Glycocalyx in Atherosclerosis-Relevant Endothelium Function and as a Therapeutic Target
Curr Atheroscler Rep
Glycocalyx in Atherosclerosis-Relevant Endothelium Function and as a Therapeutic Target
Ronodeep Mitra 0 1 2 4
Gerard Leland O'Neil 0 1 2 4
Ian Chandler Harding 0 1 2 4
Ming Jie Cheng 0 1 2 4
Solomon Arko Mensah 0 1 2 4
Eno Essien Ebong 0 1 2 4
0 Department of Chemical Engineering, Northeastern University , 360 Huntington Avenue 313 Snell Engineering Building, Boston, MA 02115 , USA
1 Department of Biology, Northeastern University , Boston, MA , USA
2 Department of Bioengineering, Northeastern University , Boston, MA , USA
3 Eno Essien Ebong
4 Department of Neuroscience, Albert Einstein College of Medicine , New York, NY , USA
Purpose of Review The cell surface-attached extracellular glycocalyx (GCX) layer is a major contributor to endothelial cell (EC) function and EC-dependent vascular health and is a first line of defense against vascular diseases including atherosclerosis. Here, we highlight our findings regarding three GCX-dependent EC functions, which are altered when GCX is shed and in atherosclerosis. We discuss why the GCX is a viable option for the prevention and treatment of atherosclerosis. Recent Findings GCX regulated EC activities such as barrier and filtration function, active cell-to-cell communication, and vascular tone mediation contribute to function of the entire vascular wall. Atheroprone vessel regions, including bifurcation sites, exhibit breakdown in GCX. This GCX degradation allows increased lipid flux and thereby promotes lipid deposition in the vessel walls, a hallmark of atherosclerosis. GCX degradation also alters EC-to-EC communication while increasing EC-to-inflammatory cell interactions that enable inflammatory cells to migrate into the vessel wall. Inflammatory macrophages and foam cells, to be specific, appear in early stages of atherosclerosis. Furthermore, GCX degradation deregulates vascular tone, by causing ECs to reduce their expression of endothelial nitric oxide synthase (eNOS) which produces the vasodilator, nitric oxide. Loss of vasodilation supports vasoconstriction, which promotes the progression of atherosclerosis. Summary Common medicinal atherosclerosis therapies include lipid lowering and anti-platelet therapies. None of these treatments specifically target the endothelial GCX, although the GCX is at the front-line in atherosclerosis combat. This review demonstrates the viability of targeting the GCX therapeutically, to support proper EC functionality and prevent and/ or treat atherosclerosis.
Atherosclerosis; Endothelial dysfunction; Endothelial glycocalyx; Cardiovascular disease treatment
Zona occludin 1
Connexin isotype 43
Endothelial nitric oxide synthase
Endothelium derived hyperpolarizing factor
Rapid freezing/freeze substitution
Transmission electron microscopy
Nicotinamide adenine dinucleotide phosphate
NADPH oxidase 4
Cardiovascular disease (CVD), the leading cause of death
worldwide, affects 92 million adults in the United States
(US) . Over 600,000 of these people die annually from
subsequent heart disease, accounting for 25% of US deaths
. When separately considered from other CVDs, stroke is
5th among all causes of death in the US, killing nearly
133,000 people per year . The precursor to most CVDs is
atherosclerosis, which occurs because of dysfunction of the
vasculoprotective endothelial cell (EC) lining of the inner
blood vessel wall . Both atherogenesis and EC dysfunction
have been noted to coincide with the loss of the cell
surfaceattached glycocalyx (GCX) that coats ECs [
]. Therefore, a
potential approach to restoring normal EC functionality to
prevent or treat atherosclerosis is to target and regenerate the
GCX layer in compromised areas of blood vessel walls.
The primary aim of this review is to highlight the role of the
GCX as a contributor to three key EC functions relevant to
vascular health and atherosclerosis. Additionally, we will
evaluate its potential as a target for therapies that treat atherosclerosis.
Atherosclerosis is a chronic arterial vessel disease,
characterized by the accumulation of plaque and subsequent erosion or
rupture of some “vulnerable” or “high risk” plaques . The
disease is preferentially located in regions of the vasculature
where blood flow is disturbed by geometric complexity such
as bends or branch points . The aortic arch curvature and
carotid sinus bifurcation, for example, cause blood flow
disruptions and irregularities that can be characterized by
recirculating flows and steep spatial variation in the
magnitude and direction of wall shear stress . Atherosclerosis
generally does not form in straight structures of the
vasculature such as the descending thoracic aorta, where blood flow is
more uniform and unidirectional [8, 9].
Dysfunction of the flow sensitive vasculoprotective
endothelium is a first step in the atherosclerosis process . The
endothelium is the innermost cell layer of the arterial wall and,
therefore, lies at the crucial interface between the blood and
vascular tissue compartments . Endothelium dysfunction
permits low-density lipoprotein (LDL) and its apolipoprotein
B to leave the blood compartment and accumulate in the
subendothelial space . The retained lipoproteins are
susceptible to modification by oxidation, enzymatic cleavage, and
aggregation [10, 11], all of which stimulate inflammation .
The inflammatory response includes the recruitment of
monocytes, which transmigrate across the endothelial monolayer
into the intima, where they proliferate and differentiate into
macrophages . The macrophages then uptake the
lipoproteins, developing into foam cells . Lesions continue to
expand due to the migration of new mononuclear cells, which
are then accompanied by cell proliferation and further
accumulation of extracellular lipids [13–15]. A change in the
nature of extracellular matrix production also occurs [13–15],
characterized by a switch from mostly elastin (and consequent
reduction in vessel wall elasticity) to collagen (causing vessel
wall hardening). Eventually, atherosclerotic lesions undergo
the formation of an overlying scar, called the fibrous cap
[16, 17]. The fibrous cap, which is in constant dynamic
equilibrium, provides a protective barrier between platelets in the
blood stream and pro-thrombotic plaque contents .
An increase in plaque size can cause narrowing of the
vessel lumen or complete obstruction of blood flow, causing
hypoxic conditions to specific organs in the body like the brain or
heart. Alternatively, large plaques “hidden” in the vessel wall
in regions of outward remodeling can disrupt on the luminal
surface to form a thrombus . This is common for advanced
plaques in which foam cells die and contribute their
lipidfilled contents to destabilizing necrotic cavities within the
plaques  while matrix degradation enzymes expand the
cavities to create large voids. Following plaque destabilization
and subsequent rupture, pro-thrombotic material on the plaque
remnant surface is exposed to flowing blood. Local occlusion
of the pro-thrombotic plaque remnant, primarily by
recruitment and adhesion of circulating platelets, can cause
obstruction of blood flow. Additionally, embolism can occur when
ruptured plaque fragments travel to and block distant blood
vessels. Rupture of atherosclerotic plaques is the most
common cause of strokes and myocardial infarctions .
The Endothelium and its Protective Glycocalyx
Because the condition of the vascular endothelium is a major
contributor to the balance between vascular health and the
progression of atherosclerosis , understanding EC function
has been the focus of intense research for many decades [19,
20]. Vascular ECs line the entire circulatory system and were
once thought to be inactive  but are now known to have
very distinct and unique activities that are essential to vascular
biology [22, 23]. Vascular ECs are directly exposed to and
able to sense changes in hemodynamic forces and
biochemistry of flowing blood [24, 25]. In turn, ECs respond genetically,
morphologically, and functionally [12, 26, 27] to mediate
modification of vasomotion, homeostasis, angiogenesis, and
vascular growth [24, 25].
A well-known endothelium response to the flow
environment involves synthesis and release of various vasoactive
substances, including the predominant vasodilator nitric oxide
. ECs subjected to uniform and unidirectional flows
constantly release nitric oxide [29, 30]. Nitric oxide has several
anti-atherosclerotic effects in the cardiovascular system, such
as inhibition of platelet aggregation, prohibition of excessive
smooth muscle cell proliferation, prevention of leukocyte
adhesion, and vessel dilation capabilities . The endothelium
also releases the vasodilators prostacyclin and endothelium
derived hyperpolarizing factor (EDHF) . In general,
EDHF-mediated responses include an increase in the
intracellular calcium concentration. This results in an
endotheliumdependent hyperpolarization of smooth muscle cells, which
evokes electrical coupling through myoendothelial junctions
and accumulation of potassium ions in the intercellular space
. Additionally, the endothelium releases vasoconstrictive
factors such as thromboxane and endothelin-1 .
Another well-characterized EC response to healthy blood
flow is the tightening of junctional interconnections between
neighboring ECs in order to (i) reinforce the barrier between
the blood and the underlying tissue [32, 33] and (ii) link
neighboring ECs so that they can communicate (Fig. 1). Barrier
function keeps unwanted molecules and cells from entering
and accumulating in vessel walls. EC-to-EC communication
is important for the inter-cytoplasmic exchange of ions,
metabolites, and other small molecules (< 1 kDa) [34, 35] that
mediate many vasculoprotective EC functions [36–38].
Endothelial cells become dysfunctional in disturbed blood
flow conditions, particularly blood flow conditions that
recirculate with high shear stress gradients as described above. This
endothelial dysfunction results in impairment of nitric oxide
production, re-distribution of inter-EC junctions, altered
communication, and loss of barrier function  (Fig. 1). Thus, disturbed
flow provides predisposition for atherogenic tendencies by
inducing EC dysfunction whereas uniform flow shields against
atherosclerosis by enhancing endothelium integrity [40–42].
In favorable blood flow conditions, endothelium protection
and functionality are governed by expression of the GCX [
19, 32, 43–51
] that acts as both a buffer and a force transmitter.
The GCX is a negatively charged, heavily hydrated,
polysaccharide mesh layer that coats ECs [
]. When GCX is
intact, its pores block infiltration of large blood components
while permitting absorption of plasma proteins such as
7-nmsized albumin (Table 1) as well as smaller solutes [
The plasma proteins contribute to the thickness of the GCX
(Table 1) [
]. In the end, GCX thickness is 0.02 to 8.9 μm
in vivo (Fig. 2), and due to preservation artifacts it is usually
thinner in vitro (0 to 3 μm). We were the first to use rapid
freezing/freeze substitution transmission electron microscopy
(RF/FS TEM) in vitro to visualize a cultured EC GCX of
several micron thickness (Fig. 2) .
GCX is connected to the ECs via its several glycoprotein and
proteoglycan backbone molecules . The glycoproteins,
which are at the base of the GCX (and typically buried by other
GCX components (Fig. 2)), are protein-glycan conjugates
(Table 1) [
]. They are adhesion molecules that can contribute
to shifting physiological conditions to a pathological state when
]. The three families of adhesive molecules that are
important to the GCX structure include the selectin family, the
integrin family, and immunoglobulin superfamily (Fig. 2 and
Table 1) [
]. Selectins, predominantly found in the
endothelium include E-selectin and P-selectin, both contributing to
leukocyte-EC interactions (Fig. 2 and Table 1) [
Integrins control the interaction of platelets with the apical EC
surface (Fig. 2 and Table 1) and attach to collagen, fibronectin,
and laminin in the subendothelium (Table 1) [
immunoglobulins include intercellular adhesion molecules 1 and 2,
vascular cell adhesion molecule 1, and platelet endothelial cell
adhesion molecule 1 . They act as ligands for integrins on
leukocytes and platelets and participate as crucial mediators of
adhesion to the endothelium (Fig. 2 and Table 1) [
20, 44, 54
GCX proteoglycans are given more attention than the
glycoproteins, due to the role that proteoglycan core proteins play in
incorporating the extracellular GCX into the EC body (Table 1)
]. Glypican core proteins are glycosylphosphatidylinositol
anchored to the caveolae compartment of the cell membrane
(Fig. 2 and Table 1) [20, 54]. Syndecan core proteins are
transmembrane and connected to the cytoskeleton (Fig. 2 and Table 1)
]. The core proteins covalently bond glycosaminoglycan
(GAG) chains and sialoglycoproteins [
], including heparan
sulfate, chondroitin sulfate, hyaluronic acid, and sialic acid
(Fig. 2 and Table 1) [
20, 44, 54
]. These structures extend into
the extracellular space . The GAGs are lengthy due to their
hundreds to thousands of disaccharide units [
]. In addition,
they carry strong, negative charges that create inter-GAG
repulsion forces [
]. GAG length and negative charge both
significantly contribute to the extension of the GCX into the vascular
GCX integrity is dependent on blood chemistry and flow
patterns along the walls of the vasculature. When blood
chemistry includes the heavy presence of various hormones,
neurotransmitters, and vasoactive factors, the GCX can be
degraded to expose its glycoprotein components that facilitate
thrombosis and inflammation . The GCX is most notably
impacted by blood flow patterns. Along vessel walls exposed
to uniform blood and in relatively good health, the GCX is
robust and thus protects the endothelium [
geometrically complicated vessels where portions of the vessel wall are
exposed to non-uniform flow patterns and predisposed to
atherogenesis, GCX thinning has been noted to occur on the
resident ECs [
2, 4, 5
]. Van den Berg et al. [
] determined that
the endothelial GCX thickness at the sinus region of a mouse
internal carotid artery, located at the arterial bifurcation, was
significantly less than the thickness of the GCX layer located
on the endothelium lining the common carotid artery. They
also concluded that impaired barrier properties of GCX
contribute to the enhanced LDL accumulation in the intima at the
carotid artery bifurcation of mice [
]. These results provide
evidence suggesting that flow patterns and associated shear
stresses contribute to varying GCX dimensions, modifying
GCX ability to protect the endothelium and guard against
atherosclerosis [8, 19, 20].
Vessel Bifurcation Region
Spatial shear stress gradients
Dysfunction of Endothelium
Increased Impaired Vessel Tone
Permeability Communication Deregulation
GCX, connective tissue
Straight Vessel Region
Healthy Vessel Wall
GCX, connective tissue
Fig. 1 The local flow patterns of the straight or bifurcation vessel regions
in which ECs reside impact GCX expression and, subsequently,
endothelium functionality and vascular health or atherosclerosis
progression. Non-uniform flow regions (red) are found in geometrically
complex vessels and are prone to degraded GCX and atherosclerosis.
Uniform flow regions (green) are found in linear vessels and have
healthy GCX expression and are less prone to atherosclerosis. These
concepts are summarized using a schematic of the carotid bifurcation.
GCX images show sialic acid labeled in green and EC nuclei labeled in
blue. A similar code is used for images of oxidative LDL permeability,
which is labeled in red. Communication images show transfer of green
gap junction permeability dye, to show the extent of cellular coupling.
Vascular tone images show intracellular eNOS (the activated form)
labeled in green. Finally, the plaque images show hyaluronic acid and
other connective tissue labeled in red, with the nuclei of luminal ECs
along with other cells labeled in blue. GJ: gap junction, LY: Lucifer
yellow. EC: endothelial cell, GCX: glycocalyx, eNOS: active
endothelial-type nitric oxide
Pro-Atherogenic Endothelial Dysfunction as a Result
of Glycocalyx Loss
In this section, we briefly report original and current findings
obtained by our research group. These findings contribute to the
body of knowledge about the role of the GCX in EC-dependent
atherosclerosis onset and progression, summarized above.
Increased Trans-Endothelial Permeability
It has been demonstrated in recent studies [
2, 46, 62
others that endothelial GCX helps to reinforce the barrier
between the blood and underlying vascular tissue, filtering
molecules and cells from crossing this barrier. Our research group
is currently studying the impact of GCX loss on impairment of
the barrier in vitro  and in vivo .
In vitro, we have examined cultured rat ECs that
naturally express robust GCX. We have confirmed that, as
expected, the intact GCX has a pore size that accepts
7 nm albumin but excludes 10 nm particles coated with
neutrally charged polyethylene glycol . We modified
the biochemical environment of the cultured ECs by
applying heparan sulfate-specific GCX cleavage enzymes or
via reduction of protein content in the culture media .
Degradation enzyme thinned the GCX by 33% and
reduced heparan sulfate content by 59% . Protein
content reduction led to 31% thinner GCX, with heparan
sulfate content shrinking by 34% . In both modified
biochemical environment cases, degraded GCX permitted EC
uptake of six- to sevenfold more 10 nm particles than the
low level of uptake observed in healthy EC with intact
GCX. This study confirms the importance of the GCX
in regulating uptake of small molecules .
This table summarizes the most widely known GCX constituents and their function
Albumin transports spingosine-1-phosphate (S1P), which binds to S1P receptors and, as a
result, inactivates matrix degradation enzymes and subsequently protects against GCX
SDC HA Binding GPC HA Integrin
Selectin Receptor GPI Anchor Immunoglobulin
Fig. 2 The protective GCX of the endothelium is shown here. a This
drawing (adapted from [
]) shows selected components of the EC GCX,
which include the integrins, selectins, and immunoglobulins. Syndecan
and glypican are also shown. They are bound to chondroitin sulfate and
heparan sulfate GAGs. Another GAG, hyaluronic acid is also shown.
Lastly, we show absorbed plasma proteins such as albumin and other
molecules, which are important components of GCX. b This electron
micrograph shows a left ventricular myocardial capillary explanted
In other in vitro experiments, we assessed the impact of
GCX integrity on EC uptake of LDL, which range in size
from 60 to 80 nm [
]. Rather than manipulating the GCX
through biochemical intervention, we modified it via the
fluid mechanics environment surrounding the cultured
ECs. Specifically, rat ECs were conditioned by 6 h of
laminar flow. To create a physiological model, a region
of a single monolayer of ECs was in direct contact with a
non-uniform flow pattern, which translates to spatially
variable shear stresses. An adjacent region of the same
monolayer was directly exposed to a uniform flow pattern of
15 dyne/cm2 shear stress. In uniform shear stress
conditions, the cells expressed their most abundant and
continuous GCX, and oxidized LDL uptake was low.
Nonuniform shear stress conditions impacted specific
subcomponents of the GCX differentially. We found that
nonuniform flow decreased the thickness of the heparan sulfate
of the GCX by 11%, and we observed a 48% reduction in
the extent to which heparan sulfate covered ECs. For the
sialic acid component of the GCX, on the other hand,
nonuniform flow resulted in a 21% decrease in thickness and a
44% decrease in coverage. These disturbed flow-induced
c h a n g e s t o t h e G C X c o m p o n e n t s c o r r e l a t e d t o
from a rat and stained with Alcian blue 8GX [
]. Bar = 1 μm. c, d
We used RF/FS TEM to visualize a cultured EC GCX of several
micron thickness on rat fat pad ECs, shown in c, and bovine aortic
ECs, shown in d [
]. Bar = 2 μm applies to both c and d. Heparan
sulfate (HS) GAG, chondroitin sulfate (CS) GAG, hyaluronic acid
(HA) GAG, syndecan (SDC) core protein, glypican (GPC) core
protein, Glycosylphosphatidylinositol (GPI)
heterogeneity in cellular uptake of oxidized LDLs.
Oxidized LDL can be defined as a particle derived from
circulating LDL that may include peroxides or their
degradation products generated within the LDL molecule or
elsewhere in the body associated with the particle [
LDL can be oxidized by vascular endothelial cells, smooth
muscle cells or macrophages [
]. Oxidized LDL binds to
its lectin-like receptor LOX-1 in endothelial cells which in
turn triggers the CD40/CD40L signaling pathways, which
then synthesizes chemokines and cell adhesion molecules
]. Class B scavenger receptor CD36 mediates
macrophage uptake and degradation of oxidized LDL, which
transforms them into foam cells . Many cells per
monolayer, not all of them, internalized oxidized LDL in
the cytoplasmic compartments. A lack of GCX was
a p p a r e n t i n a m a j o r i t y o f t h e c e l l s t h a t c o n t a i n e d
c yt op l a s m i c o xi di ze d L D L . T h es e r e s u l t s p r o v i d e
evidence to support the notion that disturbed
flowi n d u c e d G C X d e g r a d a t i o n i s a n i n i t i a t o r o f p r o
atherosclerotic EC behavior [Harding et al., unpublished
results, and manuscript in preparation].
To complement in vitro studies, we demonstrated GCX
integrity implications for barrier function and molecular
permeability in vivo. This study examined atherosclerotic
animals, which we re yo ung C57BL/6 J–backg ro und
apolipoprotein-E deficient mice on a high-fat diet, rich in
saturated fats and cholesterol, for 10 weeks . The diet leads to
significantly high levels of total cholesterol, including LDL
and cholesterol elevation, which were observed in the blood
circulation of the mice . However, lipid was only retained
in the blood vessel walls exposed to disturbed flow conditions,
such as the lower curvature of the aortic arch and the outer
wall of the brachiocephalic artery branch. GCX covering the
plaque-filled wall was found to be discontinuous, spanning
only 71% of the wall surface at a thickness of 0.85 μm .
Lipids were excluded from the blood vessel walls exposed to
uniform flow, including the common carotid arteries and the
descending thoracic aorta. In these non-plaque regions, GCX
covered 97% of the blood vessel wall and had a thickness of
1.2 μm . This work reinforces the hypothesis that GCX
shedding correlates to blood vessel wall retention of lipids
and other molecules, propagating atherosclerosis.
Altered Inter-Endothelial Communication
The role of the GCX in regulating inter-endothelial
communication is understudied. We find these junctions to be of great
interest because they are complex and mediate a number of
vasculoprotective EC functions [36–38]. In the previous
work, it was noted that the transmembrane syndecan core
proteins of the GCX are connected to the EC cytoskeleton,
which interacts with intercellular junctions through zona
occludin 1 (ZO-1) [
]. ZO-1 is important for gap junction
docking at the cell membrane [
]. Subsequently, our
research group has been one of the few to assess GCX function
by probing its control of communicating gap junctions in
cultured rat ECs.
We first established a baseline integrity of the GCX to
ensure a healthy GCX layer was present. We then either
enzymatically degraded the heparan sulfate component of
the GCX, or repaired the GCX by allowing the cells to
self-recover the heparan sulfate. Baseline and repaired
GCX coverage of EC monolayers were statistically
similar. Enzymatically degraded GCX covered 35% less of the
endothelium. We correlated the relative GCX coverage to
expression of the proteins that make up communicating
gap junctions, called connexins. We also confirmed the
extent to which connexin expression enabled
communication activity of the gap junctions, by counting the number
of gap junction-coupled cells. With baseline GCX, 60% of
EC borders expressed connexin isotype 43 (Cx43), and
individual cells could communicate to neighboring cells
through a line of up to three cells. Heparan sulfate
degradation decreased Cx43 expression to 30% and impaired
the ability of individual cells to communicate. This is the
first evidence that GCX effectively regulates the level of open
communication between ECs [Mensah et al., full manuscript
has been submitted for publication and is in review].
Human ECs are of interest for our communication studies
because human ECs have been shown to represent a more
realistic in vitro model for studying human vascular tissue
health and disease [
]. We recently started to map the human
cultured EC GCX and communication with experimental flow
conditions that replicate the flow patterns that are characteristic
of the atherosclerosis-resistant and atherogenic blood vessel
regions. To date, we have characterized the sialic acid
component of the GCX and showed that ECs express a substantial
amount of sialic acid when exposed to anti-atherosclerotic
uniform flow conditions of 15 dynes/cm2 shear stress.
Proatherosclerotic non-uniform flow conditions lead to 54%
reduction in the EC surface covered by sialic acid. At the same time,
sialic acid thickness is reduced by 60% in non-uniform flow
conditions [these are unpublished results from Mensah et al.
The manuscript is currently in preparation].
We hypothesized that heightened GCX expression
induced by uniform flow correlates to elevated
communication between neighboring human ECs, based on the earlier
EC studies that we conducted using rat cells. We
postulated that degraded GCX caused by non-uniform flow
attenuates human EC communication. Unexpectedly, we
learned that the overall level of human EC-to-EC gap
junctional communication is high regardless of the flow
]. These results were clarified when we
separated the communication into connexin-specific
components. We showed for the first time in vitro that flow is
correlated to the type of connexins underlying gap
junctional communication in cultured human EC monolayers
]. In uniform flow (lesion-resistant) locations, which
are home to GCX-rich ECs, Cx43 function is low while
connexin isotype 40 is the dominant communicator [
Cx43 only plays a major role in communication in
nonuniform flow regions, where GCX-deficient ECs reside
]. These results confirm that connexin protein
specificity of gap junctional communication is determined by
flow pattern [
], which has a synergistic effect with
Collectively, these studies imply that biochemistry and
fluid mechanics of blood environment determine connexin
protein participation in gap junctional communication, via GCX
restructuring. The connexin isoforms lead to different gap
junction properties, adapting the inter-cytoplasmic flux of
ions, metabolites, and other small molecules [
] that regulate
endothelium phenotype relevant to vascular health as well as
Deregulation of Nitric Oxide
Nitric oxide plays several anti-atherosclerotic roles—i.e.,
inhibition of platelet aggregation, prohibition of excessive
smooth muscle cell proliferation, prevention of leukocyte
adhesion, and vessel dilation capabilities —as previously
mentioned. Therefore, impairment of EC production of nitric
oxide can be detrimental at every stage of atherosclerosis
development. Impairment of nitric oxide can occur by a number
of mechanisms. One major mechanism involves a decrease in
nitric oxide production as a consequence of reduced eNOS
expression, eNOS inactivation, or dislocation of active
eNOS from the caveolae rafts that are located in the apical
EC membrane [
]. Another mechanism involves diminished
nitric oxide bioavailability via the action of nicotinamide
adenine dinucleotide phosphate (NADPH) oxidases, which
contribute to oxidative stress [
]. The balance between nitric
oxide production by eNOS and inhibition of its bioavailability
by NADPH oxidases determines the ability of nitric oxide to
bring about vasodilation.
We previously elucidated the contribution of the GCX to
shear stress regulation of the activity of eNOS, an enzyme that
synthesizes nitric oxide [
]. In brief, in bovine ECs we
confirmed that loss of the heparan sulfate component of the GCX
abolished conversion of uniform flow into eNOS activation
]. We probed deeper and found that the heparan
sulfatebound glypican-1 core protein is a key player in the
mechanism by which heparan sulfate transduces uniform
flowderived forces into eNOS activity [
]. Silencing glypican-1
blocked flow-induced eNOS activation [
]. This result can
be explained by the fact that glypican is normally anchored to
and functional in the caveolae that contains eNOS and other
signaling molecules [
]. Glypican-1 removal and its
discontinued interaction with eNOS apparently interferes with
the process by which eNOS is activated.
Expanding upon the previously published work,
experiments are ongoing to determine the differential effects of
uniform laminar shear stress versus non-uniform laminar shear
stress on GCX involvement in flow-regulated eNOS
activation. These experiments, once again, involve exposing
monolayers of rat ECs to 6 h of side-by-side uniform and
nonuniform flow. As mentioned above, the GCX is abundant
and continuous in uniform flow conditions, while impaired
in non-uniform flow. Specifically, the heparan sulfate
component of the GCX is 11% thinner and spans 48% less
endothelium surface area. The sialic acid component is 21% thinner
and covers 44% less endothelium. This GCX impairment
reduced eNOS activation by 50% in non-uniform flow as
compared to uniform flow. We also examined caveolin-1, a protein
that is a main component of caveolae. We confirmed
caveolin1 and eNOS co-localization in uniform flow and, for the first
time, showed that non-uniform flow disrupts 49% of the
colocalization in a GCX dependent manner. These experiments,
taken together, imply that in blood vessel regions with
atherogenic disturbed flow conditions, loss of GCX results in eNOS
deactivation and eNOS-caveolae separation. These outcomes
show that GCX degradation will have detrimental
consequences for regulation of vascular tone [Harding et al.,
unpublished results, and manuscript in preparation].
To date, there has been no report of studies clarifying
whether the GCX plays a role in EC control of nitric oxide
bioavailability in response to flow. In a previous study, upon
exposure to uniform flow ECs isolated from different human
subjects expressed similar levels of eNOS and nitric oxide
]. However, compared to ECs from subjects who were
more at risk for cardiovascular disease, ECs from subjects
with lower risk exhibited a lesser amount of flow-induced
NADPH oxidase 4 (NOX4) [
]. This previous work implies
that in disturbed flow conditions, NOX4 expression would be
significantly higher than in uniform flow settings. Elevated
NOX4 will diminish nitric oxide bioavailability, an accepted
marker of endothelial dysfunction . Future studies are
needed to clarify if there exists GCX-mediated mechanisms
by which EC under various flow stimuli differentially regulate
nitric oxide bioavailability via NOX.
Can Atherosclerosis Treatment Be Improved
by Targeting Glycocalyx?
Standard-of-Care Atherosclerosis Treatments
Numerous commercially available standard-of-care
treatments are utilized to reduce atherosclerosis and cardiovascular
events such as myocardial infarction, stroke, and transient
ischemia attacks [
]. They are largely focused on
alleviation of hyperlipidemia and thrombotic complications .
Statins, the most well-known atherosclerosis treatment,
lower lipid levels in the blood and help stabilize
atherosclerotic plaques [
]. Crisby et al. and others have shown the
significant benefits of pravastatin use [
]. Following the
treatment period, atherosclerotic plaques were surgically
removed from both treated and untreated (control) patient
groups for histological analysis. The analysis showed that
plaques removed from patients who were given pravastatin
had significantly less lipid content and, consequently, less
inflammatory cell infiltration [
]. Additional beneficial effects
of pravastatin treatment included reduction in matrix
degradation enzymes [
], which were correlated with higher collagen
content in pravastatin-treated plaques [
]. Another side effect
was inhibition of cell death [
]. Reduced matrix degradation
and lower cell death, taken together, fend off formation of
necrotic cavities and large voids in atherosclerotic lesions.
This led Crisby to conclude that pravastatin-treated patients
have more stable atherosclerotic plaques [
]. The implication
is that pravastatin reduces risk of plaque rupture, which is the
most common cause of strokes and myocardial infarctions .
Aspirin is another well-known anti-atherosclerotic drug.
Aspirin has multiple effects, including anti-inflammation and
reduction of thrombus formation [
]. Aspirin’s primary
Atherosclerosis Most common precursor to cardiovascular diseases such as strokes and
Initiated by excessive accumulation of LDLs in luminal region of blood vessel
Disturbed flow regions of vessel bifurcations atheroprone, with degraded or
compromised GCX in these regions
GCX structure and location Negatively charged heterogeneous polysaccharide that lines the luminal wall
of blood vessels
Primarily consists of heparan sulfate, hyaluronic acid, sialic acid, and
GCX-mediated endothelium functions
Barrier Function GCX acts as a barrier between the blood and vessel walls, filtering small
molecules, lipoproteins, and circulating blood cells that seek to permeate
A degraded or collapsed GCX has been shown to increase permeability of
molecules and inflammatory cells
Healthy GCX reduces permeability
Cell-to-cell communication GCX attached to endothelial cell cytoskeleton which has a link to
communicating gap junctions
Degraded GCX showed a decrease in gap junction protein (connexin)
expression as well as communication activity
Vascular tone GCX has a role in the production of vasodilatory factor nitric oxide
eNOS, the enzyme that produces nitric oxide, can be significantly decreased
by non-uniform flow and/or when GCX is also degraded
Standard atherosclerosis Statins (lipid lowering therapy)
treatment options Aspirin (anti-platelet therapy)
GCX as therapeutic Strengthening the GCX to counteract its degradation can restore barrier
function, cell-to-cell communication, and vascular tone
Viable preventative and treatment option for addressing atherosclerosis
mechanism of action involves platelet deactivation, reducing
platelets’ ability to release the bioactive substances that
promote platelet aggregation and thrombus formation [
Platelet activity is a physiological requirement to control
bleeding and is not considered effective for primary
prevention of atherosclerosis [
]. Aspirin-induced platelet
inhibition has been shown to be effective only as a secondary
atherosclerosis preventative measure for patients who exhibit
more advanced stages of the disease. In a case-control
analysis, the combination of aspirin with the aforementioned statin
proved to yield significant reduction in mortality [
]. It has
also been shown that aspirin’s effect can be enhanced by
coadministration with, clopidrogel, prasugrel, or ticagrelor,
agents that block platelet surface receptors as a means of
inhibiting platelet aggregation [
Emerging Glycocalyx-Targeted Therapies
A very promising new approach is to develop therapies that
will promote GCX health in order to reverse endothelial
dysfunction, an early hallmark for atherosclerosis. With stabilized
GCX structure and function, we expect that ECs will block
lipoprotein deposits and macrophage uptake in the blood
vessel wall, arresting progression of early plaque. Additionally,
GCX therapy could potentially stimulate anti-atherosclerotic
EC cell-to-cell communication and nitric oxide signaling
pathways. GCX-targeted therapy would enable early
atherosclerosis treatment. Unfortunately, there are currently no drug
therapies on the market that are approved for the purpose of
restoring or protecting the GCX. Also, there are few
commercially available drugs for which the benefits to GCX are
known, beyond the officially approved indications.
Emerging GCX-targeted approaches include stabilizing its
structure, replacing it with substitutes, blocking GCX
degradation enzymes, and enhancing GCX synthesis. A
comprehensive summary of these approaches is described in a recent
review paper written by Tarbell and Cancel [
]. Our lab has
explored the replacement and structural stabilization
approaches briefly described below.
Albumin has long been known to stabilize the GCX, and
albumin deficiency has been known to lead to GCX collapse
]. Clinically, it has been used to restore hemostasis after
]. However, the impact of albumin treatment on
GCX has been overlooked clinically, until recent cell culture
and pre-clinical studies of spingosine-1-phosphate (S1P),
which is transported by albumin [
]. S1P has been found
to modulate the structure of the GCX by binding to S1P
receptors, which has the effect of inactivating matrix
degradation enzymes and subsequently protecting against GCX
]. This allows de novo synthesis of GCX to
outweigh its shedding, stabilizing the GCX structure [
Success with GAG replacement has been reported in cell
culture models, pre-clinically, and in studies conducted in
human subjects. Our group successfully repaired the GCX with
exogenous heparan sulfate and rendered the EC impermeable
to small particles (10 nm in size); counteracting the effect of
GCX degradation enzymes to which the ECs were also
]. Other groups have rebuilt the GCX using
commercial sulodexide, which contains a combination of heparin
(80%) and dermatan sulfate (20%). It has also been
demonstrated that sulodexide use leads to restoration of endothelium
barrier function, inhibition of GCX degradation enzymes,
restraint of inflammatory activity, and deceleration of EC
agerelated deterioration and programmed cell death [
recent study demonstrated that treating obese atherosclerotic
mice with chondroitin sulfate inhibited the expression of
proinflammatory cytokines, decreased the number of monocytes
migrating to inflamed cells, and reduced macrophage presence
in arterial plaques . Rhamnan sulfate, a non-animal
polysaccharide that mimics heparan sulfate, has been successfully
used in other studies on blood vessel ECs to prevent and treat
conditions associated with transendothelial permeability,
including inflammation and atherosclerosis [
GAG replacement strategies are all promising approaches to
prevent atherosclerosis and related cardiovascular diseases.
Our l ab ha s r ec entl y f ound it nec e ssa r y to
coadminister a GCX structural stabilization agent, S1P, with
a GCX replacement compound, exogenous heparan
sulfate, in order to repair GCX in a manner that reverses
highly complicated endothelial dysfunction, impaired
gap junctional communication. We found that stabilizing
GCX structure alone did not lead to recruitment of
sufficient gap junction proteins (connexins) to the EC cell
borders, limiting gap junction formation and
intercytoplasmic communication. GCX component
replacement, alone, aided in gap junction protein (connexin)
expression and docking to EC cell borders but did not result
in open lines of communication between neighboring
ECs. Ultimately, GCX repair by treating cells with the
exogenous GCX component and the GCX stabilizing
cofactor restored gap junction protein placement, which
translated to the reactivation of gap junction channel
activity. The results of this study are encouraging. [Mensah
et al., full manuscript has been submitted for publication,
and a provisional patent application entitled “GlycoFix
(Structurally and Functionally Repaired Endothelial
Glycocalyx)” was filed in July 2017]. Pre-clinical studies
in animals are now underway to test the efficacy and
plausibility of heparan sulfate and S1P as a preventative
and therapeutic measure to combat atherosclerosis.
The endothelium forms an essential component of the
vasculature and is crucial for atheroprotection because of
its structural barrier function and its biological activities.
The latter includes actions of endothelial cell-derived
vasoactive factors such as vasodilators, via suppression of
smooth muscle cell growth, and by inhibition of
inflammatory responses, among a number of other functions [
] (Table 2). Endothelial dysfunction can lead to a
disruption in vascular homeostasis causing arterial wall
damage and contributing to early stages of atherosclerosis.
Proper functionality of the endothelium is highly
dependent on the condition and expression of its GCX, which
contributes to barrier functionality, cell-to-cell
communication, and vascular tone regulation (Table 2). Our
knowledge of GCX structure and function could potentially
o p e n n e w a v e n u e s f o r p r e v e n t i n g a n d t r e a t i n g
Acknowledgements Support was provided by the Northeastern
Departments of Chemical Engineering and Bioengineering. Irina Ahn
provided technical support, designed, and collected preliminary data for
the oxidative low-density lipoprotein experiments, and we are
appreciative of her contribution to this report.
Author Contribution The contributions of the first and second
authors, Ronodeep Mitra and Gerard Leland O’Neil, were equal.
Funding Statement We are also pleased to acknowledge that this work
was funded by the National Institutes of Health (K01 HL125499 awarded
to E. Ebong), the National Science Foundation (DGE-145070 awarded to
S. Mensah), and Northeastern University (startup funds; Tier 1 Provost
Compliance with Ethical Standards
Conflict of Interest A provisional patent application entitled
“GlycoFix (Structurally and Functionally Repaired Endothelial
Glycocalyx)” was filed by R.M., M.J.C., S.A.M., and E.E.E. in
July 2017. The authors have no other conflicts of interest to declare.
The funders had no role in data or information collection and analysis,
decision to publish, or preparation of the manuscript.
Human and Animal Rights and Informed Consent This article does
not contain any studies with human or animal subjects performed by any
of the authors.
Open Access This article is distributed under the terms of the Creative
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Creative Commons license, and indicate if changes were made.
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