Clinical Science (1994) 86, 359-374 (Printed in Great Britain)
359
Editorial Review
Role of the endothelium in modulating the vascular
response to sepsis
Nicholas P. CURZEN, Mark J. D. GRIFFITHS and Timothy W. EVANS
Unit of Critical Care, National Heart and lung Institute, Royal Brompton National
Heart and lung Hospital, London. U.K.
SEPSIS THE CLINICAL SYNDROME
Introduction
Sepsis and its associated syndromes (Table 1, Fig.
1) represent a potentially devastating systemic
inflammatory response estimated to occur in 1% of
hospitalized patients, 10-20% of whom die. Mortality approaches 60-90% in those who develop septic
shock [l-31, a figure that has changed little since
intensive care was first developed, despite improvements in monitoring and supportive techniques.
Recent re-evaluation of the clinical definitions of
sepsis has emphasized the importance of the host
response to the insult rather than the virulence of
an infecting organism. This constellation of clinical
and haematological findings occurring in critically
ill patients is associated with positive identification
of an infecting causative agent in less than 50% of
cases [4]. The term systemic inflammatory response
syndrome (SIRS) has therefore emerged as a useful
definition of the resulting clinical state [S]. Apart
from redefining sepsis, recent clinical investigations
have emphasized the central role of sepsis-induced
circulatory failure in the pathophysiology of multiple organ failure (MOF) (Table 2), which carries an
especially poor prognosis [6, 71. Our aim is to
highlight the part played by the endothelium in this
process.
Oxygen is the most supply-dependent substrate in
the circulation, but stores relative to consumption
are the lowest of any metabolite. This knowledge,
combined with technology allowing invasive haemodynamic monitoring and measurement of blood
oxygen saturation, has led to the use of oxygen
transport variables (Table 3) as indices of cardiovascular and metabolic function [8]. In critically ill
patients with sepsis [9] there is thought to be an
abnormal relationship between oxygen uptake ( VO,)
and delivery (DO,), resulting in a state of pathological ‘supply-dependency’ [lo] (Fig. 2). Patients
admitted to an intensive care unit who demonstrate
this pattern of supply-dependency have a higher
mortality than those who do not [ l l , 121. This
tendency and the apparent inability of the peripheral tissues to extract oxygen has led a number of
investigators to advocate active manipulation of
Do, to achieve pre-determined target levels of VO,.
It has subsequently been shown [13, 141 that critically ill patients in whom supraphysiological targets
for cardiac output (CO), Do, and VO, are attained
have a lower incidence of MOF and mortality.
Pathophysiological mechanisms that may account
for impaired peripheral oxygen uptake associated
with sepsis include: loss of microvascular control
with shunting, tissue oedema, compression of capillaries and microembolization [l5, 161. One major
problem with the strategy of increasing DO, in
order to increase VO, is that regional differences in
blood flow probably occur [17, 181. Hence the
expected increase in VO, in areas with the greatest
oxygen debt may be prevented by a disrupted
microcirculation.
The effects of failure of the functional and structural integrity of the endothelium that characterize
SIRS are most obvious in the adult respiratory
distress syndrome (ARDS), which complicates up to
Key words: endothelin. endothelium. nitric oxide, sepsis, vasculature.
Abbreviations: ARDS, adult respiratory distress syndrome; cNOS, constitutive form of nitric oxide synthase; CO, cardiac output; Do,, oxygen delivery; ECE, endothelin-convcrting enzyme; EDRF, endothelially derived relaxant factor($ ET, endothelin; HPV, hypoxic pulmonary vasoconstriction; IL. interleukin; iNOS. inducible form of nitric oxide
synthase; MOF, multiple organ failure; L-NAA, N-nitrN-arginine; L-NAME, N-nitreL-arginine methyl ester; L-NMMA, NG-monomethyl-L-arginine; NOS, nitric oxide synthase;
PAF, plateletactivating factor; PVR, pulmonary vascular resistance; SIRS, systemic inflammatory response syndrome; SVR, systemic vascular resistance; TNF, tumour necrosis
factoru; VO,, oxygen consumption.
Correspondence: Dr T. W. Evans, Unit of Critical Care, National Heart and Lung Institute, Royal Brompton National Heart and Lung Hospital, Sydney Street, London SW3 6WP,
U.K.
N. P. Curzen et al.
360
Table 1. Definitions of sepsis and septic shock. Definitions are from
Bone RC. Balk RA. Cerra FB, et al. Chest 1992: 101: 16444, except that
for sepsis syndrome, which is adapted from (261. Abbreviations: Pao,.
arterial partial pressure of oxygen: Paco,, arterial partial pressure of carbon
dioxide: Fio,, fraction of inspired oxygen.
I. Sepsis
'The systemic response to infection'
Includes two or more of the following:
Temperature > 38°C or < 36°C
Heart rate > 90 beats/min
Respiratory rate > 20 breaths/min or Paco, < 4.3 kPa
Leucocyte count > 12000/mm3 < 4000/mm1, or > 10% band (immature) forms
2. Sepsis syndrome
'Sepsis with evidence of altered organ perfusion'
Altered organ perfusion includes one or more of the following:
Pao,/Fio, 280 (without other cardiopulmonary disease)
Elevated lactate level ( > upper limit of normal for the laboratory)
Oliguria < 0.5 ml/kg body weight
3. SIRS
'The response to a variety of severe clinical insults (not necessarily infective), which
is indistinguishable from sepsis'
4. Septic shock
'Sepsis with hypotension (sustained decrease in systolic blood pressure < 90 mmHg,
or drop > 40 mmHg, for at least I h) despite adequate fluid resuscitation, in the
presence of perfusion abnormalities that may include, but not limited to lactic
acidosis, oliguria or an acute alteration in mental status." Patients who are on
inotropic or vasopressor agents may not be hypotensive at the time that perfusion
abnormalities are measured.
Fig. I. Relationships between SIRS, sepsis and ARDS. Abbreviations:
F. fungi: P, protozoa: V, viruses. Adapted from Bone RC, Balk, RA, Cerra
FB, et al. Chest 1992: 101: 164455.
25% of cases with SIRS [19], and has a mortality
approaching 65%. ARDS is characterized by nonhydrostatic pulmonary oedema and refractory
hypoxia [20]. Pulmonary hypertension is a common
complication of acute lung injury, and is associated
with an increased mortality [21]. Pulmonary vascular resistance (PVR) is increased, even after correction for arterial hypoxaemia [22], and this results
from both functional (vasoconstriction) and structural (embolization and vascular remodelling)
changes in the pulmonary vasculature [23].
Pathogenesis of sepsis
The initiating factor in the complex cascade of
inflammatory events leading to clinical sepsis is the
release of endotoxin or certain other comparable
substances derived from yeasts, viruses, fungi or
Gram-positive bacteria [24-261. Endotoxin is a
lipopolysaccharide component of the cell wall of
Gram-negative bacteria. The presence of endotoxin
in the circulation can result from exogenous bacteria or via a process of translocation from intestinal flora through the wall of the gastrointestinal
tract [27, 281. Endotoxin can activate both humoral
and cellular pathways in the inflammatory process
(Fig. 3) and is detectable frequently in the blood of
patients with septic shock [29, 301 whether or not
blood cultures are positive for a specific pathogen.
Evidence that endotoxin is important as an initiator
of the inflammatory response to sepsis is compelling,
as it produces similar haemodynamic changes in
humans to those observed in experimental septic
shock [31]. Animal studies suggest that endotoxin
stimulates the release of tumour necrosis factor-a
(TNF), interleukins (IL-1, -6, -8) and plateletactivating factor (PAF) from macrophages [32-351.
T N F is a 17 kDa polypeptide that can activate
neutrophils leading to the production of elastase
[36], as well as the reactive oxygen species superoxide and hydrogen peroxide [37]. It promotes the
attachment of neutrophils to endothelium [38] leading to endothelial cell destruction [39]. Radiolabelling of neutrophils has demonstrated their rapid
sequestration in the lungs after the onset of sepsis in
both animals and humans [40], and bronchoalveolar lavage fluid from patients with ARDS is rich in
neutrophils [41, 421. Injection of endotoxin causes
T N F levels to rise acutely in both animals [43] and
humans [44], eventually leading to hypotension and
increased pulmonary capillary permeability [45, 461.
Interestingly, C3H/HeJ mice, which cannot synthesize TNF, are resistant to otherwise lethal doses of
endotoxin [47]. Levels of T N F are elevated in
patients with sepsis and may correlate with prognosis [48-501.
IL-1 shares many of the properties of TNF,
producing hypotension and pulmonary oedema in
rabbits [Sl]. IL-6 production is stimulated by other
cytokines, acts by regulating lymphocyte function
and rises later in the inflammatory response [52].
IL-8 is a potent chemotactic agent for neutrophils
[53], and continues to be produced during the
subsequent inflammatory response by, among other
tissues, endothelium.
After the activation of neutrophils by TNF, PAF
36 I
Endothelium i n sepsis
Table 2 Cardiovascular pathophysiology of sepsis. Abbreviations: DIC, disseminated intravascular coagulation; SVR, systemic
vascular resistance.
Site
Pathophysiology
Clinical correlate
Resistance and capacitance vessels
Decreased tone and response t o vasoconstrictors
Decreased SVR and hypotension
Microvasculature
Shunting
Increased permeability
Microembolism
Decreased oxygen extraction
Non-hydrostatic oedema
Microinfarction/DIC
Heart
Decreased contractility
Relatively low CO and DO,
Table 3. Derivation of physiological parameters, Abbreviations: OER,
oxygen extraction ratio; MPAP, mean pulmonary artery pressure; PCWP,
pulmonary capillary wedge pressure; MAP, mean arterial pressure; CVP,
central venous pressure; Cao,. arterial oxygen content; Cvo,, mixed venous
oxygen content.
PVR (dyn s cm3)=
SVR (dyn s cm-)
[(MPAP
= [(MAP
-
PCWP) x eO]/CO
- CVP)
/-
CYTOKINES
Mrrophaga
PHN
Platclett
Endothelium
x eO]/CO
ENWDERIVED
MEDIATORS
NO
ETs
TX, PGs. LTs
CLOTTING
SYSTEMS
COM~~EMENT
I
~- .
.
Do, (ml min-’ m7) = CO x Cao,
Yo, = CO x (Cao, - Cvo,)
(ml min-’ m 9
OER = (Cao, - Cvo,)/Cao, = Vo,/oO,
Dilatation
Fyruia
Increased permeability
300
I
Clotting/thrombotic dkturbrnca
Turue damage
f
200
_E
Hypotension
fever
Fig. 3. Activation of humoral and cellmediated pathways b y endot o x i n resulting in inflammation and tissue damage. Abbreviations:
endoderived. endotheliumderived; PMN, polymorphonuclear leucocytes;
TX, thromboxane; PGs, prostaglandins; LTs, leukotrienes.
I
.-c
E
-E
400
800
I200
Do, (mlmin-lm-l)
Fig. 2 Relationship between D+ and & in health and in critical
illness. Normal subject = A B C critically ill patient = DEF. In normal
subjects Vo, is independent of Do, at rest (line BC), provided it is
maintained above a critical level (point B). In some critically ill septic
patients, however, Vo, becomes DoAependent (line DE), so that the
oxygen extraction ratio remains constant. Critically ill patients who manifest this pathological ‘supplydependency’ have a higher mortality rate than
those who do not. Adapted from Fig. 2 in [lo].
cascade as well as the contact system which generates bradykinin. This pathway, as well as neutrophilgenerated proteases, stimulates the fibrinolytic
systems, and this can result in disseminated intravascular coagulopathy [61].
One of the integral features of this complex
inflammatory response is that it involves endothelial
damage, leading to mediator/endothelial cell interaction, loss of function and increased capillary permeability [62-641. It is the investigation of these
interactions which is rapidly uncovering the role of
the endothelium in modulating the vascular response to sepsis, and thus determining the extent of
tissue injury.
THE ENDOTHELIUM
[54], leukotrienes [ 5 5 ] and prostanoids, including
thromboxane A, [56], are released. Platelet activation ensues, leading to the release of numerous
vasoactive, chemoattractant and endotheliumdamaging substances [57, 581. Endotoxin is also a
potent activator of the complement cascade [59].
In addition, there is activation of both intrinsic
and extrinsic coagulation cascades by the combination of cytokines, endotoxin and activated
endothelial cells and platelets [60]. When activated,
Factor XI1 can initiate the intrinsic coagulation
The endothelium is an intimal layer of simple
squamous cells which provides a continuous fluent
surface for circulating blood. Until recently, it was
perceived as a passive, metabolically inert permeability barrier whose function was to contain blood
and plasma. However, the endothelium is now
known to be a metabolically and physiologically
dynamic tissue with multiple functions [65]. The
remainder of this review is concerned principally
with the vasoactive substances produced by the
endothelium, how they are influenced in the res-
362
N. P. Cunen et al.
ponse to sepsis, and the manner in which they take
part in a dynamic interaction with other elements of
the response.
Anticoagulant, antithrombotic and metabolic properties
In order to maintain patency of blood vessels and
the fluidity of the circulating surface for blood,
endothelial cells synthesize and release various anticoagulant and antithrombotic substances, including
thrombomodulin, which binds thrombin to lower its
affinity for fibrinogen [66]. Tissue plasminogen activator is also synthesized by the endothelium and
activated by a variety of stimuli in the circulation
[67] to initiate the production of plasmin. The
endothelial cell surface is rich in heparan sulphates,
which contribute to the inactivation of circulating
thrombin [68]. Nevertheless, the synthesis and production of prostacyclin [69] and endothelially derived relaxant factor(s) (EDRF) is an important
component of its antithrombotic armoury, since
both mediators are vascular smooth muscle dilators
and potent inhibitors of platelet aggregation [70,
711.
Role in inflammation
The endothelium plays an integral role in the
acute inflammatory response [72], in responding to
early (non-specific) mediators such as histamine and
bradykinin, and in facilitating the adherence and
subsequent migration from blood to tissue of activated neutrophils [73]. This sequence of events
depends upon the expression of a group of surface
glycoprotein leucocyte adhesion molecules, known
collectively as selectins [74, 751 after the endothelium is itself activated by cytokines. Two selectins
are involved specifically in leucocyte/endothelial cell
adherence [76]: endothelial-leucocyte adhesion
molecule-1 (E-selectin), and granulocyte-associated
membrane protein 140 (P-selectin) [77]. E-selectin is
expressed on endothelium exposed to cytokines
(including TNF and IL- 1) and lipopolysaccharide
[78]. The kinetics of its production in cell culture
imply that it is protein synthesis-dependent. Pselectin is found in the granules of platelets and
endothelium, and expression is stimulated by thrombin and histamine [79], thereby providing a mechanism by which neutrophil adhesion is achieved early
in the inflammatory response before protein synthetic pathways have been stimulated. The vascular cell
adhesion molecule (VCAM-1) is a member of the
immunoglobulin family and is also expressed in the
presence of lipopolysaccharide, TNF and IL-1 [80].
The intercellular adhesion molecules (ICAM-1 and
-2) are also constitutively expressed by endothelium
and are therefore available for binding of neutrophils at the initiation of inflammation. Simultaneously, the activated neutrophil expresses a complementary sequence of surface adhesion receptors
termed integrins [76, 811, which have multiple speci-
ficities. The most important of these are the CD11/
CD18 complex [82]. The initial adherence of neutrophil to endothelium is followed by neutrophil
migration into the interstitium. C D l l and CD18
binding appears to be essential for this process [83],
which is also promoted by T N F [84]. Endothelial
cell gene expression has been shown recently in
response to various cytokines, including TNF [85]
and IL-1 [86].
The endothelium and vascular tone
EDRF. In 1980 the vascular relaxation induced
by acetylcholine was shown to be dependent on the
presence of intact endothelium [87] and to be
mediated via the release of a non-prostanoid, labile,
relaxant subsequently termed EDRF [88]. Evidence
has since accumulated that the chemical and pharmacological properties of EDRF are shared to a
great extent by nitric oxide (NO). Specifically, both
agents are chemically unstable and have half-lives
under assay conditions of less than 5 s [89, 901.
Secondly, both are inactivated by the superoxide
anion and both are stabilized by superoxide dismutase [91, 921. They both stimulate cyclic G M P
formation in vascular smooth muscle [93, 941 by
activating guanylate cyclase [95], and via this
mechanism both act as dilators of arterial and
venous smooth muscle. Finally, they are both inhibitors of platelet aggregation and adhesion [96, 971.
N O has been shown to be synthesized in vitro
from the semi-essential amino acid, L-arginine, by
the membrane-bound enzyme NO synthase (NOS)
[98, 991, a process that can be inhibited by Larginine analogues, such as NG-monomethyl-Larginine (L-NMMA) [100, 1011. Several distinct
NOS genes have been identified [102, 1031 and
NOS exists in two forms (Table 4): a constitutive
calcium- and calmodulin-dependent enzyme (cNOS)
[104, 1051, which is probably responsible for basal
release of NO, and an inducible calcium- and
calmodulin-independent enzyme (iNOS) [1061 (see
below). Both require NADPH and tetrahydrobiopterin as cofactors [107].
N O activates soluble guanylate cyclase, after
binding to its haem moiety, which in turn causes an
increase in intracellular cyclic GMP content causing
vascular smooth muscle to relax [lo81 (Fig. 4), a
chain of events confirmed by several studies. Thus,
L-arginine administration has been shown to
produce vasodilatation in newborn lambs [1091, an
effect blocked by the guanylate cyclase inhibitor,
Methylene Blue, or by L-NMMA. The vasodilatation was augmented, however, by a cyclic G M P
phosphodiesterase inhibitor, whose ability to raise
intracellular cyclic G M P levels has also been shown
to produce vasodilatation in other models [1 lo],
which demonstrates the importance of this mechanism as a mediator of endothelium-dependent vasodilatation. A rise in cyclic G M P may produce
vascular smooth muscle relaxation via several
Endothelium in sepsis
363
Table 4. Properties of vascular NOS isoformr. Abbreviation: LPS. lipopolvsaccharidelendotoxin.
NOS isoforms
Type II
Type 111
Response in sepsis
Constitutive
Immediate increase in NO activity
Enzyme synthesis induced by L K and cytokines
Massive NO production after 2-6 h
Location
Endothelial cell
Membranebound
Membrane smooth muscle
Cytosolic
Regulation
Induction prevented by corticosteroids and inhibitors of protein
synthesis
Activation
Calciumdependent
Non-selective inhibitors
1-Arginine analogues (e.g. L-NMMA)
Selective inhibitors
None known
Flow
EFS
Hypoxia
ACh
BK
5-HT
SubsP
u u
L-Citrulline
Endothelium
Smooth muscle
Fig. 4. Synthesis of NO in the endothelium by NOS and its
mechanismof action in smooth muscle. Abbreviations: ACh, acetylchw
line; BK, bradykinin; SHT, Ulydroxytryptamine; SubsP, substance P; BH,,
tetnhydrobiopterin; cGMP, cyclic GMP: GC, guanylate cyclase; PK, phosphokinase; [Ca2r, intracellular free calcium concentration; R, receptor;
EFS, electrical field stimulation; ANP, atrial natriuretic peptide.
mechanisms which lower intracellular free calcium
levels, including a reduction in calcium (Ca2+)
influx, a reduction in its release from intracellular
stores, an increase in Caz+ sequestration in intracellular stores, or by stimulation of Ca2+ATPase-dependent extrusion of Ca2+ [l 1 13.
The administration of NO synthase inhibitors,
such as L-NMMA, has been shown in both animals
[112-1141 and man [llS, 1161 to provoke increases
in mean arterial blood pressure and decreases in
regional blood flow, implying that a continual basal
release of NO exists which may provide physiological regulation of tissue blood flow [117]. Irnportantly, NO can also attenuate cardiac myocyte
contraction [118].
Calcium-independent
Aminoguanidine
L-Canavanine
Diphenyleneicdonium
Regulation of NO release. Local blood flow is an
important regulator of NO activity. Thus, increased
systemic blood flow can augment agonist-evoked
endothelium-dependent relaxations via an increase
in NO release [119, 1201. The size of the hypertensive response induced by intravenous L-NMMA in
animal models is dependent on basal vascular tone
[121]. Evidence also points to an important role for
NO in pulmonary vascular regulation, in that inhibitors of NOS cause dose-dependent increases in
pulmonary artery pressure in awake lambs, whereas
systemic arterial pressure remains unaffected [1223.
Similar results have been found in rabbits [123], in
which infusion of the NOS inhibitor, N-nitro+
arginine methyl ester (L-NAME), causes an increase
in respiratory rate and arterial hypoxaemia. NO is
also released during exposure to acute hypoxia, may
modulate hypoxic pulmonary vasoconstriction
(HPV) [124], and is capable of influencing the
pulmonary vascular response to chronic hypoxia
[125-1293. Disruption of normal regulatory mechanisms of the lung, especially HPV, is known to be a
feature of sepsis-associated lung injury and these
effects of NO may play a crucial role in this
pathophysiology.
The release of NO has been demonstrated in
response to many other pharmacological and physiological stimuli, including histamine, thrombin, ATP,
bradykinin, calcium ionophore and substance P
[130, 1311.
NO release in sepsis. Patients and animals with
septic shock lose peripheral vascular tone, and the
responsiveness of vessels to constricting agents, both
in vitro and in uiuo, is diminished [132-1341. The
incubation of bovine aortic endothelial cells with
lipopolysaccharide causes the rapid release of an
NO-like factor [135, 1361. In patients with septic
shock, the levels of NO metabolites in plasma are
significantly elevated [137], and the infusion of
NOS inhibitors in such cases [138] and in animal
models of septic shock [139-1421 can lead to a
rapid and reproducible rise in systemic vascular
364
N. P. Cunen et al.
resistance (SVR) where other vasoconstrictors are
ineffective. Thus, both the synthesis and release of
NO are stimulated by the inflammatory process.
Endotoxin leads to induction of an iNOS in
endothelium [143] and vascular smooth muscle, as
well as myocardium [144-1471. T N F and IL-1 can
also stimulate the expression of iNOS in both
endothelium and vascular smooth muscle [117,
1481. Interestingly, T N F can also inhibit the N O
release stimulated in isolated pulmonary vessels by
the specific agonists acetylcholine and bradykinin,
although basal NO release is unaffected [149].
Patients who are treated with IL-2 chemotherapy
excrete high levels of NO metabolites, implying that
an induction of NO synthesis occurs in response to
this treatment [lSO]. L-NMMA can inhibit TNFinduced hypotension in animals [15 11.
Further studies have demonstrated the possibility
of a two-stage release of NO from the vessel wall
during sepsis. In isolated endotoxin-treated rat main
pulmonary arteries, NOS inhibitors reverse vascular
hyporesponsiveness to phenylephrine [1521. The
NO-mediated hyporeactivity to noradrenaline starts
within 60 min in a rat model of sepsis in uivo [153],
and so is too rapid to be explained by the induction
of iNOS. This implies that the endothelium
responds immediately to the septic insult by releasing N O produced by the constitutive enzyme cNOS.
In another study, however, the use of L-NAME after
1 h in endotoxin-treated pithed rats did elevate
blood pressure and enhance vascular responsiveness
to both noradrenaline and sympathetic stimulation,
but not to a significantly greater degree than in
saline-treated animals [1541. This does not therefore
support the hypothesis that after endotoxin insult
an increase in N O release explains the early loss of
vascular responsiveness in vivo, and suggests that
some other factor(s) must be involved. However,
from about 3 h after the endotoxic insult, there is
massive N O production as a result of induction of
iNOS, probably mostly from the underlying vascular smooth muscle [145]. There is also evidence that
endothelium is required for the N O response to be
maximal. Thus, endothelial removal caused a significant delay in the onset of vascular hyporesponsiveness (6 h compared with 4 h) and reduced the
sensitivity of rat aorta exposed to lipopolysaccharide in vitro [l55, 1561. Selective inhibitors for iNOS
are now available and are the subject of intense
investigation [1571 (see below).
Endothelially derived constrictor factors
Endothelins (ETs). This subject has been covered
in detail in a recent Editorial Review in this journal
[1581. This article will therefore emphasize in particular the role of ETs in sepsis.
In 1988 an endothelially derived vasoconstrictor
was cloned and sequenced after its isolation from
the culture medium of porcine aortic endothelial
cells [159], and termed ET. This substance was
found to elicit a slow onset, sustained contraction of
isolated arteries from many different species. Three
similar, but distinct, ET-related genomic loci have
now been identified which encode for three similar,
but distinct, ET molecules (ET-1, ET-2, ET-3) [160],
all of which are derived from prepropeptides, and
consist of 21 amino acids with considerable
sequence identity. The conversion of the propeptide,
big ET-1, to ET-1 was postulated to be due to the
activity of an endothelin-converting enzyme (ECE),
since identified in several animal models as a
phosphoramidon-sensitive neutral metalloproteinase
[161-1641. Two neutral proteases with ECE activity
have now also been demonstrated [165]. One is
membrane-bound, phospharamidon-sensitive and
can utilize all three ETs as substrates; the other is
soluble and phosphoramidon-insensitive. Three ET
receptor subtypes probably exist [1661, although so
far only two have been cloned and expressed [167,
1681. ETA has the highest affinity for ET-1 compared with the other ETs, and although it has
widespread expression in humans, in particular in
vascular smooth muscle, this does not include
endothelial cells [1691. ETB is non-selective, binding
all three ETs, which are equipotent in displacing
1251-ET-l [170]. ETB is also widespread and is
expressed on endothelial cells.
ET-1 appears to act by increasing the intracellular free calcium concentration [1711, by activating
phospholipase C [172], which in turn leads to
increases in inositol trisphosphate [173] and diacylglycerol [174] synthesis. Both are implicated in the
initial rise in intracellular free calcium concentrations, and probably underlie the initiation of ET1-induced vascular contraction [1751. Protein kinase
C is also implicated in a second-messenger system
mediating ET-1-induced contraction [176, 1771, particularly as staurosporine, a protein kinase C inhibitor, attenuates contractions to ET-1 in vitro.
Release of endothelins. ET- 1 immunoreactivity
cannot be demonstrated in homogenates of capillary
endothelial cells [178], and ET release from cultured endothelial cells can be prevented by the
protein synthesis inhibitor cycloheximide [179],
suggesting that ETs are not stored but are synthesized de novo in the endothelium. Endothelial cells
exposed to agents such as adrenaline and thrombin
express ET-1 mRNA [159]. ET-1 production has
also been demonstrated, both at the level of mRNA
transcription and at the level of protein release, in
response to angiotensin 11, thrombin and transforming growth factor-p [180, 1811. Mechanical shear
stresses, similar to haemodynamic stress in vivo
[182], and hypoxia [183, 1841 also induce ET-1
production, suggesting that it has a role in modulating local blood flow in response to changes in these
factors. Finally, platelets have been found to stimulate expression of ET mRNA and ET biosynthesis
in cultured endothelial cells [l85].
Endothelins in the control of vascular tone. ET-1 is
a potent vasoconstrictor in humans [186] and many
365
Endothelium in sepsis
animal species [187, 1881. The endothelium modulates the vascular response to ETs. Thus, ET-1- and
-3-induced contraction of rat pulmonary arteries is
enhanced by endothelial removal [187] and both
agents elicit vasodilatation in preconstricted vessels
of isolated mesentery [189, 1901. N O release has
been demonstrated in rat mesentery [189, 1911 in
response to ET, an effect inhibited by L-NMMA
[192]. ET-3 is a more potent vasodilator than ET-1,
probably because the receptor involved on the
endothelium is of the ETB type, which has equal
affinity for all ETs, whereas the ETA (vasoconstrictor) receptor is predominant in vascular smooth
muscle and has a higher affinity for ET-1 [193-1961.
There is a uniform haemodynamic response to ET
infusion in most species, characterized by initial
transient hypotension followed by sustained hypertension [159, 197, 1981. The hypotensive response
can be at least partially inhibited by NOS inhibitors
[199], but does not recur if the ET infusion is
continuous rather than bolus [200]. ET-1 [201] and
ET-3 [202] are both coronary and pulmonary [198]
vasoconstrictors. However, ET-3 is a powerful vasodilator in isolated rat lungs under prior conditions
of hypoxic vasoconstriction [203]. ET-1 has been
shown to have inotropic [204] and chronotropic
[205] effects on cardiac tissue. ET-1 can induce
cultured smooth muscle cell proliferation [206], and
ET-1 and ET-3 stimulate fibroblast growth and
chemotaxis [207], suggesting a role in vascular
re-modelling.
Endothelins in sepsis. ETs seem to be important
mediators of vascular tonic responses under physiological conditions, since they are released in response to a variety of local factors including hypoxia.
Their potent effects on vascular tone are likely to
play an important role in the widespread changes
associated with endotoxaemia. ET release in response to endotoxin has been confirmed by radioimmunoassay in uitro and in uiuo [208], and in
endothelial cell cultures in response to TNF,
interferon-y, IL- 1, transforming growth factor-fi
[209, 2101 and free-radical species [211]. ET levels
increase during endotoxaemia in many animal
models [208, 212, 2131 and are elevated, possibly in
parallel with indicators of illness severity, in patients
with septic shock [214-2161.
The role played by the ETs in the inflammatory
response to sepsis is not yet clear. In endothelial
cells and vascular smooth muscle, N O [217] leads
to the activation of soluble guanylate cyclase with
the formation of cyclic GMP, and in vascular
smooth muscle this stimulates relaxation [218]. In
endothelial cells, however, cyclic G M P can inhibit
ET production [219]. In human and canine models
ET-induced contractions can also be inhibited by
NO released in response to acetylcholine or bradykinin [220, 2211. The release, and subsequently high
circulating levels, of such a potent vasoconstrictor
substance would be expected to antagonize the
observed vasodilator response, and it is possible
Membrane phospholipid
L
Phospholipase A,
I
Arachidonic acid
Acetyltransferase
1
NSAID
Lipoxygenase
Cyclwxygenase
Cyclic
endoperoxides
Throm boxane
synthetase
I
Prostacyclin
synthetase
I
Fig. 5. Cellular pathways of phospholipid metabolism. Abbreviation:
NSAID, non-steroidal anti-inflammatory drugs.
that the interaction between ETs and N O explains
this paradox.
Other endotheliumderived vasoactive factors
The endothelium produces various prostanoids
via the cyclo-oxygenase pathway of arachidonic acid
metabolism (Fig. 5), principal among which is the
vasodilator prostacyclin, which activates adenylate
cyclase, thus increasing intracellular cyclic AMP
levels and causing smooth muscle relaxation [222,
2231. Inhibitors of prostaglandin synthesis, such as
indomethacin and meclofenamate, augment HPV
[224, 2251, implying that vasodilator prostaglandins
may modulate vascular tone during hypoxic episodes. Furthermore, in a rat model of skeletal
muscle microcirculation, increases in arteriolar
blood flow elicited by the occlusion of parent
arteriolar branches induced an endotheliumdependent vasodilatation distal to the occlusion
inhibited by cyclo-oxygenase inhibitors, but not by
L-NMMA [226, 2273. Cytokines, such as TNF and
IL- 1, can stimulate prostanoid release, the vasodilator action of which is augmented by the presence of
endothelium in the porcine coronary circulation
[228].
Cyclo-oxygenase metabolism of arachidonic acid
also produces vasoconstrictor agents, such as
366
N. P. Cunen et al.
thromboxane A2 and endoperoxides [229, 2303.
Thromboxane is a potent constrictor of pulmonary
arterioles after endotoxin infusion, and it is also
capable of increasing capillary permeability [23 11.
ET-1 stimulates the release of the vasodilators
prostacyclin and prostaglandin E,, as well as the
vasoconstrictor, thromboxane, possibly via ETinduced activation of protein kinase C [232]. In
isolated rat mesenteric arteries, ET- 1 infusion
produces endothelium-dependent relaxation that can
be abolished by indomethacin, but not L-NMMA
[233]. The ET-mediated release of these two prostanoids has also been demonstrated in other animal
models [234]. In isolated human internal mammary
artery, prostacyclin reverses ET- 1-induced vasoconstriction [235]. Indomethacin pretreatment of isolated rat or guinea-pig lung preparations potentiates
ET-induced contractions [236] and increases ET-3induced ET- 1 formation in cultured endothelial cells
[237]. This implies that prostacyclin is able to
inhibit ET production and provides further evidence
of negative feedback on ET activity.
ENDOTHELIUM-DERIVED VASOACTIVE FACTORS
IN THE INFLAMMATORY RESPONSE TO SEPSIS
The response to sepsis can be seen as a cascade,
each component of which amplifies the inflammatory response. The interactions between individual
mediators of sepsis are highly complex, but determine the extent of endothelial, and subsequently
tissue, damage (Fig. 3).
After endotoxin exposure, macrophage activation
leads to cytokine release. T N F and IL-1 and -2 are
able to damage endothelial cells and increase their
permeability [39, 51, 581, activate neutrophils and
endothelial cells and facilitate adhesion between
them [38], and stimulate endothelial cells (and
macrophages) to synthesize and release N O [ 1 17,
1481, ETs [209, 2101 and prostanoids [226]. However, the activity of T N F and IL-1 to disrupt
pulmonary vascular endothelium is synergistic
[238]. T N F administration only produces features
of septic shock when PAF is present [239, 2401. The
inflammatory response may then be amplified by
endothelially derived products of arachidonic acid
metabolism [231]. Furthermore, TNF, PAF and IL1 can promote their own and each others’ release
from several cells, including the endothelium and
macrophages [241, 2421. Activated platelets express
ET mRNA and ET biosynthesis [l85] in endothelial
cell cultures, but the potency of this reaction is
multiplied several-fold by endotoxin. The ETs
released can modulate the activation of macrophages [243] and also affect the clotting cascades
via release of tissue plasminogen activator and von
Willebrand factor [244] from endothelial cells.
The endothelium also has a role in the downregulation of the inflammatory response. NO, in
particular, appears to have some anti-inflammatory
properties, although available results are conflicting,
and it is therefore not possible to extrapolate with
any confidence from one experimental preparation
either to another or to the clinical setting. For
example, there is evidence from studies in uitro and
in uiuo, utilizing N O synthesis inhibitors, that N O
can modulate the increase in vascular permeability
and protein leakage that is seen during the inflammatory response [245-2471. There is, however,
conflicting evidence that after endotoxin challenge, administration of L-NMMA dose-dependently
reduced the increase in vascular permeability in the
jejunum and colon in an ex uiuo rat preparation
[248]. It has also been shown to be able to reduce
leucocyte cell adherence to endothelial cells [249,
2501. A further anti-inflammatory property of N O
in endotoxaemia is its antithrombotic potential,
illustrated by the high rate of glomerular thrombosis in rats treated with endotoxin and L-NMMA
when compared with endotoxin without an N O
synthesis inhibitor [25 11. Finally, studies in uitro
suggest a role for N O as a scavenger of superoxide
radicals [252], which are released by activated
neutrophils and are not only cytotoxic [253] but
can also alter pulmonary vascular reactivity in their
own right [254]. This remains controversial, however, since there is also evidence that N O reacts
with superoxide radicals in pathological states, particularly those associated with hypoxia, to produce
even more cytotoxic species, such as peroxynitrites
[255]. In one rat model in uiuo immune complex
lung or dermal injury has been shown to be Larginine-dependent and could be prevented by
L-NMMA 112561. The mechanism by which N O
causes tissue damage may be related to its ability to
cause DNA strand breakage [257].
Prostacyclin can also inhibit T N F synthesis
[258], as can prostaglandin E, when macrophages
are activated [259]. Prostaglandin E, also inhibits
the fibroblast proliferation stimulated by the cytokines and ET-1 [260]. In addition, prostaglandin E,
and iloprost (a prostacyclin analogue) have been
shown to inhibit lipopolysaccharide-stimulated
induction of N O in murine macrophages [261].
Although activated platelets stimulate ET production, there appears to be regulatory negative feedback since ET can also inhibit platelet function ex
viuo in the rabbit [262].
MANIPULATION OF THE INFLAMMATORY
RESPONSE TO SEPSIS
The treatment of established septic shock with or
without M O F has achieved only limited success
[263]. At present, treatment centres around supportive care and attempts to maximize tissue oxygenation, with little attempt to limit the underlying
process. Treatment strategies have now diversified,
aiming to prevent the inflammatory cascade from
activating and damaging the endothelium by target-
Endothelium in sepsis
ing specific components in the pathway, or their
receptors.
NO inhibitors
L-NMMA was shown to correct refractory hypotension in two patients with septic shock [138], and
L-arginine and N-nitro-L-arginine (L-NNA) have
been administered to critically ill patients with
sepsis [264]. L-NNA caused a significant increase in
SVR and PVR as well as in systemic blood pressure,
and an accompanying decrease in cardiac index. LArginine administration in the same patients was
found to reverse these changes, but caused significant decreases in SVR, PVR and systemic blood
pressure and an elevation in cardiac index, with an
increase in the oxygen consumption index in
patients who had not received L-NNA.
The opportunity to utilize an agent that appears
to selectively inhibit iNOS has emerged recently. In
comparative studies between aminoguanidine and LNMMA, a non-selective NOS inhibitor, the former
was estimated to be at least seven times more
potent at inhibiting iNOS [157]. L-NMMA, however, was around 15 times more potent at inhibiting
the constitutive form of the enzyme in one model.
The potential value for such a specific inhibitor of
iNOS is great, since it may allow basal NO production, which is probably important in regulating
blood flow in the microcirculation, to continue,
whilst diminishing the surge in production of NO
that occurs during sepsis. Recent work using aminoguanidine has provided results [265] that confirm
it as a selective inhibitor of iNOS. Thus, aminoguanidine caused a dose-dependent increase in
phenylephrine-induced tension in intact and
endothelium-denuded pulmonary artery rings from
endotoxin-treated rats, but it had no effect on shamtreated controls. The effects of aminoguanidine were
not altered by cyclo-oxygenase inhibitors or by
histamine receptor blockers. The contraction caused
by aminoguanidine in vessels from endotoxintreated rats from which the endothelium had been
removed was abolished by L-arginine and LNMMA, suggesting that its action involves the Larginine/NO pathway. In addition, aminoguanidine
competitively inhibited the relaxation of artery rings
from endotoxin-treated rats by L-arginine.
inhaled NO
The attraction of NO as an inhaled treatment for
pulmonary hypertension associated with acute lung
injury, lies in its potential for selective dilatation of
pulmonary vessels that are supplying ventilated
alveoli [266], decreasing the amount of intrapulmonary shunting. A reduction in PVR also carries the additional theoretical advantage of a consequent increase in right ventricular ejection fraction
[267]. In a recently published study of septic pigs,
inhaled N O was indeed shown to attenuate pulmon-
367
ary hypertension [268]. In this study, NO had no
effect on SVR or CO, in contrast to L-arginine
infusion which attenuated pulmonary hypertension,
but also affected the systemic circulation. Inhaled
NO has also been shown to reverse pulmonary
vasoconstriction in the hypoxic and acidotic conditions in lambs in uiuo [269]. In patients with
ARDS, inhaled NO reduced the mean pulmonary
artery pressure, and increased the efficiency of arterial oxygenation as estimated by the ratio of the
arterial partial pressure of oxygen to the fraction of
inspired oxygen [270]. Significantly, the mean systemic arterial pressure and CO were unchanged.
Other therapies
Steroids. In animal models of sepsis C144, 2711,
pretreatment with steroids has been beneficial. However, this in part may derive from the inhibition of
gene expression and protein synthesis of inflammatory mediators such as NO and TNF. In fact,
corticosteroids have been shown to inhibit the
induction of iNOS, with no effect on cNOS [145].
However, in clinical studies [272, 2731 high-dose
steroid treatment has shown no benefit in reducing
mortality among patients with established septic
shock, and has not been shown to be beneficial
either as a treatment or a prophylactic in those
patients with acute lung injury or established ARDS
[274, 2751.
Prostanoids. Infusion of vasodilating prostanoids
has the theoretical benefits of reducing PVR and
right ventricular afterload, and hence increasing CO
and Do,. In one double-blind study [276], a significant reduction in mortality was seen when prostaglandin El was administered to patients with
ARDS, but only in those who were free of severe
organ dysfunction. In another study [277], prostaglandin E infusion increased intra-pulmonary shunt
with a resultant fall in arterial oxygen saturation.
Even in a study in which systemic DO, was
improved and PVR reduced by prostaglandin E,
administration, there was no improvement in mortality rate in patients with ARDS [278]. Inhaled
prostacyclin shares the theoretical benefits of
inhaled NO, and the evidence so far available does
demonstrate an improvement in arterial oxygenation in patients who are critically ill with this
treatment [279].
Cyclo-oxygenase inhibitors. Survival rates are
improved in septic animals treated with cyclooxygenase inhibitors [280, 2811, which inhibit the
effects of infused TNF and IL-1 [282] in animal
models. Studies are currently being conducted into
the clinical effects of these agents.
Anti-oxidants. Toxic oxygen species, such as
superoxide anion (O,-), hydrogen peroxide (H,O,),
hydroxyl (OH) and hypochlorous acid (HOCI), are
all capable of increasing vascular permeability in
endothelial cell monolayers [283], isolated perfused
lungs [284] and animals in uioo [285, 2861, when
368
N.
P. Cunen et al.
they can cause an ARDS-like acute lung injury.
Anti-oxidants, particularly N-acetylcysteine [287,
2881, can neutralize oxygen free radicals producing
improved gas exchange, haemodynamics and survival in animal models. A preliminary study in
patients with ARDS [289] does suggest haemodynamic benefit after N-acetylcysteine, but further studies
with anti-oxidants are required.
Antibodies directed against adhesion molecules. In
animals, monoclonal antibodies directed at components of the adhesion receptor system have achieved
success in attenuating the acute lung injury produced by activated neutrophils in septic conditions
[290, 2911. However, neutrophil activation and
adhesion are essential in immune defence, particularly for the phagocytocis of bacteria, making a
clinical trial potentially hazardous. Nevertheless, a
clinical trial using the monoclonal antibody 60.3,
which binds CD-18, is underway to assess the
therapeutic benefit of this treatment in preventing
MOF in trauma patients.
Anti-ET antibodies, ET receptor antagonists and
ECE inhibitors. Anti-ET antibodies are available
and have been used experimentally, mainly in
models of ischaemia [292, 2931, and have been
shown to reduce infarct size after coronary artery
ligation with reperfusion in rats. Since ETs have
vasoconstrictor and several apparently proinflammatory properties, it is tempting to speculate
that such treatment could be beneficial in patients
with sepsis. Several ET receptor antagonists have
also been developed, including BQ-153 [193], BQ123 [294] and FR 139317 [295]. All are selective for
the ETA receptor and inhibit vascular contraction.
Antagonists of this type therefore have therapeutic
potential, and newer agents that act both on ETA
and ETB receptor sub-types are also under evaluation [296].
Attenuation of the part played by ETs in sepsis
could also theoretically be achieved using ECE
inhibitors, although few data exist regarding the
feasibility or desirability of blocking ECE. Phosphoramidon pretreatment has been shown to successfully inhibit the haemodynamic effects of proETs [297, 2981, but more work is required, particularly in models of sepsis, to assess its potential
therapeutic value.
SUMMARY AND CONCLUSION
The endothelium is a dynamic layer of cells that
acts as a selectively permeable barrier between the
circulation and tissues. It is metabolically active,
producing a wide range of anti-coagulant and
vasoactive substances that help to maintain local
blood flow and haemostasis. The way in which it
achieves this regulatory role differs in various tissue
beds. In sepsis, which is extended as a concept to
include all conditions of profound systemic inflammatory response, a cascade of events is initiated,
usually by endotoxin, leading to the activation of
macrophages and systemic release of potent proinflammatory cytokines. These are capable of amplifying the inflammatory response and are responsible
for initial endothelial damage. The endothelial cell is
capable of activation, and responds to inflammation
by releasing agents, some of which are pro- and
some anti-inflammatory. Whether the inflammatory
process escalates to cause the extensive tissue
damage seen in conditions such as SIRS, ARDS or
MOF probably depends largely on the balance of
the interaction between the pro- and antiinflammatory mediators and the cells that produce
them. The endothelium thus plays a central part in
the modulation of the vascular response to sepsis.
Current areas of research in sepsis include scrutiny
of mediator and cell interactions in uitro and in uiuo,
and this has already led to the development of
potentially valuable clinical treatments, such as
specific inhibitors of iNOS and ET receptor antagonists. Further research will aim to continue to
increase understanding of the endothelial role in
sepsis and that of its vasoactive mediators.
ACKNOWLEDGMENTS
N.P.C. is an MRC Training Fellow, and M.J.G. is
a Wellcome Research Fellow.
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