Download Secreted protein acidic and rich in cysteine facilitates age

Am J Physiol Cell Physiol 308: C972–C982, 2015.
First published April 15, 2015; doi:10.1152/ajpcell.00402.2014.
CALL FOR PAPERS
Cellular Mechanisms of Tissue Fibrosis
Secreted protein acidic and rich in cysteine facilitates age-related cardiac
inflammation and macrophage M1 polarization
Hiroe Toba,1,2 Lisandra E. de Castro Brás,1,3 Catalin F. Baicu,4 Michael R. Zile,4,5 Merry L. Lindsey,1,6
and Amy D. Bradshaw4,5
1
Submitted 17 December 2014; accepted in final form 12 April 2015
Toba H, de Castro Brás LE, Baicu CF, Zile MR, Lindsey ML,
Bradshaw AD. Secreted protein acidic and rich in cysteine facilitates
age-related cardiac inflammation and macrophage M1 polarization.
Am J Physiol Cell Physiol 308: C972–C982, 2015. First published
April 15, 2015; doi:10.1152/ajpcell.00402.2014.—To investigate the
role of secreted protein acidic and rich in cysteine (SPARC) in
age-related cardiac inflammation, we studied six groups of mice:
young (3–5 mo old), middle-aged (10 –12 mo old), and old (18 –29 mo
old) C57BL/6 wild-type (WT) and SPARC-null (Null) mice (n ⫽
7–10/group). Cardiac function and structure were determined by
echocardiography. The left ventricle was used for cytokine gene array
and macrophage quantification by immunohistochemistry. Macrophage infiltration increased with age in WT (n ⫽ 5– 6/group, P ⬍ 0.05
for young vs. old), but not in Null. Proinflammatory markers (Ccl5,
Cx3cl1, Ccr2, and Cxcr3) increased in middle-aged and old WT,
whereas they were increased only in old Null compared with respective young (n ⫽ 5– 6/group, P ⬍ 0.05 for all). These results suggest
that SPARC deletion delayed age-related cardiac inflammation. To
further assess how SPARC affects inflammation, we stimulated peritoneal macrophages with SPARC (n ⫽ 4). SPARC treatment increased expression of proinflammatory macrophage M1 markers and
decreased anti-inflammatory M2 markers. Echocardiography (n ⫽
7–10/group) revealed an age-related increase in wall thickness of the
left ventricle in WT (0.76 ⫾ 0.02 mm in young vs. 0.91 ⫾ 0.03 mm
in old; P ⬍ 0.05) but not in Null (0.78 ⫾ 0.01 mm in young vs. 0.84
⫾ 0.02 mm in old). In conclusion, SPARC deletion delayed age-related
increases in macrophage infiltration and proinflammatory cytokine expression in vivo and in vitro. SPARC acts as an important mediator of
age-related cardiac inflammation by increasing the expression of
macrophage M1 markers and decreasing M2 markers.
secreted protein acidic and rich in cysteine; aging; heart; inflammation; macrophage
a leading cause of death in the
elderly (10). While advanced age increases the chance of
exposure to cardiovascular risk factors, accumulating studies
show that aging itself is an independent risk factor for cardioCARDIOVASCULAR DISEASE IS
Address for reprint requests and other correspondence: A. D. Bradshaw,
Gazes Cardiac Research Institute, Div. of Cardiology, Dept. of Medicine,
Medical Univ. of South Carolina; 171 Ashley Ave., Charleston, SC 29425
(e-mail: [email protected]).
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vascular disease (18, 29). With advancing age, the left ventricle
(LV) develops concentric remodeling and diastolic dysfunction, which is associated with myocyte loss and an increase in
extracellular matrix (ECM) proteins, such as fibrillar collagen
(18, 30, 38). In addition to ECM accumulation, inflammation is
a key component of cardiac aging (13, 48). Higher levels of
circulating proinflammatory cytokines are observed in elderly
people, even in the absence of chronic disease (3, 8, 16, 50). In
old mice, the cardiac expression of inflammatory genes increases
compared with that in young mice (56). Cardiac inflammation
triggers the release of fibrogenic cytokines and growth factors,
leading to excessive accumulation of fibrillar collagens and adverse remodeling that, in turn, may cause cardiac dysfunction (51,
54). Understanding the mechanisms of age-related inflammation
in the heart is an important step to prevent or delay cardiac aging
and improve quality of life in the elderly.
Procollagen, biosynthesized by the fibroblast, is secreted
into the extracellular space and processed to become a mature
insoluble collagen fibril (41). Secreted protein acidic and rich
in cysteine (SPARC), a collagen-binding matricellular protein,
is involved in procollagen processing and promotes the formation of cross-linked collagen fibrils (4, 45). The expression of
SPARC is observed primarily in cardiac fibroblasts, but is also
expressed at lower levels by endothelial cells, cardiomyocytes,
and macrophages. Expression of SPARC is particularly induced during morphogenesis and tissue remodeling (7).
SPARC-null mice show less age-related increases in cardiac
stiffness and collagen content than wild-type (WT) mice (5).
SPARC also participates in various biological processes, such
as cellular adhesion, proliferation, and migration by regulating
cell-matrix interactions, growth factor signaling, and ECM
assembly (6, 7).
Previous reports have demonstrated a role for SPARC in
inflammation. Compared with WT mice, young SPARC-null
mice exhibit less renal inflammation and fibrosis induced by
angiotensin II infusion, as well as less inflammation in response to lipopolysaccharide (LPS)-induced inflammation in a
footpad model (44, 49). Similarly, SPARC deletion decreases
colonic inflammatory symptoms in dextran sodium sulfateinduced murine colitis (39). The function of SPARC in reguhttp://www.ajpcell.org
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Mississippi Center for Heart Research and San Antonio Cardiovascular Proteomics Center, Department of Physiology and
Biophysics, University of Mississippi Medical Center, Jackson, Mississippi; 2Department of Clinical Pharmacology, Division
of Pathological Sciences, Kyoto Pharmaceutical University, Kyoto, Japan; 3Department of Physiology, East Carolina
University, Greenville, North Carolina; 4Gazes Cardiac Research Institute, Division of Cardiology, Department of Medicine,
Medical University of South Carolina, Charleston, South Carolina; 5Ralph H. Johnson Department of Veterans Affairs
Medical Center, Charleston, South Carolina, and 6G. V. (Sonny) Montgomery Veterans Affairs Medical Center,
Jackson, Mississippi
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SPARC MEDIATES CARDIAC INFLAMMATION
A
young WT
middle-aged WT
100 µm
old WT
100 µm
old Null
100 µm
100 µm
p<0.05
B
p<0.05
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0.10
0.05
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Fig. 1. Secreted protein acidic and rich in cysteine (SPARC) protein expression in the left ventricle (LV) increased with age. A: representative images of
SPARC-positive cells in the LVs of young, middle-aged, and old wild-type (WT) mice. Old SPARC-null mice served as the negative control. B: percentage of
SPARC-positive area per total area showing age-dependent increase in SPARC in WT mice. Values are means ⫾ SE (n ⫽ 5– 6/group).
lating inflammatory processes likely depends on cellular origin
of SPARC expression. Sangaletti et al. (47) reported distinct
effects on lung inflammation dependent on the expression of
SPARC by bone marrow versus stromal cells. Plasma levels of
SPARC positively correlate with hypersensitive C-reactive
protein and white blood cell numbers in gestational diabetes
mellitus (55). These reports support the idea that SPARC may
be a key factor to promote inflammation. Since animal models
exhibit increased SPARC levels in aged hearts (5, 21), it is
possible that SPARC plays an important role in age-related
cardiac inflammation. However, up to date, there are no studies
investigating the role of SPARC in cardiac age-related inflammation. Accordingly, the aim of this study was to investigate if
SPARC deletion may delay age-related cardiac inflammation.
MATERIALS AND METHODS
Animals. Mice colonies were maintained at the Medical University
of South Carolina (MUSC) animal care facility. All procedures were
performed in strict accordance with the Guide for the Care and Use of
Laboratory Animals (National Research Council, The National Academies Press, Washington, DC, 2011) and were approved by the
Institutional Animal Care and Use Committee at MUSC (approval ID:
ACORP 511). We used C57BL/6 WT and SPARC-null (Null) mice to
study age and genotype differences in the LV. Null mice were
maintained on a C57BL/6 strain (⬎12 back-crossed generations). WT
and Null mice were bred as homozygous breeding pairs and housed
under identical conditions since birth in the same room and fed
identical diets at MUSC. Environmental factors that might potentially
influence phenotype with age were minimized. Three age groups were
Table 1. Body weight and echocardiography results
WT
BW, g
LVEDV, ␮l
LVESV, ␮l
LVEF, %
LVSV, ␮l
Mean wall thickness, mm
Relative wall thickness
Null
Young
Middle-aged
Old
Young
Middle-aged
Old
26 ⫾ 1
60 ⫾ 3
25 ⫾ 2
59 ⫾ 2
35 ⫾ 2
0.76 ⫾ 0.02
0.40 ⫾ 0.01
35 ⫾ 2*
65 ⫾ 4
30 ⫾ 4
55 ⫾ 4
35 ⫾ 2
0.81 ⫾ 0.03
0.42 ⫾ 0.02
44 ⫾ 2*‡
61 ⫾ 4
24 ⫾ 3
61 ⫾ 3
37 ⫾ 3
0.91 ⫾ 0.03*‡
0.48 ⫾ 0.03*‡
26 ⫾ 1
57 ⫾ 3
24 ⫾ 1
58 ⫾ 2
33 ⫾ 2
0.78 ⫾ 0.01
0.42 ⫾ 0.01
30 ⫾ 2
59 ⫾ 3
26 ⫾ 1
56 ⫾ 2
33 ⫾ 2
0.77 ⫾ 0.04
0.40 ⫾ 0.01
32 ⫾ 2*†
66 ⫾ 4
29 ⫾ 3
56 ⫾ 4
37 ⫾ 3
0.84 ⫾ 0.02
0.42 ⫾ 0.01
Values are means ⫾ SE (n ⫽ 7–10/group). WT, wild-type; BW, body weight; LV, left ventricle; EDV, end-diastolic volume; ESV, end-systolic volume; EF,
ejection fraction; SV, stroke volume; mean wall thickness, the average of the interventricular septal wall thickness and LV posterior wall thickness; relative wall
thickness, the mean LV wall thickness divided by the LV internal dimension at end diastole. *P ⬍ 0.05 vs. respective young. ‡P ⬍ 0.05 vs. respective
middle-aged. †P ⬍ 0.05 vs. age-matched WT.
AJP-Cell Physiol • doi:10.1152/ajpcell.00402.2014 • www.ajpcell.org
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SPARC (% area)
0.20
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SPARC MEDIATES CARDIAC INFLAMMATION
studied: young (3–5 mo old), middle-aged (10 –12 mo old), and old
(18 –29 mo old), and both male and female mice were included in
each group (n ⫽ 7–10/age/genotype). The generation and phenotype of Null mice have been previously described by Norose and
colleagues (40).
Echocardiography and necropsy. Echocardiographic measurements were performed to examine LV structure and function, using a
Cdkn2a
Cdkn2a mRNA
8
p<0.05
p<0.05
6
4
2
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Null
Fig. 3. The cellular senescent marker, cyclin-dependent kinase inhibitor 2A
(Cdkn2a), increased in the LV with age. Cdkn2a expression was similarly
increased with age in the LVs of both WT (solid bars) and SPARC-null
(open bars) mice. Expression was normalized to hypoxanthine-guanine
phosphoribosyltransferase (Hprt1) levels and is shown as 2⫺⌬Ct ⫻ 100
units, where ⌬Ct is change in threshold cycle. Values are means ⫾ SE
(n ⫽ 5– 6/group).
40-MHz mechanical scanning transducer (707B) and a Vevo 770
echocardiograph (VisualSonics, Toronto, Canada), as described previously (58). LV volumes, ejection fraction, and wall thickness were
measured and calculated using the American Society of Echocardiography recommendations for chamber quantification (31). Animals
were anesthetized with isoflurane and hearts were excised. The LV
was separated from the right ventricle and divided into two equal
pieces. One piece was snap frozen and used for RNA extraction, and
the rest of the LV was fixed in zinc formalin and used for histology.
RNA extraction and quantitative real-time RT-PCR. Total RNA
was isolated using the TRIzol reagent (Invitrogen), and cDNA was
synthesized using the RT2 First Strand Kit (Qiagen, 330401). To
assess mRNA expression of cytokine molecules, quantitative realtime RT-PCR was performed using RT2 SYBR green Rox qPCR
Master Mix (Qiagen, 330523) and 96-well cytokine Array plates
(Qiagen, PAMM-011E). For cyclin-dependent kinase inhibitor 2A
(Cdkn2a), chemokine (C-X3-C motif) ligand 1 (Cx3cl1), chemokine
(C-X-C motif) ligand 5 (Cxcl5), interleukin-6 (IL-6), tumor necrosis
factor-␣ (Tnf␣), and Fizz-1 expression, Taqman gene expression
assays were performed using specific primers (Mm00494449_m1,
Mm00436454_m1, Mm00436451_g1, Mm00446190_m1, Mm00443260_
g1, and Mm00445109_m1; Applied Biosytems). The relative gene
expression of individual target molecules was calculated by normalization of the threshold cycle (Ct) values of the target genes to the Ct
values of the housekeeping gene hypoxanthine-guanine phosphoribosyltransferase 1 (Hprt1), which did not change in expression in our
samples. Hprt1 has been identified as the most stable housekeeping
gene during the aging process (11).
Histology. Paraffin-embedded LV sections were deparaffinized in
Citric-Solv (Fisherbrand), rehydrated, and then boiled for 15 min in
Antigen Retrieval Solution (Dako). The sections were stained with
hematoxylin and eosin or incubated in 3% hydrogen peroxide to
quench endogenous peroxide activity for immunohistochemistry anal-
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Fig. 2. SPARC deletion decreased age-related myocyte hypertrophy. A: representative images of hematoxylin-eosin-stained sections from the LVs of young,
middle-aged, and old WT and SPARC-null mice.
Scales indicate 50 ␮m. B: myocyte cross-sectional
areas were quantified by measuring the area of five
random cells per section. Arbitrary units show agedependent increase in myocyte area in WT, but not in
SPARC-null mice. Values are means ⫾ SE (n ⫽
5– 6/group). *P ⬍ 0.05 vs. age-matched WT.
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SPARC MEDIATES CARDIAC INFLAMMATION
Ccr2
Cx3cl1
p<0.05
p<0.05
p<0.05
25
10
p<0.05
p<0.05
100
p<0.05
0.4
0.2
0.5
WT
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p<0.05
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6
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2
0
0.0
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1.0
Tnfrsf1b mRNA
Ccl4 mRNA
0.6
p<0.05
10
1.5
p<0.05
0.8
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Tnfrsf1b
Ccl4
1.0
1
0
0
Cxcl5
2
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200
3
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300
m
Mif mRNA
0.6
*
p<0.05
Ccr1 mRNA
p<0.05
0.8
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p<0.05
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Cxcl5 mRNA
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1.0
1
Ccr1
Mif
p<0.05
2
Null
Fig. 4. Age-related changes in the expression of proinflammatory cytokines differed in WT vs. SPARC-null myocardium. Age-dependent increases in cardiac
expression of chemokine (C-C motif) ligand 5 (Ccl5), chemokine (C-X-C motif) ligand 1 (Cxcl1), C-C chemokine receptor type 2 (Ccr2), and chemokine (C-X-C
motif) receptor 3 (Cxcr3) were delayed in SPARC-null (open bars) mice compared with WT (solid bars). Migration inhibitory factor (Mif) was increased with
age in both WT and SPARC-null mice. C-C chemokine receptor type 1 (Ccr1) was decreased with age in WT mice. chemokine (C-X-C motif) ligand 5 (Cxcl5),
chemokine (C-C motif) ligand 4 (Ccl4), and tumor necrosis factor receptor superfamily member 1b (Tnfrsf1b) were increased with age in SPARC-null mice. Each
gene expression was normalized to Hprt1 and shown as 2⫺⌬Ct ⫻ 100 units. Values are means ⫾ SE (n ⫽ 5– 6/group). *P ⬍ 0.05 vs. age-matched WT.
ysis. A rat anti-mouse Mac-3 antibody (Cedarlane CL8943AP at
1:100 dilution) and a goat anti-mouse SPARC antibody (R&D AF942
at 1:100 dilution) were used to detect macrophages and SPARC,
respectively, by the avidin-biotin complex method using the 3,3=diaminobenzidine chromogen with eosin counterstain. Five images
were acquired randomly from each sample, and the dimensions of five
myocytes were measured using Image Pro Analyzer 7.0 (Media
Cybernetics). Macrophage numbers and SPARC expression were
determined by Mac-3- and SPARC-positive area per total stained
(Image-Pro Analyzer 7.0).
Peritoneal macrophage isolation and SPARC treatment. Peritoneal
macrophages were isolated from young male WT, as described
previously (57). After overnight incubation at 37°C, the cells were
treated with or without recombinant mouse SPARC (20 ␮g/ml,
R&D, lot: DJW0314031) in serum-free medium for 24 h. The
medium was removed, and cells were washed with PBS and lysed
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Cxcr3
*
3
0
0
0
Ccxr3 mRNA
5
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p<0.05
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Ccr2 mRNA
Cx3cl1 mRNA
Ccl5 mRNA
p<0.05
6
p<0.05
5
p<0.05
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p<0.05
8
p<0.05
p<0.05
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Ccl5
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SPARC MEDIATES CARDIAC INFLAMMATION
RESULTS
SPARC expression increased with age in LV. Our laboratory
previously reported that old mice have greater levels of myocardial SPARC expression than young mice by immunoblot
analysis (5). To confirm age-dependent increases in SPARC in
this study, we performed immunohistochemical analysis of the
LV. In accordance with our previous data, SPARC protein
expression significantly increased in the old WT LV compared
with both young and middle-aged WT LV (Fig. 1). These
results support the concept that aging is associated with increased SPARC expression in the LV.
SPARC deletion suppressed the age-dependent increase in
wall thickness. Previously, our laboratory reported that senescent WT mice (31.8 ⫾ 0.4 mo old) exhibit LV dilation and
decreased ejection fraction (32). In the present study, echocardiographic analysis showed no significant differences in enddiastolic volume or end-systolic volume among the groups.
Our data support the concept that LV dilation generally occurs
after 29 mo of age for healthy C57BL/6 mice. There were no
significant differences in ejection fraction among the groups,
indicating that myocyte contractility and global LV function
are still preserved at the ages evaluated. Mean and relative wall
thicknesses were increased in an age-related manner in WT,
but not in Null (Table 1, n ⫽ 7–10/group), indicating that the
WT mice had age-associated LV hypertrophy.
SPARC deletion diminished age-related myocyte hypertrophy.
Myocyte hypertrophy develops with advancing age (32, 56).
We performed hematoxylin-eosin staining and measured individual myocyte sizes for each group. Myocyte size was increased in the middle-aged and old WT mice compared with
young WT (Fig. 2). This age-dependent increase in myocyte
size was blunted in Null mice. Our results indicated that
SPARC promoted age-related myocyte hypertrophy.
Cdkn2a, a biomarker of cellular senescence, was increased
in old mice. Cdkn2a has been used widely as a marker of
mammalian aging (26). Cdkn2a mRNA was significantly increased in old mice compared with young, for both WT and
Null (Fig. 3). These results indicate that Cdkn2a is a useful
marker of cardiac aging.
SPARC deletion affected the age-dependent changes in the
expression of proinflammatory cytokines. Inflammatory cytokines are important contributors to cardiac aging (12, 56). To
assess whether SPARC mediates cardiac inflammation, we
measured the LV expression of inflammatory cytokines and
receptors by gene array (Fig. 4). Of the 84 genes measured, 9
showed differences between age or genotype. The levels of
Ccl5, Cx3cl1, C-C chemokine receptor type 2 (Ccr2) and
chemokine (C-X-C motif) receptor 3 (Cxcr3) were significantly increased in middle-aged and old WT compared with
young WT (P ⬍ 0.05 for all). Ccl5, Cx3cl1, Ccr2, and Cxcr3
demonstrated increases in middle-age over young myocardium, while SPARC expression was found to increase in old
myocardium only. This result suggested that 1) proinflammatory cytokines might increase SPARC expression, and 2)
age-dependent changes in cytokine profiles might be induced
by both SPARC-dependent and -independent factors. In the
absence of SPARC, only old mice showed a greater expression
of these molecules compared with young mice. We validated
the gene array results using a specific TaqMan gene expression
assay for Cx3cl1 and confirmed the results from gene array
(Fig. 5, left). These results suggest that SPARC deletion
delayed age-dependent increases in the expression of these
proinflammatory cytokines.
p<0.05
Cx3cl1
Cxcl5
p<0.05
p<0.05
p<0.05
0.15
*
p<0.05
Cxcl5 mRNA
0.6
0.4
0.2
0.0
*
0.10
0.05
WT
Null
AJP-Cell Physiol • doi:10.1152/ajpcell.00402.2014 • www.ajpcell.org
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Fig. 5. SPARC deletion shifted the inflammatory phenotype in the aging heart. Agerelated increase in Cx3cl1 was delayed in
SPARC-null (open bars) mice compared
with WT (solid bars). Cxcl5 was increased
with age in SPARC-null mice. Each gene
expression was normalized to Hprt1 and
shown as 2⫺⌬Ct ⫻ 100 units. Values are
means ⫾ SE (n ⫽ 5– 6/group). *P ⬍ 0.05
vs. age-matched WT.
Cx3cl1 mRNA
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directly in the plate for RNA extraction with TRIzol. All cell
culture experiments were performed under a sterile tissue culture
hood to avoid contamination. The cDNA was synthesized using a
high-capacity RNA to cDNA kit (ABI, 4387406). Taqman gene
expression assays (Applied Biosystems) were used to perform
quantitative real-time RT-PCR for chemokine (C-C motif) ligand 5
(Ccl5; Mm01302427_m1), chemokine (C-C motif) ligand 3 (Ccl3;
Mm00441259_g1), Tnf␣ (Mm00443260_g1), IL-12 (Mm00434174_
m1), IL-6 (Mm00446190_m1), arginase 1 (Arg1; Mm00475988_m1),
and mannose receptor C type 1 (Mrc1; Mm00485148_m1). Gene
levels were normalized to Hprt1 (Mm01545399_m1). SPARC treatment had no effect on the expression of IL-1␤ (P ⫽ 0.73), which
confirmed that changes in cytokine synthesis driven by SPARC were
not due to LPS contamination.
Statistics. Data are expressed as means ⫾ SE. Genotype comparisons of WT and Null across ages were performed using two-way
ANOVA followed by the Bonferroni multiple test. One-way ANOVA
followed by the Student Newman-Keuls post hoc test was used for
comparisons among young, middle-aged, and old mice. Two group
comparisons of peritoneal macrophages were performed using paired
t-test. A P ⬍ 0.05 was considered significant.
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SPARC MEDIATES CARDIAC INFLAMMATION
array data for the Cxcl5 gene levels. There were no significant
differences in Cxcl5 among the three age groups in WT, but
there was an age-dependent increase in the Cxcl5 gene levels in
the Null mice (Fig. 5, right). Hence, an increase in Cxcl5, Ccl4,
and Tnfrsf1b might compensate for a delay in other chemokine/
receptors, including Ccl5, Cx3cl1, Ccr2, and Cxcr3.
Macrophage infiltration was suppressed in Null mice. Proinflammatory cytokines recruit macrophages, and macrophages release a variety of proinflammatory cytokines,
which lead to a repetitive cycle of inflammation. To test if
SPARC deletion could suppress macrophage infiltration, we
quantified the expression of the macrophage marker, Mac-3.
Levels of Mac-3 were enhanced with age in WT; however,
SPARC deletion abolished age-dependent increase in macrophage numbers (Fig. 6). Our data suggest that SPARC
A
young
middle-aged
old
WT
Null
B
0.06
p<0.05
mac-3 (% area)
WT
Null
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0.02
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Fig. 6. SPARC deletion inhibited age-related increase in macrophage infiltration. A: representative images of Mac-3-positive cells in the LV of young,
middle-aged, and old WT and SPARC-null mice. B: percentage of Mac-3-positive area per total area showing age-dependent increase in macrophage infiltration
in WT, but not in SPARC-null mice. Values are means ⫾ SE (n ⫽ 5– 6/group).
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In contrast, other inflammatory cytokines exhibited altered
patterns of age and genotype-dependent expression. Expression of macrophage migration inhibitory factor (Mif) was
increased in middle-aged and old mice compared with young
mice in both WT and Null hearts. The expression of C-C
chemokine receptor type 1 (Ccr1), one of the receptors of Ccl5,
in old WT was lower than young and middle-aged WT, while
there was no age-related change in Null mice. The increase in
Ccr1 may be due to a negative feedback subsequent to the
increase in Ccl5 (17, 22). No significant differences were found
in the expression of Cxcl5, chemokine (C-C motif) ligand 4
(Ccl4), and tumor necrosis factor receptor superfamily member
1b (Tnfrsf1b) among all three groups in WT, although, in old
Null conditions, higher levels of Cxcl5, Ccl4, and Tnfrsf1b
were detected compared to young and middle-aged Null mice.
Using a TaqMan gene-specific assay, we validated the gene
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SPARC MEDIATES CARDIAC INFLAMMATION
Ccl5 (M1)
2
1
p<0.05
-
+
3
2
1
IL-12 (M1)
SPARC
p<0.05
2.0
1.5
1.0
0.5
0.0
0
SPARC
-
+
SPARC
-
+
IL-6 (M1)
p<0.05
p=0.23
0.020
IL6 mRNA
0.04
IL12 mRNA
Tnfα (M1)
Tnfα mRNA
Ccl3 mRNA
Ccl5 mRNA
3
0
0.03
0.02
0.01
0.015
0.010
0.005
0.000
0.00
-
+
Arg1 (M2)
-
SPARC
+
Mrc1 (M2)
p<0.05
p<0.05
0.5
Mrc1 mRNA
1.0
Arg1 mRNA
Fibrosis and inflammation are major components of cardiac
aging. SPARC plays a role in cardiac aging by facilitating the
deposition of fibrotic ECM proteins (5). In the present study,
4
4
0.8
0.6
0.4
0.2
0.0
SPARC
DISCUSSION
Ccl3 (M1)
p<0.05
5
SPARC
SPARC increased M1 macrophage polarization in vivo. To
confirm our in vitro results that SPARC promotes M1 macrophage polarization, we investigated the expression of IL-6 and
Tnf␣, M1 macrophage markers, in vivo. Levels of mRNA
encoding IL-6 and Tnf␣ were higher in old WT vs. young and
middle-aged WT. No age-dependent changes were found in
Null mice, suggesting that SPARC, which increases in old age,
enhanced M1 macrophage polarization in vivo (Fig. 8). The
expression of Fizz-1, an M2 macrophage marker, was lower in
middle-aged and old WT LV compared with young WT LV,
whereas no age-related changes in Fizz-1 expression were
detected in Null mice. This suggests that SPARC decreased
M2 macrophage polarization in vivo (Fig. 8).
0.4
0.3
0.2
0.1
0.0
-
+
SPARC
-
+
Fig. 7. SPARC treatment increased macrophage M1 polarization and decreased M2 polarization in peritoneal macrophages. Peritoneal macrophages from young
(3– 6 mo old) WT mice were stimulated with SPARC (20 ␮g/ml) for 24 h. SPARC increased the gene expression of macrophage M1 markers [Ccl5, chemokine
(C-C motif) ligand 3 (Ccl3), tumor necrosis factor-␣ (Tnf␣), and interleukin-12 (IL-12)] and decreased macrophage M2 markers [arginase 1 (Arg1) and mannose
receptor C type 1 (Mrc1)]. SPARC treatment had no effect on the expression of IL-6, another M1 macrophage marker. Expression of each gene was normalized
to Hprt1 levels and shown as 2⫺⌬Ct units. Values are means ⫾ SE (n ⫽ 4/group).
AJP-Cell Physiol • doi:10.1152/ajpcell.00402.2014 • www.ajpcell.org
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facilitates age-related cardiac inflammation by promoting
macrophage infiltration.
SPARC-stimulated peritoneal macrophages have increased
M1 and decreased M2 macrophage polarization. To further
investigate the relationship between SPARC and inflammation,
we isolated peritoneal macrophages from young mice and
treated cells with SPARC (Fig. 7) to determine whether
SPARC was sufficient to generate a proinflammatory status.
Macrophage M1 markers, Ccl5, Ccl3, Tnf␣, and IL-12, were
enhanced, and M2 markers, Arg1 and Mrc1, were reduced by
SPARC treatment. SPARC treatment had no effect on expression of IL-6 in vitro, another M1 macrophage marker, indicating that the major source of IL-6 in aged myocardium is not
likely macrophages. Our results showed that SPARC treatment
increased M1 and decreased M2 macrophage polarization in
peritoneal macrophages, which could contribute to the proinflammatory effects of SPARC in advancing age. Thus SPARC
may influence cardiac aging by suppressing M2 polarization,
resulting in a proinflammatory phenotype.
C979
SPARC MEDIATES CARDIAC INFLAMMATION
IL-6
Tnfα
p<0.05
p<0.05
p<0.05
0.4
6
p<0.05
Tnfα mRNA
0.2
0.1
0.0
4
2
Null
Fizz-1 mRNA
p<0.05
ol
d
ed
ag
dl
e-
yo
un
g
id
m
m
m
WT
p<0.05
30
ol
d
ed
dl
id
dl
id
id
Fizz-1
Fig. 8. IL-6 and Tnf␣, markers of M1 macrophage polarization, increased, and Fizz-1,
a marker of M2 macrophage polarization,
decreased with age only in the WT mice.
Age-related increases in IL-6 and Tnf␣ were
blunted in SPARC-null (open bars) mice
compared with WT (solid bars). Gene expression was normalized to Hprt1 and shown
as 2⫺⌬Ct ⫻ 100 units. Values are means ⫾
SE (n ⫽ 4 – 6/group).
20
10
ol
d
ed
ag
e-
dl
ol
d
yo
un
g
id
m
m
id
dl
e-
ag
yo
un
g
ed
0
we examined the roles of SPARC in aging heart with respect to
inflammation. A recent clinical study reports the positive
correlation between plasma SPARC and hypersensitive C-reactive protein and white blood cell numbers in gestational
diabetes mellitus (55). Another recent report by Ng et al. (39)
demonstrated that SPARC-null mice showed less inflammatory
symptoms in a mouse model of colitis. These studies suggest
that SPARC might enhance inflammation in certain settings.
The significant findings of our study were as follows: 1)
SPARC deletion suppressed age-related LV hypertrophy; 2)
SPARC deletion shifted the inflammatory phenotype by affecting age-dependent changes in gene expression of some inflammatory cytokines; 3) macrophage infiltration was delayed in
Null mice; and 4) SPARC treatment increased the expression
of proinflammatory M1 markers and reduced the expression of
anti-inflammatory M2 markers in isolated macrophages from
young mice. This study shows for the first time that SPARC
facilitates inflammation in cardiac aging. Although further
studies are needed, suppression of SPARC may be a potential
therapeutic target for cardiac aging.
Various changes in LV structure and function occur in the
aging heart (30). These changes affect mechanical load and
wall stress, leading to collagen accumulation. We found that
wall thickness and myocyte size increased with age in the WT,
but not in the Null, which provides evidence that lack of
SPARC during cardiac aging decreases cardiac hypertrophy.
Our laboratory previously reported that SPARC deletion de-
creases age-dependent cardiac collagen accumulation, which
supports the idea that SPARC participates in cardiac remodeling through, at least in part, the regulation of procollagen
processing (5). Cardiac ECM not only provides structural
support for the myocardium, but also serves as a reservoir for
the sequestration of cytokines and growth factors (23). Further
investigation into age-dependent changes in the structure and
composition of ECM is needed to clarify the role of ECM in
age-dependent inflammatory cell recruitment and cardiac remodeling.
Low levels of chronic inflammation are associated with
age-related diseases, including cardiovascular disease (13, 15).
Inflammatory cytokines, therefore, are predicted to play a role
in cardiac aging. Once inflammatory homeostasis is disturbed,
inflammatory cytokines are released from diverse cells, such as
myocytes, macrophages, endothelial cells, and fibroblasts (52).
These cytokines will in turn recruit leukocytes. Ccl5, Cx3cl1,
Ccr2, Cxcr3, and Mif were all elevated in the old WT mice
compared with young. Ccl5, also known as regulated upon
activation, normal T-cell expressed and secreted (RANTES),
plays an important role in recruiting inflammatory cell subsets
to inflammatory sites though the binding to Ccr1, Ccr3, or
Ccr5 (17, 22). Cx3cl1, also called fractalkine, can function
both as a membrane-bound form and a cleaved circulating
form. The membrane-bound form mainly acts as an adhesion
molecule and enables leukocyte adhesion (1). Ccr2 is a seventransmembrane G-protein-coupled receptor for monocyte che-
AJP-Cell Physiol • doi:10.1152/ajpcell.00402.2014 • www.ajpcell.org
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m
ag
yo
un
g
ol
d
ed
e-
ag
yo
un
g
ol
d
dl
e-
ag
yo
un
g
ed
0
e-
IL6 mRNA
0.3
C980
SPARC MEDIATES CARDIAC INFLAMMATION
For example, macrophage treatment with LPS (1 ␮g/ml) and
interferon-␥ (20 ng/ml) increased Tnf␣ and Ccl3 expression
100-fold, whereas SPARC induced 2-fold increases in these
two genes (35). Of note, in addition to SPARC increasing Tnf␣
and Ccl3, SPARC also decreased Mrc1 expression by 62%,
whereas IL-4, an M2 driver, induces a 3.5-fold increase (35).
Therefore, the proinflammatory effects of SPARC might be
related more to the decrease in M2 macrophage polarization
rather than an increase in M1 polarization.
In the present study, we did not determine the precise
mechanisms by which SPARC affected inflammation in the
aging heart. Goldblum et al. (19) reported that SPARC reduces
cultured endothelial cell adhesion to form a rounded conformation with intercellular gaps that could allow the passage of
macromolecules. Treatment of human umbilical endothelial
cells with SPARC similarly enables leukocytes to more easily
transmigrate through the monolayer (24). SPARC itself interacts with several receptors, including ␤1-integrin, vascular cell
adhesion molecule-1, and stabilin-1 (42, 53). The interaction of
SPARC and vascular cell adhesion molecule-1 triggers leukocyte transmigration (24). Stabilin-1, a scavenger receptor, is
expressed by M2 macrophages and transiently by microvascular endothelial cells during inflammation (27, 46). Stabilin-1 is
required for receptor-mediated endocytosis of SPARC in macrophages (28). Although further studies are needed, one possibility is that binding of SPARC to stabilin-1 might influence
macrophage polarization.
While we found evidence of colocalization of SPARC and
macrophages in the myocardium, we also observed macrophages that did not stain positive for SPARC, suggesting
age-dependent increases in macrophage infiltration might be
caused by both SPARC-dependent and -independent mecha-
Aging
SPARC
Macrophage M1>M2 polarization
Pro-inflammatory cytokines
Macrophage infiltration
ECM synthesis
Cardiac fibrosis
Fig. 9. The role of SPARC in age-related cardiac inflammation. The diagram
summarizes the present working hypothesis of the influence of SPARC in
age-related cardiac inflammation. SPARC levels increase with advancing age,
which induces proinflammatory macrophage M1 polarization. M1 macrophages produce proinflammatory chemokines, promoting macrophage infiltration. Increases in macrophage infiltration lead to a cycle wherein release of
proinflammatory chemokines enhances further cardiac inflammation (boxed
area highlights results from the present study). Cardiac inflammation induces
the production of fibrogenic cytokines and growth factors, leading to excessive
accumulation of extracellular matrix (ECM) and adverse remodeling.
AJP-Cell Physiol • doi:10.1152/ajpcell.00402.2014 • www.ajpcell.org
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motactic protein-1, which induces migration of monocytes to
inflammatory sites (14). Cxcr3, a receptor for Cxcl9, Cxcl10,
and Cxcl11, is known to recruit proinflammatory Th1 cells to
sites of inflammation (20, 33). Mif regulates the release of
proinflammatory mediators (9). In the heart, Mif is rapidly
released in response to a cellular stressor, such as a brief
hypoxia (25, 42). Plasma Mif levels are increased in old healthy
volunteers compared with young controls, and dietary nitrate
supplementation caused a reduction of plasma Mif levels and
improvement in vascular function (43). The age-dependent
elevated expression of these cytokines and receptors could,
therefore, cause the age-dependent increase in macrophage
infiltration observed in WT. Our data suggest that these cytokines play important roles in cardiac aging. In support, plasma
monocyte chemotactic protein-1 and Ccl5 have been reported
to be increased with age in healthy subjects (36).
Interestingly, in WT hearts, expression of Ccl5, Cx3cl1,
Ccr2, and Cxcr3 were increased in middle-aged mice, whereas
increases in macrophage numbers and expression of the senescence-associated Cdkn2a were detected only in old hearts.
Hence, an increase in the expression of these cytokines precedes recruitment of significant numbers of macrophages. One
interpretation is that the process of cardiac aging begins in
middle age and slowly progresses with age. We also found that,
with the exception of Mif, the age-dependent increase in these
proinflammatory cytokines/receptors was delayed in Null
hearts. Accordingly, Null mice do not exhibit increased collagen in aged myocardium and do not exhibit age-dependent
increases in stiffness (5). As cardiac expression of SPARC is
shown to increase with age (5, 21), we propose the novel
concept that, with age, SPARC acts to enhance macrophage
infiltration, which eventually leads to increased cardiac remodeling and dysfunction.
We further evaluated the effects of SPARC on macrophage
phenotypes to determine whether SPARC might have a direct
effect on macrophage activation in vitro. Macrophages are
classified into two major subtypes: M1 (proinflammatory or
classically activated) and M2 (anti-inflammatory or alternatively activated) macrophages (34, 37). We hypothesized that
SPARC would increase M1 and/or decrease M2 polarization.
Supporting our hypothesis, SPARC treatment increased macrophage polarization toward an M1 phenotype. Remarkably,
SPARC-treated macrophages displayed decreased M2 polarization. In agreement with these results, Arnold et al. (2)
reported an increase in the number of M2, alternatively activated, macrophages in tumors grown in Null mice. Although
the precise mechanisms are unclear, our data suggest that
increased levels of SPARC in aged hearts leads to suppression
of M2 macrophage polarization and augmentation of M1 macrophage polarization. Supporting our in vitro results, SPARC
deletion blunted in vivo age-dependent increases in Tnf␣, an
M1 macrophage marker, and age-dependent decreases in
Fizz-1, an M2 macrophage marker. SPARC treatment had no
effect on IL-6 levels in cultured peritoneal macrophages,
whereas levels of IL-6 mRNA were increased with age in WT
LV, but not in Null LV. These results suggest that the major
source of IL-6 in aged myocardium is likely not macrophages;
other candidate cell types that express IL-6 include cardiomyocytes, fibroblasts, and endothelial cells.
The effect of SPARC was less than that of the positive
control, the well-characterized M1 driver LPS ⫹ interferon-␥.
SPARC MEDIATES CARDIAC INFLAMMATION
GRANTS
We acknowledge support from the American Heart Association for
14SDG1886005, from the Biomedical Laboratory Research, Development
Service of the Veterans Affairs Office of Research and Development Awards
1I01BX001385 and 5I01BX000505, and from the National Institutes of Health
for HHSN 268201000036C (N01-HV-00244) for the San Antonio Cardiovascular Proteomics Center, HL-075360, HL-051971, and GM-104357.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
H.T., L.E.C.B., M.R.Z., M.L.L., and A.D.B. conception and design of research;
H.T. and C.F.B. performed experiments; H.T., L.E.C.B., C.F.B., M.R.Z., M.L.L.,
and A.D.B. analyzed data; H.T., L.E.C.B., M.R.Z., and M.L.L. interpreted results
of experiments; H.T. prepared figures; H.T. and M.L.L. drafted manuscript; H.T.,
L.E.C.B., C.F.B., M.R.Z., M.L.L., and A.D.B. edited and revised manuscript;
H.T., L.E.C.B., C.F.B., M.R.Z., M.L.L., and A.D.B. approved final version of
manuscript.
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