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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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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

Printed by Ipskamp PrintPartners, Enschede

Cover: Detail of the Pacific Ocean near the Galapagos Islands, Ecuador

All rights reserved. No part of the material protected by this copyright may be reproduced or

utilized in any form or by any means, electronic or mechanical, including photocopying,

recording or by any information stirage and retrieval system, without permission of the

author.

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.

Chapter 1

General Introduction

Chapter 1 General Introduction

12


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).


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.


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.


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.


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.


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 )


4.7 µm5.4 µm


4.7 µm5.4 µm5.4 µm5.4 µm











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


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


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


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


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).


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.


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


Syndecan Glypican

Cell membrane


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.


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


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, posttranslational

Table 1.1

Examples of proteins that bind to glycosaminoglycans (GAGs).

Reprinted with permission from Varki et al.


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.


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.


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


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


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.


32

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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.

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.

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


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.


42


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,


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.


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


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.


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.


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).


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)


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).


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).


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).


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


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


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.


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.


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.


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.


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.


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.


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.


23. Woolf N. The arterial endothelium. In: Pathology of Atherosclerosis, edited by Crawford ST. Butterworths & Co Ltd. London, England., 1982, p. 25-45.

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


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.


62


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.


Chemicals


BRL, PBS pH: 7.4 from Fresenius Kabi and Fetal Bovine Serum (FBS) from Biowhittaker.


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.


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






For statistical analysis, two-way unpaired t-tests were used. A value of P < 0.05 was

considered statistically significant. Values are means ± SE.


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


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


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


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).


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


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)


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)


*

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)


*


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


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).


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.


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.


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.


71


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.


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.


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.



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.


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


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.


76


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.


Chemicals


BRL, PBS pH: 7.4 from Fresenius Kabi and Fetal Bovine Serum (FBS) from Biowhittaker.


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.


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%


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





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).


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).


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


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)


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).


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


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


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.


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.


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.


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.


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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.


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.


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

90


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.


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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


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).


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


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).


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


97

immuno-luminimetric assay (ILMA) (Immulite insuline®) on Immulite 2000 (DPC,

Diagnostic Product Corporation).


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.


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 %).


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.


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.


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.


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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.


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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.


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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


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.


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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.


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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

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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

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


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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.


114


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.


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


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





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).


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


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.


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..


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.


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


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.


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).


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.


126

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Summary

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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


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


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.


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.

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



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.


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



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

Mirella Gouverneur Notes

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