The

endothelial

glycocalyx

composition

functions

and

visualization

This

review

aims

at

presenting

state-of-the-art

knowledge

on

the

composition

and

functions

of

the

endothelial

glycocalyx

The

endothelial

glycocalyx

is

a

network

of

membrane-bound

proteoglycans

and

glycoproteins

covering

the

endothelium

luminally

Both

endothelium

and

plasma-derived

soluble

molecules

integrate

into

this

mesh

Over

the

past

decade

insight

has

been

gained

into

the

role

of

the

glycocalyx

in

vascular

physiology

and

pathology

including

mechanotransduction

hemostasis

signaling

and

blood

cell

vessel

wall

interactions

The

contribution

of

the

glycocalyx

to

diabetes

ischemia/reperfusion

and

atherosclerosis

is

also

reviewed

Experimental

data

from

the

micro

and

macrocirculation

alludes

at

a

vasculoprotective

role

for

the

glycocalyx

Assessing

this

possible

role

of

the

endothelial

glycocalyx

requires

reliable

visualization

of

this

delicate

layer

which

is

a

great

challenge

An

overview

is

given

of

the

various

ways

in

which

the

endothelial

glycocalyx

has

been

visualized

up

to

now

including

first

data

from

two-photon

microscopic

imaging

Introduction

The

endothelial

glycocalyx

was

already

visualized

some

years

ago

by

Luft

using

electron

microscopy

-LSB-

-RSB-

Still

relatively

little

is

known

of

the

composition

and

function

of

this

layer

Over

the

past

decades

it

has

been

increasingly

appreciated

as

an

important

factor

in

vascular

physiology

and

pathology

as

described

in

in

a

review

by

Pries

et

al.

-LSB-

-RSB-

and

in

other

more

recent

reviews

-LSB-

-RSB-

The

interest

in

the

-LRB-

patho

-RRB-

physiological

role

of

the

glycocalyx

started

with

the

observation

of

low

and

variable

capillary

tube

hematocrit

which

depended

on

the

level

of

metabolic

and

pharmacological

activation

of

the

vascular

system

-LSB-

-RSB-

The

relation

between

metabolic

and

agonist-induced

increases

in

red

blood

cell

velocity

on

the

one

hand

and

tube

hematocrit

on

the

other

could

partly

be

explained

by

plasma

skimming

as

direct

continuation

of

the

Fåhraeus

effect

-LSB-

-RSB-

However

this

relation

dissociated

upon

local

treatment

of

the

microvessels

with

heparinase

an

enzyme

which

breaks

down

heparan

sulfates

in

the

glycocalyx

-LSB-

-RSB-

This

finding

was

in

agreement

with

theoretical

estimates

predicting

a

thick

slow-moving

plasma

layer

over

the

endothelium

-LSB-

-RSB-

In

vivo

studies

have

revealed

the

glycocalyx

in

muscle

capillaries

to

be

a

layer

of

about

μm

thick

covering

endothelial

cells

and

determining

luminal

domains

for

macromolecules

red

and

white

blood

cells

-LSB-

-RSB-

More

recent

studies

indicate

that

glycocalyx

thickness

increases

with

vascular

diameter

at

least

in

the

arterial

system

ranging

from

to

μm

in

small

arteries

-LSB-

-RSB-

to

μm

in

carotid

arteries

-LSB-

-RSB-

To

date

many

studies

indicate

a

variety

of

-LRB-

patho

-RRB-

physiological

roles

for

the

endothelial

glycocalyx

in

addition

to

modulating

capillary

red

blood

cell

filling

the

glycocalyx

may

affect

many

other

-LRB-

dys

-RRB-

functions

of

the

vascular

system

Whereas

the

vascular

endothelium

is

currently

believed

to

be

actively

involved

in

every

pathology

presenting

vascular

projections

-LSB-

-RSB-

the

same

saying

might

well

prove

true

for

the

glycocalyx

Assessing

this

possible

involvement

of

the

endothelial

glycocalyx

requires

reliable

visualization

of

this

delicate

layer

which

is

a

great

challenge

This

review

provides

basic

insight

into

the

present

knowledge

of

composition

and

functions

of

the

endothelial

glycocalyx

and

gives

an

overview

of

the

various

ways

in

which

it

has

been

visualized

up

to

now

Composition

The

endothelial

glycocalyx

is

a

carbohydrate-rich

layer

lining

the

vascular

endothelium

It

is

considered

to

be

connected

to

the

endothelium

through

several

backbone

molecules

mainly

proteoglycans

and

also

glycoproteins

These

form

a

network

in

which

soluble

molecules

either

plasma

or

endothelium-derived

are

incorporated

More

luminally

the

glycocalyx

is

formed

by

soluble

plasma

components

linked

to

each

other

in

a

direct

way

or

via

soluble

proteoglycans

and/or

glycosaminoglycans

-LRB-

which

will

be

discussed

below

-RRB-

A

dynamic

equilibrium

exists

between

this

layer

of

soluble

components

and

the

flowing

blood

continuously

affecting

composition

and

thickness

of

the

glycocalyx

Furthermore

the

glycocalyx

suffers

from

enzymatic

or

shear-induced

shedding

The

dynamic

balance

between

biosynthesis

and

shedding

makes

it

hard

to

define

the

glycocalyx

geometrically

-LSB-

-RSB-

The

composition

of

the

membrane-bound

mesh

of

proteoglycans

glycoproteins

and

glycosaminoglycans

and

the

composition

of

associated

plasma

proteins

and

soluble

glycosaminoglycans

can

not

be

viewed

as

a

static

picture

Instead

the

layer

as

a

whole

also

known

as

endothelial

surface

layer

-LRB-

ESL

-RRB-

-LSB-

-RSB-

is

very

dynamic

with

membrane-bound

molecules

being

constantly

replaced

and

no

distinct

boundary

between

locally

synthesized

and

associated

elements

membrane-bound

hyaluronan

may

reach

lengths

of

μm

Direct

visualization

techniques

-LRB-

see

section

on

Visualization

techniques

-RRB-

fail

to

demonstrate

clear

compositional

differences

within

the

glycocalyx

from

endothelial

membrane

towards

vascular

lumen

but

rather

indicate

that

the

endothelial

glycocalyx

resembles

an

intricate

self-assembling

mesh

of

various

polysaccharides

Enzymatic

removal

of

any

of

its

constituents

dramatically

affects

glycocalyx

properties

which

exemplifies

the

importance

of

considering

the

synergetic

interaction

of

all

glycocalyx

constituents

as

a

whole

In

this

review

we

will

therefore

use

the

term

-LRB-

endothelial

-RRB-

glycocalyx

for

the

total

layer

-LRB-

Fig.

-RRB-

and

be

as

specific

as

possible

when

addressing

its

various

elements

Fig.

representation

of

the

endothelial

glycocalyx

showing

its

main

components

Left

The

endothelial

glycocalyx

can

be

observed

in

vivo

as

a

red

blood

cell

exclusion

zone

located

on

the

luminal

side

of

the

vascular

endothelium

It

consists

of

membrane-bound

and

soluble

molecules

Right

Components

of

the

endothelial

glycocalyx

Bound

to

the

endothelial

membrane

are

proteoglycans

with

long

unbranched

glycosaminoglycan

side-chains

-LRB-

GAG-chain

-RRB-

and

glycoproteins

with

short

branched

carbohydrate

side-chains

Incorporated

in

and

on

top

of

this

grid

are

plasma

and

endothelium-derived

soluble

components

including

hyaluronic

acid

and

other

soluble

proteoglycans

-LRB-

e.g.

thrombomodulin

-RRB-

and

various

proteins

such

as

extracellular

superoxide

dismutase

-LRB-

ec-SOD

-RRB-

and

antithrombin

III

-LRB-

AT

III

-RRB-

Together

these

components

form

the

endothelial

glycocalyx

that

functions

as

a

barrier

between

blood

plasma

and

the

endothelium

and

exerts

various

roles

in

plasma

and

vessel

wall

homeostasis

Note

that

this

figure

is

not

drawn

to

scale

its

purpose

is

to

illustrate

glycocalyx

composition

Below

state-of-the-art

knowledge

on

the

various

components

of

the

endothelial

glycocalyx

will

be

provided

Although

many

molecules

have

been

identified

as

being

part

of

the

glycocalyx

information

on

their

distribution

is

still

scarce

if

present

such

knowledge

was

mostly

obtained

indirectly

and

nonquantitatively

Proteoglycans

Proteoglycans

are

generally

considered

to

function

as

the

most

important

backbone

molecules

of

the

glycocalyx

They

consist

of

a

core

protein

to

which

one

or

more

glycosaminoglycan

chains

are

linked

There

is

a

notable

variation

among

the

proteoglycan

core

proteins

with

regard

to

their

size

number

of

attached

glycosaminoglycan

chains

and

whether

or

not

they

are

bound

to

the

cell

membrane

-LRB-

Table

-RRB-

The

core

protein

groups

of

syndecans

-LRB-

n

-RRB-

and

glypicans

-LRB-

n

-RRB-

have

a

firm

connection

to

the

cell

membrane

via

a

membrane-spanning

domain

-LRB-

syndecans

-RRB-

or

a

glycosylphosphatidylinositol

anchor

-LRB-

glypicans

-RRB-

-LSB-

-RSB-

The

other

proteoglycans

such

as

mimecan

perlecan

and

biglycan

are

secreted

after

their

assembly

and

glycosaminoglycan

chain

modification

-LSB-

-RSB-

This

leads

to

production

of

soluble

proteoglycans

which

reside

in

the

glycocalyx

or

diffuse

into

the

blood

stream

Table

of

proteoglycan

core

proteins

in

the

vascular

endothelial

glycocalyxCore

protein

groupCore

protein

size

-LRB-

kDa

-RRB-

Number

of

subtypesNumber

of

GAG-chains

linkedType

of

GAG-chains

linkedStructural

relation

to

cell

Glycosaminoglycan

HS

heparan

sulfate

CS

chondroitin

sulfate

DS

dermatan

sulfate

KS

keratan

sulfate

GPI

glycosylphosphatidylinositol

Proteoglycans

are

promiscuous

in

their

binding

of

glycosaminoglycan

chains

meaning

that

one

core

protein

can

contain

different

types

of

glycosaminoglycan

chains

The

proportion

of

the

various

chains

may

change

under

different

circumstances

and

stimuli

-LSB-

-RSB-

Therefore

naming

of

proteoglycans

after

one

type

of

glycosaminoglycan

is

somewhat

deluding

For

example

proteoglycan

is

often

addressed

as

a

heparan

sulfate

proteoglycan

while

in

fact

it

usually

contains

similar

numbers

of

heparan

sulfate

and

chondroitin

sulfate

chains

-LSB-

-RSB-

There

are

five

types

of

glycosaminoglycan

chains

heparan

sulfate

chondroitin

sulfate

dermatan

sulfate

keratan

sulfate

and

hyaluronan

-LRB-

or

hyaluronic

acid

-RRB-

They

are

linear

polymers

of

disaccharides

with

variable

lengths

that

are

modified

by

sulfation

and/or

-LRB-

de

-RRB-

acetylation

to

a

variable

extent

The

disaccharides

are

each

composed

of

a

uronic

acid

and

a

hexosamine

classification

of

the

glycosaminoglycans

depends

on

which

uronic

acid

or

hexosamine

is

incorporated

and

on

the

pattern

of

sulfation

-LRB-

Table

-RRB-

Each

of

the

five

glycosaminoglycans

has

been

investigated

and

reviewed

extensively

-LSB-

-RSB-

Dermatan

sulfate

is

often

regarded

as

a

separate

class

of

glycosaminoglycans

although

it

actually

is

type

B

chondroitin

sulfate

The

difference

between

the

two

is

possible

epimerization

of

the

glucuronic

acids

into

iduronic

acids

in

dermatan

sulfate

which

has

important

consequences

for

functionality

Whenever

possible

we

will

try

to

separate

the

two

as

precisely

as

possible

elsewhere

they

will

be

referred

to

as

chondroitin

sulfate/dermatan

sulfate

Table

of

the

disaccharides

of

various

glycosaminoglycan

chains

Heparan

sulfateChondroitin

sulfateDermatan

sulfateaHyaluronanKeratan

sulfateUronic

acidGlcA

-LRB-

-RRB-

IdoA

-LRB-

-RRB-

GlcAGlcA

IdoA

-LRB-

-RRB-

GlcAGal

-LRB-

-RRB-

Disaccharide

-LRB-

NS

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

GlcNAcGlcNAc

-LRB-

-RRB-

Polymerization

the

various

possibilities

of

sulfation

in

heparan

sulfate

These

may

coincide

-LRB-

e.g.

in

heparan

sulfate

the

hexosamine

-RRB-

A

rare

but

possible

hexosamine

in

heparan

sulfate

is

the

N-unsubstituted

glucosamine

which

has

been

left

out

of

the

table

for

convenient

reading

Also

note

the

presence

of

IdoA

in

dermatan

sulfate

in

contrast

to

the

other

chondroitin

sulfates

making

it

more

alike

to

heparan

sulfate.GlcA

Glucuronic

acid

IdoA

iduronic

acid

Gal

galacturonic

acid

GlcNAc

N-acetyl-glucosamine

GalNAc

N-acetyl-galactosamine

NS

N-sulfatedaThere

are

three

types

of

chondroitin

sulfate

Type

A

only

has

N-acetyl-galactosamines

type

B

is

known

as

dermatan

sulfate

and

type

C

only

has

N-acetyl-galactosamines

In

vasculature

heparan

sulfate

proteoglycans

represent

roughly

of

the

total

amount

of

proteoglycans

present

in

the

glycocalyx

-LSB-

-RSB-

However

this

figure

is

variable

as

the

expression

of

proteoglycans

by

endothelial

cells

depends

on

various

stimuli

Syndecans

for

example

have

a

tightly

regulated

expression

pattern

which

varies

with

endothelial

cell

activation

or

stimulation

with

different

chemokines

-LSB-

-RSB-

The

second

most

common

glycosaminoglycan

in

the

endothelial

cell

glycocalyx

is

chondroitin

sulfate/dermatan

sulfate

The

presence

of

heparan

sulfate

and

chondroitin

sulfate

is

reported

to

have

a

typical

ratio

of

for

the

vascular

endothelium

-LSB-

-RSB-

Expression

of

keratan

sulfate

glycosaminoglycans

in

vasculature

and

its

importance

in

-LRB-

patho

-RRB-

physiology

is

less

well

understood

Another

important

glycosaminoglycan

in

the

glycocalyx

is

hyaluronan

This

long

polymeric

molecule

-LRB-

up

to

kDa

-RRB-

differs

from

other

glycosaminoglycans

in

that

it

is

not

linked

to

a

core

protein

Its

exact

link

to

the

cell

membrane

is

unknown

but

it

can

be

bound

to

the

receptor

-LSB-

-RSB-

Alternatively

hyaluronan

may

be

attached

to

its

assembly

proteins

the

hyaluronan

synthases

-LSB-

-RSB-

which

are

located

at

the

cytosolic

side

of

the

cell

membrane

Another

possibility

is

that

hyaluronan

is

-LRB-

in

part

-RRB-

not

directly

bound

to

the

membrane

at

all

It

is

capable

of

forming

strikingly

viscous

solutions

-LSB-

-RSB-

More

recently

intracellular

hyaluronan

binding

proteins

such

as

-LSB-

-RSB-

and

-LSB-

-RSB-

have

been

identified

suggesting

a

role

for

this

glycosaminoglycan

within

the

cell

-LSB-

-RSB-

Heparan

sulfate

and

chondroitin

sulfate/dermatan

sulfate

containing

proteoglycans

are

produced

in

the

endoplasmic

reticulum

and

Golgi

apparatus

of

the

endothelial

cell

After

the

ribosomal

translation

of

the

core

protein

a

xylosyltransferase

will

transfer

xylose

-LRB-

Xyl

-RRB-

from

uracildiphosphate

xylose

to

specific

serine

residues

-LRB-

Ser

-RRB-

in

the

core

protein

The

xylose-enriched

core

protein

is

transported

to

the

cis-Golgi

and

galactosyltransferases

types

I

and

II

will

add

two

galactose-groups

-LRB-

Gal

-RRB-

to

the

xylose

after

which

glucuronosyltransferase

type

I

adds

glucuronic

acid

thus

completing

the

primary

linker

for

glycosaminoglycan

chains

GlcA

β3

Gal

β3

Gal

β4

-LSB-

Ser

-RSB-

After

formation

of

the

primary

linker

the

following

step

determines

the

type

of

glycosaminoglycan

chain

that

will

be

formed

In

the

case

of

heparan

sulfate

an

is

added

in

the

case

of

chondroitin

sulfate

and

dermatan

sulfate

a

is

added

and

both

galactose-residues

from

the

linker

may

be

sulfated

From

this

point

onward

glucuronic

acids

and

glucosamines

are

linked

to

the

core

protein

After

chain

polymerization

the

growing

glycosaminoglycan

chain

will

undergo

modifications

including

N-sulfation

O-sulfation

and

epimerization

The

latter

changes

glucuronic

acid

residues

into

iduronic

acid

residues

and

will

change

any

chondroitin

sulfate

chain

into

its

type

B

dermatan

sulfate

These

chain

modifications

take

place

in

both

the

cis

and

the

trans-Golgi

determining

the

final

type

and

functionality

of

the

proteoglycan

and

each

of

its

side

chains

In

contrast

hyaluronan

is

assembled

at

the

cytosolic

side

of

the

cell

membrane

and

it

is

not

modified

afterwards

As

a

consequence

it

has

no

sulfate

groups

or

modification

pattern

As

glycosaminoglycan

chains

contain

numerous

specific

binding

sites

for

plasma-derived

proteins

small

chain

modifications

can

have

great

functional

consequences

Sequential

enzymatic

modifications

of

the

individual

saccharide

units

within

glycosaminoglycan

chains

endow

proteoglycans

with

unique

functions

Typically

different

sulfation

patterns

may

exist

per

disaccharide

and

as

functional

domains

are

assumed

to

be

usually

penta

to

deca-saccharide

long

at

least

different

sulfation

patterns

on

a

hexasaccharide

backbone

are

theoretically

possible

and

such

a

structural

diversity

corresponds

to

diversified

biological

functions

of

glycosaminoglycans

Indeed

it

has

been

shown

that

modification

patterns

vary

in

time

and

under

different

-LRB-

patho

-RRB-

physiological

stimuli

-LSB-

-RSB-

The

diversity

of

glycosaminoglycan

sulfation

patterns

and

its

effect

on

specific

protein

binding

and

modulation

of

protein

function

suggest

that

conditions

that

diminish

glycocalyx

thickness

or

modulate

protein

specific

glycosaminoglycan

sulfation

patterns

and

charge

are

likely

to

modulate

vascular

permeability

and

alter

specific

protein

binding

and

activity

Glycoproteins

Besides

the

proteoglycans

with

their

long

linear

side

chains

certain

glycoproteins

are

also

regarded

as

backbone

molecules

connecting

the

glycocalyx

to

the

endothelial

cell

membrane

This

group

of

endothelial

glycoproteins

characterized

by

relatively

small

-LRB-

sugar

residues

-RRB-

and

branched

carbohydrate

side

chains

comprises

a

number

of

molecules

that

all

have

been

studied

intensively

major

classes

that

will

be

discussed

in

more

detail

below

are

the

endothelial

cell

adhesion

molecules

and

components

of

the

coagulation

and

fibrinolysis

system

It

is

beyond

the

scope

of

this

review

to

categorically

discuss

all

glycoproteins

that

can

be

expressed

by

endothelial

cells

Furthermore

one

should

realize

that

the

level

of

glycoprotein

expression

on

the

endothelial

cell

membrane

varies

considerably

with

cell

activation

or

stimulation

The

endothelial

cell

adhesion

molecules

are

well-defined

glycoproteins

that

play

a

major

role

in

cell

recruitment

from

the

bloodstream

and

in

cell

signaling

The

three

families

of

cell

adhesion

molecules

present

in

the

endothelial

glycocalyx

are

the

selectin

family

the

integrin

family

and

the

immunoglobulin

superfamily

Glycoproteins

from

the

selectin

family

contain

a

cytoplasmic

tail

a

transmembrane

domain

several

consensus

repeats

an

epidermal

growth

factor-like

domain

and

a

terminal

lectin

domain

which

is

primarily

responsible

for

binding

of

carbohydrate

groups

on

glycosylated

proteins

or

lipids

However

the

epidermal

growth

factor-like

domain

is

involved

in

selectin-ligand

recognition

as

well

-LSB-

-RSB-

Selectins

found

on

the

vascular

endothelium

are

E-selectin

and

P-selectin

both

involved

in

leukocyte

endothelial

cell

interactions

-LSB-

-RSB-

P-selectin

is

constitutively

produced

and

subsequently

stored

in

the

Weibel

Palade

bodies

of

the

endothelial

cells

Exocytosis

of

Weibel

Palade

bodies

as

induced

by

stimuli

such

as

thrombin

and

histamine

allows

a

rapid

translocation

of

P-selectin

to

the

cell

surface

-LSB-

-RSB-

However

this

expression

is

short-lived

due

to

P-selectin

internalization

and

redirection

to

lysosomal

granules

or

the

Golgi

apparatus

where

it

is

restored

in

newly

formed

Weibel

Palade

bodies

-LSB-

-RSB-

E-selectin

is

not

stored

in

granules

but

requires

de

novo

mRNA

and

protein

synthesis

to

be

expressed

on

the

cell

surface

Stimulation

of

endothelial

cells

by

cytokines

such

as

tumor

necrosis

factor

α

and

lipopolysaccharide

upregulates

E-selectin

expression

this

typically

requires

h

-LSB-

-RSB-

In

some

tissues

like

the

skin

P

and

E-selectin

appear

to

be

constitutively

expressed

on

endothelial

cells

-LSB-

-RSB-

Integrins

are

heterodimeric

molecules

composed

of

non-covalently

bound

α

and

β

subunits

Both

subunits

have

a

cytoplasmic

tail

and

a

transmembrane

domain

and

together

they

constitute

an

integral

membrane

protein

To

date

different

α-subunits

and

β-subunits

have

been

identified

which

means

that

each

integrin

is

characterized

by

the

specific

combination

of

its

subunits

-LSB-

-RSB-

Integrins

are

found

on

many

cell

types

including

endothelial

cells

leukocytes

and

platelets

In

their

luminal

membrane

endothelial

cells

express

integrin

αVβ3

which

is

an

important

mediator

of

platelet

endothelial

cell

interactions

-LSB-

-RSB-

Most

other

endothelial

cell

integrins

are

involved

in

binding

to

the

basement

membrane

These

integrins

such

as

α2β1

α5β1

and

α6β1

bind

to

multiple

extracellular

matrix

ligands

and

are

as

such

responsible

for

interactions

with

laminin

fibronectin

and

collagen

Many

studies

have

focused

and

still

focus

on

the

interactions

between

these

integrins

and

the

subendothelial

matrix

during

angiogenesis

-LSB-

-RSB-

The

immunoglobulin

superfamily

of

glycoproteins

is

characterized

by

a

cytoplasmic

tail

a

transmembrane

domain

and

a

variable

number

of

immunoglobulin-like

domains

that

protrude

luminally

Best

known

examples

are

intercellular

adhesion

molecule

and

-LRB-

and

-RRB-

vascular

cell

adhesion

molecule

-LRB-

-RRB-

and

platelet/endothelial

cell

adhesion

molecule

-LRB-

-RRB-

which

act

as

ligand

for

integrins

on

leukocytes

and

platelets

and

are

crucial

mediators

of

leukocyte

homing

to

the

endothelium

and

subsequent

diapedesis

and

and

have

a

baseline

expression

whereas

is

only

present

after

endothelial

cell

stimulation

by

cytokines

which

also

upregulates

expression

-LSB-

-RSB-

Paradoxically

the

constitutive

expression

of

is

decreased

after

cytokine

treatment

-LSB-

-RSB-

The

role

of

in

inflammation

is

still

unclear

as

it

is

also

downregulated

by

inflammatory

stimuli

Recently

it

was

shown

that

is

involved

in

regulation

of

angiogenesis

-LSB-

-RSB-

Besides

the

cell

adhesion

molecules

the

endothelial

glycocalyx

harbors

glycoproteins

with

functionality

in

coagulation

fibrinolysis

and

hemostasis

A

good

example

is

the

glycoprotein

Ib-IX

V

complex

which

is

expressed

on

endothelial

cells

and

also

on

platelets

It

consists

of

four

glycoproteins

Ibα

Ibβ

IX

and

V

that

are

each

membrane-spanning

polypeptides

Glycoprotein

Ibα

and

Ibβ

are

covalently

linked

via

a

disulfide

group

whereas

IX

and

V

are

non-covalently

attached

to

the

Ib

heterodimer

The

Ib-IX

V

complex

binds

von

Willebrand

factor

-LRB-

vWf

-RRB-

and

is

primarily

known

as

the

platelet

vWf-receptor

-LSB-

-RSB-

Furthermore

the

complex

also

binds

P-selectin

mediating

the

interaction

of

platelets

with

activated

endothelial

cells

-LSB-

-RSB-

Like

platelets

endothelial

cells

express

all

components

of

the

Ib-IX

V

complex

-LSB-

-RSB-

which

on

one

hand

allows

binding

to

the

vWf

substrate

of

the

subendothelium

and

on

the

other

binds

Weibel

Palade

body

derived

vWf

secreted

luminally

by

activated

endothelial

cells

Soluble

components

Embedded

within

and

layered

on

top

of

the

mesh

of

proteoglycans

and

glycoproteins

are

soluble

components

of

various

types

such

as

proteins

and

soluble

proteoglycans

These

components

are

either

derived

from

the

endothelium

or

from

the

bloodstream

such

as

albumin

and

orosomucoid

which

are

pivotal

in

preserving

the

-LRB-

charge

-RRB-

selectivity

of

the

permeability

barrier

-LSB-

-RSB-

The

soluble

components

of

the

glycocalyx

contribute

greatly

to

the

functional

importance

of

the

glycocalyx

as

will

be

described

below

The

structural

attribution

of

these

soluble

components

is

less

well

established

At

least

some

of

the

soluble

components

may

contribute

to

the

structural

organization

of

the

luminal

glycocalyx

although

this

is

hard

to

prove

There

are

indications

that

proteoglycans

bind

to

each

other

and

to

proteins

-LSB-

-RSB-

Protein

glycosaminoglycan

protein

complexes

have

been

identified

although

not

in

the

glycocalyx

in

particular

-LSB-

-RSB-

Still

it

is

presumable

that

interactions

between

membrane-bound

proteoglycans

soluble

proteins

and

soluble

proteoglycans

create

a

cross-linked

mesh

and

provide

some

stability

to

the

luminal

glycocalyx

Hyaluronan

might

play

an

important

role

in

this

respect

being

a

very

large

linear

molecule

and

possibly

unbound

to

the

endothelial

cell

membrane

-LRB-

Fig.

-RRB-

It

has

been

shown

to

interact

with

itself

forming

stable

hyaluronan

hyaluronan

complexes

-LSB-

-RSB-

Still

the

glycocalyx

is

a

delicate

layer

and

removal

of

one

specific

component

may

result

in

loss

of

function

of

the

total

-LSB-

-RSB-

Functional

importance

The

endothelial

glycocalyx

as

the

endothelial

gatekeeper

Located

between

the

blood

stream

and

the

endothelium

the

endothelial

glycocalyx

is

an

important

determinant

of

vascular

permeability

-LSB-

-RSB-

It

is

able

to

limit

access

of

certain

molecules

to

the

endothelial

cell

membrane

as

has

been

demonstrated

in

small

rat

mesenteric

arteries

with

the

use

of

fluorescently

labeled

dextrans

of

various

molecular

weights

showing

increasing

permeability

for

smaller

molecules

-LSB-

-RSB-

Enzymatic

-LRB-

partial

-RRB-

removal

and

subsequent

loss

of

permeability

barrier

function

of

the

glycocalyx

in

rat

myocardial

capillaries

leads

to

myocardial

edema

-LSB-

-RSB-

Not

only

size

and

steric

hindrance

play

a

role

in

glycocalyx

dependent

permeability

but

also

electrostatic

charges

of

the

glycocalyx

and

the

permeating

substance

With

many

of

the

glycosaminoglycan

chains

being

highly

sulfated

the

glycocalyx

presents

a

net

negatively

charged

surface

to

the

bloodstream

Accordingly

neutralization

of

the

glycocalyx

induces

an

increase

in

albumin

uptake

by

cultured

endothelial

cells

-LSB-

-RSB-

and

an

increased

permeability

for

fluorescently

labeled

dextrans

in

rat

mesenteric

arteries

-LSB-

-RSB-

The

classical

model

used

to

describe

microvascular

fluid

exchange

is

that

of

Starling

-LSB-

-RSB-

stating

that

fluid

filtration

rate

across

capillary

endothelium

is

determined

by

the

hydraulic

and

colloid

osmotic

pressures

in

the

vascular

lumen

and

in

the

surrounding

tissue

This

balance

has

been

applied

across

the

entire

transendothelial

barrier

with

the

different

pressures

being

assessed

globally

The

discovery

of

a

relatively

thick

endothelial

glycocalyx

and

its

influence

on

e.g.

edema

formation

has

led

to

a

major

revision

of

the

Starling

principle

by

Weinbaum

-LSB-

-RSB-

and

Michel

-LSB-

-RSB-

who

proposed

to

apply

the

pressure

gradients

to

the

endothelial

glycocalyx

only

Hu

and

Weinbaum

used

this

idea

to

generate

a

model

of

permeability

over

different

regions

of

the

endothelial

layer

such

as

the

glycocalyx

the

endothelial

clefts

and

the

tight

junctions

-LSB-

-RSB-

Recently

this

model

was

simplified

into

a

description

of

the

varying

tissue

concentration

gradients

and

subsequent

permeability

of

the

endothelium

-LSB-

-RSB-

Another

model

known

as

the

glycocalyx-junction-break

model

applies

the

Starling

mechanism

over

the

glycocalyx

and

describes

its

effects

on

solute

and

water

transport

over

the

endothelium

based

on

theoretical

pores

in

the

endothelium

-LSB-

-RSB-

Recently

Curry

has

written

a

review

on

this

model

and

the

influences

of

phenotypical

changes

on

microvascular

permeability

-LSB-

-RSB-

The

importance

of

the

endothelial

glycocalyx

in

controlling

extravasation

of

colloids

and

fluids

is

also

stressed

by

studies

of

Rehm

et

al.

-LSB-

-RSB-

and

Jacob

et

al.

-LSB-

-RSB-

who

show

impaired

endothelial

barrier

function

after

glycocalyx

degradation

in

an

isolated

perfused

heart

model

Infusion

of

albumin

or

hydroxethyl

starch

a

natural

and

an

artificial

colloid

led

to

decreased

fluid

extravasation

However

after

mins

of

warm

ischemia

only

albumin

infusion

prevented

vascular

leakage

This

underscores

the

importance

of

an

intact

glycocalyx

and

the

role

of

plasma-derived

proteins

for

competent

glycocalyx

functioning

The

revised

Starling

principle

has

provided

more

detailed

insight

into

vascular

permeability

and

stresses

the

importance

of

the

endothelial

glycocalyx

as

a

major

determinant

Besides

its

capacity

to

restrict

molecules

from

reaching

the

endothelium

the

glycocalyx

also

influences

blood

cell

vessel

wall

interactions

It

repulses

red

blood

cells

from

the

endothelium

In

the

microcirculation

a

red

blood

cell

exclusion

zone

flanking

the

endothelium

can

be

observed

in

vivo

which

is

decreased

upon

light

dye-induced

breakdown

of

the

glycocalyx

-LSB-

-RSB-

Similarly

platelets

are

not

often

observed

interacting

with

the

endothelium

in

control

conditions

whereas

partial

glycocalyx

removal

by

infusion

with

oxidized

low-density

lipoprotein

-LRB-

ox-LDL

-RRB-

is

accompanied

by

an

increase

in

platelet

vessel

wall

interactions

-LSB-

-RSB-

The

role

of

the

glycocalyx

in

leukocyte

vessel

wall

interactions

seems

dual

on

the

one

hand

it

harbors

the

endothelial

cell

adhesion

molecules

such

as

P-selectin

and

on

the

other

hand

it

attenuates

adhesion

of

leukocytes

to

these

molecules

In

healthy

mouse

cremaster

muscle

venules

breakdown

of

heparan

sulfate

side

chains

through

heparitinase

leads

to

an

increase

in

leukocyte

adhesion

to

the

endothelium

in

a

dose-dependent

manner

-LSB-

-RSB-

Administration

of

ox-LDL

or

TNFα

also

induces

leukocyte

rolling

and

adhesion

-LSB-

-RSB-

Steric

hindrance

seems

to

play

a

role

in

this

process

The

endothelial

glycocalyx

is

much

thicker

-LRB-

ranging

from

μm

in

capillaries

-LSB-

-RSB-

to

μm

in

small

arteries

-LSB-

-RSB-

and

μm

in

carotid

arteries

-LSB-

-RSB-

-RRB-

than

the

length

of

the

various

cell

adhesion

molecules

P-selectin

for

example

the

molecule

initiating

leukocyte

rolling

only

extends

about

nm

from

the

endothelial

surface

-LSB-

-RSB-

On

cultured

cells

transfected

with

P-selectin

constructs

with

two

to

six

consensus

repeats

that

are

consequently

shorter

than

normal

P-selectin

-LRB-

nine

consensus

repeats

-RRB-

the

number

of

attaching

neutrophils

decreased

with

decreasing

P-selectin

length

Cells

defective

of

glycosylation

having

a

thinner

glycocalyx

showed

increased

attachment

of

neutrophils

-LSB-

-RSB-

Hence

in

normal

conditions

the

glycosaminoglycan

chains

and

soluble

components

of

the

glycocalyx

seem

to

shield

adhesion

molecules

thereby

preventing

interaction

Stimuli

which

degrade

the

glycocalyx

or

induce

a

more

open

mesh

such

as

enzymes

cytokines

or

ischemia

and

reperfusion

appear

to

uncover

the

adhesion

molecules

which

in

turn

allows

blood

cells

to

interact

with

the

endothelium

-LSB-

-RSB-

One

should

realize

that

ligands

for

endothelial

adhesion

molecules

are

not

uniformly

distributed

over

the

cell

membrane

of

leukocytes

but

show

association

with

microvilli

and

membrane

ruffles

-LSB-

-RSB-

As

the

glycocalyx

is

estimated

to

have

a

low

stiffness

-LSB-

-RSB-

it

is

likely

that

the

ligand

bearing

leukocyte

membrane

extensions

protrude

relatively

easily

into

the

glycocalyx

to

reach

their

receptor

and

enable

leukocyte

vessel

wall

interaction

In

contrast

to

other

tissues

leukocyte

vessel

wall

interactions

occur

spontaneously

in

venules

in

the

skin

even

in

normal

conditions

without

preceding

trauma

or

inflammation

-LSB-

-RSB-

Whether

this

is

associated

with

a

deviating

glycocalyx

structure

and/or

composition

in

skin

microvessels

remains

to

be

elucidated

The

presence

of

a

relatively

thick

endothelial

glycocalyx

in

vivo

has

great

consequences

for

rheology

especially

in

the

microvasculature

-LSB-

-RSB-

In

this

part

of

the

circulation

local

blood

viscosity

and

hematocrit

appear

to

be

modulated

by

the

glycocalyx

Using

a

physical

model

based

on

hemodynamic

and

hematocrit

measurements

in

microvascular

networks

in

vivo

Pries

and

Secomb

recently

demonstrated

that

incorporation

of

realistic

estimates

of

glycocalyx

dimensions

in

reconstructed

mesenteric

microvascular

networks

introduces

about

a

twofold

increase

in

the

apparent

viscosity

of

blood

These

changes

are

sufficient

to

minimize

discrepancies

between

experimentally

determined

and

theoretically

predicted

microvascular

network

resistances

in

previous

studies

which

were

based

on

the

apparent

viscosity

of

blood

in

glycocalyx-devoid

glass

capillaries

-LSB-

-RSB-

The

endothelial

glycocalyx

as

mechanotransducer

The

endothelium

is

exposed

to

mechanical

forces

induced

by

blood

flow

It

has

long

been

recognized

that

these

forces

in

particular

shear

stress

determine

endothelial

cell

morphology

and

function

-LSB-

-RSB-

Endothelial

cells

exposed

to

shear

stress

produce

nitric

oxide

-LRB-

NO

-RRB-

-LSB-

-RSB-

which

is

an

important

determinant

of

vascular

tone

However

the

molecule

-LRB-

s

-RRB-

responsible

for

the

translation

of

biomechanical

forces

into

biochemical

signals

-LRB-

mechanotransduction

-RRB-

have

not

been

identified

as

yet

Recently

the

glycocalyx

has

been

added

to

the

list

of

possible

candidates

In

studies

with

cultured

endothelial

cells

Florian

and

colleagues

-LSB-

-RSB-

showed

that

treatment

with

heparitinase

to

specifically

break

down

heparan

sulfate

glycosaminoglycans

results

in

inadequate

responses

to

shear

variations

and

impaired

NO

production

Similarly

ex

vivo

experiments

on

canine

femoral

arteries

conducted

by

Mochizuki

and

coworkers

exhibited

reduced

shear-induced

NO

production

after

infusion

with

hyaluronidase

which

degrades

the

hyaluronan

glycosaminoglycans

in

the

glycocalyx

-LSB-

-RSB-

Thus

both

heparan

sulfate

and

hyaluronan

appear

to

play

a

role

in

detecting

and

amplifying

flow-induced

shear

forces

-LSB-

-RSB-

Interestingly

exposure

of

human

umbilical

vein

endothelial

cells

to

shear

stress

was

found

to

increase

the

amount

of

hyaluronan

in

the

glycocalyx

approximately

twofold

which

may

represent

a

positive

feedback

for

shear

stress

sensing

by

endothelial

cells

-LSB-

-RSB-

Another

recent

study

showed

a

correlation

between

shear

stress

profile

and

glycocalyx

dimensions

in

the

mouse

carotid

artery

the

laminar

flow

profiles

in

the

common

carotid

were

found

to

coincide

with

a

glycocalyx

thickness

of

nm

whereas

disturbed

laminar

flow

in

the

sinus

region

of

the

carotid

bifurcation

coincided

with

a

thinner

glycocalyx

of

nm

-LSB-

-RSB-

Moreover

the

flow

divider

region

of

the

carotid

bifurcation

supposedly

having

an

undisturbed

high

laminar

flow

profile

was

covered

with

a

glycocalyx

of

nm

comparable

to

the

common

carotid

glycocalyx

thickness

From

these

data

it

seems

likely

that

the

glycocalyx

plays

an

important

role

in

mechanotransduction

and

that

its

composition

is

in

turn

-LRB-

at

least

partly

-RRB-

shear-dependent

The

different

components

of

the

glycocalyx

probably

operate

together

which

means

that

the

glycocalyx

as

a

whole

is

responsible

for

its

role

as

mechanotransducer

This

idea

is

confirmed

by

theoretical

models

based

on

a

regular

hexagonal

distribution

of

core

proteins

over

the

endothelial

cell

membrane

-LSB-

-RSB-

Recently

Tarbell

and

Pahakis

reviewed

the

current

concepts

on

mechanotransduction

by

the

-LRB-

membrane-bound

-RRB-

glycocalyx

-LSB-

-RSB-

They

conclude

that

the

glycocalyx

core

proteins

are

responsible

for

transmission

of

shear

stress

signals

into

specific

cell

signaling

processes

e.g.

NO

production

and

cytoskeletal

reorganization

At

the

same

time

shear

stress

is

transmitted

to

other

regions

of

the

endothelial

cell

as

well

such

as

intercellular

junctions

and

basal

adhesion

plaques

which

are

responsible

for

additional

shear

sensing

even

in

the

absence

of

a

glycocalyx

The

endothelial

glycocalyx

as

control

center

for

the

microenvironment

The

proteoglycans

in

the

glycocalyx

contribute

greatly

to

its

functional

importance

The

glycosaminoglycan

chain

variety

arising

from

chain

epimerization

elongation

and

most

notably

chain

sulfation

gives

rise

to

a

heterogeneous

surface

to

which

a

lot

of

plasma-derived

molecules

can

dock

Table

lists

a

number

of

molecules

which

depend

on

interaction

with

the

glycocalyx

for

their

functionality

Table

dependent

on

interaction

with

the

endothelial

glycocalyx

for

proper

functioningInteracting

moleculePrimary

function

in

vasculatureReference

numberAntithrombin

IIIPotent

inactivator

of

pro-coagulant

proteases

such

as

thrombin

factor

Xa

and

factor

IXa

activity

enhanced

by

heparin

or

heparan

cofactor

IIInactivator

of

the

procoagulant

protease

thrombin

activated

by

dermatan

sulfate

in

the

endothelial

protein

blocking

activated

factor

VII

and

involved

in

breakdown

of

low

density

cholesterol

and

triglycerides

through

the

stimulator

of

angiogenesis

production

of

which

is

triggered

by

factor

known

to

mediate

in

a

lot

of

signaling

pathways

including

smooth

muscle

cell

differentiation

and

vascular

tone

and

-LRB-

r

-RRB-

Growth

factor

-LRB-

receptor

-RRB-

involved

in

endothelial

cell

proliferation

and

quencher

of

reactive

oxygen

RANTESChemotaxis

of

leukocytes

to

the

subendothelium

involved

in

arrest

and

Tissue

factor

pathway

inhibitor

LPL

lipoprotein

lipase

LDL

low

density

lipoprotein

VEGF

vascular

endothelial

growth

factor

transforming

growth

factor

β1

or

β2

FGF

-LRB-

r

-RRB-

fibroblast

growth

factor

-LRB-

receptor

-RRB-

ec-SOD

extracellular

superoxide

dismutase

IL

interleukin

RANTES

Regulated

on

Activation

Normal

T

Expressed

and

Secreted

also

known

as

chemokine

Docking

of

plasma-derived

molecules

can

influence

the

local

environment

in

several

ways

-LRB-

-RRB-

Binding

of

receptors

or

enzymes

and

their

ligands

to

the

endothelial

glycocalyx

causes

a

localized

rise

in

concentration

of

these

substances

which

enables

proper

signaling

or

enzymatic

modification

Fibroblast

growth

factor

-LRB-

FGF

-RRB-

signaling

is

mediated

in

this

way

and

is

known

to

depend

completely

on

the

interaction

of

both

ligand

and

receptor

with

the

glycocalyx

-LSB-

-RSB-

Similarly

the

glycocalyx

is

involved

in

the

lipolytic

system

binding

both

lipoprotein

lipase

and

its

ligand

low-density

lipoprotein

-LRB-

LDL

-RRB-

-LSB-

-RSB-

-LRB-

-RRB-

Binding

of

plasma-derived

molecules

to

the

glycocalyx

can

lead

to

a

local

concentration

gradient

which

is

often

seen

in

growth

factor-regulated

gene

transcription

and

developmental

processes

-LSB-

-RSB-

-LRB-

-RRB-

Docking

of

enzymes

and

their

agonists

or

inhibitors

to

the

glycocalyx

adds

a

vasculoprotective

role

to

glycocalyx

functionality

Several

important

anticoagulant

mediators

can

bind

to

the

glycocalyx

such

as

antithrombin

III

heparin

cofactor

II

thrombomodulin

and

tissue

factor

pathway

inhibitor

-LRB-

TFPI

-RRB-

Antithrombin

III

is

a

strong

inhibitor

of

procoagulant

enzymes

like

thrombin

and

activated

factors

X

and

IX

-LRB-

FXa

and

FIXa

-RRB-

-LSB-

-RSB-

It

is

known

to

bind

to

specific

regions

in

heparan

sulfate

which

enhances

its

anticoagulant

activity

-LSB-

-RSB-

Heparin

cofactor

II

is

a

thrombin-specific

protease

inhibitor

-LSB-

-RSB-

which

is

activated

by

dermatan

sulfate

in

the

glycocalyx

-LSB-

-RSB-

Thrombomodulin

is

a

chondroitin

sulfate

containing

protein

expressed

by

endothelial

cells

and

is

able

to

convert

thrombin

from

a

procoagulant

enzyme

to

an

activator

of

the

protein

C

pathway

thus

becoming

anticoagulant

-LSB-

-RSB-

TFPI

is

a

potent

inhibitor

of

FVIIa

and

FXa

TFPI

is

believed

to

bind

to

the

glycocalyx

via

heparan

sulfates

but

other

proteins

could

be

involved

as

well

-LSB-

-RSB-

Furthermore

uptake

and

degradation

of

TFPI

FXa

complexes

depends

on

heparan

sulfates

in

the

glycocalyx

-LSB-

-RSB-

All

these

anticoagulant

molecules

present

in

the

glycocalyx

contribute

to

the

thromboresistant

nature

of

healthy

endothelium

-LSB-

-RSB-

The

endothelial

glycocalyx

also

modulates

inflammatory

responses

by

binding

cytokines

and

attenuating

binding

of

cytokines

to

cell

surface

receptors

Shedding

of

heparan

sulfate

from

the

glycocalyx

results

in

increased

endothelial

cell

sensitivity

to

activation

by

cytokines

-LSB-

-RSB-

Another

aspect

of

the

vasculoprotective

role

of

the

endothelial

glycocalyx

is

its

capacity

to

bind

quenchers

of

oxygen

radicals

such

as

extracellular

superoxide

dismutase

-LRB-

SOD

-RRB-

-LSB-

-RSB-

These

enzymes

help

to

reduce

the

oxidative

stress

and

keep

up

NO

bioavailability

thus

preventing

the

endothelium

from

becoming

dysfunctional

The

endothelial

glycocalyx

in

pathophysiology

In

healthy

vessels

the

endothelial

glycocalyx

determines

vascular

permeability

attenuates

blood

cell

vessel

wall

interactions

mediates

shear

stress

sensing

enables

balanced

signaling

and

fulfills

a

vasculoprotective

role

But

when

it

is

disrupted

or

modified

these

properties

are

lost

as

has

been

shown

through

direct

targeting

of

the

glycocalyx

in

experimental

settings

In

the

last

few

years

evidence

is

emerging

that

-LRB-

damage

to

-RRB-

the

glycocalyx

plays

a

pivotal

role

in

several

vascular

pathologies

Here

we

will

discuss

its

suspected

roles

in

diabetes

ischemia/reperfusion

and

atherosclerosis

Diabetes

Diabetes

is

a

clinically

well-known

disease

with

far-reaching

complications

such

as

retino

and

nephropathy

and

elevated

risks

for

atherothrombotic

cardiovascular

events

One

of

the

hallmarks

of

diabetes

is

insulin

absence

or

resistance

and

subsequent

hyperglycemia

impairing

the

protective

capacity

of

the

vessel

wall

-LSB-

-RSB-

and

resulting

in

enhanced

endothelial

permeability

-LSB-

-RSB-

and

impaired

NO

synthase

function

-LSB-

-RSB-

However

a

common

pathway

leading

to

these

vascular

dysfunctions

has

not

been

identified

Recently

it

was

shown

that

the

systemic

glycocalyx

volume

of

healthy

volunteers

as

assessed

by

comparing

the

intravascular

distribution

volume

of

a

glycocalyx-permeable

and

a

glycocalyx-impermeable

tracer

was

halved

within

h

after

induction

of

acute

hyperglycemia

-LSB-

-RSB-

Using

the

same

methodology

the

systemic

glycocalyx

volume

in

type

diabetics

was

found

to

be

about

half

of

that

of

healthy

controls

it

was

further

reduced

in

diabetics

with

microalbuminuria

-LSB-

-RSB-

In

the

same

study

plasma

levels

of

hyaluronan

and

hyaluronidase

were

found

to

be

elevated

in

patients

with

diabetes

reflecting

increased

synthesis

and

shedding

of

hyaluronan

under

hyperglycemic

conditions

Both

studies

show

that

acute

and

long-standing

hyperglycemia

is

associated

with

profound

reduction

of

glycocalyx

dimensions

It

is

tempting

to

speculate

that

this

damage

to

the

glycocalyx

contributes

to

endothelial

dysfunction

in

hyperglycemic

conditions

which

can

be

measured

in

nondiabetic

subjects

as

well

-LSB-

-RSB-

Further

studies

are

needed

to

investigate

whether

glycocalyx

perturbation

is

responsible

for

the

-LRB-

micro

-RRB-

vascular

complications

in

diabetes

Ischemia/reperfusion

Damage

to

tissues

during

a

period

of

absent

or

decreased

flow

-LRB-

total

or

partial

ischemia

-RRB-

can

paradoxically

be

exaggerated

by

restoration

of

blood

flow

-LRB-

reperfusion

-RRB-

Although

the

severity

of

damage

resulting

from

ischemia/reperfusion

varies

between

tissues

a

common

component

of

this

pathologic

process

for

all

organs

is

microvascular

dysfunction

-LSB-

-RSB-

Endothelial

cells

play

a

central

role

and

exhibit

swelling

and

detachment

from

the

basement

membrane

after

ischemia/reperfusion

-LSB-

-RSB-

Especially

in

postcapillary

venules

endothelial

cells

suffer

from

increased

oxidative

stress

-LSB-

-RSB-

leukocytes

adhere

and

transmigrate

-LSB-

-RSB-

and

vascular

permeability

increases

-LSB-

-RSB-

These

endothelial

consequences

of

ischemia/reperfusion

allude

at

involvement

of

the

endothelial

glycocalyx

Indeed

it

was

recently

shown

by

Mulivor

and

Lipowsky

-LSB-

-RSB-

that

intestinal

ischemia/reperfusion

led

to

significant

reduction

of

glycocalyx

thickness

in

rat

mesenteric

venules

most

likely

due

to

shedding

of

glycosaminoglycan

chains

The

effects

of

ischemia/reperfusion

on

the

glycocalyx

could

be

attenuated

by

blockade

of

xanthine-oxidoreductase

which

is

an

endogenous

reactive

oxygen

species

-LRB-

ROS

-RRB-

producing

enzyme

bound

to

heparan

sulfate

domains

in

the

glycocalyx

This

way

a

central

role

for

ROS

in

disruption

and

shedding

of

the

glycocalyx

in

ischemia/reperfusion

was

established

-LSB-

-RSB-

Infusion

of

exogenous

hyaluronan

to

restore

the

glycocalyx

or

administration

of

pertussis

toxin

which

inhibits

G-protein-mediated

shedding

of

glycosaminoglycan

chains

also

reduced

ischemia

reperfusion-induced

damage

-LSB-

-RSB-

Together

these

data

hint

at

a

role

for

the

endothelial

glycocalyx

in

the

pathophysiology

of

ischemia

reperfusion-induced

tissue

damage

However

its

relative

contribution

to

this

process

and

the

impact

of

possible

therapeutical

interventions

are

yet

to

be

established

-LSB-

-RSB-

Atherosclerosis

Atherosclerosis

is

a

large

artery

disease

and

typically

requires

high

plasma

levels

of

LDL

to

develop

at

predilection

sites

-LSB-

-RSB-

that

are

marked

by

disturbed

flow

profiles

Subendothelial

retention

of

atherogenic

lipoproteins

and

subsequent

inflammatory

responses

lead

to

the

formation

of

subendothelial

plaques

-LSB-

-RSB-

The

role

of

the

endothelial

glycocalyx

in

atherogenesis

is

as

yet

not

well-established

but

there

are

some

interesting

observations

which

point

at

its

involvement

In

Vink

and

colleagues

-LSB-

-RSB-

showed

that

administration

of

clinically

relevant

doses

of

ox-LDL

leads

to

a

disruption

of

the

glycocalyx

in

hamster

cremaster

muscle

microcirculation

and

evokes

local

platelet

adhesion

Co-infusion

with

SOD

and

catalase

enzymes

catalyzing

the

dismutation

of

superoxide

anion

and

the

decomposition

of

hydrogen

peroxide

abolished

this

effect

of

ox-LDL

implicating

a

role

for

oxygen-derived

free

radicals

Indeed

loss

of

glycocalyx

results

in

shedding

of

endogenous

protective

enzymes

such

as

extracellular

SOD

and

increases

the

oxidative

stress

on

endothelial

cells

This

was

further

illustrated

by

a

recent

study

by

van

den

Berg

et

al.

-LSB-

-RSB-

showing

a

reduction

in

glycocalyx

dimensions

due

to

a

high-fat

high-cholesterol

diet

Furthermore

an

inverse

relation

between

glycocalyx

thickness

and

intima

media

ratio

was

found

reflecting

a

reduction

of

vasculoprotective

capacity

of

the

endothelial

glycocalyx

at

sites

with

higher

atherogenic

risk

In

healthy

mice

regional

variations

were

found

showing

a

thinner

glycocalyx

in

the

internal

carotid

sinus

region

compared

to

the

common

carotid

artery

Together

these

data

suggest

that

the

endothelial

glycocalyx

is

involved

in

the

initiation

and

progression

of

the

atherosclerotic

process

-LSB-

-RSB-

In

summary

the

endothelial

glycocalyx

appears

to

be

perturbed

in

several

vascular

diseases

It

remains

to

be

elucidated

whether

glycocalyx

perturbation

is

causally

involved

in

the

pathophysiology

of

these

diseases

If

so

restoration

of

the

glycocalyx

may

be

a

therapeutic

target

of

interest

Furthermore

identification

of

specific

glycosaminoglycan

domains

involved

in

these

diseases

as

a

platform

for

other

substances

or

signaling

pathways

might

also

prove

to

be

of

therapeutic

value

-LSB-

-RSB-

Visualization

techniques

Because

of

the

functional

importance

of

the

endothelial

glycocalyx

development

of

direct

visualization

techniques

is

crucial

to

establish

its

exact

role

The

glycocalyx

can

be

labeled

by

administration

of

specific

markers

that

attach

to

one

or

more

of

its

components

making

them

fluorescent

or

detectable

Preparation

of

-LRB-

parts

of

-RRB-

the

vessel

would

then

allow

specific

microscopic

imaging

of

the

endothelial

glycocalyx

Regretfully

the

glycocalyx

is

very

vulnerable

and

easily

disturbed

or

dehydrated

during

vessel

handling

and

preparation

protocols

As

a

result

glycocalyx

dimensions

are

easily

underestimated

which

is

illustrated

by

the

first

images

of

the

glycocalyx

made

by

transmission

electron

microscopy

-LRB-

TEM

-RRB-

in

with

the

use

of

the

probe

ruthenium

red

glycocalyx

thickness

measured

this

way

approximated

nm

in

capillaries

-LSB-

-RSB-

Since

then

many

other

attempts

were

made

to

image

the

glycocalyx

using

TEM

On

bovine

aorta

endothelial

cells

under

shear

stress

conditions

of

Pa

the

glycocalyx

was

reported

to

be

nm

thick

-LSB-

-RSB-

These

dimensions

did

not

comply

with

theoretical

estimates

predicting

the

glycocalyx

to

be

up

to

μm

thick

-LSB-

-RSB-

Using

a

new

staining

protocol

with

Alcian

blue

van

den

Berg

et

al.

-LSB-

-RSB-

recently

applied

TEM

to

measure

endothelial

glycocalyx

dimensions

in

rat

myocardial

capillaries

which

revealed

that

endothelial

cells

are

covered

by

a

to

thick

glycocalyx

-LRB-

Fig.

-RRB-

Hyaluronidase

treatment

before

fixation

and

staining

resulted

in

significant

reduction

of

this

layer

to

nm

The

groups

of

Haraldsson

-LSB-

-RSB-

and

of

Rostgaard

and

Qvortrup

-LSB-

-RSB-

improved

the

TEM

staining

protocol

using

fluorocarbon-based

oxygen

carrying

fixatives

revealing

glycocalyces

as

thick

as

nm

in

glomerular

capillaries

and

nm

in

intestinal

fenestrated

capillaries

Apparently

the

new

staining

and

preparation

protocols

improved

glycocalyx

conservation

in

TEM

experiments

However

TEM

can

not

be

used

in

the

in

vivo

situation

Fig.

of

the

endothelial

glycocalyx

with

different

microscopic

techniques

a

Endothelial

glycocalyx

of

a

rat

left

ventricular

myocardial

capillary

stained

with

Alcian

blue

and

visualized

using

electron

microscopy

Bar

represents

μm

Reproduced

with

permission

from

reference

number

-LSB-

-RSB-

b

Intravital

microscopic

recording

of

the

endothelial

glycocalyx

of

a

hamster

cremaster

muscle

capillary

The

anatomical

diameter

of

μm

is

larger

than

the

red

blood

cell

column

width

-LRB-

left

pane

-RRB-

or

the

plasma

column

width

-LRB-

right

pane

-RRB-

labeled

with

fluorescent

dextran

-LRB-

kD

-RRB-

This

difference

is

caused

by

the

presence

of

the

endothelial

glycocalyx

The

bar

in

the

left

pane

represents

μm

Reproduced

with

permission

from

reference

number

-LSB-

-RSB-

c

Endothelial

glycocalyx

of

a

mouse

common

carotid

artery

of

a

series

of

optical

slices

obtained

with

two-photon

laser

scanning

microscopy

showing

part

of

the

vessel

wall

The

intact

vessel

was

perfused

with

FITC-labeled

lectin

-LRB-

WGA

-RRB-

to

stain

the

endothelial

glycocalyx

-LRB-

green

-RRB-

and

SYTO

to

label

cell

nuclei

-LRB-

blue

-RRB-

The

arrows

indicate

the

direction

of

the

X

Y

and

Z

axis

The

scanned

volume

approximates

For

details

on

methodology

see

also

reference

number

-LSB-

-RSB-

Some

years

after

the

first

TEM

images

were

made

Vink

et

al.

-LSB-

-RSB-

used

intravital

microscopy

to

visualize

the

endothelial

glycocalyx

in

hamster

cremaster

muscle

capillaries

in

vivo

using

indirect

approaches

The

glycocalyx

was

recognized

as

a

red

blood

cell

exclusion

zone

or

gap

between

the

flowing

red

blood

cells

and

the

endothelium

In

addition

the

plasma

was

labeled

by

a

fluorescent

dextran

and

the

glycocalyx

then

appeared

as

a

plasma

exclusion

zone

-LRB-

Fig.

-RRB-

Interestingly

no

exclusion

zone

was

found

for

rolling

white

blood

cells

suggesting

that

they

have

the

ability

to

compress

the

glycocalyx

in

these

vessels

which

complies

with

the

estimated

low

stiffness

of

the

glycocalyx

-LSB-

-RSB-

Subtraction

of

the

diameter

of

the

plasma

column

from

the

anatomical

internal

diameter

revealed

the

dimensions

of

the

glycocalyx

which

appeared

to

be

μm

thick

-LSB-

-RSB-

This

method

has

been

used

in

many

studies

since

primarily

in

the

cremaster

muscle

microcirculation

of

hamsters

-LSB-

-RSB-

or

mice

-LSB-

-RSB-

This

tissue

is

suited

for

intravital

microscopy

because

it

is

thin

and

translucent

allowing

clear

visualization

of

microvascular

endothelial

cells

and

flowing

blood

cells

with

low

or

absent

vessel

wall

motion

-LRB-

Fig.

-RRB-

In

addition

local

flow

velocities

can

be

measured

However

the

estimation

of

the

glycocalyx

thickness

using

intravital

microscopy-based

methods

is

indirect

Furthermore

intravital

microscopy

can

not

be

applied

to

image

the

endothelial

glycocalyx

in

larger

vessels

Direct

visualization

of

the

glycocalyx

has

been

performed

via

several

approaches

mostly

using

lectins

which

are

proteins

that

bind

specific

disaccharide

moieties

of

glycosaminoglycan

chains

-LSB-

-RSB-

Other

labels

include

antibodies

for

heparan

sulfate

or

hyaluronan

-LSB-

-RSB-

When

attaching

these

markers

to

a

fluorescent

probe

advanced

microscopic

techniques

can

be

applied

to

visualize

the

glycocalyx

Confocal

laser

scanning

microscopy

-LRB-

CLSM

-RRB-

enables

optical

sectioning

with

good

optical

resolution

allowing

reconstructions

of

the

specimen

Lectin

labeling

of

the

glycocalyx

of

cultured

human

umbilical

vein

endothelial

cells

and

subsequent

CLSM

imaging

revealed

a

surface

layer

as

thick

as

μm

-LSB-

-RSB-

CLSM

has

also

been

used

to

detect

concentration

changes

of

fluorescently

labeled

lectins

in

the

glycocalyx

of

fixated

rat

mesentery

postcapillary

venules

in

case

of

ischemia/reperfusion

and

inflammation

-LSB-

-RSB-

Because

larger

vessels

have

thicker

walls

which

results

in

lower

light

penetration

depths

with

significant

loss

of

resolution

at

higher

depths

-LRB-

μm

-RRB-

-LSB-

-RSB-

due

to

increased

scattering

of

signal

CLSM

is

less

suitable

for

imaging

of

the

glycocalyx

in

arteries

A

promising

technique

to

directly

visualize

the

glycocalyx

in

larger

vessels

both

ex

vivo

and

in

vivo

is

two-photon

laser

scanning

microscopy

-LRB-

TPLSM

-RRB-

TPLSM

depends

on

excitation

of

a

fluorophore

by

simultaneous

uptake

-LRB-

i.e.

within

s

-RRB-

of

two

red

photons

instead

of

one

blue

photon

as

in

conventional

fluorescence

excitation

The

use

of

long

wavelength

red

photons

reduces

scattering

and

hence

increases

penetration

depth

into

tissue

Excitation

of

the

fluorophore

and

consequent

fluorescence

only

occurs

at

the

focal

point

of

the

illumination

cone

as

the

probability

of

two-photon

excitation

depends

on

the

squared

intensity

of

the

excitatory

photons

Any

light

received

by

the

photomultipliers

has

to

originate

from

the

focal

position

so

scattering

of

the

emitted

photons

does

not

influence

resolution

and

no

pinholes

are

required

As

a

consequence

TPLSM

offers

good

resolution

and

optical

sectioning

at

reasonable

acquisition

speed

while

bleaching

and

phototoxicity

of

the

dyes

is

limited

to

the

focal

position

The

combination

of

enhanced

penetration

depth

good

resolution

optical

sectioning

and

low

phototoxicity

makes

TPLSM

a

suitable

technique

to

visualize

the

delicate

endothelial

glycocalyx

in

larger

vessels

This

idea

was

confirmed

by

a

recent

study

by

Megens

and

colleagues

-LSB-

-RSB-

in

which

the

endothelial

glycocalyx

was

imaged

with

TPLSM

in

intact

mouse

carotid

arteries

-LRB-

Fig.

-RRB-

the

glycocalyx

thickness

was

found

to

be

μm

As

TPLSM

is

also

applicable

in

vivo

in

rodents

-LSB-

-RSB-

it

might

prove

to

be

a

good

approach

of

in

vivo

visualization

of

the

glycocalyx

in

the

macrocirculation

of

these

animals

Conclusions

Overlying

the

vascular

endothelium

the

glycocalyx

is

a

membrane-bound

mesh

in

which

plasma-derived

molecules

integrate

It

exerts

a

variety

of

functions

important

in

normal

vascular

physiology

and

also

in

vascular

disease

Although

data

from

experiments

in

microcirculation

and

more

recently

in

macrocirculation

strongly

suggests

a

vasculoprotective

role

for

the

glycocalyx

research

on

this

subject

is

hampered

by

lack

of

a

good

visualization

technique

Two-photon

laser

scanning

microscopy

may

prove

to

be

a

successful

tool

in

achieving

direct

visualization

of

the

glycocalyx

in

larger

arteries

in

rodents

both

ex

vivo

and

in

vivo

with

the

possibility

to

analyze

focal

variations

in

the

composition

or

integrity

of

this

layer

O B I O O O O O O O O O O O O O O O O O O O O O B I O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O B I O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

endothelial glycocalyx
endothelial surface layer
heparan sulfate
hyaluronic acid
vascular disease