Articles in PresS. Am J Physiol Heart Circ Physiol (May 20, 2011). doi:10.1152/ajpheart.01070.2010
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SPARC Mediates Early Extracellular Matrix Remodeling
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Following Myocardial Infarction
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Sarah M. McCurdy1*; Qiuxia Dai1*; Jianhua Zhang1; Rogelio Zamilpa1, Trevi A. Ramirez1, Tariq
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Dayah1; Nguyen Nguyen2, Yu-fang Jin2, Amy D. Bradshaw3; and Merry L. Lindsey1
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San Antonio, San Antonio TX; 2 Department of Electrical and Computer Engineering, The
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University of Texas at San Antonio, San Antonio, TX; and 3Gazes Cardiac Research Institute,
Cardiology Division, Department of Medicine, The University of Texas Health Science Center at
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Division of Cardiology, Department of Medicine and the Ralph H. Johnson Veteran’s
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Administration, Charleston, SC.
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*Both authors contributed equally to this work.
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Running Title: Post-MI Effects of SPARC Deletion
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Address for Correspondence
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Merry L. Lindsey, Ph.D.
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Cardiology Division, Department of Medicine
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The University of Texas Health Science Center at San Antonio
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7703 Floyd Curl Drive, Mail Code 7872
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San Antonio, TX 78229-3900
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(phone) 210-567-4673
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email: [email protected]
(fax) 210-567-6960
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Word Count- 6174
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key words: myocardial infarction, SPARC, extracellular matrix, cardiac fibroblasts, LV
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remodeling, mice
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1
Copyright © 2011 by the American Physiological Society.
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Abstract
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Secreted protein, acidic, and rich in cysteine (SPARC) is a matricellular protein that
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functions in the extracellular processing of newly synthesized collagen. Collagen deposition to
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form a scar is a key event following a myocardial infarction (MI). Because the roles of SPARC in
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the early post-MI setting have not been defined, we examined age-matched wild type (WT;
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n=22) and SPARC deficient (Null; n=25) mice at day 3 post-MI. Day 0 WT (n=28) and Null
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(n=20) mice served as controls. Infarct size was 52±2% for WT and 47±2% for SPARC Null
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(p=n.s.), indicating that the MI injury was comparable in the two groups. By echocardiography,
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WT mice increased end diastolic volumes from 45±2 to 83±5 µL (p<0.05). SPARC Null mice
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also increased end diastolic volumes, but to a lesser extent than WT (39±3 to 63±5 µL, p<0.05
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vs day 0 controls and vs WT day 3 MI). Ejection fraction fell post-MI in WT mice, from 57±2% to
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19±1%. The decrease in ejection fraction was attenuated in the absence of SPARC (65±2% to
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28±2%). Fibroblasts isolated from SPARC Null left ventricle (LV) showed differences in the
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expression of 22 genes encoding extracellular matrix and adhesion molecule genes, including
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fibronectin, connective tissue growth factor (CTGF; CCN2), matrix metalloproteinase-3 (MMP-
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3), and tissue inhibitor of metalloproteinase (TIMP-2). The change in fibroblast gene expression
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levels was mirrored in tissue protein extracts for fibronectin, CTGF, and MMP-3 but not TIMP-2.
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Combined, the results of this study indicate that SPARC deletion preserves LV function at day 3
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post-MI but may be detrimental for the long-term response due to impaired fibroblast activation.
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Introduction
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Matricellular proteins are extracellular matrix components that contribute accessory, but
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not structural, roles to modify cell-extracellular matrix interactions.(12) Secreted protein, acidic,
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and rich in cysteine (SPARC, osteonectin, BM40) is a collagen binding matricellular protein that
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is robustly expressed in fibroblasts and endothelial cells and at low levels in cardiac myocytes.
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In addition, it has also been reported that SPARC expression is associated with α-smooth
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muscle actin positive myofibroblasts and CD45 positive leukocytes.
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signaling, adhesion, survival, proliferation, and migration in several cell types.(4, 14)
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regulates post-synthetic pro-collagen processing and assembly into fibrils, providing a third layer
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of regulation for collagen deposition in addition to mechanisms involving transcription or
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degradation.(14)
SPARC stimulates cell
SPARC
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SPARC levels increase in hearts of rats subjected to β-adrenergic receptor stimulation,
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(11) and in the left ventricles of patients with LV hypertrophy.(15) Bradshaw et al has reported
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that SPARC deletion attenuates pressure-overload induced collagen accumulation, resulting in
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improved diastolic function.(3)
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Schellings and colleagues have shown that SPARC deletion results in a four-fold higher
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incidence of mortality following MI, due to increased rates of rupture and heart failure over the
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first 14 days.(16) The increased incidence of rupture was associated with decreased deposition
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of mature collagen fibers. Further, an infusion of TGFβ in SPARC Null mice at day 2 pre-MI
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decreased rupture rates and increased collagen deposition. At the same time, TGFβ infusion did
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not alter infarct healing, suggesting that SPARC works through TGFβ dependent and
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independent pathways. However, the roles of SPARC in the early post-MI setting have not
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been defined. In this study, we evaluated the early MI response in SPARC null mice to isolate
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critical SPARC-dependent functions in remodeling and to explore the hypothesis that SPARC
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deletion alters the cardiac fibroblast response to MI, which might contribute to a more complete
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understanding of factors contributing to the higher incidence of cardiac rupture associated with
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MI.
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Materials and Methods
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All animal procedures were conducted according to the “Guide for the Care and Use of
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Laboratory Animals” (NIH Publication No. 85-23, revised 1996) and were approved by the
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Institutional Animal Care and Use Committees at the Medical University of South Carolina and
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the University of Texas Health Science Center at San Antonio.
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Mice
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We used male and female C57/BL6/SV129 wild type (WT n=23) and SPARC Null (n=29)
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mice at 4-6 months of age for the MI study. Day 0 control WT (n=28) and SPARC Null (n=20)
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mice were used as naïve controls. To induce MI, the mice were anesthetized with 2% isoflurane
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and the left anterior descending coronary artery was permanently ligated using minimally
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invasive surgery as described previously.(20) Because rupture predominantly occurs at days 3-
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7 post-MI in mice,(7) we evaluated the 3 day post-MI time point.
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At 3 days post-MI, the mice were sacrificed under isoflurane and the heart and lung were
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removed. The left and right ventricles were divided and weighed individually. The left ventricle
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was divided into apex, mid cavity, and base, and the three LV sections and RV were stained
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with 1% 2,3,5 triphenyltetrozolium chloride (Sigma) and photographed for infarct size
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determination by measuring infarct length as a percentage of the entire LV length.(5) Infarct
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and remote regions were taken from the apex and base, respectively, and were individually
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snap frozen and stored at -80°C. The mid cavity section was fixed in 10% zinc-formalin and
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paraffin-embedded for histological examination. The lungs were removed, and wet and dry
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weights were determined.
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Echocardiographic Measurements
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For the echocardiography analysis, 0.5-2% isoflurane in a 100% oxygen mix was used
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to anesthetize the mice. Electrocardiogram and heart rate were monitored using a surface
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electrocardiogram. Images were acquired using the Vevo 770TM High-Resolution In Vivo
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Imaging System (Visual Sonics) and were taken at heart rates >400 bpm to achieve
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physiologically relevant measurements. Measurements were taken from the two-dimensional
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parasternal long-axis and short axis (m-mode) recordings from the mid-papillary region.
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Echocardiographic studies were performed prior to sacrifice for day 0 control mice, and at day 0
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and 3 days post-MI for mice in the MI groups. For each parameter, three images from
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consecutive cardiac cycles were measured and averaged. End diastolic radius to free wall
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(infarct) thickness was calculated as an estimate of LV wall stress.(1)
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Histology
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The LV mid-cavity section was embedded in paraffin, sectioned at 5 µm and stained
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using hematoxylin and eosin for routine assessment. Myocyte cross sectional area was
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determined by measuring myocyte circumference of at least 10 myocytes per section in the
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remote region of hematoxylin and eosin stained sections. From the circumference, areas were
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calculated. Macrophages were stained using a rat anti-Mac-3 monoclonal antibody (Cedarlane
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Laboratories Limited; clone M3/84; 1:100 dilution) followed with the Vectastain Elite ABC kit
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(Vector Laboratories).
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Fibroblast Isolation and ECM Arrays
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Cardiac fibroblasts were isolated by enzymatic digestion with liberase blendzyme 1
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(Roche). Fibroblasts were incubated in DMEM with 10% fetal bovine serum and 1% antibiotic-
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antimycotic solution (Cellgro, 30-004-CI).
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phenotypic conversion due to prolonged culturing. To obtain basal ECM levels, fibroblasts were
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serum starved for 48 hours.
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Dissociation Reagent (Invitrogen; 12604) and snap frozen until analysis.
Cells were used at passages 2-4 to reduce
Fibroblasts were collected using TrypLE™ Express Cell
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For the ECM arrays, total RNA was isolated from the cells using TRIzol reagent plus
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Total RNA purification kit (Invitrogen). The cDNA was synthesized using SABiosciences RT2
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first strand kit (C-03).
The RT2 qPCR Primer Array for Extracellular Matrix and Adhesion
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Molecules (SuperArray APMM-013A) was used for the gene array. This array uses SYBR®
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Green-based quantitative real-time PCR assay to determine the gene expression of 84 ECM
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and adhesion molecule genes in one 96-well plate. Results were analyzed based on the ΔΔCt
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method with normalization of raw data to three housekeeper genes (Gusb, Hprt1, and
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Hsp90ab1) and were reported as 2-ΔCT values.
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Fibroblast ECM Gene Pattern Analysis
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Fibroblast extracellular matrix (ECM) gene array data collected from the 6 groups of
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mouse tissue described above was analyzed using Agilent Genespring GX 11.0, to identify
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differentially expressed genes and organize the genes into clusters. All data was combined as
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a single dataset and loaded to Genespring GX 11.0. The dataset (expressed as 2-ΔCT units) was
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inputted into Genespring, without any normalization algorithm or baseline transformation. By
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default, Genespring converts the raw data into log scale if no normalization algorithm is
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selected. A total of 22 significantly differentially expressed genes were found, based on ANOVA
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analysis of the dataset. Hierarchical clustering was performed with the Linkage set as Centroid,
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i.e, the distance between two clusters was calculated as the average distance between their
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respective centroids. All 22 differentially expressed genes were clustered in the dendrogram.
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For pattern analysis, expression levels of the 22 identified differentially expressed genes
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were normalized with respect to the averaged expression levels of the WT control group for WT
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samples and the Null Day 0 group for Null samples. Normalized expression was defined as
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expression levels in LVC or LVI divided by
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Normalized gene expression of LVC and LVI samples from WT tissues were grouped into 6
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patterns:
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increased, decreased-sustained, and decreased-decreased. Expression levels of these genes
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from Null samples were also analyzed to illustrate the effects of SPARC deletion.
increased-decreased,
expression levels in the respective control.
increased-sustained,
increased-increased,
decreased-
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LV Protein Extraction
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Soluble proteins were extracted from the infarct region of the LV (LVI) and non-infarct
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region (LVC) by homogenizing the sample in PBS containing 1x Complete Protease Inhibitor
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Cocktail (Roche). After centrifugation, the soluble supernatant was collected. The insoluble
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proteins present in the pellet were extracted by further homogenization in Sigma Reagent 4 (7
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M urea, 2 M thiourea, 40 mM Trizma® base and the detergent 1% C7BzO) and 1x complete
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protease inhibitor cocktail (Roche). Protein concentrations were determined using the Bradford
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Assay. Insoluble protein extracts were diluted 1:40 with water for Bradford assay compatibility.
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Total protein (10 µg) for each fraction of all samples were run on one-dimensional SDS gels,
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and the gels were stained with Coomassie blue to confirm protein concentration and loading
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accuracy.
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Immunoblotting of LV tissue extracts
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Soluble or insoluble protein differences were determined by immunoblotting using
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antibodies against alpha smooth muscle actin (Abcam; ab5694; 1:1,000), CD31 (PECAM-1;
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Abcam; ab28364; 1:1,000), connective tissue growth factor (R&D System; AF660; 1:
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1,000),fibronectin (Millipore; AB1954; 1:10,000), heat shock protein 47 (hsp-47; Epitomics;
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3198; 1:1,000), matrix metalloproteinase-3 (MMP-3; Abcam; ab53015; 1:1,000), matrix
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metalloproteinase-9 (MMP-9; Abcam; ab38898; 1:1,000), periostin (Abcam; ab14014; 1:1,000),
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SPARC (R&D Systems; MAB942; 1:1,000), tissue inhibitor of metalloproteinase-2 (TIMP-2;
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Chemicon; AB8107; 1:1,000), and transforming growth factor β (Sigma; AV37156, 1:1,000).
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Total protein (10 µg for each sample) was loaded on 26-well 4–12% Criterion Bis-Tris gels (Bio-
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Rad). Equal protein transfer was verified using reversible total membrane stain (Pierce; 24580)
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on the nitrocellulose membranes. Immunoblotting was performed as previously described.(6)
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Molecular Imaging Software (Kodak) was used to measure densitometry, which was normalized
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to the mean intensity of the total membrane stain.
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Statistical Analyses
Data are reported as mean±SEM and groups were analyzed using One-Way ANOVA
followed by Student Newman Kewls post-hoc test. A p<0.05 was considered significant.
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Results
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Survival, Echocardiographic, and Necropsy Analyses
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By immunoblotting, SPARC protein levels were increased to approximately two-fold in
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the infarct region of WT mice at day 3 (Figure 1). The 3 day post-MI survival rate was 96% for
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WT (n=1 mouse died) and 86% for SPARC Null mice (n=4 mice died; p=0.37 by Fisher’s exact
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t-test). One Null death could be attributed to rupture, while one WT and 3 Null deaths were
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attributed to arrhythmias or acute congestive heart failure.
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Echocardiographic, necropsy and infarct size analyses for WT and Null mice are shown
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in Table 1. Day 0 naïve WT and Null mice were used as controls.
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demonstrated reduced body weight (BW), compared to age-matched WT mice at baseline. Both
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WT and Null post-MI groups showed increased dilation compared to the respective day 0
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controls; however, the increased dilation in the Null mice post-MI was attenuated compared to
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the WT post-MI group. Likewise, ejection fraction decreased in both MI groups, with the
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decrease in ejection fraction being attenuated in the Null post-MI mice compared to the WT
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post-MI group. The improved ejection fraction indicates that SPARC Null mice showed better
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LV function at day 3 post-MI compared to the WT at day 3 post-MI.
SPARC Null mice
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The left ventricle mass to body weight ratios increased in both WT and Null post-MI
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groups, and there was no difference between the MI groups. The decrease in lung wet weight to
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body wet ratios in the Null MI, compared to the WT MI, indicate that the Null mice have less
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edema post-MI. Wall thinning and infarct sizes were similar between the two groups, indicating
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that functional responses were not due to initial differences in the severity of injury.
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Morphometric Analyses
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We measured the outside circumference and myocyte cross sectional areas from
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hematoxylin and eosin stained sections (Figure 2). The left ventricles in the WT MI group
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showed increased dilation compared to the Null, consistent with the echocardiographic findings.
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Myocyte cross sectional areas were 137±5 μm2 for WT day 0 (n=28) and increased to 169±4
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μm2 for WT day 3 (n=22; p<0.05). Similarly, myocyte areas were 132±6 μm2 for Null day 0
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(n=20) and increased to 163±3 μm2 for Null day 3 (n=25; p<0.05). Compensatory hypertrophic
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response appeared normal in Null mice, consistent with findings from studies of pressure-
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overload induced hypertrophy in SPARC Null mice.(3) Macrophages were quantified by Mac-3
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immunohistochemistry. WT macrophage levels in the infarct region were 2.22±0.17% (n=22)
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and Null macrophage levels in the infarct region were 2.47±0.15% (n=24; p=n.s.).
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Fibroblast ECM Gene Array
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Excluding SPARC (which was absent in the Null mice), there were 22 genes encoding
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ECM and adhesion molecules that were differentially expressed in fibroblasts isolated from day
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0 and the remote and infarct regions of day 3 post-MI LV.
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statistically significant changes.
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regions for either set showed similar expression patterns, indicating that fibroblasts from these
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two regions share similar ECM and adhesion molecule responses at the individual cell level.
Table 2 summarizes the 22
Of interest, fibroblasts isolated from the remote vs infarct
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In SPARC null day 0 fibroblasts ECM gene array, MMP-3, VCAM1, and CD31 (PECAM-
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1) levels were reduced, whereas Col3a1, Col4a1, Fn1, Lamc1, Postn, TIMP-2, and several
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integrins (α3, αv, and β1) were elevated in comparison to gene expression levels in WT day 0
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fibroblasts. We conclude that regulation of these genes is influenced by SPARC deletion.
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We measured collagens Iα1. IIα1. IIIα1, IVα1, IVα2, IVα3, Vα1, and VIα1 in our screen.
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Of these collagen subtypes, collagens IIIα1 and IVα1 were the only collagens that were
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statistically different among groups. Collagen III mRNA levels were actually decreased, while
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collagen IV mRNA levels were increased, in WT post-MI fibroblasts. Both collagen III and
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collagen IV levels decreased in post-MI fibroblasts from the Null mice. The fact that SPARC
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deletion was accompanied by a decrease in collagen expression may have a negative impact
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on scar formation at later time points.
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By pathway analysis, six patterns of change emerged (Figure 3B) from the fibroblast
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ECM gene array results, from which the following conclusions were drawn: 1) SPARC regulates
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collagen production (particularly collagen III and IV), in addition to deposition; 2) SPARC effects
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the expression of other ECM genes, particularly fibronectin and periostin, which has not been
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previously reported in cardiac fibroblasts; 3) SPARC regulates different ECM genes in separate
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patterns, as we see 6 distinct patterns of change; and 4) CD31 (PECAM-1), Collagen III, and
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CCN2 (CTGF) are likely indirectly regulated by SPARC, as the pathway analysis showed these
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genes to be the least integrated in the cluster (Figure 3A). Based on differential expression of
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genes found in the array analysis, we measured myocardial tissue levels of TIMP-2, fibronectin,
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CTGF, and MMP-3 by immunoblotting (Figure 4).
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Immunoblotting of Post-MI LV Tissue
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TIMP-2 protein levels in the LV tissue did not differ among any of the groups, which
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contrasted with the changes in mRNA levels seen in the remote and infarct fibroblasts.
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Normalized densitometry values for TIMP-2 were 119±14 units for WT day 0 controls (n=12),
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128±10 units for the WT LVC remote region (n=11), 143±13 units for the WT LVI infarct region
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(n=11), 125±14 units for Null day 0 controls (n=11), 105±12 units for the Null LVC remote region
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(n=9), and 95±20 units for the Null LVI infarct region (n=9; p=n.s.).
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Soluble fibronectin levels were lower in SPARC Null infarct regions, compared with WT
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infarct regions (Figure 4A).
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compared to the day 0 controls, the response was blunted in the absence of SPARC. Insoluble
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fibronectin was higher at baseline in Null hearts, consistent with the increased gene expression
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detected in SPARC Null fibroblasts over that of WT fibroblasts taken from day 0 hearts (Table 2
While levels were significantly elevated in both infarct regions
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and Figure 3). These results suggest that in addition to influencing expression and deposition of
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collagens, SPARC may also play a significant role in the regulation of fibronectin post-MI.
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Levels of CTGF were higher in the remote region in SPARC Null hearts post-MI,
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compared to levels in the remote region of WT hearts post-MI (Figure 4B). Although levels of
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CTGF significantly and linearly increased in WT hearts post-MI, CTGF levels at day 0 and in the
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remote region of the Null mice were found to be higher than that of WT. The fact that WT
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fibroblasts did not show an increase in CTGF gene expression post-MI suggests that the
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primary source of CTGF is another cell type present in vivo, for example, macrophages. In the
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insoluble fraction, CTGF levels were increased in the infarct region of WT, but not Null, LV. The
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lack of an increase in levels of CTGF in response to MI in SPARC Null heart might contribute to
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subsequent ruptures observed at later time points post-MI.
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MMP-3 levels decreased in WT and Null infarct LV, consistent with the observed
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decrease in gene expression found in fibroblasts (Figure 4C). In fibroblasts, MMP-3 gene levels
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were significantly lower in Null day 0 compared with WT day 0 cells; however, samples from LV
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day 0 showed increased active MMP-3 levels in Null vs WT LV tissue. MMP-3 is an upstream
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activator of several MMPs, including MMP-9.(13) Therefore, baseline ECM turnover might be
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higher in the absence of SPARC, whereas the decrease in MMP-3 post-MI is predicted to favor
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a net accumulation of ECM at day 3.
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Because SPARC has been shown to associate with MMP-9 and MMP-3 levels were
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found to be altered in cardiac fibroblasts and LV tissue of Null mice, we also measured levels of
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MMP-9. MMP-9 was found to be increased in the infarct regions, but levels were not different
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between WT and Null groups (p=n.s.).
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To determine if SPARC deletion influenced fibroblast and endothelial responses post-MI,
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we measured α smooth muscle actin, hsp-47, periostin, and transforming growth factor β for
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fibroblast activation markers and CD31 (PECAM-1; for endothelial cell numbers). Levels of α
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smooth muscle actin and transforming growth factor β did not change among groups, indicating
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that the 3 day post-MI time point is before fulminant fibroblast activation. Levels of hsp-47 and
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CD31 were decreased in the infarct region of WT but not Null mice (Figure 5), suggesting that
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the early decrease in ECM and angiogenic responses are attenuated by SPARC deletion.
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Periostin levels increased from 9044±586 units in WT day 0 samples to 12922±585 and
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15191±768 units in WT day 3 post-MI remote and infarct regions, respectively (both p<0.05).
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Periostin similarly increased post-MI LV of SPARC Null mice from 10514±712 units in day 0
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controls to 14009±794 and 15328±759 units in Null day 3 post-MI remote and infarct region,
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respectively (both p<0.05). Although periostin levels trended higher in the SPARC Null heart
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tissue, similar to findings in SPARC Null fibroblasts, differences in periostin levels did not reach
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statistical significance.
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Discussion
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The goal of this study was to examine the role of SPARC in early remodeling events
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post-MI. SPARC has been shown to be a key co-factor in collagen assembly, and previous
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work has shown the SPARC deletion results in increased rupture rates post-MI.(16) We
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examined the functional consequences of SPARC gene deletion on the first 3 days of
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remodeling with respect to echocardiographic parameters and extracellular matrix synthesis in
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cardiac fibroblasts. The significant and unique findings of this study were that MI induction in
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SPARC Null mice resulted in 1) improved functional remodeling parameters, including
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attenuated decreases in ejection fraction and less severe dilation; and 2) altered fibroblast
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phenotypes in terms of ECM and cell adhesion molecule expression.
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demonstration that SPARC regulates fibroblast function in the early post-MI setting.
This is the first
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In our study, the SPARC Null mice showed improved remodeling parameters at day 3
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post-MI. In the previous study by Schellings et al, they observed no difference in LV function
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between the day 3 post-MI WT and SPARC Null mice.(16) There are two key differences in our
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experimental designs that may explain this difference. Our mice were on a mixed background
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strain (C57/BL6 and SV129) while the mice in the Schellings study were on a pure C57/BL6
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background. The C57/BL6 strain has been shown to be a fibrosis-prone strain,(10) and
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differences in rupture rates among strains has been reported.(8, 18) In addition, the mice in our
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study were 4-6.5 months of age (the equivalent of 30-45 year old humans) while the mice in the
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Heymans study were 2.5-4.5 months of age (the equivalent of 20-30 year old humans). The
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strain and age differences, therefore, could account for the slight differences in MI response
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seen in the SPARC Null mice between the two studies. Additional studies comparing the effect
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of SPARC deletion on post-MI remodeling across species and ages are warranted. Bradshaw
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and colleagues recently reported that SPARC Null mice show attenuated aging in periodontal
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ligaments, indicating that age superimposed on MI in the setting of SPARC deletion is likely to
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have a different outcome.(17) It would be interesting to determine whether the absence of
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SPARC switches from a positive early regulator to a negative late regulator of remodeling, given
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that LV rupture is prevalent later. If so, the causes of this transition, while currently not clear,
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would potentially be very interesting. For instance, these studies may help to discern if cardiac
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function parameters early post-MI predict cardiac rupture or whether rupture is caused strictly by
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sudden structural failure.
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SPARC deletion altered cardiac fibroblast phenotypes. Our results suggest that the
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effect of SPARC may be to alter the remodeling kinetics by changing cell response, perhaps in
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addition to or independent of a direct ECM role. In our study, we define impaired fibroblast
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activation as the decreased ability of the fibroblast to increase ECM production in the post-MI
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setting. The fibroblast CTGF results suggest that SPARC deletion takes CTGF out of the MI
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wound healing equation.
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baseline, which suggests that collagen synthesis is higher at baseline and is confirmed by the
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higher gene expression of Col III α1 and colIV α1 in table 2. In WT mice, CTGF was only
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needed post-MI, while in SPARC Null mice CTGF was needed all of the time. One explanation
SPARC deletion was compensated by increased CTGF levels at
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for this decline may be that cardiac fibroblasts in SPARC Null hearts are constantly trying to
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assemble more ECM as collagen levels are reduced in uninjured hearts of SPARC-null mice,
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but are not successful. High levels of CTGF in day 0 and remote Null fibroblasts suggest that
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collagen and CTGF may be important mediators of myocyte-fibroblast interactions.(2, 9)
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The switch in integrin isoforms in the remote region is interesting. The wild type mice
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showed increased integrin αm in the remote region post-MI, whereas the Null mice showed
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decreased β1 and decreased α3. This result is consistent with the fact that macrophage numbers
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were not different between WT and Null Post-MI infarcts, which is consistent with results
339
reported by Schellings et al for the day 7 time point.(16) By day 14, Schellings and colleagues
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do report a decreased number of macrophages in the Nulls compared to the WT, indicating that
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SPARC may regulate macrophage viability and chronic immune responses at later times post-
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MI.(17)
343
In addition to altering fibroblast responses, SPARC deletion likely also affects
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macrophage and endothelial cell responses, which would contribute to the attenuated
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remodeling phenotype observed. SPARC is expressed in macrophages and endothelial cells,
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and SPARC has been shown to interact with the scavenger receptor stabilin 1 to regulate
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macrophage clearance.(19) Therefore, SPARC deletion likely influences the inflammatory and
348
angiogenic responses in the MI setting. CD31 levels decreased at day 3 post-MI in the WT LV,
349
but this decrease was attenuated in the absence of SPARC.
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deletion serves to preserve blood vessel numbers in the infarct region, rather than stimulating
351
an angiogenic response. Additional studies are needed to clarify the role of SPARC in these
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two cell types.
This indicates that SPARC
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In conclusion, this study is the first to examine the role of SPARC on fibroblast ECM
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production in the post-MI setting. While SPARC deletion resulted in improved function at day 3
355
post-MI, the absence of SPARC also resulted in a blunted fibroblast ECM response. Differential
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timing of SPARC inhibition, therefore, is predicted to yield different outcomes in terms of infarct
357
scar phenotype.
358
359
Acknowledgements
360
The authors acknowledge grant support from NIH 1R03EB009496 and SC2HL101430 to YJ,
361
from NIH 2P01HL48788, HL094517, and a Veteran’s Administration Merit Award to ADB, and
362
from NIH R01HL75360, the American Heart Association AHA 0855119F, and the Max and
363
Minnie Tomerlin Voelcker Fund to MLL.
364
Physiological Society Summer Undergraduate Research Fellowship.
SMM was supported in 2008 by the American
365
366
References
367
368
369
370
371
372
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
1.
Bevan AK, Hutchinson KR, Foust KD, Braun L, McGovern VL, Schmelzer L, Ward
JG, Petruska JC, Lucchesi PA, Burghes AHM, and Kaspar BK. Early heart failure in the
SMN{Delta}7 model of spinal muscular atrophy and correction by postnatal scAAV9-SMN
delivery. Hum Mol Genet 19: 3895-3905, 2010.
2.
Bowers SL, Borg TK, and Baudino TA. The dynamics of fibroblast-myocyte-capillary
interactions in the heart. Annals of the New York Academy of Sciences 1188: 143-152, 2010.
3.
Bradshaw AD, Baicu CF, Rentz TJ, Van Laer AO, Boggs J, Lacy JM, and Zile MR.
Pressure overload-induced alterations in fibrillar collagen content and myocardial diastolic
function: role of secreted protein acidic and rich in cysteine (SPARC) in post-synthetic
procollagen processing. Circulation 119: 269-280, 2009.
4.
Bradshaw AD, and Sage EH. SPARC, a matricellular protein that functions in cellular
differentiation and tissue response to injury. The Journal of clinical investigation 107: 10491054, 2001.
5.
Chiao YA, Zamilpa R, Lopez EF, Dai Q, Escobar GP, Hakala KW, Weintraub ST, and
Lindsey ML. In vivo Matrix Metalloproteinase-7 Substrates Identified in the Left Ventricle PostMyocardial Infarction Using Proteomics. Journal of proteome research 9: 2649-2657, 2010.
6.
Dai Q, Escobar GP, Hakala KW, Lambert JM, Weintraub ST, and Lindsey ML. The
Left Ventricle Proteome Differentiates Middle-Aged and Old Left Ventricles in Mice. Journal of
proteome research 7: 756-765, 2008.
7.
Gao X-M, Ming Z, Su Y, Fang L, Kiriazis H, Xu Q, Dart AM, and Du X-J. Infarct size
and post-infarct inflammation determine the risk of cardiac rupture in mice. International journal
of cardiology In Press, Corrected Proof: 2009.
8.
Gao X-M, Xu Q, Kiriazis H, Dart AM, and Du X-J. Mouse model of post-infarct
ventricular rupture: time course, strain- and gender-dependency, tensile strength, and
histopathology. Cardiovascular research 65: 469-477, 2005.
9.
Goldsmith EC, Hoffman A, Morales MO, Potts JD, Price RL, McFadden A, Rice M,
and Borg TK. Organization of fibroblasts in the heart. Dev Dyn 230: 787-794, 2004.
10.
Kolb M, Bonniaud P, Galt T, Sime PJ, Kelly MM, Margetts PJ, and Gauldie J.
Differences in the Fibrogenic Response after Transfer of Active Transforming Growth Factor15
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
{beta}1 Gene to Lungs of "Fibrosis-prone" and "Fibrosis-resistant" Mouse Strains. Am J Respir
Cell Mol Biol 27: 141-150, 2002.
11.
Masson S, Arosio B, Fiordaliso F, Gagliano N, Calvillo L, Santambrogio D,
D'Aquila S, Vergani C, Latini R, and Annoni G. Left ventricular response to beta-adrenergic
stimulation in aging rats. J Gerontol A Biol Sci Med Sci 55: B35-41; discussion B42-33, 2000.
12.
McCurdy S, Baicu CF, Heymans S, and Bradshaw AD. Cardiac extracellular matrix
remodeling: fibrillar collagens and Secreted Protein Acidic and Rich in Cysteine (SPARC).
Journal of molecular and cellular cardiology 48: 544-549, 2010.
13.
Ogata Y, Enghild JJ, and Nagase H. Matrix Metalloproteinase 3 (Stromelysin)
Activates the Precursor for the Human Matrix Metalloproteinase 9. J of Biological Chemistry
267: 3581-3584, 1992.
14.
Rentz TJ, Poobalarahi F, Bornstein P, Sage EH, and Bradshaw AD. SPARC
Regulates Processing of Procollagen I and Collagen Fibrillogenesis in Dermal Fibroblasts. J Biol
Chem 282: 22062-22071, 2007.
15.
Ridinger H, Rutenberg C, Lutz D, Buness A, Petersen I, Amann K, and Maercker C.
Expression and tissue localization of beta-catenin, alpha-actinin and chondroitin sulfate
proteoglycan 6 is modulated during rat and human left ventricular hypertrophy. Experimental
and molecular pathology 86: 23-31, 2009.
16.
Schellings MW, Vanhoutte D, Swinnen M, Cleutjens JP, Debets J, van Leeuwen
RE, d'Hooge J, Van de Werf F, Carmeliet P, Pinto YM, Sage EH, and Heymans S. Absence
of SPARC results in increased cardiac rupture and dysfunction after acute myocardial infarction.
The Journal of experimental medicine 206: 113-123, 2009.
17.
Trombetta JM, and Bradshaw AD. SPARC/osteonectin Functions to Maintain
Homeostasis of the Collagenous Extracellular Matrix in the Periodontal Ligament. J Histochem
Cytochem jhc.2010.956144, 2010.
18.
van den Borne SWM, van de Schans VAM, Strzelecka AE, Vervoort-Peters HTM,
Lijnen PM, Cleutjens JPM, Smits JFM, Daemen MJAP, Janssen BJA, and Blankesteijn
WM. Mouse strain determines the outcome of wound healing after myocardial infarction.
Cardiovascular research 84: 273-282, 2009.
19.
Workman G, and Sage EH. Identification of a sequence in the matricellular protein
SPARC that interacts with the scavenger receptor stabilin-1. Journal of Cellular Biochemistry
112: 1003-1008, 2011.
20.
Zamilpa R, Lopez EF, Chiao YA, Dai Q, Escobar GP, Hakala K, Weintraub ST, and
Lindsey ML. Proteomic Analysis Identifies In vivo Candidate Matrix Metalloproteinase-9
Substrates in the Left Ventricle Post-Myocardial Infarction. Proteomics 10: 2214-2223, 2010.
16
434
Tables
Table 1. Echocardiography and Necropsy Results.
Wild Type
Echocardiography
Day 0
Sample Sizes (n)
SPARC Null
Day 0
28
Day 3
MI
22
20
Day 3
MI
25
Heart Rate(bpm)
465±9
494±11
445±9
452±7 ¥
End Diastolic Volume (ml)
45±2
83±5 *
39±3
63±5 ‡¥
End Systolic Volume (ml)
20±2
67±4 *
14±2
46±4 ‡¥
Ejection Fraction (%)
57±2
19±1 *
65±2 *
28±2 ‡¥
Stroke Volume (ml)
25±1
16±2 *
25±2
16±1 ‡
Free Wall (Infarct) Wall Thickness (systole; mm)
1.05±0.03
0.54±0.04 *
1.02±0.05
0.56±0.04 ‡
End Diastolic Radius to Infarct Wall Thickness (mm/mm)
2.55±0.05
4.34±0.20 *
2.33±0.08
3.90±0.21 ‡
Necropsy
Day 0
Control
25.1±0.7
Day 3
MI
22.7±1.1 *
Day 0
Control
20.1±0.6 *
Day 3
MI
18.0±0.7 ¥
86±3
102±4 *
67±2 *
86±3 ‡¥
LV to BW (mg/ g)
3.4±0.1
4.8±0.1 *
3.4±0.1
4.8±0.1 ‡
Lung wet weight to BW (mg/ g)
5.0±0.1
9.3±0.7 *
5.0±0.2
8.3±0.7 ‡
Lung dry weight to BW (mg/g)
1.1±0.1
2.0±0.2 *
1.2±0.1
1.8±0.2
Body Weight (BW; g)
LV Mass (mg)
Infarct Size (%)
52±2
47±2
Data are mean±SEM. LV- left ventricle
*p<0.05 vs WT Day 0 Control; ‡p<0.05 vs Null Day 0 Control; ¥p<0.05 vs WT Day 3 MI
17
Table 2. ECM genes differentially expressed between wild type and SPARC Null cardiac
fibroblasts. The arrows indicate direction of significantly different changes at p<0.05 for the
group listed.
Gene
WT LVC
WT LVI
Null Day 0
Null LVC
Null LVI
(compared to WT Day 0)
(compared to Null Day 0)
↓
↓
Adamts2
↓
↓
Cdh2
↓
↓
↓
Vcam1
↓
↓
↓
Mmp3
↓
↓
Sgce
↓
↓
Ctnnb1
↑
Itgav
↑
Tgfb1
↑
Itgam
↑
↓
↓
Col4a1
↑
↓
↓
Itgb1
↓
Col4a2
↑
↓
↓
Fn1
↑
↓
↓
Timp2
↓
↓
Adamts5
↓
↓
↓
↓
Lama2
↑
↓
Lamc1
↑
↓
↓
Postn
↑
↓
Itga3
↑
Col3a1
↓
CCN 2 (CTGF)
↓
↓
↓
CD-31
(PECAM-1)
435
436
18
437
Figure Legends
438
Figure 1. SPARC protein levels increase in the left ventricles at day 3 post-MI. Representative
439
immunoblot images are shown on the top and quantification of WT day 0 (n=12), WT day 3
440
remote (LVC; n=11), and WT day 3 infarct (LVI; n=11) is shown below. The soluble fraction is
441
shown on the left and the insoluble fraction is shown on the right. *p<0.05 vs control day 0 and #
442
p<0.05 vs remote LVC.
443
444
Figure 2. SPARC deletion attenuates LV dilation at day 3 post-MI. A) Representative
445
photomicrographs of hematoxylin and eosin stained sections from WT and SPARC Null day 0
446
and day 3 post-MI left ventricles. B) Quantification of the outside circumference, showing that
447
absence of SPARC attenuates dilation at day 3 post-MI. Sample sizes are n=28 for WT day 0,
448
n=22 for WT day 3, n=20 for Null day 0, and n=25 for Null day 3. *p<0.05 vs control (WT or Null
449
at day 0) and + p<0.05 vs WT day 3.
450
451
Figure 3. A) Clustered dendrogram display of gene levels from the cardiac fibroblast ECM
452
gene array. Results were analyzed based on the ΔΔCt method with normalization of raw data to
453
three housekeeper genes (Gusb, Hprt1, and Hsp90ab1) and are reported as the log ratios of the
454
2-ΔCT values. Cardiac fibroblasts were isolated from day 0 controls and the remote (LVC) and
455
infarct (LVI) regions of day 3 MI samples from both WT and Null mice. Of the 84 extracellular
456
matrix and adhesion molecule genes evaluated, 22 were statistically different among groups by
457
ANOVA analysis, and these were used for the cluster analysis. The dendrogram and colored
458
image were produced by Genespring GX 11.0. The color scale ranges from blue for log ratios -
459
6.9 and below to red for log ratios 6.9 and above. Each gene is represented by a single row of
460
colored boxes. Each tissue is represented by a single column. B) Gene levels in LVC and LVI
461
were normalized to day 0 control values and plotted according to six patterns. The WT levels
19
462
are shown on the top graph and the Null levels are shown on the bottom graph for each section.
463
Sample sizes are n=4 for WT day 0, n=6 for WT day 3 remote LVC, n=3 for WT day 3 infarct
464
LVI, n=7 for Null day 0, n=6 for Null day 3 remote LVC, and n=5 for Null day 3 infarct LVI.
465
466
Figure 4. Myocardial Levels of Fibronectin, CCN2 (CTGF), and MMP-3 by Immunoblotting.
467
Soluble fraction levels are shown on the left, insoluble fraction level are shown on the right. A)
468
Soluble fibronectin levels increase five-fold in the infarct region of WT LV, and this increase is
469
attenuated in the Null. Insoluble fibronectin levels also increase in the infarct region of the WT
470
mice. The Null mice show increased levels in the day 0 controls compared to the WT controls.
471
B) Soluble CCN2 (CTGF) levels increase in both the remote and infarct regions of WT LV, while
472
CTGF levels in the Null are highest at day 0 and in the remote region. Insoluble CTGF levels
473
also increase in the infarct region of the WT mice, while the Null mice show increased levels
474
only in the day 0 control group. C) Soluble active MMP-3 levels decrease in the infarct regions
475
of both WT and Null mice. Interestingly, active MMP-3 levels in the Null are highest at the day 0
476
control time point. Insoluble active MMP-3 is reduced in the infarct region of the Null mice
477
compared with the Null day 0 values. Sample sizes are n=12 for WT day 0, n=11 for WT day 3
478
LVC, n=11 for WT day 3 LVI, n=11 for Null day 0, n=9 for Null day 3 LVC, and n=9 for Null day 3
479
LVI. *p<0.05 vs control (WT or Null at day 0), + p<0.05 vs WT at that same time point, and #
480
p<0.05 vs remote at that same time point.
481
482
Figure 5. HSP-47 and CD31 levels decrease in the infarct region of WT, but not SPARC Null
483
mice at day 3 post-MI by immunoblotting. A) The soluble fraction of the LV extracts were
484
blotted for Hsp 47. B) The insoluble fraction of the LV extracts were blotted by CD31. For
485
both, levels decreased in the infarct region of the WT LV, and this decrease was attenuated in
486
the Null LV extracts. Sample sizes are n=12 for WT day 0, n=11 for WT day 3 LVC, n=11 for
20
487
WT day 3 LVI, n=11 for Null day 0, n=9 for Null day 3 LVC, and n=9 for Null day 3 LVI. *p<0.05
488
vs control (WT or Null at day 0) and # p<0.05 vs remote at that same time point.
489
21
Figure 1
SPARC
soluble
43kD
insoluble
43kD
*##
WT
d0
WT
d3
LVC
WT
d3
LVI
*#
WT
d0
WT
d3
LVC
WT
d3
LVI
Figure 2
A
Null d0
WT d3 MI
Null d3 MI
Outsidee Circumference (mm
m)
WT d0
B
*+
*
WT
d0
WT
d3
Null
d0
Null
d3
Figure 3
A
WT
Day 0
2-ΔCT values
WT
Day 3
LVC
WT
Day 3
LVI
Null
Day 0
Null
Day 3
LVC
Null
Day 3
LVI
Adamts2
Cdh2
Vcam1
MMP3
Sgce
Ctnnb1
Itgav
Tgfb1
Itgam
Col4a1
g
Itgb1
Col4a2
Fn1
TIMP2
Adamts5
Lama2
Lamc1
Postn
Itga3
Col3a1
Ctgf
Pecam1
B
9
WT
1
9
9
1. Lamc1
2. Ctnnb1
3. Itgam
4. Timp2
5. Itgb1
Null
Day 0 Day 3
LVC
Day 3
LVI
WT
1
5
1. Itga3
2. Itgav
1
Null
1
9
Day 3
LVI
1. Fn1
9 Null
Null
1
Day 0 Day 3
LVC
Day 3
LVI
Day 0 Day 3
LVC
WT
0.2
Null
1
Day 3
LVI
WT
1
1. Cdh2
2. Col3a1
3. Vcam1
0.2
Day 0 Day 3
LVC
WT
1
1
1. Postn
2. Col4a2
g
3. Tgfb1
4. Ctgf
5. Sgce
6. Adamts2
9
2 Col4a1
2.
C l4 1
1
1
5
WT
1. MMP3
da ts5
2.. Adamts5
3. Lama2
4. Pecam1
0.2
1
Null
0.2
Day 0 Day 3
LVC
Day 3
LVI
Day 0 Day 3
LVC
Day 3
LVI
Figure 4
A. Fibronectin
soluble
insoluble
265kD
k
265kD
k
*#
*
+
WT
d3
LVI
Null
d0
*#+
WT
d0
WT
d3
LVC
WT
d3
LVI
Null
d0
Null
d3
LVC
Null
d3
LVI
WT
d0
WT
d3
LVC
Null
d3
LVC
Null
d3
LVI
B. CCN2 (CTGF)
soluble
WT
d0
insoluble
39.7kD
*
*
*
WT
d3
LVC
WT
d3
LVI
+
Null
d0
39.7kD
+
Null
d3
LVC
Null
d3
LVI
WT
d0
WT
d3
LVC
*
+
WT
d3
LVI
Null
d0
Null
d3
LVC
Null
d3
LVI
C MMP 3
C.MMP-3
Active
45kD
soluble
+
WT
d3
LVC
WT
d3
LVI
Active
45kD
insoluble
*#
*##
WT
d0
Pro
54kD
insoluble
Null
d0
Null
d3
LVC
Null
d3
LVI
*
WT
d0
WT
d3
LVC
WT
d3
LVI
Null
d0
Null
d3
LVC
Null
d3
LVI
WT
d0
WT
d3
LVC
WT
d3
LVI
Null
d0
Null
d3
LVC
Null
d3
LVI
Figure 5
A. Hsp47
p
soluble
47kD
*
WT
d0
WT
d3
LVC
WT
d3
LVI
Null
d0
Null
d3
LVC
Null
d3
LVI
B. CD31 (PECAM-1)
insoluble
100kD
*#
WT
d0
WT
d3
LVC
WT
d3
LVI
Null
d0
Null
d3
LVC
Null
d3
LVI