11
Review
Endothelium and haemostasis
W. C. Aird
Department of Medicine, Division of Molecular and Vascular Medicine, Beth Israel Deaconess Medical Center, Boston,
USA
Keywords
Schlüsselwörter
Endothelium, haemostasis
Endothel, Hämostase
Summary
Zusammenfassung
The endothelium is a widely distributed
organ system that plays an important role in
health and disease. The endothelium is remarkably heterogeneous in structure and
function. One vital function of the endothelium is to maintain blood in its fluid state,
and to provide controlled haemostasis at
sites of vascular injury. In keeping with the
theme of endothelial cell heterogeneity, endothelial cells from different sites of the vascular employ different strategies to mediate
local haemostatic balance. These differences
are sufficient to explain why systemic imbalances of haemostatic components invariably
lead to local thrombotic phenotypes. An important goal for the future is to identify diagnostic markers that reflect phenotypic
changes at the level of individual vascular
beds, and to develop therapies that target
one or another site of the vasculature.
Das Endothel ist ein weit verbreitetes Organsystem, das eine wichtige Rolle bei Gesundheit und Krankheit spielt. Seine Struktur und
Funktion sind bemerkenswert heterogen. Eine vitale Funktion besteht darin, das Blut
flüssig zu halten und im Bereich von Gefäßverletzungen eine kontrollierte Blutstillung
zu ermöglichen. Einhergehend mit der Heterogenität von Endothelzellen ist zu erwähnen, dass sich die aus unterschiedlichen Regionen des Gefäßsystems stammenden Endothelzellen unterschiedlichen Strategien bei
der Vermittlung des lokalen hämostatischen
Gleichgewichts bedienen. Diese Unterschiede erklären, warum ein systemisches Ungleichgewicht bei hämostatischen Faktoren
unweigerlich zu lokalen thrombotischen Phänotypen führt. Wichtige künftige Ziele sind
die Identifikation diagnostischer Marker, die
phänotypische Änderungen auf der Ebene
einzelner Gefäßbetten widerspiegeln, und
die Entwicklung von Therapien, die auf Regionen des Gefäßsystems zielen.
Correspondence to:
William C. Aird, M.D.
Beth Israel Deaconess Medical Center, Molecular and
Vascular Medicine
RN-227, 330 Brookline Ave., Boston MA 02215, USA
E-mail: [email protected]
Endothel und Hämostase
Hämostaseologie 2015; 35: 11–16
http://dx.doi.org/10.5482/HAMO-14-11-0075
received: November 24, 2014
accepted: November 27, 2014
The endothelium, which forms the inner
lining of blood vessels and lymphatics, is a
systemically distributed organ. It reaches
all expanses of the human body with the
exception of avascular tissues (e. g., the epidermis, nails, cornea and cartilage). The
endothelium was once considered to be
little more than an inert layer of nucleated
cellophane. However, endothelial cells are
now understood to participate in a multitude of physiological functions, including
• haemostasis,
•
•
•
•
permeability,
leukocyte trafficking,
innate and acquired immunity, and
vasomotor tone.
Moreover, endothelial cell phenotypes vary
greatly between different vascular beds. Indeed, rather than forming one giant monopoly of homogeneous cells, the endothelium represents a consortium of smaller
enterprises, each adapted to the needs of
the underlying tissue.
As an important corollary, endothelial
dysfunction leads to vascular bed-specific
pathology.
The goal of this review is to consider the
scope and nature of endothelial phenotypes
and to apply these principles to an understanding of the endothelium in haemostasis and thrombosis.
Endothelial heterogeneity
Cells
The notion that endothelial cells are heterogeneous is by no means new. In 1966,
Sir Howard Florey, who was awarded the
1945 Nobel Prize in Physiology or Medicine for the discovery of penicillin, wrote
(1): „A few years ago the endothelial cell was
thought of as a structure so thin that few details could be made out in its cytoplasm,
while now it is recognized that there are
many kinds of endothelial cell which differ
from one another substantially in structure,
and, to some extent, in function.“
During the 1960s, most of our information about endothelial cell heterogeneity
was derived from ultrastructural studies.
For example, electron microscopy revealed
that endothelial cells of arteries and veins
form a continuous uninterrupted monolayer, whereas endothelial cells that line
certain capillaries are permeated by fenestrae or large gaps. Moreover, structural
variation was shown to correlate with functional differences. For example, fenestrated
endothelium was noted in those organs
that are involved in secretion (e. g., endocrine glands, gastric and intestinal mucosa,
and choroid plexus) or filtration (i. e., kidney glomeruli).
The introduction of endothelial cell culture in the early 1970s provided investigators with a powerful new tool to study
relatively pure populations of living cells
under tightly controlled conditions (2, 3).
This technique led to an exponential in-
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W. C. Aird: Endothelium and haemostasis
crease in the number of publications related to endothelial cells and to enormous
advances in our understanding of endothelial cell biology (4). Among these advances
was the recognition that endothelial cells
express a number of procoagulants and
anticoagulants and that endothelial cell
activation is associated with a shift in the
haemostatic balance to the procoagulant
side.
A disadvantage of endothelial cell culture is that it encourages investigators to
approach the endothelium as a monopoly
of identical cells, rather than as a transcendent organ with emergent site-specific
properties, whose whole is greater than the
sum of the parts. Indeed, the very notion of
endothelial cell heterogeneity was largely
forgotten or ignored during the heyday of
cell culture. The introduction of histochemical techniques in the 1980s and
1990s led to the discovery that endothelial
cell proteins are differentially distributed in
the intact vasculature. For example, in
1992, a survey of the distribution of von
Willebrand factor (VWF) in human tissues
found that expression was higher in large
vessels compared with capillaries, leading
the authors to conclude (5): „A surprising
result from this study was the heterogeneous
expression of VWF, which is thought to be
produced in vascular endothelial cells as part
of the coagulation system.“
The reason this finding was surprising:
VWF was (and still is) widely touted as a
universal marker for endothelial cells in
culture. Such discordance in gene expression between in vitro and in vivo settings is not unique to VWF. Indeed, only a
small minority of endothelial-restricted
genes (e. g., CD31, VE-cadherin and Erg)
are uniformly expressed in the endothelium. Previous in vivo phage display studies
have uncovered site-specific repertoires of
endothelial cell surface proteins, which
have been referred to as “vascular zip
codes” (6, 7). This metaphor nicely captures the therapeutic potential of targeting
drugs to one or another vascular bed.
Mechanisms
Every biological trait, including endothelial
cell heterogeneity, requires both a proximate and evolutionary explanation (8).
•
•
Proximate explanations (how?) employ
traditional approaches of cell biology
and molecular biology to determine the
anatomy, physiology and ontogeny (developmental history) of a trait at the
level of a single organism.
Evolutionary explanations (why?) draw
on the fossil record and comparative
morphology and DNA sequences to uncover the phylogeny (evolutionary history) of a trait as well as the fitness advantage that the trait provides at the
level of a population or species.
Proximate mechanisms
Like all living cells, the endothelial cell may
be considered an input-output device.
Input arises from the extracellular environment and may include any number of biomechanical and biochemical signals arising
from the blood vessel lumen, the abluminal
side and/or neighbouring endothelial cells.
The output represents the phenotype of the
endothelial cell and varies from single-celllevel changes (e. g., calcium flux, migration,
proliferation, apoptosis) to larger scale
changes (e. g., angiogenesis, fibrin deposition). Input is coupled to output by a nonlinear array of signalling pathways that
typically begin at the cell surface and end at
the transcriptional or post-transcriptional
level. Since endothelial cells are distributed
throughout the body, they are exposed to
enormously diverse input signals. For
example, in the blood brain barrier, endothelial cells receive cues from astroglial
cells that are critical for maintaining barrier function, whereas endothelial cells that
line the hepatic sinusoids are exposed to
nutrient-rich blood on the luminal side
and stellate cell/hepatocyte-derived paracrine factors in the space of Disse. Since
input varies across the vascular tree, and in
so far as the endothelial cell is capable of
transducing signals, then output must also
vary throughout the vasculature. In other
words, the broad distribution of the endothelium is sufficient to explain phenotypic heterogeneity.
So far, this description paints the endothelium as a blank slate, marching
blindly to the tune of the microenvironment. However, endothelial cells also express properties that are epigenetically
fixed and impervious to changes in the
cell’s microenvironment. Viewed from this
perspective, endothelial cell heterogeneity
is mediated by a combination (9) of
• imminently reversible environmentally
responsive signal transduction pathways
(nurture) and
• stable epigenetic mechanisms that “lock
in” gene expression regardless of the environment (nature).
Understanding the relative roles of nurture
and nature has important therapeutic implications. If one wishes to target a phenotype that is mediated by the microenvironment, then treatment may be best directed
towards that environment. By contrast,
those properties that are more fixed may
require targeting of the cell itself.
Evolutionary mechanisms
Comparative studies have revealed that endothelial cells are present in all vertebrates,
and absent in all invertebrates (10). Thus,
the endothelium evolved in a common ancestor of all extant vertebrates, which lived
some time more than 510 million years
ago. The hagfish is the oldest extant vertebrate. Previous studies have revealed that
hagfish endothelium is heterogeneous in
structure and function (11, 12). Thus, endothelial cell heterogeneity evolved as an
early feature of this cell lineage. What is the
selective advantage of having an endothelium in the first place, and why is it so heterogeneous? These are difficult questions
to answer, but it seems likely that the endothelium evolved to optimize flow dynamics and barrier function, and/or to localize immune and coagulation functions,
while phenotypic heterogeneity reflects the
role of the endothelium in meeting the diverse needs of body tissues, as well as its
need to adapt to, and survive in, many different extracellular environments.
Endothelial cell activation
and dysfunction
The two most common descriptors for the
endothelium in disease are activation and
dysfunction. The term endothelial cell activation first appeared in the 1980s when it
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W. C. Aird: Endothelium and haemostasis
was observed that exposure of cultured endothelial cells to inflammatory mediators
resulted in the expression of new antigens
(so-called activation antigens) on the surface of cultured endothelial cells, and was
correlated with the expression of pro-adhesive, antigen-presenting and procoagulant activities; reviewed in (13). Today, the
term activation is used more widely to describe the response of endothelial cells to
an inflammatory stimulus in vitro or in
vivo (some investigators in the angiogenesis field employ the term to describe proliferating endothelial cells). The activation
phenotype varies according to the stimulus
and the vascular bed of origin. However, it
often includes some combination of increased permeability and net procoagulant
and/or proinflammatory activity. It is important to recognize that while endothelial
cell activation describes a phenotype, it
does not refer to the cost of that phenotype
to the host. In other words, endothelial cell
activation may be adaptive or non-adaptive.
In contrast, endothelial cell dysfunction
is by definition maladaptive. The term dysfunction was initially applied to the phenotype of the endothelium overlying atherosclerotic lesions in animal models of hyperlipidaemia. Today, the term is almost always used to describe abnormalities in endothelium-dependent vasomotor tone in
atheromatous conduit arteries. However,
because the endothelium is widely distributed, and because it is involved in so
many different functions (over and above
the control of arteriolar diameter), the term
dysfunction should be used more broadly
to describe situations in which the endothelial phenotype – whether or not it
meets some predetermined definition of
activation – represents a net liability to the
host.
Diagnosis and therapy
Fig. 1 The liver synthesizes a relatively constant supply of clotting factors (serine proteases II through
XII) and fibrinogen. In addition, hepatocytes synthesize several natural anticoagulants including
protein C, protein S and antithrombin IIII (AT). The bone marrow produces and releases a relatively constant number of monocytes and platelets. Monocytes are capable of expressing tissue factor (TF),
whereas activated platelets provide a cell surface for assembly of clotting reactions. Endothelial cells
(ECs) also express procoagulants and anticoagulants. However, the repertoire of EC-derived haemostatic factors varies between vascular beds (shown are reported differences between arteries, veins and
capillaries, though differences also exist between different types of arteries, veins and capillaries). The
systemic mix of liver-derived soluble mediators and bone marrow-derived blood cells is integrated into
the unique haemostatic balance of each vascular bed. It follows that changes in the systemic balance
(as occurs for example in patients with congenital deficiency of protein C, protein S or AT, or with factor
V Leiden) will have different local effects, giving rise to site-specific thrombotic phenotypes. Recent
evidence suggests that ECs in regions of disturbed flow in arteries are primed for activation (they have
increased levels of NF-κB in their cytoplasm) and that systemic imbalances (e. g. associated with sepsis
or cardiac risk factors) may result in the translocation of NF-kB to the nucleus and increased expression
of procoagulants such as tissue factor (TF) and adhesion molecules (adapted with permission from Aird
WC. Circ Res 2007; 100: 158–173). TM: thrombomodulin; t-PA: tissue-type plasminogen activator;
EPCR: endothelial protein C receptor; TFPI: tissue factor pathway inhibitor; VWF: von Willebrand factor
The endothelium has remarkable, yet
largely untapped diagnostic and therapeutic potential. From a diagnostic standpoint, the endothelium is hidden from
view and difficult to access. Owing to its
thinness and disseminated nature, the endothelium is not amenable to conventional
radiological imaging. The most commonly
used assays for the endothelium are flow
studies, which measure endothelium-dependent vasodilation; reviewed in (14). Abnormalities in endothelium-dependent
flow have been correlated with atherosclerosis and risk for acute coronary syndrome. However, these studies are highly
operator dependent, and they measure
only a single function of the endothelium.
In recent years, progress has been made in
developing novel diagnostic tools for assaying endothelial function, including
• ELISA-based quantitation of circulating
biomarkers, e. g., endothelin-1, VWF,
tissue-type plasminogen activator
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W. C. Aird: Endothelium and haemostasis
•
•
(t-PA), soluble thrombomodulin, soluble vascular cell adhesion molecule 1
(VCAM-1), soluble intercellular adhesion molecule 1 (ICAM-1), and soluble E-selectin,
enumeration and phenotyping of endothelial-derived microparticles and
circulating endothelial cells, and
molecular imaging; e. g. (15).
However, these various assays remain investigational and have yet to reach the
clinical mainstream.
The endothelium is a highly attractive
therapeutic target: It is rapidly and preferentially exposed to systemically delivered
agents.
Given the capacity of endothelial cells to
sense and respond to the local environment, it is hard to imagine that there exists
any treatment for any disease that does not
affect endothelial cell phenotypes in one
way or another. From a therapeutic standpoint, the combination of phenotypic heterogeneity and modulability offers both
opportunities and challenges.
• On one hand, the identification and
characterization of vascular bed-specific
“zip codes” should provide a foundation
for site-specific targeting.
• On the other hand, drugs that lack such
specificity are likely to exert mixed effects on the vasculature with
– protective effects in some vascular
beds and
– neutral or deleterious consequences
in others.
Endothelium in haemostasis
The vast majority of thrombotic disorders
in humans and genetically modified mice
are associated with focal clots in characteristic sites of the vasculature (16–18).
How does a systemic change in a circulating procoagulant or anticoagulant lead
to site-specific thrombosis?
The answer lies, at least in part, in the endothelium. Endothelial cells are minifactories for the production of anticoagulant
and procoagulant molecules. For example,
endothelial cells express on the
• anticoagulant side thrombomodulin,
endothelial protein C receptor (EPCR),
heparan, tissue-type plasminogen activator (t-PA), tissue factor pathway inhibitor (TFPI), endothelial nitric oxide
synthase (eNOS), and CD39,
• procoagulant side tissue factor (at least
in vitro), VWF, factor VIII, and plasminogen activator inhibitor.
In keeping with the theme of heterogeneity,
each of these molecules is differentially expressed across the vascular tree (19–21),
for example,
• TFPI is expressed predominantly in
capillary endothelium,
• EPCR in large veins and arteries,
• eNOS on the arterial side of the circulation,
• VWF in veins,
• t-PA in pulmonary and cerebral arteries,
• thrombomodulin in blood vessel types
of every caliber in all organs except the
brain.
In mice, factor VIII is preferentially expressed in endothelial cells of the liver and kidney,
compared with brain and heart (22). Thus,
the picture that emerges is one of heterogeneity layered upon heterogeneity, such that
endothelial cells from different sites of the
vascular tree employ site-specific “formulas”
of haemostatic proteins to maintain blood in
its fluid state and to promote limited clot
formation when there is a breech in the integrity of the vascular wall (▶Fig. 1). As a result, any change in the systemic balance of
haemostatic factors (as occurs for example
with hereditary hypercoagulable states, liver
disease, warfarin-induced skin necrosis, and
sepsis) is integrated into the local haemostatic balance in ways that differ between vascular beds. In other words, the normal spectrum of endothelial heterogeneity may explain why systemic hypercoagulable states
are channeled into focal thrombotic lesions.
On top of these baseline differences in
the expression of endothelial procoagulants
and anticoagulants, pathological states may
further alter local haemostatic balance. For
example, in a mouse model of endotoxaemia, VWF mRNA levels were decreased
in lung, aorta, brain, and adipose tissue, but
increased in the heart and kidney (19). Tissue factor is not detectable in normal endothelium. However, in a baboon model of
sepsis, it was shown to be up-regulated in a
subset of endothelial cells in the marginal
zone of splenic follicles, and in regions of
disturbed flow (23, 24). Moreover, in transgenic mice with moderate-severe sickle cell
disease, tissue factor expression was reported to be selectively up-regulated in
pulmonary vein endothelium (25). These
various disease-associated changes in protein expression are likely to further influence the propensity to develop site-specific thrombi.
Conclusion
The endothelium is a widely distributed
organ system that plays an important role
both in physiology and disease. The endothelium has many functions, over and
above its well-recognized role in mediating
vascular tone. One of these critical activities is maintenance of blood fluidity and
repair of injured blood vessels. The endothelium is remarkably heterogeneous in
structure and function. Indeed, endothelial
cells regulate haemostatic balance by sitespecific formulas of anticoagulants and
procoagulants. These different equations
are sufficient to explain why systemic imbalances in coagulation factors invariably
lead to local thrombotic phenotypes. The
propensity to develop clots in specific vascular beds is further accentuated by local
endothelial dysfunction. An important goal
for the future is to identify diagnostic
markers that reflect phenotypic changes at
the level of individual vascular beds, and to
develop therapies that target one or another site of the vasculature.
Conflict of interest
The author has no conflict of interest to
declare.
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