Mechanisms of Cardiac Fibrosis Induced by Urokinase Plasminogen Activator
2006; Elsevier BV; Volume: 281; Issue: 22 Linguagem: Inglês
10.1074/jbc.m512818200
ISSN1083-351X
AutoresApril Stempien‐Otero, Abigail Plawman, Jessica Meznarich, T. U. Dyamenahalli, Goro Otsuka, David A. Dichek,
Tópico(s)Peptidase Inhibition and Analysis
ResumoHuman hearts with end-stage failure and fibrosis have macrophage accumulation and elevated plasminogen activator activity. However, the mechanisms that link macrophage accumulation and plasminogen activator activity with cardiac fibrosis are unclear. We previously reported that mice with macrophage-targeted overexpression of urokinase plasminogen activator (SR-uPA+/o mice) develop cardiac macrophage accumulation by 5 weeks of age and cardiac fibrosis by 15 weeks. We used SR-uPA+/o mice to investigate mechanisms through which macrophage-expressed uPA causes cardiac macrophage accumulation and fibrosis. We hypothesized that: 1) macrophage accumulation and cardiac fibrosis in SR-uPA+/o mice are dependent on localization of uPA by the uPA receptor (uPAR); 2) activation of plasminogen by uPA and subsequent activation of transforming growth factor-β1 (TGF-β1) and matrix metalloproteinase (MMP)-2 and -9 by plasmin are critical pathways through which uPA-expressing macrophages accumulate in the heart and cause fibrosis; and 3) uPA-induced cardiac fibrosis can be attenuated by treatment with verapamil. To test these hypotheses, we bred the SR-uPA+/o transgene into mice deficient in either uPAR or plasminogen and measured cardiac macrophage accumulation and fibrosis. We also measured cardiac TGF-β1 protein (total and active), Smad2 phosphorylation, and MMP activity after the onset of macrophage accumulation but before the onset of cardiac fibrosis. Finally, we treated mice with verapamil. Our studies revealed that plasminogen is necessary for uPA-induced cardiac fibrosis and macrophage accumulation but uPAR is not. We did not detect plasmin-mediated activation of TGF-β1, MMP-2, or MMP-9 in hearts of SR-uPA+/o mice. However, verapamil treatment significantly attenuated both cardiac fibrosis and macrophage accumulation. Human hearts with end-stage failure and fibrosis have macrophage accumulation and elevated plasminogen activator activity. However, the mechanisms that link macrophage accumulation and plasminogen activator activity with cardiac fibrosis are unclear. We previously reported that mice with macrophage-targeted overexpression of urokinase plasminogen activator (SR-uPA+/o mice) develop cardiac macrophage accumulation by 5 weeks of age and cardiac fibrosis by 15 weeks. We used SR-uPA+/o mice to investigate mechanisms through which macrophage-expressed uPA causes cardiac macrophage accumulation and fibrosis. We hypothesized that: 1) macrophage accumulation and cardiac fibrosis in SR-uPA+/o mice are dependent on localization of uPA by the uPA receptor (uPAR); 2) activation of plasminogen by uPA and subsequent activation of transforming growth factor-β1 (TGF-β1) and matrix metalloproteinase (MMP)-2 and -9 by plasmin are critical pathways through which uPA-expressing macrophages accumulate in the heart and cause fibrosis; and 3) uPA-induced cardiac fibrosis can be attenuated by treatment with verapamil. To test these hypotheses, we bred the SR-uPA+/o transgene into mice deficient in either uPAR or plasminogen and measured cardiac macrophage accumulation and fibrosis. We also measured cardiac TGF-β1 protein (total and active), Smad2 phosphorylation, and MMP activity after the onset of macrophage accumulation but before the onset of cardiac fibrosis. Finally, we treated mice with verapamil. Our studies revealed that plasminogen is necessary for uPA-induced cardiac fibrosis and macrophage accumulation but uPAR is not. We did not detect plasmin-mediated activation of TGF-β1, MMP-2, or MMP-9 in hearts of SR-uPA+/o mice. However, verapamil treatment significantly attenuated both cardiac fibrosis and macrophage accumulation. Cardiac fibrosis, the accumulation of excess extracellular matrix in the heart, is a common feature of end-stage heart disease independent of etiology. Cardiac fibrosis may contribute to impaired systolic and diastolic function and is associated with both atrial and ventricular arrhythmias (1Weber K.T. Brilla C.G. Janicki J.S. Cardiovasc. Res. 1993; 27: 341-348Crossref PubMed Scopus (459) Google Scholar, 2Pogwizd S. McKenzie J. Cain M. Circulation. 1998; 98: 2404-2414Crossref PubMed Scopus (208) Google Scholar). Fibrotic cardiac tissue is relatively avascular (3Wang B. Ansari R. Sun Y. Postlethwaite A.E. Weber K.T. Kiani M.F. Am. J. Physiol. 2005; 289: H108-H113Crossref PubMed Scopus (35) Google Scholar), and cardiac fibroblasts are unable to propagate cardiac action potentials (for review see Ref. 4Kamkin A. Kiseleva I. Lozinsky I. Scholz H. Basic Res. Cardiol. 2005; 100: 337-345Crossref PubMed Scopus (68) Google Scholar). For these reasons, cardiac fibrosis will likely interfere with implementation of cell-based therapies for heart disease (5Schulze P.C. Lee R.T. Circ. Res. 2004; 95: 552-553Crossref PubMed Scopus (11) Google Scholar). Despite the importance of cardiac fibrosis, the mechanisms through which it develops are incompletely understood. Human and animal studies suggest that both macrophage accumulation and increased plasminogen activator (PA) 2The abbreviations used are: PA, plasminogen activator; uPA, urokinase plasminogen activator; uPAR, uPA receptor; TGF-β1, transforming growth factor β1; MMP, matrix metalloproteinase. activity contribute to the pathogenesis of cardiac fibrosis. Macrophage accumulation is present in fibrotic, end-stage human hearts (6Kuhl U. Noutsias M. Schultheiss H.P. Eur. Heart J. 1995; 16: 100-106Crossref PubMed Google Scholar, 7Azzawi M. Kan S.W. Hillier V. Yonan N. Hutchinson I.V. Hasleton P.S. Histopathology. 2005; 46: 314-319Crossref PubMed Scopus (32) Google Scholar). Macrophages express urokinase-type plasminogen activator (uPA) (8Chapman Jr., H.A. Stone O.L. Vavrin Z. J. Clin. Investig. 1984; 73: 806-815Crossref PubMed Scopus (117) Google Scholar), and increased PA activity is present, along with macrophages and fibrosis, in failing human hearts (9Tyagi S. Kumar S. Alla S. Reddy H. Voelker D. Janicki J. J. Cell Physiol. 1996; 167: 137-147Crossref PubMed Scopus (58) Google Scholar). Mice with increased macrophage PA activity have early cardiac macrophage accumulation and develop cardiac fibrosis later in life (10Moriwaki H. Stempien-Otero A. Kremen M. Cozen A.E. Dichek D.A. Circ. Res. 2004; 95: 637-644Crossref PubMed Scopus (112) Google Scholar). Moreover, mice that lack uPA are resistant to the development of cardiac fibrosis (11Heymans S. Luttun A. Nuyens D. Theilmeier G. Creemers E. Moons L. Dyspersin G.D. Cleutjens J.P.M. Shipley M. Angellilo A. Levi M. Nube O. Baker A. Keshet E. Lupu F. Herbert J.-M. Smits J.F.M. Shapiro S.D. Baes M. Borgers M. Collen D. Daemon M.J. Carmeliet P. Nat. Med. 1999; 5: 1135-1142Crossref PubMed Scopus (718) Google Scholar, 12Heymans S. Lupu F. Terclavers S. Vanwetswinkel B. Herbert J.M. Baker A. Collen D. Carmeliet P. Moons L. Am. J. Pathol. 2005; 166: 15-25Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). The pathways through which macrophage accumulation and increased cardiac PA activity could lead to cardiac fibrosis in both mice and humans are unknown. These pathways could include PA-mediated conversion of plasminogen to plasmin. Alternatively, cardiac fibrosis could be caused by plasminogen-independent actions of either PAs or macrophages. Definition of the pathways through which increased cardiac macrophage accumulation and PA activity lead to cardiac fibrosis may clarify the basic mechanisms of cardiac fibrosis and suggest new therapeutic approaches. Here we report the use of mice with macrophage-targeted expression of uPA (SR-uPA+/o mice (13Cozen A.E. Moriwaki H. Kremen M. DeYoung M.B. Dichek H.L. Slezicki K.I. Young S.G. Veniant M. Dichek D.A. Circulation. 2004; 109: 2129-2135Crossref PubMed Scopus (83) Google Scholar)) to investigate the mechanisms through which increased macrophage PA activity causes cardiac macrophage accumulation and fibrosis. SR-uPA+/o mice are an appropriate animal model for these investigations because, in the absence of infarction or any other overt cardiac injury, they develop cardiac macrophage accumulation by 5 weeks of age and cardiac fibrosis by 15 weeks (10Moriwaki H. Stempien-Otero A. Kremen M. Cozen A.E. Dichek D.A. Circ. Res. 2004; 95: 637-644Crossref PubMed Scopus (112) Google Scholar). We hypothesized that the uPA receptor (uPAR) (which can facilitate cell migration by focusing uPA and plasmin proteolytic activity to the leading edge of migrating cells (14Blasi F. Carmeliet P. Nat. Rev. Mol. Cell Biol. 2002; 3: 932-943Crossref PubMed Scopus (1089) Google Scholar)) is required for both cardiac macrophage accumulation and the subsequent development of fibrosis in SR-uPA+/o mice. Moreover, because transforming growth factor β1 (TGF-β1) and matrix metalloproteinases (MMPs) are plasmin substrates that are implicated as causes of cardiac fibrosis in other settings (15Lijnen P.J. Petrov V.V. Fagard R.H. Mol. Genet. Metab. 2000; 71: 418-435Crossref PubMed Scopus (409) Google Scholar, 16Kim H.E. Dalal S.S. Young E. Legato M.J. Weisfeldt M.L. D'Armiento J. J. Clin. Investig. 2000; 106: 857-866Crossref PubMed Scopus (238) Google Scholar), we hypothesized that plasmin-mediated activation of TGF-β1 and MMPs causes cardiac fibrosis in SR-uPA+/o mice. Finally, because uPA can cause arterial constriction, (17Falkenberg M. Tom C. DeYoung M.B. Wen S. Linnemann R. Dichek D.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10665-10670Crossref PubMed Scopus (50) Google Scholar), we hypothesized that treatment with the calcium channel inhibitor verapamil would reduce uPA-induced cardiac fibrosis and macrophage accumulation. Animals and Tissue Harvest—The SR-uPA+/o mice were back-crossed into the C57BL/6 background for at least eight generations and then bred with nontransgenic C57BL/6 mates to obtain experimental and control mice. C57BL/6 mice deficient in uPAR or heterozygous for plasminogen deficiency (Plaur –/– or Plg+/–) (18Bugge T.H. Flick M.J. Daugherty C.C. Degen J.L. Genes Dev. 1995; 9: 794-807Crossref PubMed Scopus (380) Google Scholar, 19Bugge T.H. Suh T.T. Flick M.J. Daugherty C.C. Romer J. Solberg V. Dano K. Degen J.L. J. Biol. Chem. 1995; 270: 16886-16894Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar) were purchased (The Jackson Laboratory). Mice were housed under specific pathogen-free conditions. Pups were weaned at 4 weeks of age and genotyped for the SR-uPA transgene by Southern blot or PCR of tail DNA. Genotyping of the Plg and Plaur alleles was performed by PCR of tail DNA using published primers (18Bugge T.H. Flick M.J. Daugherty C.C. Degen J.L. Genes Dev. 1995; 9: 794-807Crossref PubMed Scopus (380) Google Scholar, 19Bugge T.H. Suh T.T. Flick M.J. Daugherty C.C. Romer J. Solberg V. Dano K. Degen J.L. J. Biol. Chem. 1995; 270: 16886-16894Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Some of the SR-uPA+/o mice (n = 9) were treated with verapamil (1 mg/ml in 10% dextrose as drinking water) beginning at 5 weeks of age. Littermate controls (n = 8) received 10% dextrose alone. After 60 days, the mice were killed and their hearts processed for histologic analysis of macrophage and collagen content as described below. All animal protocols were approved by the University of Washington Office of Animal Welfare. To obtain hearts for histologic and biochemical analysis, deeply anesthetized mice were exsanguinated, and hearts were excised, placed in phosphate-buffered saline, transferred to phosphate-buffered saline with 5% dextrose and 25 mm KCl to produce cardiac arrest, and then placed in sucrose formalin fixative. Hearts were sectioned into three pieces (base, midventricle, and apex) and processed into a single paraffin block. Histologic Analyses—Macrophages were detected with a rat monoclonal antibody (anti-Mac-3, clone M3/84, 2.5 μg/ml; Pharmingen) (10Moriwaki H. Stempien-Otero A. Kremen M. Cozen A.E. Dichek D.A. Circ. Res. 2004; 95: 637-644Crossref PubMed Scopus (112) Google Scholar). Bound antibody was detected with peroxidase-conjugated goat anti-rat IgG (Kirkegaard and Perry) and diaminobenzidine substrate. Control slides were incubated with isotype-matched primary antibodies (Pharmingen). We quantified cardiac macrophages by counting Mac-3-stained cells in each of two or three sections spaced at least 1 mm apart. Cells were counted in 10 random high power (×400) microscopic fields per section (20–30 fields per heart), and the average macrophage density in each heart was calculated. Collagen accumulation was quantified by picrosirius red staining of a single section from the midventricle of each heart. Computer-assisted image analysis (Image Pro 3.0 software, Media Cybernetics) was used to quantify the red-stained area of each section. Quantification of cardiac macrophages and collagen was done by observers blinded to genotype. Measurement of Plasminogen Activator Activity of Bone Marrow-derived Macrophages—Cultured macrophages were used for this assay, which was performed not as a direct measurement of in vivo uPA activity but to determine whether the SR-uPA transgene was still expressed by macrophages of Plg–/– mice. Bone marrow was harvested from the femurs of 8–10-week-old mice by flushing with RPMI 1640 with 2% fetal bovine serum and 5 IU/ml heparin. The marrow cells were washed in Hanks' balanced salt solution and resuspended in Dulbecco's modified Eagle's medium with 10% heat-inactivated fetal calf serum, 1% each penicillin/streptomycin and l-glutamine, and 10% L-cell conditioned medium as a source of granulocyte-macrophage colony-stimulating factor (20Hume D.A. Gordon S. J. Cell. Physiol. 1983; 117: 189-194Crossref PubMed Scopus (76) Google Scholar). Culture media and nonadherent cells were removed, and new medium added at days 4 and 8. This protocol yields 104-106 macrophages/femur. On day 10, the medium was changed to M199. Conditioned medium was collected after 20 h and stored at –80 °C. Cells were counted and lysed, and total lysate protein was measured using the DC protein assay (Bio-Rad). Plasminogen activator activity was detected by incubating aliquots of macrophage-conditioned medium with Glu-plasminogen (0.4 μm; American Diagnostica) and the plasmin substrate S-2251 (0.9 mm; Chromogenix) and measuring the change in absorbance at 405 nm. PA activity was calculated with reference to a standard curve constructed with human single-chain uPA (American Diagnostica). Measurement of TGF-β1 Secreted by Explanted Hearts—The apical half of a heart was minced and placed in M199. After two 30-min incubations in fresh M199, the pieces were transferred to fresh M199 and incubated overnight at 37 °C. Conditioned media were collected and stored at –80 °C. Active TGF-β1 (no acid activation) and total TGF-β1 (acid activated) were measured by enzyme-linked immunosorbent assay of conditioned media (Promega). Immunoblot for Phosphorylated Smad2—The basal half of a heart was snap-frozen in liquid nitrogen, ground over ice, homogenized with a Polytron, and extracted in lysis buffer (50 mm Tris, pH 7.5, 150 mm NaCl, 0.5% Nonidet P-40, Halt protease inhibitor mixture, 10 μl/ml (Pierce)). Negative and positive controls for the presence of phospho-Smad2 consisted of lysates of AML12 cells treated with vehicle or TGF-β1 (21Romero-Gallo J. Sozmen E.G. Chytil A. Russell W.E. Whitehead R. Parks W.T. Holdren M.S. Her M.F. Gautam S. Magnuson M. Moses H.L. Grady W.M. Oncogene. 2005; 24: 3028-3041Crossref PubMed Scopus (113) Google Scholar). 3W. T. Parks (University of Washington), personal communication. Samples were resolved by SDS-polyacrylamide gel electrophoresis on 10% gels and transferred to polyvinylidene difluoride membranes in 25 mm Tris, 192 mm glycine, 5% methanol at 85 V for 3 h at 4 °C. Filters were blocked overnight with TBS-T (10 mm Tris-HCl, pH 7.4, 150 mm NaCl, 0.05% Tween 20) containing 5% skim milk. Immunoblots were analyzed for phospho-Smad-2 (Cell Signaling, catalog no. 3101) using the antibody at a 1:1000 dilution or a β-actin antibody (Abcam, catalog no. 8227-50) at a 1:5000 dilution. Immunoreactive proteins were detected according to the enhanced chemiluminescence protocol (Amersham Biosciences) using 1:10,000 horseradish peroxidase-linked anti-rabbit secondary antiserum (Abcam, catalog no. 6721-1). Blots were exposed to film for 1–10 min. MMP Detection—The basal half of a heart was snap-frozen in liquid nitrogen, ground with a mortar and pestle over liquid nitrogen, homogenized with a Polytron, and placed in lysis buffer (10 mmol/liter cacodylic acid, 0.15 mol/liter NaCl, 20 mmol/liter ZnCl, 1.5 mmol/liter NaN3, and 0.01% Triton X-100, pH 5.0) on ice for 30 min (22Coker M.L. Thomas C.V. Clair M.J. Hendrick J.W. Krombach R.S. Galis Z.S. Spinale F.G. Am. J. Physiol. 1998; 274: H1516-H1523PubMed Google Scholar). Extracted protein was measured using the DC protein assay kit (Bio-Rad), and equivalent amounts of protein were loaded with nonreducing sample buffer into precast 10% polyacrylamide gels containing gelatin or casein (Bio-Rad). Culture medium from HT-1080 cells stimulated with PMA was loaded as a positive control for gelatinolytic and caseinolytic activity. Gels were run at 4 °C, renatured in renaturation buffer (Bio-Rad) for 90 min at room temperature with shaking, rinsed, and placed in development buffer (Bio-Rad) at 37 °C overnight. The next day gels were stained with Coomassie Blue for 8 h, washed, and placed on a gel-drying rack. Statistical Analysis—Because much of the data were not normally distributed, data are presented as median (25–75% range) and group medians are compared with the Mann-Whitney rank-sum test. uPAR Is Not Required for uPA-induced Cardiac Fibrosis or Macrophage Accumulation—To test whether uPAR is a critical mediator of macrophage accumulation and cardiac fibrosis in SR-uPA+/o mice, we began by breeding SR-uPA+/o mice with nontransgenic mice deficient in uPAR (Plaur–/– mice). At 15 weeks of age, SR-uPA+/o Plaur–/– hearts had significantly more fibrosis than hearts of SR-uPAo/o Plaur–/– littermates (8.5 (3.6–13%) versus 0.45 (0.25–1.5%) picrosirius red-positive area; p = 0.001 (Fig. 1A)). To test whether uPAR is a critical mediator of macrophage accumulation in hearts of SR-uPA+/o mice, we counted macrophages in hearts of 15-week-old SR-uPA+/o Plaur–/– and SR-uPAo/o Plaur–/– littermates. SR-uPA+/o Plaur–/– mice had significantly more cardiac macrophages than SR-uPAo/o Plaur–/– littermates (51 (33–85) versus 1.8 (1.2–2.1) Mac-3-positive cells/mm2; p = 0.002 (Fig. 1B)). SR-uPA+/o Plaur–/– mice had a similar degree of cardiac fibrosis and macrophage accumulation as our previously reported SR-uPA+/o Plaur+/+ mice (10Moriwaki H. Stempien-Otero A. Kremen M. Cozen A.E. Dichek D.A. Circ. Res. 2004; 95: 637-644Crossref PubMed Scopus (112) Google Scholar). Plasminogen Is Required for uPA-induced Cardiac Fibrosis—To test whether plasminogen is a critical mediator of uPA-induced cardiac fibrosis, we began by breeding SR-uPA+/o Plg+/+ mice with nontransgenic Plg+/– mice. F1 mice were then intercrossed to generate SR-uPA+/o Plg+/+, SR-uPA+/o Plg–/–, SR-uPAo/o Plg+/+, and SR-uPAo/o Plg–/– littermates. Hearts of 15-week-old SR-uPA+/o Plg–/– mice had significantly less fibrosis than littermate SR-uPA+/o Plg+/+ mice (0.56 (0.45–0.63%) versus 5.9 (2.4–14%) picrosirius red-positive area; p = 0.01 (Fig. 2)). In addition, SR-uPA+/o Plg–/– mice had the same amount of cardiac fibrosis as nontransgenic Plg–/– littermates (0.56 (0.45–0.63%) versus 0.60 (0.46–0.79%) p = 0.68 (Fig. 2)). Plasminogen Is Required for Macrophage Accumulation in SR-uPA+/o Hearts—We measured macrophage accumulation in hearts of 15-week-old SR-uPA+/o Plg+/+, SR-uPA+/o Plg–/–, SR-uPAo/o Plg+/+, and SR-uPAo/o Plg–/– littermates. In Plg–/– mice, the SR-uPA+/o transgene did not increase cardiac macrophage accumulation (0.69 (0–0.9) Mac-3-positive cells/mm2 for SR-uPA+/o Plg–/– mice versus 0.6 (0.34–0.79) for nontransgenic Plg–/– littermates; p = 1.0 (Fig. 3)). Moreover, there were significantly more macrophages in hearts of SR-uPA+/o Plg+/+ mice (21 (13–38); p = 0.008 (Fig. 3)) than in hearts of SR-uPA+/o Plg–/– mice. PA Activity of SR-uPA+/o Macrophages Is Not Affected by the Absence of Plasminogen—To exclude the possibility that SR-uPA transgene expression in SR-uPA+/o mice might be altered in the Plg–/– background or that SR-uPA transgene expression might have been lost over time, we collected conditioned media from bone marrow-derived macrophages harvested from SR-uPA+/o Plg+/+, SR-uPA+/o Plg–/–, and SR-uPAo/o Plg–/– mice. Media from SR-uPA+/o Plg–/– macrophages had PA activity equal to the PA activity of media from SR-uPA+/oPlg+/+ macrophages (1.5 (1.1–2.0) versus 0.9 (0.7–1.5) IU/106 cells/20 h; p = 0.29 (Fig. 4)). Media from SR-uPA+/o Plg–/– macrophages had significantly greater PA activity than media conditioned by macrophages from nontransgenic Plg–/– mice, in which PA activity was uniformly below the limit of detection (p < 0.02; Fig. 4). Cardiac Fibrosis in SR-uPA+/o Mice Is Not Associated with Increased TGF-β1 Activity—Because plasmin can convert latent TGF-β1 to active TGF-β1 (23Lyons R.M. Gentry L.E. Purchio A.F. Moses H.L. J. Cell Biol. 1990; 110: 1361-1367Crossref PubMed Scopus (674) Google Scholar), and active TGF-β1 can cause cardiac fibrosis (24Rosenkranz S. Flesch M. Amann K. Haeuseler C. Kilter H. Seeland U. Schluter K.D. Bohm M. Am. J. Physiol. 2002; 283: H1253-H1262Crossref PubMed Scopus (253) Google Scholar), we tested the hypothesis that uPA-induced cardiac fibrosis is associated with increased cardiac TGF-β1 protein and activity. Total and active TGF-β1 protein were measured in media conditioned by hearts explanted from 5-week-old SR-uPA+/o Plg+/+, SR-uPA+/o Plg–/–, SR-uPAo/o Plg+/+, and SR-uPAo/o Plg–/– littermates. We chose the 5-week time point because it is after the onset of macrophage accumulation but before the onset of fibrosis in SR-uPA+/o mice (10Moriwaki H. Stempien-Otero A. Kremen M. Cozen A.E. Dichek D.A. Circ. Res. 2004; 95: 637-644Crossref PubMed Scopus (112) Google Scholar). Neither total nor active TGF-β1 protein was increased in explant cultures of hearts of 5-week-old SR-uPA+/o mice (Fig. 5, A and B). To gain confidence that we had not missed an increase in cardiac TGF-β1, we repeated this assay with hearts from 7–8-week-old SR-uPA+/o Plg+/+ and SR-uPAo/o Plg+/+ mice. Again, neither total nor active TGF-β1 was increased in media conditioned by SR-uPA+/o hearts (Fig. 5, C and D). Finally, to determine whether cardiac TGF-β1 signaling was increased even in the absence of detectable differences in TGF-β1 protein in conditioned media, we assayed extracts of hearts of 7–8-week-old SR-uPA+/o Plg+/+ and SR-uPAo/o Plg+/+ mice for phosphorylated Smad2 (phospho-Smad2). Phospho-Smad2 was detected in all heart extracts; however, there was no increase in phospho-Smad2 in hearts of SR-uPA+/o mice (Fig. 6).FIGURE 6Immunoblot for phosphorylated Smad2. Immunoblot of protein extracts of hearts from 7–8-week-old SR-uPA+/o or nontransgenic mice. Blots were probed with antibodies to phospho-Smad2 (P-Smad2) and β-actin.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Gelatinase Activity Is Not Increased in Hearts of SR-uPA+/o Mice—To test whether activity of the gelatinases MMP-2 and -9 was increased in hearts of SR-uPA+/o mice at the onset of macrophage accumulation and before the onset of cardiac fibrosis, we performed gelatin zymography of extracts of hearts from 5-week-old mice. Extracts of hearts of SR-uPA+/o Plg+/+, SR-uPA+/o Plg–/–, and SR-uPAo/o Plg+/+ mice had equivalent activity of pro-MMP-2 and no detectable active MMP-2 or MMP-9 (Fig. 7). To measure activity of MMP-3, -7, and -13, aliquots of the same samples were electrophoresed into casein-containing gels. Culture medium from HT-1080 cells was used as a positive control. No caseinolytic activity was detected in any of the samples (data not shown). Treatment with Verapamil Attenuates uPA-induced Cardiac Fibrosis and Macrophage Accumulation—To test whether verapamil could prevent cardiac fibrosis, we treated SR-uPA+/o mice with verapamil from 5 to 13.5 weeks of age. We chose the 5-week time point because it is after the onset of macrophage accumulation but before the onset of fibrosis in SR-uPA+/o mice (10Moriwaki H. Stempien-Otero A. Kremen M. Cozen A.E. Dichek D.A. Circ. Res. 2004; 95: 637-644Crossref PubMed Scopus (112) Google Scholar). Hearts of 13.5-week-old SR-uPA+/o mice treated with verapamil had significantly less fibrosis than littermate SR-uPA+/o mice treated with placebo (3.0 (2.0–3.4%) versus 10 (8.8–15%) picrosirius red-positive area; p = 0.008 (Fig. 8A)). Verapamil-treated mice also had significantly less cardiac macrophage accumulation (37 (21–48) versus 132 (88–181) Mac-3-positive cells/mm2; p = 0.005 (Fig. 8B)). We used a mouse model of macrophage-targeted uPA overexpression (SR-uPA+/o mice) to identify downstream mediators of uPA-induced macrophage accumulation and cardiac fibrosis. Our major findings were as follows. 1) uPAR is not required for uPA/plasmin-induced cardiac macrophage accumulation and fibrosis. 2) Plasminogen is necessary for macrophage accumulation and for the development of cardiac fibrosis. 3) TGF-β1 is not a critical mediator of uPA/plasmin-induced cardiac fibrosis. 4) The activity of gelatinolytic or caseinolytic cardiac MMPs is not increased before the onset of uPA/plasmin-induced cardiac fibrosis. 5) Treatment with verapamil after the onset of macrophage accumulation substantially limits further cardiac macrophage accumulation and fibrosis. Thus, although plasmin is a crucial mediator of uPA-induced cardiac fibrosis, plasmin substrates previously associated with cardiac fibrosis (TGF-β1 and MMPs) do not appear to contribute to either cardiac macrophage accumulation or fibrosis in SR-uPA+/o mice. However, uPA-induced cardiac macrophage accumulation and fibrosis depend substantially on verapamil-sensitive pathways. Increased cardiac macrophage accumulation and PA activity are associated with cardiac fibrosis in humans and in animal models of human cardiac disease (7Azzawi M. Kan S.W. Hillier V. Yonan N. Hutchinson I.V. Hasleton P.S. Histopathology. 2005; 46: 314-319Crossref PubMed Scopus (32) Google Scholar, 9Tyagi S. Kumar S. Alla S. Reddy H. Voelker D. Janicki J. J. Cell Physiol. 1996; 167: 137-147Crossref PubMed Scopus (58) Google Scholar, 25Tyagi S. Kumar S. Haas S. Reddy H. Voelker D. Hayden M. Demmy T. Schmaltz R. Curtis J. J. Mol. Cell. 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