The proinflammatory phenotype of PECAM-1-deficient mice results in atherogenic diet-induced steatohepatitis

Article (PDF Available)inAJP Gastrointestinal and Liver Physiology 293(6):G1205-14 · January 2008with7 Reads
DOI: 10.1152/ajpgi.00157.2007 · Source: PubMed
Abstract
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 macrovesicular 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-alpha 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.

Figures

The proinflammatory phenotype of PECAM-1-deficient mice results
in atherogenic diet-induced steatohepatitis
Reema Goel,
1
Brian Boylan,
1
Lynn Gruman,
2,3
Peter J. Newman,
1,4,5,7
Paula E. North,
2,3
and Debra K. Newman
1,6,7
1
Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, Wisconsin;
2
The Department of Pathology and Laboratory
Medicine, Children’s Hospital of Wisconsin, Milwaukee, Wisconsin; Departments of
3
Pathology,
4
Cell Biology, Neurobiology
and Anatomy,
5
Pharmacology, and
6
Microbiology and Molecular Genetics, and
7
The 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: reema.goel@bcw.edu).
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.org G1205
future examination of recently identified PECAM-1 allelic
isoforms as potential risk factors for developing NASH in
humans.
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 histologyofWT(A, C,
E, and G) and KO (B, D, F, and H) mice fed an ND (A and B)oranADfor3(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].
G1206 STEATOHEPATITIS IN PECAM-1 KO MICE
AJP-Gastrointest Liver Physiol VOL 293 DECEMBER 2007 www.ajpgi.org
5– 8 mice in each group using commercially available reagents (Wako
Diagnostics, Richmond, VA). Plasma levels of the proinflammatory
cytokines IL-6, IL-10, monocyte chemoattractant protein-1 (MCP-1),
IFN-, TNF-, and IL-12p70 were measured using the mouse inflam-
mation Cytometric Bead Array (BD Biosciences, San Jose, CA)
according to the manufacturer’s protocol. Plasma levels of aspartate
aminotransferase (AST) and lactic acid dehydrogenase (LDH), as well
as direct and total serum bilirubin and alanine aminotransferase (ALT)
levels, were determined by the clinical laboratory at Children’s
Hospital of Wisconsin.
Liver histology. Livers were collected and immediately divided into
three portions. One portion was fixed in 10% zinc formalin and
embedded in paraffin, and a second portion was embedded in optimal
cutting temperature (OCT) and snap-frozen; both of these portions
were used for histology. The third portion was snap frozen and used
for fluorescence microscopy studies (see below). Sections (4 min
lengeth) of paraffin-embedded tissue were deparaffinized and stained
with hematoxylin and eosin (H&E), reticulin/nuclear fast red (Dako,
Carpinteria, CA) or Masson’s trichrome (Richard Allan, Kalamazoo,
MI). Five- to 10-m sections of frozen tissue were stained with Oil
Red O (Sigma, St. Louis, MO) for 15 min, washed, and counterstained
with hematoxylin (Dako) for 45 s. Liver histology was semiquantita-
tively scored in a blinded manner as previously described (36). In each
specimen, at least 50 random microscopic fields were examined under
40 high-power (HP) magnification. Specifically, microvesicular and
macrovesicular steatosis were each individually evaluated in H&E-
stained sections and scored on a scale of 0 4 as follows: 0, no
steatosis; 1, 5% steatosis; 2, 5–33% steatosis; 3, 33– 66% steatosis; 4,
66% steatosis. Lobular and sinusoidal inflammation were each
individually evaluated in H&E-stained sections and scored on a scale
of 0–3 as follows: 0, no inflammatory foci; 1, 1 inflammatory
focus/HP field; 2, 2–3 inflammatory foci/HP field; 3, 4 inflamma-
tory foci/HP field. Fibrosis was evaluated in reticulin- and trichrome-
stained sections and scored on a scale of 0 4 as follows: 0, no
fibrosis; 1, mild fibrosis (excess connective tissue within the portal
tracts, but no extension into the adjacent parenchyma); 2, moderate
portal/periportal fibrosis (fibrous tissue occupies the portal tracts and
periportal region partially or completely with connective tissue ex-
tending into the neighboring parenchyma); 3, bridging fibrosis; 4,
cirrhosis. The average mean scores for microvesicular steatosis, ma-
Fig. 3. Quantitative analysis of the effect of platelet
endothelial cell adhesion molecule-1 (PECAM-1)
deficiency on AD-induced hepatic steatosis, inflam-
mation, and fibrosis. Histological scores for mi-
crovesicular steatosis (A), macrovesicular steatosis
(B), lobular inflammation (C), sinusoidal inflamma-
tion (D), reticulin fiber deposition (E), and collagen
deposition (F) were assigned as described in
MATERIALS AND METHODS for liver sections from
WT (open bars) and KO (solid bars) mice fed an
ND or an AD for 9 or 18 wk. Results are expressed
as mean histological scores SE determined in the
number of mice indicated at the base of each bar.
Statistically significant differences between WT
and KO mice at each time point are indicated by
asterisks (*P 0.05, ***P 0.001). Statistically
significant differences between KO and WT mice
fed an AD relative to KO and WT mice, respec-
tively, fed a normal diet, are indicated by daggers
(†P 0.05, ††P 0.01, †††P 0.001).
G1207STEATOHEPATITIS IN PECAM-1 KO MICE
AJP-Gastrointest Liver Physiol VOL 293 DECEMBER 2007 www.ajpgi.org
crovesicular steatosis, sinusoidal inflammation, lobular inflammation,
reticulin fiber staining, and trichrome staining were summed and
divided by six to obtain a mean total histological score for steato-
hepatitis.
Fluorescence microscopy for assessment of superoxide production.
The oxidation-dependent fluorescent dye dihydroethidium (DHE; Mo-
lecular Probes, Invitrogen Detection Technologies, Eugene, OR) was
used to evaluate production of superoxide. DHE is freely permeable to
cells and, in the presence of superoxide, is oxidized to 2-hy-
droxyethidium, which fluoresces at 585 nm. Unfixed frozen livers
were cut into 5–10- m thick sections, which were placed on glass
slides. Sections were pretreated for 1 h with 150 M Mn(III)tetrakis(4-
benzoic acid)porphyrin (MnTBAP) chloride (Alexis Biochemicals,
Lausen, Switzerland) to scavenge extracellular superoxide (17, 28).
DHE (5 M) was then applied to the surface of each tissue section,
and the slides were incubated in a light-protected, humidified chamber
at 37°C for 30 min. Images were obtained with a fluorescent micro-
scope (Zeiss Axioskop MC100; Carl Zeiss, Thornwood, NY)
equipped with a krypton-argon laser. Identical laser settings were used
for acquisition of images from specimens that were processed and
imaged in parallel. Fluorescence was detected with a 585-nm filter.
Macrophage recruitment assay. To obtain macrophages, mice were
injected intraperitoneally with 1 ml of 3% aged thioglycollate
(Sigma). After 5 days, peritoneal exudate was obtained from thiogly-
collate-treated mice by peritoneal lavage using ice-cold 30% sucrose
in phosphate-buffered saline. For adoptive transfer experiments, mac-
rophages were labeled with 5 M 5-(and-6)-carboxyfluorescein di-
acetate(CFDA-SE; Invitrogen Detection Technologies) (5, 51) for 20
min, and the efficiency of CFDA-SE labeling was verified by immu-
nofluorescence. Macrophages (3 10
6
)in200l of PBS were
injected retro-orbitally into mice that had received the atherogenic diet
for 8 wk. After 48 h, the recipient mice were killed and perfused, and
their livers, spleens, and lungs were isolated and frozen in OCT. Serial
tissue cryosections were stained with 4-6 diamidino-2 phenylindole
(DAPI) and H&E and examined by light and fluorescence microscopy
(Zeiss Axioskop MC100; Carl Zeiss) using identical laser settings.
Statistical analyses. Data are presented as means SE. Differ-
ences between means were analyzed by two-way ANOVA for
PECAM-1 WT and KO mice over the time course of atherogenic diet
using GraphPad Prism 4 software (GraphPad Software, San Diego,
CA). When the interaction was significant (P 0.05), the Bonferroni
posthoc test was applied to test for differences between groups.
RESULTS
PECAM-1-deficient mice develop marked atherogenic diet-
induced steatosis, inflammation, and fibrosis. The strong asso-
ciation between obesity, NAFLD, and NASH has prompted
study of the effects of diet on the development of steatosis and
steatohepatitis in mouse models (3, 37). The C57BL6 mouse is
a particularly good model of the human metabolic syndrome
because, like humans, C57BL6 mice develop obesity, hyper-
lipidemia, hyperinsulinemia, hypertension (30), and steato-
hepatitis (31, 65) when allowed ad libitum access to a high-fat
atherogenic diet. To determine whether PECAM-1 deficiency
affects the development of steatohepatitis in C57BL6 mice, 6-
to 8-wk-old male and female KO and WT C57BL6 littermates
were allowed ad libitum access to a normal chow diet or to a
high-fat atherogenic diet (21.2% fat, 0.15% cholesterol, 0.5%
sodium cholate) for up to 18 wk. PECAM-1 deficiency did not
affect weight gain on the normal chow diet (data not shown) or
on the atherogenic diet (supplemental Fig. 1; supplemental
figures for this article are available online at the American
Journal of Physiology Gastrointestinal and Liver Physiology
website) at any time point on the diet, suggesting that dietary
intake did not differ systematically between KO and WT mice.
We also examined the effect of the atherogenic diet on lipid
profile in these mice (Fig. 1). Whereas levels of total triglyc-
erides did not increase at any time on the atherogenic diet in
either WT or KO mice (Fig. 1A), total cholesterol levels were
Fig. 4. Histological characterization of AD-induced lipid accumulation and fibrosis in the liver. Representative photomicrographs of liver sections from WT
(A, C, and E) and KO (B, D, and F) mice fed an AD for 9 wk, after staining with Oil Red O as a measure of lipid accumulation (A and B), reticulin as a measure
of fibrosis (C and D), and trichrome as a measure of collagen deposition (E and F) (original magnification: 40).
G1208 STEATOHEPATITIS IN PECAM-1 KO MICE
AJP-Gastrointest Liver Physiol VOL 293 DECEMBER 2007 www.ajpgi.org
significantly higher (P 0.0001) in both WT and KO mice
after 9 and 18 wk on the atherogenic diet relative to genetically
identical littermates fed a normal diet. However, the higher
levels of total cholesterol observed after 9–18 wk on the
atherogenic diet did not differ significantly between WT and
KO littermates (Fig. 1B). These results suggest that PECAM-1
deficiency does not affect the development of hypercholester-
olemia on the atherogenic diet.
Histological analysis of H&E-stained liver sections re-
vealed that after 3 wk on an atherogenic diet, WT and KO
mice developed mild microvesicular steatosis (Fig. 2, C and
D), predominantly in hepatic parenchymal cells in the cen-
trilobular region. After 9 and 18 wk on the atherogenic diet,
prominent steatosis and mild inflammation were seen in
livers of WT mice (Figs. 2E and 3), whereas significantly
more severe steatosis, lobular, and sinusoidal inflammation
developed in the livers of KO mice (Figs. 2F and 3),
consistent with the typical histological features of steato-
hepatitis. The presence of multinucleated hepatocytes and
apoptotic cells was also observed. Histological features
progressed to prominent steatosis and mild inflammation in
WT mice (Figs. 2G and 3), whereas severe steatosis and
conspicuous inflammation was evident in KO mice (Figs.
2H and 3) after 18 wk on the atherogenic diet. Bile duct
hyperplasia was noted in some of the KO mice that were fed
the atherogenic diet, but this did not correlate with increased
bilirubin levels (data not shown).
Consistent with the histological analysis of H&E-stained
liver sections, neutral lipid accumulation was markedly more
pronounced in KO (Fig. 4B, supplemental Fig. 2) relative to
WT (Fig. 4A) livers after 9 wk of atherogenic diet. Also, in
contrast to WT mice, reticulin fiber (Fig. 4D vs. 4C,3E,
supplemental Fig. 3) and collagen deposition (Fig. 4F vs. 4E,
3F, supplemental Fig. 4) were markedly increased in the livers
of KO mice after 9 and 18 wk on the atherogenic diet. The
fibrosis was concentrated in the centrilobular area with no
bridging between portal tracts, indicating focal fibrosis had
developed by this time in KO but not WT mice. Our results
indicate that PECAM-1 deficiency exacerbates the rate and
extent of steatohepatitis in animals fed an atherogenic diet for
at least an 18-wk time period. It remains possible that signif-
icant steatohepatitis eventually does develop in WT mice fed
an atherogenic diet, but that requires a period of time longer
than 18 wk.
Fig. 5. Effect of PECAM-1 deficiency on hepatocellular damage in response
to ingestion of an AD. Plasma alanine aminotransferase (ALT) levels were
determined in WT (open bars) and KO (solid bars) mice fed an ND or an AD
for 9 or 18 wk. Results are expressed as mean ALT levels SE with the
number of mice in each group indicated at the base of each bar. Plasma ALT
levels were significantly increased in KO relative to WT mice fed an AD for
9wk(**P 0.01) and between KO mice fed an AD relative to KO mice fed
an ND for 9 wk (†††P 0.001).
Fig. 6. Histological characterization of hepatic oxidative stress. WT (A, C, E, and G)orKO(B, D, F, and H) mice fed an ND (A, B, E, and F)oranAD
(C, D, G, and H) for 9 wk were stained with the superoxide-sensitive fluorescent dye, dihydroethidine, either alone (AD) or following pretreatment with the
superoxide scavenger MnTBAP (EH). Note that superoxide levels were markedly higher in KO mice on an AD than in WT mice on an AD and in either KO
or WT mice fed an ND.
G1209STEATOHEPATITIS IN PECAM-1 KO MICE
AJP-Gastrointest Liver Physiol VOL 293 DECEMBER 2007 www.ajpgi.org
PECAM-1-deficient mice have significant hepatocellular
damage on an atherogenic diet. A number of blood tests are
commonly used to assess hepatic damage and liver disease,
including measurement of AST, ALT, LDH, alkaline phospha-
tase, and direct and total serum bilirubin, albumin, and pro-
thrombin time (7). Studies show that in NASH plasma liver
enzyme abnormalities are primarily restricted to elevations in
ALT and/or AST (1, 23). Correspondingly, we observed no
diet-induced changes in the levels of AST, direct bilirubin, or
LDH in either WT or KO (data not shown). However, we
found that plasma levels of ALT were significantly increased in
KO mice after 9 wk on an atherogenic diet, relative to WT
mice, and these levels return to normal after 18 wk as hepato-
cytes become depleted of enzyme content and are replaced by
fibrotic tissue (21) (Fig. 5). These data indicate that the athero-
genic diet induces more severe liver injury in PECAM-1-
deficient mice than in WT mice.
PECAM-1-deficient mice exhibit pronounced hepatic oxida-
tive stress and increased production of proinflammatory cyto-
kines in response to an atherogenic diet. Oxidative stress and
coincident or consequent production of proinflammatory cyto-
kines play a central role in the pathogenesis of NASH and
hepatic fibrosis (2, 3, 11, 20, 27, 43, 53). To understand the
pathophysiological mechanisms responsible for the marked
steatohepatitis that develops in PECAM-1-deficient mice upon
ingestion of an atherogenic diet, we looked for evidence of
oxidative stress and proinflammatory cytokines in these ani-
mals. We probed for superoxide production as a marker of
oxidative stress. As shown in Fig. 6, we found that livers of KO
mice fed an atherogenic diet for 9 wk exhibited strikingly
higher levels of superoxide anion compared with livers of WT
mice, and the majority of superoxide in the livers could be
scavenged by superoxide scavenger, MnTBAP. We next mea-
sured plasma levels of some of the proinflammatory cytokines
and chemokines, including IL-6, IL-10, MCP-1, IFN-,
TNF-, IL-12p70 in WT and KO mice . We found no evidence
for diet-induced changes in levels of IL-10, IFN-, or IL-12p70
in either WT or KO mice throughout the study (data not
shown). However, in both WT and KO mice fed an atherogenic
diet for 9 wk or 18 wk, plasma IL-6 levels were significantly
increased relative to littermates fed a normal diet, with no
significant differences between KO and WT mice (Fig. 7A).
This finding is consistent with previous studies, which showed
that the atherogenic diet, in general, leads to a state of chronic,
subacute inflammation (4, 8, 19). Increases in plasma levels of
MCP-1 (Fig. 7B) and TNF- (Fig. 7C) were also evident in
both WT and KO mice fed an atherogenic diet relative to
genetically identical mice fed a normal diet for 18 wk; how-
ever, plasma levels of MCP-1 and TNF- were significantly
higher in KO relative to WT mice fed an atherogenic diet for
9 wk. Taken together, these results indicate that, upon inges-
tion of an atherogenic diet for 9 wk, hepatic oxidative stress
and production of the proinflammatory cytokines MCP-1 and
TNF- are markedly exacerbated in KO mice.
Macrophage accumulation in the liver is enhanced in KO
mice on an atherogenic diet. The activation and recruitment of
leukocytes from blood into a site of inflammation is critical in
the pathogenesis of liver diseases (34, 55). PECAM-1 ho-
mophilic interactions have been reported to play an important
role in leukocyte transendothelial migration (22, 59, 64); how-
ever, our finding that liver inflammation is increased in KO
relative to WT mice is inconsistent with these previous find-
ings. We injected hypercholesterolemic recipient mice with
fluorescently labeled peritoneal macrophages from donor mice
to directly determine leukocyte recruitment and the fate of
recruited leukocytes in KO and WT mice. No noticeable
difference in macrophage accumulation was evident in the
spleens and lungs of WT relative to KO mice (data not shown).
However, as shown in Fig. 8, marked macrophage accumula-
tion in the liver was evident 48 h after injection when either
WT or KO macrophages were introduced into KO mice rela-
tive to WT recipient mice, suggesting that leukocyte recruit-
ment or retention into inflamed liver tissue is enhanced in the
absence of PECAM-1.
Fig. 7. Effect of PECAM-1 deficiency on production of proinflammatory
cytokines in response to ingestion of an atherogenic diet. Levels of IL-6 (A),
monocyte chemoattractant protein (MCP)-1 (B), and TNF- (C) were mea-
sured in plasma from WT (open bars) and KO (solid bars) mice fed an ND or
an AD for 9 or 18 wk. Results are expressed as mean cytokine levels SE
determined in the number of mice indicated at the base of each bar. MCP-1 and
TNF- levels were significantly increased in KO relative to WT mice after 9
wk on the AD (*P 0.05). Statistically significant differences between KO
and WT mice fed an AD relative to KO or WT mice, respectively, fed an ND,
are indicated by daggers (†P 0.05, ††P 0.01).
G1210 STEATOHEPATITIS IN PECAM-1 KO MICE
AJP-Gastrointest Liver Physiol VOL 293 DECEMBER 2007 www.ajpgi.org
DISCUSSION
The progression of NAFLD to NASH is thought to be
determined by an interaction between genetic and environmen-
tal factors, both of which are difficult to control in human
populations. Consequently, it is important to identify the genes
and environmental variables that influence this disease process.
In the present studies, we provide evidence that PECAM-1
deficiency, in combination with ingestion of an atherogenic
diet, results in the development of pronounced steatohepatitis
in mice. Specifically, histological evaluation of liver sections
revealed development of severe microvesicular and macrove-
sicular steatosis, lobular and sinusoidal inflammation, and focal
fibrosis; the rate and the extent of which were markedly
enhanced in KO compared with WT mice fed an atherogenic
diet and coincident with elevated plasma ALT levels, increased
hepatic oxidative stress, and increased plasma levels of the
proinflammatory cytokines TNF- and MCP-1. PECAM-1
deficiency contributed to higher levels of oxidative stress and
proinflammatory cytokine production, which have been iden-
tified as second hits capable of inducing positive feedback for
further hepatocyte steatosis, along with hepatocyte injury and
inflammation responsible for progression of simple steatosis to
NASH (3, 12, 16, 33, 42).
PECAM-1 is expressed on the surfaces of endothelial cells
as well as most bone marrow-derived hematopoietic cells,
including platelets, monocytes, granulocytes, and some lym-
phocytes (32). The liver is no exception to this paradigm,
wherein PECAM-1 is expressed on liver sinusoidal endothelial
cells (LSEC), Kupffer cells, and intrahepatic lymphocytes
(IHL) but not on hepatocytes (45). The architectural organiza-
tion of the liver allows for close proximity and even direct
interaction between these cell groups (67), making it possible
for soluble mediators produced by LSEC or leukocytes to
affect nearby hepatocytes. Previous studies have shown that
excessive exposure to either oxidative stress or inflammatory
cytokines can induce hepatocyte apoptosis and dysfunction,
which can exacerbate acute and chronic liver injury (15, 73).
Of particular relevance to the present study, it has been spec-
ulated that reactive oxygen species (ROS) and proinflamma-
tory cytokines are among the “second hits” that cause simple
steatosis in the liver to progress to NASH, and ultimately, to
fibrotic disease (3, 12, 18, 20, 53). On the basis of our findings,
therefore, we propose that loss of PECAM-1 from LSEC,
Kupffer cells, and/or IHL results in production of high levels of
ROS and inflammatory cytokines, which, in turn, profoundly
affect, albeit indirectly, the function of hepatocytes.
The finding in the present studies that PECAM-1 deficiency
is associated with heightened levels of oxidative stress and
proinflammatory cytokine production is consistent with two
sets of previous observations. First, we have previously re-
ported that ROS, such as superoxide anion, hydrogen peroxide,
and peroxynitrite, are overproduced in PECAM-1-deficient
relative to WT coronary microvessels (39). Second, several
recent studies have provided evidence that PECAM-1 sup-
presses production of proinflammatory cytokines. Specifically,
plasma concentrations of IL-1, TNF-, MCP-1, IFN-, and
IL-6 were all found to be significantly increased 24 h following
injection of lipopolysaccharide into PECAM-1-deficient rela-
tive to WT mice (9, 41), whereas IFN- production by
PECAM-1-deficient T cells was found to be four times that of
WT T cells in mice in which collagen-induced arthritis had
been experimentally induced (63). Although little is known
about the mechanism by which PECAM-1 normally suppresses
ROS production, Cepinskas et al. (10) have provided evidence
that the mechanism by which PECAM-1 suppresses production
of proinflammatory cytokines may involve interference with
translocation of nuclear factor (NF)-B into the nucleus of
inflamed endothelial cells. Whether this mechanism of action is
responsible for overproduction in PECAM-1-deficient mice of
MCP-1 and TNF- in response to ingestion of an atherogenic
Fig. 8. Distribution of fluorescently labeled
macrophages in mice fed the AD. Representa-
tive overlay photomicrographs of 4-6
diamidino-2 phenylindole (DAPI) (white
staining) and 5-(and-6)-carboxyfluorescein di-
acetate (CFDA-SE) (green staining) in liver
sections from WT (A and B)orKO(C and D)
mice fed an AD for 9 wk and injected with WT
(A and C)orKO(B and D) macrophages for
48 h. Note that livers from KO mice show
abundant infiltrated WT and KO macrophages
compared with WT mice injected with either
WT or KO macrophages.
G1211STEATOHEPATITIS IN PECAM-1 KO MICE
AJP-Gastrointest Liver Physiol VOL 293 DECEMBER 2007 www.ajpgi.org
diet remains to be determined. Likewise, the source of over-
produced ROS in PECAM-1-deficient mice and the mechanism
by which PECAM-1 suppresses ROS production remain fruit-
ful areas of future research.
The recruitment of activated inflammatory cells into the liver
is crucial for the pathogenesis of liver disease, and cell adhe-
sion molecules play an important role in this process (34).
PECAM-1 homophilic interactions have been reported to be
important for leukocyte transendothelial migration (22, 59, 64).
Nevertheless, we demonstrate in the present study that macro-
phages readily home into the inflamed livers of PECAM-1-
deficient mice fed an atherogenic diet, which indicates that
PECAM-1 homophilic interactions are not required for mac-
rophage recruitment into the liver under these conditions.
Our observation is consistent with the previous finding that
PECAM-1 does not play a major role in transmigration of
neutrophils into the liver during endotoxemia (13) and with the
finding, in at least four different in vivo models of inflamma-
tion, that the blood vessels of PECAM-1-deficient mice are
particularly susceptible to vascular leakage (9, 29, 41, 63, 71).
Together, these results suggest either that PECAM-1 ho-
mophilic interactions are not as important for leukocyte trans-
migration into the liver as they are in other tissues or that
increased vascular permeability in PECAM-1-deficient vessels
overcomes the need for PECAM-1 homophilic interactions in
transmigration of inflammatory cells into damaged tissue. The
extent to which either mechanism contributes to the enhanced
severity of NAFLD observed in PECAM-1-deficient mice fed
an atherogenic diet remains to be determined.
The NAFLD phenotype is broad, affecting males and fe-
males, children and adults, and different ethnic populations,
with widely variable outcomes ranging from a benign nonpro-
gressive course to cirrhosis and liver failure (43, 44). Familial
clustering of NASH and cirrhosis support a role for genetic
polymorphisms in factors that predispose to NASH (43, 44,
46). PECAM-1 deficiency has thus far not been described in
humans; however, the human PECAM-1 gene is polymorphic.
A number of single nucleotide polymorphisms (SNPs) in the
human PECAM-1 gene sequence have been reported (48,
60 62). Certain of these SNPs exist in linkage disequilibrium,
resulting in the existence of at least four alleles within the
human population (50). Although studies have not yet ad-
dressed the extent to which PECAM-1 alleles are associated
with human disease, several human PECAM-1 SNPs have
been found to correlate with progression of coronary artery
disease (24, 26, 68, 69) and/or myocardial infarction (24, 38,
58). These findings suggest that PECAM-1 plays a role in the
development of cardiovascular disease in humans. Since recent
studies have led to the conclusion that the factors that predis-
pose for development of cardiovascular disease are also risk
factors for development of NASH (6, 11, 35, 40, 54, 56, 66), it
will be important in future studies to determine whether any of
the PECAM-1 SNPs or alleles are associated with an increased
propensity to develop severe NAFLD.
In summary, we demonstrate in the present study that
PECAM-1 deficiency in mice fed an atherogenic diet contrib-
utes to development of more advanced stages of inflammatory
liver disease. Whether PECAM-1 normally inhibits progres-
sion of NAFLD by suppressing oxidative stress and inflamma-
tion or by maintaining an intact vascular permeability barrier
are interesting and important questions that our present data
cannot distinguish between and that remain to be addressed.
It is also important to keep in mind that these mechanisms
are not mutually exclusive. Nevertheless, our study identi-
fies PECAM-1 as a potentially attractive target to elucidate the
mechanisms leading to fatty liver disease pathogenesis and
progression.
ACKNOWLEDGMENTS
The authors thank Marjorie Kipp for help in maintaining the mouse colony.
GRANTS
This work was supported by HL-40926 (to P. J. Newman and D. K.
Newman) from National Heart, Lung, and Blood Institute of the National
Institutes of Health, and by a Postdoctoral Fellowship (to R. Goel) from the
American Heart Association.
REFERENCES
1. Adams LA, Talwalkar JA. Diagnostic evaluation of nonalcoholic fatty
liver disease. J Clin Gastroenterol 40: S34 –S38, 2006.
2. Albano E, Mottaran E, Occhino G, Reale E, Vidali M. Review article:
role of oxidative stress in the progression of non-alcoholic steatosis.
Aliment Pharmacol Ther 22, Suppl 2: 71–73, 2005.
3. Anstee QM, Goldin RD. Mouse models in non-alcoholic fatty liver
disease and steatohepatitis research. Int J Exp Pathol 87: 1–16, 2006.
4. Arkan MC, Hevener AL, Greten FR, Maeda S, Li ZW, Long JM,
Wynshaw-Boris A, Poli G, Olefsky J, Karin M. IKK- links inflam-
mation to obesity-induced insulin resistance. Nat Med 11: 191–198, 2005.
5. Becker HM, Chen M, Hay JB, Cybulsky MI. Tracking of leukocyte
recruitment into tissues of mice by in situ labeling of blood cells with the
fluorescent dye CFDA SE. J Immunol Methods 286: 69 –78, 2004.
6. Brea A, Mosquera D, Martin E, Arizti A, Cordero JL, Ros E.
Nonalcoholic fatty liver disease is associated with carotid atherosclerosis:
a case-control study. Arterioscler Thromb Vasc Biol 25: 1045–1050, 2005.
7. Burke MD. Liver function: test selection and interpretation of results. Clin
Lab Med 22: 377–390, 2002.
8. Cai D, Yuan M, Frantz DF, Melendez PA, Hansen L, Lee J, Shoelson
SE. Local and systemic insulin resistance resulting from hepatic activation
of IKK- and NF-B. Nat Med 11: 183–190, 2005.
9. Carrithers M, Tandon S, Canosa S, Michaud M, Graesser D, Madri
JA. Enhanced susceptibility to endotoxic shock and impaired STAT3
signaling in CD31-deficient mice. Am J Pathol 166: 185–196, 2005.
10. Cepinskas G, Savickiene J, Ionescu CV, Kvietys PR. PMN transendo-
thelial migration decreases nuclear NFB in IL-1-activated endothelial
cells: role of PECAM-1. J Cell Biol 161: 641– 651, 2003.
11. Chalasani N, Deeg MA, Crabb DW. Systemic levels of lipid peroxida-
tion and its metabolic and dietary correlates in patients with nonalcoholic
steatohepatitis. Am J Gastroenterol 99: 1497–1502, 2004.
12. Choi S, Diehl AM. Role of inflammation in nonalcoholic steatohepatitis.
Curr Opin Gastroenterol 21: 702–707, 2005.
13. Chosay JG, Fisher MA, Farhood A, Ready KA, Dunn CJ, Jaeschke H.
Role of PECAM-1 (CD31) in neutrophil transmigration in murine models
of liver and peritoneal inflammation. Am J Physiol Gastrointest Liver
Physiol 274: G776 –G782, 1998.
14. Clark JM. The epidemiology of nonalcoholic fatty liver disease in adults.
J Clin Gastroenterol 40: S5–S10, 2006.
15. Cogger VC, Muller M, Fraser R, McLean AJ, Khan J, Le Couteur
DG. The effects of oxidative stress on the liver sieve. J Hepatol 41:
370 –376, 2004.
16. Cortez-Pinto H, de Moura MC, Day CP. Non-alcoholic steatohepatitis:
from cell biology to clinical practice. J Hepatol 44: 197–208, 2006.
17. Day BJ, Shawen S, Liochev SI, Crapo JD. A metalloporphyrin super-
oxide dismutase mimetic protects against paraquat-induced endothelial
cell injury, in vitro. J Pharmacol Exp Ther 275: 1227–1232, 1995.
18. Day CP. Pathogenesis of steatohepatitis. Best Pract Res Clin Gastro-
enterol 16: 663– 678, 2002.
19. Day CP. From fat to inflammation. Gastroenterology 130: 207–210, 2006.
20. Day CP, James OF. Steatohepatitis: a tale of two “hits”? Gastroenterol-
ogy 114: 842– 845, 1998.
21. Dufour DR, Lott JA, Nolte FS, Gretch DR, Koff RS, Seeff LB.
Diagnosis and monitoring of hepatic injury. Recommendations for use of
G1212 STEATOHEPATITIS IN PECAM-1 KO MICE
AJP-Gastrointest Liver Physiol VOL 293 DECEMBER 2007 www.ajpgi.org
laboratory tests in screening, diagnosis, and monitoring. Clin Chem 46:
2050 –2068, 2000.
22. Duncan GS, Andrew DP, Takimoto H, Kaufman SA, Yoshida H,
Spellberg J, Luis de la Pompa J, Elia A, Wakeham A, Karan-Tamir
B, Muller WA, Senaldi G, Zukowski MM, Mak TW. Genetic evidence
for functional redundancy of Platelet/Endothelial cell adhesion molecule-1
(PECAM-1): CD31-deficient mice reveal PECAM-1-dependent and
PECAM-1-independent functions. J Immunol 162: 3022–3030, 1999.
23. Duvnjak M, Virovic L. [Non-alcoholic steatohepatitis]. Acta Med
Croatica 57: 189 –199, 2003.
24. Elrayess MA, Webb KE, Bellingan GJ, Whittall RA, Kabir J, Hawe E,
Syvanne M, Taskinen MR, Frick MH, Nieminen MS, Kesaniemi YA,
Pasternack A, Miller GJ, Humphries SE. R643G polymorphism in
PECAM-1 influences transendothelial migration of monocytes and is
associated with progression of CHD and CHD events. Atherosclerosis
177: 127–135, 2004.
25. Falati S, Patil S, Gross PL, Stapleton M, Merrill-Skoloff G, Barrett
NE, Pixton KL, Weiler H, Cooley B, Newman DK, Newman PJ, Furie
BC, Furie B, Gibbins JM. Platelet PECAM-1 inhibits thrombus forma-
tion in vivo. Blood 107: 535–541, 2006.
26. Fang L, Wei H, Chowdhury SH, Gong N, Song J, Heng CK, Sethi S,
Koh TH, Chatterjee S. Association of Leu125Val polymorphism of
platelet endothelial cell adhesion molecule-1 (PECAM-1) gene & soluble
level of PECAM-1 with coronary artery disease in Asian Indians. Indian
J Med Res 121: 92–99, 2005.
27. Farrell GC, Larter CZ. Nonalcoholic fatty liver disease: from steatosis
to cirrhosis. Hepatology 43: S99 –S112, 2006.
28. Gauuan PJ, Trova MP, Gregor-Boros L, Bocckino SB, Crapo JD, Day
BJ. Superoxide dismutase mimetics: synthesis and structure-activity rela-
tionship study of MnTBAP analogues. Bioorg Med Chem 10: 3013–3021,
2002.
29. Graesser D, Solowiej A, Bruckner M, Osterweil E, Juedes A, Davis S,
Ruddle NH, Engelhardt B, Madri JA. Altered vascular permeability and
early onset of experimental autoimmune encephalomyelitis in PECAM-1-
deficient mice. J Clin Invest 109: 383–392, 2002.
30. Grubb SC, Churchill GA, Bogue MA. A collaborative database of
inbred mouse strain characteristics. Bioinformatics 20: 2857–2859, 2004.
31. Isoda K, Sawada S, Ayaori M, Matsuki T, Horai R, Kagata Y,
Miyazaki K, Kusuhara M, Okazaki M, Matsubara O, Iwakura Y,
Ohsuzu F. Deficiency of interleukin-1 receptor antagonist deteriorates
fatty liver and cholesterol metabolism in hypercholesterolemic mice.
J Biol Chem 280: 7002–7009, 2005.
32. Jackson DE. The unfolding tale of PECAM-1. FEBS Lett 540: 7–14,
2003.
33. Jaeschke H. Reactive oxygen and mechanisms of inflammatory liver
injury. J Gastroenterol Hepatol 15: 718 –724, 2000.
34. Jaeschke H. Cellular adhesion molecules: regulation and functional sig-
nificance in the pathogenesis of liver diseases. Am J Physiol Gastrointest
Liver Physiol 273: G602–G611, 1997.
35. Jepsen P, Vilstrup H, Mellemkjaer L, Thulstrup AM, Olsen JH,
Baron JA, Sorensen HT. Prognosis of patients with a diagnosis of fatty
liver—a registry-based cohort study. Hepatogastroenterology 50: 2101–
2104, 2003.
36. Kleiner DE, Brunt EM, Van NM, Behling C, Contos MJ, Cummings
OW, Ferrell LD, Liu YC, Torbenson MS, Unalp-Arida A, Yeh M,
McCullough AJ, Sanyal AJ. Design and validation of a histological
scoring system for nonalcoholic fatty liver disease. Hepatology 41: 1313–
1321, 2005.
37. Koteish A, Mae DA. Animal models of steatohepatitis. Best Pract Res
Clin Gastroenterol 16: 679 690, 2002.
38. Listi F, Candore G, Lio D, Cavallone L, Colonna-Romano G, Caruso
M, Hoffmann E, Caruso C. Association between platelet endothelial
cellular adhesion molecule 1 (PECAM-1/CD31) polymorphisms and acute
myocardial infarction: a study in patients from Sicily. Eur J Immunogenet
31: 175–178, 2004.
39. Liu Y, Bubolz AH, Shi Y, Newman PJ, Newman DK, Gutterman DD.
Peroxynitrite reduces the endothelium-derived hyperpolarizing factor
component of coronary flow-mediated dilation in PECAM-1-knockout
mice. Am J Physiol Regul Integr Comp Physiol 290: R57–R65, 2006.
40. Loria P, Leonardo A, Bellentani S, Day CP, Marchesini G, Carulli N.
Non-alcoholic fatty liver disease (NAFLD) and cardiovascular disease: an
open question. Nutr Metab Cardiovasc Dis. 17: 684 698, 2007.
41. Maas M, Stapleton M, Bergom C, Mattson DL, Newman DK, New-
man PJ. Endothelial cell PECAM-1 confers protection against endotoxic
shock. Am J Physiol Heart Circ Physiol 288: H159 –H164, 2005.
42. Machado M, Cortez-Pinto H. Non-alcoholic steatohepatitis and meta-
bolic syndrome. Curr Opin Clin Nutr Metab Care 9: 637– 642, 2006.
43. McCullough AJ. Pathophysiology of nonalcoholic steatohepatitis. J Clin
Gastroenterol 40: S17–S29, 2006.
44. Merriman RB, Aouizerat BE, Bass NM. Genetic influences in nonalco-
holic fatty liver disease. J Clin Gastroenterol 40: S30 –S33, 2006.
45. Neubauer K, Wilfling T, Ritzel A, Ramadori G. Platelet-endothelial cell
adhesion molecule-1 gene expression in liver sinusoidal endothelial cells
during liver injury and repair. J Hepatol 32: 921–932, 2000.
46. Neuschwander-Tetri BA, Caldwell SH. Nonalcoholic steatohepatitis:
summary of an AASLD Single Topic Conference. Hepatology 37: 1202–
1219, 2003.
47. Newman PJ. Switched at birth: a new family for PECAM-1. J Clin Invest
103: 5–9, 1999.
48. Newman PJ, Berndt MC, Gorski J, White GC, Lyman S, Paddock C,
Muller WA. PECAM-1 (CD31) cloning and relation to adhesion mole-
cules of the immunoglobulin gene superfamily. Science 247: 1219 –1222,
1990.
49. Newton-Nash DK, Newman PJ. A new role for Platelet-Endothelial Cell
Adhesion Molecule-1 (CD31): inhibition of TCR-mediated signal trans-
duction. J Immunol 163: 682– 688, 1999.
50. Novinska MS, Pietz BC, Ellis TM, Newman DK, Newman PJ. The
alleles of PECAM-1. Gene 376: 95–101, 2006.
51. Patel SS, Thiagarajan R, Willerson JT, Yeh ET. Inhibition of alpha4
integrin and ICAM-1 markedly attenuate macrophage homing to athero-
sclerotic plaques in ApoE-deficient mice. Circulation 97: 75– 81, 1998.
52. Patil S, Newman DK, Newman PJ. Platelet endothelial cell adhesion
molecule-1 serves as an inhibitory receptor that modulates platelet re-
sponses to collagen. Blood 97: 1727–1732, 2001.
53. Pessayre D, Fromenty B. NASH: a mitochondrial disease. J Hepatol 42:
928 –940, 2005.
54. Qiao Q, Gao W, Zhang L, Nyamdorj R, Tuomilehto J. Metabolic
syndrome and cardiovascular disease. Ann Clin Biochem 44: 232–263,
2007.
55. Racanelli V, Rehermann B. The liver as an immunological organ.
Hepatology 43: S54 –S62, 2006.
56. Rana JS, Nieuwdorp M, Jukema JW, Kastelein JJ. Cardiovascular
metabolic syndrome an interplay of, obesity, inflammation, diabetes
and coronary heart disease. Diabetes Obes Metab 9: 218 –232, 2007.
57. Rathore V, Stapleton MA, Hillery CA, Montgomery RR, Nichols TC,
Merricks EP, Newman DK, Newman PJ. PECAM-1 negatively regu-
lates GPIb/V/IX signaling in murine platelets. Blood 102: 3658 –3664,
2003.
58. Sasaoka T, Kimura A, Hohta SA, Fukuda N, Kurosawa T, Izumi T.
Polymorphisms in the platelet-endothelial cell adhesion molecule-1
(PECAM-1) gene, Asn563Ser and Gly670Arg, associated with myocardial
infarction in the Japanese. Ann NY Acad Sci 947: 259 –269, 2001.
59. Schenkel AR, Chew TW, Muller WA. Platelet endothelial cell adhesion
molecule deficiency or blockade significantly reduces leukocyte emigra-
tion in a majority of mouse strains. J Immunol 173: 6403– 6408, 2004.
60. Shibata Y, Juji T, Tohyama H, Sakamoto H, Ozawa N, Kano K. Mixed
passive hemagglutination with soluble platelet antigens. Int Arch Allergy
Appl Immunol 74: 93–96, 1984.
61. Simmons DL, Walker C, Power C, Pigott R. Molecular cloning of
CD31, a putative intercellular adhesion molecule closely related to carci-
noembryonic antigen. J Exp Med 171: 2147–2152, 1990.
62. Stockinger H, Gadd SJ, Eher R, Majdic O, Schreiber W, Kasinrerk
W, Strass B, Schnabl E, Knapp W. Molecular characterization and
functional analysis of the leukocyte surface protein CD31. J Immunol 145:
3889 –3897, 1990.
63. Tada Y, Koarada S, Morito F, Ushiyama O, Haruta Y, Kanegae F,
Ohta A, Ho A, Mak TW, Nagasawa K. Acceleration of the onset of
collagen-induced arthritis by a deficiency of platelet endothelial cell
adhesion molecule 1. Arthritis Rheum 48: 3280 –3290, 2003.
64. Thompson RD, Noble KE, Larbi KY, Dewar A, Duncan GS, Mak TW,
Nourshargh S. Platelet-endothelial cell adhesion molecule-1 (PECAM-
1)-deficient mice demonstrate a transient and cytokine-specific role for
PECAM-1 in leukocyte migration through the perivascular basement
membrane. Blood 97: 1854 –1860, 2001.
G1213STEATOHEPATITIS IN PECAM-1 KO MICE
AJP-Gastrointest Liver Physiol VOL 293 DECEMBER 2007 www.ajpgi.org
65. Vergnes L, Phan J, Strauss M, Tafuri S, Reue K. Cholesterol and
cholate components of an atherogenic diet induce distinct stages of hepatic
inflammatory gene expression. J Biol Chem 278: 42774 42784, 2003.
66. Villanova N, Moscatiello S, Ramilli S, Bugianesi E, Magalotti D,
Vanni E, Zoli M, Marchesini G. Endothelial dysfunction and cardiovas-
cular risk profile in nonalcoholic fatty liver disease. Hepatology 42:
473– 480, 2005.
67. Warren A, Le Couteur DG, Fraser R, Bowen DG, McCaughan GW,
Bertolino P. T lymphocytes interact with hepatocytes through fenestrations in
murine liver sinusoidal endothelial cells. Hepatology 44: 1182–1190, 2006.
68. Wei H, Fang L, Chowdhury SH, Gong N, Xiong Z, Song J, Mak KH,
Wu S, Koay E, Sethi S, Lim YL, Chatterjee S. Platelet-endothelial cell
adhesion molecule-1 gene polymorphism and its soluble level are associ-
ated with severe coronary artery stenosis in Chinese Singaporean. Clin
Biochem 37: 1091–1097, 2004.
69. Wenzel K, Baumann G, Felix SB. The homozygous combination of
Leu125Val and Ser563Asn polymorphisms in the PECAM1 (CD31) gene
is associated with early severe coronary heart disease. Hum Mutat 14: 545,
1999.
70. Wilkinson R, Lyons AB, Roberts D, Wong MX, Bartley PA,
Jackson DE. Platelet endothelial cell adhesion molecule-1 (PECAM-
1/CD31) acts as a regulator of B-cell development, B-cell antigen
receptor (BCR)-mediated activation, and autoimmune disease. Blood
100: 184 –193, 2002.
71. Wong MX, Hayball JD, Hogarth PM, Jackson DE. The inhibitory
co-receptor, PECAM-1 provides a protective effect in suppression of
collagen-induced arthritis. J Clin Immunol 25: 19 –28, 2005.
72. Wong MX, Roberts D, Bartley PA, Jackson DE. Absence of platelet
endothelial cell adhesion molecule-1 (CD31) leads to increased severity of
local and systemic IgE-mediated anaphylaxis and modulation of mast cell
activation. J Immunol 168: 6455– 6462, 2002.
73. Wullaert A, van LG, Heyninck K, Beyaert R. Hepatic tumor necrosis
factor signaling and nuclear factor-kappaB: effects on liver homeostasis
and beyond. Endocr Rev 28: 365–386, 2007.
G1214 STEATOHEPATITIS IN PECAM-1 KO MICE
AJP-Gastrointest Liver Physiol VOL 293 DECEMBER 2007 www.ajpgi.org
    • "As previously reported, PECAM-1 showed a protective role against endotoxin shock in mice [25] . Similarly, lack of PECAM-1 resulted in amplified inflammation followed by liver injury in a nonalcoholic steatohepatitis mouse model [42]. Hepatocytes are the major liver cell type and main source of positive acute phase-proteins [43]. "
    [Show abstract] [Hide abstract] ABSTRACT: Platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) is known to play an important role in hepatic inflammation. Therefore, we investigated the role of PECAM-1 in wild-type (WT) and knock-out (KO)-mice after single-dose liver irradiation (25 Gy). Both, at mRNA and protein level, a time-dependent decrease in hepatic PECAM-1, corresponding to an increase in intercellular cell adhesion molecule-1 (ICAM-1) (6 hrs) was detected in WT-mice after irradiation. Immunohistologically, an increased number of neutrophil granulocytes (NG) (but not of mononuclear phagocytes) was observed in the liver of WT and PECAM-1-KO mice at 6 hrs after irradiation. The number of recruited NG was higher and prolonged until 24 hrs in KO compared to WT-mice. Correspondingly, a significant induction of hepatic tumour necrosis factor (TNF)-α and CXC-chemokines (KC/CXCL1 interleukin-8/CXCL8) was detected together with an elevation of serum liver transaminases (6-24 hrs) in WT and KO-mice. Likewise, phosphorylation of signal transducer and activator of transcription-3 (STAT-3) was observed in both animal groups after irradiation. The level of all investigated proteins as well as of the liver transaminases was significantly higher in KO than WT-mice. In the cell-line U937, irradiation led to a reduction in PECAM-1 in parallel to an increased ICAM-1 expression. TNF-α-blockage by anti-TNF-α prevented this change in both proteins in cell culture. Radiation-induced stress conditions induce a transient accumulation of granulocytes within the liver by down-regulation/absence of PECAM-1. It suggests that reduction/lack in PECAM-1 may lead to greater and prolonged inflammation which can be prevented by anti-TNFα. © 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.
    Full-text · Article · Jul 2015
    • "Platelet/endothelial cell adhesion molecule 1 (Pecam1) is a glycoprotein located near a QTL for fatty liver [32] and diabetes [30]. Previous work has suggested that Pecam1 is involved in regulating inflammation and higher expression of this gene protects the liver from the effect of high dietary fat and NAFLD [57]. High-fat fed SM/J displayed significantly higher Pecam1 expression levels when compared to low-fat fed individuals. "
    [Show abstract] [Hide abstract] ABSTRACT: The liver plays a major role in regulating metabolic homeostasis and is vital for nutrient metabolism. Identifying the genetic factors regulating these processes could lead to a greater understanding of how liver function responds to a high-fat diet and how that response may influence susceptibilities to obesity and metabolic syndrome. In this study we examine differences in hepatic gene expression between the LG/J and SM/J inbred mouse strains and how gene expression in these strains is affected by high-fat diet. LG/J and SM/J are known to differ in their responses to a high-fat diet for a variety of obesity- and diabetes-related traits, with the SM/J strain exhibiting a stronger phenotypic response to diet. Dietary intake had a significant effect on gene expression in both inbred lines. Genes up-regulated by a high-fat diet were involved in biological processes such as lipid and carbohydrate metabolism; protein and amino acid metabolic processes were down regulated on a high-fat diet. A total of 259 unique transcripts exhibited a significant diet-by-strain interaction. These genes tended to be associated with immune function. In addition, genes involved in biochemical processes related to non-alcoholic fatty liver disease (NAFLD) manifested different responses to diet between the two strains. For most of these genes, SM/J had a stronger response to the high-fat diet than LG/J. These data show that dietary fat impacts gene expression levels in SM/J relative to LG/J, with SM/J exhibiting a stronger response. This supports previous data showing that SM/J has a stronger phenotypic response to high-fat diet. Based upon these findings, we suggest that SM/J and its cross with the LG/J strain provide a good model for examining non-alcoholic fatty liver disease and its role in metabolic syndrome.
    Full-text · Article · Feb 2014
    • "CIA in DBA/1 mice Enhanced arthritis (Tada et al., 2003; Wong et al., 2005) Exposure to the bacterial endotoxin LPS Septic shock (Maas et al., 2005) Laser-induced and FeCl 3 endothelial injury Accelerated vascular occlusion (thrombosis) (Falati et al., 2006) Diet-induced non-alcoholic steatohepatitis Progressive liver disease (Goel et al., 2007) LDLR KO (hypercholesterolemic) mice Accelerated atherosclerosis (Goel et al., 2008) ApoE-deficient (hypercholesterolemic) mice Inhibited atherosclerosis (Harry et al., 2008) Bone marrow hematopoietic cell engraftment Hypersensitivity to macrophage CSF and receptor activator of NF-kB ligand; osteoclastic bone loss (Wu et al., 2009) Lipopolysaccharide (LPS)-induced endotoxemia Cytokine storm and acute respiratory distress syndrome due to accumulation of cytokine-producing leukocytes at sites of inflammation (Privratsky et al., 2010). "
    [Show abstract] [Hide abstract] ABSTRACT: Although it is expressed by all leukocytes, including T-, B-lymphocytes and dendritic cells, the immunoglobulin-like receptor CD31 is generally regarded by immunologists as a marker of endothelial cell lineage that lacks an established functional role in adaptive immunity. This perception has recently been challenged by studies that reveal a key role for this molecule in the regulation of T-cell homeostasis, effector function and trafficking. The complexity of the biological functions of CD31 results from the integration of its adhesive and signaling functions in both the immune and vascular systems. Signaling by means of CD31 is induced by homophilic engagement during the interactions of immune cells and is mediated by phosphatase recruitment or activation through immunoreceptor tyrosine inhibitory motifs (ITIMs) that are located in its cytoplasmic tail. Loss of CD31 function is associated with excessive immunoreactivity and susceptibility to cytotoxic killing. Here, we discuss recent findings that have brought to light a non-redundant, complex role for this molecule in the regulation of T-cell-mediated immune responses, with large impact on our understanding of immunity in health and disease.
    Full-text · Article · Jun 2013
Show more

Recommended publications

Discover more