REVIEWS

Evolving functions of endothelial cells

in inflammation
Jordan S. Pober and William C. Sessa
Abstract | Inflammation is usually analysed from the perspective of tissue-infiltrating
leukocytes. Microvascular endothelial cells at a site of inflammation are both active
participants in and regulators of inflammatory processes. The properties of endothelial
cells change during the transition from acute to chronic inflammation and during the
transition from innate to adaptive immunity. Mediators that act on endothelial cells also
act on leukocytes and vice versa. Consequently, many anti-inflammatory therapies
influence the behaviour of endothelial cells and vascular therapeutics influence
inflammation. This Review describes the functions performed by endothelial cells at
each stage of the inflammatory process, emphasizing the principal mediators and
signalling pathways involved and the therapeutic implications.

Ectopic lymphoid structures
Organized lymphocytic
aggregates that form in sites of
chronic inflammation. Typically,
Tcell- and Bcell-rich zones are
segregated, and dendritic cells
(DCs), germinal centres with
follicular DC (FDC) networks
and specialized endothelia are
present. These structures are
also known as the tertiary
lymphoid organs and their
formation is termed lymphoid
neogenesis.
Interdepartmental Program
in Vascular Biology and
Therapeutics, Amistad
Research Building,
Yale University School of
Medicine, 10Amistad Street,
New Haven, Connecticut
06509, USA.
Correspondence to J.S.P.
e-mail:
jordan.pober@yale.edu
doi:10.1038/nri2171
Acute inflammation is a rapid response to infectious
microbes or injured tissues that involves local recruit
ment and activation of neutrophils. It serves to eradicate
the eliciting stimulus by killing microbes and removing
cellular debris. If successful, acute inflammation is
resolved, restoring normal tissue architecture or form
ing a connective tissue scar. If the stimulus is not elimi
nated then the inflammatory process will persist and
evolve. The composition of the infiltrating leukocytes
changes from neutrophils to a mixture of mononuclear
phagocytes and Tcells. Concomitantly, the inflammatory
stimulus changes from one sensed by patternrecognition
receptors of innate immune cells to one recognized as
an antigen by activating receptors on T and Bcells
of adaptive immunity. Antigen-activated Tcells can
enhance effector functions of mononuclear phagocytes
or recruit alternative effector cells, such as eosinophils.
The specialized effector cells of adaptive immunity often
succeed in eradicating a stimulus that resists clearance by
innate immunity, which allows for resolution and repair.
Prolonged antigenic stimulation by resistant microbes or
tissue-derived autoantigens leads to chronic inflamma
tion with formation of an inflammatory neo-tissue (such
as the pannus in a rheumatoid joint) and may assume the
form of ectopic lymphoid structures with organized T and
Bcell areas. Although the historical focus of inflamma
tion research has been on the identities and functions of
infiltrating leukocytes, vascular endothelial cells have a
major role in these processes, changing their phenotypes
to support various phases of the inflammatory process.
Here, we describe the role of endothelial cells during the
inflammatory response and the signalling pathways that
are involved and discuss how endothelial cells could be
therapeutic targets.
The endothelium at rest
In non-inflamed tissues, vascular endothelial cells main
tain blood fluidity, regulate blood flow, control vessel-
wall permeability and quiesce circulating leukocytes
(FIG.1). These basal functions of endothelial cells have
been recently reviewed in depth elsewhere16.
In brief, blood fluidity is actively maintained by several
endothelial-cell-dependent mechanisms that inhibit coagu
lation throughout the vascular system3. Among the most
important anti-coagulant mechanisms are the expression
of tissue factor pathway inhibitors (TFPIs) that block
the initiation of coagulation; the expression of heparan
sulphate proteoglycans that bind anti-thrombinIII and
inactivate thrombin; and the expression of thrombo
modulin, a membrane protein that alters the specificity
of thrombin from a pro-coagulant converter of fibrino
gen to fibrin to an anti-coagulant activator of protein C.
Activated protein C, in concert with proteinS, inactivates
several components of the clotting cascade. Activation
of coagulation requires a phospholipid surface, enriched
in phosphatidylserine, which is normally provided by
activated platelets. Resting endothelial cells mask or
degrade signals that activate platelets. Endothelial cells
produce nitric oxide (NO)7 (BOX1) and prostaglandin I2
(PGI2; also known as prostacyclin), which synergistically

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a All vessels Thrombomodulin Protein C

Activated ATP Platelets Blood
Thrombin protein C or ADP
Anti-thrombin III AMP NO
TFPI vWF
HS WPB
Endothelial cell NOS3

Collagen ATPase or ADPase

b Arteriole c Capillary d Venule

Tight junctions and
Endothelial cell adherens junctions
Lymphocyte

NOS3 NOS3 Caveolae Neutrophil

Smooth NO
muscle cell NO
NO
NNO
OSS
3
NO
Endothelial cell
WPB (P-selectin and vWF)
Figure 1 | Functions of resting endothelial cells. a | All endothelial cells inhibit coagulation of the blood. Endothelial
cells bind and display tissue factor pathway inhibitors (TFPIs) that prevent the initiation of coagulation by blocking the
actions of the factor-VIIatissue-factor complex. Endothelial cells synthesize and display heparan Nature Reviews
sulphate | Immunology
proteoglycans
(HS) on their cell surface, which have anti-coagulant properties that cause bound anti-thrombin III to be capable of
inhibiting any thrombin molecules generated by the coagulation cascade. Endothelial cells also synthesize and display the
protein thrombomodulin, which binds thrombin and converts its substrate specificity from cleavage of fibrinogen (the key
step in forming a blood clot) to cleavage and activation of protein C. Activated protein C is an enzyme that destroys
certain clotting factors and inhibits coagulation. Key processes to prevent platelet activation (and therefore coagulation)
include inactivation of thrombin, conversion of ATP to inert AMP through the action of ATPases and ADPases, and blocking
the physical interaction between platelets and collagen, which can activate platelets. Endothelial cells also sequester von
Willebrand factor (vWF), a protein that strengthens the interaction of platelets with the basement membrane, by keeping
it within their storage granules, known as WeibelPalade bodies (WPB). Nitric oxide (NO), generated by nitric-oxide
synthase 3 (NOS3)-mediated conversion of arginine, further inhibits platelet activation. b | Arterial endothelial cells have a
major role in regulating blood flow by controlling the tone of smooth muscle cells in the medial layer of the vessel wall.
Although a variety of mediators are involved, the principal regulator is the vasodilator NO produced by NOS3 in
endothelial cells. c | Capillary endothelial cells are the principal regulators of transendothelial extravasation of plasma
proteins. Although the extent of this barrier varies among different tissues, most continuous capillary endothelial cells
prevent proteins from escaping from the blood by forming intercellular junctions containing elements of both tight
junctions and adherens junctions, closing off the paracellular pathway between cells. Caveolae may initiate and control
the passage of plasma proteins across the capillary layer via vesicular transport. d | Venular endothelial cells form the
principal site of leukocyte trafficking from the blood into the tissues. Efficient recruitment of leukocytes requires that the
endothelial cells are activated; resting endothelial cells actively inhibit this process by failing to express adhesion
molecules that mediate leukocyte attachment. Pselectin, which is expressed basally in human endothelial cells, is
sequestered into WPBs along with vWF; Eselectin, vascular cell-adhesion molecule 1 and intercellular adhesion
molecule1 are minimally synthesized; and NO may contribute to suppressing the synthesis of these molecules,
but also has direct effects on leukocytes, preventing their activation to motile forms capable of entering the tissues.

inhibit platelet adhesion and aggregation. Blood flow is
regulated by a balance of signals that increase or decrease
the tone of the surrounding layers of vascular smooth
muscle cells1. The specific blood-flow mediators released
by endothelial cells vary depending on the vascular bed
(and also depending on the species), but the principal
vasorelaxant is NO2.
Plasma proteins are prevented from moving from
blood to tissues by the endothelial cell lining of the
capillaries the vascular segment with the largest
surface area4. The structure of capillary endothelial cells
varies with their anatomical location8. In continuous
capillaries, the junctions between adjacent endothelial
cells contain spatially intermingled tight junctions and
adherens junctions that are composed of the same fami
lies of proteins that form such junctional structures in
epithelial cells5. These inter-endothelial cell junctional
complexes are most extensive in the capillaries of the
central nervous system. The capillaries of the liver and
spleen, known as sinusoidal capillaries, are lined with
endothelial cells, the intercellular junctions of which are
often disrupted or discontinuous, permitting the blood
to contact underlying cells. Some capillary endothelial
cells form transendothelial pores (known as fenestrae)
that allow efflux of fluid and proteins through the cell
body in addition to passage between cells. Fenestrated

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Box 1 | Activation and functions of NOS3-derived NO
Nitric-oxide synthase 3 (NOS3) is the major isoform of NOS in endothelial cells and
is responsible for endothelium-dependent relaxation of large blood vessels as
described by Furchgott etal. in 1980 (Ref.99). NOS3 is a constitutively expressed
gene that can be upregulated by increases in fluid shear stress or by exercise training.
NOS3 activity is tightly regulated to yield nanomolar bursts of nitric oxide (NO) in
response to acute changes in shear stress, Gprotein-coupled receptor (GPCR)
agonists and growth factors. NOS3 is a peripheral membrane protein that localizes
primarily in the Golgi complex and in plasma membrane caveolae of endothelial cells.
While in caveolae, NOS3 is negatively regulated by caveolin1, the coat protein of
endothelial caveolae. Following activation of the enzyme by shear stress or growth
factors (such as vascular endothelial growth factor (VEGF)), caveolin1 dissociates
from the NOS complex and this is followed by the recruitment of additional positive
regulators of NOS function such as Ca2+, calmodulin, heat-shock protein (HSP90) and
dynamin2. In addition to being regulated by its subcellular localization and protein
protein interactions, NOS3 can be regulated by multi-site phosphorylation, most
notably by the protein kinases AKT and AMP kinase. Phosphorylation of NOS3 by
these kinases increases its catalytic efficiency to increase NO release. NOS3 produces
low levels of NO and is considered to be anti-inflammatory, as basal levels of NO can
reduce platelet and leukocyte adhesion and von Willebrand factor secretion. On the
other hand, in response to mediators released during an acute inflammatory response
(namely bradykinin, histamine and platelet-activating factor), NOS3-derived NO also
can contribute to acute inflammation by increasing blood flow, vascular permeability
and promoting angiogenesis.
(ICAM1). The basal production of NO may contribute to
the quiescence of resting endothelium by several mecha
nisms, including the inhibition of pro-inflammatory
gene expression by endothelial cells13 (although studies
of endothelial cells from nitric oxide synthase 3 (NOS3)-
deficient mice do not support this idea14), inhibition of
WPB fusion with the surface of the endothelial cell15 and
inhibition of leukocyte activation. The principal signal
for basal production of NO by resting endothelial cells
is shear stress produced by flowing blood.
Failure of endothelial cells to adequately perform
any of these basal functions constitutes endothelial cell
dysfunction. Most commonly, this term describes the
failure of arterial endothelial cells to produce sufficient
NO, leading to vessel constriction. Failure to control
coagulation, failure to control permeability, or failure to
quiesce leukocytes are also instances of endothelial cell
dysfunction. Local disturbances in blood flow reduce
endothelial cell NO production and may thus contribute
to vasoconstriction as well as to local leukocyte or platelet
activation. Diminished NO secretion explains why regions
of disturbed flow, such as arterial branch points, are par
ticularly prone to develop atheromas (BOX2), achronic
inflammatory lesion within the arterial wall16.

capillaries may be covered by a protein-rich diaphragm The endothelium in acute inflammation

Lipid rafts
Cholesterol and
glycosphingolipid-rich regions
of the plasma membrane that
provide ordered structure to
the lipid bilayer.
Vesicularvacuolar
organelles
A collection of caveolae-like
structures, typically located
near the inter-endothelial
junctions, that may participate
in transcytosis of plasma
proteins and fluid during
certain types of inflammation
or in tumour vasculature. VVOs
may be distinguished from true
caveolae because they are
found in caveolin1 gene
deficient mice, which otherwise
lack endothelial caveolae.
Angiogenesis
The process of the
development of new blood
vessels from existing
blood vessels. It is frequently
associated with tumour
development and
inflammation. In recent years,
it has been appreciated that
angiogenesis may be
supplemented by the local
recruitment of circulating
endothelial progenitor cells.
The formation of new blood
vessels from progenitors is
called vasculogenesis.
(as in the capillary endothelial cells of the renal glomeru
lus), which filters the transuding blood plasma, or may
be truly open (as in the endothelial cells of the liver
sinusoidal capillaries). Capillary endothelial cells in
most tissues form a reasonably tight continuous barrier
that excludes proteins as large as or larger than albumin
(including antibodies). Concomitantly, endothelial cells
mediate a controlled passage of certain proteins via
vesicular transport4. Endothelial cells are particularly
rich in caveolae, which are flask-shaped membrane
invaginations that may occupy up to 50% of the luminal
plasma membrane9. Caveolae may pinch off from the
luminal membrane, ferry macromolecules across the
cell, and discharge them at the albuminal side of the
endothelial cell monolayer. Caveolae are organized by a
scaffold formed by oligomers of the cholesterol-binding
proteins caveolin1 and caveolin2. Consequently, the
lipid composition of the caveolae is enriched in cho
lesterol and glycosphingolipids. Caveolar membranes
are, in essence, regionally organized aggregations
of lipidrafts , and various receptors and signalling
systems are concentrated within them. In addition to
true caveolae, venular endothelial cells may contain
complex caveolaelike structures known as vesicular
vacuolar organelles (VVOs), which have been implicated
in vascular leakage associated with allergic inflammation
and tumour angiogenesis10.
Resting endothelial cells do not interact with leuko
cytes6. This is because they sequester leukocyteinteractive
proteins, such as P-selectin and chemokines, within
specialized secretory vesicles known as WeibelPalade
bodies (WPBs)11,12. Resting endothelial cells also sup
press transcription of other adhesion molecules, such as
E-selectin, vascular cell-adhesion molecule 1 (VCAM1)
and, to a large extent, intercellular adhesion molecule 1
The process of acute inflammation involves the rapid
(over a period of hours) recruitment of neutrophils and
results from endothelial cell activation, defined as the
acquisition of new capacities by resting endothelial cells.
Endothelial cell activation may be divided into rapid
responses that are independent of new gene expression
(typeI activation; also called stimulation) and somewhat
slower responses that depend on new gene expression
(typeII activation)17. In both types of activation response,
there are three principal components that underlie the
four cardinal signs of inflammation: an increase in local
blood flow, accounting for the red colour (rubor) and
warmth (calor) of inflamed tissues; a localized leakage of
plasma-protein-rich fluid (known as an exudate) into the
tissue, accounting for the swelling (tumor) of inflamed
tissues; and a localized recruitment and activation of cir
culating leukocytes such that they are induced to enter
the infected or damaged tissues. Pain (dolor), the fourth
cardinal sign of inflammation, is caused by mediators
released by leukocytes on Ctype sensory nerve fibres.
Increased blood flow augments leukocyte delivery to the
site, increased leaking of plasma-protein-rich fluid cre
ates a provisional matrix to support leukocyte entry into
the tissue, and increased adhesion facilitates leukocyte
capture and extravasation.
TypeI activation of endothelial cells. TypeI activation
is typically mediated by ligands that bind to the extra
cellular domains of heterotrimeric Gprotein-coupled
receptor (GPCRs), such as histamine H1 receptors, that
signal through the intracellular G-protein q subunit17.
Signalling results from receptor-catalysed exchange of
GDP for GTP on the heterotrimeric G-protein q sub
unit of the receptor-associated heterotrimeric G protein.
The GDP to GTP exchange triggers the dissociation of

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Box 2 | Atheromas and chronic inflammation
The atheroma, also known as an atherosclerotic plaque, is a focal lesion of the arterial
wall characteristic of the disease process atherosclerosis. The lesion is principally
located within the intimal compartment of the vessel, formed beneath an intact
endothelial cell lining and superficial to the internal elastic lamina. The core of the
lesion is a collection of necrotic cell debris and degraded plasma lipoproteins; some of
this material is contained within macrophages, the lipid-filled endocytic vesicles of
which have led to the description of these cells as foam cells. There is often a fibrous
cap of collagen and smooth muscle cells located between the necrotic core and the
luminal endothelial cell lining. The edges of the atheroma, sometimes called the
shoulder region, contain numerous inflammatory cells, mostly T helper 1 (TH1) cells and
macrophages along with some natural killer T cells. Mononuclear-cell infiltrates may
also accumulate around the outside of the vessel in the adventitial compartment,
typically sparing the smooth muscle cell-rich medial compartment of the artery
located between the intima and the adventitia. Larger atheromas often contain a
microvascular network within the intima that arises from the angiogenesis of vessels
within the adventitia or outer portions of the media. The presence of mononuclear
inflammatory cells and angiogenic blood vessels have led to the interpretation that the
atheroma is a form of chronic inflammation. Endothelial cell dysfunction, especially
alterations in permeability and inadequate production of nitric oxide, predisposes to
the development of atheromas. Arteries affected by atheromas normally dilate (that is,
they outwardly remodel) to accommodate the presence of the atheroma, preserving
the size of the arterial lumen and blood flow. In some cases, outward remodelling is
inadequate, or the vessel may actually constrict, creating a situation of inadequate
blood flow and syndromes of chronic ischaemia (for example angina in the heart or
claudication in the legs). Acute activation of Tcells and macrophages, perhaps in
response to autoantigens generated by the oxidation of lipoproteins within the lesion,
may trigger macrophage-mediated degradation of the fibrous cap and erosion or
fissure of the endothelial cell lining. This form of chronic-active inflammation, which
brings blood in contact with activated macrophages that express tissue factor and
other pro-coagulants, can trigger thrombotic occlusion of the artery, producing acute
infarction of the organ fed by that artery (for example myocardial infarction of the
heart or stroke in the brain). Anti-inflammatory therapies with cyclooxygenase2
inhibitors may predispose to these thrombotic complications by reducing the
production of prostaglandin I2, which promotes vasodilation and inhibits platelets.
Anti-angiogenic therapy, for example with antibody to vascular endothelial growth
factor A, may limit the growth of atheromas but may also trigger acute ischaemic
changes, precipitating thrombotic complications.
the G-protein q subunit from the G-protein dimer
and activates isoforms of phospholipase C (PLC),
catalysing the release of inositol1,4,5-trisphosphate
(InsP3) from the membrane lipid phosphatidylinosi
tol4,5-bisphosphate (PtdIns(4,5)P2). InsP3 induces
transient intracellular elevations in cytosolic free Ca2+
by releasing Ca2+ from endoplasmic reticulum stores.
In endothelial cells, these changes may take the form of
oscillatory, transient Ca2+ levels, or may, at greater signal
strength, produce an elevated plateau of cytosolic Ca2+
levels18. Elevated cytosolic Ca2+ levels may be sustained
by entry of extracellular Ca2+ (Ref.19). Activated GPCRs
also facilitate the exchange of GDP for GTP on the
small G protein RHO (RAS homologue) by activating
a RHO guanine-nucleotide exchange factor (RHO-
GEF) through the subunits of the heterotrimeric
Gprotein20. RHO activation and elevation in cytosolic
free Ca 2+ combine to mediate the typeI activation
response (FIG.2). Specifically, increased blood flow results
from Ca2+-mediated activation of cellular phospholipase
A2 (cPLA2), an enzyme that cleaves membrane phosphati
dylcholine into arachadonic acid and lysophosphatidyl
choline. Free arachadonic acid is rapidly and sequentially
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converted by cyclooxygenase1 (COX1; also known as
prostaglandin H (PGH) synthase1) to PGH2 and then
by prostacyclin synthase to PGI2, which is a potent
vasodilator that relaxes smooth muscle vascular tone
in terminal arterioles21. Antagonizing PGI2-dependent
increases in blood flow may be a major mechanism by
which COX inhibitors reduce inflammation22. Cytosolic
Ca2+ ions also form a complex with the adaptor protein
calmodulin that activates nitric-oxide synthase 3 (NOS3)
to produce NO, which synergizes with PGI2.
Vascular leakiness of plasma proteins involves
interactions between the Ca 2+ and RHO pathways.
The transient rise in cytosolic Ca2+ and the formation
of a Ca2+calmodulin complex leads to the activa
tion of myosin-light-chain kinase (MLCK)23, which
phosphorylates myosin light chain (MLC). At the same
time, RHO-GTP activates a RHO-dependent kinase
that phosphorylates and thereby inhibits a phosphatase
that removes phosphate groups from MLC 23. The
combination of MLCK activation (mediated by the
Ca2+calmodulin complex) and MLC phosphatase inhi
bition (mediated by RHO-dependent kinase) increases
MLCphosphorylation (FIG.2). Phosphorylated MLC
initiates contraction of actin filaments that are attached
to tight junction and adherens junction proteins, and
this results in the opening of gaps between adjacent
endothelial cells. These responses are most evident in
post-capillary venules, where the relevant GPCRs are
maximally expressed24 and tight junction elements are
least abundantly expressed5. NO and PGI2 produced by
venular endothelial cells may enhance the leakiness of
venules25. Plasma proteins leak from the blood into the
tissues and assemble into a provisional matrix13 that sup
ports the attachment, survival and migration of invading
neutrophils26.
The rise in intracellular Ca2+ in endothelial cells
also has a major role in leukocyte recruitment. The
activation of MLC in venular endothelial cells, in addi
tion to causing plasma protein leakage, initiates the
exocytosis of WPBs, bringing Pselectin to the luminal
cell surface18,27. At the same time, lysophosphatidyl
choline, a by-product of arachadonic acid generation,
is rapidly acetylated, generating an endothelial-cell-
derived acyl form of platelet-activating factor (PAF)28.
The combined display of Pselectin and PAF on the
endothelial luminal plasma membrane provides a
spatially linked, dual signal (sometimes called a jux
tacrine signal) that causes the tethering of circulating
neutrophils (due to Pselectin) followed by integrin
activation and cell regulation (due to PAF), initiating
neutrophil extravasation29.
The majority of leukocytes appear to cross through
the vessel wall by passing between adjacent endothe
lial cells30. The endothelial cell membranes at these
junctions not only contain adherens and tight junc
tion proteins, but are also enriched in the expres
sion of platelet-endothelial cell adhesion molecule 1
(PECAM1; also known as CD31) and CD99, two pro
teins that participate in homophilic interactions with
neutrophils and monocytes in a manner that is essen
tial for transmigration31. It has also been observed that,
 REVIEWS

Ligand
GPCR
DAG PtdIns(4,5)P2

GDP GDP GDP

GTP GTP GTP RHO
Endothelial cell
RHOGEF
GTP
PLC GDP RHO
InsP3 RHO-dependent
kinase

ER (active) P
Ca2+ Nucleus MLC MLC
phosphatase phosphatase Opening
Ca

of tight and
2

(inactive)
+

adherens
junctions
Ca2+
cPLA2 P
Tight and/or
MLC MLC Actin adherens
(inactive) (active) junction
PC Arachadonic acid Arginine MLCK
WPB
COX1
PAF Fusion
acetylase
Prostacyclin Ca2+
synthase NOS3 Calmodulin
Lysophosphatidylcholine PAF

P-selectin
PGI2 NO

Figure 2 | TypeI activation of endothelial cells. In response to binding of a ligand to a heterotrimeric

Nature ReviewsGprotein-
| Immunology
coupled receptor (GPCR), the q subunit of the heterotrimeric G protein exchanges bound GDP for GTP and
subsequently activates phospholipase C (PLC). This enzyme cleaves phosphatidylinositol4,5-bisphosphate
(PtdIns(4,5)P2) in the plasma membrane, releasing inositol1,4,5-trisphosphate (InsP3), which in turn triggers the
release of Ca2+ ions from the endoplasmic reticulum (ER). Diacylglycerol (DAG) remains in the membrane as a by-
product of this reaction. The rise in cytosolic free Ca2+ activates the enzyme cellular phospholipase A2 (cPLA2), which
cleaves phosphatidylcholine (PC) in the plasma membrane to yield arachadonic acid and lysophosphatidylcholine.
Arachadonic acid is converted by cyclooxygenase1 (COX1; also known as prostaglandin H synthase 1) and
prostacyclin synthase into prostaglandin I2 (PGI2; also known as prostacyclin), a potent vasodilator than relaxes
vascular smooth muscle. Elevated levels of free Ca2+ may also bind to the cytosolic adaptor protein calmodulin.
The Ca2+calmodulin complex activates nitric-oxide synthase 3 (NOS3), producing nitric oxide (NO), which can
synergize with PGI2 to relax smooth muscle cells. Production of these vasodilators by arteriolar endothelial cells
increases blood flow (and leukocyte delivery) to the tissues. At the same time, the Ca2+calmodulin complex also
activates the enzyme myosin-light-chain kinase (MLCK), which phosphorylates myosin light chain (MLC). Normally,
this reaction is rapidly terminated by an MLC phosphatase, but this enzyme can be inactivated by a pathway
involving the subunits of the heterotrimeric G protein. Specifically, free G-protein subunits activate RAS
homology (RHO)GEF (guanine nucleotide exchange factor), which converts RHO from its inactive GDP-bound
state to its active GTP-bound state. RHOGTP activates a RHO-dependent kinase that phosphorylates and
inactivates the MLC phosphatase. Phosphorylated MLC contracts actin filaments, which attach to the proteins
forming the tight junction and adherens junction complexes. This opens the junctions, especially in venular
endothelial cells, promoting vascular leakage of plasma proteins. Phosphorylated MLC also initiates the exocytosis
of WeibelPalade bodies (WPB), bringing Pselectin to the cell surface. Lysophosphatidylcholine, produced as a
byproduct of the reaction that liberated arachadonic acid from PC, may be rapidly acetylated by platelet-activating
factor (PAF) acetylase, generating an acyl form of PAF that may reside in the plasma membrane and deliver activating
signals to Pselectin-band leukocytes initiating diapedesis.

on occasion, leukocytes may instead cross through the
endothelial cell body32; a recent analysis of this proc
ess invitro suggests that the same junctional proteins
that are involved in passage between endothelial
cells may be recruited to ring-like apertures that
form in response to probing by cellular projections
(podosomes) that extend from activated leukocytes33.
The relative importance of the inter-endothelial cell
compared with the transendothelial cell pathway of
extravasation is unknown.
TypeII activation of endothelial cells. Signals by hetero
trimeric GPCRs last for 1020 minutes, after which the
receptors become desensitized, preventing restimula
tion34. This transience of signalling limits the degree of
inflammation and neutrophil extravasation that may
be caused by typeI activation alone. A more sustained
inflammatory response requires a more persistent form
of endothelial cell activation that is provided by typeII
activation. The prototypic mediators of this response
are tumour-necrosis factor (TNF; also known as TNF)

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REVIEWS

IL-1 TNF and interleukin1 (IL1) derived principally from acti

vated leukocytes17. The signalling pathways used by
IL-1R1 TNF in endothelial cells have been recently reviewed35.
TNFR1
Endothelial In brief, TNF binds to the extracellular domains of TNF
cell receptor1 (TNFR1; also known as CD120a) and recruits
TNFR-associated via death domain protein (TRADD) to
TIRAP

the intracellular death domain of the receptor. TRADD

MyD88

in turn recruits the serine/theonine kinase receptor

TRADD
interacting protein 1 (RIP1) and TNFR-associated fac
IRAK4
IRAK1

RIP1 tor2 (TRAF2), an E3 ubiquitin ligase. This complex,

TRAF2
TRAF6
Tight and/or
adherens junction
IB
p50 p65
AP1 AP1 Actin
NF-B
E-selectin
ICAM1
Arachadonic acid
VCAM1
Chemokines
COX2 COX2
Nucleus
Heparan E-selectin ICAM1 VCAM1
sulphate
proteo-
glycan PGI2
Chemokine
Figure 3 | TypeII activation of endothelial cells. In response to binding of
inflammatory cytokines, such as interleukin1 (IL1) or tumour-necrosis factor (TNF)
to relevant receptors type1 IL1 receptor (IL1R1) and TNF receptor 1 (TNFR1),
respectively signalling complexes are formed within the cell. In the case of IL1,
this complex is initiated by binding of myeloid differentiation primary-response
gene88 (MyD88) and Toll/IL1 receptor accessory protein (TIRAP; also known as
MAL) to the cytosolic regions of the activated receptor. MyD88 rapidly dissociates
from the receptor and interacts with IL1R-associated kinase 1 (IRAK1) and IRAK4,
and with TNFR-associated factor 6 (TRAF6). In the case of TNF, stimulated TNFR1
initially recruits TNFR-associated via death domain protein (TRADD), which, in turn,
binds receptor-interacting protein 1 (RIP1) and TRAF2; the NatureTRADDRIP1TRAF2
Reviews | Immunology
complex then dissociates from internalized TNFR1. Both complexes that are formed
as a result of IL-1 or TNF binding to their respective receptors appear to activate the
same sets of mitogen-activated kinase kinase kinases (MAPKKKs) that are involved in
the initiation of signals that lead to the activation of the transcription factor nuclear
factor-B (NF-B) and activating protein 1 (AP1). These factors initiate the
transcription of specific genes within the nucleus leading to expression of pro-
inflammatory proteins. Among these are adhesion molecules that bind leukocytes,
such as Eselectin, intercellular adhesion molecule 1 (ICAM1) and vascular cell-
adhesion molecule 1 (VCAM1); chemokines; enzymes, such as cyclooxygenase2
(COX2); and unknown effector proteins that reorganize actin filaments. COX2
increases the efficiency of COX1 of converting arachadonic acid to prostaglandin I2
(PGI2) in arteriolar endothelial cells, but this response still requires a type-I-activation
response to liberate arachadonic acid from membrane phosphatidylcholine. Actin
filament reorganization in venular endothelial cells can open intercellular junctions,
promoting vascular leakage of plasma proteins. Chemokines synthesized by
endothelial cells or other cell types may be displayed on the venular endothelial cell
surface, bound to heparan sulphate proteoglycans, where they can act on leukocytes
interacting with cytokine-induced adhesion molecules, promoting leukocyte entry
into the tissue. IB, inhibitor of NF-B.
sometimes called a signalosome, initiates various kinase
cascades that lead to activation of the transcription factors
nuclear factor-B (NF-B) and activator protein1 (AP1)
(FIG.3). The binding of IL1 to the type1 IL1 receptor
(IL-1R1) induces a signalling pathway mediated by a
signalling complex comprised of myeloid differen
tiation primaryresponse gene 88 (MyD88), Toll/IL1
receptor accessory protein (TIRAP; also known as
MAL), IL1R-associated kinase 1 (IRAK1), IRAK4, and
TRAF6, and activates the same transcription factors as
does TNF36. The proinflammatory responses induced
by these cytokines arise from new gene transcription
mediated by NFB and AP1. Because these responses
require transcription and translation of new proteins,
the responses of typeII activation require a longer time
to be initiated compared with those of typeI activation
(hours as opposed to minutes).
Similar to typeI activation, typeII activation leads
to increased blood flow, increased vascular leakage of
plasma proteins, and increased leukocyte recruitment at
the site of inflammation. Type-II-activated endothelial
cells display enhanced PGI2 synthesis due to the induc
tion of COX2, which, like COX1, can initiate prosta
glandin synthesis by converting arachadonic acid to
PGH2 but at a much higher level of throughput. Because
COX2 is a higher throughput enzyme than COX1 and
because it is induced at sites of inflammation, COX2-
selective inhibitors are effective as anti-inflammatory
agents despite their limited effects on COX1 (Ref.22).
The increase in blood flow produced by type-II-activated
endothelial cells still depends on a rise in cytosolic Ca2+
as a result of typeI activation in order to activate cPLA2
and release arachadonic acid37.
TNF and IL1 induce the leakage of plasma proteins
by stimulating venular endothelial cells to reorganize
their actin and tubulin cytoskeletons and thereby open
up gaps between adjacent cells38,39. Leakage of plasma pro
teins in response to TNF is triggered in an NFB- and
protein-synthesis-dependent manner, although the precise
protein or proteins responsible for actin reorganization
are not known40. The sustained leakage of plasma pro
teins leads to a particularly firm provisional matrix, often
described as an induration (hard swelling), rather than
the soft and transient oedema characteristic of the fluid-
rich leakage observed in typeI activation. The hardness
is due to the leakage of very large plasma proteins such as
fibrinogen, which is converted into a fibrin-rich clot. This
response is the basis of a positive tuberculin skin test.
Leukocyte recruitment is much more effective follow
ing typeII activation than typeI activation. Neutrophil
recruitment is triggered by the synthesis and display

808 | october 2007 | volume 7 www.nature.com/reviews/immunol

2007 Nature Publishing Group

E3 ubiquitin ligase
An enzyme that is required to
attach the molecular tag
ubiquitin to proteins.
Depending on the position and
number of ubiquitin molecules
that are attached, the ubiquitin
tag can target proteins for
degradation in the
proteasomal complex, create a
scaffold for assembly of
signalling complexes, sort them
to specific subcellular
compartments or modify their
biological activity.
Diapedesis
The last step in the leukocyte
endothelial cell adhesion
cascade. This cascade includes
tethering, triggering, tight
adhesion and transmigration.
Diapedesis is the migration of
leukocytes across the
endothelium, which generally
occurs by squeezing through
the junctions between adjacent
endothelial cells, although in
some settings, leukocytes have
been observed to pass through
transiently formed gaps in the
cytoplasm of endothelial cells.
nature reviews | immunology volume 7 | october 2007 | 809
of chemokines, such as CXC-chemokine ligand 8
(CXCL8; also known as IL8) and new leukocyte adhe
sion molecules, such as Eselectin6. Endothelial cells
can also capture chemokines made by other cells and
display them on their luminal surface bound to heparan
sulphate proteoglycans. Eselectin (which is induced
by inflammatory cytokines) and Pselectin (which
is mobilized by binding of ligands to heterotrimeric
GPCRs) are functionally similar. In mice, but not in
humans, Pselectin synthesis is also increased as a
result of TNFR1 or IL1R1 signalling (Ref.41). CXCL8,
similar to PAF, triggers firm attachment of neutrophils
to endothelial cells and induces diapedesis into the tissue.
It can only act efficiently on neutrophils that are tethered
or rolling on the endothelial cell surface; that is, CXCL8
and Eselectin, similar to PAF and Pselectin, provide a
juxtacrine signal.
TypeII activation may be modified by the actions
of typeI activators. As mentioned earlier, typeI signals
allow type-II-activated endothelial cells to maximally
synthesize PGI2 by providing a Ca2+ signal to activate
cPLA2. TypeI agonists, such as histamine, also trigger
the activation of TNF-converting enzyme, a metallo
proteinase that can remove TNFR1 from the cell surface.
The shed receptor is soon replaced by spare receptor
molecules stored in the Golgi, restoring the response.
However, TNF sensitivity is reduced for about 30 min
utes until surface receptor levels are replenished42. This
transient desensitization may account for the temporal
lag of TNF-mediated late-phase reactions that follow his
tamine-mediated immediate hypersensitivity responses
in allergic inflammation.
Evolution of typeII endothelial cell activation. Once
initiated, the TNF and/or IL1 responses are not only
more sustained than that of heterotrimeric GPCR sig
nalling, but they are programmed to evolve over time.
Eselectin synthesis is spontaneously shut off, despite
the continued presence of the activating cytokine; this
response correlates with the inactivation of AP1 (Ref.43).
With somewhat slower kinetics than those for Eselectin
expression, cytokine-activated venular endothelial
cells increase their expression of VCAM1 and ICAM1
(Ref.44). In combination with the synthesis of other
chemokines, such as CCchemokine ligand 2 (CCL2),
these changes in the expression of adhesion molecules
favours a transition from neutrophil-rich infiltrates
to mononuclear-cell-rich infiltrates, which typically
occurs between 6 and 24 hours after cytokine-mediated
activation44.
By 24 hours, leukocyte-mediated endothelial cell
injury may also contribute to inflammation, especially
in the capillaries45. TNF and IL1, when combined
with other mediators such as interferon (IFN), may
also exacerbate endothelial cell injury by triggering
endothelial cell death46,47. IFN may facilitate TNF-
induced endothelial cell death by at least two potential
mechanisms of action. First, IFN increases the level of
pro-caspase8 expressed in endothelial cells48. Activation
of TRADD by TNF will, after a delay of several hours,
recruit FAS-associated death domain protein (FADD),
2007 Nature Publishing Group
REVIEWS
creating a signalling complex that can activate pro-
caspase8 (Ref.49). This is normally prevented by the
presence of cellular caspase8 (FLICE)-like inhibitory
protein (cFLIP) which competes with pro-caspase8 for
binding to FADD50. The increased ratio of pro-caspase8
to cFLIP in IFN-treated cells allows caspase8 activa
tion to occur despite the presence of cFLIP. Second, IFN
mobilizes cathepsin B from lysosomes into the cytosol,
where it can be activated in response to TNF-induced
signals, thereby triggering a mitochondrial-dependent
cell-death pathway that is independent of caspase8
(Ref.50).
Endothelial cell injury and death also favour
thrombosis51. Apoptotic endothelial cells lose their
anti-coagulant functions, for example, by shedding
the anti-coagulant heparan sulphate proteoglycan, and
acquire pro-coagulant functions, such as exocytosis of
microparticles with exposed phosphatidylserine that may
support coagulation. In addition, exposed sub-endothelial
basement membrane collagen by retracted or desqua
mated endothelial cells support the adhesion and acti
vation of platelets. TNF may exacerbate endothelial cell
dysfunction by shutting off the synthesis of NOS3 (Ref.52)
and of thrombomodulin53 through posttranscriptional
and transcriptional mechanisms, respectively. TNF and
IL1 also induce the synthesis of tissue factor, the prin
cipal initiator of coagulation54. However, tissue factor
is normally sequestered within the endothelial cell and
is unable to start the coagulation cascade. In apoptotic
endothelial cells, tissue factor is de-encrypted by being
exposed to the blood as a component of microparticles
that are shed from the cell surface. The formation of
thrombus, which is an aggregation of intravascular fibrin
and platelets, is an important component of acute inflam
mation, serving to wall off an infected tissue to limit the
spread of microbes. At the same time, thrombosis can
further damage the vessel and induce haemorrhage that
often accompanies local inflammation. When ischaemic
injury and infarction due to thrombosis are superim
posed on tissue damage caused by the initial process of
inflammation, the repair process is more likely to result
in scarring rather than full recovery.
Whereas typeI activation spontaneously resolves
due to receptor desensitization, typeII activation in its
evolved forms can persist so long as activating cytokines
are present. One cause of resolution of typeII activation
may be the elimination of cytokine production due to
the removal of the inflammatory stimulus, for example,
by eradication of an infection. In addition, the expression
of endothelial cell proteins associated with typeII activa
tion may be actively suppressed by a variety of feedback
mechanisms that shut off inflammatory gene expression,
for example by terminating NFB activation55.
The endothelium in chronic inflammation
Endothelial cells in adaptive immune responses. If acute
inflammatory reactions fail to eradicate the triggering
stimulus, and especially if an adaptive immune response
is activated by persistent stimulation, the inflammatory
process will evolve to a more chronic form, in which
specialized effector cells are involved. Endothelial cells
REVIEWS
might participate in this process by presenting antigens
to circulating effector and memory Tcells (reviewed
in Ref.56), although this hypothesis is controversial.
Antigen presentation by human endothelial cells lining
microvessels was first suggested by the insitu expres
sion of high levels of both MHC class I and class II
molecules the only known function of which is to
present antigen to Tcells by these cells57,58. Human
endothelial cells also express co-stimulators such as
ligands for CD2, ICOS (inducible Tcell co-stimulator),
41BB and OX40 that are involved in the formation
and activation of Tcell memory59. Consistent with their
invivo phenotype, cultured human endothelial cells,
when reactivated by IFN to express MHC molecules,
effectively activate both cytokine production and prolif
eration by CD4+ and CD8+ memory Tcells, but not by
naive T cells59. (The failure of human endothelial cells to
activate naive Tcells may arise from a failure to express
co-stimulators that can engage CD28.) This is an impor
tant difference compared with mouse endothelial cells,
which typically do not express MHC class II molecules
insitu and which fail to activate CD4+ Tcells in culture,
even when induced to express MHC class II molecules60.
The putative function of antigen presentation by human
endothelial cells is unknown, but two possible explana
tions are: that the periodic low-affinity engagement of
a Tcell receptor (TCR) on a circulating memory Tcell
by an MHC molecule bearing a self-peptide displayed
on an endothelial cell serves to keep memory T cells
alive, or that the high-affinity engagement of a TCR
on a circulating effector memory Tcell by an MHC
molecule bearing a foreign, microbe-derived peptide
on an endothelial cell signals the reappearance of a
pathogenic microbe within the surrounding tissue and
thereby initiates a localized recall response. Consistent
with the second idea, we have recently observed that
antigen presentation by cultured human microvascular
endothelial cells can stimulate transendothelial migra
tion of effector memory CD4+ Tcells (T. D. Manes and
J. S. P., unpublished observations).
Other changes within the endothelial cells of a
chronically inflamed tissue may have roles in the polari
zation of inflammatory responses associated with adap
tive immunity. For example, in a T helper 1 (TH1)cell
dominant response, which favours the recruitment of
IFN-producing Tcells, endothelial cells will synthesize
and express the chemokine CXCL10 (Ref.61), which
binds to the CXC-chemokine receptor 3 (CXCR3)
expressed by effector and effector-memory Tcells, and
sustain the expression of certain adhesion molecules,
such as Eselectin46, which favours the recruitment
of TH1 cells62. These changes in endothelial cells are
induced by IFN, creating a positive feedback loop that
sustains TH1-cell responses. By contrast, if the inflamma
tory response is dominated by TH2 cells, characterized
by CD4+ Tcellsthat secrete IL4, IL5 and/or IL13, through tyrosine kinase receptor 2 (TIE2). As an aside,
endothelial cells respond to IL4 or IL13 by synthesizing
chemokines, such as CCL26 (also known as eotaxin3),
and expressing adhesion molecules, such as VCAM1,
that favour the local recruitment of TH2 cells, as well
as eosinophils63. Although IL4 and IL13 can enhance
810 | october 2007 | volume 7 www.nature.com/reviews/immunol
2007 Nature Publishing Group
VCAM1 expression alone, they are most effective when
synergizing with TNF64. It is as yet unknown whether
endothelial cells at the site of inflammation can display
a phenotype that favours the recruitment of effector
TH17cells or of regulatory Tcells.
The process of selective recruitment of different
memory Tcell subsets by inflammatory signals may
overlap with but is distinct from the process of tis
sue-specific memory Tcell recruitment. For example,
Tcells that express ligands for Eselectin (also known as
cutaneous lymphocyte-associated antigen1) may pref
erentially home to the skin because endothelial cells of
skin microvessels may display more sustained expression
of Eselectin than do endothelial cells in other tissues65.
Most regulatory Tcells in the peripheral circulation of
humans also display Eselectin ligands and thus also may
home selectively to the skin66; however, this property
does not distinguish skin-homing regulatory Tcells
from effector Tcells.
Angiogenesis. If the specialized effector cells of the
adaptive immune system fail to eradicate the antigen,
then two additional changes associated with chronic
inflammation may take place: angiogenesis and tertiary
lymphoid organogenesis. Angiogenesis is initiated by
the migration of endothelial cells lining the venules into
the tissue, and ultimately results in a new plexus of capil
laries67. The generation of new blood vessels is necessary
for the sustained survival of inflammatory cells within
the tissue and supports the conversion by mesenchymal
cells of the initial provisional matrix into a more long-
lasting connective tissue stroma28. Such inflammatory
neo-tissues may become prominent features of certain
chronic disease states, such as the pannus of rheuma
toid arthritis68 or the complicated atheromaof advanced
atherosclerosis69. The principal sources ofangiogenic
factors in the adaptive immune response are likely to
be activated Tcells and mononuclear phagocytes70.
Inother words, the failure of the inflammatory response
to resolve inflammation leads to the production of fac
tors that increase the blood flow to the inflamed tissue,
which sustains the viability of the inflammatory cells
that produce these factors. Inhibition of factors that pro
mote angiogenesis may reduce inflammatory neotissue
growth and prevent disease progression.
The precise mediators of angiogenesis released by
Tcells or macrophages in the setting of chronic inflamma
tion are unknown and may vary with species, anatomical
site and stimulus. The best described angiogenic factors
are cytokines that signal through receptor tyrosine kinases,
such as vascular endothelial growth factor A (VEGFA),
which signals through VEGF receptor1 (VEGFR1) and
VEGFR2, (basic) fibroblast growth factor2 (FGF2),
which signals through FGF receptor 1 (FGFR1), and
angiopoietin1 and angiopoietin2, which signal
angiopoietin-2 is a partial agonist of the TIE2 receptor
and may act to block the full agonist actions of angio
poietin1. These receptors dimerize upon ligand bind
ing, enabling their intracellular tyrosine kinase domains
to phosphorylate tyrosine residues on their homodimeric
 REVIEWS

a b
Growth factor
LT LTR
Plasma
membrane RTK Plasma membrane
PtdIns(4,5)P2 PtdIns(3,4,5)P3 PDK AKT P
P Y Y P PI3K
P Y Y P Canonical Non-canonical
RAS PLC pathway pathway
GEF NIK
MEKK3 IKK
GDP GTP
RAS RAS InsP3 TAK1 P IKK IKK
Ca2+ Calmodulin
TORC1
Calcineurin
IKK P
RAF Ca2+ Ca2+ REL-B
IKK IKK P
ER
p105
NFAT
P P I B P
ERK p51 REL-B
p50 p65 p50 p65
P
NF-B

Nucleus Nucleus
Cell cycle Increased
proteins translation E-selectin HEC-GlcNAc6ST

Figure 4 | | Chronic inflammatory responses of endothelial cells. a | Angiogenesis. AlthoughNature the process
Reviewsof| Immunology
angiogenesis is complex, these responses are typically initiated by growth factors, such as vascular endothelial growth
factor A (VEGFA), binding to receptor tyrosine kinases (RTKs), such as VEGFR2. Ligand-occupied RTKs multimerize and
cross-phosphorylate tyrosine residues on the other receptors in the complex. These various phosphotyrosine residues
recruit proteins that form a RASGEF (guanine nucleotide exchange factor) complex that catalyses the conversion of
inactive RASGDP to active RASGTP. RASGTP activates RAF, which in turn activates MEK1 (mitogen-activated protein
kinase (MAPK)/ extracellular-signal-regulated kinase (ERK) kinase 1), a kinase that activates ERK1 and ERK2 (not shown).
Activated ERKs enter the nucleus and phosphorylate and activate certain transcription factors. In parallel, activated
RTKs can also recruit, phosphorylate and activate phospholipase C (PLC). PLC cleaves phosphatidylinositol-4,5-
bisphosphate (PtdIns(4,5)P2) to release inositol1,4,5-trisphosphate (InsP3), which in turn signals the release calcium ions
(Ca2+) from intracellular storage compartments such as the endoplasmic reticulum (ER). Cytosolic Ca2+ binds to
calmodulin, which can then activate calcineurin. Calcineurin removes phosphate moieties from the nuclear localization
signal of nuclear factor of activated Tcells (NFAT), thereby allowing its entry into the nucleus. Both ERK and NFAT
contribute to the transcription of genes encoding proteins that are involved in cell cycle entry and progression. A third
signalling pathway involves the recruitment of the regulatory subunits of phosphoinositide 3kinase (PI3K) to RTK. PI3K
converts PtdIns(4,5)P2 to phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) within the plasma membrane, which
can then serve as a docking site for several enzyme complexes, including phosphatidylinositol-dependent kinases (PDKs),
that phosphorylate and activate AKT. Phosphorylated AKT can block several apoptotic pathways, thereby promoting cell
survival, and can also lead to the phosphorylation and activation of mammalian target of rapamycin complex 1 (TORC1),
which increases the capacity of the growing cell to synthesize new proteins. b | Conversion to high endothelial venules.
A second aspect of chronic activation is the conversion of flat venular endothelial cells to a phenotype that resembles
the high endothelial venules of lymph nodes. A key element of this change is the sulphation of certain macromolecules
so that they may serve as ligands for Lselectin expressed by naive lymphocytes and by central memory Tcells.
Lymphotoxin (LT) binds to the LT receptor (LTR) and seems to be a major signal for this conversion event. The key
enzyme in this pathway is under the control of the non-canonical nuclear factor-B (NF-B) pathway. In the canonical
NFB pathway, which is activated by tumour-necrosis factor (TNF) binding to TNFR1, certain MAPK kinase kinases
(MAPKKKs), such as MEKK3 and transforming-growth-factor--activated kinase 1 (TAK1), activate inhibitor of NK-B
(IB) kinase (IKK). In conjunction with IKK, activated IKK phosphorylates IB proteins, such as IB, IB or IB,
that normally bind to and sequester NF-B transcription factors, typically composed of p65 (also known as REL-A) and
p50 (also known as NFB1) subunits. Phosphorylated IB proteins are rapidly ubiquitylated and degraded by the
proteasome, releasing the NFB factors to enter the nucleus and initiate new gene transcription. This may result in the
synthesis of proteins involved in typeII activation, such as Eselectin. In the non-canonical NFB pathway, the LT signal
additionally activates a different MAPKKK NFB inducing kinase (NIK) and, in turn, NIK activates IKK. This enzyme acts
on a different form of NFB, typically composed of p105 and REL-B subunits. When p105 is phosphorylated by IKK, it is
proteolytically converted to p51 (also known as NFB2), and the p51REL-B complex enters the nucleus and activates
certain genes not affected by p50p65 dimers of NFB, including a high-endothelial-cell-associated factor
Nacetylglucosamine 6-sulphotransferase (HEC-GIcNAc6ST) involved in synthesis of Lselectin ligands.

nature reviews | immunology volume 7 | october 2007 | 811

2007 Nature Publishing Group
REVIEWS
Epitope spreading
The denovo activation of
autoreactive Tcells by self-
antigens that have been
released after T or Bcell-
mediated bystander damage.
812 | october 2007 | volume 7 www.nature.com/reviews/immunol
partner, aprocess known as crossphosphorylation. The
various phosphotyrosine residues serve as docking sites
for specific adaptor proteins, which recognize phospho
tyrosine in the context of a specific short target sequence
through SRC homology 2 (SH2) domains. Three signal
ling pathways are typically activated in endothelial cells
for effective angiogenesis: the RASextracellularsignal
regulated kinase 1 (ERK1)/ERK2 pathway, the phos
phoinositide 3kinase (PI3K)AKT pathway and the
Ca2+PLC pathway71 (FIG.4a). The effects of the Ca2+
pathway activated by growth factors, such as VEGFA,
overlaps with those activated through heterotrimeric
GPCRs by typeI activation, which explains why VEGFA
is an inducer of vascular leakage72, but it also has unique
effects, such as the activation of nuclear factor of activated
Tcells (NFAT), a transcription factor that has a role in
endothelial cell proliferation73. These three signalling
pathways interact to promote cell growth, survival and
migration; three crucial processes in the angiogenic
response. Ligand binding to VEGFR2 or FGFR1 acti
vates all three signalling pathways. By contrast, angi
opoietin1 binding to TIE2 selectively activates the
PI3K pathway. Angiopoietin1 normally stabilizes new
vessels formed by angiogenesis whereas angiopoietin2
destabilizes existing vessels, participating in the initia
tion of angiogenesis74. Certain other types of mediators
can also contribute to angiogenesis. For example, TNFcan
act as a pro-angiogenic cytokine by binding to TNF
receptor 2 (TNFR2) and signalling through cytosolic
epithelial/endothelial tyrosine kinase (ETK; also known
as bone-marrow Xlinked kinase or BMX)75. Activated
ETK phosphorylates a subset of tyrosine residues in the
cytoplasmic domain of VEGFR2 selectively activating
the PI3KAKT pathway. COX2 also contributes to
angiogenesis through the production of prostaglandins
that act on endothelial cells through heterotrimeric
GPCRs76. As COX2 is induced in endothelial cells by
signals from TNFR1 or IL1R1, these receptors may also
contribute to angiogenesis. Certain chemokines, such
as CXCL8, also have angiogenic properties77. However,
some chemokines can inhibit angiogenesis. CXCL10,
for example, is anti-angiogenic, and may account for
some of the anti-angiogenic activities attributed to IFN
and IL12 (Ref.78).
Tertiary lymphoid organogenesis and endothelial cells.
Additional endothelial cell responses occur as chroni
cally inflamed tissues acquire the characteristics of a
secondary lymphoid organ, sometimes referred to as
tertiary lymphoid organs (TLOs)79. The formation of
a TLO involves the recruitment of lymphocytes not
normally associated with inflammatory infiltrates,
notably naive Tcells, central memory Tcells and
Bcells. These cells express Lselectin and CCR7, which
allows them to respond to chemokines such as CCL21
(also known as SLC) and CXCL19 (also known as
ELC). The venular endothelial cells in TLOs undergo
a conversion from flat venules to tall endothelial cells,
similar to those found in the high endothelial venules
(HEVs) of lymph nodes. Concomitantly with shape
change, endothelial cells express HEC-GIcNAc6ST
2007 Nature Publishing Group
(highendothelialcellassociated factor Nacetyl
glucosamine 6sulphotransferase), which can complete
the synthesis of Lselectin ligands. These responses are
driven by lymphotoxin (LT) and HEV formation
is defective in LT or LT-receptor (LTR)-deficient
mice. LTR is a member of the TNFR superfamily and,
like TNFR1, activates p50 (also known as NFB1)- and
p65 (also known as REL-A)-containing forms of NFB
(the canonical pathway) (FIG.4b). In addition, LTR
activates NFB-inducing kinase (NIK), a MAPKKK
that initiates the alternative (noncanonical) NFB
pathway (FIG.4b) by activating p51 (also known as
NFB2)- and REL-B-containing forms of NFB. This
form of NFB initiates transcription of a distinct subset
of genes, including HEC-GIcNAc6ST. The expression of
Lselectin ligands by the endothelial cells of HEVs per
mits the recruitment of naive Tcells, central memory
Tcells and Bcells not normally recruited by type-II-
activated venular endothelial cells. The newly recruited
leukocyte populations reorganize themselves into dis
tinct B and Tcell areas, each with characteristic acces
sory cells: follicular dendritic cells for the Bcell areas
and myeloid dendritic cells for the Tcell areas72. Other
TNF family members along with VEGFC contribute to
the formation of new lymphatic vessels, a process that
parallels blood-vessel angiogenesis at sites of chronic
inflammation80. This conversion of a chronic inflamma
tory infiltrate into an organized lymphoid tissue may
facilitate the process of epitope spreading associated
with autoimmunity. In organ-specific autoimmune
conditions, such as Hashimotos thyroiditis or Sjgrens
Syndrome, TLO formation may thus sustain the proc
ess of autoimmune organ damage. TLO formation is
inhibited in mice with defects in the alternative NFB
pathway or by blockade of LT signalling79.
The endothelium in chronic-active inflammation
The distinction between acute and chronic inflamma
tion is heuristically useful but may oversimplify real
disease processes in which both stages of inflamma
tion may be observed in the same tissues. This may
result from overlapping activities and interactions
of the mediators of these processes. For example,
TNF signals through TNFR1 to induce endothelial
cell responses that are characteristic of typeII acute
inflammation, but can also signal through TNFR2 and
ETK to (partly) activate VEGFR2, and thereby pro
mote angiogenesis37 a feature of chronic inflamma
tion. VEGFA, the prototypic mediator of angiogenesis,
also causes vascular leakage and promotes provisional
matrix assembly to support neutrophil entry into tis
sues. VEGFA also enhances acute inflammatory effects
mediated by TNF (for example, co-induction of adhe
sion molecules and tissue factor) 81 or by IFN (for
example by co-inducing CXCL10)82. These responses,
which are more characteristic of acute than chronic
inflammation, may involve the activation of PI3K 83.
In the lung, VEGFA appears to selectively promote
TH2-type inflammation84. Angiopoietin2, which sig
nals through TIE2, enhances the induction of adhesion
molecules by TNF especially when TNF is present at
 REVIEWS

Table 1 | Endothelium-targeted actions of selected agents that reduce inflammation

Therapeutic agents (examples) Molecules targeted Phases of endothelial activation affected Clinical indications
Non-steroidal anti-inflammatory COX1 and COX2 TypeI and II (reduces local blood flow) Pain, fever and inflammation
agents (ibuprofen, ASA, celecoxib*)
Anti-histamines H1-histamine receptors TypeI (reduces local blood flow and local Allergic rhinitis and others
(diphenhydramine, fexofenadine) vascular permeability)
TNF-specific antibodies or soluble TNF TypeII (all aspects) and chronic (reduces Arthritis, psoriasis and
TNF receptor (etanercept) inflammatory angiogenesis) inflammatory bowel disease
VEGF-specific antibodies or VEGF VEGFA TypeI and II (reduces vascular permeability Cancer and macular
receptor antagonists (bevacizumab||) and blood flow), and chronic (reduces degeneration (wet)
angiogenesis)
Statins (simvastatin, atorvastatin#) HMG-CoA reductase TypeII; decreases IFN induced MHC class II Hyperlipidaemia and
and protein prenylation expression and adhesion molecules atherosclerosis
IL-1RA (anakinra**) IL1 TypeII (all aspects) systemic-onset juvenile
idiopathic arthritis and
autoinflammatory disorders
Rapamycin (sirolimus) mTOR Angiogenesis Immunosuppression in
transplantation
*Celebrex; Pfizer. Allegra; Aventis. Enbrel; Amgen, Wyeth. || Avastin; Genentech. Zocor; Merck. # Lipitor; Pfizer. **Kineret; Amgen. Rapamune; Wyeth.
ASA, acetylsalicylic acid; COX, cyclooxygenase; HMG-CoA, 3hydroxy3-methylglutaryl coenzyme A; IFN, interferon-; IL1, interleukin-1; IL-1RA, IL-1 receptor
antagonist; mTOR, mammalian target of rapamycin; TNF, tumour-necrosis factor; VEGF, vascular endothelial growth factor.

low concentrations85 and promotes oxidant-induced
injury and inflammation86; by contrast, angiopoietin1,
which also signals through TIE2, reduces VEGFA-
induced vascular leakage87 as well as the expression
of tissue factor88 thereby negatively regulating pro-
inflammatory signalling. The precise mechanisms of
signal transduction in these responses are unknown.
In some cell types, pro-inflammatory signalling could
involve an AKTmediated augmentation of NFB acti
vation, but this has not been observed in endothelial
cells89. The role of chemokines (which were first identi
fied as mediators of leukocyte recruitment) as regulators
of angiogenesis is yet another example of this conver
gence80. The key point is that the overlapping features of
acute and chronic inflammation, sometimes described
as chronic-active inflammation, arise because many
of the mediators commonly associated with either the
responses of endothelial cells in acute inflammation (for
example TNF and adhesion-molecule or chemokine
induction) or chronic inflammation (for example VEGF
in angiogenesis) have effects in both processes.
The endothelium as a therapeutic target
Many agents have been developed to inhibit inflamma
tion, usually with leukocytes as the intended targets.
In this Review, we have described the central role of
endothelial cells in the inflammatory process and
described a number of mediators and signalling path
ways in this cell type that contribute to inflammation.
Many of these are shared with leukocytes. Consequently,
many anti-inflammatory drugs in common use target
endothelial cells as well as leukocytes. Similarly, some
agents introduced to treat vascular diseases have effects
on inflammatory processes. Additionally, the overlap
between mediators of acute and chronic inflammation
means that drugs introduced for one or the other may
have effects on both. Some examples of the effects of
drugs on endothelial cells and the consequences for
inflammation are listed in Table 1. A few additional
points are highlighted here. TNF is both a typeII
activator of endothelial cells (through TNFR1) and a
pro-angiogenic factor (principally through TNFR2).
Neutralizing TNF may be advantageous for chronic-
active disorders such as rheumatoid arthritis in which
angiogenesis contributes to inflammation 68, but may
explain why TNF can exacerbate congestive heart fail
ure, in which angiogenesis is part of the recovery and
repair process90,91. The adverse effects of COX2 inhibi
tors on heart disease may arise from the reduction in
PGI2 synthesis and the anti-platelet and vasodilatory
effects of this agent92.
Statins, an important class of lipid-lowering drugs,
may exert part of their beneficial actions independ
ent of lowering cholesterol by promoting low-level,
sustained NO production from NOS3 and thereby
reduce inflammation93. In addition, statins are being
tested in the settings of autoimmune conditions, such
as multiple sclerosis, in part because of their actions
in reducing typeII endothelial cell activation94. It will
be important to also understand the effects of NFB
inhibitors that are being developed for cancer therapies
on the vasculature95. Examples of preventing the inter
actions of leukocytes with endothelial cells have largely
focused on antibody blockade of adhesive interactions.
A clear success in this field has been the use of an
4integrin-specific antibody for the treatment of mul
tiple sclerosis96. Certain statins and their derivates may
also show activity and therapeutic benefit as LFA1 (lym
phocyte function-associated antigen 1) antagonists97.
A less appreciated agent may be heparin, which, by com
petition with endothelial cell surface proteoglycans for
binding of chemokines, may reduce chemokine display
and leukocyte recruitment at sites of type-II-activated
endothelial cells98.

nature reviews | immunology volume 7 | october 2007 | 813

2007 Nature Publishing Group
REVIEWS

Concluding remarks cell responses such as angiogenesis, for example in

The central theme of this Review is that inflamma
tion can be analysed as a response to changes in the
endothelial cells that line blood microvessels. Acute
inflammation results from typeI or typeII activation
responses of endothelial cells, which are mediated in
type I activation by ligands of heterotrimeric GPCRs,
such as histamine, or in type II activation by inflamma
tory cytokines, such as TNF or IL1. Time-dependent
changes in molecules expressed by type-II-activated
endothelial cells underlie the change in inflammation
from neutrophil-dominated to mononuclear-cell-
dominated responses. Adaptive immunity, through
cytokines such as IFN or IL4, may superimpose
changes on type-II-activated endothelial cells that
enhance TH1-type or TH2-type polarized inflammatory
reactions. Chronic inflammation involves endothelial
response to VEGFA, and TLO formation in response to
LT. Although each of these endothelial cell behaviours
is largely associated with specific classes of mediators,
the same mediators may contribute to different phases
of the endothelial cell response. This overlap blurs the
heuristic division between acute and chronic inflamma
tion. Targeting promiscuous mediators such as TNF
or VEGFA has led to unintended effects, sometimes
helpful and sometimes harmful. We encourage the
reader to consider endothelial cell responses induced
by existing and new leukocyte-derived agents for con
trolling inflammation, as well as to consider the effects
of agents commonly used to treat vascular diseases on
inflammation. We also encourage the development of a
new therapeutic approach to inflammation that directly
focuses on vascular endothelial cell responses.

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Acknowledgements
The authors are supported by grants from the National
Institutes of Health, USA.
Competing interests statement
The authors declare no competing financial interests
DATABASES
Entrez Gene:
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene
CD99 | COX1 | COX2 | E-selectin | FGFR1 | ICAM1 | IL-1R1 |
MLCK | NOS3 | PECAM1 | P-selectin | TIE2 | TNFR1 | TRADD |
VCAM1 | VEGFR1 | VEGFR2
all links are active in the online pdf.