Am J Physiol Gastrointest Liver Physiol 299: G833–G843, 2010.
First published August 5, 2010; doi:10.1152/ajpgi.00065.2010.
HIF-1 mediates pathogenic inflammatory responses to intestinal
ischemia-reperfusion injury
Rena Feinman,1 Edwin A. Deitch,1 Anthony C. Watkins,1 Billy Abungu,1 Iriana Colorado,1
Kolenkode B. Kannan,1 Sharvil U. Sheth,1 Francis J. Caputo,1 Qi Lu,1 Madhuri Ramanathan,1
Shirhan Attan,1 Chirag D. Badami,1 Danielle Doucet,1 Dimitrios Barlos,1 Marta Bosch-Marce,2
Gregg L. Semenza,2 and Da-Zhong Xu1
1
Department of Surgery, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, New
Jersey; and 2Vascular Program, Institute for Cell Engineering, and McKusick-Nathans Institute of Genetic Medicine, John
Hopkins University School of Medicine, Baltimore, Maryland
Submitted 17 February 2010; accepted in final form 27 July 2010
Feinman R, Deitch EA, Watkins AC, Abungu B, Colorado I,
Kannan KB, Sheth SU, Caputo FJ, Lu Q, Ramanathan M, Attan S,
Badami CD, Doucet D, Barlos D, Bosch-Marce M, Semenza GL,
Xu D. HIF-1 mediates pathogenic inflammatory responses to
intestinal ischemia-reperfusion injury. Am J Physiol Gastroin-
test Liver Physiol 299: G833–G843, 2010. First published August
5, 2010; doi:10.1152/ajpgi.00065.2010.—Acute lung injury (ALI)
and the development of the multiple organ dysfunction syndrome
(MODS) are major causes of death in trauma patients. Gut inflam-
mation and loss of gut barrier function as a consequence of splanchnic
ischemia-reperfusion (I/R) have been implicated as the initial trigger-
ing events that contribute to the development of the systemic inflam-
matory response, ALI, and MODS. Since hypoxia-inducible factor
(HIF-1) is a key regulator of the physiological and pathophysiological
response to hypoxia, we asked whether HIF-1 plays a proximal role in
the induction of gut injury and subsequent lung injury. Utilizing
partially HIF-1␣-deficient mice in a global trauma hemorrhagic shock
(T/HS) model, we found that HIF-1 activation was necessary for the
development of gut injury and that the prevention of gut injury was
associated with an abrogation of lung injury. Specifically, in vivo
studies demonstrated that partial HIF-1␣ deficiency ameliorated
T/HS-induced increases in intestinal permeability, bacterial translo-
cation, and caspase-3 activation. Lastly, partial HIF-1␣ deficiency
reduced TNF-␣, IL-1␤, cyclooxygenase-2, and inducible nitric oxide
synthase levels in the ileal mucosa after T/HS whereas IL-1␤ mRNA
levels were reduced in the lung after T/HS. This study indicates that
prolonged intestinal HIF-1 activation is a proximal regulator of
I/R-induced gut mucosal injury and gut-induced lung injury. Conse-
quently, these results provide unique information on the initiating
events in trauma-hemorrhagic shock-induced ALI and MODS as well
as potential therapeutic insights.
hemorrhagic shock; inflammation; multiple organ dysfunction syn-
drome; acute lung injury
IN PATIENTS SUSTAINING major trauma, the development of the
systemic inflammatory response syndrome (SIRS) and multi-
ple organ dysfunction (MODS) is a major clinical problem
resulting in 50 – 80% of all deaths in surgical intensive care
units. Since the pathophysiology of this syndrome remains
incompletely understood and therapy remains largely support-
ive (16), studies focusing on the basic biology of trauma-
induced SIRS, organ injury/dysfunction, and MODS have been
major areas of investigation. These mechanistic studies have
Address for reprint requests and other correspondence: R. Feinman,
UMDNJ-New Jersey Medical School, Dept. of Surgery, 185 South Orange
Ave. MSB-G594, Newark, NJ 07103 (e-mail: feinmarr@umdnj.edu).
http://www.ajpgi.org 0193-1857/10 Copyright © 2010 the American Physiological Society
generated several working hypotheses, one of which is the gut
hypothesis of MODS. A key element in the gut hypothesis of
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MODS is that a splanchnic ischemia-reperfusion (I/R) insult
leading to gut inflammation and loss of barrier function is the
initial triggering event that turns the gut into the “motor” of
MODS (19). However, the exact mechanisms by which gut I/R
leads to intestinal injury and how an intestinal ischemic insult
is transduced into a systemic inflammatory response remains
incomplete. To date, the majority of the molecular and cellular
studies investigating shock-induced gut injury and gut-induced
MODS have focused primarily on the reperfusion phase of the
intestinal I/R insult and the consequent production of proin-
flammatory mediators such as inducible nitric oxide synthase
(iNOS)-derived nitric oxide (22), reactive oxygen species,
cytokines (IL-6) (48), transcription factors (NF-␬B, AP-1)
(72), cyclooxygenase-2 (COX-2) (31), and poly(ADP-ribose)
polymerase (44). Yet, because the induction of many of these
factors is secondary to or accentuated by hypoxia and because
ischemia precedes reperfusion, it seemed likely that the mo-
lecular response triggered by the ischemic component of the
I/R insult is a critical step in initiating the sequence of events
that lead to the development of gut injury and MODS.
The adaptive response to hypoxia or ischemia has been
shown to be primarily regulated by hypoxia-inducible factor
(HIF-1). The HIF-1 heterodimer consists of an oxygen-labile
HIF-1␣ subunit and a constitutively expressed HIF-1␤ subunit
that mediates a wide spectrum of physiological and cellular
adaptive responses such as angiogenesis, metabolic adaption,
erythropoiesis, and vascular tone (67). In the presence of
oxygen, prolyl hydroxylation of the oxygen-dependent degra-
dation domain of HIF-1␣ marks it for ubiquitin-proteasomal
degradation (34, 36) and asparaginyl hydroxylation of the
COOH-terminal transactivation domain of HIF-1␣ prevents
interaction with the transcriptional coactivator p300 (42, 47).
In the context of ischemic injury, HIF-1 was originally
shown to protect organs that are highly sensitive to energy
deprivation such as the brain, heart, and kidney from ischemic
damage in I/R preconditioning models (23). Thus one accepted
role for HIF-1 is that it acts as an adaptive and survival factor
for cells exposed to hypoxia or cells undergoing stress such as
ischemic injury, especially in models of ischemic precondition-
ing (23, 45). However, in response to prolonged ischemic
stress as well as in various nonpreconditioning models, HIF-1
may be deleterious because of its ability to augment both
apoptotic (1, 26) and inflammatory processes (13, 45), espe-
cially via upregulation of iNOS. In fact, our earlier studies
G833
G834 HIF MEDIATES GUT ISCHEMIA-REPERFUSION INJURY

demonstrated that the in vivo intestinal mucosal response to gut
I/R was associated with a prolonged increase in HIF-1␣ ex-
pression that was not rapidly lost during reperfusion when the
gut becomes reoxygenated (41). These results were surprising
since cellular HIF-1 levels rapidly decrease to normoxic levels
within minutes of reoxygenation (32). This relatively unique
prolonged HIF-1␣ response to I/R in the intestine appeared to
be mediated by bacteria and bacterial products within the gut
lumen coming into direct contact with the stressed intestinal
mucosa (41). In this study, we hypothesized that prolongation
of the intestinal HIF-1 response could be potentially maladap-
tive or injurious and contribute to loss of gut barrier function
and the development of distant organ injury. Using partially
HIF-1␣-deficient mice, we tested the functional significance of
HIF-1 in a trauma hemorrhagic shock (T/HS)-mediated gut I/R
injury model.
vein until a mean arterial pressure (MAP) between 35 and 40 mmHg
was obtained and maintained for 60 min. After 60 min, the mice were
resuscitated with their shed blood for 3 h. Sham-shock animals (T/SS)
underwent cannulation of the femoral artery and jugular vein followed
by a laparotomy; however, no blood was withdrawn and the MAP was
kept within normal limits.
Morphological analysis of gut and lung injury. At euthanasia,
terminal ileum or lung was fixed in 10% buffered formalin, processed,
stained with hematoxylin and eosin, and examined by light micros-
copy. For each animal, the percentage of villous injury was a gross
assessment of the number of damaged villus tips per 200 villus tips
(⬃20 –25 visual fields at ⫻100 magnification), which ranged from
blunting and vacuolation of villus tips to denuded villi and mucosal
ulceration. According to the grading systems for villous injury as
described by Refs. 9, 49, 74, the numerical scores were the following:
0 ⫽ normal mucosa, 1 ⫽ development of subepithelial Gruenhagen’s
space and vacuolization at the villus tip, 2 ⫽ extension of the
subepithelial space with moderate lifting of epithelial layer from the

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MATERIALS AND METHODS
Mice. The generation and genotyping of HIF-1␣⫹/⫺ and HIF-
⫹/⫹
1␣ mice on a C57B6/129 genetic background was described
previously (35) and provided by G. L. Semenza (John Hopkins
University School of Medicine, Baltimore, MD). All animal proce-
dures were approved by the University of Medical and Dentistry of
New Jersey Animal Care and Use Committee and maintained in
accordance with the recommendations of the “National Institutes of
Health Guide for Care and Use of Laboratory Animals.”
T/HS model. Male mice were anesthetized with pentobarbital
(60 – 80 mg/kg ip) and under strict asepsis, a 2.5-cm midline laparot-
omy was performed. Isoflurane was given if needed to maintain
surgical level of anesthesia. Blood was withdrawn from the jugular
lamina propria, 3 ⫽ massive subepithelial lifting/sloughing and in-
creased vacuolization from the tip to midportion of villi, 4 ⫽ epithelial
lifting and vacuolization from the tip to lower portion of villi, and 5 ⫽
mucosal ulceration and disintegration of the lamina propria (Fig. 1).
Similarly, lung injury was determined by using a modified lung injury
grading scale (10) that assessed the magnitude of interstitial edema
(0 –2), pulmonary edema (0 –2), and alveolar integrity (0 –1). In this
grading system, 0 indicates no injury and the lung injury score is
calculated by adding the injury grades of each component (maximal
lung injury score is 5). All slides were evaluated by two examiners in
a blinded fashion.
In vivo intestinal permeability assay. Under anesthetized condi-
tions, at 30 min before euthanasia, a 5-cm segment of distal ileum was
isolated, beginning at 3 cm proximal to the cecum and bilateral end of

Fig. 1. Histological criteria used to grade villous damage following trauma hemorrhagic shock (T/HS). 0 ⫽ normal mucosa, 1 ⫽ development of subepithelial
Gruenhagen’s space and vacuolization at the villus tip, 2 ⫽ extension of the subepithelial space with moderate lifting of epithelial layer from the lamina propria,
3 ⫽ massive subepithelial lifting/ sloughing and increased vacuolization from the tip to midportion of villi, 4 ⫽ epithelial lifting and vacuolization from the tip
to lower portion of villi, and 5 ⫽ mucosal ulceration and disintegration of the lamina propria. The shorter arrows depict the presence of vacuoles, the longer
arrows depict the subepithelial and epithelial lifting, and the stars depict mucosal ulceration. Magnification ⫻400.

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 HIF MEDIATES GUT ISCHEMIA-REPERFUSION INJURY
ileum, and then ligated with a 4-0 silk suture to prevent leakage of
4.4-kDa fluorescein isothiocyanate-dextran (FD-4; Sigma-Aldrich).
PBS (200 ␮l) containing 25 mg/ml FD-4 was injected into the lumen,
which was returned to the abdominal cavity and then closed. After 30
min, the mouse was euthanized and a blood sample (100 ␮l) was taken
by cardiac puncture. The concentration of FD-4 in the plasma was
measured by a fluorescence spectrophotometer.
MPO activity. Frozen ileal mucosa and lung tissue was homoge-
nized and processed for myeloperoxidase (MPO) activity measure-
ment as previously described (73). The MPO activity in these super-
natants was determined by measuring the H2O2-mediated oxidation of
o-dianisidine hydrochloride at 460 nm. MPO activity was determined
by using a standard curve from MPO derived from human leukocytes
(Sigma Aldrich) and normalized to milligrams of protein determined
by bicinchoninic acid protein assay (Pierce).
Bacterial translocation. The mesenteric lymph node (MLN) com-
plex was harvested and the level of translocating bacterial quantified
as previously described (17). Briefly, using sterile technique, the MLN
complex was harvested, weighed, and homogenized in 0.2 ml of
sterile saline. Aliquots were plated onto both blood and MacConkey
agar plates. These plates were examined at 24 h of aerobic incubation
AJP-Gastrointest Liver Physiol • VOL
G835
at 37°C. The colonies were counted on the plates and recorded as the
number of colony forming units per gram of MLN.
Real-time PCR. Total RNA was prepared from ileal mucosa or lung
tissue by using the RNeasy kit (Qiagen) and cDNA synthesis was
reverse transcribed by using the high-capacity cDNA reverse tran-
scription kit (Applied Biosystems) according to manufacturer’s pro-
tocols. cDNA was then amplified with TaqMan gene expression
Master Mix and predesigned TaqMan probes for murine GLUT1,
COX-2, TNF-␣, IL-1␤, IL-6, and IL-10 as recommended by Applied
Biosystems. Within each experimental group, mRNA expression was
normalized to 18S amplification (⌬Ct) and then fold changes in
expression relative to wild-type (WT) T/SS (⌬⌬Ct) was determined
by use of 2⫺(⫺⌬⌬Ct) (54).
Western blotting. Whole cell extracts prepared from ileal mucosal
scrapings as described (41) were resolved by SDS-PAGE and detected
by Western blotting. The following antibodies were used: iNOS,
HIF-1␣ (BD Biosciences), cleaved Asp175 caspase-3, p42/p44 (Cell
Signaling), and HIF-1␣ (Novus Biologicals). As reported by others
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(38), total p42/p44 was used as a our loading control since the
expression of commonly used loading controls such as actin and
tubulin have been shown to be altered in tissues of animals subjected
Fig. 2. Induction of hypoxia-inducible factor
(HIF)-1␣- and HIF-1-dependent genes in the
ileal mucosa after T/HS. Wild-type (WT) and
HIF-1␣⫹/⫺ mice were subjected to T/HS or
sham shock (T/SS) for 60 min and 3 h reper-
fusion. A: relative levels of HIF-1␣, GLUT1,
and cyclooxygenase-2 (COX-2) mRNA levels
in the ileal mucosa were determined by real-
time PCR using the ⌬⌬Ct method by using a
log2 scale. Mean values ⫾ SE are shown (n ⫽
4 –15 mice/group). B: whole cell extracts
(WCEs) prepared from mucosal scrapings
from the distal ileum were examined for HIF-
1␣, inducible nitric oxide synthase (iNOS),
and total MAPK expression by Western blot
analysis. A nonspecific (NS) band (70 kDa)
recognized by iNOS antibody also confirmed
equal loading of our samples. Numbers refer to
individual mice.
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G836 HIF MEDIATES GUT ISCHEMIA-REPERFUSION INJURY

to I/R injury (63) and intestinal cytoskeleton degradation has been
shown to precede tight junction loss (64). Ponceau S staining of the
membranes was also done to validate transfer efficiency as well as
equal protein loading. The Western blots were developed with Immo-
bilon Western chemiluminescent HRP substrate (Millipore) and ana-
lyzed by densitometry using an AlphaImager 3400 imaging system
and AlphaEase FC software (Alpha Innotech).
Caspase-3 activity and caspase-3/7 activity assays. For in vivo
studies, caspase-3 activity was determined according to the instruc-
tions provided by the caspase-3/CPP32 colorimetric assay kit with
DEVD-pNA substrate (Biovision). For in vitro studies, caspase-3/7
was determined according to the instructions provided by the
sensoLyte homogenous Rh110 caspase-3/7 assay kit (AnaSpec).
Generation of HIF-1␣ stable transfectants. Using the Nucleofector
T kit (Amaxa), Caco-2 cells (ATCC) were nucleofected with p
(HA)HIF-1␣ or pcDNA3 (empty) expression vector provided by H. F.
Bunn (Harvard Medical School, Boston, MA). Pools of HIF-1␣ or
control Caco-2 stable transfectants were selected in the presence of
ANOVA analysis followed by Tukey-Kramer or Newman-Keuls. For
lung injury score and real-time PCR analysis, data were analyzed by
Mann-Whitney test and bacterial translocation by ␹2 analysis with
Fisher exact test. P values less than 0.05 were considered statistically
significant.
RESULTS
Partial HIF-1␣ deficiency attenuates gut and lung I/R
injury. To establish the functional significance of HIF-1 in gut
I/R injury, we utilized mice that were heterozygous for a
knockout allele at the HIF-1␣ locus (HIF-1␣⫹/⫺) in a com-
bined T/HS model. The T/HS model (laparotomy plus 60 min
of hemorrhagic shock at 35– 40 mmHg) and 3 h of reperfusion
represents a global I/R injury. The addition of a laparotomy is
important because tissue injury is a component of traumatic
hemorrhage and the combined insult of hemorrhage and tissue

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G418 (1 mg/ml) for 14 days. To confirm HIF-1␣ expression, control
and HIF-1␣ transfectants were starved overnight in DMEM contain-
ing 0.1% FBS and exposed to normoxia or hypoxia (1% O2) for 3 h
as described (41).
Statistical analysis. Statistical significance among groups for vil-
lous injury, in vivo intestinal permeability, polymorphonuclear cell
(PMN) infiltration, and MPO activity was determined by one-way
injury results in an inflammatory response that more closely
mimics the clinical situation than hemorrhage alone (15). We
first determined the effects of T/HS on the induction of HIF-1␣
mRNA and protein expression in the ileal mucosa of WT and
HIF-1␣⫹/⫺ mice. As shown in Fig. 2A, real-time PCR analysis
demonstrated a modest increase in mucosal HIF-1␣ expression

Fig. 3. Partial HIF-1␣ deficiency attenuates T/HS-induced gut injury. WT and HIF-1␣⫹/⫺ mice were subjected to T/HS or T/SS for 60 min and 3 h reperfusion.
A: representative sections of hematoxylin and eosin (H&E) staining of the distal ileum (⫻200). Arrows depict subepithelial lifting sloughing and mucosal edema
in the villi. B: percentage of villous injury. C: histological scoring of villous damage. Values in B and C are expressed as means ⫾ SE (n ⫽ 7–11 mice/group).
D: MPO activity (U/mg of protein) was measured in the ileal mucosa. Values are expressed as means ⫾ SE (n ⫽ 5–10 mice/group).

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 HIF MEDIATES GUT ISCHEMIA-REPERFUSION INJURY
in WT mice subjected to T/HS compared with their T/SS
counterparts. In contrast, HIF-1␣ expression was not evident in
HIF-1␣⫹/⫺ mice after T/HS. T/HS was also capable of induc-
ing the expression of two HIF-1 targets, GLUT1 and COX-2,
in WT but not HIF-1␣⫹/⫺ mice after T/HS. In agreement with
earlier studies (5, 35, 52), the baseline mRNA levels of HIF-1␣
were significantly lower in the T/SS group of HIF-1␣⫹/⫺
relative to the T/SS WT group. Consistent with these findings,
both HIF-1␣ and iNOS protein levels were increased in the
ileal mucosa of WT mice after T/HS compared with T/SS-
operated mice (Fig. 2B). In contrast, the HIF-1␣ and iNOS
responses were markedly lower in the ileal mucosa of HIF-
1␣⫹/⫺ mice after T/HS. Negligible HIF-1␣ and iNOS protein
levels were detected in WT or HIF-1␣⫹/⫺ mice subjected to
T/SS. We chose to characterize the iNOS response since iNOS
has been identified as a HIF-1 target and is a key effector in the
pathophysiology of gut barrier failure during shocked states.
G837
subepithelial lifting, mucosal edema, and sloughing in WT
mice subjected to T/HS whereas the ileal mucosa of the
T/HS-challenged HIF-1␣⫹/⫺ mice had minimal mucosal dam-
age and resembled the T/SS groups, regardless of genotype.
The magnitude of T/HS-induced villous injury was approxi-
mately fourfold greater in WT mice than in HIF-1␣⫹/⫺ mice
(Fig. 3B). Although the percentage of villous injury in HIF-
1␣⫹/⫺ mice was the same for T/SS and T/HS groups, the
percentage of villous injury was twofold higher in the HIF-
1␣⫹/⫺ mice subjected to T/SS as compared its WT counterpart.
The villous injury score was also markedly higher in WT than
HIF-1␣⫹/⫺ mice after T/HS (Fig. 3C). We also determined
whether HIF-1 modulated the sequestration of PMN into the
ileal mucosa after T/HS. Although MPO levels were extremely
low in the ileal mucosa, T/HS elevated MPO levels in WT
mice by approximately threefold compared with their T/SS
counterpart (P ⬍ 0.02; Fig. 3D). However, there was no

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These findings are in agreement with earlier studies demon-
strating reduced expression of endogenous HIF-1␣ protein and
downstream targets in HIF-1␣⫹/⫺ cells under hypoxic condi-
tions (5, 35, 52) and HIF-1␣⫹/⫺ mice manifesting impaired
physiological responses to ischemia and hypoxia (5, 52, 60).
Since our findings demonstrated that T/HS induced an in-
testinal HIF-1␣ response, we assessed the effect of partial
HIF-1␣ deficiency on T/HS-induced villous injury. Histologi-
cal sections of the ileal mucosa (Fig. 3A) showed evidence of
difference between MPO levels in HIF-1␣⫹/⫺ mice subjected
to T/SS and T/HS.
On the basis of earlier studies demonstrating that T/HS-
induced gut injury leads to the development of a systemic
inflammatory response and lung injury, we asked whether
HIF-1 played a maladaptive role in T/HS-induced lung injury.
As a consequence of T/HS, loss of alveolar integrity, increased
inflammation, and red blood cell congestion are evident in the
lungs of WT mice but not of HIF-1␣⫹/⫺ mice (Fig. 4A). No

Fig. 4. Partial HIF-1␣ deficiency attenuates T/HS-induced lung injury. WT and HIF-1␣⫹/⫺ mice were subjected to T/HS or T/SS for 60 min and 3 h reperfusion.
A: representative section of H&E staining of the lung. B: lung injury score. C: number of infiltrating polymorphonuclear cells (PMNs) per high-power field (hpf).
D: MPO activity (U/mg of protein) was measured in the lung. Values in B–D are expressed as means ⫾ SE (n ⫽ 6 –13 mice/group).

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G838 HIF MEDIATES GUT ISCHEMIA-REPERFUSION INJURY
evidence of lung injury is seen in WT or HIF-1␣⫹/⫺ mice
subjected to T/SS. The lung injury score (Fig. 4B), the number
of infiltrating PMN per high-power field (Fig. 4C), and MPO
levels (Fig. 4D) were markedly elevated in WT mice subjected
to T/HS compared with T/SS. In contrast, there was no signif-
icant difference in lung injury and PMN influx in the lungs of
HIF-1␣⫹/⫺ mice subjected to T/HS or T/SS. Thus partial
HIF-1␣ deficiency significantly attenuated T/HS-induced gut
and lung injury, thereby supporting the concept that elevation
of HIF-1␣ after gut I/R is deleterious and contributes to both
local gut and distant lung organ damage.
Persistent HIF-1 activation is linked to loss of gut barrier
function. Since functional studies of intestinal barrier function
have demonstrated that intestinal permeability increases after
gut I/R (57, 70), we tested the hypothesis that HIF-1␣ activa-
tion contributes to T/HS-induced increased intestinal perme-
ability. With use of an in vivo FD-4 intestinal permeability
assay, T/HS increased intestinal permeability by 3.8-fold in
WT mice compared with their T/SS counterparts (Fig. 5A).
However, T/HS was only capable of increasing intestinal
permeability by 1.8-fold in HIF-1␣⫹/⫺ mice vs. their T/SS
counterparts. The baseline level of intestinal permeability for
the T/SS HIF-1␣⫹/⫺ group was approximately twofold higher
than the WT T/SS group (P ⬍ 0.05).
Consistent with these results, bacterial translocation to the
mesenteric lymph node was markedly increased in WT mice
subjected to T/HS relative to their T/SS group (Fig. 5B). In
Fig. 5. HIF-1 directly impairs gut barrier function. A: in vivo intestinal
permeability was assessed in WT and HIF-1␣⫹/⫺ mice subjected to T/SS or
T/HS for 60 min and 3 h reperfusion by quantifying 4.4-kDa fluorescein
isothiocyanate-dextran (FD-4) concentration (␮g/ml) in the serum. Values are
expressed as means ⫾ SE (n ⫽ 5– 8 mice/group). B: bacterial translocation to
the mesenteric lymph nodes (MLN) was measured in WT and HIF-1␣⫹/⫺ mice
subjected to T/SS or T/HS. Number of colony forming units of bacteria per
gram MLN (CFU/g) and values are expressed as means ⫾ SD (n ⫽ 6 – 8
mice/group).
AJP-Gastrointest Liver Physiol • VOL
contrast, no translocation was evident in HIF-1␣⫹/⫺ mice after
T/HS and as expected no translocation was found in either WT
or HIF-1␣⫹/⫺ mice after T/SS. Taken together, our findings
suggest that partial HIF-1␣ deficiency ameliorated intestinal
barrier dysfunction during T/HS, thereby supporting the con-
cept that HIF-1 mediates gut injury.
HIF-1 promotes apoptosis during gut I/R injury. We next
studied caspase-3 activation since it has been implicated in gut
injury models of T/HS (39, 40), hypoxia (20), and ischemia (7,
25) and the caspase-3 promoter contains a functional HIF-1
response element (65). A significant decrease in the cleavage
of caspase-3 (Fig. 6, A and B) and caspase-3/7 activity (Fig.
6C) was found in the ileal mucosa of HIF-1␣⫹/⫺ mice sub-
jected to T/HS compared with their WT counterparts. Simi-
larly, a progressive increase in caspase-3 activity was found in
our HIF-1␣ Caco-2 transfectants exposed to normoxia, hyp-
oxia, or hypoxia/reoxygenation compared with control Caco-2
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transfectants (Fig. 6D). These findings suggest that HIF-1 also
manifests a proapoptotic role during T/HS-induced gut injury.
HIF-1 differentially regulates proinflammatory cytokines in
the ileal mucosa and lung after T/HS. In T/HS models, loss of
gut barrier function has been associated with a gut inflamma-
tory response (18, 21). The translocation of bacteria or their
products have also been shown to trigger or sustain gut inflam-
mation (21, 62), and our recent studies have demonstrated that
bacteria and LPS upregulated HIF-1 expression in intestinal
epithelial cells under normoxic conditions (41). Given that
HIF-1 is capable of modulating the innate immune and inflam-
matory responses in hematopoietic and nonhematopoietic cells,
we determined whether HIF-1 activation modulated the intes-
tinal and lung inflammatory response after T/HS. As shown in
Fig. 7, T/HS increased TNF-␣ and IL-1␤ mRNA levels in the
ileal mucosa of WT mice compared with their T/SS group (P ⬍
0.007 and P ⬍ 0.05, respectively). In contrast, partial HIF-1␣
deficiency attenuated the induction of mucosal TNF-␣ and
IL-1␤ gene expression after T/HS compared with WT mice
after T/HS (P ⬍ 0.05). Ileal IL-6 gene expression was not
modulated after T/HS in either genotype, but its baseline level
of expression as well as IL-1␤ were significantly lower in
HIF-1␣⫹/⫺ mice than in WT mice (P ⬍ 0.05). We then asked
whether HIF-1 activation affected the pulmonary inflammatory
response. Lung TNF-␣, IL-1␤, and IL-6 levels were markedly
higher in WT mice after T/HS. However, in contrast to the
intestinal inflammatory response, only the increase in T/HS-
induced lung IL-1␤ expression was prevented in HIF-1␣⫹/⫺
mice (P ⬍ 0.03). IL-10 levels were also measured to determine
whether T/HS induced higher levels of IL-10 in HIF-1␣⫹/⫺
mice compared with WT mice since IL-10 was described as an
anti-inflammatory cytokine (46, 68). IL-10 levels were equally
elevated in both the ileum and lungs of WT and HIF-1␣⫹/⫺
mice subjected to T/HS compared with their T/SS counterparts.
Thus the attenuation of gut and subsequent lung injury by
partial HIF-1␣ deficiency was not due to an increased IL-10
response. Taken together, our results suggest that HIF-1 dif-
ferentially regulates intestinal and lung-derived inflammation
after T/HS.
DISCUSSION
The major observation of the present work is that prolonged
HIF-1 activation significantly contributes to splanchnic ische-
299 • OCTOBER 2010 • www.ajpgi.org
 HIF MEDIATES GUT ISCHEMIA-REPERFUSION INJURY
mia-reperfusion-induced loss of gut barrier function, bacterial
translocation, apoptosis, and gut-derived inflammatory response,
subsequently resulting in villous injury. A second major observa-
tion is that resistance to gut injury was associated with resistance
to lung inflammation and injury in the HIF-1␣⫹/⫺ mice. These
observations expand our earlier studies demonstrating that gut
ischemia in vivo induces a prolonged intestinal mucosal HIF-1
response that does not disappear upon reoxygenation of the
intestine and that this persistence of the HIF-1 response ap-
pears to be mediated, at least in part, by intestinal bacteria
and/or bacterial products, such as LPS (41). Thus the present
study provides evidence that a prolonged intestinal mucosal
HIF-1 response, which persists after gut reperfusion, plays a
role in the pathogenesis of gut-mediated I/R injury. These
results are of potential clinical importance because gut injury
and loss of normal intestinal barrier function have been asso-
ciated with the development of SIRS, acute respiratory distress
syndrome (ARDS), and MODS as well as worse clinical
outcomes in severely injured as well as intensive care unit
AJP-Gastrointest Liver Physiol • VOL
G839
Fig. 6. HIF-1 promotes apoptosis during gut isch-
emia-reperfusion (I/R) injury. A and B: WCEs
were prepared from ileal mucosa of WT and HIF-
1␣⫹/⫺ mice subjected to T/HS or T/SS for 60 min
and 3 h reperfusion. A: cleaved caspase-3 and total
Downloaded from http://ajpgi.physiology.org/ at EBSCO on May 6, 2013
p42/p44 expression was determined by Western
blotting. B: densitometry was performed to quan-
tify cleaved caspase-3 and total p42/p44 expres-
sion. Data are represented as means ⫾ SE (4 – 6
mice/group). C: caspase-3 activity is expressed as
absorbance at 405 nm per 50 ␮g of protein. Values
are expressed as means ⫾ SE (n ⫽ 4 –5/group).
D: caspase-3/7 activity assay using WCEs derived
from HIF-1␣ and control (Ctl) Caco-2 transfec-
tants exposed to normoxia (N), hypoxia (H) for 3
h, or H followed by reoxygenation for 3 h (H/R).
RFU, relative fluorescence units. Mean values ⫾
SE are shown (n ⫽ 3– 4/condition).
patient populations (18). The reason why gut injury and loss of
barrier function appear to be clinically important in these
patient populations is related to its increased susceptibility to
injury during stress states (11, 18) and the fact that the gut and
its contents are a major source of factors that contribute to the
development of a systemic inflammatory state and distant
organ including acute lung injury (19).
Although cytokines (58), bacteria (41, 51), and LPS (41, 59)
as well as hypoxia have been shown to activate HIF-1 in
enterocytes, our knowledge of HIF-1 activation and its func-
tional sequelae in gut injury models is limited and to some
extent controversial. For example, in contrast to our acute gut
I/R results, HIF-1␣ was found to be protective in more chronic
gut injury models utilizing colon-specific HIF-1␣-deficient
mice. These models included 2,4,6-trinitrobenzene sulfonic
acid -induced colitis (12, 37, 56), chronic hypoxia (24), and
Yersinia enterocolitica orogastric infection (28). In the context
of gut I/R injury, one study found that mice administered the
hydroxylase inhibitor, dimethyloxalglycine (DMOG) was pro-
299 • OCTOBER 2010 • www.ajpgi.org
G840 HIF MEDIATES GUT ISCHEMIA-REPERFUSION INJURY

Downloaded from http://ajpgi.physiology.org/ at EBSCO on May 6, 2013

Fig. 7. HIF-1 differentially regulates proin-
flammatory cytokines in the ileal mucosa
and lung after T/HS. RNA was prepared
from ileal mucosa and lung of WT and
HIF-1␣⫹/⫺ mice subjected to T/HS or T/SS
for 60 min and 3 h reperfusion. Relative
mRNA expression for TNF-␣, IL-1␤, IL-6,
and IL-10 was quantified by real-time PCR
using the ⌬⌬Ct method using a log2 scale.
Mean values ⫾ SE are shown (n ⫽ 4 –7
mice/group).

tective in a localized gut I/R injury model (superior mesenteric
artery occlusion for 15 min and 3 h reperfusion), and HIF-1
mediated its protective effects via CD73 (27). One possible
explanation for this discrepancy may be due to differences in
our models of gut I/R injury and a second explanation is that
the pretreatment of mice with DMOG may have precondi-
tioned their intestine to subsequent gut I/R injury. However,
consistent with our studies, HIF-1␣ was found to be detrimental
in necrotizing enterocolitis (2) and in dextran sulfate sodium-
induced colitis in von Hippel-Lindau-deficient mice character-
ized for their constitutive HIF-1 expression (61). Thus the role
of HIF-1, as protective or deleterious, may depend on whether
the insult is acute or chronic, the segments of the intestine
injured, as well as on whether the loss of HIF-1␣ is partial or
total. For example, the chronic studies, where HIF-1 was
protective, utilized conditional intestinal and colonic epithelial

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 HIF MEDIATES GUT ISCHEMIA-REPERFUSION INJURY
HIF-1␣ mice (28, 37, 56), whereas our studies were performed
in mice with a partial HIF-1␣ deficiency that involves the
entire gut. These differences may be important, since the
segment of the gut that is susceptible to acute gut I/R-mediated
injury is the distal small intestine (33, 69). This increased
susceptibility of the ileum to I/R-mediated injury is consistent
with the observation that the levels of proinflammatory genes,
such as iNOS, and apoptotic cell death were markedly in-
creased after hemorrhagic shock in the ileum compared with
duodenum, jejunum, and colon (33). Thus acute and chronic
gut injury models differ not only in their mechanism of injury,
but also in the segments of the guts that are at highest risk of
injury. Additionally, the use of partially HIF-1␣⫹/⫺-deficient
mice has the advantage that only a partial HIF-1␣ deficiency
exists whereas complete deficiency of HIF-1␣ in a specific cell
type, as with the conditional deletion of HIF-1␣ in intestinal
and colonic cells, may result in a phenotype that is not as
clinically relevant, since such a state is unlikely to exist in any
human disease state. Thus although HIF-1 appears to be
involved in the regulation of intestinal homeostasis, it appears
to have dichotomous roles in gut inflammatory diseases in that
it can be injurious or protective depending on the exact phys-
iological conditions studied as well as the nature and duration
of the insult.
This dichotomous effect of HIF-1 activation has also been
observed in studies focusing on the heart and brain as well as
the gut. For example, HIF-1 has been implicated as a cardio-
protective factor in ischemic preconditioning models, since
cardioprotection by intermittent ischemia was impaired in
partially deficient HIF-1␣ mice (6). However, while HIF-1 is
cardioprotective in preconditioning models, where HIF-1 is
only transiently elevated, chronic activation of HIF-1 signaling
was found to be deleterious in ischemic hearts (43). Similarly,
HIF-1␣ was found to mediate ischemic tolerance in the neo-
natal rat brain (3) as well as in transient (55) and permanent (4)
focal cerebral ischemia models. However, in contrast to its
protective effects in models of preconditioning or tolerance,
ablation of HIF-1 in the brain was associated with neuropro-
tection to an acute ischemic insult (30) and loss of HIF-1␣ in
astrocytes markedly protected neurons from hypoxia-induced
neuronal death (8, 66). Thus HIF-1␣ activation has been shown
to be either beneficial or deleterious in several organ systems
depending on the physiological context of the model.
There is evidence that the relationship between HIF-1 acti-
vation and inflammation appears to be bidirectional since
numerous studies have shown that HIF-1 is a central regulator
of inflammation and innate immunity (50), whereas at the same
time proinflammatory mediators such as TNF-␣, IL-1␤, nitric
oxide, and LPS have been shown to induce HIF-1␣ expression
in cells, including enterocytes, even under normoxic conditions
(29, 41, 58, 59). In this context, our findings suggest that the
diminished intestinal TNF-␣, IL-1␤, and iNOS responses as
well as the reduced pulmonary IL-1␤ response in partially
deficient HIF-1␣ mice after T/HS may be linked to the atten-
uation of villous and lung injury. Support for this concept
stems from earlier studies linking gut-derived factors such as
IL-1␤, TNF-␣, and iNOS as well as enteric bacteria to intes-
tinal barrier dysfunction and the development of both sepsis-
and T/HS-mediated distant organ injury (14, 70). Additionally,
HIF-1 activation has been implicated in the proinflammatory
response of myeloid cells during sepsis and deletion of HIF-1␣
AJP-Gastrointest Liver Physiol • VOL
G841
in myeloid cells afforded protection against LPS-induced mor-
tality (53). Although ileal IL-6 levels were not significantly
elevated in WT mice subjected to T/HS as reported previously
(71), the baseline levels of intestinal IL-6 level and IL-1␤ were
significantly lower in HIF-1␣⫹/⫺ mice than WT mice. Whether
partially HIF-1␣ deficient mice have a less primed or less
active immune status remains to be determined since we only
profiled a limited number of proinflammatory cytokines. Al-
though our findings suggest that HIF-1 activation exacerbates
the T/HS-induced gut-and lung-derived inflammatory response
and HIF-1 differentially regulates cytokine expression in the
ileal mucosa and lung, a more extensive analysis of the HIF-
1-driven intestinal and lung inflammatory response is needed to
resolve this issue.
Since SIRS, ARDS, and MODS remain common causes of
morbidity and mortality, understanding the mechanisms by
which shock and gut ischemic states, such as trauma, lead to
Downloaded from http://ajpgi.physiology.org/ at EBSCO on May 6, 2013
gut injury and gut-induced acute lung injury are critical to
advancing the care of these patients. In this context, our study
showing for the first time that prolonged HIF-1 activation is the
proximal regulator of T/HS-induced gut mucosal injury and
inflammation provides novel insights into the biology of this
clinically important syndrome as well as potential therapeutic
insights.
GRANTS
This research was supported by National Institutes of Health grants
1P50GM069790 (to R. Feinman and E. A. Deitch), T32-GM069330 (to F. J.
Caputo and D. Doucet) and R01-HL55338 (to G. L. Semenza).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author (s).
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