PLoS ONEplosplosonePLoS ONE1932-6203Public Library of ScienceSan Francisco, USAPONE-D-10-0255110.1371/journal.pone.0018668Research ArticleBiologyImmunologyImmune systemCytokinesMolecular cell biologyMedicineCardiovascularHeart failureImmunologyLack of Chemokine Signaling through CXCR5 Causes Increased Mortality,
Ventricular Dilatation and Deranged Matrix during Cardiac Pressure
OverloadCXCL13/CXCR5 in Heart FailureWaehreAnne11011*HalvorsenBente210YndestadArne210HusbergCathrine111SjaastadIvar151011NygårdStåle11112DahlChristen P.2411AhmedM. Shakil31011FinsenAlexandra V.2411ReimsHenrik6LouchWilliam E.111Hilfiker-KleinerDenise7VingeLeif E.2411RoaldBorghild6AttramadalHåvard31011LippMartin8GullestadLars241011AukrustPål2910ChristensenGeir11011Institute for Experimental Medical Research,
Oslo University Hospital Ullevål, Oslo, NorwayResearch Institute for Internal Medicine, Oslo
University Hospital Rikshospitalet, Oslo, NorwayInstitute for Surgical Research, Oslo
University Hospital Rikshospitalet, Oslo, NorwayDepartment of Cardiology, Oslo University
Hospital Rikshospitalet, Oslo, NorwayDepartment of Cardiology, Oslo University
Hospital Ullevål, Oslo, NorwayDepartment of Pathology, Oslo University
Hospital Ullevål, Oslo, NorwayDepartment of Cardiology and Angiology,
Hanover Medical School, Hanover, GermanyDepartment of Molecular Tumor Genetics and
Immunogenetics, Max-Delbrück-Center for Molecular Medicine, Berlin,
GermanySection of Clinical Immunology and Infectious
Diseases, Oslo University Hospital Rikshospitalet, Oslo, NorwayFaculty of Medicine, University of Oslo,
Oslo, NorwayCenter for Heart Failure Research,
University of Oslo, Oslo, NorwayBioinformatics Core Facility, Institute for
Medical Informatics, Oslo University Hospital Rikshospitalet, Oslo,
NorwayKellyKathleen A.EditorUniversity of California Los Angeles, United States of America* E-mail: anne.waehre@medisin.uio.no
Conceived and designed the experiments: AW GC BH AY PA. Performed the
experiments: AW BH AY CH AVF HR MSA DH-K WEL. Analyzed the data: AW BH GC PA
IS SN HA MSA BR HR LEV. Contributed reagents/materials/analysis tools: ML.
Wrote the paper: AW GC PA. Collected humane data and commented on the
manuscript: CPD LG.
The authors have declared that no competing interests exist.
2011184201164e18668229201015320112011Waehre et alThis is an open-access article distributed under the
terms of the Creative Commons Attribution License, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original author and
source are credited.Rationale
Inflammatory mechanisms have been suggested to play a role in the development
of heart failure (HF), but a role for chemokines is largely unknown. Based
on their role in inflammation and matrix remodeling in other tissues, we
hypothesized that CXCL13 and CXCR5 could be involved in cardiac remodeling
during HF.
Objective
We sought to analyze the role of the chemokine CXCL13 and its receptor CXCR5
in cardiac pathophysiology leading to HF.
Methods and Results
Mice harboring a systemic knockout of the CXCR5
(CXCR5−/−) displayed increased mortality during a
follow-up of 80 days after aortic banding (AB). Following three weeks of AB,
CXCR5−/− developed significant left ventricular
(LV) dilatation compared to wild type (WT) mice. Microarray analysis
revealed altered expression of several small leucine-rich proteoglycans
(SLRPs) that bind to collagen and modulate fibril assembly. Protein levels
of fibromodulin, decorin and lumican (all SLRPs) were significantly reduced
in AB CXCR5−/− compared to AB WT mice. Electron
microscopy revealed loosely packed extracellular matrix with individual
collagen fibers and small networks of proteoglycans in AB
CXCR5−/− mice. Addition of CXCL13 to cultured
cardiac fibroblasts enhanced the expression of SLRPs. In patients with HF,
we observed increased myocardial levels of CXCR5 and SLRPs, which was
reversed following LV assist device treatment.
Conclusions
Lack of CXCR5 leads to LV dilatation and increased mortality during pressure
overload, possibly via lack of an increase in SLRPs. This study demonstrates
a critical role of the chemokine CXCL13 and CXCR5 in survival and
maintaining of cardiac structure upon pressure overload, by regulating
proteoglycans essential for correct collagen assembly.
Norwegian Research Council, Anders Jahre's Fund for the Promotion of
Science, Joh. H. Andresen's Medical Fund. The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the
manuscript.Introduction
Heart failure (HF) is a disorder associated with low-grade immune activation and
inflammation, as evidenced by elevated circulating and myocardial levels of
inflammatory cytokines, including tumor necrosis factor (TNF)α, interleukin
(IL)-1β and IL-18, and chemokines such as monocyte chemoattractant protein
(MCP)-1 and fractalkine [1]–[5]. A range of experimental studies have also suggested a
pathogenic role for several of these inflammatory mediators in the development and
progression of HF [4], [6]–[8]. However, the role of inflammation in HF remains
incompletely understood. Identification of the most important mediators of the
inflammatory pathways involved in the pathogenesis of HF and their mechanism of
action are issues that need to be further clarified.
While most chemokines have been linked to inflammatory processes in peripheral
tissue, the homeostatic chemokines (i.e., CCL19, CCL21, and CXCL13) and their
corresponding receptors (i.e., CCR7 for [CCL19 and CCL21] and CXCR5 for
[CXCL13]) have been associated with development and maintenance of
secondary lymphoid organs [9]–[12], as well as the entry of lymphocytes and dendritic cells
to secondary lymphoid tissue [13]–[15]. Recently, however, reports have pointed to a broader
role for these homeostatic chemokines, including modulation of inflammatory and
anti-inflammatory responses in lymphoid and non-lymphoid tissue. Thus, while CXCL13
was known to dictate homing and motility of B cells in lymphoid tissue, more recent
studies suggest that CXCL13 is involved in the formation of ectopic lymphoid tissue
in chronic inflammation [16], [17]. This chemokine has also been linked to T cell [9], [18], [19] and monocyte
activation [20]
and apoptosis [21]. In line with its newly discovered role in the immune
system, CXCL13 has been suggested to be involved in the pathogenesis of rheumatoid
arthritis [22],
Sjögren syndrome [23]–[25], inflammatory bowel disease [26], and multiple sclerosis [27]. CXCR5 is a G
protein-coupled seven transmembrane receptor and belongs to the CXC chemokine
receptor family [28]. Recently, CXCR5 has been found to be involved in
remodeling of extracellular matrix (ECM) in various types of cancer, including colon
[29] and
prostate cancer [30]. However, the potential role for CXCL13 and CXCR5 in the
pathogenesis of myocardial remodeling has not been studied.
Based on their potential role in inflammation and matrix remodeling, we hypothesized
that CXCL13 and its receptor CXCR5 are involved in cardiac remodeling and
development of HF. We examined this hypothesis by studying the cardiac morphology,
function and molecular alterations in CXCR5 deficient
(CXCR5−/−) mice exposed to left ventricular (LV) pressure
overload induced by aortic banding (AB).
ResultsExpression of CXCR5 and CXCL13 in murine hearts
We first examined if the CXCL13/CXCR5 dyad was regulated during AB in mice. Both
CXCL13 and CXCR5 were expressed within the murine heart, and as shown in Fig. 1A and
B, we found significantly enhanced myocardial
expression of CXCR5, but not of CXCL13, in mice that underwent AB as compared
with sham operated mice. Within the myocardium, we found mRNA expression of
CXCL13 and CXCR5 in cardiomyocytes, fibroblasts and endothelial cells with the
highest expression of both CXCR5 and CXCL13 in myocardial fibroblasts (Fig. 2A,
B).
10.1371/journal.pone.0018668.g001
Myocardial gene expression of CXCL13 and CXCR5 in mice.
Myocardial gene expression of (A) CXCL13 and
(B) CXCR5 in wild type (WT) Sham
(n = 7) and WT aorta banded (AB)
(n = 14) group. The results are mean ± SEM.
**p<0.01 vs. Sham group.
10.1371/journal.pone.0018668.g002
Gene expression of CXCL13 and CXCR5 in myocardial cells in
mice.
Gene expression of (A) CXCL13 and (B) CXCR5 in
cardiomyocytes, fibroblasts and endothelial cells from wild type mice
(n = 3). mRNA levels were assesssed by quantitaive
real time PCR. AU = Arbitrary unit. The results are
mean ± SEM. * p<0.05, **p<0.01,
***p<0.001 vs. cardiomyocytes.
††p <0.01 vs. fibroblasts.
Survival during LV pressure overload
As depicted in Kaplan-Meier survival curves (Fig. 3), CXCR5−/− mice
exhibited significantly higher mortality rates than WT mice during an 80-day
follow-up after AB induction. The differences in mortality emerged after 40 days
of AB.
10.1371/journal.pone.0018668.g003
Kaplan-Meier survival curves of wild type (WT)
(n = 20) and CXCR5−/− mice
(n = 20) after aortic banding (AB).
Echocardiographic assessment showed comparable increases in flow
velocities across the banded region of the aorta in WT and
CXCR5−/− twenty-one days after primary
surgery. Differences in survival between WT and
CXCR5−/− mice were tested with the log-rank
test. *p<0.05 vs. WT.
Severe LV dilatation in CXCR5−/− mice following
pressure overload
To investigate a potential role for CXCR5 in cardiac remodeling and development
of HF, we evaluated cardiac morphology and function in WT and
CXCR5−/− mice. Two-dimensional and M-mode
echocardiography performed in non-operated WT (n = 6) and
CXCR5−/− (n = 6) mice showed no
significant differences in LV function or dimensions (Table S1).
Twenty-one days after AB, echocardiographic assessment showed comparable
increases in flow velocities across the banded region of the aorta in the WT and
CXCR5−/− groups. However, LV fractional shortening
was reduced by 65% in CXCR5−/− mice, but only by
13% in WT (Table S1). The mean LV diastolic dimension
was significantly larger and the LV posterior wall thickness was significantly
lower in CXCR5−/− mice compared to WT mice at the same
time point (Fig. 4A,
B).
10.1371/journal.pone.0018668.g004
Left ventricular (LV) dilatation in CXCR5−/−
mice 3 weeks following aortic banding (AB).
LV inner diameter (A) and thickness of the posterior wall
(B) were measured in the wild type (WT) Sham
(n = 6), CXCR5−/− Sham
(n = 6), WT AB (n = 6), and
CXCR5−/− AB (n = 6)
groups. The results are mean ± SEM. *p<0.05 and
***p<0.001 vs. Sham groups;
††p<0.01 and †††p<0.001
vs. WT AB group.
CXCR5−/− mice show increased expression of hypertrophy
maker genes in response to pressure overload
Despite similar increases in heart weight to tibial length (HW/TL) ratio in both
genotypes after AB, CXCR5−/− mice exhibited a more marked
increase in expression of ANP (3.3-fold increase), BNP (2.3-fold increase) and
β-MHC (2.5-fold increase) than WT (Fig. S1A–C). The
marked increase in ANP and BNP in CXCR5−/− mice following
AB might suggest increased myocardial wall stress in these mice.
CXCR5−/− mice exhibit major alterations in ECM in
response to pressure overload
Since alterations in extracellular matrix (ECM) might be responsible for LV
dilatation, we examined the quality and composition of the ECM following AB.
CXCR5−/− mice exhibited increased myocardial collagen
content following AB as compared with WT mice, as illustrated by both Masson
trichrome staining (Fig.
5A) and hydroxyproline measurement by HPLC
(Fig.
5B). This increase in collagen content in
CXCR5−/− mice was accompanied by a significant
increase in total matrix metalloproteinase (MMP) activity (Fig. 5C) and gelatinolytic
activity (Fig. 5D,
E). This combination of increased collagen content and
increased MMP activity suggest enhanced matrix remodeling in CXCR5 deficient
mice following AB.
10.1371/journal.pone.0018668.g005
Extracellular matrix remodeling in wild type (WT) and
CXCR5−/− mice following aortic banding
(AB).
Collagen content in myocardium examined with (A) Masson
trichrome staining in representative sections from AB WT and
CXCR5−/− mice and (B) by
hydroxyproline measurement in WT Sham (n=13),
CXCR5−/− Sham (n=12), WT AB
(n=13) and CXCR5−/− AB (n=14)
groups. (C) Total MMP activity in percentage of the WT Sham
group (n=11) measured in the CXCR5−/−
Sham (n=13), WT AB (n=9), and
CXCR5−/− AB groups (n=9).
(D) Gelatinolytic activity of MMP‐2 relative to
the WT Sham group (n=5) measured in the
CXCR5−/− Sham (n=5), WT AB
(n=5), and CXCR5−/− AB groups
(n=5) and of (E) MMP‐9 relative to the WT
Sham group (n=6) measured in the
CXCR5−/− Sham (n=6), WT AB
(n=8), and CXCR5−/− AB groups
(n=9). The results are mean ± SEM. *p<0.05,
**p<0.01 and ***p<0.001 vs.
Sham groups; †p<0.05 and ††p<0.01
vs. WT AB groups.
In addition to collagen, the quantity and quality of other ECM constituents also
importantly influence cardiac function [31]. We therefore performed
microarray analysis (Affymetrix) of the myocardium from WT and
CXCR5−/− mice 3 weeks after AB. The seeded Bayesian
network method [32] was used to explore interactions between
differentially expressed genes. This analysis identified a cluster of genes
encoding ECM proteins. Interestingly, this cluster contained fibromodulin which
belongs to a family of small leucine-rich repeat proteoglycans (SLRPs), which
are known to influence ECM assembly [33]. Microarray data are
accessible through GEO Series accession number GSE22295 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE22295).
Protein levels of SLRPs following AB in CXCR5−/− and
WT mice
To further examine the regulation of SLRPs following AB, we measured protein
levels of fibromodulin and other members in the SLRP family, including decorin,
lumican and biglycan. With the exception of biglycan, all of these SLRPs were
differently regulated in CXCR5 −/− mice compared to WT
mice following AB (Fig. 6).
While decorin and lumican markedly increased during AB in WT mice, this was not
observed in CXCR5−/− mice (Fig. 6A, B). Moreover, while
fibromodulin decreased following AB in WT and CXCR5 −/−
mice, the decrease was significantly more pronounced in the
CXCR5−/− mice (Fig. 6C).
10.1371/journal.pone.0018668.g006
Protein levels of small leucine-rich repeat proteoglycans (SLRPs) and
transmission electron microscopic analysis in wild type (WT) and
CXCR5−/− mice following aortic banding
(AB).
Protein levels of (A) decorin and (B) lumican
in (WT) Sham (n = 7),
CXCR5−/− Sham (n = 5),
WT AB (n = 6), and CXCR5−/−
AB (n = 5), and (C) fibromodulin in WT
Sham (n = 13), CXCR5−/− Sham
(n = 12), WT AB (n = 12), and
CXCR5−/− AB (n = 11)
groups as assessed by western blotting. The upper panels show
representative blots from two mice in each group. § Glycosylated
forms of decorin and lumican, respectively. The results are mean
± SEM. *p<0.05, **p<0.001 and
***p<0.001 vs. Sham groups;
†p<0.05 and ††p<0.01 vs. WT
AB group. The lower panels show representative transmission electron
micrographs of the LV free wall at ×440 and ×23000
magnification in WT (D and F) and
CXCR5−/− (E and G)
AB mice.
Electron microscopic analysis revealed loosely packed ECM in
CXCR5−/− mice following AB
SLRPs are capable of binding to different types of collagen [34], [35], thereby regulating
fibril assembly and organization, degradation, and quantitative and functional
aspects of the collagen network [33]. To further elucidate the
effect of decreased levels of SLRPs during pressure overload, LV tissue sections
were examined by electron microscopic analysis in WT and CXCR5-deficient mice.
As shown in Fig.
6D and E, a
considerable increase in the extracellular space was observed in LVs from
CXCR5−/− as compared to WT mice. In addition, banded
WT mice exhibited densely packed collagen fibers of variable thickness and
orientation as well as proteoglycan particles associated with fine filaments
(Fig.
6F). In contrast, the ECM in
CXCR5−/− mice exhibited large areas with a coarse
network of individual collagen fibrils, and finer networks of proteoglycans with
associated filaments (Fig.
6G), suggesting deranged ECM.
No difference in apoptosis and leukocyte infiltration between
CXCR5−/− and WT mice after pressure overload
CXCL13 has been suggested to exhibit anti-apoptotic properties [36], [37]. However,
analysis of cardiac apoptosis by in situ TUNEL staining
revealed that, after either sham-operation or AB, the number of apoptotic cells
was similar in WT and CXCR5-deficient mice (Fig. S2).
Moreover, although CXCL13 is known to influence lymphocyte trafficking [38], [39], we found no
significant difference in infiltration of CD3+ or
CD45+ cells between CXCR5−/− and WT
mice after either sham operation or AB (Fig. S3A and
B).
CXCL13 stimulates expression of SLRPs
The changes in ECM in CXCR5−/− mice following AB,
consisting of enhanced MMP activity and decreased expression of several SLRPs,
could potentially reflect direct effects of CXCL13 on myocardial fibroblasts. In
fact, both CXCL13 and CXCR5 were strongly expressed within myocardial
fibroblasts, and CXCR5 showed enhanced myocardial expression during AB in WT
mice (Fig. 1). In addition,
fibroblasts are important producers of ECM proteins, including SLRPs.
Stimulation of cardiac neonatal rat fibroblasts with CXCL13 did indeed enhance
the expression of fibromodulin, biglycan and lumican, and in particular of
decorin, and at the same time down-regulated total MMP activity (Fig. 7A,
B).
10.1371/journal.pone.0018668.g007
Effects of CXCL13 on gene expression of small leucine-rich repeat
proteoglycans (SLRPs), total MMP activity and the intracellular
signalling pathway ERK 1/2 in neonatale rat fibroblasts (FB).
The effect of CXCL13 (200 ng/ml) stimulation on gene expression of
(A) decorin, biglycan, lumican and fibromodulin
(n = 4) in myocardial FB after 20 hours of
stimulation in relation to GAPDH and (B) total MMP activity
in cell-free supernatant (n = 7) after 20 hours of
stimulation. The ratio of phosphorylated ERK 1/2 (p-ERK 1/2) to total
ERK 1/2 assessed by (C) western blotting in WT Sham
(n = 13), CXCR5−/− Sham
(n = 12), WT AB (n = 11), and
CXCR5−/− AB (n = 10)
groups, and by (D) BioPlex in WT Sham
(n = 6), CXCR5−/− Sham
(n = 5), WT AB (n = 7), and
CXCR5−/− AB (n = 6)
groups. (E) The effect of blocking ERK 1/2 activation with
UO126 (10 µM) on the CXCL13-mediated induction of fibromodulin
gene expression in myocardial FB (n = 4). The
results are mean ± SEM. *p<0.05 and **p<0.01
vs. unstimulated FB or
CXCR5−/− Sham; †p<0.05
vs. WT AB group, ‡p<0.05 vs. CXCL13
stimulated FB without inhibitor (UO126).
CXCL13-CXCR5 mediate their effects on matrix modulation via ERK1/2
signaling
We next examined alterations in the ERK1/2 pathway in
CXCR5−/− following AB, as this pathway is of
importance in cardiac remodeling and since CXCL13 signaling through CXCR5 is
known to activate the MAPK pathway via ERK 1/2 [40], [41]. As shown in Fig. 7C and
D, LV from CXCR5−/− mice showed decreased
levels of phosphorylated ERK1/2 as compared with WT mice. In cardiac
fibroblasts, blocking the ERK1/2 pathway by UO126, a highly selective inhibitor
of MEK 1 and 2, significantly attenuated up-regulation of fibromodulin following
CXCL13 treatment (Fig.
7E).
Expression of CXCR5 and SLRPs in patients with HF
Assessments of CXCR5 mRNA levels in myocardial tissue from 9 HF patients (all
with advanced HF, NYHA class IV) and 5 controls (non-failing hearts) showed that
HF patients had markedly enhanced gene expression of CXCR5 (84% increase,
p<0.005). As shown in Fig.
7, the 9 HF patients also had significantly enhanced gene expression
of biglycan, lumican and fibromodulin. When the HF patients were treated with
continuous-flow LV assist device (LVAD) for 8±1.7 months, improvement in
hemodynamic parameters (LV end diastolic volume decreased from 294.9 mL to 237.8
mL, LV diastolic diameter from 7.6 cm to 6.8 cm, and LV end systolic volume from
244.8 mL to 183.4 mL, reflected in an increase in LVEF from 18.2 to
29.6%, p<0.05 for all) was accompanied by a marked decrease in mRNA
levels of CXCR5 as well as biglycan and fibromodulin, although the decrease in
fibromodulin did not reach statistical significance (Fig. 8).
10.1371/journal.pone.0018668.g008
Gene expression of CXCR5, biglycan, lumican and fibromodulin in
patients with heart failure (HF) (n = 9) and
controls (n = 5) as assesssed by quantitaive real
time PCR.
In the HF patients, the mRNA levels were measured before and after
treatment with continuous-flow left ventricular assist device
(LVAD).
Discussion
Despite observations of enhanced levels of chemokines and their corresponding
receptors in human HF [5], [42], [43], the role of chemokines in maintaining cardiac structure
and function has never been established. The present work clearly demonstrates that
the chemokine CXCL13 and its receptor, CXCR5, are critically involved in cardiac
remodeling. The key results of this study were increased mortality and severe LV
dilatation in CXCR5-deficient mice in response to pressure overload, potentially
resulting from impaired quality of ECM. These ECM alterations derived, at least
partly from decreased SLRP levels and enhanced MMP activity within the myocardium.
Our in vitro findings showed that CXCL13 can promote SLRP
expression and attenuate MMP activity in myocardial fibroblasts. Therefore, the
opposite pattern seen in CXCR5-deficient mice could reflect the inability of their
myocardial fibroblasts to respond to CXCL13. These data indicate that the
CXCL13/CXCR5 interaction is involved in myocardial remodeling following pressure
overload, possibly by regulating proteoglycans crucial for the quality of myocardial
ECM. Our findings of a strong expression of CXCL13 and CXCR5 in fibroblasts within
murine hearts and enhanced myocardial expression of CXCR5 during AB further support
such a notion.
The possible role of chemokines in the pathogenesis of HF has, at least in part, been
attributed to the ability of these molecules to promote leukocyte infiltration in
failing myocardium. However, in the present study we did not detect altered
infiltration of CD3+ and CD45+ cells in
CXCR5−/− mice compared to the WT mice following AB.
Previously, various chemokines (e.g., CXCL16, MCP-1 and CX3CL1) have been shown to
promote direct effects on myocardial fibroblasts and cardiomyocytes in
vitro[5], [43]–. Also, the
lack of CXCR4 has been associated with severe myocardial developmental defects
(i.e., ventricular septum defects) [46]. In contrast to CXCR4, CXCR5
deficient mice are viable and display normal morphology and function of the adult
heart. However, our present data showing a markedly dilating myocardial phenotype in
CXCR5−/− mice exposed to pressure overload, without any
significant changes in myocardial leukocyte infiltration, may suggest a direct
involvement of CXCL13/CXCR5 activation in myocardial remodeling. The ability of
CXCL13 to attenuate MMP activity and increase SLRP expression in myocardial
fibroblasts as well as the up-regulation of CXCR5 during AB in WT mice further
supports such a notion.
Collagen synthesis, fibrillogenesis, and matrix degradation must be finely tuned, as
an imbalance in these processes might result in cardiac dilatation, cardiac
hypertrophy and fibrosis. Although we observed increased total collagen content in
CXCR5−/− mice, these mice were also characterized by
massively disturbed structural frameworks after AB compared to WT. Our molecular
analysis suggested that this distorted ECM structure derives from a failure in the
regulation of SLRPs in pressure overloaded CXCR5-deficient mice. SLRPs are known to
bind to collagens, and in so doing, regulate the self-assembly process of
pro-collagen into fibrils [33]–[35]. This assembly is necessary for covalent cross-linking
which is required for reinforcement of the collagen fibrils. SLRPs have been shown
to be up-regulated in the infarcted area in rats and mice following myocardial
infarction (MI) [47],
[48].
Studies in SLRP deficient mice have shown abnormal fibril organization and loose
fibril packing in the MI scar [49], [50]. In the current study we show an attenuated up-regulation
(i.e., decorin and lumican) and a more pronounced decrease (i.e., fibromodulin) in
SLRP expression following AB in CXCR5 deficient mice as compared with WT mice.
Interestingly, enhanced MMP activity has been found to impair SLRP function [44], and we
suggest that the combination of decreased SLRP expression and increased MMP activity
could be of major importance for the premature LV dilatation and HF in
CXCR5−/− mice following AB.
Our in vitro data suggest that CXCL13 via activation of CXCR5 on
myocardial fibroblasts induces the expression of SLRPs through the ERK1/2 pathway.
This mechanism appears to be absent in fibroblasts from
CXCR5−/− mice. ERK is one of the key protein kinases that
regulate growth and proliferation of cardiac fibroblasts [40]. In line with a crucial role
of CXCL13/CXCR5-mediated ERK1/2 activation for cardiac remodeling in response to
pressure overload, ERK1/2 phosphorylation was substantially lower in
CXCR5−/− mice compared to WT after AB. These findings
further support previous reports of a central role of the ERK pathway for myocardial
remodeling [51],
[52]. In this
regard, the present study adds a novel component to this pathway by linking
CXCR5-mediated effects to SLRPs and subsequent ECM remodeling.
Our studies in patients with advanced HF suggest that our findings in experimental HF
may have relevance to clinical HF. We showed enhanced myocardial expression of CXCR5
and certain SLRPs (i.e., biglycan, lumican and fibromodulin) in the failing
myocardium, and notably, biglycan and lumican were down-regulated following the
clinical and hemodynamic improvement during treatment with LV assist device. Based
on our experimental data, it is tempting to speculate that CXCR5 activation promotes
protective responses in ECM in failing myocardium involving enhanced expression of
SLRPs. As myocardial function improves, this response is attenuated. At present,
however, the stimuli for enhanced myocardial CXCR5 expression in HF and the
interpretation of our human data will require further investigation.
In conclusion, we have found that CXCR5 plays an important role in cardiac remodeling
during pressure-overload. Loss of CXCR5 in this situation adversely affects matrix
remodeling and causes LV dilatation, possibly through altered regulation of
proteoglycans crucial for the quality of myocardial ECM (i.e., SLRPs) as well as
through enhanced MMP activity. These changes could, at least in part, be attributed
to loss of CXCL13 mediated effects on myocardial fibroblasts in CXCR5 deficient
mice. The identification of molecular and structural changes causing LV dilatation
during pressure overload is of major importance for the development of new treatment
strategies in HF related to hypertension and aortic stenosis. Future studies should
examine how the CXCL13/CXCR5 dyad could be utilized therapeutically in clinical
HF.
Materials and MethodsEthics
All animal experiments were approved by the Norwegian Animal Research Committee
(ID 1902) and conform to the Guide for the Care and Use of Laboratory Animals
published by the US National Institutes of Health (NIH Publication No.
85–23, revised 1996). The part of the study that involved humans was
approved by the local ethics committee (REK Helse Sør-Øst) and
conducted according to the ethical guidelines outlined in the Declaration of
Helsinki for use of human tissue and subjects. Informed written consent was
obtained from all subjects. The authors had full access to the data and take
responsibility for its integrity. All authors have read and agree to the
manuscript as written.
Data analysis
All data are expressed as group means ± SEM unless indicated otherwise.
For comparisons of 2 groups, the Mann-Whitney U test was
employed. The Wilcoxon test was employed when analysing the effect of LVAD
treatment. Differences between the WT Sham, WT AB,
CXCR5−/− Sham, and CXCR5−/− AB
groups were determined by one-way ANOVA with post-hoc Tukey test. Differences in
survival between wild type and CXCR5−/− mice in Fig. 3 were compared using
Kaplan- Meier survival curves and tested with the log-rank test. All tests were
employed using a 5% significance level.
Animals and AB protocol
Mice were housed in M2 or M3 cages with Bee Kay bedding (Scanbur BK, Nittedal,
Norway) in 55% humidity on a 12 h light/dark cycle. Food pellets (RM1,
801151, Scanbur BK) and water were freely available. All mice utilized in this
study were male and had a weight 20–30 g. WT C57BL/6 mice were obtained
from Taconic (Skensved, Denmark). The generation of
CXCR5−/− mice (C57BL/6 background, now accessible at
the Jackson Laboratory, stock number 006659, strain name B6.129S2
(Cg)-Cxcr5tm1Lipp/J has been described
previously [14]. Briefly, gene targeting was performed in
129S2/SvPas-derived D3 embryonic stem cells, replacing the coding region of the
CXCR5 gene with a neomycin resistance gene. Mutant mice were backcrossed to
C57BL/6 mice for 8 generations. AB was induced in C57BL/6 and
CXCR5−/− mice as previously described [53]. Briefly,
after being anesthetized with 5% isoflurane and ∼98% oxygen in
a gas chamber, the animals where endotracheal intubated and the cannula was
connected to a volume cycled rodent ventilator (Harvard Apparatus) on supplement
of a mixture of ∼1.75% isoflurane and ∼98% oxygen. A
thoracotomy was performed in the second intercostal space on the left side, and
the aortic constriction was created by placing a ligature securely around the
ascending aorta and a 26-gauge needle and then removing the needle. Sham
operated animals underwent the same procedure except for aortic constriction.
The animals were extubated after getting a dose of analgesic (buprenorphine, 0.1
mg/kg) subcutaneously and allowed to recover. Doppler echocardiography was
performed 21 days after primary surgery under general anaesthesia with
isoflurane as described above. This time point was selected since previous
studies in our laboratory have shown marked hypertrophy in WT mice at this
stage. AB mice in both groups with a flow velocity across the aortic banding
site greater than 3.5 m/s were included in the study. The same mice anesthetized
with isoflurane were euthanized by dislocation of the neck, and the hearts and
lungs were removed and blotted dry. The right ventricle and atria were removed.
The LV, right ventricular free wall and lungs were weighed and normalized to
tibial length.
Doppler Echocardiography
Mice were examined while sedated in the supine position with the chest closed, as
previously described [54]. Echocardiography was performed using a i13L 13 MHz
linear array transducer (GE Healthcare Technologies, Oslo, Norway) and data were
analyzed with EchoPac PC software (GE Healthcare Technologies, Oslo, Norway) as
described [55]. The data were recorded and analyzed by a cardiologist
(IS), blinded for the genotype.
Isolation of adult myocardial murine cardiomyocytes, fibroblasts and
endothelial cells
Mouse cardiomyocytes were isolated as previously described [56]. Endothelial cells were
enriched from the non-cardiomyocyte fraction by labeling the cells with a rat
anti-mouse CD31 antibody (eBioscience, San Diego, CA) and subsequent extraction
using an anti-rat secondary antibody coupled to magnetic beads (Miltenyi Biotec,
Auburn, CA) and further column purifications according to the protocols provided
by the manufacturer (http://www.miltenyibiotec.com). The cell fraction remaining
after extraction of cardiomyocytes and endothelial cells contains predominantly
fibroblasts.
RNA isolation
Total RNA was isolated from the LV in WT and CXCR5−/− mice
(SV total RNA isolation system, Promega, Inc., Madison, WI), mouse and neonatal
rat cardiomyocytes and fibroblasts, and mouse endothelial cells (RNeasy mini
kit, Qiagen, Valencia, CA) as previously described [5].
Quantitative real-time PCR (qRT-PCR)
Reverse transcription reactions were performed with iScript Select cDNA Synthesis
Kit (Bio-Rad Laboratories, Inc., Hercules, CA). Pre-designed TaqMan assays
(Applied Biosystems, Foster City, CA) were used to determine gene expression of
CXCL13(Mm00444534_m1), CXCR5 (Mm00432086_m1), ANP (Mm01255748_g1), BNP
(Mm00435304_g1), β-MHC (Mm01319006_g1), biglycan (Rn00567229_m1),
fibromodlin (Rn00589918_m1), lumican (Rn00579127_m1) and decorin
(Rn01503161_m1). The results were detected on an ABI PRISM 7900 Sequence
Detection System (Applied Biosystems) as described previously [5]. In the
human studies, quantification of gene expression was performed using the ABI
Prism 7500 (Applied Biosystems), Power SYBR Green Master Mix (Applied
Biosystems), and sequence-specific PCR primers were designed using the Primer
Express software, version 3.0 (Applied Biosystems). List of the real-time PCR
assays used in the human study is shown in Table
S2.
Perfusion fixation and histology
After the hearts were excised and rinsed in cold NaCl solution, the aorta was
cannulated, and the hearts were mounted on a Langendorff setup, and retrogradely
perfused with warm (37°C) oxygenated Thyrodes solution (5 mM Hepes, pH 7.4,
140 mM NaCl, 5.4 mM KCl, 0.4 MgH2PO4, 0.5
MgCL2) with 1.8 mM Ca2+. Hearts were stopped in
diastole by aortic perfusion of Thyrodes solution with high KCl (10.8 mM) for 3
min and fixated for 10 min by perfusion of 4% phosphate-buffered
formalin. After the cannulas were removed, fixation by immersion continued for 2
h. Each heart was transected at the midventricular level, and both halves were
routinely processed and embedded in paraffin. Paired 3.5 µm sections were
prepared, mounted on glass slides, and stained with hematoxylin and eosin and
Masson tricrome stain.
Hydroxyproline analysis
Quantitative analysis of tissue levels of hydroxyproline was performed by HPLC
using the AccQ-Fluor reagent kit (Waters Corporation, Milford, MA) essentially
as previously described [57]. Briefly, cardiac tissue samples (5 mg dry weight)
were hydrolyzed in 6 M HCl for 16 h at 110°C and subsequently dried under
vacuum and redissolved in the AccQ-Fluor borate buffer. Derivatization was
initiated by addition of the AccQ-Fluor reagent at 55°C and terminated after
10 min. The samples were finally subjected to HPLC-chromatography using a
20×3.9 mm Sentry Guard column (Nova-Pak C18 bonded silica)
connected to a 150×3.9 mm AccQ-Tag reversed-phase column (both from
Waters) according to the manufacturer's instructions. Derivatized
hydroxyproline was detected by fluorescence signal following excitation at 250
nm and recording of emission at 395 nm. Elution of hydroxyproline from
myocardial tissue samples was verified and quantified by co-elution with known
amounts of derivatized hydroxyproline standards (Fluka, Buchs SG, Switzerland).
The relation of myocardial hydroxyproline contents to myocardial collagen has
previously been reported [58].
Measurements of total MMP activity and gelatinolytic activity
Total MMP activity in the LV was measured by a fluorogenic peptide substrate
(R&D Systems) used to assess broad-range MMP activity (MMP-1, -2, -7, -8,
-9, -12 and -13 can cleave the peptide). Gelatinolytic activity was assessed by
gelatine zymography. Briefly, the MMP substrate was diluted in TCN buffer (50 mM
Tris HCl, 150 mM NaCl, 10 mM CaCl2; pH 7.5) and added to the
supernatants before incubation at 37°C. After 120 min the total MMP activity
was determined on a fluorimeter (FLX 800 Microplate Fluorescence Reader, Bio-Tek
Instruments, Winooski, VT).
Gene expression profiling and microarray data analysis
Total RNA was isolated from the LV in Sham (n = 3) and AB
(n = 4) WT and CXCR5-/- mice as described
previously [5]. Preparation of cRNA and the subsequent steps leading
to hybridization of Affymetrix GeneChip® mouse ST 1.0 arrays (Affymetrix,
Santa Clara, CA), washing, and scanning were performed according to standard
protocols (Affymetrix). Microarray preprocessing was done using robust
multi-array average [59]. Differentially expressed genes were found using
significance analysis of microarrays [60]. The seeded Bayesian
network method [32] was used to explore interactions between
differentially expressed genes. This method finds interactions in the expression
data using literature co-citations, databases of protein-protein interactions,
as well as co-regulations in the expression data. We constructed two networks;
one for the wild type situation, using the 60 most differentially expressed
genes between AB WT and SHAM WT, and similarly one for the KO situation, using
the 60 most differentially expressed between CXCR5−/− AB
and Sham AB. The set of 60 genes were based on a ranking according to fold
change (AB vs. Sham) using a subset of the genes for which
false discovery rate was less than 0.05. All data is MIAME compliant and the
following link has been created to allow review of the data in Gene Expression
Omnibus (record GSE22295): http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=xdivnmkmeyeiifg&acc=GSE22295
Western blotting
Western blotting was performed as previously described [61] with minor modifications.
Snap frozen left ventricles from WT and CXCR5−/− mice
were homogenized in cell lysis buffer and equal amounts of protein being
separated from each sample by SDS-PAGE (10%) before transferred to
polyvinylidene fluoride (PVDF) membranes. Non-specific bindings to the membrane
was blocked with 5% BSA for 1 h at room temperature, followed by
incubation with anti-fibromodulin (SC-33772; Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA), anti-decorin (AF1060; R&D Systems, Minneapolis, MN),
anti-biglycan (AF2667; R&D) or anti-lumican (AF2745; R&D) overnight at
4°C. The membranes were washed in TBS-T and followed by species-specific
horseradish peroxidase-coupled secondary antibodies in 5% BSA added for 1
h. After washing, the immune complexes were visualized by ECL (GE Healthcare,
Buckinghamshire, UK) and the membranes were exposed to x-ray film (HyperfilmTM
ECL, GE healthcare) and developed. Immunoblots were stripped and re-probed with
anti-GAPDH (C20357; R&D) for normalization. Filters with LV lysate were also
probed with antibody against P-ERK1/2 (Phospho-p44/42 MAP kinase (Thr 202/Tyr
204), Cell Signaling Technology, Danvers, MA) or p44/42 MAP kinase (Cell
Signaling Technology) followed by species-specific horseradish
peroxidase-coupled secondary antibodies (Cell Signaling). The immune complexes
were visualized with the use of Supersignal West Pico (Pierce, Rockford, IL) and
exposed films were detected by using Kodak 440 CF imaging station (Boston, MA).
The software Total Laboratory v.1*10 (Phoretix, Newcastle, UK) was used for
quantification.
Transmission electron microscopy
For transmission electron microscopy, hearts (n = 2) from
each group were perfused with 2.0% glutaraldehyde buffered in 0.2 M
cacodylate at pH 7.4 for 15 min. Small blocks (about 3 mm3 in size)
of the LV and septum were taken. The tissues were fixated in cacodylate buffer
for 2 h and washed in the same buffer 3 times. The blocks of tissue were then
transferred to a 1% OsO4 solution for 10 min on ice. After
washing in cacodylate, dehydration was carried out rapidly in graded ethanol
series, followed by embedding in Epon. Sections were cut at a thickness of
60–100 nm and collected on 200 mesh grids, and stained with uranyl acetat
for 7 minutes and lead for 3 minutes. The sections were examined and
photographed in a Tecnai G2 spirit BioTWIN 120 kV, LaB6, Transmission Electron
Microscope with 4k Eagle camera from FEI Company. We obtained micrographs of the
LV septum and the free wall at different magnifications.
TUNEL Staining
TUNEL staining was performed on paraffin-embedded sections using the In
Situ Cell Death Detection kit (Roche Diagnostics) as described
[62].
Briefly, paraffin-embedded (6 µm) sections of mouse hearts were
deparaffinized in xylene, rehydrated, and treated with 0.5% Triton X-100
in 0.1% Na-citrate for 30 min. After several washes with PBS, the
sections were permeabilized with proteinase K (20 µg/ml in TE, pH 8.0) for
30 min at 37°C. Subsequently, the sections were rinsed with PBS, and the
area around the sample was dried. TUNEL reaction mixture (50 µl)
containing terminal deoxynucleotidyl transferase was applied and tissue sections
were incubated in a dark, humidified chamber for 1 h at 37°C. After several
washes with PBS the tissue sections were analyzed with a fluorescence microscope
(515–565 nm). A quantitative analysis (number of apoptotic cells/total
number of cells counted) was performed by counting cells in a randomly selected
area of each tissue sample.
Immunohistochemistry
Paired 3.5 µm sections were immunostained using affinity-purified rabbit
polyclonal CD3 antibody (Abcam, Cambridge, U.K.), dilution 1∶400, and
anti-mouse/human CD45R (eBioscience, San Diego, CA), dilution 1∶4500. The
immune reaction was visualized using horseradish peroxidase in a Dako
Autostainer plus (Dako, Glostrup, Denmark). We obtained 32 digital images of
evenly distributed microscopic high power fields (×400) from the left
ventricular free wall and ventricular septum of six heart sections in each
group. The sections were from no less than three hearts in each group. The 32
images from each heart were studied by two investigators (AW and HMR), blinded
for mouse identity, counting the total number of CD3 and CD45R positive
lymphocytes.
Isolation and stimulation of neonatal myocardial rat fibroblasts
Primary neonatal fibroblasts were isolated from 1–3 day old Wistar rats
(Taconic, Skensved, Denmark). Briefly, fibroblasts were separated by Percoll
density gradient and transferred to plating medium and maintained in culture for
up to 96 hours. The fibroblasts were stimulated with human recombinant CXCL13,
200 ng/ml (R&D Systems, Minneapolis, MN), with or without the ERK1/2
inhibitor (10 µM final concentration UO126 (MEK inhibitor, Promega, WI)),
for 3 and 20 hours before storing cell pellet (mRNA analyses) and cell-free
supernatant (MMP activity) at −80°C until further analyses.
Un-stimulated (control) cells were also given vehicle. The toxicity in cell
cultures was examined routinely for lactate dehydrogenase leakage using a
cytotoxicity detection kit (Roche Applied Science, Mannheim, Germany).
P-ERK1/2 (Phospho-p44/42 MAP kinase) and ERK1/2 (p44/42 MAP kinase)
detection
Phospho-p44/42 MAP kinase and p44/42 MAP kinase levels in left ventricle lysate
were measured by multiplex suspension array technology using the BioPlex
(Bio-Rad, Hercules, CA). Phospho-p44/42 MAP kinase and p44/42 MAP kinase
multiplexable beads were purchased from R&D Systems. The quantification was
accomplished by using the BioPlex Manager Software (Bio-Rad).
Tissue sampling from human myocardium
In nine patients with advanced HF (NYHA class IV; 8 male, 1 female; age
29±5 years), LV tissue was available at the time of implantation and at
the time of removal (heart transplantation) of a continuous-flow LV assist
device (LVAD; EntrAssist, Ventracor Ltd, Chatswood, Australia). Average time on
LVAD was 8±1.7 months. Control (non-failing) human LV tissue was obtained
from subjects whose hearts were rejected as cardiac donors for surgical reasons
(n = 5). The cause of death of donors was cerebrovascular
accident, and none had a history of heart disease. Myocardium from these
subjects was kept on ice for 1 to 4 hours before tissue sampling was conducted.
In both failing and non-failing myocardium, LV tissue samples were snap-frozen
in liquid nitrogen, and stored at −80°C until use. None of patients
(failing and non-failing myocardium) had significant concomitant disease such as
infection, malignancy, or autoimmune disorder.
Supporting Information
Altered expression of markers of cardiac wall stress and
remodeling. Relative gene expression of (A) atrial
natriuretic peptide (ANP), (B) brain natriuretic peptide (BNP)
and (C) β-myosin heavy chain (MHC) in wild type (WT) Sham
(n = 6), CXCR5-/- Sham
(n = 6), WT aorta banded (AB)
(n = 6), and CXCR5-/- AB
(n = 6) groups. The results are mean ± SEM.
*p<0.05 and ***p<0.001 vs. Sham
groups; †p<0.05 and †††p <0.001
vs. WT AB group.
(TIF)
Fluorescent micrographs of sections of left ventricular myocardium from
wild type (WT) and CXCR5-/- mice. The arrows indicate
TUNEL-positive myocyte nucleus.
(TIF)
CD45 and CD3 positive lymphocytes in the left ventricular myocardium
from wild type (WT) and CXCR5-/- mice. Total number of
CD45R (A) and CD3 (B) postive lymphocytes was not
significantly different between CXCR5-/- and WT mice after sham
operation or AB. (n = 6 heart sections in all groups).
Cells counted from 32 digital, evenly distributed images (x400) from each
heart.The results are mean ± SEM.
(TIF)
Weights and echocardiographic measurements. Values are means
± SE. BW, body weight; TL, tibia length; LVW, left ventricular
weight; LW, lung weight; IVSd and IVSs, interventricular septum thickness in
diastole and in systole, respectively; LVDd and LVDs,
left ventricular diameter in diastole and in systole, respectively; FS,
fractional shortening in LVD; LVPWd and LVPWs, posterior wall thickness in
diastole and in systole, respectively; LAD, left atrial diameter; HR, heart
rate; AVmax, peak aortic stenosis flow velocity; TVs, peak tissue velocity
in systole; TVd, peak tissue velocity in diastole. LVW/TL and LW/TL are
mg/mm. * p<0.05, ** p<0.01, *** p<0.001 vs.
Non-operated and Sham groups. †p<0.05, ††, p
<0.01, †††p<0.001 vs. WT AB group.
‡p<0.05 vs CXCR5-/- Non-operated group. The results are
mean ± SEM.
(DOC)
Characteristics of the real-time PCR assays used in the human
study. The table shows the sequence of primers used in the
real-time PCR assays. (+), forward primers; (–), reverse primers;
Acc.nr, GenBank accession number; GAPDH, glyceraldehyde 3-phosphate
dehydrogenase.
(DOC)
We thank Bjørg Austbø, Dina Behmen, Sigurd Boye, Ulla Enger, Geir
Florholmen, Eli Wallem Gulliksen, Tove Noren, Ole Kristoffer Olstad, Anett
Hellebø Ottesen, Ellen Lund Sagen and Kalpana Sinnadurai for excellent
assistance.
ReferencesAukrustPUelandTLienEBendtzenKMüllerF1999Cytokine network in congestive heart failure secondary to
ischemic or idiopathic dilated cardiomyopathy.83376382Torre-AmioneGKapadiaSLeeJDurandJBBiesRD1996Tumor necrosis factor-alpha and tumor necrosis factor receptors
in the failing human heart.93704711DamåsJKGullestadLAassHSimonsenSFjeldJG2001Enhanced gene expression of chemokines and their corresponding
receptors in mononuclear blood cells in chronic heart
failure—modulatory effect of intravenous
immunoglobulin.38187193WoldbækPRSandeJBStrømmeTALundePKDjurovicS2005Daily administration of interleukin-18 causes myocardial
dysfunction in healthy mice.289H708H714HusbergCNygardSFinsenAVDamåsJKFrigessiA2008Cytokine expression profiling of the myocardium reveals a role
for CX3CL1 (fractalkine) in heart failure.45261269MannDL2001Recent insights into the role of tumor necrosis factor in the
failing heart.67180MatsumoriASasayamaS2001The role of inflammatory mediators in the failing heart:
immunomodulation of cytokines in experimental models of heart
failure.6129136DibbsZKurrelmeyerKKalraDSetaYWangF1999Cytokines in heart failure: pathogenetic mechanisms and potential
treatment.111423428FörsterREmrichTKremmerELippM1994Expression of the G-protein—coupled receptor BLR1 defines
mature, recirculating B cells and a subset of T-helper memory
cells.84830840FörsterRDavalos-MisslitzACRotA2008CCR7 and its ligands: balancing immunity and
tolerance.8362371MüllerGHöpkenUELippM2003The impact of CCR7 and CXCR5 on lymphoid organ development and
systemic immunity.195117135OhlLHenningGKrautwaldSLippMHardtkeS2003Cooperating mechanisms of CXCR5 and CCR7 in development and
organization of secondary lymphoid organs.19711991204EbertLMSchaerliPMoserB2005Chemokine-mediated control of T cell traffic in lymphoid and
peripheral tissues.42799809FörsterRMattisAEKremmerEWolfEBremG1996A putative chemokine receptor, BLR1, directs B cell migration to
defined lymphoid organs and specific anatomic compartments of the
spleen.8710371047CysterJGAnselKMReifKEklandEHHymanPL2000Follicular stromal cells and lymphocyte homing to
follicles.176181193GunnMDNgoVNAnselKMEklandEHCysterJG1998A B-cell-homing chemokine made in lymphoid follicles activates
Burkitt's lymphoma receptor-1.391799803WeyandCMGoronzyJJ2003Ectopic germinal center formation in rheumatoid
synovitis.987140149AnselKMHeyzer-WilliamsLJNgoVNHeyzer-WilliamsMGCysterJG1999In vivo-activated CD4 T cells upregulate CXC chemokine receptor 5
and reprogram their response to lymphoid chemokines.19011231134HaynesNMAllenCDLesleyRAnselKMKilleenN2007Role of CXCR5 and CCR7 in follicular Th cell positioning and
appearance of a programmed cell death gene-1high germinal center-associated
subpopulation.17950995108SchmutzCHulmeABurmanASalmonMAshtonB2005Chemokine receptors in the rheumatoid synovium: upregulation of
CXCR5.7R217R229QiupingZJieXYouxinJQunWWeiJ2005Selectively frequent expression of CXCR5 enhances resistance to
apoptosis in CD8(+)CD34(+) T cells from patients with
T-cell-lineage acute lymphocytic leukemia.24573584ShiKHayashidaKKanekoMHashimotoJTomitaT2001Lymphoid chemokine B cell-attracting chemokine-1 (CXCL13) is
expressed in germinal center of ectopic lymphoid follicles within the
synovium of chronic arthritis patients.166650655AmftNCurnowSJScheel-ToellnerDDevadasAOatesJ2001Ectopic expression of the B cell-attracting chemokine BCA-1
(CXCL13) on endothelial cells and within lymphoid follicles contributes to
the establishment of germinal center-like structures in Sjogren's
syndrome.4426332641SalomonssonSLarssonPTengnerPMellquistEHjelmströmP2002Expression of the B cell-attracting chemokine CXCL13 in the
target organ and autoantibody production in ectopic lymphoid tissue in the
chronic inflammatory disease Sjogren's syndrome.55336342XanthouGPolihronisMTzioufasAGPaikosSSiderasP2001“Lymphoid” chemokine messenger RNA expression by
epithelial cells in the chronic inflammatory lesion of the salivary glands
of Sjogren's syndrome patients: possible participation in lymphoid
structure formation.44408418CarlsenHSBaekkevoldESJohansenFEHaraldsenGBrandtzaegP2002B cell attracting chemokine 1 (CXCL13) and its receptor CXCR5 are
expressed in normal and aberrant gut associated lymphoid
tissue.51364371FestaEDHankiewiczKKimSSkurnickJWolanskyLJ2009Serum levels of CXCL13 are elevated in active multiple
sclerosis.Mult SclerDobnerTWolfIEmrichTLippM1992Differentiation-specific expression of a novel G protein-coupled
receptor from Burkitt's lymphoma.2227952799MeijerJZeelenbergISSiposBRoosE2006The CXCR5 chemokine receptor is expressed by carcinoma cells and
promotes growth of colon carcinoma in the liver.6695769582SinghSSinghRSinghUPRaiSNNovakovicKR2009Clinical and biological significance of CXCR5 expressed by
prostate cancer specimens and cell lines.12522882295TyagiSC1998Extracellular matrix dynamics in heart failure: a prospect for
gene therapy.68403410DjebbariAQuackenbushJ2008Seeded Bayesian Networks: constructing genetic networks from
microarray data.257HockingAMShinomuraTMcQuillanDJ1998Leucine-rich repeat glycoproteins of the extracellular
matrix.17119SvenssonLAszodiAReinholtFPFasslerRHeinegardD1999Fibromodulin-null mice have abnormal collagen fibrils, tissue
organization, and altered lumican deposition in tendon.27496369647DanielsonKGBaribaultHHolmesDFGrahamHKadlerKE1997Targeted disruption of decorin leads to abnormal collagen fibril
morphology and skin fragility.136729743HuCXiongJZhangLHuangBZhangQ2004PEG10 activation by co-stimulation of CXCR5 and CCR7 essentially
contributes to resistance to apoptosis in CD19+CD34+ B cells from
patients with B cell lineage acute and chronic lymphocytic
leukemia.1280294ChunsongHYulingHLiWJieXGangZ2006CXC chemokine ligand 13 and CC chemokine ligand 19 cooperatively
render resistance to apoptosis in B cell lineage acute and chronic
lymphocytic leukemia CD23+CD5+ B cells.17767136722LeglerDFLoetscherMRoosRSClark-LewisIBaggioliniM1998B cell-attracting chemokine 1, a human CXC chemokine expressed in
lymphoid tissues, selectively attracts B lymphocytes via
BLR1/CXCR5.187655660MoserBSchaerliPLoetscherP2002CXCR5(+) T cells: follicular homing takes center stage in
T-helper-cell responses.23250254YamazakiTKomuroIYazakiY1998Signalling pathways for cardiac hypertrophy.10693698MüllerGLippM2001Signal transduction by the chemokine receptor CXCR5: structural
requirements for G protein activation analyzed by chimeric CXCR1/CXCR5
molecules.38213871397DamåsJKEikenHGOieEBjerkeliVYndestadA2000Myocardial expression of CC- and CXC-chemokines and their
receptors in human end-stage heart failure.47778787DahlCPHusbergCGullestadLWaehreADamåsJK2009Increased production of CXCL16 in experimental and clinical heart
failure: a possible role in extracellular matrix remodeling.2624632MonfortJTardifGReboulPMineauFRoughleyP2006Degradation of small leucine-rich repeat proteoglycans by matrix
metalloprotease-13: identification of a new biglycan cleavage
site.8R26MorimotoHTakahashiMIzawaAIseHHongoM2006Cardiac overexpression of monocyte chemoattractant protein-1 in
transgenic mice prevents cardiac dysfunction and remodeling after myocardial
infarction.99891899TachibanaKHirotaSIizasaHYoshidaHKawabataK1998The chemokine receptor CXCR4 is essential for vascularization of
the gastrointestinal tract.393591594DoiMKusachiSMurakamiTNinomiyaYMurakamiM2000Time-dependent changes of decorin in the infarct zone after
experimentally induced myocardial infarction in rats: comparison with
biglycan.1962333YamamotoKKusachiSNinomiyaYMurakamiMDoiM1998Increase in the expression of biglycan mRNA expression
Co-localized closely with that of type I collagen mRNA in the infarct zone
after experimentally-induced myocardial infarction in rats.3017491756WeisSMZimmermanSDShahMCovellJWOmensJH2005A role for decorin in the remodeling of myocardial
infarction.24313324WestermannDMersmannJMelchiorAFreudenbergerTPetrikC2008Biglycan is required for adaptive remodeling after myocardial
infarction.11712691276BuenoOFDe WindtLJTymitzKMWittSAKimballTR2000The MEK1-ERK1/2 signaling pathway promotes compensated cardiac
hypertrophy in transgenic mice.1963416350SannaBBuenoOFDaiYSWilkinsBJMolkentinJD2005Direct and indirect interactions between calcineurin-NFAT and
MEK1-extracellular signal-regulated kinase 1/2 signaling pathways regulate
cardiac gene expression and cellular growth.25865878DingBPriceRLBorgTKWeinbergEOHalloranPF1999Pressure overload induces severe hypertrophy in mice treated with
cyclosporine, an inhibitor of calcineurin.84729734SjaastadISejerstedOMIlebekkABjørnerheimR2000Echocardiographic criteria for detection of postinfarction
congestive heart failure in rats.8914451454FinsenAVChristensenGSjaastadI2005Echocardiographic parameters discriminating myocardial infarction
with pulmonary congestion from myocardial infarction without congestion in
the mouse.98680689VingeLEvon LuederTGAasumEQvigstadEGravningJA2008Cardiac-restricted expression of the carboxyl-terminal fragment
of GRK3 uncovers distinct functions of GRK3 in regulation of cardiac
contractility and growth: GRK3 controls cardiac alpha1-adrenergic receptor
responsiveness.2831060110610LiuHSanuda-PenaMCHarvey-WhiteJDKalraSCohenSA1998Determination of submicromolar concentrations of neurotransmitter
amino acids by fluorescence detection using a modification of the
6-aminoquinolyl-N-hydroxysuccinimidyl carbamate method for amino acid
analysis.828383395LaurentGJCockerillPMcAnultyRJHastingsJR1981A simplified method for quantitation of the relative amounts of
type I and type III collagen in small tissue samples.113301312IrizarryRAHobbsBCollinFBeazer-BarclayYDAntonellisKJ2003Exploration, normalization, and summaries of high density
oligonucleotide array probe level data.4249264TusherVGTibshiraniRChuG2001Significance analysis of microarrays applied to the ionizing
radiation response.9851165121FinsenAVWoldbækPRLiJWuJLybergT2004Increased syndecan expression following myocardial infarction
indicates a role in cardiac remodeling.16301308CalvertJWZhouCNandaAZhangJH2003Effect of hyperbaric oxygen on apoptosis in neonatal
hypoxia-ischemia rat model.9520722080