Bucci anemo 2015 - Glicocalice endoteliale la centrale della periferia

Glicocalice endoteliale :
la centrale della periferia
Endothelial Glycocalyx:
headquarter of periphery
Lucio Bucci
UTI ‘‘Bozza’’
I SAR
A.O. Ospedale Niguarda Cà Granda
Milano
Programma DEFINITIVO
FINAL program
S. Donato Milanese
6-7 MARZO 2015
2015, 6th
-7th
March
SEDE CENTRALE
IRCCS Policlinico San Donato
P.zza Malan, 1 - San Donato Milanese
Direttore del Corso
Marco Pavesi
IRCCS - Policlinico S. Donato
www.anemo.it
SEDI REMOTE
Coordinatore: M.B. Rondinelli
Roma - Hotel Il Cantico
Coordinatore: A. Corcione
Napoli - C.T.O. Ospedali dei Colli
Strategie di Risparmio del Sangue
®
GE
e.
ngibile
al
e Verde
uliano.
.
st,
gio.
i
IVA
4 Milano
MeL
ECM RICHIESTI

the human circolatory system :
400.000 miles long… Fu BM. and Tarbell JM WIREs Syst Biol Med 2013
the microcirculation : <100 μm diameter
10 billion capillaries lined
with endothelial cells Hernandez G et al. Curr Vascular Pharmacology 2013
the endothelium : >0.5 km2 surface area
Hernandez G. et al Curr Vascular Pharmacology 2013
the endothelial glycocalyx layer (EGL): a complex,
negatively charged, fragile sponge -like mesh network,
made of complex sugars and proteins up to 2 μm thick
Kolsen-Petersen JA. Acta Anesthesiologica Scandinavica 2015

the great luminal barrier…
Kolsen-Petersen JA 2015

Endothelial
Cells of Vessel
A carbohydrate-rich layer connected to the
endothelium via backbone proteoglycans
and giycoproteins. A complex network of
plasma- and epithelium-derived soluble

Endothelial
Cells of Vessel
cans have been the most extensively studied,
ntion recently shifting toward their ability
n as signal transduction molecules.19,21
The
ular transport and cell signaling throug
molecules, most notably endothelial-type
synthase (eNOS).
--K+, Na+, Ca++, L-arginine
--Albumin, bFGF, LPL, polycationic peptides
--Caveolin-1
--Cholesterol and glycosphingolipids
--K+, Na+, Ca++, L-arginine channels
--Hyaluronic acid
--Chondroitin sulfates
--Heparan sulfates
CD44
Glycoprotein
Sialic acids
A B
C
Glypican
Syndecans
Shedding

Endothelial
Cells of Vessel
cans have been the most extensively studied,
ntion recently shifting toward their ability
n as signal transduction molecules.19,21
The
ular transport and cell signaling throug
molecules, most notably endothelial-type
synthase (eNOS).
--K+, Na+, Ca++, L-arginine
--Albumin, bFGF, LPL, polycationic peptides
--Caveolin-1
--Cholesterol and glycosphingolipids
--K+, Na+, Ca++, L-arginine channels
--Hyaluronic acid
--Chondroitin sulfates
--Heparan sulfates
CD44
Glycoprotein
Sialic acids
A B
C
Glypican
Syndecans
Shedding
Starling Principle Revisited: Influence of the Glycocalyx
, endothelial cell; HPi, hydrostatic P in interstitial space; HPvL, hydrostatic P in vascular lumen; OPesL,
e layer; OPi, oncotic P in interstitial spaoe; OPvL, oncotic P in vascular lumen.
Above is intact glycocaiyx in
myocardial vessel tissue. To the
riglit is the same vasculature
tissue after a brief period of
ischemia with major erosion of
the highly fragile glycocalyx.

Endothelial Glycocalyx Layer (EGL):
three main functions (to date…)
• mechanotransduction of fluid shear
stress to the endothelial cell (EC)
cytoskeleton with the resulting
biochemical responses
• modulation of permeability in the
transcapillary exchange of water :
Starling revisited
• regulation of red and white blood
cells interactions with EC triggering
inflammatory response and
relationships with coagulation system
Weinbaum S et al. Annu Rev Biomed Eng. 2007

Mechano-sensing and
transduction by endothelial
surface glycocalyx: composition,
structure, and function
Bingmei M. Fu∗
and John M. Tarbell
The endothelial cells (ECs) lining every blood vessel wall are constantly exposed
to the mechanical forces generated by blood flow. The EC responses to these
hemodynamic forces play a critical role in the homeostasis of the circulatory
system. To ensure proper EC mechano-sensing and transduction, there are a
variety of mechano-sensors and transducers that have been identified on the EC
surface, intra- and trans-EC membrane and within the EC cytoskeleton. Among
them, the most recent candidate is the endothelial surface glycocalyx (ESG), which
is a matrix-like thin layer covering the luminal surface of the EC. It consists
of various proteoglycans, glycosaminoglycans, and plasma proteins, and is close
to other prominent EC mechano-sensors and transducers. The ESG thickness
was found to be in the order of 0.1–1 µm by different visualization techniques
and in different types of vessels. Detailed analysis on the electron microscopy
(EM) images of the microvascular ESG revealed a quasi-periodic substructure
with the ESG fiber diameter of 10–12 and 20 nm spacing between adjacent
fibers. Atomic force microscopy and optical tweezers were applied to investigate
the mechanical properties of the ESG on the cultured EC monolayers and in
solutions. Enzymatic degradation of specific ESG glycosaminoglycan components
was used to directly elucidate the role of the ESG in EC mechano-sensing and
transduction by measuring the shear-induced productions of nitric oxide and
prostacyclin, two characteristic responses of the ECs to the flow. The unique
location, composition, and structure of the ESG determine its role in EC mechano-
sensing and transduction. © 2013 Wiley Periodicals, Inc.
How to cite this article:
WIREs Syst Biol Med 2013. doi: 10.1002/wsbm.1211
INTRODUCTION
In addition to forming a transport barrier between
the blood and vessel wall, vascular endothelial
protein expressions.1
EC responses to the hemody-
namic forces (mechano-sensing and transduction) are
critical to maintaining normal vascular functions.2,3
Failure in mechano-sensing and transduction con-
Focus Article
Pressure(a)
(b)
Stretch
Blood flow
ShearSurface glycocalyx
EC
VSMC
solutions. Enzymatic degradation of specific ESG glycosaminoglycan
was used to directly elucidate the role of the ESG in EC mechano-
transduction by measuring the shear-induced productions of nitri
prostacyclin, two characteristic responses of the ECs to the flow.
location, composition, and structure of the ESG determine its role in E
INTRODUCTION
cells (ECs) play important roles in regulating cir-
culation functions. Besides biochemical stimuli, blood
flow-induced (hemodynamic) mechanical stimuli, such
as shear stress, pressure and circumferential stretch,
modulate EC morphology and functions by activating
mechano-sensors, signaling pathways, and gene and
Conflict of interest: The authors declare no conflict of interest.
∗
Correspondence to: fu@ccny.cuny.edu
Department of Biomedical Engineering, The City College of the City
University of New York, New York, NY, USA
EC res
namic forces (mechano-sensin
critical to maintaining norm
Failure in mechano-sensing
tributes to serious vascular d
tension, atherosclerosis, aneu
to name a few.4
The hemodynamic forc
are described in Figure 1(a). T
area) perpendicular to the EC
pressure due to the hydrodyn
the heart. The human circul
miles long, and the magnitu
not uniform in all the bloo
© 2013 Wiley Periodicals, Inc.

Mechano-sensing and
Bingmei M. Fu∗
and John M. Tarbell
INTRODUCTION
INTRODUCTION
∗
EC res
to name a few.4

Mechano-sensing and
Bingmei M. Fu∗
and John M. Tarbell
INTRODUCTION
(black) to high (red). (Figure 1(b): Reprinted with permission from Ref 7.
Copyright 2004 IOS press)
body. The blood pressure ranges from almost 0 to
∼120 mmHg for a healthy adult human under resting
conditions.5
Another type of force (per unit surface
area) which is tangential to the EC surface is called
shear or shear stress. The shear is due to the friction
(e.g., VE-cadherin, PECAM-1), ion channels,
tyrosine kinase receptors (e.g., vascular endothelia
growth factor receptor 2),9
G-protein-coupled recep
tors and G-proteins,6
caveolae,12
primary cilia,13
acti
ﬁlaments,14
nesprins,15
integrins,16
and endothelia
surface glycocalyx (ESG)17
(Figure 2). Because of it
proteoglycan and glycosaminoglycan (GAG) compo
sition and structure, the ESG may cover the entir
surface of the EC as shown in Figure 2 (the yellow
Surface glycocalyx
Blood flow
PECAM-1
VE-cadherin
Actin filament
Adhesion protein
Nucleus
Nesprin
Caveola
Actin
filament
T K
G
FA
ECM
Ion channels T K receptor
GPCR
PC1
Primary cilia
FIGURE 2 | Currently identiﬁed endothelial mechano-sensors and transducers. At endothelial cell (EC) surface: surface glycocalyx, adherences
junction protein VE-cadherin, cell adhesion molecule PECAM-1, ion channels, tyrosine kinase (TK) receptor, G-protein-coupled receptors (GPCR),
INTRODUCTION
∗
EC res
to name a few.4

Mechano-sensing and
Bingmei M. Fu∗
and John M. Tarbell
INTRODUCTION
INTRODUCTION
∗
EC res
to name a few.4
vivo single microvessel and ex vivo aorta immunos-
taining, Yen et al.42
revealed that the thickness of the
ESG on rat mesenteric and mouse cremaster capillaries
and post-capillary venules is 1–1.5 µm. Surprisingly,
there was no detectable ESG in arterioles by using
fluorescence labeled anti-HS. The ESG thickness is
2–2.5 µm on rat and mouse aorta. They also observed
that the ESG is continuously and evenly distributed
Fourier transforms of EM images of frog mesenteric
microvessels, they identified a quasi-periodic substruc-
ture in the ESG, which is a 3D fibrous meshwork
(Figure 4) with characteristic spacing of ∼20 nm. The
fiber diameter was observed as 10–12 nm. They also
showed that the fibrous elements may occur in clus-
ters with a common intercluster spacing of ∼100 nm
and may be linked to the underlying actin cortical
Glycocalyx
150 nm
(a)
(c)
(e)
(d)
(b)
100 nm
20 nm
20 nm
100 nm

T
he endothelial glycocalyx (EG) is a complex and mul-
ticomponent layer of macromolecules at the luminal
surface of vascular endothelium. This concept was
proposed more than 70 years ago and its composition is
well studied as detailed in 2 reviews1,2
; however, its role
in mechanisms of endothelial protection and injury and
subsequent clinical implications have just recently become
evident. The EG consists of a variety of endothelial mem-
brane–bound molecules, including glycoproteins and pro-
teoglycans, that provide the basis for plasma–endothelial
cell interaction. EG structure, although well characterized
in vitro, is poorly defined in vivo because of its dynamically
changing composition by self-assembly and enzymatic deg-
radation or shear-dependent shedding of its elements. Its
major constituents are hyaluronic acid and the negatively
charged heparan sulfate proteoglycans. Together with gly-
cosaminoglycans (GAGs) and plasma proteins, the EG layer
as a whole forms the endothelial surface layer (ESL) that
acts as a barrier to circulating cells and large molecules.
Considerable prognostic and therapeutic promise lies with
the emergence of the EG as a key mediator of endothelial
dysfunction in pathogenic states, particularly with regard to
vascular permeability and edema formation. Several studies
have demonstrated the role of the EG in plasma/interstitial
fluid balance and solute exchange,3–5
mechanotransduction
that couples intravascular pressure and shear stress (i.e.,
biomechanical forces) to endothelial cell responses (i.e., bio-
chemical signals),6
and the inflammatory response cascade
via physical blockade of neutrophils to the endothelial cell
surface.7–9
This review explores the emerging evidence for
the role of the EG in vascular permeability, examines evi-
dence for modulation by the EG of inflammatory processes
that lead to edema formation, and provides insight into the
role of the EG in the development of pulmonary edema and
lung injury. The concept of the glycocalyx as a mechano-
transducer of pathophysiologic signals in the pathogenesis
of lung injury after pulmonary resection surgery is also
explored.
THE STARLING EQUATION AND PULMONARY
EDEMA
Our understanding of vascular permeability as well as
plasma/interstitial fluid movement and edema formation
has changed with recognition of and insight into the EG, a
meshwork of proteins and soluble components that forms
a major barrier to water and plasma protein exchange. The
fundamental principle guiding microvascular filtration and
transcapillary fluid shifts was proposed in 1896 by Starling10
;
however, this traditional model has been revised given our
current, more sophisticated view of the endothelial barrier
and its dynamic components. Starling10
initially devised a
series of experiments showing that fluid movement across
the walls of capillaries (and postcapillary venules) is pas-
sive and dependent on pressure gradients across the endo-
thelium. He suggested that fluid filtration is a balance
between opposing hydrostatic and colloid (protein) osmotic
pressures. Since hydrostatic pressure decreases along a
capillary, it follows that filtration occurs along the arterial
end of capillaries and reabsorption at the venous end of
capillaries, though this model has been challenged in more
recent years.11,12
Not until decades later did Starling’s initial
observations become expressed in mathematical format,13,14
The endothelial glycocalyx is a dynamic layer of macromolecules at the luminal surface of
vascular endothelium that is involved in fluid homeostasis and regulation. Its role in vascular
permeability and edema formation is emerging but is still not well understood. In this special
article, we highlight key concepts of endothelial dysfunction with regards to the glycocalyx and
provide new insights into the glycocalyx as a mediator of processes central to the development
of pulmonary edema and lung injury. (Anesth Analg 2013;XX:00–00)
The Endothelial Glycocalyx: Emerging Concepts
in Pulmonary Edema and Acute Lung Injury
Stephen R. Collins, MD,* Randal S. Blank, MD, PhD,* Lindy S. Deatherage, MD,†
and Randal O. Dull, MD, PhD‡
T
he endothelial glycocalyx (EG) is a complex and mul-
ticomponent layer of macromolecules at the luminal
surface of vascular endothelium. This concept was
proposed more than 70 years ago and its composition is
well studied as detailed in 2 reviews1,2
; however, its role
in mechanisms of endothelial protection and injury and
subsequent clinical implications have just recently become
evident. The EG consists of a variety of endothelial mem-
brane–bound molecules, including glycoproteins and pro-
teoglycans, that provide the basis for plasma–endothelial
cell interaction. EG structure, although well characterized
in vitro, is poorly defined in vivo because of its dynamically
changing composition by self-assembly and enzymatic deg-
radation or shear-dependent shedding of its elements. Its
major constituents are hyaluronic acid and the negatively
charged heparan sulfate proteoglycans. Together with gly-
cosaminoglycans (GAGs) and plasma proteins, the EG layer
as a whole forms the endothelial surface layer (ESL) that
acts as a barrier to circulating cells and large molecules.
the role of
dence for m
that lead to
role of the
lung injury
transducer
of lung in
explored.
THE STAR
EDEMA
Our under
plasma/in
has change
meshwork
a major ba
fundament
transcapill
however, t
vascular endothelium that is involved in fluid homeostasis a
permeability and edema formation is emerging but is still no
article, we highlight key concepts of endothelial dysfunction
provide new insights into the glycocalyx as a mediator of proc
of pulmonary edema and lung injury. (Anesth Analg 2013;X
Endothelial Glycocalyx
Figure 3. Schematic illustrating the hypothesized role of the glycocalyx in lung vascular mechanotransduction. Left: During static conditions,
the glycocalyx maintains barrier function over the intercellular junction. Right: During increased vascular pressure, the increased hydraulic
flow through the glycocalyx deforms or stresses the glycosaminoglycan (GAG) fibers, which in turn activates endothelial nitric oxide synthase
(eNOS) and leads to barrier dysfunction. ∆Pc = change in capillary pressure; Q = flow; ZO-1 and ZO-2 = zonula occludens-1 and -2; vin =
vinculin; VE-Cad = vascular endothelial cadherin; ECM = extracellular matrix. Adapted from Dull et al.25

The Circulating Glycosaminoglycan Signature of Respiratory
Failure in Critically Ill Adults*
Received for publication,November 30, 2013, and in revised form, January 21, 2014 Published, JBC Papers in Press,February 7, 2014, DOI 10.1074/jbc.M113.539452
Eric P. Schmidt‡§1
, Guoyun Li¶ʈ
, Lingyun Liʈ
, Li Fuʈ
, Yimu Yang‡
, Katherine H. Overdier§
, Ivor S. Douglas‡§
,
and Robert J. Linhardtʈ
From the ‡
Program in Translational Lung Research, Department of Medicine, Division of Pulmonary Sciences and Critical Care
Medicine, University of Colorado School of Medicine, Aurora, Colorado 80045, the §
Denver Health Medical Center, Denver,
Colorado 80204, the ¶
College of Food Science and Technology, Ocean University of China, Qingdao, Shandong 266003, China, and
the ʈ
Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer
Polytechnic Institute, Troy, New York 12180
Background: Endothelial glycocalyx degradation contributes to the pathogenesis of critical illness.
Results: Mechanically ventilated subjects exhibited plasma glycocalyx breakdown signatures (glycosaminoglycan fragments)
characteristic of direct versus indirect etiologies of respiratory failure.
Conclusion: Circulating glycosaminoglycans provide insight into respiratory failure pathophysiology.
Significance: This is the first study to characterize circulating glycosaminoglycans during critical illness, offering insight into
the mechanisms underlying respiratory failure.
Systemic inflammatory illnesses (such as sepsis) are marked
by degradation of the endothelial glycocalyx, a layer of glycos-
aminoglycans (including heparan sulfate, chondroitin sulfate,
and hyaluronic acid) lining the vascular lumen. We hypothe-
sized that different pathophysiologic insults would produce
characteristic patterns of released glycocalyx fragments. We
collected plasma from healthy donors as well as from subjects
with respiratory failure due to altered mental status (intoxica-
tion, ischemic brain injury), indirect lung injury (non-pulmo-
nary sepsis, pancreatitis), or direct lung injury (aspiration, pneu-
monia). Mass spectrometry was employed to determine the
quantity and sulfation patterns of circulating glycosaminogly-
cans. We found that circulating heparan sulfate fragments were
significantly (23-fold) elevated in patients with indirect lung
injury, while circulating hyaluronic acid concentrations were
elevated (32-fold) in patients with direct lung injury. N-Sulfa-
tion and tri-sulfation of heparan disaccharides were signifi-
cantly increased in patients with indirect lung injury. Chondroi-
tin disaccharide sulfation was suppressed in all groups with
respiratory failure. Plasma heparan sulfate concentrations
directly correlated with intensive care unit length of stay. Serial
plasma measurements performed in select patients revealed
The endothelial glycocalyx is a layer of proteoglycans and
associated glycosaminoglycans (including heparan sulfate
(HS),2
hyaluronic acid (HA), and chondroitin sulfate (CS)) that
lines the vascular lumen (1). In vivo, the glycocalyx forms a thick
endothelial surface layer (ESL) that contributes to the regula-
tion of endothelial permeability, leukocyte adhesion, and nitric
oxide production (2–4). Accordingly, degradation of the ESL
has been implicated in the pathogenesis of critical illnesses (e.g.
sepsis, major trauma) characterized by vascular hyperperme-
ability, inflammation, and aberrant vascular tone (5, 6). Increas-
ing attention has therefore been dedicated to understanding
the fate of glycocalyx/ESL integrity during the course of critical
illness. Glycocalyx protection is increasingly a goal of resusci-
tation strategies in intensive care unit (ICU) patients (7).
To date, human studies of glycocalyx/ESL degradation dur-
ing critical illness have primarily relied upon either intravital
microscopy of vascular beds of uncertain clinical relevance (e.g.
the sublingual microcirculation (8, 9)) or the detection of cir-
culating glycocalyx fragments by immunoassay (6, 9–13).
These techniques offer little insight into the mechanisms
underlying ESL loss across different vascular beds during criti-
cal illness. As glycosaminoglycan composition varies across tis-
atUniversitàdeglistudidiMilanoonFebruary17,2015http://www.jbc.org/Downloadedfrom
ating Glycosaminoglycan Signature of Respiratory
Critically Ill Adults*
vember 30, 2013, and in revised form, January 21, 2014 Published, JBC Papers in Press,February 7, 2014, DOI 10.1074/jbc.M113.539452
Guoyun Li¶ʈ
, Lingyun Liʈ
, Li Fuʈ
, Yimu Yang‡
,
dtʈ
Translational Lung Research, Department of Medicine, Division of Pulmonary Sciences and Critical Care
f Colorado School of Medicine, Aurora, Colorado 80045, the §
hemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer
Troy, New York 12180
othelial glycocalyx degradation contributes to the pathogenesis of critical illness.
cally ventilated subjects exhibited plasma glycocalyx breakdown signatures (glycosaminoglycan fragments)
irect versus indirect etiologies of respiratory failure.
ulating glycosaminoglycans provide insight into respiratory failure pathophysiology.
s is the first study to characterize circulating glycosaminoglycans during critical illness, offering insight into
nderlying respiratory failure.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 12, pp. 8194–8202, March 21, 2014
© 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
, Guoyun Li¶ʈ
, Lingyun Liʈ
, Li Fuʈ
, Yimu Yang‡
,
From the ‡
the ʈ
httpDownloadedfrom
unsulfated HS disaccharides in normal plasma, heparan sulfa-
tion increased in patients with altered mental status- or indirect
lung injury-induced respiratory failure (Fig. 4a). Patients with
indirect lung injury had increased levels of N-sulfated (Fig. 4b)
and tri-sulfated (Fig. 4c) disaccharides. As above, these statisti-
cally significant differences persisted even when excluding a
high-sulfation outlier (subject no. 9). Chondroitin sulfation was
suppressed in all patients with respiratory failure (Fig. 4, d and
e). Given its unsulfated structure, HA sulfation analyses could
not be performed. Hematologic indices (e.g. white blood cell or
tractedionchromatography(EIC)ofAMAC-taggeddisaccha-
s of HS fragments isolated from human plasma. a, disaccha-
ds; b, HS disaccharides from normal human plasma; c, HS disac-
m the plasma of a patient (no. 14) with severe acute pancreatitis.
asma glycosaminoglycan concentrations in normal donors or patients with respiratory failure due to altered mental status, indirect
injury, or direct lung injury. *, p Ͻ 0.05 compared with normal donors.
2014•VOLUME 289•NUMBER 12 JOURNAL OF BIOLOGICAL CHEMISTRY 8197
atUniversitàdeglistudidiMilanoonFebruary17,2015http://www.jbc.org/dfrom
4 control subjects and 17 mechanically
ventilated patients with respiratory failure
due to :
-altered mental status
(intoxication,ischemic brain injury)
-indirect lung injury (pancreatitis)
-direct lung injury
(aspiration,pneumonia)

, Guoyun Li¶ʈ
, Lingyun Liʈ
, Li Fuʈ
, Yimu Yang‡
,
From the ‡
the ʈ
(HS),2
Guoyun Li¶ʈ
, Lingyun Liʈ
, Li Fuʈ
, Yimu Yang‡
,
dtʈ
due to :
-direct lung injury

, Guoyun Li¶ʈ
, Lingyun Liʈ
, Li Fuʈ
, Yimu Yang‡
,
From the ‡
the ʈ
(HS),2
Guoyun Li¶ʈ
, Lingyun Liʈ
, Li Fuʈ
, Yimu Yang‡
,
dtʈ
due to :
-direct lung injury
that circulating highly sulfated heparan fragments persisted for
greater than 3 days after the onset of respiratory failure. Our
findings demonstrate that circulating glycosaminoglycans are
elevated in patterns characteristic of the etiology of respiratory
failure and may serve as diagnostic and/or prognostic biomark-
ers of critical illness.
in
t
il
t
in
m
t
c
T
u
c
s
c
F
(
c
t

Endothelial Glycocalyx Layer (EGL):
three main functions (to date…)
• mechanotransduction of fluid shear stress to
the endothelial cell (EC) cytoskeleton with the
resulting biochemical responses
•modulation of
permeability in the
transcapillary exchange of
water : Starling revisited
•regulation of red and white blood cells
interactions with EC triggering inflammatory
response and relationships with coagulation system
Weinbaum S et al. Annu Rev. Biomed Eng. 2007

The importance of being Ernest :
THE STARLING LAW (1896)
• Pc is the capillary hydrostatic pressure
• Pi is the interstitial hydrostatic pressure
• πc is the capillary oncotic pressure
• πi is the interstitial oncotic pressure
• Kf is the filtration coefficient – a proportionality constant
• σ is the reflection coefficient
Starling EH. J Physiol 1896

• endothelial glycocalyx layer
(EGL): an active working
structure to keep low hydraulic
conductivity
• fluid filtration not closely
related to interstitial protein
concentration
• the paradigmatic shift : the
oncotic pressure difference for
fluid homeostsis built up
between the intravascular space
and the protein-free zone
beneath the protein-loaded
EGL
NT
N Reappraising Starling: the physiology of
the microcirculation
Matthias Jacob and Daniel Chappell
Purpose of review
Vascular permeability is traditionally explained by Starling’s principle, describing two opposing forces
across the endothelial cell line to maintain compartments in balance. Several contradictions to this principle
have recently questioned its validity.
Recent findings
Hydraulic conductivity is kept low by a properly working endothelial surface layer, created by binding and
intercalating plasma constituents with the structural elements of an endothelial glycocalyx. Limiting fluid
filtration is not closely related to the interstitial protein concentration. Rather, the oncotic pressure difference
pertinent to fluid homeostasis is built up between the intravascular space and a small protein-free zone
beneath the protein-loaded endothelial glycocalyx. This crucial structure, and therefore the resistance of the
barrier against outflow of large molecules, is endangered by ischaemia, inflammation and intravascular
hypervolaemia. An intact endothelial surface layer retains iso-oncotic preparations of large molecules
infused to compensate for acute bleeding. Crystalloids cannot be held back sufficiently, even if preload is
warranted.
Summary
Starling’s principle requires an adaptation to recognize that there is no inward-directed oncotic pressure
gradient across the whole anatomical vessel wall. The carrier of vascular barrier competence is the intact
endothelial surface layer which might be protected by avoiding intravascular hypervolaemia and limiting
OPINION Reappraising Starling: the phys
Purpose of review
Vascular permeability is traditionally explained by Starling’s prin
across the endothelial cell line to maintain compartments in bala
Recent findings
Hydraulic conductivity is kept low by a properly working endoth
intercalating plasma constituents with the structural elements of a
filtration is not closely related to the interstitial protein concentrat
pertinent to fluid homeostasis is built up between the intravascula
beneath the protein-loaded endothelial glycocalyx. This crucial s
barrier against outflow of large molecules, is endangered by isc
hypervolaemia. An intact endothelial surface layer retains iso-on
infused to compensate for acute bleeding. Crystalloids cannot be
warranted.
Summary
Starling’s principle requires an adaptation to recognize that ther
gradient across the whole anatomical vessel wall. The carrier of
endothelial surface layer which might be protected by avoiding
inflammation.
Keywords
endothelial glycocalyx, endothelial surface layer, endothelium, v
pyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this artic
PROPAEDEUTICS OF HUMAN
PHYSIOLOGY
Protozoa consist merely of one compartment which
s directly in touch with the environment; they do
not need a circulation. During evolution, single cells
were organized to organs and higher organisms.
Germany
Correspondence to Matthias Jacob, MD, PhD, S
ate Professor, Department of Anaesthesiolo
Munich, Nußbaumstrasse 20, 80336 Munich
5160 2691; fax: +49 89 5160 4446; e-mail: m
muenchen.de
Curr Opin Crit Care 2013, 19:282–289
DOI:10.1097/MCC.0b013e3283632d5e
www.co-criticalcare.com Volume 19 Num
CURRENT
OPINION Re
th
Pu
Va
ac
ha
Re
Hy
int
filt
pe
be
ba
hy
inf
wa
Su
Sta
vealed a true thickness of the endo-
alyx of up to 1 mm [23]. Beyond
r functioning, other important pro-
ti-inflammation [2] and shear-stress
o the endothelial surface [24] have
d to this structure.
thelial glycocalyx is composed of
und glycoproteins and proteoglycans,
an and glypican, carrying negatively
hains (mainly heparan, but also der-
ondroitin sulphates) and hyaluronan
glycocalyx itself, however, is nothing
keleton. In vivo, by binding plasma
mainly albumin), it is completed to
l surface layer [5,19]. Only this entire
ologically active as a vascular barrier
s denomination, integrating the
ble hydraulic conductivity [10] and
capacity to hold back large plasma
oviding a filtration-limiting inward-
Arteriolar segments Venular sections
PI
PV
ESL
EC
IS
pI
pV
pV
pg
pg
pI
PV-PI
pV-pg
FIGURE 5. The revised model of vascular barrier
functioning. Within the high-pressure segments (PV ) PI) a
tight endothelial surface layer guarantees a low hydraulic
conductivity and the low oncotic pressure directly beneath
ds

• endothelial glycocalyx layer
(EGL): an active working
structure to keep low hydraulic
conductivity
• fluid filtration not closely
related to interstitial protein
concentration
• the paradigmatic shift : the
oncotic pressure difference for
fluid homeostsis built up
between the intravascular space
and the protein-free zone
beneath the protein-loaded
EGL
NT
Purpose of review
Recent findings
warranted.
Summary
Purpose of review
Recent findings
warranted.
Summary
inflammation.
Keywords
pyright © Lippincott Williams Wilkins. Unauthorized reproduction of this artic
PHYSIOLOGY
Germany
5160 2691; fax: +49 89 5160 4446; e-mail: m
muenchen.de
DOI:10.1097/MCC.0b013e3283632d5e
CURRENT
OPINION Re
th
Pu
Va
ac
ha
Re
Hy
int
filt
pe
be
ba
hy
inf
wa
Su
Sta
PI
PV
ESL
EC
IS
pI
pV
pV
pg
pg
pI
PV-PI
pV-pg
ds

NT
Purpose of review
Recent findings
warranted.
Summary
Purpose of review
Recent findings
warranted.
Summary
inflammation.
Keywords
PHYSIOLOGY
Germany
5160 2691; fax: +49 89 5160 4446; e-mail: m
muenchen.de
DOI:10.1097/MCC.0b013e3283632d5e
CURRENT
OPINION Re
th
Pu
Va
ac
ha
Re
Hy
int
filt
pe
be
ba
hy
inf
wa
Su
Sta
PI
PV
ESL
EC
IS
pI
pV
pV
pg
pg
pI
PV-PI
pV-pg
ds

NT
Purpose of review
Recent findings
warranted.
Summary
Purpose of review
Recent findings
warranted.
Summary
inflammation.
Keywords
PHYSIOLOGY
Germany
5160 2691; fax: +49 89 5160 4446; e-mail: m
muenchen.de
DOI:10.1097/MCC.0b013e3283632d5e
CURRENT
OPINION Re
th
Pu
Va
ac
ha
Re
Hy
int
filt
pe
be
ba
hy
inf
wa
Su
Sta
PI
PV
ESL
EC
IS
pI
pV
pV
pg
pg
pI
PV-PI
pV-pg
ds
• EGL appears to be the real
barrier against outﬂow of
large molecole to
interstitial space (i.e.:
edema)
• EGL damaging/shedding
situations : shock,
ischemia-riperfusion,
inﬂammation,
hypervolemia

• in clinical practice, consider the different distribution behaviours of crystalloids
and colloids
• crystalloids for volume replacement in ongoing extracellular losses from
extracellular compartment
• in acute bleeding setting below transfusion trigger seems reasonable the use of a
class of drugs targeting the intravascular compartment : colloids such starches
(careful with coagulation and renal impairments) or iso-oncotic human albumin
preparations
• prophylactic hypervolemia outdated and possibly threaten, as it increase ANP with
direct damage of EGL, outflow of fluids and proteins with interstitial edema and
decreasing cardiac output, with possible worse outcome
3
Role of the glycocalyx in fluid management:
Small things matter*
Daniel Chappell, M.D., Associate Professor a
,
Matthias Jacob, M.D., Associate Professor a, b, *
a
Department of Anaesthesiology, University Hospital Munich, Nussbaumstrasse 20,
80336 Munich, Germany
b
Harlaching Hospital, Munich Municipal Hospital Group, Sanatoriumsplatz 2, 81545 Munich, Germany
Keywords:
colloids
crystalloids
endothelial glycocalyx
endothelial surface layer
fluid therapy
Intravenous fluid therapy and perception of volume effects are
often misunderstood. The pharmacokinetical difference between
colloids and crystalloids depends on the condition of the vascular
permeability barrier. Its functioning is still largely based on Star-
ling's principle from 1896, realising that transport of fluid to and
from the interstitial space follows the balance between opposing
oncotic and hydrostatic pressures. In the past decade, the endo-
thelial glycocalyx, located on the luminal side of healthy vascula-
ture, has increasingly been taken into consideration around
models of transvascular fluid filtration. While crystalloids can
freely pass through the glycocalyx, colloids are held back in the
vasculature by this structure. This is reflected by a markedly higher
intravascular persistence of isooncotic colloids (80e100%) versus
crystalloids (around 20%), at least as long as the glycocalyx is
intact. Protecting this structure in surgical practice means limiting
the surgical trauma and avoiding intravascular hypervolemia.
© 2014 Published by Elsevier Ltd.
Over the past years, fluid management has been a controversially discussed topic [1]. Meanwhile,
consensus has been achieved to the effect that the composition of crystalloids should resemble plasma
concentrations of electrolytes and, therefore, balanced solutions should be preferred over isotonic
8
Volume therapy in trauma and neurotrauma
M.F.M. James, MBChB, PhD, FRCA, FFA(SA), Emeritus
Professor *
Department of Anaesthesia, University of Cape Town, Anzio Road, Observatory, Cape Town,
Western Cape 7925, South Africa
Keywords:
trauma
fluid therapy
blood transfusion
resuscitation
crystalloids
colloids
Volume therapy in trauma should be directed at the restitution
of disordered physiology including volume replacement to re-
establishment of tissue perfusion, correction of coagulation
deficits and avoidance of fluid overload. Recent literature has
emphasised the importance of damage control resuscitation,
focussing on the restoration of normal coagulation through
increased use of blood products including fresh frozen plasma,
platelets and cryoprecipitate. However, once these targets have
been met, and in patients not in need of damage control
resuscitation, clear fluid volume replacement remains essential.
Contents lists available at ScienceDirect
Best Practice Research Clinical
Anaesthesiology
journal homepage: www.elsevier.com/locate/bean
Best Practice Research Clinical Anaesthesiology 28 (2014) 227e234

Revised Starling equation and the glycocalyx model
of transvascular fluid exchange: an improved paradigm
for prescribing intravenous fluid therapy
T. E. Woodcock1* and T. M. Woodcock2
1
Critical Care Service, Southampton University Hospitals NHS Trust, Tremona Road, Southampton SO16 6YD, UK
2
The Australian School of Advanced Medicine, Macquarie University, NSW 2109, Australia
* Corresponding author. E-mail: tom.woodcock@me.com
Editor’s key points
† The classic Starling
principle does not hold
for fluid resuscitation in
clinical settings.
† The endothelial
glycocalyx layer appears
to have a major role in
fluid exchange.
† A revision of Starling
incorporating the
glycocalyx model
appears to explain better
the responses seen
clinically.
Summary. I.V. fluid therapy does not result in the extracellular volume distribution expected
from Starling’s original model of semi-permeable capillaries subject to hydrostatic and
oncotic pressure gradients within the extracellular fluid. Fluid therapy to support the
circulation relies on applying a physiological paradigm that better explains clinical and
research observations. The revised Starling equation based on recent research considers
the contributions of the endothelial glycocalyx layer (EGL), the endothelial basement
membrane, and the extracellular matrix. The characteristics of capillaries in various
tissues are reviewed and some clinical corollaries considered. The oncotic pressure
difference across the EGL opposes, but does not reverse, the filtration rate (the ‘no
absorption’ rule) and is an important feature of the revised paradigm and highlights the
limitations of attempting to prevent or treat oedema by transfusing colloids. Filtered fluid
returns to the circulation as lymph. The EGL excludes larger molecules and occupies a
substantial volume of the intravascular space and therefore requires a new interpretation
of dilution studies of blood volume and the speculation that protection or restoration of
the EGL might be an important therapeutic goal. An explanation for the phenomenon of
context sensitivity of fluid volume kinetics is offered, and the proposal that crystalloid
resuscitation from low capillary pressures is rational. Any potential advantage of plasma
or plasma substitutes over crystalloids for volume expansion only manifests itself at
higher capillary pressures.
Keywords: fluid therapy; intensive care
Twenty-five years ago, Twigley and Hillman announced ‘the
end of the crystalloid era’. Using a simplified diagram of
plasma, interstitial and intracellular fluid compartments,
and their anatomic volumes, they argued that colloids
could be used to selectively maintain the plasma volume.1
Plasma volume being about 20% of the extracellular fluid
Africa demonstrated no advantages of bolus therapy with
albumin compared with ISS, and a survival advantage for
slow ISS resuscitation without bolus therapy.8
A series of
volume kinetics experiments have demonstrated that the
central volume of distribution of ISS is much smaller than
the anatomic ECF volume,9
and an editorial had to conclude
atOSPEDALENIGUARDACA'GRANDAonJanuaryhttp://bja.oxfordjournals.org/Downloadedfrom
Revised Starling equation and the glycocalyx model
of transvascular fluid exchange: an improved paradigm
for prescribing intravenous fluid therapy
1
Critical Care Service, Southampton University Hospitals NHS Trust, Tremona Road, Southampton SO16 6YD, UK
2
The Australian School of Advanced Medicine, Macquarie University, NSW 2109, Australia
clinical settings.
† The endothelial
fluid exchange.
incorporating the
glycocalyx model
appears to explain better
the responses seen
clinically.
Summary. I.V. fluid therapy does not result in the extracellular volume distribution expected
from Starling’s original model of semi-permeable capillaries subject to hydrostatic and
oncotic pressure gradients within the extracellular fluid. Fluid therapy to support the
circulation relies on applying a physiological paradigm that better explains clinical and
research observations. The revised Starling equation based on recent research considers
the contributions of the endothelial glycocalyx layer (EGL), the endothelial basement
membrane, and the extracellular matrix. The characteristics of capillaries in various
tissues are reviewed and some clinical corollaries considered. The oncotic pressure
difference across the EGL opposes, but does not reverse, the filtration rate (the ‘no
absorption’ rule) and is an important feature of the revised paradigm and highlights the
limitations of attempting to prevent or treat oedema by transfusing colloids. Filtered fluid
returns to the circulation as lymph. The EGL excludes larger molecules and occupies a
substantial volume of the intravascular space and therefore requires a new interpretation
of dilution studies of blood volume and the speculation that protection or restoration of
the EGL might be an important therapeutic goal. An explanation for the phenomenon of
context sensitivity of fluid volume kinetics is offered, and the proposal that crystalloid
resuscitation from low capillary pressures is rational. Any potential advantage of plasma
or plasma substitutes over crystalloids for volume expansion only manifests itself at
British Journal of Anaesthesia 108 (3): 384–94 (2012)
Advance Access publication 29 January 2012 . doi:10.1093/bja/aer515
Revised Starling equation
of transvascular fluid exc
for prescribing intraveno
1
Critical Care Service, Southampton University Hospital
2
The Australian School of Advanced Medicine, Macqua
clinical settings.
† The endothelial
fluid exchange.
incorporating the
Summary. I.V. fl
from Starling’s
oncotic pressu
circulation relie
research obser
the contributio
membrane, an
tissues are re
difference acro
absorption’ rule
limitations of a
returns to the
Advance Access publication 29 January 2012 . doi:10.10
Intraoperative fluids: how much is too much?
M. Doherty1* and D. J. Buggy1,2
1
Department of Anaesthesia, Mater Misericordiae University Hospital, University College Dublin, Ireland
2
Outcomes Research Consortium, Cleveland Clinic, OH, USA
* Corresponding author. E-mail: margaretdoherty@yahoo.com
† Both too little and excessive fluid
during the intraoperative period can
adversely affect patient outcome.
† Greater understanding of fluid
kinetics at the endothelial glycocalyx
enhances insight into bodily fluid
Summary. There is increasing evidence that intraoperative fluid therapy
decisions may influence postoperative outcomes. In the past, patients
undergoing major surgery were often administered large volumes of
crystalloid, based on a presumption of preoperative dehydration and
nebulous intraoperative ‘third space’ fluid loss. However, positive perioperative
fluid balance, with postoperative fluid-based weight gain, is associated with
increased major morbidity. The concept of ‘third space’ fluid loss has been
emphatically refuted, and preoperative dehydration has been almost
Advance Access publication 1 June 2012 . doi:10.1093/bja/aes171
Downloadedfrom
Intraoperative fluids: how
1
Department of Anaesthesia, Mater Misericordiae Univ
2
Outcomes Research Consortium, Cleveland Clinic, OH,
* Corresponding author. E-mail: margaretdoherty@yaho
Sum
dec
und
Advance Access publication 1 June 2012 . doi:10.1093/
Intraoperative fluids: how much is too much?
1
Department of Anaesthesia, Mater Misericordiae University Hospital, University College Dublin, Ireland
2
Outcomes Research Consortium, Cleveland Clinic, OH, USA
* Corresponding author. E-mail: margaretdoherty@yahoo.com
during the intraoperative period can
adversely affect patient outcome.
† Greater understanding of fluid
kinetics at the endothelial glycocalyx
enhances insight into bodily fluid
Summary. There is increasing evidence that intraoperative fluid ther
decisions may influence postoperative outcomes. In the past, patie
undergoing major surgery were often administered large volumes
crystalloid, based on a presumption of preoperative dehydration
nebulous intraoperative ‘third space’ fluid loss. However, positive periopera
fluid balance, with postoperative fluid-based weight gain, is associated w
increased major morbidity. The concept of ‘third space’ fluid loss has b
emphatically refuted, and preoperative dehydration has been alm
Advance Access publication 1 June 2012 . doi:10.1093/bja/aes171

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