ALIMENTARY TRACT II
Microvascular acclimatization to hypoxia: role of
Alfred J Casillan, MD, PhD, Norberto Gonzalez, MD,
John Wood, PhD, James Thomas, MD
University of Kansas Medical Center, Kansas City, KS
INTRODUCTION: Acute systemic hypoxia initiates microvascular
inflammation within the gastrointestinal tract, and this response in-
volves reactive oxidant species (ROS) and mast cell degranulation.
After chronic hypoxia, however, this initial inflammation resolves
and the microcirculation becomes resistant to even further decreases
in oxygen levels. This study investigated the role of inducible nitric
oxide synthase (iNOS) in attenuating microvascular inflammation
during chronic hypoxia.
METHODS: Rats were acclimatized to hypobaric hypoxia for 3
weeks at a level equivalent to a reduction in inspired O2 to 10%. The
hematocrit of acclimatized rats was reduced to normal levels prior to
experiments. Leukocyte adherence/emigration, venular permeability
to albumin, ROS levels, and mast cell degranulation were measured
within the mesenteric microcirculation using intravital microscopy.
Tissue iNOS was measured using Western blots and immunohisto-
RESULTS: As shown in the table, systemic hypoxia resulted in mi-
crovascular inflammation in non-acclimatized but not in acclima-
tized rats. In acclimatized rats, the iNOS inhibitor L-NIL promoted
hypoxia-induced inflammation, and iNOS expression was increased
within mast cells.
Non-Acclimatized Rats Acclimatized Rats
Nx Hx Nx Hx Hx ؉ L-NIL
0.2Ϯ0.1 8.6Ϯ1.6ء 0.3Ϯ0.2 0.4Ϯ0.2 5.6Ϯ2.1ء
2.2Ϯ0.4 11.4Ϯ1.6ءء 2.6Ϯ1.4 2.9Ϯ1.6 9.2Ϯ0.8ء
0.16Ϯ0.04 0.74Ϯ0.07ء 0.16Ϯ0.04 0.18Ϯ0.06 0.58Ϯ0.16ء
ROS level % of
101Ϯ6 168Ϯ12ءء 102Ϯ6 104Ϯ2 148Ϯ10ء
1.02Ϯ0.06 1.48Ϯ0.1ء 1.02Ϯ0.05 1.04Ϯ0.04 1.26Ϯ0.06ء
Nx, normoxia; Hx, hypoxia.
ء indicates p Ͻ 0.05 and ءء p Ͻ 0.01 vs corresponding Nx. n ϭ 5 or 6 rats
in each group.
CONCLUSIONS: These results demonstrate that iNOS upregu-
lation contributes to microvascular acclimatization during
chronic hypoxia. Since tissue hypoxia is a common feature of
many gastrointestinal diseases, a better understanding of such
acclimatization mechanisms may lead to novel interventions in
TNF-alpha enhances Il-1-beta-mediated inducible
nitric oxide synthase (iNOS) release from rat
intestinal epithelial cells (IEC-6)
Brian Lugo, MD, Yigit Guner, MD, Anatoly Grishin, PhD,
Jeffrey Upperman, MD, Jin Wang, MS, Xiaoru Zhang, MD,
Patricia Boyle, BS, Henri Ford, MD, FACS
Childrens Hospital Los Angeles, Los Angeles, CA
INTRODUCTION: Necrotizing enterocolitis (NEC) is characterized
by an exuberant inflammatory response and accumulation of toxic
metabolites. We hypothesize that Tumor Necrosis Factor-alpha
(TNF-alpha), a cytokine that is upregulated in NEC, may contribute
to iNOS induction, which in turn mediates epithelial injury through
the production of NO.
METHODS: IEC-6 cells were cultured with IL-1b, TNF-a, IFN-g
alone or in various combinations. Levels of iNOS protein, transcrip-
tion factors, and mitogen-activated protein kinases (MAPK) activa-
tion were measured by Western Blot or using specific assays. Electro-
mobility shift assay (EMSA) was used to measure activation of NF-
kappaB, STAT-1, and AP-1. NO production was measured using the
Greiss reaction. Data expressed mean Ϯ SEM, ANOVA, pϽ.05.
RESULTS: TNF (1ng/ml) enhanced iNOS expression and NO
production in cells treated with Il-1b; IFN-g, or LPS did not. TNF
decreased IkB levels. In addition, Il-1 and TNF separately activated
ERK. Both NF-kB and ERK inhibitors independently blocked
iNOS expression and NO production. EMSA demonstrated that
Il-1, TNF, and LPS activated NF-kB. ERK inhibition did not affect
this activation. Il-1 led to complete downregulation of AP-1 binding,
and TNF had no effect.
CONCLUSIONS: TNF augments Il-1 induced iNOS expression in
enterocytes through a NF-kB and ERK dependent pathway. AP-1
appears to be a negative regulator of iNOS gene transcription which
is relieved by addition of Il-1. For the first time, we demonstrate a
novel mechanism of iNOS upregulation in enterocytes.
Critical inﬂammatory role of Egr-1 in endotoxin
Joachim Schmidt, MD,* Burkhard Stoffels, MD,
Savanh Chanthaphavong, PhD, Anthony Bauer, PhD
University of Pittsburgh, Pittsburgh, PA
INTRODUCTION: Early growth response gene-1 (Egr-1) has re-
cently been linked to the transcriptional regulation of inflammatory
mediators. Endotoxin is known to activate the dense network of
resident muscularis macrophages, which subsequently produce cyto-
kines and chemokines that recruit monocytes into the muscularis
externa resulting in sepsis induced ileus. Our objective was to mech-
anistically investigate the role of Egr-1 in endotoxin induced ileus.
METHODS: Wild-type and Egr-1 knockout mice were subjected to
intraperitoneal LPS injection (5 mg/kg) to induce ileus. RT-PCR,
Western blot and immunohistochemistry quantified and localized
Egr-1. Intralumenal transit of non-absorbable FITC labeled dextran
and calculated geometric centers measured gastrointestinal motility.
© 2007 by the American College of Surgeons ISSN 1072-7515/07/$32.00
Published by Elsevier Inc.