The proinflammatory phenotype of PECAM-1-deficient mice results
in atherogenic diet-induced steatohepatitis
Reema Goel,1Brian Boylan,1Lynn Gruman,2,3Peter J. Newman,1,4,5,7Paula E. North,2,3
and Debra K. Newman1,6,7
1Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, Wisconsin;2The Department of Pathology and Laboratory
Medicine, Children’s Hospital of Wisconsin, Milwaukee, Wisconsin; Departments of3Pathology,4Cell Biology, Neurobiology
and Anatomy,5Pharmacology, and6Microbiology and Molecular Genetics, and7The Cardiovascular Center, Medical College
of Wisconsin, Milwaukee, Wisconsin
Submitted 11 April 2007; accepted in final form 2 October 2007
Goel R, Boylan B, Gruman L, Newman PJ, North PE, Newman
DK. The proinflammatory phenotype of PECAM-1-deficient mice
results in atherogenic diet-induced steatohepatitis. Am J Physiol Gas-
trointest Liver Physiol 293: G1205–G1214, 2007. First published
October 11, 2007; doi:10.1152/ajpgi.00157.2007.—The severity of
nonalcoholic steatohepatitis (NASH) is determined by environmental
and genetic factors, the latter of which are incompletely characterized.
Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a 130-
kDa transmembrane glycoprotein expressed on blood and vascular
cells. In the present study, we provide data for the novel finding that
genetic deficiency of PECAM-1 potentiates the development and
progression of NASH. We found that the rate of development and
severity of diet-induced NASH are markedly enhanced in PECAM-
1-deficient [knockout (KO)] mice relative to wild-type (WT) mice, as
measured by histological and biochemical evaluation. Livers from KO
mice exhibited typical histological features of NASH, including ma-
crovesicular fat accumulation, hepatocyte injury with infiltration of
inflammatory cells, fibrosis, and heightened oxidative stress. Alanine
aminotransferase, a marker for liver injury, was also significantly
higher in KO compared with WT mice. Consistent with a role for
PECAM-1 as a suppressor of proinflammatory cytokines, plasma
levels of inflammatory cytokines, including TNF-? and monocyte
chemoattractant protein-1 (MCP-1), were also significantly higher in
KO compared with WT mice. These findings are the first to show that
the PECAM-1-deficient mouse develops progressive nonalcoholic
fatty liver disease (NAFLD), supporting a role for PECAM-1 as a
negative regulator of NAFLD progression. Future examination of
recently identified PECAM-1 allelic isoforms in humans as potential
risk factors for developing NASH may be warranted.
CD31; liver; NASH; inflammation
NONALCOHOLIC FATTY LIVER DISEASE (NAFLD) encompasses an
array of liver pathologies observed in individuals who do not
abuse alcohol. The disease spectrum ranges from accumulation
of fat in hepatocytes (steatosis) to the presence of an inflam-
matory infiltrate and fibrosis [nonalcoholic steatohepatitis
(NASH)], and ultimately to progressive fibrosis and cirrhosis
(14, 42–44, 46). The steatosis that characterizes NAFLD is
frequently associated with features of the metabolic syndrome,
including intra-abdominal obesity, dyslipidemia, insulin resis-
tance, type 2 diabetes, and hypertension (14, 44), which has led
to the suggestion that NAFLD is the hepatic manifestation of
the metabolic syndrome (3, 12). Because the metabolic syn-
drome is found with increasing frequency in association with a
Westernized lifestyle, NAFLD is increasingly recognized as a
frequent cause of liver dysfunction in Western societies, and is
estimated to occur with a prevalence of ?25% in Western
countries, although only a subset of affected individuals de-
velop the more advanced forms of the disease (14, 16, 42). A
two-hit model has been proposed to explain NAFLD and
NASH progression (18, 20). The first hit is steatosis, which
results from disrupted synthesis, transport, and removal of long
chain fatty acids and triglycerides and sensitizes the liver to the
occurrence of a second hit (3, 44). The second hit induces
hepatocyte injury and inflammation and is critically dependent
on oxidative stress and production of proinflammatory cyto-
kines (3, 16, 42). The nature and severity of first and second
hits can be influenced by both environmental (diet, drugs) and
genetic variables (18, 43).
Platelet endothelial cell adhesion molecule-1 (PECAM-1) is
a 130-kDa glycoprotein that is expressed at the junctions of all
continuous endothelium and on circulating blood cells (32).
There is increasing evidence that PECAM-1 regulates events
that contribute to inflammation. PECAM-1 has been shown in
a number of experimental systems to function as an inhibitory
receptor that limits agonist-induced activation of blood and
vascular cells (47). To date, PECAM-1 engagement or expres-
sion has been shown to inhibit T cell (49), B cell (70), mast cell
(72), and platelet (25, 52, 57) reactivity and to inhibit
production of proinflammatory cytokines in vivo (9, 41, 63).
PECAM-1 has also been implicated in maintaining vascular
integrity in at least four different in vivo models of inflam-
mation, including intradermal injection of histamine (29),
autoimmune encephalomyelitis (29), collagen-induced ar-
thritis (63, 71), and lipopolysaccharide-induced endotox-
emia (9, 41).
Since PECAM-1 is thought to be a negative regulator of
inflammatory responses, and since progression of NAFLD to
NASH is associated with chronic inflammation, we sought to
determine the effect of PECAM-1 deficiency on development
of NASH. Using a high-fat diet-induced mouse model of
NAFLD, we found that, whereas both PECAM-1-deficient and
wild-type (WT) mice developed steatosis on the diet, only
PECAM-1-deficient mice exhibited steatohepatitis with as-
sociated liver injury, inflammation, oxidative stress, and
fibrosis. Our studies demonstrate that PECAM-1 deficiency
places mice at risk for development of NASH and support
Address for reprint requests and other correspondence: Reema Goel, Blood
Research Institute, BloodCenter of Wisconsin, P.O. Box 2178, Milwaukee, WI
53201 (e-mail: email@example.com).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Am J Physiol Gastrointest Liver Physiol 293: G1205–G1214, 2007.
First published October 11, 2007; doi:10.1152/ajpgi.00157.2007.
0193-1857/07 $8.00 Copyright © 2007 the American Physiological Societyhttp://www.ajpgi.orgG1205
future examination of recently identified PECAM-1 allelic
isoforms as potential risk factors for developing NASH in
MATERIALS AND METHODS
Animals and diet. WT C57BL6 mice and PECAM-1-deficient
(KO) mice (22) that had been backcrossed for ?12 generations
onto a C57BL6 background, which display no evidence of liver
disease during their lifetime when fed a normal diet (R. Goel and
D. Newman, unpublished observations), were maintained in a
facility free of well-defined pathogens under the supervision of the
Biological Resource Center at the Medical College of Wisconsin.
All animal protocols were approved by the Institutional Animal
Care and Use Committee of the Medical College of Wisconsin.
Six- to 8-wk-old male and female KO and WT mice were placed on
either a normal diet (ND) (mouse chow 5010; Purina, St. Louis,
MO), containing 13.4% of calories derived from fat, or a high-fat
diet (Adjusted Calories Western Type Diet TD.05248; Harlan
Teklad, Madison, WI), containing 42% of calories derived from fat
and 0.5% sodium cholate, hereafter referred to as the atherogenic
diet (AD). The mice were housed in groups of four per cage,
maintained under alternating 12-h light-dark cycles, and had free
access to food and water.
Plasma and serum lipid, cytokine, and liver enzyme analyses.
Blood was collected by cardiac puncture of anesthetized mice, plasma
and serum were prepared, and aliquots were stored at ?80°C until
analyzed. Plasma levels of total cholesterol, HDL cholesterol, and
triglycerides were measured by enzymatic colorimetric assay per
manufacturer instructions in individual plasma samples (3–5 ?l) from
Fig. 1. Effect of an atherogenic diet on plasma
lipid levels. Nonfasting plasma levels of total
triglycerides (A) and total cholesterol (B) were
measured in wild-type (WT, open bars) and
PECAM-1-deficient (KO, solid bars) mice fed
a normal diet (ND) or an atherogenic diet (AD)
for 9 or 18 wk. Results are expressed as mean
plasma lipid levels ? SE measured in the
number of mice indicated at the base of the
relevant bar. Plasma levels of total cholesterol
but not triglycerides were significantly in-
creased in both KO and WT mice fed an AD for
9 or 18 wk and relative KO or WT mice,
respectively, fed an ND (†††P ? 0.001).
Fig. 2. Histological characterization of AD-induced hepatic steatosis and inflammation. Representative photomicrographs showing liver histology of WT (A, C,
E, and G) and KO (B, D, F, and H) mice fed an ND (A and B) or an AD for 3 (C and D), 9 (E and F), or 18 (G and H) wk. Note that steatosis and inflammation
occur earlier and are more severe in KO than in WT mice on the AD [hematoxylin and eosin (H&E) stain; original magnification: ?20].
STEATOHEPATITIS IN PECAM-1 KO MICE
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STEATOHEPATITIS IN PECAM-1 KO MICE
AJP-Gastrointest Liver Physiol • VOL 293 • DECEMBER 2007 • www.ajpgi.org