Article

CD44 Is Critically Involved in Infarct Healing by Regulating the Inflammatory and Fibrotic Response

Section of Cardiovascular Sciences, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
The Journal of Immunology (Impact Factor: 4.92). 03/2008; 180(4):2625-33. DOI: 10.4049/jimmunol.180.4.2625
Source: PubMed

ABSTRACT

Infarct healing is dependent on an inflammatory reaction that results in leukocyte infiltration and clearance of the wound from dead cells and matrix debris. However, optimal infarct healing requires timely activation of "stop signals" that suppress inflammatory mediator synthesis and mediate resolution of the inflammatory infiltrate, promoting formation of a scar. A growing body of evidence suggests that interactions involving the transmembrane receptor CD44 may play an important role in resolution of inflammation and migration of fibroblasts in injured tissues. We examined the role of CD44 signaling in infarct healing and cardiac remodeling using a mouse model of reperfused infarction. CD44 expression was markedly induced in the infarcted myocardium and was localized on infiltrating leukocytes, wound myofibroblasts, and vascular cells. In comparison with wild-type mice, CD44(-/-) animals showed enhanced and prolonged neutrophil and macrophage infiltration and increased expression of proinflammatory cytokines following myocardial infarction. In CD44(null) infarcts, the enhanced inflammatory phase was followed by decreased fibroblast infiltration, reduced collagen deposition, and diminished proliferative activity. Isolated CD44(null) cardiac fibroblasts had reduced proliferation upon stimulation with serum and decreased collagen synthesis in response to TGF-beta in comparison to wild-type fibroblasts. The healing defects in CD44(-/-) mice were associated with enhanced dilative remodeling of the infarcted ventricle, without affecting the size of the infarct. Our findings suggest that CD44-mediated interactions are critically involved in infarct healing. CD44 signaling is important for resolution of the postinfarction inflammatory reaction and regulates fibroblast function.

Full-text

Available from: Sandra B Haudek
CD44 Is Critically Involved in Infarct Healing by Regulating
the Inflammatory and Fibrotic Response
1
Peter Huebener, Tareq Abou-Khamis, Pawel Zymek, Marcin Bujak, Xia Ying, Khaled Chatila,
Sandra Haudek, Geeta Thakker, and Nikolaos G. Frangogiannis
2
Infarct healing is dependent on an inflammatory reaction that results in leukocyte infiltration and clearance of the wound from dead
cells and matrix debris. However, optimal infarct healing requires timely activation of “stop signals” that suppress inflammatory
mediator synthesis and mediate resolution of the inflammatory infiltrate, promoting formation of a scar. A growing body of evidence
suggests that interactions involving the transmembrane receptor CD44 may play an important role in resolution of inflammation and
migration of fibroblasts in injured tissues. We examined the role of CD44 signaling in infarct healing and cardiac remodeling using a
mouse model of reperfused infarction. CD44 expression was markedly induced in the infarcted myocardium and was localized on
infiltrating leukocytes, wound myofibroblasts, and vascular cells. In comparison with wild-type mice, CD44
/
animals showed en-
hanced and prolonged neutrophil and macrophage infiltration and increased expression of proinflammatory cytokines following myo-
cardial infarction. In CD44
null
infarcts, the enhanced inflammatory phase was followed by decreased fibroblast infiltration, reduced
collagen deposition, and diminished proliferative activity. Isolated CD44
null
cardiac fibroblasts had reduced proliferation upon stimu-
lation with serum and decreased collagen synthesis in response to TGF-
in comparison to wild-type fibroblasts. The healing defects in
CD44
/
mice were associated with enhanced dilative remodeling of the infarcted ventricle, without affecting the size of the infarct. Our
findings suggest that CD44-mediated interactions are critically involved in infarct healing. CD44 signaling is important for resolution
of the postinfarction inflammatory reaction and regulates fibroblast function. The Journal of Immunology, 2008, 180: 2625–2633.
P
ostinfarction cardiac repair is dependent on release of in-
flammatory mediators and subsequent infiltration of the
infarcted myocardium with leukocytes that clear the
wound from dead cells and matrix debris (1–3). However, optimal
infarct healing requires timely activation of “stop signals” that sup-
press chemokine and cytokine synthesis resulting in resolution of
the inflammatory infiltrate. The inflammatory phase is followed by
the proliferative phase as myofibroblasts accumulate in the infarct
and deposit extracellular matrix proteins forming a collagen-based
scar. The dynamic changes in the extracellular matrix network play
an important role in regulating the cellular events involved in
wound healing. Increased turnover of extracellular matrix is a hall-
mark of tissue injury and results in generation of degradation prod-
ucts. Hyaluronan, a ubiquitously present constituent of the extra-
cellular matrix (4), exists as a high m.w. polymer under
physiologic conditions, but undergoes degradation resulting in ac-
cumulation of lower m.w. species after tissue injury. Hyaluronan
fragments induce the expression of a variety of inflammatory
genes by endothelial cells and macrophages (5), including chemo-
kines and cytokines, and may play an important role in regulating
the inflammatory process. Furthermore, clearance of hyaluronan
fragments from the injured tissue is crucial for resolution of
chronic inflammation (6). Hyaluronan participates in induction and
resolution of inflammation through interactions with the trans-
membrane adhesion molecule CD44 (7), a ubiquitously distributed
glycoprotein that mediates a wide variety of cell-cell and cell-
matrix interactions. CD44-hyaluronan interactions play an impor-
tant role in regulating leukocyte extravasation into inflammatory
sites (8, 9) and mediate efficient phagocytosis (10). In addition, a
growing body of evidence suggests that CD44 serves as a key
factor in resolution of inflammation through removal of matrix
breakdown products and clearance of apoptotic neutrophils (6),
and mediates fibroblast migration and invasion in the wound pro-
visional matrix (11). Although numerous studies have explored the
significance of CD44-mediated interactions in a variety of inflam-
matory and fibrotic processes, the role of CD44 signaling in heal-
ing myocardial infarcts has not been investigated.
We hypothesized that CD44 may play an essential role in infarct
healing by regulating the inflammatory and fibrotic response. We
found that CD44
null
animals exhibit enhanced and prolonged in-
flammation in the infarcted heart followed by reduced myofibro-
blast infiltration. The healing defect in CD44
/
mice was asso-
ciated with impaired fibroblast function and markedly diminished
collagen deposition in the scar and resulted in enhanced adverse
remodeling of the infarcted ventricle.
Materials and Methods
Murine ischemia/reperfusion protocols
All animal studies were approved by the Animal Protocol Review Committee
at Baylor College of Medicine. CD44
/
mice (12) and wild-type (WT)
3
B6129PF2/J controls (purchased from The Jackson Laboratory) were used
Section of Cardiovascular Sciences, Baylor College of Medicine, Houston, TX 77030
Received for publication March 8, 2007. Accepted for publication December 3, 2007.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health R01 HL-76246, R01
HL-85440, and the American Heart Association.
2
Address correspondence and reprint requests to Dr. Nikolaos G. Frangogiannis,
Section of Cardiovascular Sciences, Baylor College of Medicine, One Baylor Plaza
BCM620, Houston, TX 77030. E-mail address: ngf@bcm.tmc.edu
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
3
Abbreviations used in this paper: WT, wild type; KO, knockout; qPCR, quantitative
PCR; RPS3, ribosomal protein S3;
-SMA,
-smooth muscle actin; PCNA, prolif-
erating cell nuclear Ag; HA, hyaluronic acid; LVEDV, left ventricular end-diastolic
volume; RPA, RNA protection assay; IP, IFN-
-inducible protein; RPA, ribonuclease
protection assay; pNS, p not significant.
The Journal of Immunology
www.jimmunol.org
Page 1
for myocardial infarction experiments. Male and female mice, 8 –12 wk of
age (18.0 –22.0 g body weight) were anesthetized by an i.p. injection of
sodium pentobarbital (60
g/g). A closed-chest mouse model of reperfused
myocardial infarction was used to avoid the confounding effects of surgical
trauma and inflammation, which may influence the baseline levels of che-
mokines and cytokines (2). The left anterior descending coronary artery
was occluded for 1 h then reperfused for6hto7days. At the end of the
experiment, the chest was opened and the heart was immediately excised,
fixed in zinc-formalin, and embedded in paraffin for histological studies, or
snap-frozen and stored at 80°C for RNA or protein isolation. Sham an-
imals were prepared identically without undergoing coronary occlusion/
reperfusion. Animals used for histology underwent 24-h, 72-h, and
7-day reperfusion protocols (eight animals per group). Mice used for
RNA extraction underwent 6 h, 24 h, and 72 h of reperfusion (eight
animals per group). To assess remodeling-associated parameters, addi-
tional mice were used for perfusion-fixation of the heart (knockout
(KO), n 10; WT, n 8) after 7 days of reperfusion. Mice used for
isolation of infarct myofibroblasts underwent 3-day-reperfusion proto-
cols (KO, n 6; WT, n 6).
Quantitative PCR (qPCR)
Total RNA was treated with DNase to remove any genomic contamination
as described by the manufacturer (DNA-free; Ambion) First-strand cDNA
was synthesized using the iScript cDNA Synthesis kit (Bio-Rad) with ran-
dom primers as described in the manufacturer’s protocol. CD44 primers
were specifically designed using the National Center for Biotechnology
Information genome database and manufactured by Sigma-Genosys. Real-
time PCR was performed and analyzed with 1/10 diluted cDNA according
to the manufacturer’s instructions on an ABI Prism 7000 Sequence Detec-
tion System (Applied Biosystems). Target gene expression was normal-
ized to an internal control, ribosomal protein S3 (RPS3). Both CD44 and Rps3
were measured using SYBR Green chemistry and the relative standard
curve method. At the end of PCR cycle, dissociation curve analysis was
performed to ascertain the amplification of a single PCR product. Se-
quences of the murine primers were as follows: CD44 forward (5-ATCA
GCAGATCGATTTGAATGTAA-3), CD44 reverse (5-CATTTCCTTCT
ATGAACCCATACC-3); RPS3 forward (5-ATCAGAGAGTTGACCGC
AGTTG-3), RPS3 reverse (5-AATGAACCGAAGCACACCATAG-3).
Immunohistochemistry and quantitative histology
Murine hearts were fixed in zinc-formalin (Z-fix; Anatech), and embedded
in paraffin. Sections were cut at 3
m and stained immunohistochemi-
cally with the following Abs: rat anti-mouse CD44 Ab (BD Pharmin-
gen), monoclonal anti-
smooth muscle actin (
-SMA) Ab (Sigma-
Aldrich), rat anti-mouse macrophage Ab clone F4/80 (Research
Diagnostics), rabbit anti-mouse proliferating cell nuclear Ag (PCNA) Ab
(Abcam), and rat anti-neutrophil Ab (Serotec). Staining was performed
using a peroxidase-based technique with the Vectastain ELITE rat, rabbit,
or goat kit (Vector Laboratories) and developed with diaminobenzidine
plus nickel (Vector Laboratories). The Mouse on Mouse (MOM) kit (Vec-
tor Laboratories) was used for
-SMA immunohistochemistry. For F4/80
and PCNA staining, unmasking with trypsin was performed. Hyaluronic
acid (HA) was detected using histochemical staining with biotinylated HA-
binding protein (Cape Cod/Seikagaku Biochemicals) as previously de-
scribed (13). Collagen was stained with picrosirius red (14). Apoptotic
cells were labeled using the CardioTacs In Situ Apoptosis Detection kit
(Trevigen). Quantitative assessment of macrophage density was performed
by counting the number of F4/80-positive cells in the infarcted area as
previously described (15). Myofibroblasts were identified as extravascular
-SMA-positive cells and counted in the infarcted myocardium. Macro-
phage, neutrophil, and myofibroblast density was expressed as cells per
millimeter-squared. Proliferative activity in the infarct was assessed by
counting the PCNA index as the percentage of cells with PCNA-positive
nuclei among all cells in the infarct. Collagen density in the infarcted area
was expressed as the percentage of the infarcted area stained with
picrosirius red.
Perfusion fixation and assessment of ventricular volumes
For assessment of postinfarction remodeling, infarcted hearts after 7 days
of reperfusion were used for perfusion-fixation as previously described (2).
The entire heart from base to apex was cross-sectioned at 250-
m inter-
vals. Ten serial 5-
m sections were obtained at each interval. The left
ventricular end-diastolic volume (LVEDV), left ventricular volume, septal
volume, and scar size were assessed with ImagePro software using meth-
ods developed in our laboratory (2). Left ventricular mass and septal mass
were derived by multiplying the left ventricular volume and septal volume,
FIGURE 1. Expression and localization of CD44 and its main ligand hyaluronan in healing mouse myocardial infarcts. A, qPCR demonstrated that CD44
mRNA levels were markedly induced in the infarcted heart, peaking after6hofreperfusion (, p 0.05). B–E, CD44 immunohistochemistry in mouse
infarcts. CD44 was localized in infiltrating leukocytes after 24 h of reperfusion (B). After 72 h (C) 7days (D) of reperfusion, many granulation tissue cells
infiltrating the infarct exhibited CD44 expression. After 72 h of reperfusion, CD44 was localized in infarct myofibroblasts, identified as nonvascular
-smooth muscle actin-expressing spindle-shaped cells (arrows, E and F). In contrast, vascular smooth muscle cells did not show CD44 immunoreactivity
(arrowhead). G–I, Affinity histochemistry for hyaluronan in murine myocardial infarcts. A thin rim of hyaluronan was identified in the endoperimysium
of the mouse heart. After 24 h of reperfusion, disruption of the hyaluronan network was observed in the infarcted area (G, arrows). Hyaluronan deposition
was noted in the infarct after 72 h of reperfusion (H, arrows) forming an organized matrix network after 7 days (I). Magnification 100, counterstained
with eosin.
2626 CD44 SIGNALING REGULATES INFARCT HEALING
Page 2
respectively, by the specific gravity of the myocardium (1.065 g/ml). The
size of the infarct was expressed as a percentage of the left ventricular
volume.
RNA extraction and RNase protection assay (RPA)
Inflammatory gene expression in murine hearts was assessed using RPA as
previously described (2, 16). The mRNA expression level of the chemo-
kines MIP-1
, MIP-1
, MIP-2, MCP-1, and IFN-
-inducible protein (IP)-
10, the cytokines TNF-
, IL-1
, osteopontin, IL-6, and IL-10, the growth
factors TGF-
1, 2, and 3, and M-CSF were determined using a RPA
(RiboQuant; BD Pharmingen) according to the manufacturer’s protocol.
Phosphorimaging of the gels was performed (Storm 860; Molecular Dy-
namics) and signals were quantified using Image QuaNT software and
normalized to the ribosomal protein L32 mRNA.
Protein extraction and Western blotting
Protein isolation and Western blot analysis were conducted as previously
described (17, 18). WT (n 4) and CD44
/
(n 5) mice undergoing
reperfused infarction protocols (1 h ischemia/24 h reperfusion) were used
for protein extraction. A total of 15
g of protein were separated on SDS-
polyacrylamide gels in a Tris-HCl buffer system, transferred onto nitrocel-
lulose membranes, and blotted according to standard procedures using a
polyclonal rabbit anti-Smad2 (1/1,000) or a polyclonal rabbit anti-phospho-
Smad2 (Ser
465/467
; 1/200) Ab (both obtained from Cell Signaling). The
specific bands of target proteins were visualized by chemiluminescence,
and band intensities were evaluated using Image QuaNT. Membranes were
then stripped and reblotted with monoclonal anti-GAPDH (1/10,000; Ad-
vanced ImmunoChemical). Target signals were normalized to GAPDH sig-
nal. The ratio of p-smad2 to total smad2 expression was used as an indi-
cator of activation of the TGF-
-signaling pathway.
Isolation and stimulation of murine cardiac fibroblasts
Fibroblasts were isolated from normal mouse hearts by enzymatic diges-
tion with a collagenase buffer as previously described (19). Three nonin-
farcted WT or CD44
/
hearts were used for each experiment. The hearts
were dissected free of vessels and atria, transferred to 1 ml of collagenase
buffer, and quickly minced into small pieces. Digestion with collagenase
buffer continued until no visible tissue fragments were left. The isolated
FIGURE 2. Effects of CD44 gene
disruption on the time course of
neutrophil and macrophage infiltra-
tion in the infarcted myocardium.
CD44
null
mice exhibited enhanced
and prolonged neutrophil (A) and
macrophage infiltration (B)inthe
infarct in comparison with WT ani-
mals (ⴱⴱ, p 0.01). However, reso-
lution of the neutrophilic infiltrate
was noted after 7 days of reperfusion
in both CD44
/
and WT infarcts.
C–F, Immunohistochemistry identi-
fies neutrophils (PMN) in WT (C and
E) and CD44
/
infarcts (D and F). G
and H, Macrophages (MP) in WT (G)
and CD44
null
(H) infarcts were identi
-
fied with F4/80 immunohistochemistry.
2627The Journal of Immunology
Page 3
cell suspensions from each round were pelleted and washed. All cell sus-
pensions were combined, plated on a T75 tissue-culture flask (Corning) in
full medium supplemented with 10% of FBS (HyClone) and antibiotic-
antimycotic solution. After overnight incubation, nonadherent cells were
removed and adherent cells were cultivated. Upon reaching confluence,
cells were detached with trypsin/EDTA, split in a 1:2 or 1:4 ratio and
recultured. Characteristic fibroblast morphology was determined visually
under a light microscope. Because the phenotype of fibroblasts can be
influenced by growth conditions such as passage and cell density (20), only
fibroblasts at passages 1–3 were used for experiments. Pure fibroblast cul-
tures were confirmed by immunocytochemistry using Abs against vimentin
(a mesenchymal cell marker),
-SMA (both obtained from Sigma-Al-
drich), and collagen type I (Rockland). To study the effects of TGF-
on
control WT and CD44
/
cardiac fibroblasts, cells were stimulated with
rTGF-
1 (100 ng/ml; R&D Systems) for 4 h. At the end of the experiment,
protein was extracted from the cell lysates and Western blotting was per-
formed to assess collagen type I expression using a rabbit anti-collagen
type I Ab (Rockland) and to quantitate Smad2 phosphorylation (18) as
previously described.
Fibroblast proliferation assay
Proliferation was determined by bromodeoxyuridine incorporation us-
ing a commercially available colorimetric kit (Roche Applied Science)
as previously described (21). To normalize data from different experi-
ments, proliferation in response to 5% serum was expressed as fold
increase to cells maintained in serum-free medium. The proliferative
response to serum was compared between fibroblasts isolated from WT
and CD44
null
hearts.
Isolation of myofibroblasts from healing infarcts
To examine the role of CD44 deficiency on the phenotypic characteristics
of fibroblasts in the healing infarcts, infarct myofibroblasts were isolated
from infarcted WT and CD44
/
hearts after1hofischemia and 72 h of
reperfusion (n 6/group). This time point represents the peak of the pro-
liferative phase, when fibroblast density in the infarcted heart is maximal.
The infarct and the border zone area were excised and used for isolation of
fibroblasts as described above. Isolated cells were used for immunocyto-
chemical studies (n 3/group) or for protein extraction (n 3/group).
Characterization of the cells was performed using immunocytochemistry
for
-SMA, vimentin, CD31, and collagen type I. To compare activation of
fibrogenic pathways in CD44
/
and WT infarct myofibroblasts, the pro
-
tein extracts were used for Western blotting to assess collagen type I ex-
pression and p-Smad2:Smad2 ratio.
Statistical analysis
Statistical analysis was performed using ANOVA followed by a t test cor-
rected for multiple comparisons (Student-Newman-Keuls). Data were ex-
pressed as mean SEM. Statistical significance was set at 0.05.
Results
Expression and localization of CD44 and its main ligand,
hyaluronan, in healing mouse infarcts
qPCR demonstrated that CD44 mRNA was markedly induced in
the infarcted mouse myocardium, peaking after6hofreperfusion
(CD44:RPS3 ratio, 0.77 0.09 in sham mice vs 4.90 0.63 after
6 h of reperfusion, p 0.01). CD44 mRNA levels remained ele-
vated after 24 h ( p 0.05) and 72 h ( p 0.05) of reperfusion
(Fig. 1A). Immunohistochemical studies showed that CD44 was
predominantly localized on infiltrating cells after 24 h of reperfu-
sion (Fig. 1B). After 72 h-7 days of reperfusion, the majority of
granulation tissue cells in the infarcted myocardium stained for
CD44 (Fig. 1, C and D), including macrophages, myofibroblasts
(identified as
-SMA-positive spindle-shaped cells) (Fig. 1, E and
F), and endothelial cells. Using affinity histochemistry, we identi-
fied a thin rim of hyaluronan in the endoperimysium of the murine
myocardium. After 24 h of reperfusion, the hyaluronan network in
the infarcted area exhibited extensive fragmentation (Fig. 1G).
Healing of the infarct was associated with hyaluronan deposition
in the wound forming an organized network after 7 days of reper-
fusion (Fig. 1I).
FIGURE 3. Cytokine mRNA ex-
pression in infarcted CD44
null
and
WT hearts. RPA analysis (A) demon-
strated that CD44
/
mice had en
-
hanced peak mRNA levels of the
proinflammatory cytokines IL-1
(B),
TNF-
(C), and IL-6 (D), the inhibi-
tory cytokine IL-10 (E), and the ma-
tricellular protein OPN (F), a marker
of monocyte to macrophage differen-
tiation (, p 0.05; ⴱⴱ, p 0.01).
However, both WT and CD44
null
in
-
farcts demonstrated timely repression
of the cytokine response (U, unpro-
tected probe).
2628 CD44 SIGNALING REGULATES INFARCT HEALING
Page 4
CD44
null
mice exhibited enhanced and prolonged inflammatory
leukocyte infiltration in the healing infarct
Mortality rates following reperfused infarction were comparable in
WT and CD44
null
mice (CD44
/
: 8.3% vs WT: 6% p not sig
-
nificant (pNS)). CD44
/
mice exhibited increased neutrophil
(862.4 34.32 cells/mm
2
vs 502.6 47.17 cells/mm
2
, p 0.05;
Fig. 2, A, C, and D) and macrophage (423.4 22.4 cells/mm
2
vs
248.9 19.4 cells/mm
2
, p 0.05; Fig. 2B) density in the infarcted
myocardium after 24 h of reperfusion. In addition, CD44
null
mice
had prolonged infiltration with inflammatory leukocytes showing
significantly higher neutrophil and macrophage density after 72 h
and 7 days of reperfusion (Fig. 2, A, B, E–H). To examine whether
prolonged neutrophil and macrophage infiltration in CD44-defi-
cient animals was due to defective clearance of apoptotic leuko-
cytes from the infarcted myocardium, we assessed the density of
TUNEL-positive cells in the infarct. CD44 deficiency was not as-
sociated with an increased number of apoptotic cells in the in-
farcted myocardium. After 24 h of reperfusion, the percentage of
apoptotic cells was not significantly different between CD44
/
and WT mice (CD44
/
: 11.8 1.5% vs WT: 12.8 1.4%,
pNS). After 72 h of reperfusion, CD44
null
infarcts had slightly
reduced percentage of apoptotic cells in comparison with WT mice
(CD44
/
: 5.5 0.5% vs WT: 9.97 0.9%; p 0.05).
CD44-deficient mice showed increased myocardial cytokine
expression but comparable chemokine mRNA levels following
myocardial infarction
Reperfused murine myocardial infarction triggers a robust but
transient up-regulation of proinflammatory cytokines and chemo-
kines (16). In comparison to their WT littermates, CD44
/
ani
-
mals showed significantly increased peak mRNA expression of the
proinflammatory cytokines, IL-1
(Fig. 3B), TNF-
(Fig. 3C),
IL-6 (Fig. 3D), and M-CSF (data not shown). Expression of the
matricellular protein osteopontin, a marker of monocyte to mac-
rophage differentiation was significantly higher in CD44
null
in
-
farcts (Fig. 3F). In addition, mRNA expression of the inhibitory
cytokine IL-10 was significantly higher in CD44
null
infarcts com
-
pared with WT animals (Fig. 3E).
In contrast, peak expression of the chemokines MCP-1, MIP-1
,
MIP-1
, and MIP-2 was comparable between CD44
/
and WT
infarcts, whereas IP-10 mRNA levels were modestly but signifi-
cantly higher in CD44
null
infarcts ( p 0.05) (Fig. 4
).
Effects of CD44 deficiency on repression of inflammatory
cytokine and chemokine synthesis
In both WT and CD44
null
infarcts the postinfarction inflammatory
response was followed by repression of cytokine and chemokine
synthesis after 24 h of reperfusion. mRNA levels of the proinflam-
matory cytokines TNF-
, IL-1
, M-CSF (Fig. 3), and the chemo-
kines MIP-1
, MIP-1
, MIP-2, and IP-10 were comparable be-
tween WT and CD44
null
animals, whereas MCP-1 (0.0755 0.01
vs 0.1402 0.02, p 0.05) and IL-6 (0.0113 0.0015 vs
0.0196 0.002, p 0.05) levels were lower in CD44
null
infarcts
indicating that the absence of CD44 did not affect the timely re-
pression of chemokines and cytokines in the infarcted myocardium
(Figs. 3 and 4).
CD44
null
mice exhibited decreased myofibroblast infiltration
and reduced collagen deposition in the healing infarct
During the proliferative phase of healing murine myocardial in-
farcts exhibit intense infiltration with myofibroblasts, phenotypi-
cally modulated fibroblasts that express
-SMA and are the main
collagen-producing cells in the infarct (22). CD44
null
mice had
significantly lower myofibroblast density in the infarcted myocar-
dium after 3 days of reperfusion (WT: 233.3 16.9 cells/mm
2
vs
/ 123.3 13.0, p 0.05) in comparison with WT animals
(Fig. 5). PCNA immunohistochemistry demonstrated that in the
absence of CD44, decreased myofibroblast density was associated
with significantly reduced proliferative activity in the infarcted
myocardium (PCNA index WT: 24.05 2.0% vs CD44
/
:
13.5 1.2%, p 0.05). After 7 days of reperfusion, mouse in-
farcts show deposition of collagen and formation of a scar.
CD44
null
mice had reduced collagen content in the infarct com
-
pared with WT mice (CD44
/
: 12.3 1.1% vs WT: 20.99
0.6%, p 0.05) (Fig. 5, F–H).
Expression of phosphorylated Smad2 in infarcted CD44
null
hearts
Because TGF-
is a key mediator of fibrosis, involved in myo-
fibroblast differentiation and extracellular matrix deposition, we
examined expression of TGF-
isoforms and activation of the
smad2/3-signaling pathway in the infarcted hearts. Infarcted
WT and CD44
null
hearts had comparable TGF-
1,
2, and
3
mRNA expression after 72 h of reperfusion (data not shown).
Western blotting experiments demonstrated that CD44
null
in
-
farcts had significantly higher Smad2 expression levels, but
markedly reduced p-Smad2:Smad2 ratio (CD44
null
: 0.14 0.03
vs WT: 0.41 0.11, p 0.05) (Fig. 6).
FIGURE 4. Effects of CD44 gene disruption on chemokine mRNA ex-
pression in the infarcted heart. A, RPA analysis was used for assessment of
chemokine mRNA expression in the infarcted heart (U, unprotected probe).
CD44
null
and WT mice had comparable peak MIP-2 (B) and MCP-1 (D)
mRNA expression in the infarcted heart, whereas peak IP-10 mRNA ex-
pression was higher in CD44
/
infarcts (C). CD44 gene disruption did not
affect the time course of chemokine mRNA repression in the infarcted heart
(ⴱⴱ, p 0.01; , p 0.05).
2629The Journal of Immunology
Page 5
The absence of CD44 enhanced dilative, but not hypertrophic,
postinfarction remodeling without a significant effect on
infarct size
After 7 days of reperfusion, CD44
null
mice exhibited increased
LVEDV compared with WT animals (WT: 33.62 1.66 mm
3
vs CD44
/
: 43.81 4.06 mm
3
, p 0.05) (Fig. 7
), although
scar size was comparable between the two groups (WT: 9.7
1.1% vs CD44
/
: 12.5 1.4% of LV volume; pNS). Although
infarcted CD44
/
animals had enhanced ventricular dilation,
they showed a trend toward lower left ventricular mass in com-
parison with WT animals (Table I), suggesting that CD44 gene
disruption enhances dilative but not hypertrophic remodeling
following myocardial infarction.
TGF-
-induced collagen type I up-regulation is attenuated in
CD44
null
cardiac fibroblasts
Fibroblasts isolated from CD44
null
and WT hearts exhibited
similar morphology. Upon stimulation with 5% serum,
FIGURE 5. CD44 gene disruption results in decreased infiltration of the infarcted myocardium with myofibroblasts and markedly reduced collagen
deposition. A, Myofibroblast density was significantly lower in CD44
null
infarcts after 3 days of reperfusion (ⴱⴱ, p 0.01). B and C, Immunohistochemistry
for
-SMA identified myofibroblasts as spindle-shaped interstitial cells in WT (B) and CD44
null
(C) mice. Myofibroblast infiltration was significantly
decreased in CD44
/
animals after 72 h of reperfusion (counterstained with eosin). D and E, Immunohistochemical staining for PCNA was used to label
proliferating cells in the infarcted myocardium (arrows). The PCNA index was calculated as the percentage of cells with PCNA positive nuclei among all cells
in the infarct (identified with hematoxylin counterstaining). At the peak of the proliferative phase (72 h of reperfusion), CD44
null
infarcts exhibited significantly
lower number of proliferating cells in comparison with WT animals. F, Quantitative assessment of the collagen-stained area in the infarcted myocardium
demonstrated that after 7 days of reperfusion, CD44
/
infarcts had lower collagen deposition than WT infarcts (ⴱⴱ, p 0.01). G and H, Sirius red-staining labels
the collagen network in WT (G) and CD44
null
infarcts (H) after 7 days of reperfusion. Dense deposition of collagen is noted in WT, but not in CD44
/
infarcts.
FIGURE 6. CD44
/
mice exhibit impaired Smad2 phosphorylation in
the infarcted heart. CD44
null
infarcts had higher Smad2 levels, lower p-
Smad2 expression, and a markedly reduced p-Smad2:Smad2 ratio in com-
parison with WT infarcts.
FIGURE 7. CD44 gene disruption resulted in enhanced dilative remod-
eling following myocardial infarction. LVEDV was significantly higher in
infarcted CD44
null
hearts compared with WT animals (, p 0.05); infarct
size was comparable between groups. Although CD44 absence enhanced
dilative remodeling, postinfarction hypertrophy was comparable between
CD44
null
and WT animals.
2630 CD44 SIGNALING REGULATES INFARCT HEALING
Page 6
CD44
null
fibroblasts had an attenuated proliferative response in
comparison to WT cardiac fibroblasts (fold increase of cell pro-
liferation in comparison to serum free-cells: WT, 4.14 0.59,
vs CD44
/
2.17 0.39, p 0.05, n 4). Furthermore,
-
broblasts isolated from WT, but not from CD44
null
, mouse
hearts exhibited significant up-regulation of collagen type I syn-
thesis upon stimulation with TGF-
(Fig. 8A). The blunted re-
sponse to TGF-
stimulation in the absence of CD44 was not
due to impaired activation of the Smad2 pathway. Both WT and
CD44
null
fibroblasts showed marked increase of the p-Smad2:
Smad2 ratio, when stimulated with TGF-
(Fig. 8B).
Infarct myofibroblasts isolated from CD44
null
animals exhibit
similar p-Smad2 expression, but reduced collagen type I
synthesis in comparison to WT mice
Infarct myofibroblasts were isolated from WT and CD44
null
in
-
farcts and were characterized as vimentin and
-SMA-positive,
CD31-negative (nonendothelial) spindle-shaped cells. A trend to-
ward lower collagen type I expression levels was noted in cells
isolated from CD44
/
infarcted hearts when compared with WT
animals (mean collagen type I levels, WT: 1.15 0.31 vs
CD44
/
: 0.66 0.35 p 0.14, n 3) (Fig. 8, C–E). In contrast,
p-Smad2 expression levels were comparable between groups (p-
Smad2:Smad2 ratio WT: 1.25 0.03 vs CD44
/
: 1.22 0.02;
pNS) (Fig. 8F).
Discussion
Diverse cellular functions have been attributed to CD44, including
involvement in leukocyte chemotaxis and activation (23), clear-
ance of apoptotic cells and matrix fragments (6, 24), resolution of
chronic inflammation (6), regulation of fibrous tissue deposition
(11), and angiogenesis (25). The present study reports the first
evidence that CD44 plays an essential role in infarct healing and
regulates postinfarction cardiac remodeling. CD44 expression was
FIGURE 8. Impaired fibrogenic response in
CD44
null
cardiac fibroblasts. A, Cardiac fibroblasts were
isolated from uninjured CD44 and WT mouse hearts.
WT but not CD44
/
cardiac fibroblasts showed up-
regulation of collagen type I (Col I) protein synthesis
upon stimulation with TGF-
(100 ng/ml, 4 h) (, p
0.05 vs control unstimulated fibroblasts, n 4). B,
TGF-
stimulation induced Smad2 phosphorylation in
both WT and CD44
/
cardiac fibroblasts (, p 0.05
vs corresponding control fibroblasts, n 4). C–F, Phe-
notypic characteristics of infarct myofibroblasts isolated
from WT and CD44
null
hearts. Myofibroblasts were iso
-
lated from WT (C) and CD44
null
infarcts (D) after 72 h
of reperfusion (at the peak of the proliferative phase)
and were characterized using immunocytochemistry.
Cells isolated from WT infarcts (C) had more intense
staining for collagen I in comparison to CD44
/
cells
(D). E and F, In additional experiments, myofibroblasts
isolated from WT and CD44
/
infarcts were used for
protein extraction (n 3/group). Col I expression levels
(E) and Smad2 phosphorylation (F) were quantitatively
assessed. E, A representative experiment (one of three
experiments with similar findings) demonstrates that
CD44
/
infarct myofibroblasts had lower Col I expres
-
sion in comparison to WT cells. Statistical analysis
showed a trend toward lower Col I expression in cells
isolated from CD44
/
infarcts (mean Col I levels, WT:
1.15 0.31 vs CD44
/
: 0.66 0.35 p 0.14). F,In
contrast, WT and CD44
/
infarct myofibroblasts had
comparable p-Smad2:Smad2 ratios.
Table I. Remodeling-associated parameters in infarcted CD44
null
and
WT animals
WT CD44
/
Significance
LVEDV (mm
3
)
33.6 1.66 43.8 4.06 p 0.05
Scar size (%) 9.7 1.1 12.5 1.4 pNS
LV mass (mg) 48.1 1.6 43.4 1.7 p 0.07
Septal mass (mg) 22.4 1.2 21 1.1 pNS
2631The Journal of Immunology
Page 7
markedly induced in the infarcted myocardium (Fig. 1) and was
localized in leukocytes, fibroblasts, and endothelial cells infiltrat-
ing the wound. CD44 deficiency resulted in enhancement of the
postinfarction inflammatory response. However, despite showing
an accentuated inflammatory phase, infarcted CD44
/
mice ex
-
hibited markedly attenuated myofibroblast infiltration and dimin-
ished collagen deposition in the wound, associated with decreased
proliferative activity and evidence of impaired TGF-
signaling.
These healing defects resulted in enhanced dilative remodeling of
the infarcted ventricle.
The role of CD44 in regulation of the postinfarction
inflammatory response
Numerous studies have explored the role of CD44 in models of
acute inflammation generating disparate and often contradictory
results that underline the complex and multifunctional role of
CD44 in the inflammatory response. Administration of anti-CD44
Abs inhibited leukocyte extravasation in a model of cutaneous de-
layed-type hypersensitivity (26) and abrogated tissue edema and
leukocyte infiltration in murine arthritis (8). Anti-CD44 treatment
induced the rapid loss of CD44 from the surface of leukocytes
presumably preventing cell-extracellular matrix interactions criti-
cal for leukocyte infiltration (8). Moreover, CD44
null
animals bred
into an atherosclerosis-prone ApoE
null
background had markedly
reduced macrophage infiltration in aortic lesions (27). In contrast,
other investigations did not support an essential role for CD44 in
mediating leukocyte infiltration in inflamed tissues, but suggested
that CD44-hyaluronan interactions are important for suppression
of inflammation and resolution of the inflammatory infiltrate.
CD44 KO animals exhibited accentuated inflammation in a model
of collagen-induced arthritis (28), were more susceptible to endo-
toxin-induced shock (29), and showed enhanced neutrophil migra-
tion and lung injury in murine bacterial pneumonia (30). In vitro
experiments demonstrated that CD44
/
neutrophils migrate
faster through matrigel than WT neutrophils, suggesting that CD44
may slow neutrophil migration through extracellular matrix (30).
These actions may be due to CD44-induced intracellular signaling
events in leukocytes bound to hyaluronan or other ligands (30). In
addition, CD44 may prevent exaggerated inflammatory responses
by promoting the expression of negative regulators of TLR-4 sig-
naling (29). Furthermore, CD44
null
animals succumb to unremit
-
ting inflammation following noninfectious lung injury (6) demon-
strating impaired clearance of apoptotic neutrophils and
hyaluronan fragments. It appears that in the absence of CD44 the
highly potent surface receptor for hyaluronan-mediated motility
compensates for the loss of CD44 supporting inflammatory leu-
kocyte migration and inducing an intense inflammatory response
(28). Our findings suggest that CD44 does not mediate recruitment
of leukocytes in the infarcted myocardium but plays an important
role in suppression of the postinfarction inflammatory response.
CD44 absence resulted in an enhanced inflammatory response, as-
sociated with increased neutrophil (Fig. 2) and macrophage infil-
tration (Fig. 2) and increased cytokine mRNA expression (Fig. 3)
in the infarcted myocardium. Enhanced neutrophil density was
not due to the persistent presence of apoptotic neutrophils in the
infarct. Defective clearance of hyaluronan fragments from the
infarcted myocardium may have resulted in enhanced inflam-
mation in CD44
/
animals. However, resolution of postinfarc
-
tion inflammation ultimately occurred in CD44
null
mice, sug
-
gesting that in the absence of CD44 other inhibitory pathways
mediate repression of inflammatory mediators and clearance of
the leukocyte infiltrate.
Role of CD44 in fibrous tissue deposition in the healing infarct
In the absence of CD44, enhanced peak expression of inflamma-
tory cytokines and accentuated leukocyte infiltration in the in-
farcted myocardium was followed by decreased myofibroblast ac-
cumulation and markedly diminished collagen deposition in the
healing wound (Fig. 5). Several mechanisms may be responsible
for decreased fibroblast infiltration in the healing infarct. First,
CD44-mediated interactions may play a direct role in fibroblast
proliferation (31). CD44
null
mice exhibited reduced proliferative
activity in the infarcted myocardium in comparison with WT an-
imals (Fig. 5). In addition, isolated CD44
null
cardiac fibroblasts
had decreased proliferative activity when stimulated with serum.
Second, CD44 deficiency may result in impaired fibroblast migra-
tion and invasion of the provisional matrix in the infarct. Svee et
al. (11) demonstrated that anti-CD44 Ab blocked fibroblast migra-
tion on the provisional matrix proteins fibronectin, fibrinogen, and
HA, all important components of the healing wound (13). Third,
CD44 may promote fibroblast survival by decreasing apoptosis
(32). Although previous investigations demonstrated that anti-
CD44 treatment induced apoptotic death in cultured fibroblasts,
our experiments showed no significant difference in the density of
apoptotic cells between CD44
null
and WT infarcts. Fourth, CD44
gene disruption may impair fibrogenic responses in stimulated fi-
broblasts. CD44-HA interactions stimulate TGF-
RI serine/threo-
nine kinase activity inducing Smad2/3 phosphorylation in meta-
static breast cancer cells (33). Because the TGF-
/Smad2/3
signaling cascade plays a crucial role in fibrous tissue deposition
(34 –36) in the infarcted heart, we examined whether CD44 defi-
ciency directly impairs TGF-
-mediated fibrogenic responses.
CD44
null
infarcts had significantly higher Smad2 expression lev
-
els, but markedly reduced the p-Smad2:Smad2 ratio (Fig. 6) rais-
ing the possibility that, in the absence of CD44, infarct fibroblasts
may exhibit defective activation of the Smad2/3 pathway. Al-
though CD44
null
fibroblasts had attenuated collagen up-regulation
upon stimulation with TGF-
(Fig. 8A), this defect was not asso-
ciated with impairment in Smad2 phosphorylation (Fig. 8B). Thus,
the mechanism responsible for the defective response of CD44
null
cardiac fibroblasts to TGF-
remains unknown. The absence of
CD44 appears to have profound effects on fibroblast proliferation
and matrix protein synthesis; these defects may result in dimin-
ished extracellular matrix deposition in the infarcted myocardium.
The role of CD44 in cardiac remodeling
In comparison with WT animals, CD44
null
mice exhibited en
-
hanced ventricular dilation following myocardial infarction (Fig.
7, Table I). However, infarct size was comparable in CD44
/
and
WT hearts suggesting that accentuated dilative remodeling in in-
farcted CD44
/
hearts was not a consequence of increased car
-
diomyocyte injury due to the enhanced inflammatory reaction.
Augmented dilation of the infarcted heart may be due to the heal-
ing defects associated with CD44 deficiency resulting in marked
decrease in collagen deposition in the scar. Formation of a defec-
tive matrix network reduces the tensile strength of the ventricle
(37) promoting chamber enlargement. In contrast, CD44 absence
did not affect hypertrophic remodeling suggesting that CD44 sig-
naling does not play a role in the development of cardiomyocyte
hypertrophy following myocardial infarction.
Conclusions
CD44-mediated interactions are critical for cardiac repair and ap-
pear to regulate both inflammatory and fibrotic responses. CD44
signaling does not mediate leukocyte infiltration, but is an impor-
tant inhibitory signal responsible for suppression of postinfarction
2632 CD44 SIGNALING REGULATES INFARCT HEALING
Page 8
inflammation. In addition, CD44 plays a key role in the formation
of scar-mediating myofibroblast infiltration and collagen deposi-
tion. Defects in the CD44-signaling cascade may be important in
the pathogenesis of adverse remodeling following myocardial in-
farction. Dissecting the pathways involved in CD44-mediated ac-
tions may facilitate design of novel therapeutic interventions to
optimize infarct healing and prevent adverse remodeling.
Disclosures
The authors have no financial conflict of interest.
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2633The Journal of Immunology
Page 9
  • Source
    • "Time course in upregulation of CD44 by Ang II infusion was consistent with enhanced extravasation of monocytes to macrophages (Fig. 6). In this respect, a previous study in cultured human umbilical vein endothelial cells has shown that adhesion of monocytes to endothelial cells by exogenous upregulation of CD44 is significantly reduced following pretreatment with the CD44 antibody [27]. HA is a widely distributed extracellular matrix component that can be produced from endothelial cells, smooth muscle cells and fibroblasts by a class of integral membrane proteins named as HA synthases (i.e., HAS1–3). "
    [Show abstract] [Hide abstract] ABSTRACT: Aim: This study tested the hypothesis that angiotensin II (Ang II) AT1 receptor is involved in development of hypertension and cardiac fibrosis via modifying ACE2 activity, eNOS expression and CD44-hyaluronan interaction. Main methods: Male Sprague-Dawley rats were subjected to Ang II infusion (500ng/kg/min) using osmotic minipumps up to 4weeks and the AT1 receptor blocker, telmisartan was administered by gastric gavage (10mg/kg/day) during Ang II infusion. Key findings: Our results indicated that Ang II enhances AT1 receptor, downregulates AT2 receptor, ACE2 activity and eNOS expression, and increases CD44 expression and hyaluronidase activity, an enzyme for hyaluronan degradation. Further analyses revealed that Ang II increases blood pressure and augments vascular/interstitial fibrosis. Comparison of the Ang II group, treatment with telmisartan significantly increased ACE2 activity and eNOS expression in the intracardiac vessels and intermyocardium. These changes occurred in coincidence with decreased blood pressure. Furthermore, the locally-expressed AT1 receptor was downregulated, as evidenced by an increased ratio of the AT2 over AT1 receptor (1.4±0.4% vs. 0.4±0.1% in Ang II group, P<0.05). Along with these modulations, telmisartan inhibited membrane CD44 expression and hyaluronidase activity, decreased populations of macrophages and myofibroblasts, and reduced expression of TGFβ1 and Smads. Collagen I synthesis and tissue fibrosis were attenuated as demonstrated by the less extensive collagen-rich area. Significance: These results suggest that the AT1 receptor is involved in development of hypertension and cardiac fibrosis. Selective activating ACE2/eNOS and inhibiting CD44/HA interaction might be considered as the therapeutic targets for attenuating Ang II induced deleterious cardiovascular effects.
    Full-text · Article · Apr 2016 · Life sciences
  • Source
    • "Reduced recruitment of CD44-null macrophages to atherosclerotic lesions (200) indicates the contribution of CD44 to monocyte/macrophage recruitment to inflammation sites. CD44-null mice also experienced reduced levels of cerebral ischemia injury, further supporting a proinflammatory role for CD44 (201, 202). Studies also revealed that treatment with anti-CD44 mAbs reduced the severity of arthritis in a collagen-induced mouse model for human rheumatoid arthritis (RA) (203204205) and reduced the diabetic activity in NOD nice (206). "
    [Show abstract] [Hide abstract] ABSTRACT: The glycosaminoglycan hyaluronan (HA), a major component of extracellular matrices, and cell surface receptors of HA have been proposed to have pivotal roles in cell proliferation, migration, and invasion, which are necessary for inflammation and cancer progression. CD44 and receptor for HA-mediated motility (RHAMM) are the two main HA-receptors whose biological functions in human and murine inflammations and tumor cells have been investigated comprehensively. HA was initially considered to be only an inert component of connective tissues, but is now known as a "dynamic" molecule with a constant turnover in many tissues through rapid metabolism that involves HA molecules of various sizes: high molecular weight HA (HMW HA), low molecular weight HA, and oligosaccharides. The intracellular signaling pathways initiated by HA interactions with CD44 and RHAMM that lead to inflammatory and tumorigenic responses are complex. Interestingly, these molecules have dual functions in inflammations and tumorigenesis. For example, the presence of CD44 is involved in initiation of arthritis, while the absence of CD44 by genetic deletion in an arthritis mouse model increases rather than decreases disease severity. Similar dual functions of CD44 exist in initiation and progression of cancer. RHAMM overexpression is most commonly linked to cancer progression, whereas loss of RHAMM is associated with malignant peripheral nerve sheath tumor growth. HA may similarly perform dual functions. An abundance of HMW HA can promote malignant cell proliferation and development of cancer, whereas antagonists to HA-CD44 signaling inhibit tumor cell growth in vitro and in vivo by interfering with HMW HA-CD44 interaction. This review describes the roles of HA interactions with CD44 and RHAMM in inflammatory responses and tumor development/progression, and how therapeutic strategies that block these key inflammatory/tumorigenic processes may be developed in rodent and human diseases.
    Full-text · Article · May 2015 · Frontiers in Immunology
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    • "RvD1 promoted CD44, which is important for the containment of chronic inflammation and enrichment of fibroblasts post-MI [40] . CD44 is associated with enhanced, prolonged neutrophil and macrophage infiltration and also an increase in expression of pro-inflammatory cytokines in the infarcted myocardium [40]. Activation of CD44 expression in the MI-saline group was associated with overactive neutrophils at d5 post-MI. "
    [Show abstract] [Hide abstract] ABSTRACT: Unresolved inflammation is a major contributor to the development of heart failure following myocardial infarction (MI). Pro-resolving lipid mediators, such as resolvins (e.g. RvD1), are biosynthesized endogenously. The role of RvD1 in resolving post-MI inflammation has not been elucidated due to its unstable nature. Here, we have tested the role for two forms of RvD1, after incorporation into liposomes (Lipo-RvD1) and its free acid form (RvD1) in the left ventricle (LV) and splenic remodeling post-MI. 8 to 12-week old male, C57BL/6J-mice were subjected to coronary artery ligation and Lipo-RvD1 or RvD1 (3 mu g/kg/day) was injected 3 h post-MI for day (d)1 or until d5. No-MI mice and saline-injected MI mice served as controls. RvD1 injected groups showed improved fractional shortening post-MI; preserving transient changes in the splenic reservoir compared to MI-saline. RvD1-groups showed an early exit of neutrophils from LV and spleen at d5 post-MI with an increased expression of lipoxin A(4) receptor (ALX; synonym formyl peptide receptor; FPR2) compared to the MI-saline group. The levels of pro-resolving mediators RvD1, RvD2, Maresin 1 (MaR1) and Lipoxin A(4) (LXA(4)) were increased in spleens from RvD1 injected mice at d5 post-MI. RvD1 administration reduced macrophage density, ccr5 and cxcl5 levels at d5 post-MI compared to saline injected mice (both, p <0.05). Increased transcripts of mrc-1, arg-1 and Ym-1 (all, p < 0.05) suggest macrophage-mediated clearance of necrotic cells in RvD1-groups. RvD1 reduced the pro-fibrotic genes (colla1, coll2a1 and tnc (all; p < 0.05)) and decreased collagen deposition, thereby reducing post-MI fibrosis and thus stabilizing the extracellular matrix. In summary, RvD1 and Lipo-RvD1 promote the resolution of acute inflammation initiated by MI, thereby delaying the onset of heart failure.
    Full-text · Article · Apr 2015 · Journal of Molecular and Cellular Cardiology
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