Vasculoprotective properties of the endothelial glycocalyx: Effects of fluid shear stress

Abstract

The endothelial glycocalyx exerts a wide array of vasculoprotective effects via inhibition of coagulation and leucocyte adhesion, by contributing to the vascular permeability barrier and by mediating shear stress-induced NO release. In this review, we will focus on the relationship 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 hyperglycaemia on whole body glycocalyx volume in humans and its effect on endothelial function.

Full text

SYMPOSIUM
Vasculoprotective properties of the endothelial glycocalyx:
effects of fluid shear stress
M. GOUVERNEUR
1
,B.VANDENBERG
2
,M.NIEUWDORP
3
,E.STROES
3
&H.VINK
1
From the
1
Department of Medical Physics, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands,
2
Department of
Molecular and Vascular Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA, and
3
Department of Vascular
Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands
Abstract. Gouverneur M, van den Berg B,
Nieuwdorp M, Stroes E, Vink H. (University of
Amsterdam, Amsterdam, the Netherlands; and
Harvard Medical School, Boston, MA, USA).
Vasculoprotective properties of the endothelial
glycocalyx: effects of fluid shear stress J Intern Med
2006; 259: 393–400.
The endothelial glycocalyx exerts a wide array of
vasculoprotective effects via inhibition of coagula-
tion and leucocyte adhesion, by contributing to the
vascular permeability barrier and by mediating
shear stress-induced NO release. In this review, we
will focus on the relationship between fluid shear
stress and the endothelial glycocalyx. We will ad-
dress 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 bifur-
cations. Finally, we will discuss the effects of known
atherogenic stimuli such as hyperglycaemia on
whole body glycocalyx volume in humans and its
effect on endothelial function.
Keywords: atherosclerosis, glycocalyx,
hyaluronan, shear stress.
Introduction
Cardiovascular disease is the major cause of mor-
tality worldwide 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 cardio-
vascular 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. As the endothelium is the first-
line defence 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, prot-
eoglycans, glycosaminoglycans (GAGs) and associ-
ated plasma proteins, is situated at the luminal side
of all blood vessels [2]. Its major constituents
comprise hyaluronic acid and the negatively
charged heparan sulphate proteoglycans. Glycoca-
lyx dimensions depend upon the balance between
biosynthesis and enzymatic or shear-dependent
Journal of Internal Medicine 2006; 259: 393–400 doi:10.1111/j.1365-2796.2006.01625.x
Ó2006 Blackwell Publishing Ltd 393

shedding of its components [3], and whereas
historically this layer was thought to be confined
to a thickness of only several nanometres, it has
recently been demonstrated to reach up to 0.5–
3lm intraluminally [4, 5]. This relatively large
dimension of the glycocalyx, which exceeds the
thickness of the endothelium and the length of
leucocyte adhesion molecules, has triggered
researchers to study its role in the course of
atherogenesis [6].
Numerous studies in both micro- and macrovas-
culature have demonstrated that constituents of the
glycocalyx, such as hyaluronan, are intimately
involved in vascular homeostasis, such as maintain-
ing the vascular permeability barrier [7] and regu-
lating the release of nitric oxide (NO) by serving as a
mechano-shear sensor for NO release [8–11]. In
addition, the glycocalyx harbours a wide array of
enzymes that might contribute to its vasculoprotec-
tive effect. Thus, extracellular superoxide dismutase
(ec-SOD), an enzyme which dismutates oxygen
radicals to hydrogen peroxide [12], is bound to
proteoglycans within the glycocalyx. Damage to the
glycocalyx is accompanied by increased shedding of
ec-SOD, which results in a dysbalance in favour of a
pro-oxidant state [13]. Collectively, these observa-
tions are of particular interest as altered vascular
permeability, attenuated NO-bioavailability and
redox dysregulation are amongst the earliest char-
acteristics of atherogenesis [14]. In spite of these
observations, it has proved difficult to show direct
relevance of the glycocalyx as a vasculoprotective
paradigm for larger vessels. The latter is predomin-
antly due to the fact that glycocalyx research has
traditionally focused at the microvasculature, in
which atherogenesis does not occur.
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 50–100 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 var-
iations in dimension and appearance [4, 16–20].
Fenestrated endothelium, in addition, was found to
have a combination of surface-bound stained struc-
tures, about 50–100 nm thick, and distinct filamen-
tous 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 (Fig. 1). In addition, co-local-
ization 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 microvascu-
lar- and systemic haematocrit [22] that could be
abrogated upon enzymatic treatment of the micro-
vascular network with heparinase [23]. From these
studies a 0.3–1 lm thick slow-moving plasma layer
on the endothelial cell surface consisting principally
of heparan-sulphate proteoglycans was thought to
be involved. The first visual evidence of a 0.4–
0.5 lm thick continuous endothelial cell surface
layer was provided by comparing the width of the
flowing plasma column containing large, anionic
fluorescein-labelled 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 pre-
dicted a glycocalyx thickness of 0.5–1 lmto
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 hyperaemia in a dog [28]. Enzymatic
degradation of the glycocalyx with hyaluronidase
has been shown to significantly increase the avail-
able intralumenal space for flowing blood [7].
Although various studies are consistent with the
concept that perturbation of the glycocalyx contri-
butes to increases in endothelial vulnerability upon
ischaemia/reperfusion [17], hypoxia [20], exposure
to low-density lipoproteins [29, 30] and atherogenic
shear stress profiles [6, 18], it has proved difficult
to show direct relevance of the glycocalyx as a
Ó2006 Blackwell Publishing Ltd Journal of Internal Medicine 259: 393–400
394 M. GOUVERNEUR et al.

vasculoprotective paradigm for larger vessels. The
latter is predominantly due to the fact that glyco-
calyx 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. [5] recently visualized a thick endothelial
glycocalyx in larger arteries in rats. The glycocalyx
in larger vessels has also been shown to decrease
extravasation of LDL particles into the subendothel-
ial space [31, 32]. Amongst others, these data imply
that also in the macrovasculature the glycocalyx
adds towards the vasculoprotective properties of the
vessel wall.
Glycocalyx at arterial bifurcations
Although reduced levels of surface-bound sialic
acids [33] and increased endothelial permeability
and susceptibility to atherosclerotic lesion forma-
tion [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 atherogen-
ic risk areas. Atherosclerotic lesions within the
arterial tree develop at predictable vessel geome-
tries, 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-to-media ratios (IMR).
Increases in IMR have been found to be associated
with increased cardiovascular risk factors and
atherosclerosis [34–36].
In a recent study, van den Berg et al. [6]
hypothesized that endothelial cells, which play a
central role in response to shear stress [37],
express a modified surface glycocalyx at high
Fig. 1 (a) Electron micrographs of goat capillary glycocalyx and (b) examples of the spatial heterogeneity of glycocalyx dimensions in the
vascular system (courtesy of Dr Bernard van den Berg).
Ó2006 Blackwell Publishing Ltd Journal of Internal Medicine 259: 393–400
SYMPOSIUM: FLUID SHEAR STRESS AND VASCULOPROTECTIVE PROPERTIES OF THE
GLYCOCALYX 395

atherogenic risk regions and, in turn, contribute to
predisposition of these arterial sites to atheroscler-
otic lesion formation. The endothelial glycocalyx
dimension was investigated by electron micro-
scopic 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 sites exposed to low-
and high-atherogenic risk, respectively [38]. As
shown in Fig. 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 IMR and
a thicker subendothelial layer, indeed confirming
that regional differences in glycocalyx dimen-
sion reflect variations in its vasculoprotective
capacity.
Previous studies have demonstrated that loss of
GAGs from the endothelial glycocalyx by enzyme
treatment is associated by oedema formation of the
subendothelial space [4], indicating that flow profile-
related modulation of the glycocalyx might contrib-
ute to the earlier observed progression from a
decreased endothelial barrier function into subse-
quent intimal oedema at vascular regions exposed to
disturbed flow [39]. Whether oedema 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 because of 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.
Mechanism of glycocalyx reduction at
high-risk regions
The fact that the glycocalyx dimension is significantly
diminished at the sinus region compared with 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 glyco-
calyx dimension are related to local variations in flow
profiles. It is well known that areas of high athero-
genic 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 endot-
helial glycocalyx indeed plays an important role in
mechanotransduction of fluid shear stress, very few
data are available on the relationship between fluid
shear stress and glycocalyx synthesis.
600
(a)
(b)
500
400
300
100
1000
800
600
400
200
0
0
C57B16
Common carotid
region
Internal carotid sinus
region
C57B*16ApoE*3
*
*
*
*
**
ApoE*3
C57B16
Common carotid
region
Internal carotid sinus
region
C57B*16ApoE*3 ApoE*3
Glycocalyx thickness (mm)
Subendothelial matrix
thickness (mm)
200
Fig. 2 (a) Glycocalyx dimension is diminished at the atheroprone
sinus region of the internal carotid artery in mice compared with
the atheroprotected common carotid artery. Systemic atherogenic
stimulation by a hyperlipidaemic, hypercholesterolaemic diet for
6 weeks in ApoE3-Leiden mice further diminishes the dimension
of the glycocalyx in the common carotid artery (from reference
van den Berg et al. [6]). *P< 0.05 compared with common region
of C57Bl6 mice on normal diet. (b) 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 van den
Berg et al. [6]). *P< 0.05 compared with common region of
C57Bl6 mice on normal diet.
Ó2006 Blackwell Publishing Ltd Journal of Internal Medicine 259: 393–400
396 M. GOUVERNEUR et al.

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 [40] and Wang et al.
[41] revealed thicker glycocalyces at high shear
regions compared with 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.
Recently, Gouverneur et al. [42] demonstrated
that exposure of cultured endothelial cells for 24 h
to a shear stress of 10 dynes cm
)2
stimulates
incorporation of glucosamine-containing GAGs in
the glycocalyx, which is accompanied by elevated
levels of glucosamine-containing GAGs in the super-
natant. These increases were confirmed by direct
demonstration of increased hyaluronan concentra-
tions in the glycocalyx and in the supernatant, as
well as by a threefold increase in the incorporation
of hyaluronan-binding protein in the glycocalyx. In
addition to its incorporation in hyaluronan, gluco-
samine is also incorporated in sulphated sugars like
heparan sulphate and chondroitin sulphate. In
addition, Arisaka et al. [43] used pig aortic endot-
helial cells exposed to shear stress levels of 15 and
40 dynes cm
)2
in a parallel flow chamber for
periods of 3, 6, 12 and 24 h. These authors
demonstrated increased synthesis of sulphated GAGs
after high shear stress of 40 dynes cm
)2
, and also a
small, but significant increase at 15 dynes cm
)2
.
Similarly, Elhadj et al. [44] exposed bovine aortic
endothelial cells for 7 days to <0.5 dynes cm
)2
prior to increasing shear rates for 3 days to 5 and
23 dynes cm
)2
. No significant increase in the net
sulphated GAG synthesis was detected, but a shift in
its size distribution was reported, indicating that
modulation of specific sulphation patterns may
occur despite limited effects on sulphated GAG
synthesis. In summary, these experiments demon-
strate that shear stress increases hyaluronan con-
tent in the endothelial glycocalyx, that shear stress
exposure alters the size distribution of endothelial
sulphated GAGs, and that high levels of shear stress
may also increase sulphated GAG synthesis.
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 hypercholester-
olaemia and/or hypertriglyceraemia on top of
pre-existing regional variations in glycocalyx-pro-
tective 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 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 tumour necrosis factor-a[45–47]. 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 addi-
tional atherogenic challenge results in endothelial
thickening and excessive swelling of the subendot-
helial space, in line with the proposed hypothesis
that vascular sites with diminished glycocalyx are
more vulnerable to pro-inflammatory and athero-
sclerotic sequelae.
Human glycocalyx measurements
To date, direct visualization of endothelial glycoca-
lyx in humans has been unsuccessful, mainly due to
the fact that the endothelial glycocalyx is a very
delicate structure depending critically on the pres-
ence of flowing plasma [2]. As a consequence, the
best way to measure the endothelial glycocalyx in
humans is to compare systemic intravascular distri-
bution volumes for glycocalyx permeable versus
glycocalyx impermeable tracers. Subtracting these
two volumes provides an estimate of whole body
glycocalyx volume [48].
At present, Nieuwdorp et al. [48] tried to answer
the question whether glycocalyx perturbation-medi-
ated vascular vulnerability contributes to the accel-
erated rate of atherogenesis in patients with type 1
diabetes. Whereas this is at least in part the
Ó2006 Blackwell Publishing Ltd Journal of Internal Medicine 259: 393–400
SYMPOSIUM: FLUID SHEAR STRESS AND VASCULOPROTECTIVE PROPERTIES OF THE
GLYCOCALYX 397

consequence of increased prevalence of traditional
cardiovascular risk factors, these cannot fully
explain the propensity towards cardiovascular com-
plications in diabetic patients [49]. Disease-specific
abnormalities, such as hyperglycaemia, may also
facilitate the development of vascular lesions in
these patients. Thus, hyperglycaemia has been
shown to induce a wide array of downstream effects,
which may adversely affect the protective capacity of
the vessel wall [50]. As increased degradation of
proteoglycans has indeed been demonstrated in
hyperglycaemic conditions [51, 52], the impact of
hyperglycaemia on the glycocalyx merits special
interest. Therefore, Nieuwdorp et al. recently set out
to evaluate the impact of hyperglycaemia on the
glycocalyx in healthy volunteers. Systemic glycoca-
lyx volume was measured before and 6 h after
normo-insulinaemic, hyperglycaemic clamping.
Interestingly, Nieuwdorp et al. demonstrated that
glycocalyx constitutes a large intravascular com-
partment of up to 2 L in healthy volunteers (Fig. 3),
which can be estimated in a reproducible fashion.
More importantly, they show that hyperglycaemic
clamping elicits a profound reduction in glycocalyx
volume coinciding with increased circulating plas-
ma levels of glycocalyx constituents like hyaluro-
nan, consistent with release of glycocalyx
constituents into the circulation upon hyperglycae-
mia. These disturbances are accompanied by im-
paired 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 hyperglycaemic conditions.
Similarly, several other research groups have repor-
ted endothelial dysfunction under hyperglycaemic
conditions [53, 54]. Whereas impaired NO bioavail-
ability has predominantly been adjudicated to direct
inactivation of NO by increased radical production
[55, 56], 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 endothel-
ial cells [9–11, 57]. Accordingly, the release of NO
by endothelial cells in response to shear stress is
abolished upon enzymatic removal of GAGs from the
endothelial glycocalyx [9, 10, 57]. It is tempting to
speculate that loss of glycocalyx may have contri-
buted to the impaired shear-mediated NO release
during hyperglycaemia.
Summary
Currently available evidence in animal models
shows that the glycocalyx exerts a wide array of
anti-atherogenic effects via inhibition of coagulation
and leucocyte 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 increa-
ses 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 dimin-
ished glycocalyx dimension appear to be more
sensitive to further provocation by systemic ather-
ogenic 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 athero-
sclerotic 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.
Effect of hyperglycaemia on human glycocalyx volume
2
1
0
Control Hyperglycaemia
P < 0.05
Glycocalyx volume (L)
Fig. 3 Effect of 6 h acute hyperglycaemia (16 mmol l
)1
) on sys-
temic glycocalyx volume in healthy human volunteers (repro-
duced from Nieuwdorp et al. [48]).
Ó2006 Blackwell Publishing Ltd Journal of Internal Medicine 259: 393–400
398 M. GOUVERNEUR et al.

Conflict of interest statement
No conflict of interest was declared.
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Correspondence: Hans Vink PhD, Department of Medical Physics,
Academic Medical Center, University of Amsterdam, Meibergdreef
15, 1105 AZ Amsterdam, The Netherlands.
(fax: 31 (0)20 6917233; e-mail: h.vink@amc.uva.nl).
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