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Vascular Pharmacology 43
The effects of nitric oxide in acute lung injury
Sanjay Mehta *
Centre for Critical Illness Research, Lawson Health Research Institute, Division of Respirology, London Health Sciences Center and Department of Medicine,
University of Western Ontario, London, Ontario, Canada N6A 4G5
Received 11 July 2005; accepted 3 August 2005
Abstract
Acute lung injury (ALI) is a common clinical problem associated with significant morbidity and mortality. Ongoing clinical and basic research
and a greater understanding of the pathophysiology of ALI have not been translated into new anti-inflammatory therapeutic options for patients
with ALI, or into a significant improvement in the outcome of ALI. In both animal models and humans with ALI, there is increased endogenous
production of nitric oxide (NO) due to enhanced expression and activity of inducible NO synthase (iNOS). This increased presence of iNOS and
NO in ALI contributes importantly to the pathophysiology of ALI. However, inhibition of total NO production or selective inhibition of iNOS has
not been effective in the treatment of ALI. We have recently suggested that there may be differential effects of NO derived from different cell
populations in ALI. This concept of cell-source-specific effects of NO in ALI has potential therapeutic relevance, as targeted iNOS inhibition
specifically to key individual cells may be an effective therapeutic approach in patients with ALI. In this paper, we will explore the potential role
for endogenous iNOS-derived NO in ALI. We will review the evidence for increased iNOS expression and NO production, the effects of non-
selective NOS inhibition, the effects of selective inhibition or deficiency of iNOS, and this concept of cell-source-specific effects of iNOS in both
animal models and human ALI.
D 2005 Elsevier Inc. All rights reserved.
Keywords: Acute lung injury; Nitric oxide; Endothelial cell; Neutrophil; Macrophage; Permeability
1. Introduction
Acute lung injury (ALI) is a major clinical problem,
contributing to the mortality of up to 100,000 critically ill
patients in the United States annually. Intensive clinical and
basic science investigation has led to a greater understanding of
the pathophysiology of ALI. However, this knowledge has yet
to be translated into new anti-inflammatory therapeutic options
for patients with ALI, or into a significant improvement in the
outcome of ALI.
ALI is characterized by up-regulation of a host of
inflammatory mediators, including an increase in the expres-
sion and activity of inducible nitric oxide (NO) synthase
(iNOS), resulting in increased NO production. Although the
1537-1891/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.vph.2005.08.013
Abbreviations: ALI, acute lung injury; ARDS, acute respiratory distress
syndrome; CLP, cecal ligation and perforation; cNOS, calcium-dependent NO
synthase; EC, endothelial cell; iNOS, calcium-independent NO synthase; LPS,
lipopolysaccharide; MPO, myeloperoxidase; 3-NT, 3-nitrotyrosine; NO, nitric
oxide; NOS, NO synthase; SP-A, surfactant association protein-A.
* Tel.: +1 519 667 6723; fax: +1 519 667 6552.
E-mail address: sanjay.mehta@lhsc.on.ca.
increased presence of iNOS and NO in ALI are thought to
contribute importantly to the pathophysiology of ALI, this
remains controversial. Indeed, non-selective inhibition of total
NO production or selective inhibition of iNOS has not been
consistently effective in the treatment of ALI. We have recently
proposed the concept of cell-source-specific effects of NO in
ALI. This idea has potential therapeutic relevance: rather than
inhibition of NO production in all cells, targeted iNOS
inhibition specifically to key individual cells may be a more
effective therapeutic approach in patients with ALI.
In this paper, we will explore the potential role for
endogenous iNOS-derived NO in ALI. We will review the
evidence for increased iNOS expression and NO production, the
effects of non-selective NOS inhibition, the effects of selective
inhibition or deficiency of iNOS, and the concept of cell-source-
specific effects of iNOS in both animal models and human ALI.
2. Acute lung injury
ALI is an important clinical problem which is characterized
by high-protein pulmonary edema and severe hypoxemia and
(2005) 390 – 403
ww
S. Mehta / Vascular Pharmacology 43 (2005) 390–403 391
which is associated with significant morbidity and mortality.
Indeed, ALI and its most severe form, the acute respiratory
distress syndrome (ARDS) have case-fatality rates of approx-
imately 40%, contributing to the mortality of up to 100,000
critically ill patients in the United States annually (Ware and
Matthay, 2000; Rubenfeld, 2003). Despite great advances in
our understanding of the inflammatory basis of the pathophys-
iology of ALI, this inflammation is not addressed by current
ALI therapy (Abraham, 1999; Glauser, 2000; Wheeler and
Bernard, 1999). Treatment of patients with ALI remains limited
to supportive care, e.g. supplemental oxygen and mechanical
ventilation, and thus, mortality has not improved significantly.
Many clinical disorders can result in ALI, either through a
direct pulmonary insult, e.g. gastric acid aspiration, or an
indirect pulmonary insult, e.g. sepsis. The most common cause
of ALI remains systemic sepsis, which occurs in 1% of
hospitalized patients (Bone et al., 1992; Angus et al., 2001).
Regardless of the initiating cause, the pathophysiology of ALI
is characterized by activation of many cells, including
endothelial cells, neutrophils, and macrophages, and increased
synthesis and release of many soluble mediators, such as TNFa
and NO (Bone et al., 1992; Bone, 1994; Neumann et al., 1999).
Neutrophils are central to the microvascular and tissue injury of
ALI (Heflin and Brigham, 1981; Tate and Repine, 1983;
Worthen et al., 1987; Downey et al., 1995; Wagner and Roth,
1999; Doerschuk, 2001). Pulmonary neutrophil infiltration is
the result of a cascade of steps including reduced neutrophil
deformability, pulmonary vascular sequestration (McCormack
et al., 2000), neutrophil–EC adhesion, and trans-EC migration
(Wagner and Roth, 1999; Doerschuk, 2001). Neutrophil
infiltration is facilitated by endothelial cell (EC) barrier
dysfunction (Furie et al., 1987; Garcia et al., 1998; Dull and
Garcia, 2002).
A key step in pulmonary neutrophil infiltration is neutro-
phil–EC adhesion, mediated through the specific interaction of
surface adhesion molecules on each cell, including neutrophil
h2-integrins (e.g. CD11b/CD18) and EC adhesions (e.g.
ICAM-1) (Hynes, 1992; Adams and Shaw, 1994; Hynes,
1992). Following neutrophil–EC adhesion, neutrophils subse-
quently migrate through inter-EC gaps in response to chemo-
taxins (e.g. IL-8 in humans, MIP-2 in mice) (Del Maschio et
al., 1996; Burns et al., 1997; Garcia et al., 1998; Haelens et al.,
1996), putatively through the homotypic interaction of
PECAM-1 on the surface of both neutrophils and EC (Muller
et al., 1993; Schenkel et al., 2004). Neutrophils may contribute
to EC barrier dysfunction and ALI through several mechan-
isms. Neutrophils release a variety of pro-inflammatory factors
(e.g. oxidants, proteolytic enzymes) that may directly injure EC
and contribute to pulmonary oxidant stress (Burg and Pillinger,
2001; Babior et al., 2002; Dull and Garcia, 2002; Allport et al.,
2002). Alternatively, neutrophil–EC adhesion may activate
intracellular signaling cascades in the EC which can result in
barrier dysfunction (Del Maschio et al., 1996; Huang et al.,
1993; Garcia et al., 1998).
The end-result of this complex, integrated inflammatory
response involving cells and soluble mediators is injury and
dysfunction of the alveolar capillary–epithelial permeability
barrier. In direct pulmonary insults, the alveolar epithelial cell
is the principal target, exhibiting features of injury and
apoptosis. In contrast, in sepsis-induced ALI, the pulmonary
microvascular EC is one of the key targets of the systemic
inflammatory response (Curzen et al., 1994; Reinhart et al.,
2002). The importance of EC injury in sepsis has recently been
highlighted, as markers of EC injury correlate with survival in
human sepsis and ALI (Ware et al., 2001).
In summary, injury and death of pulmonary microvascular
EC and alveolar epithelial cells leads to barrier dysfunction and
the leak of plasma into the pulmonary interstitium and alveolar
spaces, resulting in high-protein pulmonary edema, as well as
neutrophil infiltration into the lungs (Wang et al., 2002; Razavi
et al., 2002; Granger and Kubes, 1994; Garcia et al., 1998;
Wagner and Roth, 1999). We will consider the effects of NO on
each of the key pathophysiologic features of ALI, including EC
and alveolar epithelial cell injury and death, pulmonary oxidant
stress, pulmonary protein leak, and pulmonary neutrophil
infiltration.
3. Endogenous NO synthesis in ALI
NO in mammalian cells is produced by a family of NO
synthases (NOS). NOS isoenzymes are classified as low-
output, calcium-dependent (cNOS) or high-output, calcium-
independent, cytokine-inducible NOS (iNOS) isoforms. In the
lung, cNOS is constitutively expressed in EC (ecNOS) and in
neurones (nNOS). cNOS is important in pulmonary homeo-
stasis, including mediating direct and neurogenic pulmonary
vasodilatation, bronchodilation, and immune modulation (Scott
and McCormack, 1999a; Mehta and Drazen, 2000; Scott et al.,
1996). However, pulmonary cNOS has a lesser role in sepsis
and ALI, in which we and others have shown cNOS is down-
regulated (Razavi et al., 2002; Scott et al., 2002; Ermert et al.,
2002).
In contrast, iNOS expression is induced in the majority of
mammalian cell types upon exposure to inflammatory stimuli,
including cytokines, bacteria, and bacterial products (e.g.
lipopolysaccharide [LPS]) (Hibbs et al., 1988; Lamas et al.,
1991; Nakayama et al., 1992; Nathan, 1992; Kolls et al., 1994;
Robbins et al., 1994; Ermert et al., 2002). It should be noted
that there is evidence for constitutive expression of iNOS in
some cells, such as bronchial epithelial cells in humans (Guo et
al., 1995). With regard to ALI, all of the key cellular
participants (e.g. neutrophils, macrophages, EC, epithelial
cells) can express iNOS under inflammatory conditions
(Robbins et al., 1993; Nathan and Hibbs, 1991). Neutrophils
and macrophages express high levels of iNOS and are
important sources of iNOS-derived NO. Moreover, NO
mediates many of the actions of neutrophils and macrophages,
e.g. microbial and tumor cell killing, cytotoxicity (Hibbs et al.,
1988; McCall et al., 1989; Munoz-Fernandez et al., 1992;
Amin et al., 1995; Bratt and Gyllenhammar, 1995; MacMick-
ing et al., 1995; Evans et al., 1996; Tsukahara et al., 1998;
Boyle et al., 2000). Of relevance to ALI in humans, enhanced
iNOS expression and/or activity have been reported in
stimulated human neutrophils and macrophages (Munoz-
S. Mehta / Vascular Pharmacology 43 (2005) 390–403392
Fernandez et al., 1992; Amin et al., 1995; Bratt and
Gyllenhammar, 1995; Evans et al., 1996; Nicholson et al.,
1996; Wheeler et al., 1997; Tsukahara et al., 1998; Kobayashi
et al., 1998; Annane et al., 2000; Boyle et al., 2002; Kooguchi
et al., 2002).
3.1. Evidence for increased NO synthesis in ALI
3.1.1. Animal studies
The intense inflammatory response that characterizes ALI is
associated with increased expression and activity of inducible
nitric oxide (NO) synthase (iNOS), leading to increased NO
production (Table 1) (Mehta et al., 1999; Webert et al., 2000;
Weicker et al., 2001; Razavi et al., 2002; Wang et al., 2002;
Hickey et al., 2002; Koizumi et al., 2004). Increased NO
production in ALI and sepsis has been most commonly
assessed indirectly by measuring the levels of the oxidative
metabolites of NO (nitrites, nitrates) in plasma and bronch-
oalveolar lavage fluid. For example, induction of sepsis in rats
and mice by cecal ligation and perforation (CLP) is associated
with increased plasma nitrites/nitrates (Pheng et al., 1995; Scott
et al., 2002; Wang et al., 2002). Increased BAL nitrites/nitrates
have also been reported in various animal models of ALI, such
as intraperitoneal or intratracheal LPS injection (Pheng et al.,
1995; Li et al., 1998).
Increased NO production in ALI can also be directly
assessed by measuring levels of NO in exhaled breath (Mehta
et al., 1999; Weicker et al., 2001; Zegdi et al., 2002). For
example, systemic LPS administration was associated with
increased exhaled NO in spontaneously breathing mice and
Table 1
Evidence of increased NO production in animal models and in humans with septic
Reference Species Model of ALI
Pheng et al., 1995 Rat i.v. LPS+FNLP
Laszlo et al., 1995 Rat i.v. LPS
Hickey et al., 1997 Mouse i.v. LPS
Kobayashi et al., 1998 Human ARDS
Li et al., 1998 Rat i.t. LPS
Mehta et al., 1999 Pig i.v. LPS
Greenberg et al., 1999 Mouse i.t. LPS
Webert et al., 2000 Rat i.t. Pseudomonas
Okamoto et al., 2000 Rat CLP
Weicker et al., 2001 Mouse i.p. LPS
Sittipunt et al., 2001 Human ARDS
Zhu et al., 2001 Human ARDS
Razavi et al., 2002 Mouse CLP
Scott et al., 2002 Rat CLP
Hickey et al., 2002 Mouse i.v. LPS
Scumpia et al., 2002 Rat i.v. LPS
Soop et al., 2003 Human i.v. LPS
Mikawa et al., 2003 Rabbit i.v. LPS
Raykova et al., 2003 Rat i.p. heat-inactivated GBS
Tsubochi et al., 2003 Rat i.t. LPS
Baron et al., 2004a Mouse Nebulized LPS
Koizumi et al., 2004 Sheep i.v. LPS
Razavi et al., 2005 Mouse CLP
NO, nitric oxide; i.p., intraperitoneal; i.v., intravenous; NOS, NO synthase; iNOS
phenylalanine; ARDS, acute respiratory distress syndrome; i.t., intratracheal; BAL
streptococci; EPR, electron paramagnetic resonance; cGMP, cyclic guanosine mono
anesthetized rats (Weicker et al., 2001; Scumpia et al., 2002).
Similarly, in ALI due to intravenous hydrochloric acid
infusion, exhaled NO was significantly increased in association
with increased pulmonary iNOS expression (Haque et al.,
2003). However, exhaled NO may not necessarily correlate
with the degree of ALI, as suggested in a rat study of ALI due
to extracorporeal circulation (Zegdi et al., 2002).
In most animal models of ALI, especially in small
mammals, this increased NO production is associated with
evidence for increased iNOS expression and/or iNOS activity
in lung tissue (Table 1) (Webert et al., 2000; Razavi et al.,
2002; Wang et al., 2002; Scott et al., 1996; Ermert et al.,
2002; Hickey et al., 2002; Baron et al., 2004a), although this
may not be the case in large animals, e.g. pigs (Mehta et al.,
1999). Importantly, increased iNOS expression and/or activ-
ity have been documented in individual cell populations
including inflammatory cells, e.g. neutrophils, macrophages,
and also pulmonary parenchymal cells, e.g. epithelial,
endothelial, in animal models of ALI (Wang et al., 2001;
Hibbs et al., 1988; Haddad et al., 1994; Liu et al., 1997;
Wheeler et al., 1997; Kobayashi et al., 1998; Ermert et al.,
2002; Baron et al., 2004a). Moreover, there is evidence for
marked cell-specific differences in the regulation of iNOS vs.
cNOS expression in response to inflammatory stimuli, such
as LPS, in different pulmonary cell types (Ermert et al.,
2002).
3.1.2. Human studies
As in animals, there is evidence for increased NO
production in septic humans, as reflected by increased plasma
acute lung injury
Evidence
Increased plasma and BAL nitrites/nitrates
Increased pulmonary NOS activity
Increased pulmonary iNOS mRNA
Increased iNOS protein in alveolar macrophages
Increased BAL nitrites/nitrates
Increased exhaled NO
Increased lung homogenate nitrites/nitrates
Increased plasma nitrites/nitrates and pulmonary iNOS activity
Increased plasma nitrites/nitrates, pulmonary iNOS protein and activity
Increased exhaled NO
Increased BAL nitrites/nitrates, increased iNOS in alveolar macrophages
Increased BAL nitrites/nitrates
Increased plasma nitrites/nitrates and pulmonary iNOS activity
Increased plasma nitrites/nitrates, pulmonary iNOS mRNA and activity
Increased plasma nitrites/nitrates, pulmonary iNOS mRNA and protein
Increased exhaled NO, pulmonary iNOS mRNA and protein
Increased exhaled NO
Increased BAL and plasma nitrites/nitrates
Increased pulmonary iNOS mRNA
Increased pulmonary NO EPR signal, cGMP content, and iNOS protein
Increased iNOS protein in pulmonary parenchymal and inflammatory cells
Increased plasma and lung lymph nitrites/nitrates
Increased plasma nitrites/nitrates and pulmonary iNOS activity
, inducible NO synthase; LPS, lipopolysaccharide; FNLP, formyl–norleucly–
, bronchoalveolar lavage; CLP, cecal ligation and perforation; GBS, Group B
phosphate.
S. Mehta / Vascular Pharmacology 43 (2005) 390–403 393
levels of nitrites/nitrates (Ochoa et al., 1991; Evans et al.,
1993; Groeneveld et al., 1996). Moreover, intravenous
infusion of LPS in healthy volunteers was associated with
increased exhaled NO levels (Soop et al., 2003). In patients
with sepsis, plasma levels of nitrites/nitrates correlate with
severity of illness (APACHE III score) and with measures of
multiple organ dysfunction, such as the Sequential Organ
Failure Assessment (SOFA) score (Ochoa et al., 1991;
Groeneveld et al., 1996; Rixen et al., 1997; Mitaka et al.,
2003).
However, there are few reports of increased NO production
in humans specifically with ALI or ARDS. Two studies have
reported increased nitrites/nitrates in bronchoalveolar lavage
(BAL) fluid in humans either at risk for ARDS or with
confirmed ARDS (Table 1) (Sittipunt et al., 2001; Zhu et al.,
2001).
There is also a paucity of data directly confirming that the
increased NO production in humans with sepsis and ALI is
derived from enhanced iNOS expression and/or activity in
human cells or lung tissue. The increase in BAL nitrites/
nitrates reported in humans either at risk for ARDS or with
confirmed ARDS was associated with increased immunohis-
tochemical staining for iNOS in macrophages isolated from
BAL fluid (Sittipunt et al., 2001). This confirms other reports
of increased human macrophage iNOS expression in ARDS
and pulmonary tuberculosis (Kobayashi et al., 1998; Choi et
al., 2002). Moreover, increased macrophage iNOS was also
found by immunohistochemistry in skin and subcutaneous
tissue samples from humans with sepsis due to soft-tissue
infection (Annane et al., 2000). Although increased neutro-
phil iNOS expression has not been specifically assessed in
patients with ALI or ARDS, it has been recognized that
human neutrophils can increase iNOS expression under
inflammatory conditions, such as sepsis and urinary tract
infection (Wheeler et al., 1997; Amin et al., 1995; Tsukahara
et al., 1998).
Thus, there is evidence for increased pulmonary NO
production due to enhanced pulmonary iNOS expression and/
or activity in both animal models of ALI and in human patients
with ALI/ARDS.
4. Effects of NO in ALI
As reviewed above, there is much evidence that NO
production is increased in ALI. Based on this evidence,
many studies have assessed the effects of NOS inhibition
in animal models of sepsis and ALI (Table 2). The earliest
studies used l-arginine analogues, such as l-nitro-l-
arginine methyl ester (l-NAME) and l-monomethyl-l-
arginine (l-NMMA), which are non-isoform selective
NOS inhibitors and thus, inhibit both cNOS and iNOS.
Subsequently, the effects of iNOS-selective inhibitors, such
as aminoguanidine and 1400W, were assessed in ALI.
Moreover, the severity and characteristics of ALI have been
compared between wild-type and iNOS�/� mice in order to
address the effects of iNOS in ALI. More recently, the
concept of cell-source-specific effects of iNOS has been
supported by studies dissecting out the effects of iNOS
from different cells in ALI.
4.1. General effects of NO in ALI
4.1.1. Animal studies
The earliest studies pursued a role for NO in ALI through
assessment of the effects of non-selective NOS inhibitors. In
one of the first studies to suggest a role for NO in ALI, l-
NAME and l-NMMA attenuated pulmonary microvascular
protein leak in a rat model of ALI due to hindlimb ischemia–
reperfusion (Seekamp et al., 1993). Similarly, non-selective
NOS inhibitors attenuated pulmonary vascular permeability in
various models of ALI including pulmonary radiation, com-
plement deposition, or oxidant stress (Mulligan et al., 1991;
Nozaki et al., 1997; Akai et al., 1998). Interestingly, non-
selective NOS inhibition also attenuated the increase in
alveolar epithelial permeability following intratracheal LPS in
rats (Li et al., 1998).
However, the effects of NO in ALI are controversial, as non-
selective NOS inhibition worsened lung permeability and
injury in other animal models of ALI (Terada et al., 1996;
Pheng et al., 1995; Aaron et al., 1998; Becker et al., 1998; Liu
et al., 2001). Moreover, NOS inhibition may have differential
effects on the different pathophysiologic features of ALI. For
example, although NOS inhibition reduced pulmonary micro-
vascular protein leak and edema in several models of ALI,
pulmonary neutrophil infiltration was not reduced by NOS
inhibition in these models (Mulligan et al., 1991; Seekamp et
al., 1993; O’Donovan et al., 1995; Li et al., 1998). Such
conflicting data on the effects of NO in ALI suggested that
endogenous NO may either prevent or contribute to ALI,
depending on concentration of NO, local physicochemical
conditions, and perhaps the exact cellular sources of NO.
As a result, non-selective NOS inhibition has been of little
overall benefit in animal models of ALI, especially in sepsis.
Non-selective NOS inhibitors improve mean arterial pressure
in septic animals (Kilbourn and Belloni, 1990; Cobb et al.,
1992; Walker et al., 1995; Tracey et al., 1995), but total NOS
inhibition reduces tissue viability, impairs organ function, and
often increases mortality (Harbrecht et al., 1992; Cobb et al.,
1992; Minnard et al., 1994; Lopez et al., 2004). The adverse
effects of non-selective NOS inhibition therapy in sepsis and
ALI are likely due to loss of the beneficial, homeostatic
effects of cNOS-derived NO. Indeed, ecNOS overexpression
in transgenic mice was associated with less mortality and
hypotension, as well as less severe ALI, including reduced
pulmonary neutrophil infiltration, edema, and histologic
injury following intraperitoneal LPS administration (Yama-
shita et al., 2000).
4.1.2. Human studies
As in animal models, several studies of non-selective NOS
inhibition in humans with sepsis have been reported. A
consistent observation has been an increase in mean arterial
pressure, which may be associated with a reduced requirement
for exogenous vasopressors (Nava et al., 1991; Avontuur et al.,
Table 2
Effects of NOS inhibition or deficiency in animal models of septic acute lung injury
Reference Species Model of ALI Method of NOS inhibition/
deficiency
Effects of NOS inhibition/Deficiency
Pheng et al., 1995 Mouse i.p. LPS+FNLP i.v. AG Enhanced septic increase in BAL protein blunted septic increase in
pulmonary neutrophil infiltration
Laszlo et al., 1995 Rat i.v. LPS s.c. l-NAME/l-NMMA Attenuated LPS-induced increase in lung microvascular albumin leak
Hickey et al., 1997 Mouse i.v. LPS iNOS�/� mice Increased septic pulmonary neutrophil infiltration
Li et al., 1998 Rat i.t. LPS i.p. l-NMMA Attenuated LPS-induced increase in lung epithelial permeability
Aaron et al., 1998 Rat i.v. LPS i.v. l-NAME Greater septic increase in interstitial cellular infiltrate
Kristof et al., 1998 Mouse i.v. LPS iNOS�/� mice Attenuated LPS-induced increase in lung microvascular protein leak,
lung W/D weight ratio, and lung 3-NT
Greenberg et al., 1999 Mouse i.t. LPS+Klebsiella iNOS�/� mice, i.p. l-NIL Increased LPS- and Klebsiella-induced pulmonary neutrophil
infiltration, and enhanced Klebsiella clearance
Hinder et al., 1999 Sheep i.v. LPS i.v. l-NAME Aggravated LPS-induced increase in extravascular lung water
Okamoto et al., 2000 Rat CLP s.c. ONO-1714 Attenuated septic increase in histologic peribronchiolar edema and
interstitial area, no effect on lung W/D weight ratio
Evgenov et al., 2000 Sheep i.v. LPS i.v. AG Enhanced LPS-induced increases in lung lymph flow and protein
clearance, but unchanged lung water; improved pulmonary gas
exchange
Wang et al., 2002 Mouse CLP iNOS�/� mice, i.v. 1400W Abrogated septic increase in pulmonary microvascular albumin leak;
unchanged septic increase in pulmonary neutrophil infiltration
Volman et al., 2002 Mouse i.p. LPS+zymosan iNOS�/� mice, i.p. AG No effect on LPS/zymosan-induced increases in lung weight and
histologic ALI score
Speyer et al., 2003 Mouse i.t. LPS iNOS�/� mice Enhanced LPS-induced increases in pulmonary neutrophil infiltration
and pulmonary MCP-1 production
Mikawa et al., 2003 Rabbit i.v. LPS i.v. ONO-1714 Attenuated LPS-induced increases in lung W/D weight ratio,
histologic ALI score, and BAL neutrophils and albumin
Raykova et al., 2003 Rat i.p. heat-inactivated GBS i.p. AG Attenuated septic increases in lung IL-6 and MIP-2 mRNA expression
Tsubochi et al., 2003 Rat i.t. LPS s.c. AG Reversed the LPS-induced early decrease and late increase in alveolar
fluid clearance
Razavi et al., 2004 Mouse CLP iNOS�/� mice Attenuated septic pulmonary microvascular neutrophil sequestration;
enhanced septic increase in pulmonary neutrophil infiltration
Baron et al., 2004a Mouse Nebulized LPS iNOS�/� mice Attenuated LPS-induced increases in lung tissue resistance and
elastance attenuated LPS-induced surfactant dysfunction and loss of
SP-B expression
Koizumi et al., 2004 Sheep i.v. LPS i.v. ONO-1714 Attenuated LPS-induced impaired oxygenation and increase in lung
lymph flow
Razavi et al., 2005 Mouse CLP iNOS�/� mice Eliminated septic increase in pulmonary 8-isoprostanes and 3-NT
ALI, acute lung injury; AG, aminoguanidine; l-NAME, l-nitro-arginine methyl ester; l-NMMA, l-N-monomethyl arginine; W/D, wet-to-dry weight ratio; 3-NT, 3-
nitro-tyrosine; l-NIL, l-N6-iminoethyllysine; ONO-1714, (1S, 5S, 6R, 7R)-7-chloro-3-imino-5-methyl-2-azabicyclo[4,1,0]heptane HCl; 1400W, N-(3-
(aminomethyl)benzyl) acetamidine; MCP-1, monocyte chemoattractant peptide-1; IL-6, interleukin-6; MIP-2, macrophage inflammatory protein-2; SP-B,
surfactant-association protein-B; for others, see legend in Table 1.
S. Mehta / Vascular Pharmacology 43 (2005) 390–403394
1998; Grover et al., 1999). However, increased mean arterial
pressure appears to be mediated by systemic vasoconstriction,
often in concert with reduced blood flow, impaired cardiac
function, and possibly increased mortality (Nava et al., 1991;
Avontuur et al., 1998; Grover et al., 1999; Watson et al., 2004).
Unfortunately, there are no data on the effects of non-selective
NOS inhibition on the overall severity or the individual
pathophysiologic features of ALI in humans. A role for NO
in human pulmonary artery EC injury has been suggested in
vitro. When these EC were co-cultured with LPS-activated
monocytes, EC injury was attenuated by addition of carboxy-
PTIO, a NO scavenger (Hidaka et al., 1997). In contrast, when
stimulated neutrophils were co-cultured with these human EC,
treatment with either carboxy-PTIO or l-NAME exacerbated
neutrophil-dependent EC injury, putatively due to loss of
ecNOS-derived NO. Thus, as in animal models of ALI, NO
may worsen or improve human pulmonary EC injury, depend-
ing on the cellular sources and amount of NO released.
4.2. Specific effects of iNOS-derived NO in ALI
4.2.1. Animal studies
As reviewed above, iNOS expression is increased in many
cells in the lung and endogenous NO production is markedly
increased in ALI. The role of iNOS in ALI has been addressed
in various animal models of ALI through study of the effects of
selective iNOS inhibition and through characterization of
differences in ALI in wild-type mice vs. iNOS�/� ‘‘knockout’’
mice. Most studies demonstrate that iNOS-derived NO is
clearly involved in the pathophysiology of ALI. However, the
precise role of iNOS in ALI remains controversial. Indeed,
some studies suggest that iNOS-derived NO may not play a
role in ALI, or may attenuate the severity of ALI (Pheng et al.,
1995; Volman et al., 2002). For example, iNOS inhibition or
deficiency had no effect on increased lung weight and
macroscopic pulmonary hemorrhage in mice treated with
intraperitoneal LPS and zymosan (Volman et al., 2002).
S. Mehta / Vascular Pharmacology 43 (2005) 390–403 395
It is generally accepted that the weight of the published
evidence suggests that iNOS contributes significantly to the
key pathophysiologic features of ALI, such as protein-rich
pulmonary edema, oxidant stress, and surfactant dysfunction
(Mikawa et al., 1998; Scott and McCormack, 1999b; Mehta et
al., 1999; Bateman et al., 2001; Wang et al., 2002; MacMicking
et al., 1995; Groeneveld et al., 1996; Ruetten et al., 1996;
Kristof et al., 1998; Vincent et al., 2000; Fakhrzadeh et al.,
2002; Baron et al., 2004a; Razavi et al., 2005). Many studies
have reported that iNOS mediates microvascular injury and
protein-rich pulmonary edema in a variety of animal models of
ALI, such as endotoxemia, sepsis, ozone inhalation, intra-
pleural carrageenan, and combined cutaneous burn and smoke
inhalation (Table 2) (Kristof et al., 1998; Cuzzocrea et al.,
2000; Wang et al., 2002; Fakhrzadeh et al., 2002; Enkhbaatar et
al., 2003). For example, in mice undergoing CLP, sepsis-
induced pulmonary protein leak was completely iNOS-depen-
dent, as it was abolished both by selective iNOS inhibition with
1400W in wild-type (iNOS+/+) mice, and by iNOS deficiency
in iNOS�/� mice (Wang et al., 2002). Similarly, pulmonary
oxidant stress, reflected by lung 8-isoprostane levels, was
evident in septic wild-type but not iNOS�/� mice (Razavi et
al., 2005). Moreover, in a recent study of mice exposed to
nebulized LPS, iNOS�/� mice were protected from LPS-
induced pulmonary physiologic dysfunction (increased tissue
resistance and elastance), surfactant dysfunction, and reduced
SP-B mRNA and protein expression (Baron et al., 2004a).
The effects of iNOS inhibition or deficiency on pulmonary
leukocyte infiltration in ALI are unresolved. In our studies of
septic mice, although iNOS deficiency or inhibition reduced
the severity of ALI, e.g. decreased pulmonary microvascular
protein leak and oxidant stress, pulmonary leukocyte infiltra-
tion was not attenuated (Wang et al., 2002; Razavi et al., 2004,
2005). Indeed, some studies have even demonstrated increased
pulmonary leukocyte infiltration in ALI following iNOS
inhibition or in iNOS�/� mice (Hickey et al., 1997; Razavi et
al., 2004). These conflicting data may be due to the technique
for measurement of pulmonary neutrophil infiltration in ALI.
For example, a common technique is the measurement of
pulmonary tissue myeloperoxidase (MPO) activity. However,
recent evidence suggests that neutrophils release MPO into the
circulation or directly at the surface of EC (Baldus et al., 2001;
Eiserich et al., 2002). Thus, increased pulmonary MPO in ALI
may reflect, in part, adherence of circulating or locally released
MPO to pulmonary microvascular EC, and may not be due
solely to pulmonary neutrophil infiltration.
MPO in a homogenate of pulmonary tissue also ignores the
potential differential presence of neutrophils in discrete
compartments, such as the pulmonary microvasculature,
interstitial space, and bronchoalveolar airspace. Indeed, in
mice with CLP-induced sepsis, we have consistently found
similar pulmonary tissue MPO activity in wild-type and
iNOS�/� mice (Wang et al., 2002; Razavi et al., 2004,
2005). However, when we analyzed neutrophil presence in
the different compartments, there were striking differences
between septic wild-type and iNOS�/� mice (Razavi et al.,
2004). Blood neutrophil counts were similar in septic iNOS�/�
mice vs. wild-type mice. However, pulmonary intravital
videomicroscopy during mechanical ventilation demonstrated
a significant increase in pulmonary microvascular sequestered
neutrophils in septic wild-type mice, which was attenuated in
iNOS�/� mice. Moreover, the septic increase in bronchoalveo-
lar lavage neutrophil counts in wild-type mice was significantly
enhanced in iNOS�/� mice. Thus, the presence of iNOS in
septic ALI appears to increase pulmonary microvascular
sequestration and possibly EC adhesion, and appears to retard
the trans-EC migration and pulmonary tissue infiltration of
neutrophils.
A few studies have reported variable effects of NO and
NOS inhibition, depending on the time point at which ALI is
assessed. For example, selective inhibition of iNOS with
ONO-1714 improved oxygenation, lung mechanics, pulmo-
nary edema, and leukocyte sequestration when administered
prior to or within 2 h after induction of ALI by intravenous
LPS injection in mechanically ventilated rabbits, but not if
administered 3 or 4 h after LPS (Mikawa et al., 2003). In
contrast, iNOS inhibition with ONO-1714 in rats with CLP-
induced ALI improved survival and pulmonary histology,
including edema, leukocyte infiltration, and microvascular
thrombi, when administered 12 h after CLP, but not when
administered immediately or 6 h after CLP (Okamoto et al.,
2000). Moreover, a recent study reported that iNOS reduced
alveolar fluid clearance at 6 h, but increased it at 24 h after
intratracheal LPS in rats (Tsubochi et al., 2003). The
conflicting data on the effects of NO at different time points
in the natural history of ALI remain unresolved, but may also
reflect the different effects of iNOS-derived NO from discrete
cell populations.
The effect of iNOS inhibition or iNOS deficiency on
mortality in the setting of ALI is also controversial. For
example, in septic models of ALI, iNOS inhibition or
deficiency does not improve and may even increase mortality
(Laubach et al., 1995; MacMicking et al., 1995; Cobb et al.,
1999). In contrast, others have suggested improved mortality
due to iNOS inhibition in septic rats and mice (Hollenberg et
al., 2000; Okamoto et al., 2000; Baron et al., 2004b).
4.2.2. Human studies
iNOS-dependent pathophysiologic changes in human ALI
have been difficult to demonstrate directly. Indeed, the effects
of iNOS-selective inhibitors have not been reported in
humans with ALI/ARDS. However, there are data that
suggest that human cells mediate injury through iNOS
expression and activity, similar to animal cells. For example,
as described for animal cells, human neutrophils also mediate
bacterial killing through iNOS (Malawista et al., 1992). In
addition, in human pulmonary artery EC co-cultured with
human neutrophils, neutrophil-dependent EC injury was
iNOS-dependent (Hidaka et al., 1997). In our own studies
of microvascular EC isolated directly from human lungs and
co-cultured with human neutrophils, we have found that
cytomix-stimulated trans-EC protein leak was clearly iNOS-
dependent, being eliminated by pre-treatment with either
1400W or l-NAME.
S. Mehta / Vascular Pharmacology 43 (2005) 390–403396
Thus, iNOS-derived NO likely contributes significantly to
the pathophysiology of human ALI, although direct evidence is
not yet available.
4.3. Effects of cell-source-specific iNOS in ALI
4.3.1. Animal studies
Several lines of evidence have suggested the possibility of
differential effects of iNOS-derived NO from different cells in
ALI. As above, this includes the controversial effects of iNOS
inhibition or deficiency on the individual pathophysiologic
effects of ALI, as well as on mortality. Similarly, the effects of
iNOS-derived NO from different cells may explain the
conflicting data from iNOS inhibition at different time points
after the induction of ALI. Over the past few years, we and
other groups have designed and pursued studies to dissect out
the potential differential effects of iNOS in different cells in the
setting of murine septic ALI.
In order to investigate this concept of cell-source-specific
effects of iNOS-derived NO in ALI, we have generated and
used reciprocal bone marrow transplanted iNOS chimeric mice
(Wang et al., 2001, 2002; Razavi et al., 2004, 2005). Following
bone-marrow ablative irradiation in a wild-type mouse, the
bone marrow is reconstituted over 4–6 weeks following
intravenous infusion of donor bone marrow from an iNOS�/
� mouse. In this mouse, all parenchymal cells, e.g. endothelial
and epithelial, retain the iNOS+/+ genotype of the host.
However, all bone marrow derived inflammatory cells, e.g.
neutrophils and macrophages, were depleted and replaced by
donor iNOS�/� cells. Thus, this mouse is designated a � to +
iNOS chimera, and has iNOS expression restricted to host
parenchymal cells only. The reciprocal + to � iNOS chimera is
also generated by transplantation of iNOS+/+ bone marrow into
irradiated iNOS�/� mice, thus restricting iNOS expression to
bone marrow-derived inflammatory cells only. These two
reciprocal iNOS chimeric mice are genetically identical except
for the cellular population distribution of iNOS. In order to
assess the role of iNOS specifically in parenchymal vs.
inflammatory cells, we compared pulmonary iNOS expres-
sion/activity and the pathophysiologic features of sepsis-
induced ALI between these two reciprocal iNOS chimeric
mice.
Firstly, there is good evidence that marked differences exist
in the level of iNOS expression and NO production from these
two different cell populations. In intraperitoneal LPS-treated
mice, we found that pulmonary iNOS expression/activity was
predominantly localized to parenchymal cells (¨70%) rather
than inflammatory cells (Wang et al., 2001). Moreover,
systemic NO production, reflected by plasma levels of
nitrites/nitrates, was almost exclusively derived from paren-
chymal cell iNOS: LPS treatment was associated with a
marked increase in plasma nitrites/nitrates in � to + iNOS
chimeras (iNOS localized to parenchymal cells), similar to
LPS-treated wild-type mice, with only a negligible LPS-
induced increase in plasma nitrites/nitrates in + to � iNOS
chimeras (iNOS localized to inflammatory cells). Other groups
have confirmed and supported our findings (Hickey et al.,
2002; Baron et al., 2004a). Interestingly, in a more clinically
relevant disease model of sepsis, induced by CLP, pulmonary
iNOS expression/activity were similar in septic + to � and � to
+ iNOS chimeric mice, suggesting a significant level of iNOS
expression in both inflammatory and parenchymal cells (Wang
et al., 2002; Razavi et al., 2005). Of note, systemic NO
production was still largely dependent on parenchymal cell
iNOS in CLP-induced sepsis. Thus, both pulmonary paren-
chymal and inflammatory cells express iNOS in ALI. Asses-
sing ALI in the two reciprocal iNOS chimeric mice can then
differentiate the specific effects of iNOS from these distinct
cell populations.
We initially compared pulmonary microvascular protein
leak following CLP-induced sepsis in the reciprocal iNOS
chimeric mice (Wang et al., 2002). Sepsis was associated with
significant pulmonary microvascular protein leak in + to �iNOS chimeras (iNOS localized to inflammatory cells), similar
in degree to septic wild-type mice. In contrast, sepsis-induced
pulmonary microvascular protein leak was completely abro-
gated in � to + iNOS chimeras (iNOS localized to parenchy-
mal cells), similar to iNOS�/� mice. Moreover, we recently
showed that sepsis-induced pulmonary oxidant stress, as
reflected by tissue 8-isoprostane levels, was also present in +
to �, but not in � to + iNOS chimeras (Razavi et al., 2005).
Thus, iNOS-derived NO from inflammatory cells (e.g. neu-
trophils, macrophages) totally accounted for sepsis-induced
pulmonary protein leak and oxidant stress, key pathophysio-
logic features of ALI, with no obvious contribution of
parenchymal cell iNOS. Ongoing studies in our lab are
dissecting out the individual roles of macrophage and
neutrophil iNOS in murine ALI. Preliminary evidence suggests
that both macrophage and neutrophil iNOS contribute impor-
tantly to pulmonary microvascular EC injury and protein leak
(Fig. 1).
Based on the work of others using these same bone-marrow
transplanted iNOS chimeric mice, selective iNOS expression in
parenchymal cells may also contribute to some pathophysio-
logic features of ALI. In mice exposed to nebulized LPS, lung
physiologic dysfunction and reduced SP-B expression were
specifically dependent on iNOS expression in parenchymal
cells (Baron et al., 2004a).
We have also recently reported cell-source-specific effects
of iNOS on the trans-endothelial migration and pulmonary
infiltration of neutrophils (Razavi et al., 2004). The specific
presence of iNOS in inflammatory cells alone in + to � iNOS
chimeric mice was associated with sepsis-induced pulmonary
microvascular sequestration and increased BAL neutrophils
similar to levels seen in wild-type mice. In contrast, septic
pulmonary microvascular sequestration was reduced and BAL
neutrophilia increased in � to + iNOS chimeric mice, similar
to the findings in septic iNOS�/� mice. Thus, iNOS
specifically in inflammatory cells (e.g. neutrophils and
macrophages) promotes pulmonary microvascular neutrophil
sequestration, yet inhibits the trans-EC migration and
pulmonary infiltration of neutrophils in septic ALI. Despite
the significant levels of parenchymal cell iNOS, this does not
appear to modulate pulmonary neutrophil kinetics in septic
Fig. 1. Schematic representation of the cell-source specific effects of inducible nitric oxide synthase (iNOS) in septic acute lung injury. iNOS in neutrophils (PMN)
and in alveolar macrophages stimulates (+) pulmonary microvascular leak of plasma protein. PMN iNOS also stimulates PMN sequestration in the pulmonary
microvasculature, but inhibits (�) trans-endothelial PMN migration. iNOS in types I and II alveolar epithelial cells (AEC I and II) appears to mediate surfactant
dysfunction in ALI. The role of iNOS in endothelial cells (EC) in ALI is presently unknown.
S. Mehta / Vascular Pharmacology 43 (2005) 390–403 397
mice. It is noteworthy that previous reports have suggested a
similar inhibitory effect of neutrophil iNOS on peritoneal
neutrophil migration during peritoneal sepsis (Tavares-Murta
et al., 1998; Crosara-Alberto et al., 2002; Benjamim et al.,
2002). We have tried to corroborate our in vivo findings in
murine septic ALI by co-culturing mouse lung microvascular
EC with neutrophils in vitro (Razavi et al., 2004). Indeed,
neutrophil iNOS significantly inhibited cytokine-stimulated
trans-EC neutrophil migration, whereas EC iNOS presence
had no effect. Of note, cytokine-stimulated neutrophil
migration across a matrix in the absence of EC was similar
for wild-type and iNOS�/� neutrophils. Mechanistically, this
suggests that neutrophil iNOS inhibits trans-EC neutrophil
migration through modulation of neutrophil–EC interaction,
and not through alteration of neutrophil deformability or
intrinsic migratory capacity.
4.3.2. Human studies
It is becoming clear that the concept of cell-source-specific
effects of iNOS has relevance to human disease as well. For
example, compartmentalization of iNOS expression specifical-
ly to macrophages has been reported in necrotic skin and
muscle from humans with sepsis due to cellulitis (Annane et
al., 2000). Moreover, an immunohistochemical study has
suggested cell-source-specific nitrosative and oxidative effects
of iNOS-derived NO in human inflammatory bowel disease
(Tomobuchi et al., 2001).
It remains unknown whether this concept of cell-source-
specific effects of iNOS has clinical relevance for human
ALI/ARDS. In our ongoing studies of human lung
microvascular EC co-cultured with human neutrophils and
macrophages, we are attempting to dissect out the effects of
iNOS in neutrophils and macrophages in septic human EC
injury.
4.4. Mechanisms of NO’s effects in ALI
4.4.1. Animal studies
NO exerts its effects through many different mechanisms,
such as the activation of soluble guanylate cyclase and
generation of cGMP, nitrosation of protein thiols, and
formation of nitrosyl complexes with metal ions in the active
sites of key proteins (Ignarro, 1991; Abu-Soud and Hazen,
2000; Mohr et al., 1999). In the setting of ALI and increased
iNOS expression and enhanced NO production, NO’s effects
are largely not mediated through cGMP.
The interaction of NO with reactive oxygen species (ROS)
is a major mechanism of NO’s effects in the setting of
inflammation associated with increased production of both NO
and ROS, as in ALI. The rapid, diffusion-limited reaction of
NO with superoxide anion, generating the potent oxidants
peroxynitrite and subsequently hydroxyl radical, may be one of
the most important mechanisms by which iNOS-derived NO
exerts pro-oxidant, pathophysiologic effects in ALI (Rubbo et
al., 1994; Radi et al., 1991a,b; Beckman et al., 1990). The
nitration of tyrosine residues in specific proteins, yielding 3-
nitrotyrosine residues, is a putative marker of NO-dependent
oxidant stress and peroxynitrite action.
The enhanced presence of 3-nitrotyrosine in pulmonary
tissue and BAL fluid has been reported in several animal
models of ALI (Cuzzocrea et al., 1999; Tsuji et al., 2000;
Fakhrzadeh et al., 2002; Chen et al., 2003; Laffey et al., 2004).
Moreover, pulmonary 3-nitrotyrosine presence in ALI appears
to be iNOS-dependent (Tsuji et al., 2000; Fakhrzadeh et al.,
S. Mehta / Vascular Pharmacology 43 (2005) 390–403398
2002; Chen et al., 2003; Razavi et al., 2005). Similar to
pulmonary microvascular leak and oxidant stress, we recently
showed in reciprocal iNOS chimeric mice that pulmonary 3-
nitrotyrosine presence in septic ALI was specifically dependent
on the expression of iNOS in inflammatory cells, and not in
pulmonary parenchymal cells (Razavi et al., 2005). Finally,
exogenous peroxynitrite induces EC barrier dysfunction, in
association with actin and h-catenin nitration (Knepler et al.,
2001).
NO may also contribute to the pathophysiology of ALI
through modulation of the production of other inflammatory
mediators, including cytokines and chemokines (Walley et
al., 1999; Raykova et al., 2003; Speyer et al., 2003). As
above, the precise effects of NO on pulmonary cytokine
production are unclear. For example, selective iNOS
inhibition by aminoguanidine decreased pulmonary IL-6
and MIP-2 expression in rats with sepsis-induced ALI due
to Group B Streptococci (Raykova et al., 2003). In contrast,
l-NAME increased pulmonary IL-6 and TNF-a expression in
mice with ALI following intratracheal LPS (Walley et al.,
1999). Similarly, increased lung neutrophil infiltration was
associated with greater BAL levels of CC chemokines (e.g.
MCP-1 and MCP-3) in iNOS�/� vs. wild-type mice
following intratracheal LPS (Speyer et al., 2003). Moreover,
in vitro studies supported cell-source-specific effects of
iNOS in modulation of chemokine production: iNOS�/�
dermal microvascular EC and peritoneal macrophages both
demonstrated greater chemokine production following LPS/
IFN-gamma stimulation than wild-type cells (Speyer et al.,
2003).
4.4.2. Human studies
As in animal models of ALI, there is evidence that NO-
dependent peroxynitrite production may be the mechanism of
iNOS-dependent ALI in humans. Enhanced immunohisto-
chemical staining for 3-nitrotyrosine is found in isolated cells
and tissues from humans with ARDS (Haddad et al., 1994;
Kooy et al., 1995; Kobayashi et al., 1998; Sittipunt et al.,
2001). Increased 3-nitrotyrosine residues have also been found
in proteins isolated from BAL in humans with ALI/ARDS
(Lamb et al., 1999; Zhu et al., 2001). For example, nitration of
surfactant-associated protein (SP)-A was recently identified
(Zhu et al., 2001). Importantly, protein nitration can modify the
function of many proteins, and appears to be associated with
impairment of the immune modulatory functions of SP-A, such
as mannose binding ability and opsonization of pathogens (Zhu
et al., 1996, 1998).
There is also direct evidence for peroxynitrite production by
human neutrophils following LPS treatment (Gagnon et al.,
1998). Moreover, exogenous nitrite and peroxynitrite were
found to activate the nuclear transcription factor kB (NF-kB) in
cultured human alveolar epithelial cells, similar to the
activation seen upon treatment of these cells with BAL fluid
from ARDS patients (Nys et al., 2003). Thus, endogenous
iNOS-dependent local pulmonary production of peroxynitrite
may be responsible for alveolar epithelial and capillary
endothelial injury in ALI/ARDS.
5. Summary and future directions
Over the past few years, it has become clear that NO likely
plays a significant role in the pathophysiology of ALI in both
animals and humans. There is much evidence both in animal
models and in humans with ALI for enhanced pulmonary
expression of iNOS and increased NO production. Although
non-selective NOS inhibitors can effectively reduce NO
overproduction in animals and humans with ALI, the lack of
clinical benefit of this therapeutic approach has tempered
excitement around use of these inhibitors in human ALI/
ARDS. Similarly, complete iNOS inhibition or genetic
deficiency has not been consistently beneficial in animal
models of ALI. Rapidly evolving new ideas on the concept
of cell-source-specific effects of iNOS-derived NO in ALI may
explain in large part the lack of benefit in previous studies of
non-selective NOS inhibition or complete iNOS inhibition in
all cells.
Given the clear importance of endogenous NO in ALI, there
is an increasing focus on the measurement of increased NO
levels in human ALI using novel approaches, such as collection
and analysis of exhaled breath and exhaled breath condensate.
Such approaches are very likely to advance our understanding
of the effects of NO specifically in human ALI/ARDS.
Moreover, this increasing understanding of the role of NO in
ALI will likely also push us further towards therapeutic
manipulation of the NO system in human ALI/ARDS. Indeed,
ongoing studies will identify the specific effects of iNOS from
individual cells, e.g. neutrophils, macrophages. We suggest this
knowledge will permit targeted iNOS inhibition in these
individual cells and will be more beneficial in ALI than non-
targeted, widespread iNOS inhibition in many cells. Indeed,
this potential therapeutic approach is a reality, as novel
liposomal protein-grafting technology has already permitted
specific delivery of chemotherapeutic agents to individual
cancer cells (Hatipoglu et al., 1998; Dagar et al., 2001).
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