Download The glycocalyx is present as soon as blood flow is initiated and is

Developmental Biology 369 (2012) 330–339
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Developmental Biology
journal homepage: www.elsevier.com/locate/developmentalbiology
The glycocalyx is present as soon as blood flow is initiated and is required for
normal vascular development
Caitlin E. Henderson-Toth a, Espen D. Jahnsen a,b, Roya Jamarani a, Sarah Al-Roubaie a,
Elizabeth A.V. Jones a,b,n
a
b
McGill University, Department of Chemical Engineering, 3610 University St, Montreal, QC, Canada H3A 2B2
Lady Davis Institute, McGill University, 3755 Cote Ste Catherine, Montreal, QC, Canada H3T 1E2
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 December 2011
Received in revised form
2 July 2012
Accepted 10 July 2012
Available online 20 July 2012
The glycocalyx, and the thicker endothelial surface layer (ESL), are necessary both for endothelial
barrier function and for sensing mechanical forces in the adult. The goal of this study is to use a
combination of imaging techniques to establish when the glycocalyx and endothelial surface layer form
during embryonic development and to determine the biological significance of the glycocalyx layer
during vascular development in quail embryos. Using transmission electron microscopy, we show that
the glycocalyx layer is present as soon as blood flow starts (14 somites). The early endothelial
glycocalyx (14 somites) lacks the distinct hair-like morphology that is present later in development (17
and 25 somites). The average thickness does not change significantly (14 somites, 182 nm 733 nm; 17
somites, 218 7 30 nm; 25 somites, 212 7 32 nm). The trapping of circulating fluorescent albumin was
used to evaluate the development of the ESL. Trapped fluorescent albumin was first observed at 25
somites. In order to assess a functional role for the glycocalyx during development, we selectively
degraded luminal glycosaminoglycans. Degradation of hyaluronan compromised endothelial barrier
function and prevented vascular remodeling. Degradation of heparan sulfate down regulated the
expression of shear-sensitive genes but does not inhibit vascular remodeling. Our findings show that
the glycocalyx layer is present as soon as blood flow starts (14 somites). Selective degradations of major
glycocalyx components were shown to inhibit normal vascular development, examined through
morphology, vascular barrier function, and gene expression.
& 2012 Elsevier Inc. All rights reserved.
Keywords:
Glycocalyx
Endothelial surface layer
Mechanotransduction
Vascular remodeling
Vascular development
Introduction
Endothelial cells form a barrier within blood vessels, between the
circulating blood and surrounding tissues. In the adult, the luminal
side of endothelial cells expresses a thin, gel-like layer called the
glycocalyx. The glycocalyx is composed of membrane-bound proteoglycans and glycosaminoglycans (GAGs) such as hyaluronan
(HA), chondroitin sulfate (CS), and heparan sulfate (HS), with
terminal sialic acids (SA) (Pries et al., 2000). Blood proteins, such
as albumin and superoxide dismutase, become entrapped in the
glycocalyx and create a thicker layer known as the endothelial
surface layer (ESL) (Becker et al., 1994; Pries et al., 2000; Shimada
et al., 1991). The glycocalyx forms hair-like extensions into the
vascular lumen that are anchored directly to actin stress fibers of the
cytoskeleton and are believed to transmit shear stress, a force
created by circulating blood, to the cytoskeleton (Florian et al.,
n
Corresponding author at: Lady Davis Institute, McGill University, 3755 Cote Ste
Catherine, Montreal, QC, Canada H3T 1E2. Fax: þ1 514 398 6678.
E-mail address: [email protected] (E.A.V. Jones).
0012-1606/$ - see front matter & 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.ydbio.2012.07.009
2003; Pahakis et al., 2007; Thi et al., 2004; Yao et al., 2007).
The negatively charged glycocalyx also creates both a charge and a
size barrier for protein diffusion to the endothelial cell surface and is
vital for endothelial barrier function (Florian et al., 2003; Vink and
Duling, 2000). Selective degradation of the adult glycocalyx has been
shown to increase vascular permeability (Henry and DeFouw, 1995),
cause attenuated nitrous oxide availability (Mochizuki et al., 2003),
and increase adhesion of leukocytes and platelets to the endothelium (Constantinescu et al., 2003). Thinning of the glycocalyx has
been linked to diseases such as diabetes mellitus, where dysfunction
of the glycocalyx is believed to contribute to an increase in vascular
permeability (Nieuwdorp et al., 2006a).
Though the glycocalyx plays a significant role in adult vascular
homeostasis, the role of the glycocalyx during vascular development has not yet been investigated. During embryonic development the vasculature forms as an immature capillary plexus
that requires blood flow to mature (Chapman, 1918; le Noble
et al., 2004; Lucitti et al., 2007). Mechanical forces, such as shear
stress, must be present for vascular development to occur
normally (le Noble et al., 2004; Lucitti et al., 2007). As the heart
begins to beat, vessel permeability plays a large role in the type of
C.E. Henderson-Toth et al. / Developmental Biology 369 (2012) 330–339
flow that is present. Since liquids flow in the direction of least
resistance, if no barrier to flow into the interstitium is present, the
blood plasma will flow into surrounding tissues and reduce the
flow in the blood vessels themselves. Therefore, since the glycocalyx is important in the adult both for permeability and for
mechanotransduction, it seems likely that the glycocalyx plays a
significant role in vascular development.
The stage at which both the glycocalyx and the ESL form during
vascular development is not known. Many of the proteins that
produce the GAGs of the glycocalyx are expressed before the onset
of blood flow (Klewer et al., 2006). Lectins are proteins that bind to
specific disaccharides in glycosaminoglycan chains (Barker et al.,
2004; Florian et al., 2003; Mulivor and Lipowsky, 2004). Lectin
binding affinities change during early vascular development
(Henry and DeFouw, 1995) and therefore, there is indirect evidence
that the composition of the glycocalyx is variable during development. Beyond this, not much is known about the presence of the
glycocalyx in the embryo. Even if the glycocalyx and the ESL are
present before the onset of circulation, blood flow itself is likely to
modify both. Proteins that produce the GAGs of the glycocalyx,
such as hyaluronan synthases, are upregulated when endothelial
cells are exposed to flow in vitro (Gouverneur et al., 2006). The ESL,
on the other hand, is created by the incorporation of flowing
plasma protein and therefore the onset of circulation could alter
the composition and structure of this layer.
In this work, we have investigated the formation and role of
the glycocalyx and the ESL during vascular development using the
quail embryo as our model system. Transmission electron microscopy (TEM) was used to visualize the glycocalyx proper.
A fluorescent albumin method was used to visualize the ESL.
We found that the glycocalyx is present when blood flow begins.
Large protrusions of glycosaminoglycans are present early in
development, though these often follow irregularities in the
endothelial cell membrane. By 25 somites, the glycocasminoglycan structure shows a regular thickness and distinct hair-like
structure similar to the adult. We could detect the presence of an
ESL starting at 25 somites. By immunohistochemistry, we investigated the presence of HA, CS, HS, and SA in the glycocalyx during
vascular development. We then used intra-vascular injection of
bead-immobilized enzymes to selectively degrade the luminal
GAGs that make up the glycocalyx. We looked at the effect of
glycocalyx degradation on vascular network morphology, permeability, gene expression and smooth muscle cell recruitment.
Materials & methods
Transmission electron microscopy
Fertilized quail eggs (Coturnix japonica) were incubated at 37 1C
and approximately 60% humidity for 40–72 h as noted. Embryos were
fixed for TEM using a modified version of a previously published
protocol (van den Berg et al., 2003). For more information, please see
supplemental materials. Samples were imaged on a FEI Tecnai 12
120 kV equipped with an AMT XR80C CCD Camera. Because the
endothelial lining was irregular, the thickness of the glycocalyx was
calculated by measuring the area of the glycocalyx divided by the
length of the endothelium in regions that lacked large protrusions.
It was necessary to ignore these large protrusions since they
overstated the thickness covering most of the vessel. A protrusion
was defined as a structure greater than twice the average thickness.
Ex ovo fluorescent albumin method
Embryos were placed in culture using a modified version of a
previously published ex ovo method (Krull and Kulesa, 1998).
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Acetylated low-density lipoprotein (AcLDL, Invitrogen L35353)
was injected directly into the embryonic heart and allowed to
circulate for 15 min to label endothelial cells. AcLDL is taken up
by scavenger receptors expressed on endothelial cells (Adachi
et al., 1997). After AcLDL labeling, fluorescently-tagged albumin
from bovine serum (AlexaFluor 488; Invitrogen A13100) was
injected directly into the embryonic heart. Embryos were
returned to the incubator for one hour to allow the albumin time
to incorporate into the ESL. Embryos were imaged on a heated
confocal microscope. Due to the lack of growth media, embryos
were imaged for only 1–3 h.
Immunohistochemistry
For whole-mount, embryos were dissected and fixed in 4%
paraformaldehyde solution. Embryos were blocked and incubated
with primary antibodies. Endothelial cell were labeled with QH1
(Developmental Studies Hybridoma Bank, 1:100). The heparan
sulfate proteoglycan perlecan was labeled with a-perlecan,
(Developmental Studies Hybridoma Bank, 5C9, 1:100). Smooth
muscle cells were labeled with a-SMA-Cy3 (clone 1A4, Sigma
Aldrich; 1:100). Replicating cells were labeled with a-PH3 (New
England Biolabs, 1:100). Fluorescent secondary antibodies were
purchased from Invitrogen (AlexaFluor 488; 1:400). For sections,
embedded embryos were cut into 7–10 mm sections and antigen
retrieval was performed using citrate buffer unmasking solution
(Vector Labs). Biotinylated primary antibodies against hyaluronan
(HABP, EMD Chemicals, 1:100) and sialic acid (Flavus Limax, EY
Laboratories, 1:100) were used and detected with Cy3-Streptavidin (Sigma, 1:400). For more information, please see supplemental
materials.
Analysis of blood flow
Fluospheres (1 mm, Invitrogen) were injected into circulation
using a picospritzer and a fine pulled needle. Embryos were
transferred to a fluorescent microscope equipped with an Ultima
APX-RS camera and the arterial region of the yolk sac was imaged
for 1000 frames at 500 fps. From the series of 1000 images, the
period of systole was assessed visually and four frames were
extracted from the observed peak velocities. Several individual
vessel segments of approximately equal diameter were cropped
out of the larger images. Using uraPIV, an open source MatLab
software developed by Roi Gurka, the speckle pattern in the vessel
segments was analyzed for all image pairs. The interrogation
window size and spacing was optimized for the vessel segment
and based on a laminar flow profile the data from the best image
pair was selected. Only peak velocities in the center of the vessel
are reported.
Enzymatic degradation
Embryos were incubated until they reached 14 somites
(approximately 40 h). A small window was cut into the top of
the eggshell to expose the embryo. Embryos were injected
intravascularly using a pulled quartz needle filled with either
enzyme in solution or enzymes immobilized on beads. Enzyme
immobilization was achieved using sulfated Fluospheres (Invitrogen). Immobilization was necessary for longer exposures to the
enzymes since proteins in solution could leach out of the blood
vessels. The sulfated Fluorospheres were mixed with concentrated enzyme solution for 8 h. The suspension was then centrifuged at 3000 rpm for 10 min, washed, re-centrifuged and
resuspended in 40,000 MW Texas Red dextran. After injection,
eggs were sealed and returned to the incubator for 14–18 h. All
enzymes were purchased from Sigma (Hyaluronidase, H1136;
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Chondroitinase ABC, C2905; Heparinase III, H8891; Neuraminidase Type V, N2876). Albumin was immobilized on beads as a
control.
AnnexinV analysis
Apoptosis was analyzed after embryos had been exposed to
bead-immobilized enzymes for 18 h using AnnexinV staining.
AnnexinV binds phosphatidylserine which is expressed on the
cell surface of apoptotic cells (Koopman et al., 1994). Embryos
were injected intra-vascularly with AnnexinV-AlexaFluor555
(Invitrogen) and returned to the incubator for 30 min. Embryos
were dissected and fixed in 4% paraformaldehyde solution.
Immunohistochemistry was performed against QH1 that detects
quail endothelial cells (Pardanaud et al., 1987).
Analysis of vascular permeability
Embryos were injected in ovo with Ringer’s (control) and
enzyme solution and returned to the incubator. After one hour
of incubation, embryos were injected with FITC-dextran (Sigma,
FD40S), and observed every hour until vessels were indistinguishable from avascular regions. For more information, please see
supplemental materials.
RNA extraction and gene expression analysis
Quail embryos were injected with immobilized enzyme at 14
somites and dissected at 21–26 somites. Three embryos were
combined per sample (n ¼6–9 samples, for all treatments), and
Fig. 1. The Glycocalyx is present as soon as blood flow initiates. Transmission
electron microscopy of the endothelial glycocalyx in the dorsal aorta of 14 somite
quail embryos (A). At this stage, the thickness is variable and protrusions of
glycosaminoglycans are visible. Fewer protrusions are present at 17 somites and
the structure of the glycosaminoglycans displays a regular hair-like structure (B).
By 25 somites, these protrusions are absent and a very regular hair-like structure
is present (C). Scale bar 500 nm.
treated with collagenase to acquire a single cell suspension. QH1
conjugated Dynabeads (Invitrogen) were used to isolate endothelial cells. Primers for qPCR were designed based on regions of high
sequence conservation between mouse, rat, human and chicken,
using sequence information from the chicken. For more information, please see supplemental materials.
Results
The glycocalyx and endothelial surface layer are present from the
onset of blood flow
We investigated the formation of the glycocalyx of the dorsal aorta
by TEM at 14 somites (onset of blood flow), 17 somites (early vascular
remodeling) and 25 somites (late vascular remodeling). At 14 somites,
we observed that the glycocalyx is present, although the thickness
was not uniform (average value¼ 182 nm722 nm, n¼3 embryos)
and luminal protrusions were observed (Fig. 1A). Although the
glycocalyx is present at this stage, the structure of the glycosaminoglycans did not show a regular hair-like pattern as has been
previously observed for adult blood vessels in frogs and rats (Squire
et al., 2001; van den Berg et al., 2003). At 17 somites, the glycocalyx
was similar in thickness that of the 14-somite embryo (average
value¼218730 nm, n¼3), but fewer protrusions into the vascular
lumen were present and a hair-like morphology was present (Fig. 1B).
At 25 somites, no differences in glycocalyx thickness were observed
when compared to younger embryos and luminal protrusions were
no longer present (Fig. 1C, average value¼ 212732 nm, n¼3).
The ESL is believed to consist largely of entrapped of proteins,
particularly albumin (Pries et al., 1998). We therefore injected
fluorescently-tagged albumin into the extra-embryonic vasculature
of quail embryos to image luminal immobilized albumin (Fig. 2).
Extra-embryonic vessels were imaged since the dorsal aorta is too
Fig. 2. The Endothelial Surface Layer (ESL) is visible at 25 and 35 Somites.
The endothelial surface layer, in vessels of the extra-embryonic yolk sac of a living
embryo, was imaged using fluorescently conjugated albumin (green). The ESL is
visible by this method at 25 (A) and 35 somites (B). Endothelial cells lining vessel
wall were labeled with AF555-AcLDL (red, C & D). Large patch of immobilized
albumin (green) are present extending into the vessel lumen from the endothelial
cell surface (red). Scale bar 50 mm.
C.E. Henderson-Toth et al. / Developmental Biology 369 (2012) 330–339
deep in the tissue for confocal microscopy. Endothelial cells also took
up fluorescent albumin so we co-labeled using AcLDL (which labels
the endothelial wall) to differentiate between albumin inside
endothelial cells and luminally-trapped albumin. At 14 and 17
somites, the fluorescent albumin did not indicate the presence of
the ESL layer rather it had become incorporated into the endothelial
cells (data not shown, n¼6). At 25 and 35 somites, it was possible to
visually identify patches of fluorescence in the lumen of vessels
associated with the endothelial lining but entrapped albumin was
always observed in patches and not as a continuous layer (arrows,
n¼6, Fig. 2A, B).
Using immunohistochemistry, we investigated the presence of the
most abundant components of the glycocalyx (HA, CS, HS and SA)
during vascular development (Fig. 3). We stained whole mount and
tissue sections for these components at 14, 17, 25, and 35 somites of
development using immunohistochemistry and lectin-labeling of
extra-embryonic vessels (n¼3 for each stage and each component).
HA detection showed positive staining at all stages investigated.
For 14 and 17 somite embryos, the intensity of the staining around
blood vessels was the same as surrounding tissue (Fig. 3A–B). By 25
somites, the blood vessel wall was stained more strongly for HA than
the surrounding tissues (Fig. 3C, arrows). The strong vascular staining
333
was only present on a subset of blood vessels, which is clearly visible
in the image of the embryo after 4 days of incubation (435 somites
Fig. 3D). The staining for CS revealed this component’s presence
between 14 somites and 35 somites (Fig. 3E–H). However, the CS is
difficult to visualize by immunohistochemistry, and does not appear
to present any developmental pattern over the stages we examined.
Perlecan is a heparin sulfate proteoglycan that is localized to the
basement membrane of endothelial cells (Dziadek et al., 1985) but
that is also secreted into the ESL (Haraldsson et al., 2008). The
perlecan staining was performed in whole-mount and showed a
vascular specific staining during development in extra-embryonic
vessels (Fig. 3I–L). Thickened regions of perlecan were observed at 17
and 25 somites, during vascular remodeling (Fig. 3J–K). We stained
for the presence of SA using a lectin from Limax flavus, which is
specific for this GAG (Miller, 1982). Though staining was present at all
stages observed, the intensity was not any stronger near blood vessels
than in other parts of the tissue (Fig. 3M–P).
Degradation of the glycocalyx affects vascular morphology
In order to help identify the functional role of the glycocalyx
during vascular development, we degraded individual components of
Fig. 3. Immunohistochemical staining of components of the glycocalyx during development. The extra-embryonic yolk sac was stained for HA (A–D, sections),
CS (E–H, sections), a HS called perlecan (I–L, whole-mount) and SA (M–P, sections) at different developmental stages. Hyaluronan was present at all stages but the
intensity was the same for vascular and non-vascular tissue at 14 and 17 somites (A–B). Stronger perivascular signal was present at 25 and 35 somites for some vessels
(arrows, C–D). L¼lumen. Scale bars 100 mm.
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C.E. Henderson-Toth et al. / Developmental Biology 369 (2012) 330–339
this structure via enzyme-mediated degradation in vivo, then evaluated changes in vascular development. First, Hyaluronidase (Hyal)
from S. hyalurolyticus was used to degrade HA. This enzyme is specific
to HA, and unlike other hyaluronidases, does not degrade other GAGs
(Ohya and Kaneko, 1970). Chondroitinase ABC (ChABC) was used to
degrade both CS and dermatan sulfate. Heparinase III (HepIII) was
used to specifically degrade HS, and Neuraminidase Type V (NeurV)
was used to degrade SA. Ringer’s saline solution was injected as a
control.
All enzymes were tested for specificity in vitro prior to use in
the embryo. We tested whether any of the four other major
components were degraded at concentrations that degrade the
target GAG. The enzymes were applied to a monolayer of
endothelial cells, and then immunohistochemical analyses of all
four major components of the glycocalyx were performed postdegradation to investigate the enzymatic effects (n ¼3 for each
enzyme, please see supplemental materials Figs. 1–4). Though we
found some enzymes, such as Hyal, only caused a partial degradation of their target component (please see supplemental
materials Fig. 1E), we did not observe any non-specific enzymatic
degradation of the other major components.
We then explored whether gross cardiovascular function was
affected by enzymatic degradation of the glycocalyx. We injected
enzyme solutions into circulation and then injecting fluorescent
microspheres one hour later. We imaged the movement of these
spheres with a high-speed microscope camera (500 fps) and used
micro-Particle Image Velocimetry to calculate blood flow velocities
in the center of blood vessels at peak systole (n¼ 3 per enzyme, see
Methods). Blood flow in Hyal-treated embryos was significantly
reduced as compared to control embryos (Fig. 4, Supplemental
materials Movie 1–2). Injection of ChABC solution (1 U/mL) also
caused a decrease in the blood flow velocity (Fig. 4, Movie 3).
Furthermore, the heart of ChABC injected embryos stop beating
abruptly 1–2 h after injection of the enzyme solution. Blood flow in
both the HepIII injected embryos (Movie 4) and the NeurV injected
embryos (Movie 5) was similar to control injected embryos.
Supplementary material related to this article can be found
online at http://dx.doi.org/10.1016/j.ydbio.2012.07.009.
In order to observe changes in the vascular network morphology, embryos were exposed to vascular-specific enzyme degradation for 16 h and stained with QH1, an antibody that labels quail
endothelial cells (Pardanaud et al., 1987) (Fig. 5A–E). Many
components of the glycocalyx are also present in the extracellular matrix (ECM) and we were concerned that over a period
of 16 h, the enzymes might leach from the blood vessels and
affect GAGs in the ECM. To circumvent this problem, we immobilized the proteins on 1 mm fluorescent microspheres before
injection. This also allowed us to confirm by fluorescence that
the beads remained in circulation for the entire treatment period.
We verified that GAGs in the ECM were not degraded by
immunohistochemistry on sections (data not shown). Bead injection did not hinder normal vascular remodeling in control
embryos (n ¼3, Fig. 5A). Degradation of HA resulted in hyperfused
vasculature in which hierarchical branching was absent (Fig. 5B,
n¼7). In some embryos (n ¼4 of 7), the vitelline artery formed but
its growth was stunted and the artery did not extend far beyond
the embryo proper. Unlike injection of ChABC in solution, immobilized-ChABC did not cause the heart to stop beating. ChABCtreated embryos remodeled (Fig. 5C, n ¼6), however a lower
vascular density was observed near the large vitelline artery in
some embryos (arrow, n ¼3 of 6). HepIII treated embryos underwent vascular remodeling (Fig. 4D, n¼3) as did the embryos
treated with immobilized NeurV (Fig. 5E, n ¼4).
We looked whether endothelial proliferation and endothelial
apoptosis were affected by enzymatic degradation. The number of
replicating cells per mm2 was assessed by co-staining for QH1
and for phospho-histone 3, which marks cells during mitosis.
No statistically significant difference in endothelial replication
was found in any of the enzymatic degradations as compared to
control (please see supplemental materials). The number of
apoptotic cells was analyzed by injection of AnnexinV, a protein
that binds to phosphatidylserine, which is expressed on the
surface of apoptotic cells. There was no significant difference
between the amount of cell death in control injected and experimental embryos (please see supplemental materials Fig. 5B).
Vascular remodeling is accompanied with the recruitment of
smooth muscle cells to larger arteries and does not occur if blood
flow is stopped. The ability of endothelial cells to recruit smooth
muscle cells was examined after enzymatic degradation using the
bead-immobilized enzymes. With all treatments, we found no
effect on smooth muscle cell recruitment (please see Supplemental Fig. 6).
Degradation of hyaluronan causes an increase in vascular
permeability
Fig. 4. Blood flow velocity is reduced after injection of hyaluronidase or
chondroitinase. Embryos were injected with concentrated enzyme solution
(or albumin for control) and then injected with fluorescent microspheres one
hour later. Flow was imaged using a high-speed fluorescent microscopy (please
see supplemental movies) and the velocity at the center of vessel segments during
systole was analyzed using micro-particle image velocimetry. Both Hyal injection
and ChABC injection caused a decrease in blood flow velocity as compared to
control. Within 1–2 h of ChABC injection, the embryo’s heart stopped beating.
HepIII and NeurV injection had no effect on blood flow velocity. *p o 0.05.
One of the most important roles of the glycocalyx in the adult
vasculature is to control vascular permeability (Vink and Duling,
2000). Embryonic vessels are often described as ‘‘leaky’’.
We therefore sought to investigate the role of the glycocalyx in
controlling permeability in embryonic vessels. To establish the
stage at which endothelial barrier function could first be
observed, quail embryos between 14 and 26 somites were
injected in ovo with FITC-40K neutral dextran. Embryos were
observed each hour in order to assess the time required for the
dextran to leach out from the vessels (Fig. 6A). The endpoint was
chosen as the time at which vessels could no longer be differentiated from avascular tissues by fluorescence. At 14 somites,
dextran leached out of the blood vessels within 1–2 h. The time
required for the dye to leach from the vasculature increased until
embryos reached 16 somites, after which dextran took around 6 h
to leach out of vessels regardless of the stages observed.
C.E. Henderson-Toth et al. / Developmental Biology 369 (2012) 330–339
335
Fig. 5. Vessel branching is altered by selective degradation of the glycocalyx. Whole-mount embryos were stained with QH1 antibody following 16-hour exposure to
bead-immobilized enzymes (A–E). Hyal treatment induced regions of hyperfused vessels and stunted the growth of the vitelline artery (arrow, B). Injection of beadimmobilized ChABC resulted in a decrease in vascular density near the vitelline artery (arrow, C). Injection of HepIII and NeurV did not inhibit vascular remodeling (D, E).
Images were obtained by capturing 40–60 images of the extra-embryonic yolk sac on a confocal microscope and reassembled using Adobe Photoshop’s photomerge
function. The beads used for protein immobilization are fluorescent and create a speckle pattern in the blood vessels. Scale bar 1 mm.
Fig. 6. Hyaluronidase is the main component which controls permeability. Diagram detailing hours required for FITC-dextran to leach from embryonic vessels at
different somite stages during vascular development (A). Graph detailing the hours required for leaching of FITC-dextran in embryos after enzymatic degradation (B).
Dextran-injected embryos initially (C,E) and after 3 h (D, F) for control and Hyal embryos. Treatment with Hyal (50 U/mL) caused a decrease in the time required for dye to
leach out. ChABC treatment (0.001 U/mL) caused a small, but statistically significant increase, in the time required for dye to leach out. ***p o 0.005. Scale bar 1 mm.
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C.E. Henderson-Toth et al. / Developmental Biology 369 (2012) 330–339
To investigate the role of the various components in establishing vascular barrier function, we examined the time required
for dextran to leach from vessels after each of the enzymatic
solution degradations. Quail embryos were injected at 19–20
somites since a stable level of vascular barrier was present
by this stage (Fig. 6A). As expected from our analysis of permeability changes with somite stage, FITC-dextran needed an
average of 6 h to leach out of vessels in control-injected embryos
(n¼13, Fig. 6B). In embryos treated with Hyal at 1 U/mL,
FITC-dextran leached out much more quickly and was no longer
visible in the blood vessels within 3 h post-injection (n¼ 3,
Fig. 6E–F). At this time, control embryos still retained the dye
within the vasculature (Fig. 6D). For ChABC, we performed a serial
dilution to find a concentration that did not stop the heart from
beating. Injection of ChABC at 0.001 U/mL resulted in a small,
but statistically significant, increase in the time required for
FITC-dextran to leach out (n ¼7, 8 h versus 6 h for control).
Injections of HepIII and of NeurV did not cause statistically
significant differences in leaching times (n ¼6, for both). Therefore, only degradation of hyaluronan caused an increase in
vascular permeability.
Endothelial gene expression following selective degradation of the
glycocalyx
Because of the reported role of the glycocalyx in mechanotransduction, we investigated whether the expression of genes
known to be affected by shear stress were altered in embryos
subjected to enzymatic degradation (Fig. 7A). We also investigated whether genes known to be involved in angiogenesis were
affected since we observed defects in vascular branching patterns
(Fig. 7B). We injected the immobilized enzymes at the onset of
flow (14 somites) and isolated endothelial cells 16 h later using
magnetic beads (Dynabeads) coated with QH1, an antibody
specific to quail endothelial cells and macrophages. Dynabeads
are able to isolate cell types to 95% purity (Gomm et al., 1995).
Though the population will not be purely endothelial, the isolated
cells are highly enriched for endothelial cells. Genes used to
assess changes in mechanotransduction included: endothelial
nitrous oxide synthase (nos3), krüppel-like factor 2 (klf2), nuclear
factor kB (nfkb1), platelet derived growth factor b (pdgfb), and
endothelin-1 (edn1). Genes used to analyze effects on angiogenesis included: uncoordinated 5b (unc5b), vascular endothelial
growth factor (vegfa), delta-like 4 (dll4), and hif1a (hif1a). Vegfa
and hif1a were chosen since they are central to vascular health.
Dll4 is expressed by sprouting endothelial cells and is a marker of
angiogenesis (Hellstrom et al., 1999; Lobov et al., 2007; Suchting
et al., 2007). Unc5b, conversely, inhibits angiogenesis and sprouting (Larrivee et al., 2007).
A subset of mechanotransduced genes was affected by HepIII
and Hyal degradation (Fig. 7A), whereas the other treatments had
no effect on these mechanotransduced genes. For instance, HepIII
treatment caused a reduction in nos3 and nfkb1 expression but
did not affect the expression of klf2, pdgfb or edn1. Hyal treatment
affected only the expression of edn1. While this down-regulation
was statistically significant, the magnitude was never larger than
2-fold.
The expression of genes involved in angiogenesis was altered
by three of the enzymatic treatments with immobilized enzymes
(Fig. 7B). Degradation of CS induced a reduction in the expression
of both unc5B and vegfa. Injection of immobilized HepIII resulted
in a 2-fold decrease in expression of vegfa by endothelial cells.
NeurV treatment also decreased unc5b expression. While a
decrease in unc5b expression was also observed with Hyal and
HepIII, this decrease was not statistically significant.
Fig. 7. Expression of mechanotransduced and angiogenesis genes after degradation of the glycocalyx. Endothelial cell gene expression for shear-induced
genes (A) nos3, klf2, nfkb1, pdgfb, and edn1, and for genes linked to angiogenesis (B)
unc5b, vegfa, dll4 and hif1a after enzymatic degradation. Both nos3 and nfkb1
expression were significantly downregulated with injection of bead-immobilized
HepIII. Injection of immobilized Hyal caused a reduction in expression of edn1.
Unc5B was downregulated by ChABC and NeurV treatment. Vegfa expression was
downregulated by ChABC and HepIII treatment. Gene expression normalized to
two housekeeping genes (hprt, actb). *p o 0.05, ***p o 0.005.
Discussion
The importance of the glycocalyx in adult vasculature has
received a significant amount of attention, yet little is known
about its role during vascular development. It has been shown
that in the adult the glycocalyx is essential for maintaining
normal endothelial barrier function and for transduction of the
mechanical signals produced by blood flow (Florian et al., 2003;
Hecker et al., 1993; van den Berg et al., 2003; Vink and Duling,
2000). Interestingly, both of these functions are essential for
proper vascular development (Lucitti et al., 2007; Madri et al.,
2003). Our results establish that the glycocalyx is present in the
quail embryo as soon as blood flow begins at 14 somites. We find
that the early glycocalyx does not show a regular hair-like
structure as is seen later in development. Protrusions of glycosaminoglycans into the vascular lumen are evident at 14 and 17
somites. At these stages, however, the surface of endothelial cells
is also irregular and protrusions are often associated with irregularities in the endothelial cell surface. The structure of the
glycocalyx becomes more uniform as vascular remodeling proceeds, and fewer protrusions are visible (25 somites). The average
thickness does not change significantly during development.
C.E. Henderson-Toth et al. / Developmental Biology 369 (2012) 330–339
We were unable to detect the ESL by imaging immobilized
luminal albumin in the developing vasculature when blood flow
first starts. The entrapped albumin was present in clumps at 25 and
35 somites. We observed entrapped masses of albumin as thick as
10–12 mm, which is significantly thicker than ever observed in the
adult vasculature (Pries et al., 2000). At this point, we do not know
whether thicker regions of the ESL have a functional purpose. When
we time-lapsed embryos co-labeled with fluorescent albumin and
AcLDL, the thickened regions did not correspond to any specific
morphological events, such as angiogenic sprouting or increasing
vessel diameter (data not shown).
The glycocalyx is composed of a large array of GAGs. For the
purpose of this study we chose to test four of the most prevalent
components in the adult glycocalyx (Reitsma et al., 2007). While it is
possible that the glycocalyx composition differs between development and the adult, this possibility is outside the scope of this study.
The glycocalyx is known to shed GAGs under normal circumstances,
and blood naturally contains components that damage and degrade
parts of the glycocalyx. GAGs of the glycocalyx are experiencing
constant degradation and synthesis (Mulivor and Lipowsky, 2004).
By keeping fluorosphere beads with enzyme in circulation until just
prior to dissection, we ensure that partial degradation of the
glycocalyx component occurs continuously throughout the incubation period. We believe, however, that our treatments lead to only a
partial degradation of the glycocalyx.
ChABC in solution caused a decrease in the velocity of blood
flow and caused the heart to stop beating. When ChABC is
delivered immobilized on beads to ensure the enzyme is confined
to the luminal side of the vasculature, we observed a healthy
heartbeat and vascular remodeling occurred. This might indicate
that the CS component in the ECM of the heart is required for
cardiac contraction.
Because the glycocalyx and ESL are known to be important for
mechanotransduction, we investigated whether degradation of
the glycocalyx during vascular development affects the expression of mechanotransduced genes. Our results showed a two-fold
reduction in nos3 expression after HepIII degradation. A 44%
percent reduction in NO production after heparinase treatment
has previously been reported for endothelial cells exposed to flow
in culture (Pahakis et al., 2007). The authors also found that
degradation of HA, SA and CS influenced NO production (Pahakis
et al., 2007). Our work, conversely, did not show an effect on nos3
expression with any of the other enzymatic treatments. We did
not measure eNOS phosphorylation or NO production and it has
been reported that it is the level of phosphorylated eNOS and not
the total levels of eNOS levels that are affected by glycocalyx
degradation (Tarbell and Ebong, 2008).
Though the expression of two important mechanotransduced
genes (nos3 and nfkb1) was reduced with HepIII treatment,
vascular remodeling was normal. We cannot rule out the possibility that HS was only partially degraded and that more significant defects would be present if all luminal HS was removed.
The role of mechanotransduced genes during development is also
controversial. Mouse embryos in which all three nitric oxide
synthases are ablated are viable (Tsutsui et al., 2006) and undergo
vascular remodeling. PECAM-1 is believed to be required for
mechanotransduction (Tzima et al., 2005), however genetic ablation of this protein does not affect vascular development (Duncan
et al., 1999). Therefore, our results are in agreement with the
literature that embryonic development can still occur when
certain aspects of mechanotransduction are ablated. Since
mechanical signals from blood flow are required for vascular
development (Lucitti et al., 2007), this may indicate that redundant pathways for mechanotransduction are present or that the
effects by HepIII degradation in our system were too subtle to
induce significant defects in remodeling. While it has been
337
previously established that HA in the ECM is involved in the
migration and proliferation of smooth muscle cells, we did not
observe any down regulation in shear-induced genes after treatment with hyaluronidase. Therefore it is not surprising that shear
dependent recruitment of smooth muscle cells occurred normally.
Because we observed changes in the branching pattern of
blood vessels (Fig. 5B, C), we examined whether genes involved in
endothelial cell sprouting and guidance were affected by enzymatic degradation. HepIII is known to be anti-angiogenic (Van
Sluis et al., 2010) and we found that degrading luminal HS caused
a decrease in vegfa expression. This was not associated with any
specific vascular defects in the HepIII treated embryos. Surprisingly, the gene most affected by degradation of the glycocalyx
was unc5b, whose signaling is known to be inhibitory to angiogenesis (Larrivee et al., 2007). Only treatment with CS showed any
reduction in vascular density. In the nervous system, CS is
inhibitory to neuritic outgrowth and the ligand for Unc5B, Netrin
(Leonardo et al., 1997), has been shown to bind cell surface HS
and CS. Interestingly, these are the two treatments that led to a
statistically significant decrease in unc5b expression. This raises
the interesting possibility that CS plays a similar role in the
cardiovascular system by guiding angiogenic sprouts, similar to
its established role in the nervous system. It is not clear how
luminal GAGs could affect endothelial sprouting into tissues.
Netrin-1 is secreted into blood plasma (Ramesh et al., 2011) and
luminal GAGs could sequester Netrin and prevent its entry into
surrounding tissues (Shipp and Hsieh-Wilson, 2007).
When we looked at the importance of the glycocalyx components in establishing vascular permeability we found that only
Hyal treatment increased vascular permeability in the embryo.
Interestingly, the appearance of vascular-specific staining for HA
occurs between 17 and 25 somites (Fig. 3B–C), which coincides
with the stage at which we see vascular barrier function established in the embryonic vasculature (Fig. 6A). Fluid shear stress
has previously been shown to induce incorporation of HA into the
glycocalyx (Gouverneur et al., 2006) and the vascular-specific HA
staining in the embryo appears just shortly after the onset of
blood flow. Degradation of HA in the adult vasculature increases
vascular permeability, rendering the glycocalyx permeable to 70
and 145 kDa high-molecular weight dextrans, which are normally
excluded from the glycocalyx (Henry and Duling, 1999). Degradation of the glycocalyx through perfusion of the enzyme pronase
results in a 2.5 increase in the hydraulic permeability of blood
vessels (Adamson, 1990). Our results confirm a vital role for HA in
controlling the permeability of the embryonic vasculature.
We find that dextrans leach out of blood vessels twice as fast
after hyaluronidase treatment as compared to control.
The degradation of hyaluronan also prevents vascular remodeling and results in a hyperfused vascular plexus. The branching
patterns observed in the embryos treated with Hyal solution are
similar to what occurs when flow is stopped (Chapman, 1918)
(data not shown). Since Hyal treatment also affects blood flow, we
cannot separate effects due to permeability from those due to
decreased shear stress, though we believe the reduced flow is a
secondary defect induced by increased permeability.
We saw that treatment with ChABC caused an increase in time
required for dye to leach out of blood vessels. We do not believe
this increase is due to altered barrier function. Though the
heartbeat was normal at the concentrations of ChABC used to
test for permeability (0.001 U/mL), we cannot state that more
subtle defects in blood flow were absent. Since transmural flow is
a function of both hydrostatic pressure in the vessels and vessel
permeability, we believe that blood pressure may have been
slightly lowered, leading to a decrease in hydrostatic pressure
and therefore a decrease in the transmural flowrate out of the
vessel. Interestingly, this presents itself as a sensitive and simple
338
C.E. Henderson-Toth et al. / Developmental Biology 369 (2012) 330–339
method to investigate the presence of mild defects in heart
function during vascular development.
We did not observe an increase in permeability due to the
partial degradation of HS, which makes up the great majority of
GAGs in the endothelial glycocalyx (Oohira et al., 1983). Though it
is surprising that degradation of a major component of the
glycocalyx does not affect permeability, these results are supported by findings in the adult whereby degradation of HS has no
effect on permeability (Rehm et al., 2004).
Several diseases have been linked to the thinning of the
glycocalyx. Diabetes mellitus is a disease wherein high levels of
oxygen radicals cause the glycocalyx to become thinned
(Nieuwdorp et al., 2006b). In this disease, the thinned glycocalyx
is also made hyperpermeable. Incubation of embryos in high
glucose inhibits vascular remodeling in a manner similar to what
we observed due to HA degradation (Fig. 5B) (Madri et al., 2003).
Since HA determines barrier function in the embryo, it is likely
that it is a component affected by maternal diabetes.
Conclusions
Our results show the presence of the glycocalyx before the onset
of blood flow. Using TEM microscopy, we also observe that the early
glycocalyx (14 and 17 somites) shows significant protrusion into the
vascular lumen though these protrusions are often accompanied by
heterogeneity in the endothelial cell lining. At 14-somites, the distinct
hair-like structure of the glycocalyx is absent. Additionally, we
demonstrated the functionality of the individual glycocalyx components in the embryo through the use of selective enzymatic degradation. We have found that HA is the component mainly responsible for
controlling barrier function and that degradation of this component
causes a morphological effect similar to the effects of maternal
diabetes. We also showed that while some mechanotransduction
genes were down regulated by HepIII treatment, embryonic vascular
development remained normal. These results indicate that the
glycocalyx has a functional role in vascular development.
Sources of funding
This work was supported by grants from the Sick Kids
Foundation of Canada (NI12-029), NSERC Discovery Program
(342134), and Canadian Foundation for Innovation and the
Canada Research Chair Program. CHT was supported by the CIHR
Banting and Best Master’s Award.
Acknowledgments
A special thanks to the staff at the Facility for Electron
Microscopy Research for their kind assistance with electron
microscopy preparation and imaging. Additional thanks to Bahar
Kasaai for her kind assistance.
Appendix A. Supporting information
Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.ydbio.2012.07.009.
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