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Review article: cellular and molecular mechanisms of

NSAID-induced peptic ulcers


C. MUSUMBA* , , D. M. PRI TCHARD, & M. PI RMOHAMED* ,
*Department of Pharmacology and
Therapeutics, School of Biomedical
Sciences, University of Liverpool,
Liverpool, UK; Gastroenterology
Research Unit, School of Clinical
Sciences, University of Liverpool,
Liverpool, UK and The Royal
Liverpool and Broadgreen University
Hospitals NHS Trust, Liverpool, UK
Correspondence to:
Prof. M. Pirmohamed, Department of
Pharmacology and Therapeutics, The
University of Liverpool, Ashton Street,
Liverpool, L69 3GE, UK.
E-mail: munirp@liv.ac.uk
Publication data
Submitted 11 January 2009
First decision 3 February 2009
Resubmitted 29 June 2009
Accepted 1 July 2009
Epub Accepted Article 3 July 2009
SUMMARY
Background
Nonsteroidal anti-inammatory drugs (NSAIDs) are some of the most
prescribed drugs worldwide and have now probably overtaken Helicob-
acter pylori as the most common cause of gastrointestinal injury in
Western countries. Further understanding of the pathogenesis of
NSAID-induced ulcers is important to enable the development of novel
and effective preventive strategies.
Aims
To provide an update on recent advances in our understanding of the
cellular and molecular mechanisms involved in the development of
NSAID-induced ulcers.
Methods
A Medline search was performed to identify relevant literature using
search terms including nonsteroidal anti-inammatory drugs, aspirin,
gastric ulcer, duodenal ulcer, pathogenesis, pharmacogenetics.
Results
The mechanisms of NSAID-induced ulcers can be divided into topical
and systemic effects and the latter may be prostaglandin-dependent
(through COX inhibition) or prostaglandin-independent. Genetic factors
may play an important role in determining individual predisposition.
Conclusions
The pathogenesis of NSAID-induced peptic ulcers is complex and multi-
factorial. Recent advances in cellular and molecular biology have high-
lighted the importance of various prostaglandin-independent
mechanisms. Pharmacogenetic studies may provide further insights into
the pathogenetic mechanisms of NSAID-induced ulcers and help iden-
tify patients at increased risk.
Aliment Pharmacol Ther 30, 517531
Alimentary Pharmacology & Therapeutics
2009 Blackwell Publishing Ltd 517
doi:10.1111/j.1365-2036.2009.04086.x
I NTRODUCTI ON
Nonsteroidal anti-inammatory drugs cause gastroin-
testinal injury through both topical and systemic
effects; the latter is mediated principally by blocking
prostaglandin synthesis through inhibition of the cy-
clooxygenase (COX) enzymes, COX-1 and COX-2.
However, there is irrefutable evidence that COX inhibi-
tion is not the sole mechanism of NSAID-induced
gastrointestinal injury and that other prostaglandin-
independent mechanisms are important in ulcer patho-
genesis, as shown in Figure 1. Following mucosal
injury, NSAIDs inhibit both the early events of restitu-
tion necessary to repair the initial supercial injury as
well as the later events of cell proliferation and angio-
genesis, leading to delayed ulcer healing. Recent stud-
ies have highlighted the contribution of a number of
autacoids acting in concert with prostaglandins in
maintaining the gastric mucosal defence and inducing
healing including the gaseous mediators nitric oxide
and hydrogen sulphide (H
2
S), growth factors, neuro-
peptides (calcitonin gene related peptide; CGRP and
substance P), hormones (such as melatonin and gas-
trin), polyamines, stress proteins and matrix metallo-
proteinases. These components sub-serve similar
functions and have a dynamic interaction, such that
when one of the mediators is depressed, there is a
compensatory increase in the others. Hence, greater
mucosal injury is seen following administration of
NSAIDs when this compensatory mechanism is
impaired.
The likelihood of developing an ulcer and or its
complications depends on a complex interplay of fac-
tors that can vary between individuals such as the
pharmacokinetic and pharmacodynamic properties of a
particular NSAID, as well as the interaction of the
drug with pre-existing risk factors for NSAID-induced
ulcers. In this review, we summarize some of the
recent advances from basic and clinical research, per-
formed mainly in animal models, which have
enhanced our understanding of the molecular and cel-
lular mechanisms involved in NSAID-induced ulcers,
and highlight the pharmacogenetic studies undertaken
in humans in this eld to date.
TOPI CAL EFFECTS
Topical injury initiates the initial mucosal erosions by
disrupting the gastric epithelial cell barrier, but prosta-
glandin depletion appears essential for the development
of clinically signicant gastric and duodenal ulcers.
1
The ability of a NSAID to cause short-term topical
injury depends on its pKa (a measure of its acidity which
is also crucial for the inhibition of COX enzymes) and
lipid solubility (log P) and can be dissociated from its
ability to inhibit COX-1 and deplete gastric mucosal
prostaglandins. Most NSAIDs are weak organic acids
(pKa 35) containing a monocarboxylic acid group in
their structure. The acidity and lipid solubility of hetero-
cyclic carboxylic acids are interdependent; the carbox-
ylic group of NSAIDs also contributes signicantly to
their water solubility and confers their detergent proper-
ties (important in their interaction with surface mem-
brane phospholipids), their rate of entry into the gastric
cells and the degree of intracellular ion trapping.
2
Studies in both isolated mitochondria and various
cells have shown that NSAIDs accumulate in gastric epi-
thelial cells through ion trapping, with subsequent
uncoupling of mitochondrial oxidative phosphorylation
and inhibition of the electron transport chain. This
results in depletion of intracellular ATP, cellular Ca
2+
toxicity and generation of reactive oxygen species
(ROS) like superoxide and hydroxyl radicals. ROS may
directly oxidize cellular proteins, lipids or nucleic acids
and cause general damage or dysfunction or may initi-
ate the cell death process through affecting various sig-
nalling cascades leading to necrosis and apoptosis.
3
Uncoupling oxidative phosphorylation also dissipates
the mitochondrial transmembrane potential and may
lead to apoptosis by releasing cytochrome c.
4
Further-
more, NSAIDs have been shown to attenuate the hydro-
phobic surface barrier of the stomach by chemically
associating with extracellular phospholipids present
within and on the surface of the gastrointestinal mucus
gel layer, disrupting its hydrophobic barrier proper-
ties.
5, 6
NSAIDs also disrupt membrane phospholipids
and increase permeability by inducing changes in mem-
brane hydrophobicity, uidity, thickness, bending stiff-
ness, and pore formation. This allows acid back
diffusion, leading to cell death by apoptosis and necro-
sis and development of gastric ulcers.
6
Aspirin has been
demonstrated to produce ulcers in mice only when
given orally, in contrast to other NSAIDs such as indo-
methacin which cause injury regardless of route of
administration (despite similar decrease in mucosal
prostaglandin levels by both agents), emphasizing the
importance of topical injury in aspirin-induced ulcers.
7
Oxidative stress involvement in NSAID-induced
injury has been recently demonstrated by using gene-
expression studies in the rat stomach. These
518 C. MUSUMBA et al.
Aliment Pharmacol Ther 30, 517531
2009 Blackwell Publishing Ltd
revealed increased expression of genes involved in oxi-
dative stress such as those involved in glutathione
metabolism.
8
Chattopadhyay et al. showed that
indomethacin treatment induced oxidative stress in rat
gastric mucosa by the irreversible inactivation of
gastric peroxidase.
9
Several mechanisms have been pro-
posed for NSAID-induced apoptosis ranging from cas-
pase-dependent to caspase-independent effects, some of
which are summarized in Table 1. This process seems
to be independent of their COX inhibiting ability,
NSAIDs
CSE COX-1 COX-2 ODC
Polyamines
Gastric
GMBF
hypermotility,
Permeability,
Acid
cNOS
NO,H
2
S,
IL-1,
CGRP,MT
SPs,TFFs
ATL
Reduced
prostaglandins
NO
H
2
S
Ulcer
Mucus,PLs,
bicarbonate
Proinflammatory mediators
Neutrophil adherence
Disruption of
extracellular and
membrane PLs
Permeability
Uncoupling of
oxidative
phosphorylation
Topical
Aspirin
Reduced
prostaglandins
Release of growth
factors
ROS,apoptosis
and necrosis
Acute mucosal
injury,erosions
Impaired mucosal
repair and healing
Impaired mucosal
repair and healing
ECE-1
ROS and cell damage/death by
necrosis and apoptosis
Figure 1. Cellular and molecular pathogenesis of NSAID-induced ulcers: NSAIDs cause ulcers by both topical and systemic
effects, and the latter may be prostaglandin-dependent (through COX-1 and COX-2 inhibition), or prostaglandin-indepen-
dent (e.g. through inhibition of NO, H
2
S and polyamines). Depletion of mucosal prostaglandins causes a compensatory
increase (green arrows) in the other mediators of gastric mucosal defence to limit and repair injury. Ulcers develop when
these compensatory mechanisms are overwhelmed. Aspirin uniquely acetylates COX-2, which is then still able to metabolize
arachidonic acid to the gastroprotective aspirin triggered lipoxin (ATL); COX-2: cyclooxygenase-2; COX-1: cyclooxygen-
ase-1; PLs: phospholipids; CSE: cystathione-c-lyase; ECE-1: endothelin-converting enzyme-1; cNOS: constitutive nitric
oxide synthase; ODC: ornithine decarboxylase; NO: nitric oxide; H
2
S: hydrogen sulphide; IL-1b: interleukin-1b; CGRP:
calcitonin gene related peptide; MT: melatonin; SPs: stress proteins; TFFs: trefoil factors; GMBF: gastric mucosal blood
ow; ROS: reactive oxygen species.
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occurring at concentrations that are higher than those
required for inhibition of prostaglandin synthesis.
SYSTEMI C EFFECTS
Inhibition of COX-1 and COX-2 and depletion
of prostaglandins
The integrity of gastric mucosal defence depends on
continuous generation of prostaglandin E
2
(PGE
2
) and
prostacyclin (PGI
2
), mediated by COX-1 and COX-2,
which catalyse the rate-limiting step in the conversion
of arachidonic acid to prostaglandin endoperoxide
(PGG
2
) and prostanoids (Figure 2). PGE
2
and PGI
2
are
both potent vasodilators and control almost all aspects
of gastric mucosal defence and healing. The various
functions of PGE
2
are mediated by four G-protein-
coupled receptors, EP
1
to EP
4
and those of PGI
2
by IP
receptors (Table 2). EP receptors use different signal-
ling pathways; PGE
2
induces intracellular Ca
2+
or cyc-
lic adenosine monophosphate (cAMP) when it couples
and signals through EP
1
or EP
2
EP
4
receptors respec-
tively and decreases cAMP when it signals through
EP
3
receptors.
10
Table 1. Potential mechanisms of NSAID-induced apoptosis in the pathogenesis of gastrointestinal ulcers*
Protein Abbreviation Function NSAID effect
Mechanism
of apoptosis Reference
Tumour necrosis factor
alpha
TNF-a Induces apoptosis
when bound to
TNFR I and II
Upregulates
levels
Direct activation of effector
caspases or indirect activation
of mitochondrial apoptotic
pathway
(99)
Caspase 8 Initiator caspase Activation Cytochrome c release and
sequential activation of
caspases-9, and effector
caspases
(100, 101)
Caspase 9 Initiator caspase Activation Activation of effector caspases
Mediates Smac-induced
apoptosis
(100, 101)
Cytochrome c Promotes apoptosis Release via
mitochondrial
permeability
Sequential activation of
initiator caspase-9 and
effector caspases
(101)
Second mitochondria-
derived
activator of caspase
Smac Promotes apoptosis Induces release Activation of effector caspases (101)
Apoptosis inducing
factor
AIF Promotes caspase-
independent
apoptosis
Release via
mitochondrial
permeability
Caspase-independent
apoptosis-like cell death
(102)
Nuclear factor
kappa-B
NF-jB Promotes cell survival Inhibit activation Activation of effector
caspase-3
(103)
Survivin Apoptosis inhibitor Levels Effector caspase-3 mediated
apoptosis Caspase-
independent apoptosis
(104)
Ubiquitin proteosome
system
UPS Promotes cell survival Proteosome
activity
Activation of caspase- 9 and
effector caspase-3
(105)
Protein kinase C-b1 PKC-b1 Promotes cell survival Downregulates
expression
Cell survival (103)
Poly (ADP-ribose)
polymerase
PARP Mediates DNA repair Cleavage Enables effector caspase-3 (100)
C EBP homologous
protein
CHOP Controls development
of apoptosis
in response to ER stress
Upregulates
expression
Initiates apoptotic response (106)
* TNFR I and II, TNF receptor types 1 and 2; DNA, deoxyribonucleic acid; ER, endoplasmic reticulum.
520 C. MUSUMBA et al.
Aliment Pharmacol Ther 30, 517531
2009 Blackwell Publishing Ltd
Experiments in laboratory animals have shown that
COX-1 dependent PGE
2
depletion causes a decrease in
gastric mucosal blood ow while COX-2 inhibition
promotes leucocyte adherence.
11
Despite negligible
prostaglandin synthesis, COX-1 or COX-2 decient
mice or mice treated with selective COX-1 or selective
COX-2 inhibitors do not develop spontaneous gastric
damage. However, they develop severe gastric lesions
when both isoenzymes are simultaneously blocked or
when given a NSAID.
1113
Conversely, in impaired
gastric mucosa (such as during acid challenge, sup-
pression of nitric oxide synthesis or ablation of affer-
ent neurons), isolated inhibition of either COX-1 or
COX 2 is ulcerogenic.
12, 14
The ability of NSAIDs to
cause ulcers has been shown to correlate well with
their ability to suppress gastric prostaglandin synthesis
(although not invariably), and selectiveness for COX-1
rather than COX-2. Inhibition of platelet COX-1 also
leads to decreased thromboxane production causing
enhanced bleeding tendency and this may be the main
factor determining the propensity of a NSAID to cause
bleeding complications.
Whereas COX-1 is abundant in the gastric mucosa,
COX-2 is expressed at low levels in the intact stomach,
but is rapidly upregulated when COX-1 is inhibited or
following injury or pre-existing ulcers.
15
Although
COX-2 derived prostaglandins are the key mediators
during gastric ulcer healing (by promoting epithelial
cell proliferation and angiogenesis mediated by growth
factors), COX-1 derived prostaglandins become impor-
tant when COX-2 derived prostaglandins become de-
cient.
16
In humans, both COX-1 and COX-2 are
upregulated at gastric ulcer margins, in contrast to rats
where only COX-2 is upregulated.
17, 18
Using knockout
mice, Schmassmann et al. demonstrated that dual
inhibition of COX-1 and COX-2 caused greater impair-
ment of ulcer healing than selective inhibition of
COX-2, while inhibition of COX-1 had no effect on
ulcer healing.
16
More recently, Starodub et al. demon-
strated that COX-1 knockout mice with supercial gas-
tric damage had delayed onset of repair restitution,
suggesting that COX-1 derived PGE
2
is a potential
mediator of gastric protection against the early events
that lead to gastric damage progression.
19
Both selec-
tive and nonselective COX inhibitors have been shown
to delay ulcer healing in man.
20
Dikman et al. showed
naproxen delayed healing of iatrogenic ulcers in
human volunteers to a greater extent than aspirin or
celecoxib, in agreement with the ndings from animal
studies.
16, 21
Phospholipase A2
Tissue specific
isomerases
Membrane phospholipids
Arachidonic acid
Prostaglandin G
2 Prostaglandin G/H synthase 1
and 2 (COX-1 and COX-2)
Prostaglandin H
2
Prostaglandin D
2
Prostaglandin E
2
Prostaglandin F
2
Prostanoids Prostacyclin Thromboxane A
2
TP

TP

DP
1
DP
2
EP
1
,EP
2
,EP
3
,EP
4
FP

FP

IP Receptors
Figure 2. Schematic representation of prostaglandin synthetic pathways and the enzymes that catalyse specic reactions.
Prostanoids activate specic G-protein-coupled cell membrane receptors, denoted as IP: prostacyclin receptor; TP:
thromboxane receptor; DP: prostaglandin D2 receptor; EP: prostagladin E
2
receptor and FP: prostaglandin F
2a
receptor.
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Role of hypermotility
Gastric hypermotility, mediated via COX-1 inhibition,
has been shown to play a role in NSAID-induced
ulcers in animal studies. Although the mechanism of
injury is unclear, it is assumed to result from temporal
restriction of blood ow to the mucosa because of
high amplitude contractions resulting in microvascular
damage mediated through tissue hypoxia, neutrophil
endothelial interaction and decreased mucosal resis-
tance.
15, 22
Enhanced gastric hypermotility leads to
increased mucosal permeability and myeloperoxidase
activity and eventually gastric lesions. Tanaka et al.
demonstrated using rats that selective COX-1 inhibi-
tion caused gastric hypermotility and microvascular
disturbances, but an increase in myeloperoxidase
activity only occurred when both COX-1 and COX-2
were inhibited
15
in contrast to the study by Wallace
et al.
11
They hypothesized that NSAIDS cause gastric
damage via inhibition of COX-1 dependent prostaglan-
din production with consequent central nervous
system-mediated enhanced hypermotility and micro-
vascular disturbance causing neutrophil endothelial
adherence and ROS damage. As COX-2 is simulta-
neously blocked, there is no compensatory increase in
COX-2 derived prostaglandins to counteract this effect.
Whether gastric hypermotility plays any role in
NSAID-induced ulcers in man is currently not known.
Role of acid
Acid back-diffusion through the disrupted gastric
mucosal barrier plays a role in converting supercial
mucosal lesions to deeper mucosal necrosis and frank
ulcers. Other pathogenetic mechanisms attributed to
acid include interference with the process of restitution
(by damaging the basement membrane) and ulcer
healing (by altering the normal proliferative responses
Table 2. Role of prostaglan-
dins and their mediators in
gastric mucosal defence
Gastroprotective effect Receptor s (species) COX-Isoform References
Inhibit acid secretion EP
3
(mouse, rat)
IP (mouse)
COX-1 (107, 108)
Stimulate acid secretion EP
4
(rat) (107)
Stimulate mucus secretion EP
4
(rat) (109)
Stimulate bicarbonate secretion in
stomach
EP
1
(rat, mouse) COX-1 (110, 111)
Stimulate bicarbonate secretion in
duodenum
EP4 (rat, human) (112, 113)
Maintain mucosal surface
hydrophobicity
? (mouse) COX-1 (5)
Increase GMBF EP
2
EP
3
EP
4
(rat),
EP
1
(mouse)
COX-1 (109) (114)
Facilitate CGRP-mediated
hyperaemic response
EP
2
, IP (rat) (115)
Suppress increased TNF-a
expression
EP
2
, EP
4
(mouse) COX-2 (116)
Inhibit neutrophil adherence and
activation
EP
2
EP
4
COX-2 (116)
Inhibit epithelial cell apoptosis EP
2
EP
4
(guinea pig) (117)
Decrease gastric hypermotility EP
1
(rat) COX-1 (22, 109)
Decrease epithelial paracellular
permeability
? (rat) COX-1 (118)
Adaptive gastric cytoprotection EP
1
(rat, mouse) (119)
Accelerate restitution ? (rat, mouse) COX-1, COX-2 (19, 120)
Accelerate ulcer healing EP
4
(mouse) COX-2 (121)
Resist ischaemia reperfusion
induced gastric damage
IP COX-2 (122)
GMBF, gastric mucosal blood ow; CGRP, calcitonin gene related peptide; COX, cyclo-
oxygenase; EP, prostaglandin E
2
receptor; IP, prostacyclin receptor. Modied from ref
(123).
522 C. MUSUMBA et al.
Aliment Pharmacol Ther 30, 517531
2009 Blackwell Publishing Ltd
to growth factors and interfering with angiogenesis),
impairment of haemostasis (by interfering with the
ability of platelets to aggregate), and indirectly
enhancing mucosal injury by increasing the absorption
of acidic NSAIDs. The NSAIDs have been shown to
increase basal acid secretion as a result of COX-1
mediated prostaglandin depletion in rats with gastritis,
but not in those with intact stomachs.
23
In humans,
NSAID-induced acute gastric injury has been shown to
be greater in volunteers who had the lowest fasting pH
following naproxen administration.
24
Naproxen has
been demonstrated to reduce total gastric acid volume
with concomitant increase in gastric acid concentra-
tion in humans.
25
Funatsu et al. recently showed that
acid plays a key role in the development of acute gas-
tric mucosal lesions in the rat stomach and that
NSAID-induced reduction in gastric mucosal blood
ow only occurs in the presence of acid.
26
On the
other hand, administration of NSAIDs leads to early
release of interleukin-1b (IL-1b), one of the most
potent inhibitors of gastric acid secretion, which may
be a protective mechanism against NSAID-induced
mucosal injury.
Role of aspirin triggered lipoxins in the
mechanism of action of aspirin
Aspirin inhibits COX activity by covalently modifying,
through acetylation, serine residues in the active sites of
the COX enzymes. Inhibition of COX-1 is achieved by
acetylation of a serine residue (ser 530), causing a
conformational change in the enzyme, which disables it
from oxidizing arachidonic acid. Conversely, acetyla-
tion of a serine residue (Ser 516) in COX-2 still enables
it to metabolize arachidonic acid to 15-R-hydroxyeico-
satetraenoic acid (15-R-HETE). This molecule can be
subsequently metabolized by lipooxygenases to 15-
epi-Lipoxin A
4
(15 epi-LXA
4
or aspirin triggered lipoxin
ATL), a potent neutrophil inhibitor with protean anti-
inammatory effects and which has also been demon-
strated to have potent anti-inammatory effects on
neutrophils, as well as stimulating resolution of inam-
mation.
27
ATL has also been shown to dose-dependently
induce the expression of haem-oxygenase-1 (HO-1), a
ubiquitous and crucial tissue protective enzyme with
vasodilative, anti-inammatory and anti-oxidant prop-
erties, which may contribute to the impairment of
leucocyte inux during the resolution phase of inam-
mation.
28
Aspirin increases plasma
29
and urinary
30
15
epi-LXA
4
in healthy human volunteers.
The protective effects of ATL have been demonstrated
in the rat stomach, whereby blocking its production by
concurrent administration of a COX-2 inhibitor and
aspirin led to signicantly increased gastric damage.
31
This is the mechanism now thought responsible for the
abrogation of the gastrointestinal safety of COX-2
inhibitors when used in combination with aspirin.
32
Administration of LXA
4
parenterally signicantly
reduced aspirin induced damage and an LXA
4
receptor
antagonist (ALX) signicantly worsened aspirin
induced damage similar to a COX-2 inhibitor. 15
epi-LXA
4
is thought to act through eliciting nitric oxide
synthesis (from eNOS and iNOS), which ultimately
inhibits leucocyte endothelial interaction. Paul-Clark
et al. provided evidence for this by demonstrating that
aspirin inhibited leucocyte-endothelial adherence simi-
lar to nitric oxide in wild type mice, but both aspirin
and 15 epi-LXA
4
had markedly reduced effects on
leucocyte endothelial adherence in eNOS- and iNOS-
decient mice compared with wild type mice.
33
Central role of mucosal blood ow and the role
of cytokines and neutrophils
Microvascular damage plays an early and critical role
in the pathogenesis of NSAID-induced ulcers, under-
scoring the fact that the mucosal microvascular
response is possibly the most important component of
mucosal defence. The hyperaemic response to gastric
acid is predominantly mediated by extrinsic sensory
primary afferent nerves, which release CGRP and sub-
stance P (mediated by PGE
2
and PGI
2
) with subsequent
nitric oxide-mediated vasodilatation increasing
submucosal blood ow.
34
CGRP and COX-2 derived
prostaglandins may also synergistically enhance
angiogenesis and ulcer healing via upregulation of
vascular endothelial growth factor (VEGF).
35
CGRPs
protective role is gender-dependent as enhanced
expression has been shown both in female mice and
following oestrogen administration and ovariectomy
reduces its expression rendering the mucosa more vul-
nerable to stress-induced injury.
36, 37
This might
explain the observed lower frequency of peptic ulcers
in human females than in males. Most NSAID-induced
ulcers develop in the gastric antrum, which is also the
site of greatest reduction in gastric mucosal blood
ow. It has been suggested that this is because of focal
ischaemia impairing the ability of the mucosa to with-
stand acid back diffusion leading to tissue injury.
Using twenty healthy volunteers given ibuprofen for
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2009 Blackwell Publishing Ltd
7 days, Kim et al. recently demonstrated signicantly
decreased gastric antral mucosal blood ow (as mea-
sured by laser doppler owmeter) and this signi-
cantly correlated with increased gastric mucosal
injury.
38
It is now well recognized that mucosal pro-inam-
matory mediators such as tumour necrosis factor
(TNF-a), interleukin-1b (IL-1b), interleukin-8 (IL-8)
and platelet activating factor (PAF) are upregulated
following NSAIDs including aspirin and are key medi-
ators of the ensuing gastric injury and healing.
39
TNF-
a (acting via TNF receptor-1),
40
IL-8 and PAF strongly
promote inammation and subsequent tissue destruc-
tion through recruiting leucocytes, especially neu-
trophils and monocytes, by inducing adhesion
molecules on the vascular endothelium (ICAM-1) and
neutrophils (CD11b CD18), leading to their activation.
Activated neutrophils can induce injury by physically
occluding microvessels or through the ensuing ischae-
mia leading to release of ROS and proteases. In
contrast, IL-1b limits leucocyte adherence and gastro-
duodenal injury through stimulating release of
prostaglandins (via inducing COX-2 expression) and
nitric oxide (via increased iNOS expression), and via
inhibiting release of other ulcerogenic mediators (such
as PAF) from mast cells, in addition to its acid inhibi-
tory effects.
41
Role of growth factors
Growth factors are upregulated at ulcer margins fol-
lowing stimulation by PGE
2
, cytokines (such as IL-1b
and IL-6), or gastrin.
42
They contribute to gastro-
duodenal protection and healing of peptic ulcers by
stimulating angiogenesis, granulation tissue formation
and re-epithelialisation.
43
These effects are mediated
though increased COX-2 derived PGE
2
, increased NOS
expression and the prevention of apoptosis by the
upregulation and downregulation of anti-apoptotic
and pro-apoptotic proteins respectively.
4446
Epidermal
growth factor (EGF), transforming growth factor-
(TGF-a), hepatocyte growth factor (HGF) and trefoil
factors predominantly facilitate restitution, while basic
broblast growth factor (bFGF), VEGF, platelet-derived
growth factor (PDGF) and insulin-like growth factor-1
(IGF-1) all promote restoration of the connective tis-
sue and vasculature. Trefoil factor peptides also stabi-
lize surface mucous gels by interacting with mucin
core proteins, and possess anti-apoptotic and pro-
angiogenic effects.
47, 48
NSAIDs may delay repair of
gastrointestinal mucosa by suppressing PGE
2
-mediated
growth factor production. Conversely, NSAIDs such
as aspirin and indomethacin have been shown to
upregulate trefoil factor 2 (TFF2) expression in
MKN45 gastric cells through activation of peroxisome
proliferator-activated receptor c (PPARc) which may
limit the extent of mucosal injury.
49
Role of hydrogen sulphide and nitric oxide
Nitric oxide and H
2
S are two endogenously generated
gaseous mediators important in maintaining gastric
mucosal integrity that share many biological effects
with prostaglandins. They both possess antiapoptotic
and antinecrotic properties and induce expression of
HO-1.
5052
Nitric oxide has additionally been shown to
increase PGE
2
via upregulation of COX-2 in rat bro-
blasts,
53
and to inhibit gastric acid secretion.
54
Nitric
oxide is a small molecule synthesized by nitric oxide
synthetase (NOS) which exists in three isoforms; two
Ca
2+
calmodulin dependent constitutive enzymes
(cNOS) - neuronal NOS (nNOS) and endothelial NOS
(eNOS) - and an inducible form (iNOS) which is Ca
2+
-
independent. Generally, eNOS and nNOS produce low
amounts of nitric oxide that perform many physiologi-
cal functions such as mucosal repair and ulcer healing
by increasing mucosal blood ow and promoting
angiogenesis (through stimulating VEGF). On the other
hand, iNOS can promote ulcer healing (by promoting
mucosal blood ow) or conversely promote mucosal
injury (by enhancing apoptosis), depending on factors
such as tissue type, level and duration of NOS expres-
sion and probably redox status of the tissue.
55
H
2
S is
produced through several pathways, the most impor-
tant being that involving the conversion of L-cysteine
to H
2
S through two pyridoxal-5 phosphate-dependent
enzymes, cystathione B-synthetase (CBS) and cystathi-
onine-c-lyase (CSE), both of which are expressed in
the gastric mucosa. It acts at least partially by activat-
ing ATP-sensitive K
+
channels (K
+
ATP). H
2
S also has
proangiogenic effects, which unlike nitric oxide are
independent of VEGF.
56
Experimental evidence in animals and humans
suggests that nitric oxide contributes to NSAID-
induced ulcerogenesis via generation of iNOS
5759
or a
decrease in cNOS. NSAIDs have been shown to
increase endothelin-converting enzyme-1 (ECE-1)
activity, thereby upregulating endothelin-1 (ET-1),
which suppresses cNOS and endothelial nitric oxide
leading to loss of mucosal integrity in rats.
60
More
524 C. MUSUMBA et al.
Aliment Pharmacol Ther 30, 517531
2009 Blackwell Publishing Ltd
recently, Motawi et al. demonstrated a reduction in
gastric tissue nitric oxide levels and a marked increase
in serum nitric oxide in rats treated with indometha-
cin, which they attributed to a decrease in gastric
mucosal cNOS and an increase in iNOS expression in
activated circulating neutrophils respectively.
61
Like-
wise, Nagai et al. recently demonstrated high iNOS
mRNA expression and nitric oxide in indomethacin
induced gastric ulcers in arthritic rats and attributed
this to excessive production caused by activated IL-
18.
62
A study using Ibuprofen in 24 healthy human
volunteers showed unchanged eNOS levels, but
increased iNOS activity, which correlated with mucosal
lesions, although the differences were not statistically
signicant.
63
Using genetically engineered mice, Souza
et al. demonstrated that iNOS decient mice had less
severe gastric damage following indomethacin admin-
istration than wild-type mice. Moreover, wild type
mice treated with N
G
-nitro-L-arginine methyl ester (L-
NAME), a NOS inhibitor, at doses that inhibited iNOS,
or a specic iNOS inhibitor also showed less dam-
age.
40, 59
In contrast, Schmassmann et al. demon-
strated that in NSAID-impaired gastric ulcer healing,
there is a compensatory increase in nitric oxide
derived from both iNOS and eNOS which seems to
play an even greater role in healing than during regu-
lar healing.
16
Wallace et al. showed increased synthesis of H
2
S in
the ulcerated rat stomach accompanied by marked
increases in CSE and CBS.
64
NSAIDs reduce expression
of CSE gene and H
2
S production in the gastric
mucosa. NaHS (a H
2
S donor) prevented several
NSAID-induced effects such as reduction in gastric
mucosal blood ow, leucocyte adhesion to endothelial
cells in mesenteric venules, mucosal leucocyte inltra-
tion and the upregulation of TNF-a and ICAM-1.
65, 66
Pre-treatment with glibenclamide, (a K
+
ATP blocker)
and DL-propargylglycine (a CSE inhibitor) aggravated
the NSAID induced gastric injury, whereas a K
+
ATP
opener improved gastric damage.
67
Role of stress proteins
NSAIDs have been shown to upregulate various stress
proteins in in vitro and in vivo studies including heat
shock protein 72 (HSP-72), glucose-regulated protein
78 (GRP-78), and HO-1 (also known as HSP-32).
68
Stress proteins protect the gastric mucosa from
NSAID-induced apoptosis, and may therefore play an
important role in NSAID-induced cytoprotection and
adaptive gastroprotection when prostaglandins are
suppressed. Tanaka et al. recently provided direct
genetic evidence for the protective role of heat shock
proteins against irritant induced damage, by showing
an increased severity of gastric lesions induced by eth-
anol or hydrochloric acid and also more apoptotic
cells in heat shock factor-1-null mice.
69
Whereas no
change in expression of HSP-27 was observed follow-
ing exposure of healthy human gastric mucosa to
NSAIDs, expression of HSP-27 was shown to be up-
regulated at the base and margins of human ulcers,
suggesting that it is involved in the repair and regen-
eration of gastric mucosal injury, particularly in the
later stages of ulcer healing. Transgenic mice over-
expressing human HSP27 display a phenotype that is
resistant to NSAID-induced gastric erosions and
ulcers.
70
Role of polyamines
The polyamines, putrescine, spermidine and spermine
are abundant polycations in eukaryotic cells and are
essential for cell growth and proliferation. They are
also crucial for the many phases of restitution in
response to gastrointestinal injury by irritants such as
NSAIDs. Intracellular polyamine levels are tightly
regulated by the biosynthetic enzyme ornithine dacar-
boxylase (ODC) and the catabolic enzyme spermidine
spermine N
1
-acetyltransferase (SSAT). High levels lead
to rapid proliferation while lower levels have been
shown to promote apoptosis. NSAIDs have been shown
to induce toxicity in vitro in cancer cells by decreasing
intracellular polyamines (by up to 50%) through inhi-
bition of ODC.
71
It has been suggested that polyamine
depletion may contribute to NSAID-induced cellular
damage by preventing wound healing through inhibi-
tion of both restitution and epithelial cell prolifera-
tion.
72
A recent study in rats showed that exogenously
administered spermine signicantly reduced gastric
ulcer index (by up to 90%) and normalized elevated
acid secretion in rats treated with indomethacin. In
addition, spermine signicantly elevated gastric mucin
content, attenuated neutrophil inltration (evidenced
by reduced myeloperoxidase activity), and decreased
elevated serum nitric oxide.
61
Role of matrix metalloproteinases
Extracellular matrix remodelling plays an important
role in the development and healing of the ulcer
REVI EW: NSAI D-I NDUCED PEPTI C ULCERS 525
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2009 Blackwell Publishing Ltd
wound and is mainly controlled by matrix metallopro-
teinases (MMPs). MMPs are zinc-dependent endopep-
tidases found in gastric epithelial cells, macrophages
and neutrophils that are tightly regulated at multiple
levels including gene expression, spatial localisation,
zymogen activation and inhibition by tissue inhibitors
of metalloproteinases (TIMPs).
73
Whereas MMP-2
appears to be constitutively expressed by many cell
types in culture and participates in the physiological
turnover of gastric extracellular matrix, MMP-9
expression is induced by cytokines (such as TNF-a and
IL-6), growth factors, hormones and cell stroma inter-
actions and may be important in the early phase of
ulcer development.
74, 75
NSAIDs such as indomethacin increase MMP-9 and
decrease MMP-2 activity in gastric ulcer tissue com-
pared to surrounding tissue in rats, with the ratio of
MMP-9 MMP-2 increasing in parallel with the ulcer
severity index.
76
This is mediated via prostaglandin-
dependent or prostaglandin-independent pathways,
through generation of ROS which modulate MMP
expression either positively (promoting MMP-9 tran-
scription) or negatively (suppressing MMP-2 transcrip-
tion).
77, 78
Ganguli et al. showed that the decrease in
MMP-2 is transient and only occurs in acute ulcers
with levels increasing by day 3 in chronic ulcers, sug-
gesting a critical role for MMP-2 in ECM remodelling
by controlling the balance between ECM degradation
and production.
79
Increased MMP-1 concentration has
been demonstrated in human gastric ulcer biopsies,
with higher levels seen in H. pylori-induced ulcers
compared to NSAID-induced ulcers.
80
More recently,
Tomita et al. demonstrated increased production of
MMP-3 in H. pylori induced gastric ulcers, which
signicantly correlated with pro-inammatory cyto-
kine production. Higher levels were seen at the centre
of the ulcer compared to the margins, suggesting
that MMP-3 plays an important role in ulcer
healing.
81
Role of melatonin
Melatonin also plays a crucial role in gastroprotection
and ulcer healing, acting via melatonin receptors
(MT
2
R).
82
Melatonin exerts its gastroprotective actions
by upregulating MMP-2 and attenuating MMP-9 activ-
ity, downregulating TIMP-2 and directly scavenging
ROS following indomethacin-induced gastric
injury.
79, 83
In addition, melatonin dose-dependently
inhibits gastric acid secretion, attenuates neutrophil-
induced inltration and cytotoxicity, increases blood
ow at ulcer margins, stimulates duodenal bicarbonate
secretion and activates the COX-prostaglandin and
NOS-nitric oxide systems as well as CGRP.
8486
Konturek et al. demonstrated increased mRNA expres-
sion of MT
2
R and the two major enzymes involved in
melatonin biosynthesis, N-acetyltransferase (NAT) and
hydroxyindole-O-methyltransferase (HIOMT) accompa-
nied by increased gastric mucosal blood ow, gastrin
and ghrelin in healing acetic acid-induced ulcers of
rats treated with exogenous melatonin and L-trypto-
phan, suggesting that locally generated melatonin in
the ulcer bed could contribute to healing of ulcers.
87
The melatonin-enhanced ulcer healing seen was abro-
gated by blocking MT
2
R, COX-2 and cNOS and by a
reduction in iNOS, again underscoring the dynamic
interaction of these mediators in gastroprotection and
ulcer healing.
Melatonin has also been demonstrated to attenuate
dose-dependently gastrointestinal lesions induced by
indomethacin and piroxicam
88
in a rat model. Like-
wise, Konturek et al. showed that oral administration
of melatonin and its precursor, L-tryptophan, protects
against gastrointestinal mucosal injury induced by
aspirin in healthy humans. They also demonstrated
increased plasma levels of melatonin after aspirin
administration, suggesting that the aspirin-induced
acute gastrointestinal mucosal damage is accompa-
nied by increased melatonin synthesis in gastric
mucosa.
89
Inhibition of prostaglandin synthesis (by
indomethacin) and NOS (by L-NAME) has been
shown to attenuate signicantly the acceleration of
ulcer healing, the accompanying increase in mucosal
blood ow and luminal nitric oxide release seen in
rats following administration of melatonin and L-
tryptophan.
84
GENETI C CONSI DERATI ONS
Genetic polymorphisms affecting absorption, distribu-
tion, metabolism and excretion are potential sources
of pharmacokinetic variability following administra-
tion of NSAIDs and can have signicant effects on
their toxicity. It has been recognized for a long time
that genetic defects in the cytochrome P450 (CYP)
class of metabolizing enzymes such as CYP2D6,
CYP2C19 and CYP2C9 are critical determinants of the
therapeutic efcacy or toxicities of certain drugs.
90
Functional polymorphisms in the COX enzymes, being
the primary target of NSAIDs, could also be important
526 C. MUSUMBA et al.
Aliment Pharmacol Ther 30, 517531
2009 Blackwell Publishing Ltd
in the pathogenesis of peptic ulcers through inhibition
of prostaglandin synthesis, hence increasing suscepti-
bility to NSAID-induced ulcers. Fries et al. showed
that there is marked intra- and inter-individual vari-
ability in response to acute dosing with celecoxib and
rofecoxib and that single nucleotide polymorphisms
(SNPs) in both CYP2C9 and COX-1 were associated
with elements of drug response such as higher plasma
concentrations of celecoxib (in the CYP2C9*2 variant)
and a reduction in COX-1 inhibition causing failure of
inhibition of thromboxane formation with both drugs
(in the Pro17Leu variant).
91
Several studies looking at the genetic predisposition
to NSAID-induced ulcers and their complications
(upper gastrointestinal bleeding) have been performed,
with conicting results because of small numbers and
poor pharmacological phenotyping. These have
assessed genetic polymorphisms in metabolizing
enzymes,
9295
NSAID target enzymes (COX-1)
96, 97
and
eNOS
98
(Table 3). Further larger studies are required,
which look not only at pathways of drug disposition
but also at some of the other pathways that may be
important in the pathogenesis of ulcers, as highlighted
in this article.
CONCLUSI ONS
Whereas the initial mechanism proposed by Vane in the
early1970s that attributed the therapeutic and adverse
effects of NSAIDs to the inhibition of cyclooxygenase
causing reduction in prostaglandin biosynthesis largely
still holds true, it is clear that this alone is insufcient to
explain fully the pathogenesis of NSAID-induced ulcers.
The pathogenesis of NSAID-induced ulcers is complex
Table 3. Summary of studies looking at genetic predisposition to NSAID-induced toxicity (ulcers or ulcer bleeding)
Study (Design) N Outcome measures Genetic analysis Findings
Pilotto et al.
95
(case control) 78 Endoscopic UGIB CYP2C9 CYP2C9*1 *3,CYP2C9*1 *2
increase risk CYP2C9*3 allele
carriers have higher risk
Martinez et al.
94
(prospective) 218 Endoscopic UGIB CYP2C9 CYP2C9*2 mutated alleles
increase risk OR 2.5, 3.7 for
heterozygous and
homozygous careers
respectively
Martin et al.
93
(retrospective) 54 GU or GU scar CYP2C9 No association found (*1*1,
*1*2, *1*3 genotypes)
Lopez-Rodriguez et al.
92
(prospective)
69 Ibuprofen safety
pharmacokinetics
pharmacodynamics
CYP2C8
CYP2C9
CYP2C9*3 metabolism
and clearance of racemic
ibuprofen, clearance of
S- and R-ibuprofen
CYP2C8*3 clearance of
R-ibuprofen, iNOS
expression CYP2C8*3 and 4*
adverse events,
Arisawa et al.
96
(retrospective) 480 GU or DU COX-1 -1676T allele increases risk
No A-842T C50T found
Van Oijen et al.
97
(retrospective) 194 GU or DU COX-1 A-842G C50T showed inverse
association
Halushka et al.
124
(prospective) 38 Products of AA
pathway (PGF
2a
, TXA
2)
COX-1 A-842G C50T haplotype had
greater inhibition of PG
formation by Aspirin
Piazuelo et al.
98
(case-control) 354 UGIB eNOS gene a allele associated with
decreased risk
N, number of subjects in study; UGIB, upper gastrointestinal bleeding; GU, gastric ulcer; DU, duodenal ulcer; CYP, cytochrome
P450 enzyme; AA, arachidonic acid; TXA
2
, thromboxane A
2
; COX, cyclooxygenase; eNOS, endothelial nitric oxide synthetase;
iNOS, inducible nitric oxide synthetase; PG, prostaglandin.
REVI EW: NSAI D-I NDUCED PEPTI C ULCERS 527
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2009 Blackwell Publishing Ltd
and involves both prostaglandin-dependent and prosta-
glandin-independent mechanisms. Although still not
fully understood, substantial progress has been made in
recent years in elucidating the cellular and molecular
mechanisms involved. Genetic studies using genome
wide association and candidate gene approaches are
powerful investigational tools that may further enhance
our understanding of the pathogenetic pathways
involved, improve the identication of individuals at
risk and aid in the design of novel, more effective strate-
gies for prevention.
ACKNOWLEDGEMENTS
Declaration of personal interests: None. Declaration of
funding interests: We would like to thank the Depart-
ment of Health (UK) for funding the NHS Chair of
Pharmacogenetics programme.
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2009 Blackwell Publishing Ltd