initial
transient increase in glycocalyx synthesis
Mirella Gouverneur1, Max Nieuwdorp2, Jos AE Spaan1,
Erik Stroes2 and Hans Vink1
Departments of Medical Physics1 and Vascular Medicine2, Academic Medical Center,
University of Amsterdam, Amsterdam, The Netherlands
Submitted for publication
Chapter 4 Hyperglycemic glycocalyx loss secundairy to initial increase in synthesis
74
Chapter 4 Hyperglycemic glycocalyx loss secundairy to initial increase in synthesis
75
Abstract
Recently, we demonstrated that exposure of healthy volunteers to 6 hours of
normoinsulinemic hyperglycemia results in loss of systemic glycocalyx volume. In the
present study, we determined the effect of hyperglycemia on cultured endothelial cell
glycocalyx synthesis and shedding. In the present study we determined the effect of acute
hyperglycemia (25mM glucose for 6 hours) on the biochemical incorporation of 3H-
glucosamine and 35S-sulfate into the glycocalyx of venular endothelial cells. In addition, the
dynamics of hyperglycemic glycocalyx shedding is compared with the dynamics of plasma
levels of glycocalyx hyaluronan in healthy volunteers during 6 hours of hyperglycemic
clamping. Hyperglycemia stimulated shedding of glucosamine containing
glycosaminoglycans from the endothelial by 40% compared to normoglycemic endothelial
cells (P<0.05). Hyperglycemic shedding was accompanied by a rapid, transient 50% increase
in glycocalyx synthesis within the first hour of hyperglycemia (P<0.05). After 1 hour,
glycocalyx synthesis normalized to normoglycemic levels. In contrast, hyperglycemic
glycocalyx shedding maintained increased during the 6 hour exposure to hyperglycemia in a
similar fashion as recently determined plasma levels of glycocalyx hyaluronan in human
volunteers exposed to acute hyperglycemia. The elevated level of glycocalyx shedding
relative to normalized glycocalyx synthesis, resulted in a net reduction of glycocalyx
glucosamine content by 20% compared to normoglycemic controls (P<0.05). Hyperglycemia
induces a transient increase in glycocalyx synthesis, which is followed by a maintained
elevated level of glycocalyx shedding, resulted in net loss of glycocalyx from the surface of
endothelial cells after 6 hours of hyperglycemia.
Chapter 4 Hyperglycemic glycocalyx loss secundairy to initial increase in synthesis
76
Chapter 4 Hyperglycemic glycocalyx loss secundairy to initial increase in synthesis
77
4.1 Introduction
The main contributor to higher cardiovascular complication incidence in the diabetic state is
increased hyperglycemia and poor hyperglycemic control (1). Evidence has shown that
glucose serum levels 2 hours after oral challenge (“hyperglycemic spikes”) is a powerful
mediator of cardiovascular risk (4) Infarction meta analysis show a continuous correlation
between glucose serum levels and cardiovascular complications even in subjects without
diabetes (3). Furthermore, hyperglycemia has been associated with increased vascular
permeability and increased platelet and leukocyte adhesion (4; 12). The in vivo endothelial
glycocalyx is a highly negatively charged protective barrier between endothelial cells and
flowing blood consisting of glycoproteins, proteoglycans and associated glycosaminoglycans
and plasma proteins (21). Its functions include establishing permeability barrier (20), binding
anticoagulation factors (15), modulating leukocyte interactions with the endothelium (6; 13),
limiting myocardial edema (19) and it plays a role in mechano shear sensing (23). Recently
we demonstrated that 6 hours of hyperglycemia decreases systemic glycocalyx volume in
healthy volunteers (14). In this study we tested the effect of acute hyperglycemia on
glycocalyx of cultured endothelial cells and compared the dynamics of hyperglycemic
glycocalyx shedding with dynamics of plasma hyaluronan concentration in hyperglycemic
humans.
4.2 Materials and Methods
Chemicals
M199 media, L-glutamine, antibiotic-antimycotic and trypsin were obtained from Gibco-
BRL, PBS pH: 7.4 from Fresenius Kabi and Fetal Bovine Serum (FBS) from Biowhittaker.
The following chemicals were obtained from Sigma; heparin, endothelial cell growth
supplement (ECGS). D(+)glucose was obtained from Merck. The radiochemicals 6-
[3H]glucosamine (specific activity: ~25-40Ci/mmol) and carrier-free Na[35S]O4 (specific
activity: ~43Ci/mgS) were purchased from ICN. Fibronectin was a kind gift from Central
Laboratory for Blood transfusion (CLB), Amsterdam, The Netherlands.
Chapter 4 Hyperglycemic glycocalyx loss secundairy to initial increase in synthesis
78
Cell culture
EC-RF24 cell line, human umbilical vein endothelial cells immortalized with an
amphotrophic replication-deficient retrovirus containing human papilloma virus 16 E6/E7
DNA (8). The cells were grown on 10µg/ml fibronectin-coated cell culture flasks in M199
media supplemented with 20% heat-inactivated fetal bovine serum, 50µg/ml heparin and
12.5µg/ml endothelial cell growth supplement, 0.2mmol/l L-glutamine and 100U/ml
Penicillin-G, 100U/ml Streptomycin sulfate, 25µg/ml Amphotericin-B at 37oC in 5% CO2.
Radioactive incorporation studies and hyperglycemic stimulus
The cells were seeded on fibronectin coated thermanox cover slips and attached to
confluency for 2 hours in M199 media without antibiotics (approximately
2x105cells/thermanox). The cells were incubated for 24 hours in complete media M199
without antibiotics, supplemented with 40µCi/ml 6-[3H] glucosamine and 50µCi/ml
Na2[35S]O4 (7). After 24 hours the cells were exposed to normoglycemic (5mM) and
hyperglycemic (25mM) media for 6 hour, during which samples were taken at time points 5,
27, 80, 180, 360min. After 6 hours, the cells were treated with 0.05% trypsin for 30min at RT
and additional 10min at 37oC and centrifuged at 1500g for 5 min to separate cells from
trypsinated proteoglycan fraction (16). The cell pellet was resuspended in 2M NaOH and
radioactive counts in time point samples, trypsin and cell fractions were determined using
scintillation counter.
Hyperglycemic clamping human subjects
A hyperglycemic clamp was applied for 6 hours with a target glucose concentration of 16
mmol/L (300 mg/dL) (24). To prevent hypokalemia 10mmol/L KCl was added to the glucose
solutions. Octreotide (Sandostatin ® kindly provided by Novartis, Switzerland) was dissolved
in saline and albumin and administered at final concentration of 30ng/kg/min to attenuate the
increase in endogenous insulin production in order to minimize potential confounding effects
of hyperinsulinemia (11). At this dose, octreotide has no significant vasoactive or haemostatic
side effects (24; 25) During the clamping protocol, blood glucose concentration was
measured by the glucose oxidase method (YSI 2300 STAT, Yellow Spring Inc., USA).
Target value of glucose was maintained by adjusting the infusion rate of glucose 20%
(Baxter, USA). As a time and osmolality control, the octreotide protocol was repeated on a
separate study day, during which glucose 20% was replaced with equimolar mannitol 20%
Chapter 4 Hyperglycemic glycocalyx loss secundairy to initial increase in synthesis
79
(Baxter, USA) infusion. Venous samples were obtained throughout the protocol to document
the achieved level of osmolality. Osmolality was determined by measurement of freezing
point depression on the Osmo Station (Menarini, Benelux). All samples for glucose, insulin,
and osmolality were performed in duplicate. The glutathione donor N-Acetylcysteine
(clinically graded manufactured by Department of Pharmacy, AMC) was administered as a
bolus of 100mg/kg in 15 minutes before start of glucose infusion and thereafter as a
continuous infusion of 60mg/kg throughout the identical hyperglycemia study protocol. The
rate of infusion and total amount of infused NAC in our experiment was similar to that used
for the treatment of paracetamol intoxication (18) Plasma samples were taken at time points,
0, 1, 2, 3 and 6hr during hyperglycemic clamping for hyaluronan content measurements.
Hyaluronan content
Hyaluronan mass was determined using enzyme linked immunosorbent assay kit,
commercially available from Echelon biosciences incorporated. The principle is based on
competitive ELISA assay in which the colorimetric signal is inversely proportional to the
amount of hyaluronan present in the sample.
Statistics
For statistical analysis, two-way unpaired t-tests were used. A value of P < 0.05 was
considered statistically significant. Values are means ± SE.
4.3 Results
Cummulative glucosamine and sulfate glycocalyx shedding: Effect of hyperglycemia
Figure 4.1 shows cumulative levels of glucosamine (top) and sulfate (bottom) shedded in the
media of normo- and hyperglycemic endothelial cells. Following a rapid increase in
shedding, hyperglycemic cells maintain increased levels of glucosamine shedding, 30%
higher than normoglycemic condition (32645±9773 versus 22837±630 cpm/10000cells;
P<0.05) (Figure 4.1A). Cumulative sulfate shedding shows no significant change under
hyperglycemic condition after 6 hours hyperglycemia (981±218 versus 874±181; NS) (Figure
4.1B).
Chapter 4 Hyperglycemic glycocalyx loss secundairy to initial increase in synthesis
80
Cummulative glycocalyx Glucosamine shedding
0
10000
20000
30000
40000
50000
0 100 200 300 400
T ime (min)
cpm
(10(
4)ce
lls)
NGHG
A
B Cummulative glycocalyx Sulfate shedding
0
200
400
600
800
1000
1200
1400
0 100 200 300 400
Time (min)
cpm
(10(
4)ce
lls)
NGHG
Figure 4.1: Cumulative levels of glucosamine (A) and sulfate (B) shedded in the media of normo- and
hyperglycemic endothelial cells.
Following a rapid increase in shedding, hyperglycemic cells maintain increased levels of glucosamine shedding,
30% higher than normoglycemic condition (P<0.05) (Figure 4.1A). Cumulative sulfate shedding shows no
significant change under hyperglycemic condition after 6 hours hyperglycemia (NS) (Figure 4.1B).
Chapter 4 Hyperglycemic glycocalyx loss secundairy to initial increase in synthesis
81
Glycocalyx glucosamine and sulfate content: Effect of hyperglycemia
Figure 4.2A shows an initial increase in glycocalyx glucosamine content after 1 hour of
exposure to hyperglycemia. After 6 hours, the glycocalyx glucosamine content has decreased
under hyperglycemic condition. Figure 4.2B shows an initial increase in glycocalyx sulfate
content after 1 hour of exposure to hyperglycemia. After 6 hours, the glycocalyx sulfate
content has normalized to the normoglycemic condition. After the initial increase in
glycocalyx glucosamine content under hyperglycemic condition (160±24% of
normoglycemic condition; P<0.05), after 6 hours the glycocalyx glucosamine content is
significantly decreased (81±14% of normoglycemic condition; P<0.05) (Figure 4.2C).
Figure 4.2: After an initial increase in glycocalyx sulfate content after 1 hour of exposure to hyperglycemia, at 6
hours, the glycocalyx sulfate content has normalized to the normoglycemic condition (Figure 4.2A).
glycocalyx sulfate content
0
510
1520
25
3035
40
0 100 200 300 400
Time (min)
% o
f tot
al p
ool
NGHG
A
Chapter 4 Hyperglycemic glycocalyx loss secundairy to initial increase in synthesis
82
B
C
Figure 4.2 cont: An initial increase in glycocalyx glucosamine content after 1 hour of exposure to
hyperglycemia. After 6 hours, the glycocalyx glucosamine content has decreased under hyperglycemic
condition (Figure 4.2B). After the initial increase in glycocalyx glucosamine content under hyperglycemic
condition (P<0.05), after 6 hours the glycocalyx glucosamine content is significantly decreased (P<0.05)
(Figure 4. 2C).
glycocalyx glucosamine content
0
5
10
15
20
25
30
35
40
0 100 200 300 400Time (min)
% o
f tot
al p
ool
NGHG
Effect of HG on glycocalyx content
0
50
100
150
0 100 200 300 400
Duration of hyperglycemia (min)
Glu
cosa
min
e gl
ycoc
alyx
co
nten
t (%
of N
G)
Chapter 4 Hyperglycemic glycocalyx loss secundairy to initial increase in synthesis
83
Effect of hyperglycemia on in vitro glycocalyx glucosamine shedding and human plasma
hyaluronan concentration dynamics.
Cells under hyperglycemia show an initial increase in burst in glucosamine shedding
compared to cells under normoglycemic condition (140±12%; P<0.05). After 6 hours the
glucosamine shedding is significantly higher in cells under hyperglycemia compared to
normoglycemia (140±18%; P<0.05) (Figure 4.3A). The glucosamine shedding dynamics of
cultured endothelial cells show a resemblance to the hyaluronan shedding dynamics in human
plasma of subjects exposed to 6 hours of hyperglycemic clapping (Figure 4.3B).
Chapter 4 Hyperglycemic glycocalyx loss secundairy to initial increase in synthesis
84
Figure 4.3: Cells under hyperglycemia show an initial increase in burst in glucosamine shedding compared to
cells under normoglycemic condition (P<0.05). After 6 hours the glucosamine shedding is significantly higher
in cells under hyperglycemia compared to normoglycemia (P<0.05) (Figure 4.3A). The glucosamine shedding
dynamics of cultured endothelial cells show a resemblance to the hyaluronan shedding dynamics in human
plasma of subjects exposed to 6 hours of hyperglycemic clapping, which after 6 hours of hyperglycemic
clamping the hyaluronan shedding is significantly increased. (Figure4. 3B).
A
B
Effect of HG on glycocalyx shedding
0
50
100
150
0 100 200 300 400
Duration of hyperglycemia (min)
Glu
cosa
min
e sh
eddi
ng(%
of N
G)
[HA] in plasma during acute hyperglycemia
40
60
80
100
120
140
160
180
-100 0 100 200 300 400
[HA
] in
plas
ma
Chapter 4 Hyperglycemic glycocalyx loss secundairy to initial increase in synthesis
85
4.4 Discussion
Recently, we reported on the loss of systemic glycocalyx volume in healthy volunteers during
acute exposure to 6 hours of hyperglycemia. Loss of glycocalyx volume was reflected by
significantly elevated plasma hyaluronan levels after 6 hours. In the current study, we
monitored the dynamics of glycocalyx synthesis and shedding using cultured endothelial cells
exposed to hyperglycemic conditions for 6 hours. Consistent with the clinical findings,
hyperglycemia reduced incorporation of glucosamine and sulfate into the glycocalyx of
hyperglycemic endothelial cells compared to normoglycemic paired controls. Furthermore,
the dynamics of supernatant glycocalyx shedding was remarkebly similar to the dynamics of
human plasma hyaluronan levels, showing an initial transient rise within the first hour,
followed by a more slowly, steady increase in glycocalyx shedding. Our current findings
demonstrate that the initial transient shedding increase reflects a transient hyperglycemic
increase in glycocalyx synthesis, while the later increases in glycocalyx shedding are
responsible for the net hyperglycemic loss of glycocalyx after 6 hours.
Short-term hyperglycemia has been shown to increase endothelial glycocalyx permeability
with acute capillary shutdown in C57BL/6 mice treated by normoglycemic, acutely
hyperglycemic (25 mM) for 60 min due to infusion of glucose, or hyperglycemic (25 mM)
for 2-4 wk (db/db mice). The data indicate that short-term hyperglycemia causes a rapid
decrease of the ability of the glycocalyx to exclude 70kDa dextran. No change in the vascular
permeation of 40kDa dextran was observed. These data indicate that the described increased
vascular permeability with hyperglycemia can be ascribed to an increased permeability of the
glycocalyx, identifying the glycocalyx as a potential early target of hyperglycemia (26)
Additionally studies have shown that diabetic mice display an increase in hyaluronan in
plasma compared to healthy subjects (5) and Type 2 diabetic rats show 2 fold increase in
hyaluronidase in plasma (9).
Mechanism of acute hyperglycemia induced glycocalyx damage
The mechanisms by which hyperglycemia affect hyaluronan metabolism are incompletely
understood. Brownlee (2) hypothesizes on the possibility that hyperglycemia causes diabetic
complications through an increased hexosamine pathway flux. So far, the effect of high
Chapter 4 Hyperglycemic glycocalyx loss secundairy to initial increase in synthesis
86
glucose on increased amounts of glycosaminoglycan substrates, such as glucosamine and
glucuronic acid, and its effect on glycosaminoglycan metabolism has remained undiscussed
in the literature. Since the enzyme hyaluronan synthase is situated in the cells membrane and
utilizes glucosamine and glucuronic acid precursor building blocks directly from the cytosol
without entering cellular posttranslational modification pathways, the increased substrate
pool could very well have a direct effect on the hyaluronan metabolism (17), and indirectly
stimulate hyaluronidase activity by virtue of the close association of hyaluronan synthesis and
its degradation. Additionally, studies by Wang and Hascall shows hyaluronic structures
synthesized by rat mesengial cells in response to hyperglycemia induced monocyte adhesion
(22) Studies on the effect of AGEs on vitreous of diabetic patients showed that in addition of
light exposure, AGEs promotes the decrease of MW hyaluronan (10).
Summary
Like in humans, hyperglycemia results in loss of glycocalyx on cultured cells. Loss is
secondary to an initial, transient in glycocalyx synthesis, which is reflected by a transient
supernatant peak of shedded glycocalyx constituents after 1 hour of hyperglycemia. The
initial increase in glycocalyx synthesis and shedding is followed by a maintained elevated
shedding level, resulting in a net reduction in glycocalyx content of glucosamine and sulfate.
Further studies need to determine whether activation of the glycocalyx biosynthetic pathways
by high glucose may contribute to net loss of glycocalyx protective properties during acute
exposure to hyperglycemia.
Chapter 4 Hyperglycemic glycocalyx loss secundairy to initial increase in synthesis
87
References
1. Algenstaedt P, Schaefer C, Biermann T, Hamann A, Schwarzloh B, Greten H, Ruther W and Hansen-Algenstaedt N. Microvascular alterations in diabetic mice correlate with level of hyperglycemia. Diabetes 52: 542-549, 2003.
2. Brownlee M. Biochemistry and molecular cell biology of diabetic complications. Nature 414: 813-820, 2001.
3. Capes SE, Hunt D, Malmberg K and Gerstein HC. Stress hyperglycaemia and increased risk of death after myocardial infarction in patients with and without diabetes: a systematic overview. Lancet 355: 773-778, 2000.
4. Ceriello A. Impaired glucose tolerance and cardiovascular disease: The possible role of post-prandial hyperglycemia. American Heart Journal 147: 803-807, 2004.
5. Chajara A, Raoudi M, Delpech B, Leroy M, Basuyau JP and Levesque H. Circulating hyaluronan and hyaluronidase are increased in diabetic rats. Diabetologia 43: 387-388, 2000.
6. Constantinescu AA, Vink H and Spaan JA. Endothelial Cell Glycocalyx Modulates Immobilization of Leukocytes at the Endothelial Surface. Arterioscler Thromb Vasc Biol 23: 1541-1547, 2003.
7. Esko JD, Stewart TE and Taylor WH. Animal cell mutants defective in glycosaminoglycan biosynthesis. Proceedings of the National Academy of Sciences of the United States of America 82: 3197-3201, 1985.
8. Fontijn R, Hop C, Brinkman HJ, Slater R, Westerveld A, van MJ and Pannekoek H. Maintenance of vascular endothelial cell-specific properties after immortalization with an amphotrophic replication-deficient retrovirus containing human papilloma virus 16 E6/E7 DNA. Exp Cell Res 216: 199-207, 1995.
9. Ikegami-Kawai M, Okuda R, Nemoto T, Inada N and Takahashi T. Enhanced activity of serum and urinary hyaluronidases in streptozotocin-induced diabetic Wistar and GK rats. Glycobiology 14: 65-72, 2004.
10. Katsumura C, Sugiyama T, Nakamura K, Obayashi H, Hasegawa G and Ikeda T. Effects of advanced glycation endo products on hyaluronan proteolysis: A new mechanism of diabetic vitreopathy. Ophthalmic research 36: 327-331, 2004.
11. Krentz AJ, Boyle PJ, Macdonald LM and Schade DS. Octreotide - A Long-Acting Inhibitor of Endogenous Hormone-Secretion for Human Metabolic Investigations. Metabolism-Clinical and Experimental 43: 24-31, 1994.
12. Morigi M, Angioletti S, Imberti B, Donadelli R, Micheletti G, Figliuzzi M, Remuzzi A, Zoja C and Remuzzi G. Leukocyte-endothelial interaction is augmented by high glucose concentrations and hyperglycemia in a NF-kB-dependent fashion. J Clin Invest 101: 1905-1915, 1998.
Chapter 4 Hyperglycemic glycocalyx loss secundairy to initial increase in synthesis
88
13. Mulivor AW and Lipowsky HH. Role of glycocalyx in leukocyte-endothelial cell adhesion. Am J Physiol Heart Circ Physiol 283: H1282-H1291, 2002.
14. Nieuwdorp M, van Haeften TM, Gouverneur MCLG, Mooij HL, van Lieshout MHP, Levi M, Meijers JCM, Holleman F, Hoekstra JBL, Vink H, Kastelein JJP and Stroes ESG. Loss of endothelial glycocalyx during acute hyperglycemia coincides with endothelial dysfunction and coagulation activation in vivo. Diabetes 55: 480-486, 2006.
15. Rosenberg RD. Redesigning heparin. N Engl J Med 344: 673-675, 2001.
16. Shworak NW. High-specific-activity 35S-labeled heparan sulfate prepared from cultured cells. Methods Mol Biol 171: 79-89, 2001.
17. Stern R. Hyaluronan catabolism: a new metabolic pathway. European Journal of Cell Biology 83: 317-325, 2004.
18. Vale JA and Proudfoot AT. Paracetamol (Acetaminophen) Poisoning. Lancet 346: 547-552, 1995.
19. van den Berg BM, Vink H and Spaan JA. The endothelial glycocalyx protects against myocardial edema. Circ Res 92: 592-594, 2003.
20. Vink H and Duling BR. Capillary endothelial surface layer selectively reduces plasma solute distribution volume. American Journal of Physiology - Heart & Circulatory Physiology 278: H285-H289, 2000.
21. Vink H, Wieringa PA and Spaan JA. Evidence that cell surface charge reduction modifes capillary red cell velocity-flux relationships in hamster cremaster muscle. Journal of Physiology 489: 193-201, 1995.
22. Wang A and Hascall VC. Hyaluronan structures synthesized by rat mesangial cells in response to hyperglycemia induce monocyte adhesion. J Biol Chem 279: 10279-10285, 2004.
23. Weinbaum S, Zhang X, Han Y, Vink H and Cowin SC. Mechanotransduction and flow across the endothelial glycocalyx. Proc Natl Acad Sci U S A 100: 7988-7995, 2003.
24. Williams SB, Goldfine AB, Timimi FK, Ting HH, Roddy MA, Simonson DC and Creager MA. Acute hyperglycemia attenuates endothelium-dependent vasodilation in humans in vivo. Circulation 97: 1695-1701, 1998.
25. Witzig TE, Kvols LK, Moertel CG and Bowie EJW. Effect of the Somatostatin Analog Octreotide Acetate on Hemostasis in Humans. Mayo Clinic Proceedings 66: 283-286, 1991.
26. Zuurbier CJ, Demirci C, Koeman A, Vink H and Ince C. Short-term hyperglycemia increases endothelial glycocalyx permeability and acutely decreases lineal density of capillaries with flowing red blood cells. Journal of Applied Physiology 99: 1471-1476, 2005.
Chapter 5
Loss of Endothelial Glycocalyx during Acute
Hyperglycemia Coincides with Endothelial Dysfunction and
Coagulation Activation in vivo
Max Nieuwdorp1, Timon W. van Haeften2, Mirella C.L.G. Gouverneur3, Hans L. Mooij1,
Miriam H.P. van Lieshout1, Marcel Levi4, Joost C.M. Meijers1,
Frits Holleman4, Joost B.L. Hoekstra4, Hans Vink3,
John J.P. Kastelein1, and Erik S.G. Stroes1
Departments of Vascular Medicine1, Medical Physics3 and Internal Medicine4 , Academic
Medical Center, Amsterdam, the Netherlands
Department of Internal Medicine2, University Medical Center Utrecht, Utrecht, the
Netherlands
Diabetes. 2006 Feb;55(2):480-6.
Chapter 5 Loss of glycocalyx during acutae hyperglycemia
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Chapter 5 Loss of glycocalyx during acutae hyperglycemia
91
Abstract
Hyperglycemia is associated with increased susceptibility towards athero-thrombotic stimuli.
The glycocalyx, a layer of proteoglycans covering the endothelium, is involved in the
protective capacity of the vessel wall. We therefore evaluated whether hyperglycemia affects
the glycocalyx, thereby increasing vascular vulnerability. Systemic glycocalyx volume was
estimated by comparing the distribution volume of a glycocalyx permeable tracer (Dextran
40) to that of a glycocalyx impermeable tracer (labeled erythrocytes) in 10 healthy males.
Measurements were performed in randomized order on 5 occasions: 2 control measurements,
two measurements during normo-insulinemic hyperglycemia with or without N-Acetyl
cysteine (NAC) infusion, respectively, and one during mannitol infusion. Glycocalyx
measurements were reproducible (1.7 ± 0.2 versus 1.7 ± 0.3 liters). Hyperglycemia reduced
glycocalyx volume (0.8 ± 0.2 liters, p<0.05), which could be prevented by NAC (1.4 ± 0.2
liters). Mannitol infusion had no affect on glycocalyx volume (1.6 ± 0.1 liters).
Hyperglycemia resulted in endothelial dysfunction, increased plasma hyaluronan levels (70 ±
6 to 112 ± 16 ng/mL, p<0.05) and coagulation activation (F1+2: 0.4 ± 0.1 to 1.1 ± 0.2
nmol/L; D-Dimer: from 0.27 ± 0.1 to 0.55 ± 0.2 g/L, p< 0.05). Taken together, these data
indicate a potential role for glycocalyx perturbation in mediating vascular dysfunction during
hyperglycemia.
Chapter 5 Loss of glycocalyx during acutae hyperglycemia
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Chapter 5 Loss of glycocalyx during acutae hyperglycemia
93
5.1 Introduction
Patients with diabetes mellitus have increased vulnerability to atherogenic insults, leading to
accelerated atherogenesis. Athought atherogenesis is in part due to the increased prevalence
of traditional cardiovascular risk factors, these factors cannot fully explain the propensity
towards vascular complications in diabetic patients (1). Hyperglycemia itself has been shown
to induce a wide array of downstream effects that adversely affect the protective capacity of
the vessel wall (2). Hyperglycemia has been associated with enhanced endothelial
permeability, increased leukocyte-endothelium adhesion and impaired nitric oxide (NO) bio-
availability (3-5). Despite clear progress in understanding the underlying pathophysiological
mechanisms contributing to this vascular dysfunction, it has proven difficult to unravel a final
common pathway for the increased vascular vulnerability under hyperglycemic conditions
(6).
The glycocalyx covers the endothelium and consists of endothelial cell-derived
proteoglycans, glycoproteins and adsorbed plasma proteins. This layer has been shown to
orchestrate vascular homeostasis (7). Its thickness (up to 1µm) micrometers may explain its
potent antiadhesive effects on leukocytes and platelets (8,9). Hyaluronan
glycosaminoglycans, one of the major constituents of the glycocalyx, are crucial for
maintaining endothelial barrier properties for plasma macromolecules (10). The glycocalyx
also serves as mechanosensor of shear stress, mediating shear-induced release of NO by
endothelial cells (11-13). In fact, selective perturbation of the glycocalyx leads to increased
vascular permeability, attenuated NO availability and increased adhesion of leukocytes and
platelets. Reconstitution of the glycocalyx results in restoration of its barrier and antiadhesive
properties (10,14). In view of the intricate relation between glycocalyx integrity and vascular
homeostasis in experimental models, it has been postulated that glycocalyx derangement
could contribute to increased vascular vulnerability in humans (15). Because increased
degradation of proteoglycans has previously been demonstrated in hyperglycemic conditions
(16,17), the impact of hyperglycemia on the glycocalyx merits special interest. In the present
study we set out to evaluate the impact of hyperglycemia on the glycocalyx in healthy
volunteers. We measured changes in systemic glycocalyx volume before and 6 h after
hyperglycemic-normoinsulinemic clamping. We simultaneously assessed changes in plasma
Chapter 5 Loss of glycocalyx during acutae hyperglycemia
94
hyaluronan, endothelial function, and coagulation parameters. To elucidate the role of
reactive oxygen species, we repeated the hyperglycemic clamp in conjunction with infusion
of the anti-oxidant N-acetylcysteine (NAC).
5.2 Materials and Methods
Subjects for this study were 10 Caucasian male volunteers. Measurements were performed on
five separate occasions in random order, with a minimum interval of 2 weeks between
measurements. Two baseline measurements were performed after saline infusion to estimate
the intersession coefficient of variance (CV) of the glycocalyx volume estimates. Two
measurements were performed after hyperglycemic clamping and mannitol infusion,
respectively. To gain insight into the mechanistic properties, a fifth measurement was
performed after hyperglycemic-normoinsulinemic protocol with concomitant infusion of
NAC in a random subgroup (n=6). The protocol was approved by the internal review board
and written informed consent was obtained from all volunteers prior to the investigation. The
study was carried out in accordance with the principles of the Declaration of Helsinki.
Estimation of glycocalyx volume
The endothelial glycocalyx provides limits access to plasma macromolecules and erythrocytes
(18,19). Hence the glycocalyx volume can be estimated by comparing the circulating blood
volume using a glycocalyx impermeable tracer such as labelled erythrocytes (18,20) with the
total intravascular volume using a glycocalyx permeable tracer such as neutral Dextran 40 (MW
40 kD). For the study, centrifuged erythrocytes were mixed with sodium fluorescein (250
mg/mL) for 5 minutes. After being washed, the labeled erythrocytes were resuspended in
saline to the initial volume and reinfused. Blood was subsequently drawn at 4, 5, 6, and 7
minutes after infusion. The fraction of labeled erythrocytes versus the total erythrocyte pool
was used to estimate circulating erythrocyte volume. Unlabeled erythrocytes (t = -1) served
as negative controls. Labeled erythrocytes were measured using a FACScan analyzer (FACS
Calibur ®, Becton Dickinson, Mountain View, USA), with at least 100,000 cells being
counted to measure the circulating fraction of labeled erythrocytes. Data were analyzed using
Cellquest (Becton Dickinson, San Jose, CA, USA). The circulating plasma volume was
calculated as [(1-Hsys) x Vrbc]/Hsys, where Vrbc is the circulating erythrocyte volume and
Hsys is the large vessel hematocrit (20). Dextran 40 is used as a probe for intravascular
Chapter 5 Loss of glycocalyx during acutae hyperglycemia
95
glycocalyx volume (19). Before Dextran 40 was injected, a single bolus of 10 ml Dextran 1
(Promiten ®, NPBI International BV, Emmercompascuum, the Netherlands) was injected to
attenuate the risk for anaphylactic reactions. At least 1 h later, 100 ml Dextran 40
(Rheomacrodex ®, NPBI International BV, Emmercompascuum, the Netherlands) was
injected intravenously, after which repeated blood sampling at 5, 7, 10, 15, 20 and 30 minutes
was performed. The dextran 40 concentration was calculated by measuring the increase in
glucose polymers (21). The glucose concentration per time point was assessed in duplicate
using the hexokinase method (Gluco-quant ®, Hitachi 917; Hitachi Corporation). The
procedure was calibrated with dextran 40 added to plasma in vitro. To determine the initial
intravascular distribution volume of Dextran 40, the concentration of dextran 40 at the time
of injection was estimated by exponential fitting of the measured dextran 40 concentrations.
Hyperglycemic-normo-insulinemic clamp
A hyperglycemic clamp was applied for 6 hours with a target glucose concentration of 16
mmol/L (300 mg/dL) (22). To prevent hypokalemia 10mmol/L KCl was added to the glucose
solution. Octreotide (Sandostatin ®; Novartis, Basel, Switzerland) was dissolved in saline
and albumin and administered at final concentration of 30ng/kg/min to attenuate the increase
in endogenous insulin production and minimize potential confounding effects of
hyperinsulinemia (23). At this dose, octreotide has no significant vasoactive or haemostatic
side effects (22,24). During the clamping protocol, blood glucose concentration was
measured by the glucose oxidase method (YSI 2300 STAT, Yellow Spring Inc., USA).
Target value of glucose was maintained by adjusting the infusion rate of glucose 20%
(Baxter, USA). As a time and osmolality control, the octreotide protocol was repeated on a
separate study day, during which glucose 20% was replaced with equimolar mannitol 20%
(Baxter, USA) infusion. Venous samples were obtained throughout the protocol to document
the achieved level of osmolality. Osmolality was determined by measuring the freezing point
depression on the Osmo Station (Menarini, Benelux). All samples for glucose, insulin, and
osmolality were performed in duplicate. The glutathione donor N-Acetylcysteine (NAC)
(clinically graded manufactured by Department of Pharmacy, AMC) was administered as a
bolus of 100mg/kg in 15 minutes before start of glucose infusion and thereafter as a
continuous infusion of 60mg/kg throughout the identical hyperglycemia study protocol. The
rate of infusion and total amount of infused NAC in our experiment was similar to that used
for the treatment of acetominophen intoxication (25).
Chapter 5 Loss of glycocalyx during acutae hyperglycemia
96
Flow mediated dilation
Flow-mediated dilation (FMD) was assessed before each glycocalyx volume measurement
(26). With the subjects in the supine position, a blood pressure cuff was placed just below the
elbow of the right arm. The brachial artery in the right antecubital fossa was visualized using
a 7.5-Mhz transducer. A wall tracking system was used to measure lumen diameter. After
two baseline vessel diameter measurements were obtained, reactive hyperemia was induced
by inflating the lower-arm blood pressure cuff to 200 mmHg. Upon release of the cuff after 4
minutes, ultrasonography continued for 5 minutes to allow for lumen diameter measurements
at 30-s second intervals. Images were stored digitally and analyzed off-line using the Wall
Track System software analysis package. All measurements were performed by the same
person, who was unaware of clinical details and the stage of the experiment. At our
institution, intra- and intersession CVs for baseline diameter assessment using the Wall Track
System are 1.1 % and 3.8 %, respectively. Intersession variability of the FMD measurement
is 13.9% (26).
Blood sampling and laboratory methods
Blood samples were drawn from the subjects after a 12-h overnight fast and 2, 4, 6 and 30 h
after start of infusion. Aliquots were centrifuged within 1 hour after being collected, snap-
frozen in liquid nitrogen and stored at -80oC. Hematocrit was measured after centrifugation of
heparinized blood in a Hettich-Haematokrit centrifuge (Hettich, Tuttlingen, Germany) at
10000 rpm during 5 minutes. Total cholesterol, HDL-cholesterol, and triglycerides were
measured by standard enzymatic methods (Roche Diagnostics, Basel, Switzerland). LDL
cCholesterol was calculated using the Friedewald formula. Alanine and aspartate
aminotransferase (ALAT and ASAT) were measured by pyridoxalphosphate activation assay
(Roche Diagnostics, Basel, Switzerland). HbA1c was measured on HPLC (Reagens Bio-Rad
Laboratories B.V., the Netherlands) on Variant II (Bio-Rad Laboratories). Quantitative
plasma hyaluronan levels were measured in duplicate by enzyme-linked immunosorbent
assay (ELISA) (Echelon Biosciences, Salt Lake City, USA). As a measure of thrombin
generation, the prothrombin activation fragment F1+2 (Dade Behring, Marburg, Germany)
was assessed by ELISA. D-Dimer levels were used as a reflection of endogenous fibrinolysis
in the presence of coagulation activation and were measured with an automated quantitative
latex particle immunoassay (Biomerieux, Durham, NC). Plasma insulin was measured by
Chapter 5 Loss of glycocalyx during acutae hyperglycemia
97
immuno-luminimetric assay (ILMA) (Immulite insuline®) on Immulite 2000 (DPC,
Diagnostic Product Corporation).
Statistical analysis
All data except Table 1 are presented as means ± SE. Differences between treatment groups
were tested by ANOVA. Comparisons within groups were done with the Wilcoxon’s signed-
rank test. P < 0.05 was considered significant.
5.3 Results
Reproducibility of glycocalyx measurement
Baseline characteristics of volunteers are listed in Table 5.1. Throughout all infusion
protocols, blood pressure and heart rate remained unaffected (data not shown). Infusion of the
Dextran 40 solution had no significant effect on hematocrit values. Circulating plasma
volumes (3.0 ± 0.1 versus 3.0 ± 0.1 liters, n.s.) and systemic Dextran 40 distribution volumes
(4.7 ± 0.2 versus 4.7 ± 0.3 liters, n.s.) were similar during the baseline study visits.
Accordingly, glycocalyx volumes were reproducible between visits (1.7 ± 0.2 versus 1.7 ±
0.3 liters, n.s., figure 5.1a and b; inter-session coefficient of variance of 15.2 ± 9.8%).
Table 5.1: Clinical characteristics of the volunteers.
Chapter 5 Loss of glycocalyx during acutae hyperglycemia
98
Glycocalyx volume after 6h infusion of glucose, N-acetylcysteine with glucose or mannitol
During hyperglycemic clamping, glucose concentration was raised to target levels within 15
minutes and remained stable throughout the hyperglycemic period (~16 mmol/L). Glucose
levels during mannitol infusion were unchanged (4.8 ± 0.1 versus 5.3 ± 0.1 mmol/L, n.s..).
Despite coinfusion of octreotide, plasma insulin levels rose during hyperglycemia but
remained within the physiological range (from 35 ± 5 to 116 ± 10 pmol/L, p<0.001).
Mannitol infusion with octreotide was associated with a decrease in insulin levels (42 ± 5
pmol/L to 15 ± 1 pmol/L, p<0.01). The plasma osmolality levels during hyperglycemia and
mannitol were comparable (284 ± 2 versus 288 ± 2 mMol/kg, n.s.). Glycocalyx volumes were
profoundly decreased during hyperglycemia compared to mannitol (0.8 ± 0.2 versus 1.6 ± 0.1
liters, p< 0.05, figure 5.1b), predominantly due to a reduction in Dextran 40 distribution
volume (4.0 ± 0.3 vs. 4.7 ± 0.3 liters, hyperglycemia vs. mannitol, p< 0.05). There were no
changes in circulating plasma volumes (3.2 ± 0.1 versus 3.1 ± 0.1 liters, hyperglycemia vs.
mannitol,n.s.) nor in hematocrit values (0.40 ± 0.001 versus 0.41 ± 0.001 %, hyperglycemia
vs. mannitol n.s.). Co-infusion of N-acetylcysteine during the hyperglycemic clamp abolished
the reduction in glycocalyx volume (1.4 ± 0.2 liters, p<0.05 compared to hyperglycemia
alone) due to normalization of Dextran 40 distribution volume (4.4 ± 0.2 liters) without
affecting circulating plasma volume (3.0 ± 0.1 liters) and hematocrit (0.41 ± 0.01 %).
Chapter 5 Loss of glycocalyx during acutae hyperglycemia
99
Figure 5.1. A: Plasma dextran 40 (D40) clearance curves under baseline (_, [Dex40]C1 _0.057e_0.000481 t;
_, [Dex40]C2 _ 0.058e_0.000671 t), glucose infusion (F, [Dex40]HG _ 0.073e_0.001718 t), glucose-NAC
infusion (OE, ([Dex40]NAc _ 0.064e_0.000320 t), and mannitol infusion (f, [Dex40]Man _ 0.056e_0.000190
t). During glucose infusion, the rate of dextran 40 plasma clearance was increased as compared with the
mannitol infusion. Depicted values on each time point are expressed as means _ SE. B: Systemic glycocalyx
volumes were determined in random order before and after infusion with glucose (bars 1 and 2) or mannitol
(bars 3 and 4) and after glucose-NAC (bar 5). Systemic glycocalyx volumes were identical at baseline;
glucose infusion resulted in a statistically significant decrease in systemic glycocalyx volume compared with
baseline, mannitol, and glucose-NAC. Data are means _ SE. *P < 0.05.
Chapter 5 Loss of glycocalyx during acutae hyperglycemia
100
Endothelial function
Flow mediated dilation showed good reproducibility between saline visits (8.8 ± 0.8 versus
8.2 ± 0.5 %, n.s; inter-session coefficient of variance of 16.8 ± 8.2%). Flow mediated dilation
was attenuated during hyperglycemia (5.8 ± 0.6 versus 8.8 ± 0.8 %, hyperglycemia vs.
baseline, p<0.05). Mannitol infusion had no effect on FMD (7.1±1.0 versus 8.2 ± 0.5%,
mannitol vs. baseline, n.s., figure 5.2). There was no difference in nitroglycerine response
after hyperglycemia vs. mannitol infusion (data not shown). Of note, due to the small sample
size, we did not determine FMD in the NAC hyperglycemia protocol.
Figure 5.2. FMD determined in a random order before and after infusion with glucose (bars 1and 2) or mannitol
(bars 3 and 4). FMD was identical before the interventions; glucose infusion resulted in a statistically significant
decrease in FMD compared with mannitol infusion and baseline. Data are means _ SE. *P < 0.05.
Chapter 5 Loss of glycocalyx during acutae hyperglycemia
101
Laboratory parameters
Plasma hyaluronan levels rose significantly during hyperglycemia (112 ± 16 versus 70 ± 6
ng/mL, HG vs. baseline p< 0.05) returning towards baseline values within 24 hours (81 ± 6
ng/mL) (figure 5.3).
Figure 5.3. Shedding of endothelial glycocalyx compounds (as assessed by plasma hyaluronan) in subjects
infused with glucose (F), mannitol (f), or glucose-NAC (OE). Data are means _ SE. *P < 0.05 vs. baseline; #P
< 0.05 among groups.
Chapter 5 Loss of glycocalyx during acutae hyperglycemia
102
Activation of the coagulation system (as indicated by an increase in F1+2 levels 1.1 ± 0.2
versus 0.4 ± 0.1 nmol/L, p<0.05) and fibrinolytic system (increase in D-Dimer levels 0.55 ±
0.2 versus 0.27 ± 0.1 g/L, p< 0.05 see figure 5.4 a and b) occurred during hyperglycemia. No
effect of mannitol was seen on these parameters. Hyperglycemia with concomitant NAC
infusion resulted in blunting of plasma hyaluronan shedding (69 ± 8 ng/mL, p<0.05, figure
5.3) and coagulation activation compared to hyperglycemia.
(F1+2: 0.8 ± 0.15 nmol/L and D-dimer: 0.44 ± 0.07 g/L,n.s. see figure 5.4a and b).
Figure 5.4. A: Activation of
coagulation system (as assessed
by prothrombin fragments 1 _ 2)
in human volunteers infused with
glucose (F), mannitol (f), or
glucose-NAC (OE). Data are
means _ SE. *P < 0.05 vs.
baseline. B: Activation of the
fibrinolytic system (as assessed by
D-dimer levels) in subjects
infused with glucose (F), mannitol
(f), or glucose- NAC (OE). Data
are means _ SE. *P < 0.05 vs.
baseline.
Chapter 5 Loss of glycocalyx during acutae hyperglycemia
103
5.4 Discussion
In the present study, we showed that the glycocalyx constitutes a large intravascular
compartment in healthy volunteers that can be estimated in a reproducible fashion in vivo.
More importantly, we showed that hyperglycemic clamping elicits a profound reduction in
glycocalyx volume that coincides with increased circulating plasma levels of glycocalyx
constituents like hyaluronan, an observation that is consistent with release of glycocalyx
constituents into the circulation. These disturbances are accompanied by impaired FMD as
well as activation of the coagulation system. Infusion of the antioxidant NAC prevented this
glycocalyx perturbation, indicating that generation of reactive oxygen species contributes to
the glycocalyx perturbation under hyperglycemic conditions.
Hyperglycemia and glycocalyx volume
Previously we validated glycocalyx measurements in isolated vessels by comparison of
erythrocyte- and Dextran 40 distribution volumes as markers of glycocalyx- impermeable and
permeable tracers, respectively (18,19). Consistent with these experimental data, we now find
comparable values for glycocalyx volume in healthy volunteers with good reproducibility of
the measurement between sessions (CV < 20%). The size of the glycocalyx volume in the
present study is in line with predictions of glycocalyx dimension in vivo, based on a thickness
of 0.5 to 3.0 µm combined with a total endothelial surface area between 1000 and 7000 m2
(27,28). After 6 h of hyperglycemic clamping, systemic glycocalyx volume is reduced to
~50% of the baseline value. This reduction coincides with a rapid increase in circulating
plasma hyaluronan levels, an important constituent of the glycocalyx. Similarly,
hyperglycemia has been associated with increased hyaluronidase activity and concomitant
increased plasma hyaluronan concentrations in animal models (17, 29). Hyaluronan has been
shown to be a principal determinant of vascular permeability, since selective removal of
hyaluronan from the vessel wall is accompanied by a profound increase in macromolecular
glycocalyx permeation (10). We presently show in vivo that loss of glycocalyx volume and
shedding of hyaluronan into the plasma is indeed accompanied by a significant increase in the
rate of Dextran 40 clearance from the circulation (figure 5.1a). This correlation between
glycocalyx volume reduction and increased permeability suggests a potential contribution of
the glycocalyx, particularly hyaluronan, to the preservation of the systemic vascular
permeability barrier.
Chapter 5 Loss of glycocalyx during acutae hyperglycemia
104
Increased formation of reactive oxygen species and glycocalyx
Several mechanisms may contribute to loss of glycocalyx volume during acute
hyperglycemia. First, hyperglycemia per se constitutes a potent pro-oxidant and pro-
inflammatory stimulus, which has been linked to enhanced degradation of the glycocalyx as
well as to shedding of hyaluronan (30). Therefore, glycocalyx loss may be secondary to a
direct effect of oxygen radicals on the synthesis of glycosaminoglycans. Indeed, in our study
infusion of the potent antioxidant NAC was able abolish the reduction in glycocalyx during
hyperglycemia. On the other hand, increased shedding of glycosaminoglycans may follow
vascular injury resulting in an upregulation of glycosaminoglycan synthesis to compensate
for stimulated increased degradation (6, 31).
Endothelial function
In conjunction with glycocalyx loss we observed a loss of flow mediated dilation after
hyperglycemic clamping. In line, several research groups have reported endothelial
dysfunction under hyperglycemic conditions (22, 32). Whereas impaired NO bioavailability
has predominantly been attributed to direct inactivation of NO by increased radical
production (33, 34), the present finding provides us with an alternative explanation. It has
been demonstrated that the endothelial glycocalyx plays an important mechano-sensory role
translating intravascular shear stress into biochemical activation of endothelial cells (12).
Accordingly, the release of NO in response to shear stress is abolished upon enzymatic
removal of glycosaminoglycans from the endothelial glycocalyx (15).
Coagulation activation
Hyperglycemia elicited coagulation and fibrinolysis, reflected by increased thrombin
generation (F1+2) as well as increased fibrinolysis (D-dimer). Our data are in line with
studies showing that induction of acute hyperglycemia in healthy volunteers increased plasma
levels of coagulation factor VIIa and stimulated tissue factor-dependent activation of
coagulation (35). The endothelial glycocalyx is a crucial compartment for binding and
regulation of enzymes involved in the coagulation cascade. In addition, the most important
inhibitor of thrombin and factor Xa, i.e. antithrombin, is firmly attached to the endothelial
glycocalyx (36). In agreement, we and others have previously demonstrated that glycocalyx
perturbation has direct effects on coagulation and fibrinolytic responses (9, 37). It is therefore
Chapter 5 Loss of glycocalyx during acutae hyperglycemia
105
not surprising that hyperglycemia-induced loss of glycocalyx is accompanied by activation of
coagulation. The subsequent increase in endogenous fibrinolysis can thus be explained by
counterbalancing the increased thrombin generation during hyperglycemia.
Study limitations
Firstly the accuracy of glycocalyx volume estimates is determined by the accuracy of Dextran
40 distribution volume estimates. Because of its relatively small size and neutral charge,
Dextran 40 is slowly cleared from the circulation. Extrapolation of measured plasma Dextran
40 concentrations to the time of its initial intravascular injection is used to estimate
intravascular Dextran 40 concentration prior to leakage. However, as can be appreciated from
the average clearance curves in figure 1a, the error of the estimated initial Dextran 40
concentration is relatively small and will therefore have no major impact on the estimates of
glycocalyx volume. Second, the stable circulating blood volumes during hyperglycemic
clamping cannot exclude changes of microcirculatory volume. Due to anatomical dimensions
the largest part of the erythrocyte volume is located in the macrovasculature, whereas
measured dextran 40 volume is mainly situated in the microvasculature. In fact, we recently
reported that hyperglycemia reduces perfused murine capillary density up to 38% (38).
Hence, in addition to glycocalyx shedding, impaired microcirculatory perfusion may also
have contributed to the reduction of systemic glycocalyx. Finally, during hyperglycemic
clamping, an increase in insulin levels was observed in spite of the concomitant infusion of
octreotide. Higher octreotide administration was not feasible due to gastro-intestinal side
effects (23). Under these circumstances, plasma insulin increases are inextricably entangled
with hyperglycemic clamping in humans (22). Although the insulin levels were within
physiological range, we cannot exclude a potential confounding effect of insulin.
Chapter 5 Loss of glycocalyx during acutae hyperglycemia
106
Clinical implications
Experimental studies have shown that the glycocalyx constitutes a crucial intravascular
compartment, which mediates transduction of shear-stress induced NO release, modulates
vascular permeability and harbors a wide array of anticoagulant proteins. In a time course
comparable to the loss of glycocalyx volume, we find loss of shear-stress induced NO-
release, increased vascular permeability and activation of coagulation during hyperglycemic
clamping in healthy volunteers. The prevention of glycocalyx damage by antioxidant infusion
confirms other research in this area on the role of oxidative stress in hyperglycemia-induced
vascular damage (6, 39). Therefore, glycocalyx measurement may hold a promise as a tool to
estimate cardiovascular risk and the impact of cardiovascular risk lowering therapies in
patients with diabetes.
Chapter 5 Loss of glycocalyx during acutae hyperglycemia
107
References
1. Haffner SM, Lehto S, Ronnemaa T, Pyorala K, Laakso M. Mortality from coronary heart disease in subjects with type 2 diabetes and in nondiabetic subjects with and without prior myocardial infarction. N Engl J Med 1998; 339(4):229-34.
2. Nathan DM, Lachin J, Cleary P, Orchard T, Brillon DJ, Backlund JY, O’Leary DH, Genuth S.
Intensive diabetes therapy and carotid intima-media thickness in type 1 diabetes mellitus. N Engl J Med. 2003; 348(23):2294-303.
3. Algenstaedt P, Schaefer C, Biermann T, Hamann A, Schwarzloh B, Greten H, Ruther W, Hansen-
Algenstaedt N. Microvascular alterations in diabetic mice correlate with level of hyperglycaemia. Diabetes. 2003;52(2):542-9.
4. Morigi M, Angioletti S, Imberti B, Donadelli R, Micheletti G, Figliuzzi M, Remuzzi A, Zoja C,
Remuzzi G. Leukocyte-endothelial interaction is augmented by high glucose concentrations and hyperglycaemia in a NF-kB-dependent fashion.. J Clin Invest. 1998;101(9):1905-15.
5. Du XL, Edelstein D, Dimmeler S, Ju Q, Sui C, Brownlee M. Hyperglycaemia inhibits endothelial nitric
oxide synthase activity by posttranslational modification at the Akt site. J Clin Invest. 2001;108(9):1341-8.
6. Brownlee M. Biochemistry and molecular cell biology of diabetic complications. Nature.
2001;414(6865):813-20. 7. Nieuwdorp M, Meuwese MC, Vink H, Hoekstra JB, Kastelein JJ, Stroes ES. The endothelial
glycocalyx: a potential barrier between health and vascular disease. Curr Opin Lipidology;16(5):507-11.
8. Mulivor AW, Lipowsky HH. Role of glycocalyx in leukocyte-endothelial cell adhesion. Am J Physiol
Heart Circ Physiol. 2002;283(4):H1282-91 9. Vink H, Constantinescu AA, Spaan JA. Oxidized lipoproteins degrade the endothelial surface layer :
implications for platelet-endothelial cell adhesion. Circulation 2000; 101(13):1500-2. 10. Henry CB, Duling BR. Permeation of the luminal capillary glycocalyx is determined by hyaluronan.
Am J Physiol 1999;277(2Pt2):H508-14. 11. Weinbaum S, Zhang X, Han Y, Vink H, Cowin SC. Mechanotransduction and flow across the
endothelial glycocalyx. Proc Natl Acad Sci U S A. 2003;100(13):7988-95. 12. Mochizuki S, Vink H, Hiramatsu O, Kajita T, Shigeto F, Spaan JA, Kajiya F. Role of hyaluronic acid
in shear induced endothelium derived nitric oxide release. Am J Phys 2003;285(2):H722-6 13. Florian JA, Kosky JR, Ainslie K, Pang Z, Dull RO, Tarbell JM. Heparan eparin proteoglycan is a
mechanosensor on endothelial cells. Circ Res. 2003 ;93(10) :e136-42. 14. Constantinescu AA, Vink H, Spaan JA. Endothelial cell glycocalyx modulates immobilization of
leucocytes at the endothelial surface. Art Thromb Vasc Biol 2003;23(9):1541-7.
Chapter 5 Loss of glycocalyx during acutae hyperglycemia
108
15. Thi MM, Tarbell JM, Weinbaum S, Spray DC. The role of the glycocalyx in reorganization of the actin cytoskeleton under fluid shear stress: a bumper-car model. Proc Natl Acad Sci U S A. 2004;101(47):16483-8.
16. Ceriello A, Giugliano D, Dello Russo P, Passariello N, Saccomanno F, Sgambato S.
Glycosaminoglycans in human diabetes. Diabetes Metab. 1983;9(1):32-4. 17. Ikegami-Kawai M, Okuda R, Nemoto T, Inada N, Takahashi T. Enhanced activity of serum and urinary
hyaluronidases in streptozotocin-induced diabetic Wistar and GK rats. Glycobiology. 2004;14(1):65-72 18. Vink H, Duling BR. Identification of distinct luminal domains for macromolecules, erythrocytes, and
leukocytes within mammalian capillaries. Circ Res. 1996;79(3): 581-9. 19. Vink H, Duling BR. Capillary endothelial surface layer selectively reduces plasma solute distribution
volume. Am J Physiol Heart Circ Physiol. 2000; 278(1):H285-9. 20. Orth VH, Rehm M, Thiel M, Kreimeier U, Haller M, Brechtelsbauer H, Finsterer U. First clinical
implications of perioperative red cell volume measurement with a nonradioactive marker (sodium fluorescein). Anesth Analg. 1998 ;87(6):1234-8.
21. van Kreel BK, van Beek E, Spaanderman ME, Peeters LL. A new method for plasma volume
measurements with unlabeled Dextran-70 instead of 125I-labeled albumin as an indicator. Clin Chim Acta. 1998 ;275(1):71-80.
22. Williams SB, Goldfine AB, Timimi FK, Ting HH, Roddy MA, Simonson DC, Creager MA. Acute
hyperglycemia attenuates endothelium-dependent vasodilation in humans in vivo. Circulation 1998; 97(17): 1695-701.
23. Krentz AJ, Boyle PJ, Macdonald LM, Schade DS. Octreotide: a long-acting inhibitor of endogenous
hormone secretion for human metabolic investigations. Metabolism. 1994 ;43(1):24-31. 24. Witzig TE, Kvols LK, Moertel CG, Bowie EJ. Effect of the somatostatin analogue octreotide acetate on
hemostasis in humans. Mayo Clin Proc. 1991;66(3):283-6. 25. Vale JA, Proudfoot AT. Paracetamol (acetaminophen) poisoning. Lancet 1995;346:547-52. 26. Hijmering ML, Stroes ES, Pasterkamp G, Sierevogel M, Banga JD, Rabelink TJ. Variability of flow
mediated dilation: consequences for clinical application. Atherosclerosis. 2001;157(2):369-73. 27. Klitzman B and Duling BR. Microvascular hematocrit and red cell flow in resting and contracting
striated muscle. Am.J.Physiol 1979;237:H481-H490, 28. Desjardins C, Duling BR. Microvessel hematocrit: measurement and implications for capillary oxygen
transport. Am.J.Physiol 1987; 252:H494-H503 29. Wang A, Hascall VC. Hyaluronan structures synthesized by rat mesangial cells in response to
hyperglycemia induce monocyte adhesion. J Biol Chem. 2004 279:10279-85. 30. Mulivor AW, Lipowsky HH. Inflammation- and ischemia-induced shedding of venular glycocalyx. Am
J Physiol Heart Circ Physiol. 2004 ;286(5):H1672-80. 31. Henry CB, Duling BR. TNF-alpha increases entry of macromolecules into luminal endothelial cell
glycocalyx. Am J Physiol Heart Circ Physiol 2000; 279:H2815–H2823.
Chapter 5 Loss of glycocalyx during acutae hyperglycemia
109
32. Title LM, Cummings PM, Giddens K, Nassar BA. Oral glucose loading acutely attenuates
endothelium-dependent vasodilation in healthy adults without diabetes: an effect prevented by vitamins C and E. J Am Coll Cardiol. 2000 ;36(7):2185-91.
33. Timimi FK, Ting HH, Haley EA, Roddy MA, Ganz P, Creager MA.Vitamin C improves endothelium-
dependent vasodilation in patients with insulin-dependent diabetes mellitus. J Am Coll Cardiol. 1998 ;31(3):552-7
34. Cosentino F, Luscher TF. Tetrahydrobiopterin and endothelial function. Eur Heart J. 1998;19 Suppl
G:G3-8. 35. Rao AK, Chouhan V, Chen X, Sun L, Boden G: Activation of the tissue factor pathway of blood
coagulation during prolonged hyperglycemia in young healthy men. Diabetes 1999 ; 48 :1156-1161 36. Esmon CT. Inflammation and thrombosis. J Thromb Haemost 2003; 1:1343–1348 37. Pearson MJ, Lipowsky HH. Effect of fibrinogen on leukocyte margination and adhesion in
postcapillary venules. Microcirculation 2004; 11:295–306. 38. Zuurbier CJ, Demirci C, Koeman A, Vink H, Ince C. Short-term hyperglycemia increases endothelial
glycocalyx permeability and acutely decreases lineal density of capillaries with flowing RBC’s. J Appl Physiol. 2005; 99:1471-1476
39. Kurzelewski M, Czarnowska E, Beresewicz A: Superoxide- and nitricoxygen-derived species mediate
endothelial dysfunction, endothelial glycocalyx disruption, and enhanced neutrophil adhesion in the post-ischemic guinea-pig heart. J. Physiol Pharmacol. 2005; 56:163-178
110
Chapter 6
Vasculoprotective Properties of the Endothelial
Glycocalyx: Effects of Fluid Shear Stress
Mirella Gouverneur1, Bernard van den Berg2, Max Nieuwdorp3,
Erik Stroes3, & Hans Vink1
Department of Medical Physics1, Academic Medical Center, University of Amsterdam,
Amsterdam, the Netherlands, Department of
Molecular and Vascular Medicine2, Beth Israel Deaconess Medical Center, Harvard Medical
School, Boston, MA, USA, and Department of Vascular
Medicine3, Academic Medical Center, University of Amsterdam, Amsterdam, the
Netherlands
J Intern Med. 2006 Apr;259(4):393-400
Chapter 6 Vasculoprotective properties of the endothelial glycocalyx
112
Chapter 6 Vasculoprotective properties of the endothelial glycocalyx
113
Abstract The endothelial glycocalyx exerts a wide array of vasculoprotective effects via inhibition of
coagulation and leukocyte adhesion, by contributing to the vascular permeability barrier and
by mediating shear stress induced NO release. In this review we will focus on the relation
between fluid shear stress and the endothelial glycocalyx. We will address the hypothesis that
modulation of glycocalyx synthesis by fluid shear stress may contribute to thinner
glycocalyces, and therefore more vulnerable endothelium, at lesion prone sites of arterial
bifurcations. Finally, we will discuss the effects of known atherogenic stimuli such as
hyperglycemia on whole body glycocalyx volume in humans and its effect on endothelial
function.
Chapter 6 Vasculoprotective properties of the endothelial glycocalyx
114
Chapter 6 Vasculoprotective properties of the endothelial glycocalyx
115
6.1 Introduction
Cardiovascular disease is the major cause of mortality world wide and notwithstanding many
efforts to reduce cardiovascular disease burden, current strategies aimed at lowering systemic
risk factors have only achieved a 20-30% reduction in cardiovascular event rate (1). The
remaining 70 – 80% of events highlights the need for novel strategies to improve
cardiovascular outcome. The insight that all cardiovascular risk factors inflict loss of anti-
atherogenic properties of the vessel wall, has shifted attention from only treating systemic
risk factors towards augmenting vasculoprotective properties of the vessel wall itself. Since
the endothelium is the first line defense mechanism against atherosclerosis, much research
effort has focused at novel strategies to improve endothelial function.
Over the past several years, it is recognized that the endothelial glycocalyx may contribute to
the protection of the vascular wall against disease. The glycocalyx, consisting of a negatively
charged, organized mesh of membranous glycoproteins, proteoglycans, glycosaminoglycans
and associated plasma proteins, is situated at the luminal side of all blood vessels (2). Its
major constituents comprise hyaluronic acid (HA) and the negatively-charged eparin
sulphate proteoglycans. Glycocalyx dimensions depend upon the balance between
biosynthesis and enzymatic or shear-dependent shedding of its components (3), and whereas
historically this layer was thought to be confined to a thickness of only several nanometers, it
has recently been demonstrated to reach up to 0.5 – 3 µm intraluminally (4, 5). This relatively
large dimension of the glycocalyx, which exceeds the thickness of the endothelium and the
length of leukocyte adhesion molecules, has triggered researchers to study its role in the
course of atherogenesis (6).
Numerous studies in both micro- and macrovasculature have demonstrated that constituents
of the glycocalyx, such as hyaluronan, are intimately involved in vascular homeostasis, such
as maintaining the vascular permeability barrier (7) and regulating the release of nitric oxide
(NO) by serving as a mechano-shear sensor for NO-release (8-11). In addition, the
glycocalyx harbors a wide array of enzymes that might contribute to its vasculoprotective
effect. Thus, extracellular superoxide dismutase (ec-SOD), an enzyme which dismutates
oxygen radicals to hydrogen peroxide (12), is bound to proteoglycans within the glycocalyx.
Chapter 6 Vasculoprotective properties of the endothelial glycocalyx
116
Damage to the glycocalyx is accompanied by increased shedding of ec-SOD, which results in
a dysbalance in favor of a pro-oxidant state (13). Collectively, these observations are of
particular interest since altered vascular permeability, attenuated NO-bioavailability and
redox dysregulation are amongst the earliest characteristics of atherogenesis (14). In spite of
these observations, it has proven difficult to show direct relevance of the glycocalyx as a
vasculoprotective paradigm for larger vessels. The latter is predominantly due to the fact that
glycocalyx research has traditionally focused at the microvasculature, in which atherogenesis
does not occur.
6.2 Structural properties of the endothelial glycocalyx
The first visualization of the endothelial glycocalyx was performed by conventional electron
microscopy using the cationic dye ruthenium red, which has a high affinity for acidic
mucopolysaccharides (15). Electron micrographs revealed a small irregular shaped layer
extending approximately 50- to 100 nm into the vessel lumen. Subsequent approaches with
varying perfusate contents or fixatives revealed stained structures on continuous endothelial
cell surfaces throughout diverse microvascular beds, arterial- and venular macrovessels with
large variations in dimension and appearance (4, 16-20). Fenestrated endothelium, in
addition, was found to have a combination of surface bound stained structures, about 50 –
100 nm thick, and distinct filamentous plugs composed of 20 to 40 filaments with a length of
about 350 nm on the surface of the endothelial fenestrae (21). These studies, especially when
specific approaches were applied to stabilize anionic carbohydrate structures to prevent loss-
and or collapse of these structures, gave evidence for a thick endothelial surface layer
throughout the whole vascular tree (Figure 6.1). In addition, co-localization of lectins to the
observed stained structures confirmed its saccharine nature in several of these studies (4, 16,
18).
Intravital microscopy studies on cremaster muscle showed dramatic differences between
microvascular- and systemic hematocrit (22) that could be abrogated upon enzymatic
treatment of the microvascular network with heparinase (23). From these studies a 0.3 – 1
µm thick slow-moving plasma layer on the endothelial cell surface consisting principally of
eparin-sulfate proteoglycans was thought to be involved. The first visual evidence of a 0.4
Chapter 6 Vasculoprotective properties of the endothelial glycocalyx
117
– 0.5 µm thick continuous endothelial cell surface layer was provided by comparing the
width of the flowing plasma column containing large, anionic fluorescein-labeled dextrans
with the anatomic capillary diameter as defined by the position of the luminal endothelial cell
boundaries (24). Based on observations in this study, theoretical studies predicted a
glycocalyx thickness of 0.5 – 1 µm to account for observed variations in red-cell motion
through microvessels and the discrepancy between in vivo and in vitro estimates of resistance
to blood flow (25-27). Indeed, such differences in blood flow resistance have been observed
between control- and hyaluronidase treated vessels in a study of coronary reactive hyperemia
in a dog (28). Enzymatic degradation of the glycocalyx with hyaluronidase has been shown
to significantly increase the available intralumenal space for flowing blood (7).
2 µm2 µm
EndothelialEndothelialEndothelialcellcellcell
GlycocalyxGlycocalyxGlycocalyx
0.2 µm0.2 µm2 µm2 µm
EndothelialEndothelialEndothelialcellcellcell
GlycocalyxGlycocalyxGlycocalyx
EndothelialEndothelialEndothelialcellcellcell
GlycocalyxGlycocalyxGlycocalyx
0.2 µm0.2 µm
Figure 6.1a: electron micrographs of goat capillary glycocalyx (Courtesy of Dr. Bernard van den Berg)
Lumen
0.5 µm
EC
Lumen
0.5 µm
EC
Figure 6.1b: examples of the spatial heterogeneity of glycocalyx dimensions in the vascular system (Courtesy of Dr. Bernard van den Berg).
Chapter 6 Vasculoprotective properties of the endothelial glycocalyx
118
Although various studies are consistent with the concept that perturbation of the glycocalyx
contributes to increases in endothelial vulnerability upon ischemia/reperfusion (17), hypoxia
(20), exposure to low-density lipoproteins (29, 30) and atherogenic shear stress profiles (6,
18), it has proven difficult to show direct relevance of the glycocalyx as a vasculoprotective
paradigm for larger vessels. The latter is predominantly due to the fact that glycocalyx
research has traditionally focused at the microvasculature, in which atherogenesis does not
occur. However, several studies have emphasized that the relevance of the glycocalyx is not
confined to smaller vessels (6, 17). For example, van Haaren et al recently visualized a thick
endothelial glycocalyx in larger arteries in rats (5). The glycocalyx in larger vessels has also
been shown to decrease extravasation of LDL particles into the subendothelial space (31, 32).
Amongst others, these data imply that also in the macrovasculature the glycocalyx adds
towards the vasculoprotective properties of the vessel wall.
6.3 Glycocalyx at arterial bifurcations
Although reduced levels of surface bound sialic acids (33) and increased endothelial
permeability and susceptibility to atherosclerotic lesion formation (18) have been found to
coincide with arterial branch points and curvatures, little is known about the contribution of
glycocalyx perturbation to the increased vascular vulnerability of high atherogenic risk areas.
Atherosclerotic lesions within the arterial tree develop at predictable vessel geometries, e.g.
arterial branching and curvatures, and constraints on vessel motion by the surrounding
tissues, which lead to local flow instabilities and separations. Such lesions can be detected
and visualized as changes in vascular wall properties and quantified as intima media ratios
(IMR). Increases in IMR have been found to be associated with increased cardiovascular risk
factors and atherosclerosis (37, 38, 39).
In a recent study, van den Berg et al. (6) hypothesized that endothelial cells, which play a
central role in response to shear stress (40), express a modified surface glycocalyx at high
atherogenic risk regions and, in turn, contribute to predisposition of these arterial sites to
atherosclerotic lesion formation. The endothelial glycocalyx dimension was investigated by
electron microscopic observation at low- and high risk regions of the C57Bl/6J mouse carotid
artery, using the common- and internal carotid bifurcation (sinus) area as a model for arterial
Chapter 6 Vasculoprotective properties of the endothelial glycocalyx
119
sites exposed to low- and high atherogenic risk, respectively (41). As shown in Figure 6.2, it
is clear that the dimension of the endothelial glycocalyx at the sinus region of the mouse
internal carotid artery is significantly smaller than the glycocalyx dimension on the luminal
surface of the common carotid artery. This finding is in support of the hypothesis that
perturbation of the glycocalyx contributes to the increased vascular vulnerability of regions
that are at high atherogenic risk. Furthermore, this thinner glycocalyx is accompanied by
greater intima media ratios and a thicker subendothelial layer, indeed confirming that
regional differences in glycocalyx dimension reflect variations in its vasculo-protective
capacity.
0
100
200
300
400
500
600
C57Bl6 ApoE*3 C57Bl6 ApoE*3
Gly
coca
lyx
thic
knes
s (n
m)
Common carotidregion
Internal carotid sinusregion
* * *
Figure 6.2a: Glycocalyx dimension is diminished at the atheroprone sinus region of the internal carotid
artery in mice compared to the atheroprotected common carotid artery. Systemic atherogenic stimulation by
a hyperlipidemic, hypercholesterolemic diet for 6 weeks in ApoE3-Leiden mice further diminishes the
dimension of the glycocalyx in the common carotid artery (From reference 6).
* = P < 0.05 compared to common region of C57Bl6 mice on normal diet.
Chapter 6 Vasculoprotective properties of the endothelial glycocalyx
120
0
200
400
600
800
1000
C57Bl6 ApoE*3 C57Bl6 ApoE*3
Sube
ndot
helia
l mat
rix th
ickn
ess
(nm
)
Common carotidregion
Internal carotid sinusregion
*
*
*C
Figure 6.2b: Greater dimensions of the subendothelial intima layer result in greater intima-to-media ratios (IMR) at
vulnerable sites of the carotid arterial bifurcation with diminished glycocalyx dimensions (From reference 6).
* = P < 0.05 compared to common region of C57Bl6 mice on normal diet..
Chapter 6 Vasculoprotective properties of the endothelial glycocalyx
121
Previous studies have demonstrated that loss of glycosaminoglycans from the endothelial
glycocalyx by enzyme treatment is associated by edema formation of the subendothelial
space (4), indicating that flow profile related modulation of the glycocalyx might contribute
to the earlier observed progression from a decreased endothelial barrier function into
subsequent intimal edema at vascular regions exposed to disturbed flow (42). Whether
edema formation contributed to the increased IMR in the present study remains to be
explored. However, the site specific differences in IMR occurred in the absence of changes
in the dimension of the media layer, and were predominantly due to increases in the
dimension of the subendothelial space. Furthermore, no evidence was found for
accumulation of blood cells or monocytes in the intima layer, indicating that the contribution
of the inflammatory response was minimal at this stage.
6.4 Mechanism of glycocalyx reduction at high risk regions
The fact that the glycocalyx dimension is significantly diminished at the sinus region
compared to the glycocalyx dimension at the opposite site of the internal carotid near the
flow divider as well as at the common carotid area just proximal to the carotid bifurcation,
suggests that spatial differences in glycocalyx dimension are related to local variations in
flow profiles. It is well known that areas of high atherogenic risk are located close to regions
of disturbed flow at arterial bifurcations. Therefore, it is tempting to speculate that
undisturbed flow patterns and the associated stimulation of vascular endothelium by fluid
shear stress are essential to obtain optimal glycocalyx protective properties. However,
although studies have recently demonstrated that the endothelial glycocalyx indeed plays an
important role in mechanotransduction of fluid shear stress, very few data are available on the
relation between fluid shear stress and glycocalyx synthesis.
Earlier studies, using sialic acid binding lectins (33) and alcian blue (18), showed that
reduced dimensions of the endothelial glycocalyx at arterial sites exposed to disturbed flow
patterns associate with increases in endothelial permeability and susceptibility to
atherosclerotic lesion formation. Additionally, studies by Woolf (43) and Wang et al. (44)
revealed thicker glycocalyces at high shear regions compared to low shear regions and
demonstrated that glycocalyx dimension is reduced when rabbits are fed an atherogenic diet.
Chapter 6 Vasculoprotective properties of the endothelial glycocalyx
122
Steady state glycocalyx dimension is the result of local synthesis and degradation of its
constituents and it is important to know the factors that determine this balance.
Recently, Gouverneur (45) demonstrated that exposure of cultured endothelial cells for 24 h
to a shear stress of 10 dynes/cm2 stimulates incorporation of glucosamine-containing
glycosaminoglycans (GAGs) in the glycocalyx, which is accompanied by elevated levels of
glucosamine-containing GAGs in the supernatant. These increases were confirmed by direct
demonstration of increased hyaluronan concentrations in the glycocalyx and in the
supernatant, as well as by a 3 fold increase in the incorporation of hyaluronan binding protein
in the glycocalyx. In addition to its incorporation in hyaluronan, glucosamine is also
incorporated in sulfated sugars like eparin sulfate and chondroitin sulfate. In addition,
Arisaka et al.(46) used pig aortic endothelial cells exposed to shear stress levels of 15 and 40
dynes/cm2 in a parallel flow chamber for periods of 3, 6, 12 and 24 h. These authors
demonstrated increased synthesis of sulfated GAGs after high shear stress of 40 dynes/cm2,
and also a small, but significant increase at 15 dynes/cm2. Similarly, Elhadj et al (47) exposed
bovine aortic endothelial cells for 7 days to < 0.5 dynes/cm2 prior to increasing shear rates for
3 days to 5 and 23 dynes/cm2. No significant increase in the net sulfated GAG synthesis was
detected, but a shift in its size distribution was reported, indicating that modulation of specific
sulfation patterns may occur despite limited effects on sulfated GAG synthesis. In summary,
these experiments demonstrate that shear stress increases hyaluronan content in the
endothelial glycocalyx, that shear stress exposure alters the size distribution of endothelial
sulfated GAGs, and that high levels of shear stress may also increase sulfated GAG synthesis.
6.5 Glycocalyx and systemic atherogenic stimuli
In addition to the spatial differences in glycocalyx dimension at arterial bifurcations, van den
Berg et al. (6) also reported that the glycocalyx is diminished upon systemic atherogenic
challenge by a high fat- high cholesterol diet. Systemic perturbation of the glycocalyx by
hypercholesterolemia and/or hypertriglyceremia on top of pre-existing regional variations in
glycocalyx protective properties, introduced further increases in vascular vulnerability. The
mechanism by which the glycocalyx is diminished in atherogenic mice remains to be
elucidated, but the present finding is consistent with previous studies demonstrating rapid
Chapter 6 Vasculoprotective properties of the endothelial glycocalyx
123
shedding of glycocalyx from the endothelial surface upon acute stimulation with elevated
plasma levels of Ox-LDL or by acute exposure of the endothelium to inflammatory agents
like thrombin or TNF-α (48, 49, 50). In conclusion, both regional and risk factor induced
increases in atherogenic risk are associated by smaller glycocalyx dimensions and greater
IMR. Exposure of the high risk sinus area to an additional atherogenic challenge results in
endothelial thickening and excessive swelling of the subendothelial space, in line with the
proposed hypothesis that vascular sites with diminished glycocalyx are more vulnerable to
pro-inflammatory and atherosclerotic sequelae.
6.6 Human glycocalyx measurements
To date, direct visualization of endothelial glycocalyx in humans has been unsuccessful,
mainly due to the fact that the endothelial glycocalyx is a very delicate structure depending
critically on the presence of flowing plasma (2). As a consequence, the best way to measure
the endothelial glycocalyx in humans is to compare systemic intravascular distribution
volumes for glycocalyx permeable versus glycocalyx impermeable tracers. Subtracting these
two volumes provides an estimate of whole body glycocalyx volume (51).
At present, Nieuwdorp et al. (51) try to answer the question whether glycocalyx perturbation
mediated vascular vulnerability contributes to the accelerated rate of atherogenesis in patients
with type 1 diabetes. Whereas this is at least in part the consequence of increased prevalence
of traditional cardiovascular risk factors, these cannot fully explain the propensity towards
cardiovascular complications in diabetic patients (52). Disease-specific abnormalities, such as
hyperglycemia, may also facilitate the development of vascular lesions in these patients.
Thus, hyperglycemia has been shown to induce a wide array of downstream effects, which
may adversely affect the protective capacity of the vessel wall (53). Since increased
degradation of proteoglycans has indeed been demonstrated in hyperglycemic conditions (54,
55), the impact of hyperglycemia on the glycocalyx merits special interest. Therefore,
Nieuwdorp et al. recently set out to evaluate the impact of hyperglycemia on the glycocalyx
in healthy volunteers. Systemic glycocalyx volume was measured before and 6 hours after
normo-insulinemic, hyperglycemic clamping.
Chapter 6 Vasculoprotective properties of the endothelial glycocalyx
124
Interestingly, Nieuwdorp et al. demonstrate that the glycocalyx constitutes a large
intravascular compartment of up to 2 liters in healthy volunteers (Figure 6.3), which can be
estimated in a reproducible fashion. More importantly, they show that hyperglycemic
clamping elicits a profound reduction in glycocalyx volume coinciding with increased
circulating plasma levels of glycocalyx constituents like hyaluronan, consistent with release
of glycocalyx constituents into the circulation upon hyperglycemia. These disturbances are
accompanied by impaired flow mediated dilation as well as activation of the coagulation
system. Taken in conjunction with available experimental data, the present findings imply
that glycocalyx perturbation may be a novel mechanism contributing to enhanced
vulnerability of the vessel wall under hyperglycemic conditions. Similarly, several other
research groups have reported endothelial dysfunction under hyperglycemic conditions (56,
57). Whereas impaired NO bioavailability has predominantly been adjudicated to direct
inactivation of NO by increased radical production (58, 59), the present finding provides us
with an alternative option. It has been acknowledged that the glycocalyx serves as part of the
endothelial mechanosensor, which translates intravascular shear stress into biochemical
activation of endothelial cells (9-11, 60). Accordingly, the release of NO by endothelial cells
in response to shear stress is abolished upon enzymatic removal of glycosaminoglycans from
the endothelial glycocalyx (9, 10, 60). It is tempting to speculate that loss of glycocalyx may
have contributed to the impaired shear-mediated NO release during hyperglycemia.
Effect of Hyperglycemia on Human Glycocalyx Volume
0
1
2
Control Hyperglycemia
Gly
coca
lyx
volu
me
(lite
rs)
P < 0.05
Effect of Hyperglycemia on Human Glycocalyx Volume
0
1
2
Control Hyperglycemia
Gly
coca
lyx
volu
me
(lite
rs)
P < 0.05Figure 6.3: Effect of 6h acute
hyperglycemia (16 mmol/l) on
systemic glycocalyx volume in
healthy human volunteers
(Reproduced from reference 51).
Chapter 6 Vasculoprotective properties of the endothelial glycocalyx
125
6.7 Summary
Currently available evidence in animal models shows that the glycocalyx exerts a wide array
of anti-atherogenic effects via inhibition of coagulation and leukocyte adhesion, by
contributing to the vascular permeability barrier as well as by mediating shear stress induced
NO release. In agreement with the hypothesis that glycocalyx perturbation increases
endothelial vulnerability, the dimension of the endothelial glycocalyx at atherogenic lesion
prone sites is significantly smaller than its dimension on the luminal surface of the
atheroprotected common carotid artery. Furthermore, focal sites with diminished glycocalyx
dimension appear to be more sensitive to further provocation by systemic atherogenic stimuli.
Most intriguing is the finding that relatively great systemic glycocalyx volumes in healthy
volunteers are significantly reduced upon exposure to atherogenic risk factors. As yet, this
finding does not prove causality of glycocalyx derangement in mediating elevated
atherogenic risk and future studies need therefore to address whether restoration of the
glycocalyx in itself is able to slow down or even reverse the progression of atherosclerotic
disease. Nevertheless, systemic glycocalyx measurement may hold a promise as a diagnostic
tool to estimate cardiovascular risk as well as to evaluate the impact of cardiovascular risk
lowering or even glycocalyx restoring therapeutic interventions.
Chapter 6 Vasculoprotective properties of the endothelial glycocalyx
126
References
1. Cheung BM, Lauder IJ, Lau CP, Kumana CR. Meta-analysis of large randomized controlled trials to evaluate the impact of statins on cardiovascular outcomes. Br J Clin Pharmacol. 2004; 57(5):640-51.
2. Pries AR, Secomb TW,Gaehtgens P. The endothelial surface layer. Pflugers Arch. 2000; 440 (5):653-66 3. Lipowsky HH. Microvascular rheology and hemodynamics. Microcirculation 2005; 12(1): 5-15 4. Van den Berg B, Vink H, Spaan JA. The endothelial glycocalyx protects against myocardial edema. Circ
Res 2003;(92);592-4 5. van Haaren PM, van Bavel E, Vink H, Spaan JA. Localization of the permeability barrier to solutes in
isolated arteries by confocal microscopy. Am J Physiol 2003;285(6):H2848-56. 6. van den Berg BM, Spaan JAE, Rolf TM, Vink H. Atherogenic region and diet diminish glycocalyx
dimension and increase intima media ratios at the murine carotid artery bifurcation. Am J Physiol 2006; 290:H915-20.
7. Henry CB, Duling BR. Permeation of the luminal capillary glycocalyx is determined by hyaluronan. Am J
Physiol 1999;277(2Pt2):H508-14. 8. Weinbaum S, Zhang X, Han Y, Vink H, Cowin SC. Mechanotransduction and flow across the endothelial
glycocalyx. Proc Natl Acad Sci U S A. 2003;100(13):7988-95. 9. Mochizuki S, Vink H, Hiramatsu O, Kajita T, Shigeto F, Spaan JA, Kajiya F. Role of hyaluronic acid in
shear induced endothelium derived nitric oxide release. Am J Phys 2003;285(2):H722-6 10. Florian JA, Kosky JR, Ainslie K, Pang Z, Dull RO, Tarbell JM. Heparan sulfate proteoglycan is a
mechanosensor on endothelial cells. Circ Res. 2003;93(10):e136-42. 11. Thi MM, Tarbell JM, Weinbaum S, Spray DC. The role of the glycocalyx in reorganization of the actin
cytoskeleton under fluid shear stress: a bumper-car model. Proc Natl Acad Sci U S A. 2004;101(47):16483-8.
12. Li Q, Bolli R, Qiu Y, Tang XL, Murphree SS, French BA. Gene therapy with extracellular superoxide
dismutase attenuates myocardial stunning in conscious rabbits. Circulation. 1998;98(14):1438-48. 13. Maczewski M, Duda M, Pawlak W, Beresewicz A. Endothelial protection from reperfusion injury by
ischemic preconditioning and diazoxide involves a SOD-like anti-O2- mechanism. J Physiol Pharmacol. 2004;55(3):537-50.
14. Libby P. Inflammation in atherosclerosis. Nature. 2002;420(6917):868-74. 15. Luft JH. Fine structure of capillary and endocapillary layer as revealed by ruthenium red. Microcirc Symp
Fed Proc 1966;25:1773-1783. 16. Baldwin AL, Winlove CP. Effects of perfusate composition on binding of ruthenium red and gold colloid to
glycocalyx of rabbit aortic endothelium. J Histochem Cytochem 1984;32:259-266.
Chapter 6 Vasculoprotective properties of the endothelial glycocalyx
127
17. Beresewicz A, Czarnowska E, Maczewski M. Ischemic preconditioning and superoxide dismutase protect against endothelial dysfunction and endothelium glycocalyx disruption in the postischemic guinea-pig hearts. Mol Cell Biochem 1998;186:87-97.
18. Haldenby KA, Chappell DC, Winlove CP, Parker KH and Firth JA. Focal and regional variations in the
composition of the glycocalyx of large vessel endothelium. J Vasc Res 1994;31:2-9. 19. Sims DE, Horne MM. Non-aqueous fixative preserves macromolecules on the endothelial cell surface: an in
situ study. Eur J Morphol 1993;32:59-64. 20. Ward BJ, Donnelly JL. Hypoxia induced disruption of the cardiac endothelial glycocalyx: implications for
capillary permeability. Cardiovasc Res 1993;27:384-389. 21. Rostgaard J, Qvortrup K. Electron microscopic demonstrations of filamentous molecular sieve plugs in
capillary fenestrae. Microvasc Res 1997;53:1-13. 22. Klitzman B, Duling BR. Microvascular hematocrit and red cell flow in resting and contracting striated
muscle. Am J Physiol 1979;237:H481-H490. 23. Desjardins C, Duling BR. Heparinase treatment suggests a role for the endothelial cell glycocalyx in
regulation of capillary hematocrit. Am J Physiol 1990;258:H647-H654. 24. Vink H, Duling BR. Identification of distinct luminal domains for macromolecules, erythrocytes, and
leukocytes within mammalian capillaries. Circ Res 1996;79(3): 581-589. 25. Damiano ER. The effect of the endothelial-cell glycocalyx on the motion of red blood cells through
capillaries. Microvasc Res 1998;55:77-91. 26. Feng J, Weinbaum S. Lubrication theory in highly compressible porous media: the mechanics of skiing,
from red cells to humans. J Fluid Mech 2000;422:281-317. 27. Secomb TW, Hsu R, Pries AR. Motion of red blood cells in a capillary with an endothelial surface layer:
effect of flow velocity. Am J Physiol 2001;281:H629-H636. 28. Van Teeffelen JWGE, Dekker S, Fokkema DS, Siebes M, Vink H, Spaan JAE. Hyaluronidase treatment of
coronary glycocalyx increases reactive hyperemia but not adenosine hyperemia in dog hearts. Am J Physiol 2005;289:H2508-13.
29. Constantinescu AA, Vink H, Spaan JA. Elevated capillary tube hematocrit reflects degradation of
endothelial cell glycocalyx by oxidized LDL. Am J Physiol 2001;280:H1051-H1057. 30. Vink H, Constantinescu AA, Spaan JA. Oxidized lipoproteins degrade the endothelial surface layer :
implications for platelet-endothelial cell adhesion. Circulation 2000; 101(13):1500-1502. 31. Adamson, RH. Permeability of frog mesenteric capillaries after partial pronase digestion of the endothelial
glycocalyx. J Physiol 1990;428:1-13. 32. Huxley VH, Williams DA. Role of a glycocalyx on coronary arteriole permeability to proteins: evidence
from enzyme treatments. Am J Physiol 2000; 278(4):H1177-85. 33. Gorog P, and Born GVR. Uneven distribution of sialic acids on the luminal surface of aortic endothelium.
Br J Exp Pathol 1983; 64: 418-424.
Chapter 6 Vasculoprotective properties of the endothelial glycocalyx
128
34. Allen PL, Mowbray PI, Lee AJ, Fowkes FG. Relationship between carotid intima-media thickness and
symptomatic and asymptomatic peripheral arterial disease: the Edinburgh Artery Study. Stroke 1997; 28: 348-53.
35. O’Leary DH, Polak JF, Kronmal RA, Kittner SJ, Bond MG, Wolfson SK Jr, Bommer W, Price TR, Gardins
JM, and Savage PJ. Distribution and correlates of sonographically detected carotid artery disease in the Cardiovascular Health Study. Stroke 1992; 23: 1752-1760.
36. Poli A, Tremoli E, Colombo A, Sirtori M, Pignoli P, and Paoletti R. Ultrasonographic measurement of the
common carotid artery wall thickness in hypercholesterolemic patients: a new model for the quantitation and follow-up of preclinical atherosclerosis in living human subjects. Atherosclerosis 1988; 70: 253-261.
37. Davies PF. Flow-mediated endothelial mechanotransduction. Physiol Rev 1995; 76: 519-560. 38. Liepsch D. An introduction to biofluid mechanics – basic models and applications. J Biomech 2002; 35:
415-435.
39. Fry DL. Arterial intimal-medial permeability and coevolving structural responses to defined shear-stress exposures. Am J Physiol 2002; 283: H2341-H2355.
40. Woolf N. The arterial endothelium. In: Pathology of Atherosclerosis, edited by Crawford ST. Butterworths & Co Ltd. London, England., 1982, p. 25-45.
41. Wang S, Okano M, and Yoshida. Ultrastructure of endothelial cells and lipid deposition on the flow dividers of branchiocephalic and left subclavian arterial bifurcations of the rabbit aorta. J.Jpn.Atheroscler.Soc. 1991; 19, 1089-1100.
42. Gouverneur M, Spaan JA, Pannekoek H, Fontijn RD, Vink H. Fluid shear stress stimulates incorporation of hyaluronan into the endothelial cell glycocalyx. Am J Physiol Heart Circ Physiol. 2006; 290: H458-62.
43. Arisaka T, Mitsumata M, Kawasumi M, Tohjima T, Hirose S and Yoshida Y. Effects of shear stress on glycosaminoglycan synthesis in vascular endothelial cells. Ann N Y Acad Sci 1995; 748: 543-554.
44. Elhadj S, Mousa SA and Forsten-Williams K. Chronic pulsatile shear stress impacts synthesis of proteoglycans by endothelial cells: Effect on platelet aggregation and coagulation. J Cell Biochem 2002; 86: 239-250.
45. Constantinescu, AA, Vink H, and Spaan JAE. Endothelial cell glycocalyx modulates immobilization of leukocytes at the endothelial surface. Arterioscler Thromb Vasc Biol 2003; 23: 1541-1547.
46. Henry CB, and Duling BR. TNF-α increases entry of macromolecules into luminal endothelial cell glycocalyx. Am J Physiol 2000; 279: H2815-H2823.
47. Subramanian SV, Fitzgerald, and Bernfield M. Regulated shedding of syndecan-1 and -4 ectodomains by thrombin and growth factor receptor activation. J Biol Chem 1997; 272: 14713-14720.
48. Nieuwdorp M, van Haeften TW, Gouverneur MCLG, Mooij HL, van Lieshout MHP, Levi M, Meijers JCM,
Holleman F, Hoekstra JBL, Vink H, Kastelein JJP and Stroes ESG. Loss of Endothelial Glycocalyx during Acute Hyperglycemia Coincides with Endothelial Dysfunction and Coagulation Activation in vivo. Diabetes 2006; 55:480-6.
49. Haffner SM, Lehto S, Ronnemaa T, Pyorala K, Laakso M. Mortality from coronary heart disease in subjects
Chapter 6 Vasculoprotective properties of the endothelial glycocalyx
129
with type 2 diabetes and in nondiabetic subjects with and without prior myocardial infarction. N Engl J Med 1998; 339(4):229-34.
50. Nathan DM, Lachin J, Cleary P, Orchard T, Brillon DJ, Backlund JY, O'Leary DH, Genuth S. Intensive
diabetes therapy and carotid intima-media thickness in type 1 diabetes mellitus. N Engl J Med. 2003; 348(23):2294-303.
51. Ceriello A, Giugliano D, Dello Russo P, Passariello N, Saccomanno F, Sgambato S. Glycosaminoglycans in
human diabetes. Diabetes Metab. 1983;9(1):32-4. 52. Ikegami-Kawai M, Okuda R, Nemoto T, Inada N, Takahashi T. Enhanced activity of serum and urinary
hyaluronidases in streptozotocin-induced diabetic Wistar and GK rats. Glycobiology. 2004;14(1):65-72 53. Krentz AJ, Boyle PJ, Macdonald LM, Schade DS. Octreotide: a long-acting inhibitor of endogenous
hormone secretion for human metabolic investigations. Metabolism. 1994; 43(1):24-31. 54. Title LM, Cummings PM, Giddens K, Nassar BA. Oral glucose loading acutely attenuates endothelium-
dependent vasodilation in healthy adults without diabetes: an effect prevented by vitamins C and E. J Am Coll Cardiol. 2000; 36(7):2185-91.
55. Timimi FK, Ting HH, Haley EA, Roddy MA, Ganz P, Creager MA. Vitamin C improves endothelium-
dependent vasodilation in patients with insulin-dependent diabetes mellitus. J Am Coll Cardiol. 1998; 31(3):552-7
56. Cosentino F, Luscher TF. Tetrahydrobiopterin and endothelial function. Eur Heart J. 1998;19 Suppl G:G3-
8. 57. Pohl U, De Wit C, Gloe T. Large arterioles in the control of blood flow: role of endothelium-dependent
dilation. Acta Physiol Scand. 2000; 168(4):505-10.
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Summary
131
Summary
In chapter 1 the endothelial glycocalyx is introduced as a highly negatively charged,
organized mesh on the endothelial cell surface, consisting of membranous glycoproteins,
proteoglycans, glycosaminoglycans and associated plasma proteins, situated at the luminal
side of blood vessels. It functions as a protective barrier between endothelial cells and
flowing blood by contributing to the endothelial permeability barrier, binding anticoagulation
factors, modulating leukocyte interactions with the endothelium, limiting myocardial edema,
and becoming in focus for its role as a mechano-shear sensor and its role in clinical
manifestations such as atherosclerosis and diabetes.
Chapter 2 shows that fluid shear stress stimulates incorporation of hyaluronan into the
endothelial cell glycocalyx. Exposure of cultured endothelial cells for 24 hrs to a shear stress
of 10 dynes/cm2 stimulates incorporation of glucosamine-containing GAGs in the glycocalyx,
which is accompanied by elevated levels of glucosamine-containing GAGs in the
supernatant. These increases were confirmed by direct demonstration of increased hyaluronan
concentrations in the glycocalyx and in the supernatant, as well as by a 3 fold increase in the
incorporation of hyaluronan binding protein in the glycocalyx.
Chapter 3 demonstrates that hyperglycemia attenuates flow-induced hyaluronan production
by cultured EC-RF24 endothelial cells and it is demonstrated that low shear stress levels of 1
dyne/cm2 are not sufficient to stimulate endothelial hyaluronan synthesis, which is consistent
with previous findings of very little endothelial cell hyaluronan synthesis under static
conditions. Furthermore, we report that overnight incubation of static endothelial cells with
hyperglycemic medium results in increased initial shedding of hyaluronan from the
endothelial surface upon start of the exposure of endothelial cells to fluid shear stress. The
increased hyperglycemic shedding of endothelial hyaluronan is followed by an impaired
stimulation of 10dynes/cm2 fluid shear stress to induce endothelial hyaluronan synthesis.
Hyperglycemic shear induced hyaluronan synthesis is reduced by about 50% compared to
normoglycemic controls, which may contribute to attenuation of the protective properties of
the glycocalyx in diabetes and the associated increased vascular vulnerability for disease.
Summary
132
Chapter 4 shows hyperglycemic glycocalyx loss is secondary to an initial transient increase in
glycocalyx synthesis, by monitoring the dynamics of glycocalyx synthesis and shedding in
cultured endothelial cells exposed to hyperglycemic conditions for 6 hours. Consistent with
the clinical findings, hyperglycemia reduced incorporation of glucosamine and sulfate into
the glycocalyx of hyperglycemic endothelial cells compared to normoglycemic paired
controls. In addition, the dynamics of supernatant glycocalyx shedding was remarkably
similar to the dynamics of human plasma hyaluronan levels, showing an initial transient rise
within the first hour, followed by a more slowly, steady increase in glycocalyx shedding.
These findings demonstrate that the initial transient shedding increase reflects a transient
hyperglycemic increase in glycocalyx synthesis, while the latter increases in glycocalyx
shedding are responsible for the net hyperglycemic loss of glycocalyx after 6 hours.
Chapter 5. In this study we set out to evaluate the impact of hyperglycemia on the glycocalyx
in healthy volunteers, measuring changes in systemic glycocalyx volume before and 6 hours
after normo-insulinemic, hyperglycemic clamping, changes in plasma hyaluronan,
endothelial function as well as changes in coagulation parameters and elucidating the role of
reactive oxygen species using anti-oxidant N-acetylcysteine (NAC). Results show that the
glycocalyx constitutes a large intravascular compartment in healthy volunteers, which can be
estimated in a reproducible fashion in vivo. More importantly, we show that hyperglycemic
clamping elicits a profound reduction in glycocalyx volume coinciding with increased
circulating plasma levels of glycocalyx constituents like hyaluronan, which is consistent with
release of glycocalyx constituents into the circulation. These disturbances are accompanied
by impaired flow mediated dilation as well as activation of the coagulation system. Infusion
of the antioxidant NAC prevented this glycocalyx perturbation, indicating that generation of
reactive oxygen species contributes to the glycocalyx perturbation under hyperglycemic
conditions.
Chapter 6 Vasculoprotective Properties of the Endothelial Glycocalyx: Effect of Fluid Shear
Stress. This review gives an overview of the relation between fluid shear stress and the
endothelial glycocalyx. We addressed the hypothesis that modulation of glycocalyx synthesis
by fluid shear stress may contribute to thinner glycocalyces, and therefore more vulnerable
endothelium, at lesion prone sites of arterial bifurcations. Finally, we discussed the effects of
known atherogenic stimuli such as hyperglycemia on whole body glycocalyx volume in
humans and its effect on endothelial function.
Nederlandse Samenvatting
133
Nederlandse Samenvatting
In Hoofdstuk 1 wordt de endotheliale glycocalyx geintroduceerd als een negatief geladen,
georganiseerd netwerk op het endotheliale cel oppervlak. Het bestaat uit glycoproteinen,
proteoglycanen, glycosaminoglycanen (GAG) and geassocieerde plasma eiwitten en bevindt
zich aan de binnenzijde van de vaatwand. Het functioneert als een beschermende barriëre
tussen endotheelcellen en het stomend bloed en speelt een belangrijke rol in de permeabiliteit,
binden van coagulatiefactoren, bewerken van leukocyte interacties met het endotheel,
bescherming tegen myocardiaal oedeem and krijgt steeds meer aandacht in verband met zijn
rol als mechanosensor en in klinische settings zoals atherosclerose en diabetes.
Hoofdstuk 2 laat zien dat voeistof afschuifspanningen de inbouw van hyaluronan in de
endotheliale glycocalyx stimuleert. De blootstelling van endotheelcellen aan 24 uur
afschuifspanningen van 10 dynes/cm2 stimuleert de incorporatie van glucosamine bevattende
glycosaminoglycanen in de glycocalyx, welke vergezeld wordt met toenemende glucosamine
bevattende glycvosaminoglycanen in het supernatant. Deze toenamen werden bevestigd door
toegenomen hyaluronan concentraties in de glycocalyx en in het supernatant en door 3-
voudige toename van hyaluronan bindend eiwit in de glycocalyx.
Hoofdstuk 3 laat zien dat hyperglykemie de flow-geinduceerde hyaluronan productie van
gekweekte ECRF24 cellen verzwakt en dat lage vloeistofafschuivingen van 1 dyne/cm2 niet
voldoende zijn voor het stimuleren van endotheliale hyaluronan synthese, welke consistent is
met eerdere bevindingen van weinig endotheliale cel hyaluronan synthese onder statische
kweek condities. Verder vinden we dat een overnacht incubatie van statische endotheel cellen
met hyperglycemisch medium de de initiële hyaluronan uitscheiding van het endotheliale
oppervlak stimuleert alsvorens blootstelling aan de vloeistofschuifspanningen. De
toegenomen hyperglykemische uitscheiding van endotheliaal hyaluronan word gevolgd door
een verstoorde stimulatie van 10dynes/cm2 vloeistof afschuifspanning voor toenane van
endotheliale hyaluronan synthesis. Hyperglykemie vloeistof afschuifspanning geïnduceerde
hyaluronan synthese is 50% afgenomen vergeleken met normoglykemische controles, welke
mogelijk bijdraagt aan de verzwakking van de beschermende eigenschappen van de
Nederlandse Samenvatting
134
glycocalyx in diabetes en de geassocieerde toegenomen vasculaire kwetsbaarheid voor
ziekten.
Hoofdstuk 4 laat zien dat hyperglykemische glycocalyx verlies op de tweede plaats komt na
een initiele toename in glycocalyx synthesis door het bestuderen van de dynamiek van
synthese en uitscheiden in gekweekte endotheelcellen blootgesteld aan hyperglycemische
condities gedurende 6 uur. Consistent met de klinische bevindingen, hyperglycemia
veroorzaakte een afname van inbouw van glucosamine en sulfaat in de glycocalyx van
hyperglykemische endotheel cellen vergeleken met normoglykemische controles. Ook was de
dynamica van de uitgescheide glycocalyx in het supernatant opmerkelijk vergelijkbaar met de
dynamiek van plasma hyaluronan hoeveelheden, welke een initiële toename laat zien
gedurende het eerste uur, gevolgd door een meer langzame, constante toename in glycocalyx
uitscheiding. Deze bevindingen laten zien dat de initiële uitscheidingstoename, een toename
in hyperglycemische toename in glycocalyx synthese reflecteert, terwijl de latere toenamen in
glycocalyx uitscheiding verantwoordelijk zijn voor de netto hyperglykemisch verlies van
glycocalyx na 6 uur.
Hoofdstuk 5. In deze studie evalueren we de impact van hyperglykemie op de glycocalyx van
gezonde vrijwilligers. We meten veranderingen in systemisch glycocalyx volume voor en na
6 uur normo-insulinemisch, hyperglykemische klemming, veranderingen in plasma
hyaluronan, endotheelfunctie alswel veranderingen in coagulatie parameters en verheldering
van de rol van oxidatieve radicalen met behulp van antioxidant N-acetylcysteine (NAC).
Resultaten laten zien dat de glycocalyx beschikt over een groot intravascular compartiment in
gezonde vrijwilligers welke op een reproduceerbare manier in vivo kan worden geschat.
Belangrijker nog, we laten zien dat hyperglykemisch klemmen een duidelijke afname in
glycocalyx volume teweegbrengt, welke samen gaat met een toename in circulerende plasma
niveaus van glycocalyx bestanddelen zoals hyaluronan, wat consistent is met het vrijkomen
van glycocalyx bestanddelen in de circulatie. Deze verstoringen worden vergezeld door een
verstoorde flow-gemedieerde dilatatie alswel activatie van het coagulatie systeem. Infusie
van het antioxidant NAC voorkwam deze glycocalyx verstoringen, wat aangeeft dat
aanwezigheid van oxidatieve stress bijdraagt aan de glycocalyx verstoring onder glykemische
conditie.
Nederlandse Samenvatting
135
Hoofdstuk 6 Dit hoofdstuk geeft een overzicht van de relatie tussen voeistof
afschuifspanningen en de endotheliale glycocalyx. We behandelen de hypothese dat
modulatie van glycocalyx synthese door voeistofafschuifspanningen zou kunnen bijdragen tot
kleinere glycocalyx dimensies and daardoor meer kwetsbaar endotheel op arteriële
bifurcaties, de plaatsen gevoelig voor lesies. Tenslotte behandelen we de effecten van
bekende atherogene stimuli zoals hyperglycemie op heel lichaam glycocalyx volume in
mensen en subsequent effect op endotheliale functie.
136
Dankwoord
137
Dankwoord
Het lijkt alweer een leven geleden dat ik Hans Vink belde vanuit Boston met de vraag of hij
me misschien kon assisteren met het vinden van een gepaste wetenschappelijke baan in de
Nederland. Tot die tijd had ik het zo druk gehad met avontuurlijke buitenlandse ervaringen
dat het Nederlandse systeem me vrij onbekend was. Eenmaal terug in Nederland was het al
snel bekeken. Een afspraak met Hans, door mij ingeschatte tijd een uurtje gaf me een
ervaring wat de komende jaren een duidelijk patroon zou zijn, met Hans spreek je namelijk
geen uurtje. Zo’n vijf uur later stond ik verdwaasd weer buiten voor het AMC met het
voorstel Hans te assisteren de wonderen van de glycocalyx te ontrafelen.
‘The rest is history’ wordt dan wel eens gezegd en het resultaat van mijn aandeel in het
glycocalyx onderzoek ligt voor u in de vorm van dit proefschrift. Dit doet de afgelopen jaren
enorm tekort. Er is heel wat gebeurd en er zijn wat strijden gestreden, zowel intellectueel als
mentaal. Soms werd het zo donker maar altijd wist je me weer te wijzen op een lichtje, soms
als een speldenprik zo klein. Hans, je inspireert me. Alle dank voor je inzichten, steun en
vriendschap.
Naast mijn dagelijkse begeleiding in de vorm van copromotor Hans Vink werd de
wetenschappelijke kwaliteitscontrole overkoepeld door mijn promotoren Jos Spaan en Hans
Pannekoek. Mijn dank aan jullie dat het me is gelukt om glycocalyx promotieonderzoek met
een biochemisch tintje bij de afdeling medische fysica te kunnen voltooien.
Mijn promotiecommissie; Profs. Daemen, van Hinsbergh, Ince en Dr. Horrevoets, mijn dank
voor het kritisch beoordelen van mijn proefschrift en ik zal graag met u van gedachten
wisselen aangaande de bevindingen beschreven in dit proefschrift. Prof. Huxley, thank you
for participating in my commission and I look forward in discussing my findings with you.
Mijn paranimfen Paul en Radha. Ik ben trots dat jullie me bij gaan staan en beiden ontroerden
me met jullie reactie op mijn vraag.
Dankwoord
138
Van de afdeling medische fysica, over al die jaren: de glycocalyx unit, Alina, Paul, Bernard,
Jurgen, Andreas, Nadja, Judith, Hans Mooij, Carin, Titia; CARVAS divisie Ed, Esther, Oana,
Radha, Erik, Adriaan, Jeroen, René, Isabelle, Boaz, Dirk, Terry (Scranton USA), Zhila, Sjak,
Maria, Christina, Michiel, Jenny (Delft), Annemiek, Medtech tak Cees, Jeroen, Marcel,
Henk, Bart, Marc, Niels, Robert, Remmet, Geert, Jan Arie, Hugo, Nico, Jan, Oscar. Mijn
dank voor de adviezen, hulp, gezelligheid tijdens koffie, lunch, borrels en tijdens congressen
in exotische oorden als New Orleans, Washington DC, San Diego en Vught. Willekeurige
ontmoetingen bij de printer zorgen soms ook voor gezellige conversaties, over komende
vakantieplannen of even gezamenlijk ergeren aan de printer, computer of iets dergelijks.
Jetty, bedankt voor je assistentie en de mogelijkheid altijd even bij je binnen te kunnen lopen
bij je voor een praatje of om even een ei te leggen.
Van de afdeling biochemie Birgit, Ruud, Rob, Yvonne, Richard. Bedankt voor jullie hulp en
gastvrijheid als ik weer eens een apparaat, een stofje, cellen of advies nodig had.
Professor de Groot (UMC Utrecht), mijn dank voor het verschaffen van de flowkamer,
onmisbaar voor dit proefschrift.
MTO, Ton Meulemans voor de technische assistentie en ontwerp van de nieuwe flowkamer
aan de AIO wat soms vast niet meevalt.
Van het B-lab Frits, Guus, Henk, André, bedankt voor alle assistentie en bijstand in de
‘bunker’. Joop, ook voor alle lunches, swirls en koffie, wat hadden ik zonder gedaan?
De directe samenwerking met de afdeling vasculaire geneeskunde in het bijzonder Eric Stroes
en Max Nieuwdorp maakt het mogelijk om klinische bepalingen aangaande het glycocalyx
volume te meten. Deze bevindingen gekoppeld aan de celkweek resultaten hebben een enorm
rijke bron aan inzichten verschaft. Ik ben zeer trots betrokken te zijn geweest bij dit
onderzoek van celkweek tot kliniek.
Ook van vasculaire geneeskunde, in het beginstadium, Jan Albert, Jaap, Karin, Floreanne,
bedankt voor de hulp en assistentie.
Dankwoord
139
Vrienden, Nic & Eduard, Rebecca, Cindy, Miruna, Linda zorgen voor de nodige afleiding in
de vorm van shoppen, een filmpje, een etentje, een uitje of gewoon even een borrel.
John, my travel buddy. What would I have done without those fantastic trips? For sure I
would have had more trouble finding an appropriate cover for this dissertation. Thank you for
your friendship.
Jan & Lucy Kerstens voor fijne dineetjes en goede gesprekken, zo ook mevrouw Folkers voor
alle geduld en verhelderende observaties.
Ten slotte, mijn familie, het viel soms allemaal niet mee. Arigo, broer, bedankt voor je steun
en de nodige goede grappen om alles wat te relativeren. Mam, voor alle liefde en het altijd
klaarstaan met mijn lievelingsgerechten als troost. En lieve Pa, voor alle steun en opbeurende
telefoontjes als het weer eens mis ging. Samen komen we er altijd wel weer uit. Love you.
En als allerlaatste, Timo, voor alle knuffels.
Dankwoord
140
Curriculum Vitae
141
Curriculum vitae Mirella Gouverneur was born as Maria Cornelia Lucia Gerdina Gouverneur on May 4th 1971
in Waalwijk, The Netherlands. After primary school she attended the Walewyc MAVO for 4
years followed by 2 years HAVO on Dr. Mollercollege, both in Waalwijk. Her higher
education was conducted at the polytechnic faculty of the Hogeschool West Brabant at Dr.
Struijkeninstituut for in Etten Leur. During her internship she worked at FMC Bioproducts te
Rockland, Maine, USA on the project development of Reliant® precast gel system, after
which she finished her studies and obtained Bachelor of Science degree. She worked as a
technical teaching assistant at Cobbenhagen College in Tilburg before she started graduate
school in Medical Biotechnology in Etten Leur in collaboration with Demontfort University
in Leicester, Great Britain. Her internship she conducted research under supervision of Dr.
Erdjan Salih in the laboratory for the study of skeletal disorders and rehabilitation at the
department of Orthopedic Research of The Children’s Hospital, Harvard Medical School in
Boston, MA, USA. Her thesis was entitled: ‘Bone specific protein kinases and
phosphoproteins and their role in bone generation and repair’. After receiving the Master of
Science degree she continued her research in Boston and was involved in various projects.
After 3 years she returned to The Netherlands and worked as a biochemical cell culture
research analist at the Department of Medical Physics at the Academic Medical Center in
Amsterdam. After one year she started her PhD project, studying the biochemical properties
of the endothelial glycocalyx at the Department of Medical Physics of the University of
Amsterdam under the supervision of Hans Vink, Jos Spaan en Hans Pannekoek, resulting in
this dissertation.
Curriculum Vitae
142
Publication list
143
Publication list
2006 Vasculoprotective Properties of the Endothelial Glycocalyx: Effects of Fluid Shear
Stress
Mirella Gouverneur, Bernard van den Berg, Max Nieuwdorp, Erik Stroes, Hans Vink.
Journal of Internal Medicine (2006) 259:393-400
2006 Loss of endothelial glycocalyx during acute hyperglycemia coincides with endothelial
dysfunction and coagulation activation in vivo.
Max Nieuwdorp, Timon W van Haeften, Mirella CLG Gouverneur, Hans L Mooij,
Miriam HP van Lieshout, Marcel Levi, Joost CM Meijers, Frits Holleman, Joost BL
Hoekstra, Hans Vink, John JP Kastelein and Erik SG Stroes.
Diabetes (2006) Feb;55(2):480-6
2006 Fluid shear stress stimulates incorporation of hyaluronan into the endothelial cell
glycocalyx
Mirella Gouverneur, Jos AE Spaan, Hans Pannekoek, Ruud D Fontijn and Hans Vink
American Journal of Physiology (2006) Jan; 290(1): H458-2
2000 An investigation into the role of oxygen free radical scavengers in preventing PMMA
necrosis in an osteoblast cell culture.
J.G. Kennedy, P.O. Grady, D.R. McCarthy, S.J. Johnson, D. Hynes, M. Walsh, F.
McManus, E. Salih, M. Gouverneur, J. Fitzpatrick
Orthopedics (2000) 23(5) 481-5
1998 Protein kinases that phosphorylate the extra-cellular matrix phosphoproteins of
mineralizing tissues.
Erdjan Salih, John C. Huang, Mirella Gouverneur, Dieu Ly, James Mah and Melvin
Glimcher
Biological Mechanisms of Tooth Eruption, Resorption and Replacement by Implants.
Eds. Z. Davidovitch and J. Mah. 1998 Harvard Society for the Advancement of
Orthodontics p.133-147
Publication list
144
1998 Enamel specific protein kinases and the state of phosphorylation of purified
amelogenins.
E. Salih, J.C. Huang, E. Strawich, M. Gouverneur and M. Glimcher
Connect Tissue (1998) Vol 38 (1-4) p.225-235
1996 Isolation and identification of major protein of bovine bone responsible for the
predominant phosphorylation of bovine bone osteopontin and bone sialoprotein.
E. Salih, M. Gouverneur and M. Glimcher.
J Bone Miner Res (1996) Volume 11 supplement 1
Conference abstracts
145
Conference abstracts
2005 FASEB, Translating the genome, San Diego, USA March 2005
Short-term hyperglycemia diminishes the endothelial glycocalyx of cultured cells
Gouverneur M., Spaan J.A.E., Vink H.
Faseb Journal (2005) 18(4)A632
Selected oral & poster presentation
2004 FASEB, Translating the genome, Washington, USA March 2004
Shear stress stimulates synthesis of glucosamine containing glycosaminoglycans of
the endothelial cell glycocalyx
Gouverneur M., Spaan J.A.E., Vink H.
Faseb Journal (2004) 18(4)A632
Poster presentation
2003 FASEB, Translating the genome, San Diego, USA March 2003
Binding of lipoprotein lipase to cell surface glycocalyx is determined by
glycosaminoglycan synthesis, sugar sulfation patterns and fluid shear stress
Gouverneur M., Vink H.
Faseb Journal (2003) 18(4)A632
Selected oral & poster presentation
2002 Federation American Society of Experimental Biology (FASEB), Translating the
genome, New Orleans, USA March 2002
Oxidized low density lipoproteins attenuate biosynthesis of membrane bound sulfated
Gouverneur M., Spaan J.A.E., Vink H.
Faseb Journal (2002) 18(4)A632
Poster presentation
1996 Isolation and identification of major protein kinase of bovine bone responsible for the
predominant phosphorylation of bovine bone osteopontin and bone sialoprotein
Salih E., Gouverneur M., Glimcher M.J.
Journal of Bone and Mineral Research. (1996) Abstract M446
146
Mirella Gouverneur Notes
147
Mirella Gouverneur Notes
148
Mirella Gouverneur Notes
149
Mirella Gouverneur Notes
150
Mirella Gouverneur Notes
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Mirella Gouverneur Notes
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