Am J Physiol Gastrointest Liver Physiol 303: G281–G290, 2012.
First published May 17, 2012; doi:10.1152/ajpgi.00423.2011.
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Physiology and GI Cancer
Hyperhomocysteinemia decreases intestinal motility leading to constipation
S. Givvimani,* C. Munjal,* N. Narayanan, F. Aqil, G. Tyagi, N. Metreveli, and S. C. Tyagi
Department of Physiology and Biophysics, University of Louisville School of Medicine, Louisville, Kentucky
Submitted 18 October 2011; accepted in final form 14 May 2012
matrix metalloproteinase-9; intestinal remodeling
HYPERHOMOCYSTEINEMIA (HHcy) is well-established risk factor
for various disease conditions including ischemic heart disease,
chronic kidney disease, cerebrovascular disease, and various
vascular pathologies. It has been recognized that HHcy is a risk
factor for both arterial and venous thrombosis including mesenteric ischemia and thrombosis (2, 8, 15, 35). Recent studies
(10, 33) have explored the relation between HHcy and inflammatory bowel disease (IBD) condition. According to Centers
for Disease Control and Prevention, IBD is one of the most
common gastrointestinal diseases in the United States, with
healthcare cost averaging more than $1.7 billion. Globally, the
prevalence of IBD has reached up to 396/100,000 persons (23)
affecting the quality of life.
* S. Givvimani and C. Munjal contributed equally to this work.
Address for reprint requests and other correspondence: S. Givvimani, Dept.
of Physiology and Biophysics, Univ. of Louisville School of Medicine, 500
South Preston St., Louisville, KY 40202 (e-mail: [email protected]).
http://www.ajpgi.org
Interestingly, clinical studies (38) have reported that HHcy
is more common in IBD patients compared with healthy
controls and is associated with lower vitamin B12 levels. The
gastrointestinal tract (GIT) is a significant site of sulfur amino
acid metabolism. The gastrointestinal tract accounts for ⬃25%
of whole body transmethylation and transsulfuration pathways
and is a prominent site of net Hcy release (3, 36). Previous
studies (7) have shown that exposure of Hcy to isolated human
intestinal microvascular endothelial cells triggers surface expression of cell adhesion molecules (ICAM-1) and contributes
to the accumulation of inflammatory infiltrate and also aggravates IBD condition. HHcy due to methylene tetrahydrofolate
reductase (MTHFR) gene polymorphism was reported as one
of the risk factors of mesenteric venous thrombosis leading to
infarction of the bowel (18). In addition, increased prevalence
of MTHFR gene polymorphism has been reported in colorectal
cancer and IBD patients (4, 34). As the enzyme MTHFR plays
a critical role in the remethylation pathway of Hcy metabolism,
polymorphism/mutation of MTHFR results in elevated levels
of Hcy in the plasma, particularly in folate deficiency (43).
Furthermore, IBD patients were found to have lower levels of
folate and higher levels of Hcy in the circulation (20).
HHcy has been associated with inflammation and remodeling. The inflammatory response represents coordinative proteolytic activity in a facet of remodeling and disintegration.
Studies (31) have reported that during experimental colitis
inflammation induces endothelial-dependent arteriolar dysfunction and results in decreased blood flow. Similarly, we (14,
32) reported that during HHcy there is rarefaction and narrowing of vessels in the gut microvasculature. Furthermore, studies
(46) have shown Hcy causes upregulation of inducible nitric
oxide synthase (iNOS), which further contributes towards the
inflammatory response. iNOS was further shown to initiate complex inflammatory changes during hemorrhagic shock leading to
morphological and functional intestinal injury (16). Along the
same line, another inflammatory molecule, ICAM-1, was shown
to impair intestinal smooth muscle contractility during hemorrhagic shock (21). Interestingly, anti-inflammatory medications
such as resveratrol and aspirin have been found to have beneficial
role in lowering Hcy levels (40).
Matrix metalloproteinases (MMPs) were found to play an
important role in the pathophysiology of several intestinal
inflammatory disorders (28). MMP-9 has been associated with
mucosal damage during intestinal inflammation, whereas
MMP-2 was found to be protective (12). Previous reports (9)
suggest that inhibiting MMPs has a therapeutic potential in
targeting intestinal inflammation such as during colitis. Besides
the regular symptoms of abdominal pain and diarrhea, constipation can also be a symptom of IBD. The constipation that is
present in these cases is due to bowel obstruction and slowed
0193-1857/12 Copyright © 2012 the American Physiological Society
G281
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Givvimani S, Munjal C, Narayanan N, Aqil F, Tyagi G,
Metreveli N, Tyagi SC. Hyperhomocysteinemia decreases intestinal motility leading to constipation. Am J Physiol Gastrointest
Liver Physiol 303: G281–G290, 2012. First published May 17,
2012; doi:10.1152/ajpgi.00423.2011.—Elevated levels of plasma
homocysteine (Hcy) called hyperhomocysteinemia (HHcy) have been
implicated in inflammation and remodeling in intestinal vasculature, and
HHcy is also known to aggravate the pathogenesis of inflammatory bowel
disease (IBD). Interestingly, colon is the pivotal site that regulates Hcy levels
in the plasma. We hypothesize that HHcy decreases intestinal motility
through matrix metalloproteinase-9 (MMP-9)-induced intestinal remodeling leading to constipation. To verify this hypothesis, we used
C57BL/6J or wild-type (WT), cystathionine ␤-synthase (CBS⫹/⫺),
MMP-9⫺/⫺, and MMP-9⫺/⫺ ⫹ Hcy mice. Intestinal motility was
assessed by barium meal studies and daily feces output. Plasma Hcy
levels were measured by HPLC. Expression of ICAM-1, inducible
nitric oxide synthase, MMP-9, and tissue inhibitors of MMPs was
studied by Western blot and immunohistochemistry. Reactive oxygen
species (ROS) including super oxide were measured by the Invitrogen
molecular probe method. Tissue nitric oxide levels were assessed by
a commercially available kit. Plasma Hcy levels in the treated MMP-9
group mice were comparable to CBS⫹/⫺ mice. Barium meal studies
suggest that intestinal motility is significantly decreased in CBS⫹/⫺
mice compared with other groups. Fecal output-to-body weight ratio
was significantly reduced in CBS⫹/⫺ mice compared with other
groups. There was significant upregulation of MMP-9, iNOS, and
ICAM-1 expression in the colon from CBS⫹/⫺ mice compared with
WT mice. Levels of ROS, superoxide, and inducible nitric oxide were
elevated in the CBS⫹/⫺ mice compared with other groups. Results
suggest that HHcy decreases intestinal motility due to MMP-9induced intestinal remodeling leading to constipation.
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ROLE OF MMP-9 IN HOMOCYSTEINE-INDUCED CONSTIPATION
transit. Drug-induced mal absorption of vitamins during active
IBD results in deficiency of folate as well as vitamin B12.
Furthermore, vitamin B12 deficiency has also been implicated
as a digestive disorder resulting in constipation due to reduced
gastrointestinal motility (43).
In current study, we tried to evaluate the role of MMP-9 in
inducing intestinal remodeling during HHcy and thus leading
to decreased intestinal motility or constipation. To our knowledge, this is the first study to explore the effect HHcy on colon
transit time and its role in intestinal remodeling.
MATERIALS AND METHODS
Antibodies and reagents. Rabbit polyclonal antibodies to MMP-9,
tissue inhibitors of MMP-1 (TIMP-1), and iNOS were purchased from
Abcam (Cambridge, MA), and rabbit polyclonal antibodies to TIMP-4
were from Sigma-Aldrich (St. Louis, MO). Goat polyclonal and rabbit
polyclonal antibodies to ICAM-1 and SOD-1 antibody were procured
from Santa Cruz Biotechnology (Santa Cruz, CA). PVDF membrane
was from Bio-Rad (Hercules, CA).
Animals. Male C57BL/6J wild-type mice (8 –10 wk old), cystathionine ␤-synthase [CBS⫹/⫺] heterozygous mice (HHcy), and
MMP-9⫺/⫺ mice were obtained from Jackson Laboratory (Bar
Harbor, ME) and kept in the animal care facility of University of
Louisville where ambient environmental conditions (12:12-h lightdark cycle, 22–24°C) were maintained. CBS heterozygous knockout mice on standard chow are well-known models of HHcy (26).
The background strain for both CBS⫹/⫺ and MMP-9⫺/⫺ is
C57BL6/J, and it serves as control for both groups (Jackson mice
data reference). Recent literature also confirmed the C57BL/6
Fig. 2. Barium meal study showing the differences in intestinal transit among the
groups of mice. The farther the barium travelled, the faster is the motility. The distance
barium travelled is measured with Image Pro
software, and the data are depicted in arbitrary units (AU). Barium travelled at a much
slower rate in CBS⫹/⫺ mice representing
decreased motility or constipation. Quantified data from the image are depicted in bar
diagram. *P ⬍ 0.05, compared with WT
group. #P ⬍ 0.05, compared with CBS⫹/⫺
group. Data represent means ⫾ SE from n ⫽
5 per group.
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Fig. 1. A: gross images of colon from all the
4 groups of mice showing residual stool
content. B: bar graph showing the data from
stool/fecal weight (FWt) over body weight
(BWt) in grams. C: bar graph depicting the
data from length of the colon in all groups of
mice. *P ⬍ 0.05, compared with wild-type
(WT) group and #P ⬍ 0.05, compared with
cystathionine ␤-synthase (CBS⫹/⫺) group.
MMP-9, matrix metalloproteinase-9; Hcy,
homocysteine. Data represent means ⫾ SE
from n ⫽ 5 per group.
ROLE OF MMP-9 IN HOMOCYSTEINE-INDUCED CONSTIPATION
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Fig. 3. Hematoxylin and eosin (H&E) staining in frozen colon sections in WT, CBS⫹/⫺,
matrix metalloproteinase-9 (MMP-9⫺/⫺) and
Hcy-treated MMP-9⫺/⫺ groups. The thickness of muscularis mucosa assessed by Image Proplus software is depicted in bar diagram. *P ⬍ 0.05, compared with WT group.
#P ⬍ 0.05, compared with CBS⫹/⫺ group.
Data represent means ⫾ SE from n ⫽ 5 per
group.
transferred to PVDF, blocked with 5% fat-free milk, and blotted with
respective antibodies. As a loading control, GAPDH was used. The
bands were normalized with GAPDH controls.
Gelatin-zymography. Colon was harvested and cleaned minutely of
any fecal attachment and from blood by being flushed with PBS and
observed under microscope. For each zymogram, four colons were
homogenized in buffer containing 50 mM Tris·HCl pH 7.6, 5 mM
CaCl2, 150 mM NaCl, 0.05% Brij35 (Sigma, St. Louis, MO), and
0.02% NaN3. The supernatant was collected, and estimation of protein
was done using BCA assay kit (Pierce, Rockford, IL). The supernatant
containing eluted protein was then collected after centrifugation (10
min at 10,000 g). Each sample was mixed with an equal volume of 2⫻
zymography loading buffer (Bio-Rad), incubated for 10 min at room
temperature, and then separated on a 10% gelatin zymogram gel
(Bio-Rad). Gels were soaked in 1⫻ zymogram renaturation buffer for
15 min at room temperature; this was repeated three times. The gel
was incubated in 1⫻ zymogram developing buffer for 24 h at 37°C,
stained with Coomassie blue (1%) in a 30% methanol and 10% acetic
acid 1 h, destained in water, and developed using Gel-Doc.
Barium meal method. To study the gut motility in mice, we used
barium meal preparation. Barium meal is very popular method of
studying the gut abnormalities in pediatric and adult populations. For
the first time we used similar technique in mice to study the motility
differences between the study groups. Briefly, 1 g of barium sulfate
Fig. 4. A: elastin staining using commercially
available kit was done in colon tissue sections from all the 4 groups. Elastin fibers are
stained as brown-black color and collagen in
red color. B: elastin content was assessed by
Image Pro software, and intensity data (AU)
are represented in bar graphs. *P ⬍ 0.05,
compared with WT group. #P ⬍ 0.05, compared with CBS⫹/⫺ group. Data represent
means ⫾ SE from n ⫽ 5 per group.
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background for CBS⫹/⫺ mice. This is reported in the article under
the animal detail section in Ref. 26.
The animals were fed standard chow and water ad libitum. All
animal procedures were reviewed and approved by the Institutional
Animal Care and Use Committee of the University of Louisville,
School of Medicine in accord with Animal Care and Use Program
Guidelines of the National Institutes of Health.
Groups. Wild-type (C57BL/6J), CBS⫹/⫺, MMP-9⫺/⫺, and MMP9⫺/⫺ mice with high Hcy diet. Hcy was supplemented through
drinking water and high methionine diet. A minimum number of five
animals were used for each of the studies performed.
Body weight/stool weight. Animals were fed similar amount of
feed, and daily fecal output was measured and normalized with body
weight ratio.
Tissue extraction. Abdominal cavity was exposed by a midline
laparotomy, and the entire colon was removed from the caecum to the
anus. The length of the colon was measured, the colon was flushed
with cold PBS, and tissue obtained from each colon was processed for
further investigations.
Immunoblot studies of MMP-9, TIMP-4, iNOS, and ICAM-1. Western blot analyses were performed on colonic tissue homogenates using
10% SDS-PAGE as described previously (44). The BCA method was
used to estimate total protein, and 25 ␮g of protein were loaded in
each well of electrophoresis gels. After electrophoresis, proteins were
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ROLE OF MMP-9 IN HOMOCYSTEINE-INDUCED CONSTIPATION
Fig. 5. A: collagen content was assessed by
Mas O2-n’s trichrome staining kit. Collagen
is stained as blue color. B: bar graphs represent the quantified blue color intensity by
Image Pro software in AU. *P ⬍ 0.05, compared with WT group. #P ⬍ 0.05, compared
with CBS⫹/⫺ group. Data represent means ⫾
SE from n ⫽ 5 per group.
frozen colon sections using commercially available kits from Fisher
Scientific/Richard Allan Scientific following their staining protocol.
Immunofluorescence. Immunofluorescence staining was performed
on 5-␮m thick frozen sections of the colon tissue with MMP-9 and
TIMP-1 primary antibodies and secondarily stained with Alexa Fluor
488 (Invitrogen) to detect protein expression.
RNA isolation and real-time PCR. RNA was isolated from colon
samples using commercially available kit from Promega (catalog no.
A3800). The purity of RNA was assessed by nano drop, and only pure
RNA was used for real-time PCR by the Syber green method. PCR for
cDNA and real time was set up as mentioned elsewhere (30).
Plasma Hcy measurement. Plasma Hcy levels in all the animal
groups were measured using high-performance liquid chromatography as previously described (41).
Nitric oxide assay. Nitric oxide (NO) assay was performed as
mentioned elsewhere (26). Briefly, tissue homogenates were prepared
from the animal groups and filtered through 30-kDa molecular mass
cutoff microfuge ultrafiltration device (Amicon, Millipore, MA) and
filtrate was used for analysis. Total nitrate and nitrite was measured
using nitrate and nitrite calorimetric assay kit (780001; Cayman
Fig. 6. A: immunohistochemistry data showing MMP-9 expression secondarily stained
with Alexa fluor 488 (green intensity) in all
the 4 groups. B: bar graphs representing the
intensity (AU) data assessed by Image Pro
software. *P ⬍ 0.05, compared with WT
group. #P ⬍ 0.05, compared with CBS⫹/⫺
group. Data represent means ⫾ SE from n ⫽
5 per group. C: zymography data showing
MMP-9 and -2 activity in WT and CBS⫹/⫺
mice. MMP-9 activity is enormously increased in CBS⫹/⫺ mice compared with WT.
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(BaSo4) contrast is mixed in 5 ml of water and thoroughly mixed to
make an aqueous mixture. Mice weighing 30 g were used and were
deprived of food for overnight and water for 2 h. Under pentobarbital
anesthesia, animals were instilled with 0.3 ml of BaSo4 aqueous
mixture by oral gavage. Mice were then killed after watching for a
fixed time period of 7 h. The whole intestine from duodenum to
rectum was harvested and radiographed using Kodak In-Vivo Imaging
system FX Pro (Care stream Health). Radio opaque barium sulfate is
visualized through X-ray images. The distance barium travelled measures the intestinal motility.
Cryosectioning. The colon tissue was excised and cryopreserved in
Peel-A-Way disposable plastic tissue-embedding molds (Polysciences, Warrington, PA) containing tissue-freezing media (Triangle
Biomedical Sciences, Durham, NC). These molds were kept frozen
(⫺70°C) until serial 8-␮m tissue sections were made in Cryocut 1800
(Reichert-Jung). Cryosections were placed on Superfrost/plus microscope slides and air dried.
Histological studies. Hematoxylin and eosin staining showing
general colon histology in all groups was done using commercially
available kit. Similarly elastin and collagen staining was done on
ROLE OF MMP-9 IN HOMOCYSTEINE-INDUCED CONSTIPATION
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Fig. 7. A: Western blot data showing protein
expression of MMP-9, ICAM, and inducible
nitric oxide synthase (iNOS) and the corresponding GAPDH blots. B: bar graphs representing data from densitometry of the protein expression normalized with GAPDH assessed by Bio-Rad software. *P ⬍ 0.05,
compared with WT group. #P ⬍ 0.05, compared with CBS⫹/⫺ group. Data represent
means ⫾ SE from n ⫽ 5 per group.
Muscularis mucosa and elastin is decreased in HHcy mice.
Histological examination of the colonic sections by hematoxylin and eosin, elastin staining, and Masson’s trichrome staining suggests gross histological variation in CBS⫹/⫺ mice
compared with other group of mice. The thickness of muscularis mucosa and elastin content was significantly decreased in
CBS⫹/⫺ mice whereas collagen content assessed by Masson’s
trichrome staining was significantly increased compared with
rest of the groups. Other histological inflammatory changes
such as ulceration, crypt dilation, and goblet cell depletion are
found in CBS colonic architecture. Interestingly, muscularis mucosa and elastin were restored to normal and collagen content is
RESULTS
Fecal output is decreased in HHcy mice. Differences in
morphological parameters such as the body weight, stool, and
colon length measurement are examined in all the four groups.
We found that colon length in the CBS mice was significantly
greater than the rest of the groups with a large amount of
residual stool (Fig. 1, A and C). Although the animals used in
this study were ⬃27–30 g in weight, we used the stool weight
and body weight ratio to normalize the stool weight data. The
body weight-to-stool weight ratio was significantly less in CBS
mice compared with remaining groups and normalized in
Hcy-treated MMP-9 mice (Fig. 1B).
Intestinal motility is decreased in HHcy due to MMP-9induced intestinal remodeling. Intestinal motility was assessed
by the barium meal study with X-ray machine, and the farther
the barium travelled in given time, the faster is the intestinal
motility. The data suggest that barium travelled at a slower rate
in HHcy CBS⫹/⫺ mice, suggesting decreased intestinal motility compared with other groups. Interestingly, we found that
intestinal motility is increased in Hcy-treated MMP-9⫺/⫺ mice,
suggesting the role of MMP-9 in decreased intestinal motility
during HHcy (Fig. 2). We also report that plasma Hcy levels
assessed by HPLC method showed elevated levels of Hcy in
MMP-9⫺/⫺-treated mice that are comparable to HHcy CBS⫹/⫺
mice (see Fig. 13A).
Fig. 8. Cycle threshold (Ct) value from real time PCR data was depicted in bar
diagram. mRNA expression of MMP-9 and iNOS was significantly increased
in CBS⫹/⫺ mice compared with rest of the groups, which was normalized in
MMP-9 knockout mice treated with Hcy. *P ⬍ 0.05, compared with WT
group. #P ⬍ 0.05, compared with CBS⫹/⫺ group. Data represent means ⫾ SE
from n ⫽ 5 per group.
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chemical, Ann Arbor, MI) according to manufacturer’s instructions.
After the assay was performed, absorbance at 540 nm was measured
in the samples and standards using spectrophotometer and results
were expressed as NO in ␮M.
Superoxide and reactive oxygen species measurements. Superoxide
and reactive oxygen species (ROS) were measure in frozen tissue
sections using molecular probes from Invitrogen as described earlier
(1, 24). Fluorescence intensity was measured using confocal microscope, and images were analyzed using Image Proplus software.
Statistics. All data are expressed as means ⫾ SE. One-way
ANOVA was performed to test for treatment effects, and differences
between groups were determined using Tukey’s post hoc test. A P
value ⬍0.05 was considered to be significant.
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ROLE OF MMP-9 IN HOMOCYSTEINE-INDUCED CONSTIPATION
Fig. 9. A: immunohistochemistry data showing tissue inhibitor of MMP-1 (TIMP-1) expression secondarily stained with Alexa fluor
488 (Green intensity) in all the 4 groups. B:
bar graphs representing the intensity (AU)
data assessed by Image Pro software. *P ⬍
0.05 compared with WT group and #P ⬍
0.05 compared with CBS⫹/⫺ group. Data
represent means ⫾ SE from n ⫽ 5 per group.
mined along with SOD (Fig. Fig. 9). Levels of TIMP-1 and
-4 and SOD are decreased in CBS⫹/⫺ mice compared with
other groups, suggesting the decreased antioxidant protection in HHcy (Fig. Fig. 10). Surprisingly, these effects were
normalized in MMP-9 knockout mice that are supplemented
with Hcy treatment.
Induced NO and ROS including superoxide (O2·⫺) expressions were increased in HHcy. NO assay performed by
commercially available kit (Caymen) showed significant
increase in expression of induced NO. Interestingly measurement
of ROS including O2·⫺ done by molecular probe staining (Invitrogen) showed increased expression in CBS⫹/⫺ mice, suggesting
their role during HHcy (Figs. 11, 12, and 13B).
DISCUSSION
The significant association of HHcy to the intestinal
inflammation has been well established in previous studies
(10, 33). Studies (28, 29) have also found the role of MMP-9
in inducing the inflammation in colitis and other intestinal
pathology. Interestingly, MMP-9 inhibitors alleviated these
Fig. 10. A: Western blot data showing protein expression of SOD-1 and TIMP-4 and
the corresponding GAPDH blots. B: bar
graphs representing data from densitometry
of the protein expression normalized with
GAPDH assessed by Bio-Rad software.
*P ⬍ 0.05, compared with WT group. #P ⬍
0.05, compared with CBS⫹/⫺ group. Data
represent means ⫾ SE from n ⫽ 5 per group.
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decreased in Hcy-treated MMP-9⫺/⫺ mice, suggesting the role of
MMP-9 in inducing these changes (Figs. 3, 4, and 5).
Expressions of MMP-9, ICAM, and iNOS are increased in
CBS⫹/⫺ mice. To provide further insight into the molecular
mechanisms responsible for gross morphological changes in
the colons from HHcy mice, protein expression of inflammatory cytokine, iNOS, and adhesion molecule ICAM-1
was measured. To understand the role of MMP-9 in colonic
inflammation in CBS⫹/⫺ mice immunoblot and immunohistochemistry assessing MMP-9, ICAM and INOS were performed (Figs. 6 and 7). The results showed significant
upregulation of MMP-9 along with other inflammatory
markers like ICAM and iNOS in the colons of CBS⫹/⫺ mice
compared with WT mice, suggesting the possible role of
MMP-9 in induced inflammatory changes during HHcy.
mRNA expression assessed by real time PCR showed increased expression of MMP-9 and iNOS in HHcy CBS⫹/⫺
mice compared with other groups (Fig. 8).
Expression of TIMP-1 and -4 and SOD were decreased in
HHcy. To elucidate possible causes of upregulation of
MMP-9, TIMP-1, and TIMP-4 protein levels were deter-
ROLE OF MMP-9 IN HOMOCYSTEINE-INDUCED CONSTIPATION
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Fig. 11. Staining for reactive oxygen species
(ROS) showing elevated levels in CBS⫹/⫺
mice compared with other groups. ROS are
stained with green fluorescence intensity and
nuclei are stained with DAPI (blue). *P ⬍
0.05, compared with WT group. #P ⬍ 0.05,
compared with CBS⫹/⫺ group. Data represent means ⫾ SE from n ⫽ 5 per group.
along with SOD. It has been shown previously that colon
lengthening slowed intestinal transit due to elongated longitudinal muscle (42). Findings from our study are consistent with this finding as the length of colon in CBS⫹/⫺ mice
is significantly elongated and these mice have lot of residual
stool in their colon (Fig. 1). Fecal output is also is significantly low in CBS⫹/⫺ mice compared with rest of the
groups. In support, results from our barium meal study
suggest that there is decreased motility in HHcy CBS⫹/⫺
mice whereas the motility is much improved in MMP-9⫺/⫺
mice treated with Hcy (Fig. 2).
Elevated levels of Hcy have also been implicated in
increased cardiovascular risk and thromboembolic disorders
in IBD patients (10). Although there is a higher prevalence
Fig. 12. Superoxide staining with dihydroethidium (DHE) stain from Molecular Probes,
Invitrogen showing increased superoxide expression in CBS⫹/⫺ mice compared with rest
of the groups. O2·⫺ is stained with red fluorescence intensity and nuclei with DAPI (blue).
*P ⬍ 0.05, compared with WT group. #P ⬍
0.05, compared with CBS⫹/⫺ group. Data represent means ⫾ SE from n ⫽ 5 per group.
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intestinal inflammatory changes in preclinical studies (12).
Decreased intestinal motility leading to constipation is also
an important clinical symptom during intestinal inflammation or colitis. In our present study, we tried to evaluate the
role of MMP-9 in inducing the decreased intestinal motility
during HHcy and associated intestinal inflammatory
changes by studying histological and molecular inflammatory determinants in WT, CBS⫹/⫺, MMP-9⫺/⫺, and Hcytreated MMP-9⫺/⫺ mice.
The present study showed that the CBS⫹/⫺, HHcy mice
model has delayed intestinal transits due to elevated levels
of inflammatory cytokines and increased expression and
activity of MMP-9 in colonic mucosa. In addition, there was
a marked reduction in expression of TIMP-1 and TIMP-4
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ROLE OF MMP-9 IN HOMOCYSTEINE-INDUCED CONSTIPATION
Fig. 14. Hypothetical representation depicting mechanisms involved in MMP9-induced intestinal remodeling during hyperhomocyteinemia leading to decreased motility and thus to constipation.
Hcy-treated MMP-9 knockout mice. These findings suggest
the plausible role of MMP-9 in inducing the inflammatory
changes and thus decreased motility during HHcy.
It has been postulated that oxy radicals are key mediator
in colitis-associated intestinal injury (22). SOD-1 plays a
critical role in regulating redox balance and capable of
removing excess of oxy radicals such as H2O2. A previous
report (19) demonstrated folic acid supplementation ameliorated Hcy-induced suppression of antioxidant enzyme. In
the present study, we showed that colonic mucosa from
HHcy mice is relatively deficient in antioxidant enzymes
such as SOD-1 (Fig. 10). ROS including superoxide levels
were increased during HHcy, suggesting their mechanistic
role during MMP-9-induced inflammation (Figs. 11 and 12).
HHcy was shown to induce iNOS expression, which
further exacerbates the overall inflammation associated with
atherosclerosis (47). Furthermore, expression of iNOS and
ICAM-1 was found to be elevated in inflamed colons in IBD
(6). These reports are consistent with our findings of increased expression of iNOS and ICAM-1 in colon tissue
from HHcy, which was normalized in Hcy-treated MMP9⫺/⫺ colons. Nitric oxide levels due to iNOS were significantly increased during HHcy, suggesting inflammatory
mechanism (Fig. 13B).
To summarize, the results from our study showed that
MMP-9 plays an important role in inducing inflammatory
changes and thus decreased intestinal motility (constipation)
during HHcy. These changes were nullified in Hcy-treated
MMP-9 knockout (MMP-9⫺/⫺) mice, suggesting the role of
MMP-9. Overall hypothesis of our study is depicted in flow
diagram (Fig. 14). To our knowledge, our study is the first
to show the association of Hcy-induced constipation to
MMP-9 and this further gives insight into the mechanisms
involved in constipation during HHcy.
GRANTS
Fig. 13. A: bar graph representing the quantified data from plasma Hcy levels.
B: quantified data from nitric oxide (NO) assay are depicted in bar diagram.
*P ⬍ 0.05, compared with WT group. #P ⬍ 0.05, compared with CBS⫹/⫺
group. Data represent means ⫾ SE from n ⫽ 5 per group.
A part of this study was supported by National Institutes of Grants
HL-71010 and NS-51568.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00423.2011 • www.ajpgi.org
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of HHcy during intestinal inflammation, the mechanism by
which Hcy causes colonic injury and hence reduced colonic
motility has not been clearly elucidated. MMP/TIMP axis
have been associated to have patho-physiological role in
intestinal inflammation (28). MMP-9 expression and activity have been corelated with inflammation and ROS activation. The assessment of MMP-9 activity is a well-established marker for quantifying intestinal inflammation. Also,
MMP-9 plays a pivotal role in the Hcy-induced formation of
ROS that leads to tissue damage (45). In our study, we
showed that Hcy induces MMP-9 activation in the colonic
mucosa. Our results are consistent with previous reports (17,
32) showing that Hcy induces MMPs in heart and vascular
tissue and hence participates in the remodeling process.
Previous reports (12) demonstrated that increased expression of MMP-9 contributes to intestinal inflammation and
colitis. In addition, MMP levels have been suggested to be
marker for IBD condition (25). TIMP-1 plays a key role in
regulating MMPs in the body. Others (37) have shown that
Hcy induced upregulation of TIMP-1 in the liver tissue.
Interestingly, our study showed decreased expression
TIMP-1 and TIMP-4 in the colons of HHcy (CBS⫹/⫺) mice.
Previous research (5, 39) showed that MMP-9 inhibitor has
therapeutic potential in alleviating inflammation associated
with IBD. Similarly our study showed that the inflammatory
changes, loss of normal histological architecture, and function associated with HHcy CBS⫹/⫺ mice were abrogated in
ROLE OF MMP-9 IN HOMOCYSTEINE-INDUCED CONSTIPATION
AUTHOR CONTRIBUTIONS
Author contributions: S.G. and C.M. conception and design of research;
S.G., C.M., N.N., F.A., G.T., and N.M. performed experiments; S.G., N.N.,
F.A., and N.M. analyzed data; S.G. interpreted results of experiments; S.G.
prepared figures; S.G., C.M., and N.N. drafted manuscript; S.G. and S.C.T.
edited and revised manuscript; S.G. and S.C.T. approved final version of
manuscript.
21.
22.
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