Nonclassical Resident Macrophages Are Important Determinants in the Development of Myocardial Fibrosis
2015; Elsevier BV; Volume: 185; Issue: 4 Linguagem: Inglês
10.1016/j.ajpath.2014.11.027
ISSN1525-2191
AutoresAlec Falkenham, Roberto de Antueno, Nicole L. Rosin, Devin Betsch, Timothy D.G. Lee, Roy Duncan, Jean‐François Légaré,
Tópico(s)Immune cells in cancer
ResumoMacrophages are increasingly recognized as a potential therapeutic target in myocardial fibrosis via interactions with fibroblasts. We have characterized macrophage depletion and inhibition of nonclassical macrophage migration, in addition to direct interactions between nonclassical macrophages and fibroblasts in angiotensin II (AngII)-mediated, hypertensive myocardial fibrosis. Macrophage depletion was achieved by daily i.v. clodronate liposomes (−1 day to +3 days) during AngII infusion. Cx3cr1−/− mice were used to inhibit nonclassical macrophage migration. Macrophage phenotype (F4/80, CD11b, Ly6C) was characterized by immunofluorescence and flow cytometry. Collagen was assessed by Sirius Red/Fast Green. Quantitative real-time RT-PCR was performed for transcript levels. AngII/wild-type (WT) mice displayed significant infiltrate and fibrosis compared with saline/WT, which was virtually ablated by clodronate liposomes independent of hypertension. In vitro data supported M2 macrophages promoting fibroblast differentiation and collagen production. AngII/Cx3cr1−/− mice, however, significantly increased macrophage infiltrate and fibrosis relative to AngII/WT. AngII/Cx3cr1−/− mice also showed an M1 phenotypic shift relative to WT mice in, which the predominant phenotype was Ly6Clow, CD206+ (M2). Myocardial IL-1β was significantly up-regulated, whereas transforming growth factor β down-regulated with this M1 shift. We demonstrated that infiltrating macrophages are critical to AngII-mediated myocardial fibrosis by preventing the development of fibrosis after liposomal depletion of circulating monocytes. Our findings also suggest that some macrophages, namely M2, may confer a protective myocardial environment that may prevent excessive tissue injury. Macrophages are increasingly recognized as a potential therapeutic target in myocardial fibrosis via interactions with fibroblasts. We have characterized macrophage depletion and inhibition of nonclassical macrophage migration, in addition to direct interactions between nonclassical macrophages and fibroblasts in angiotensin II (AngII)-mediated, hypertensive myocardial fibrosis. Macrophage depletion was achieved by daily i.v. clodronate liposomes (−1 day to +3 days) during AngII infusion. Cx3cr1−/− mice were used to inhibit nonclassical macrophage migration. Macrophage phenotype (F4/80, CD11b, Ly6C) was characterized by immunofluorescence and flow cytometry. Collagen was assessed by Sirius Red/Fast Green. Quantitative real-time RT-PCR was performed for transcript levels. AngII/wild-type (WT) mice displayed significant infiltrate and fibrosis compared with saline/WT, which was virtually ablated by clodronate liposomes independent of hypertension. In vitro data supported M2 macrophages promoting fibroblast differentiation and collagen production. AngII/Cx3cr1−/− mice, however, significantly increased macrophage infiltrate and fibrosis relative to AngII/WT. AngII/Cx3cr1−/− mice also showed an M1 phenotypic shift relative to WT mice in, which the predominant phenotype was Ly6Clow, CD206+ (M2). Myocardial IL-1β was significantly up-regulated, whereas transforming growth factor β down-regulated with this M1 shift. We demonstrated that infiltrating macrophages are critical to AngII-mediated myocardial fibrosis by preventing the development of fibrosis after liposomal depletion of circulating monocytes. Our findings also suggest that some macrophages, namely M2, may confer a protective myocardial environment that may prevent excessive tissue injury. Myocardial fibrosis is a common feature in cardiovascular diseases and is suspected to be the final pathway in the development of heart failure.1Leask A. Signaling in fibrosis: targeting the TGF beta, endothelin-1 and CCN2 axis in scleroderma.Front Biosci (Elite Ed). 2009; 1: 115-122PubMed Google Scholar The characteristic feature of myocardial fibrosis is the abnormal accumulation of extracellular matrix (ECM) proteins. As ECM proteins accumulate, the heart stiffens, and myocardial dysfunction develops that can ultimately culminate in heart failure. In the United States alone, almost 6 million people are afflicted with heart failure, which contributes to an estimated >300,000 deaths per year, emphasizing the importance of understanding the mechanisms underlying the development of myocardial fibrosis.2Go A.S. Mozaffarian D. Roger V.L. Benjamin E.J. Berry J.D. Blaha M.J. et al.Heart disease and stroke statistics–2014 update: a report from the American Heart Association.Circulation. 2013; 129: e28-e292Crossref PubMed Scopus (4492) Google Scholar Fibroblasts are described as the effector cell responsible for ECM deposition; however, there is increasing evidence that macrophages may be key orchestrators of fibrosis through their interactions with fibroblasts.3Schulze P.C. Lee R.T. Macrophage-mediated cardiac fibrosis.Circ Res. 2004; 95: 552-553Crossref PubMed Scopus (9) Google Scholar, 4Wang L. Li Y.L. Zhang C.C. Cui W. Wang X. Xia Y. Du J. Li H.H. Inhibition of Toll-like receptor 2 reduces cardiac fibrosis by attenuating macrophage-mediated inflammation.Cardiovasc Res. 2014; 101: 383-392Crossref PubMed Scopus (93) Google Scholar, 5Ma F. Li Y. Jia L. Han Y. Cheng J. Li H. Qi Y. Du J. 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The macrophage F4/80 receptor is required for the induction of antigen-specific efferent regulatory T cells in peripheral tolerance.J Exp Med. 2005; 201: 1615-1625Crossref PubMed Scopus (272) Google Scholar This chemokine receptor expression is mirrored in mice; however, mice do not express CD16, and the M1 to M2 transition is characterized by the loss of Ly6C expression.9Shi C. Pamer E.G. Monocyte recruitment during infection and inflammation.Nat Rev Immunol. 2011; 11: 762-774Crossref PubMed Scopus (1839) Google Scholar, 10Lin H.H. Faunce D.E. Stacey M. Terajewicz A. Nakamura T. Zhang-Hoover J. Kerley M. Mucenski M.L. Gordon S. Stein-Streilein J. The macrophage F4/80 receptor is required for the induction of antigen-specific efferent regulatory T cells in peripheral tolerance.J Exp Med. 2005; 201: 1615-1625Crossref PubMed Scopus (272) Google Scholar Thus, the consistency of the M1/M2 paradigm between mice and humans supports the use of mouse models to characterize the roles of monocyte and macrophage subsets in disease. Although M1 macrophage-mediated inflammation can worsen tissue injury and exacerbate fibrotic responses, M2 macrophages can also produce anti-inflammatory and profibrotic factors such as IL-10, transforming growth factor β (TGF-β), connective tissue growth factor (CTGF), and platelet-derived growth factor to directly promote fibroblast proliferation and ECM production.6Shen J.Z. Morgan J. Tesch G.H. Fuller P.J. Young M.J. CCL2-dependent macrophage recruitment is critical for mineralocorticoid receptor-mediated cardiac fibrosis, inflammation, and blood pressure responses in male mice.Endocrinology. 2014; 155: 1057-1066Crossref PubMed Scopus (38) Google Scholar, 11Aoyama T. Inokuchi S. Brenner D.A. Seki E. CX3CL1-CX3CR1 interaction prevents carbon tetrachloride-induced liver inflammation and fibrosis in mice.Hepatology. 2010; 52: 1390-1400Crossref PubMed Scopus (131) Google Scholar, 12Heymann F. Trautwein C. Tacke F. Monocytes and macrophages as cellular targets in liver fibrosis.Inflamm Allergy Drug Targets. 2009; 8: 307-318Crossref PubMed Scopus (145) Google Scholar These macrophage activation states do not exist exclusively from one another. Rather, the contribution of macrophage subsets to fibrosis is part of a dynamic response determined by the demands of healing tissues and characterized by differential monocyte recruitment and the ability to shift between functional phenotypes. Macrophage phenotype change during fibrotic responses is suggested in a number of fibrosis models, including liver, lung, and kidney; however, the evidence in the myocardium is only suggestive and limited to ischemic injury.3Schulze P.C. Lee R.T. Macrophage-mediated cardiac fibrosis.Circ Res. 2004; 95: 552-553Crossref PubMed Scopus (9) Google Scholar, 12Heymann F. Trautwein C. Tacke F. Monocytes and macrophages as cellular targets in liver fibrosis.Inflamm Allergy Drug Targets. 2009; 8: 307-318Crossref PubMed Scopus (145) Google Scholar, 13Vernon M.A. Mylonas K.J. Hughes J. Macrophages and renal fibrosis.Semin Nephrol. 2010; 30: 302-317Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 14Pechkovsky D.V. Prasse A. Kollert F. Engel K.M. Dentler J. Luttmann W. Friedrich K. Müller-Quernheim J. Zissel G. Alternatively activated alveolar macrophages in pulmonary fibrosis-mediator production and intracellular signal transduction.Clin Immunol. 2010; 137: 89-101Crossref PubMed Scopus (228) Google Scholar Work in our laboratory by using a hypertension model of myocardial fibrosis has shown that myocardial fibrosis is preceded by a mass influx of circulating mononuclear cells characteristic of macrophages, expressing markers such as ED1, CD11b, and F4/80.15Sopel M.J. Rosin N.L. Lee T.D. Légaré J.F. Myocardial fibrosis in response to Angiotensin II is preceded by the recruitment of mesenchymal progenitor cells.Lab Invest. 2011; 91: 565-578Crossref PubMed Scopus (54) Google Scholar, 16Sopel M. Falkenham A. Oxner A. Ma I. Lee T.D. Legare J.F. Fibroblast progenitor cells are recruited into the myocardium prior to the development of myocardial fibrosis.Int J Exp Pathol. 2012; 93: 115-124Crossref PubMed Scopus (29) Google Scholar Infiltrating monocytes can dramatically expand the macrophage population in the heart. Recent findings have also suggested that an important resident macrophage population exists in the myocardium, thus providing an additional source of macrophage-fibroblast interactions, but their role in myocardial fibrosis is unknown.17Pinto A.R. Paolicelli R. Salimova E. Gospocic J. Slonimsky E. Bilbao-Cortes D. Godwin J.W. Rosenthal N.A. An abundant tissue macrophage population in the adult murine heart with a distinct alternatively-activated macrophage profile.PLoS One. 2012; 7: e36814Crossref PubMed Scopus (215) Google Scholar These new knowledge could mean that monocytes and macrophages may represent important therapeutic targets for myocardial fibrosis rather than targeting the final effector cell, the fibroblast. However, elucidating the contributions of the different macrophage populations and specific subsets in myocardial fibrosis needs to be better understood and is a necessary step toward macrophage-dependent therapies. Liposomal monocyte depletion and chemokine receptor knockouts are valuable tools for characterizing the contribution of macrophages subsets.18Buiting A.M. Van Rooijen N. Liposome mediated depletion of macrophages: an approach for fundamental studies.J Drug Target. 1994; 2: 357-362Crossref PubMed Scopus (38) Google Scholar, 19van Rooijen N. Liposomes for targeting of antigens and drugs: immunoadjuvant activity and liposome-mediated depletion of macrophages.J Drug Target. 2008; 16: 529-534Crossref PubMed Scopus (26) Google Scholar, 20Wynn T.A. Chawla A. Pollard J.W. Macrophage biology in development, homeostasis and disease.Nature. 2013; 496: 445-455Crossref PubMed Scopus (2761) Google Scholar Here, we use the well-established angiotensin II (AngII) model to study myocardial fibrosis, which depends on sustained hypertension and renin angiotensin aldosterone system activation as is seen clinically in patients.15Sopel M.J. Rosin N.L. Lee T.D. Légaré J.F. Myocardial fibrosis in response to Angiotensin II is preceded by the recruitment of mesenchymal progenitor cells.Lab Invest. 2011; 91: 565-578Crossref PubMed Scopus (54) Google Scholar, 16Sopel M. Falkenham A. Oxner A. Ma I. Lee T.D. Legare J.F. Fibroblast progenitor cells are recruited into the myocardium prior to the development of myocardial fibrosis.Int J Exp Pathol. 2012; 93: 115-124Crossref PubMed Scopus (29) Google Scholar, 21Falkenham A. Sopel M. Rosin N. Lee T.D. Issekutz T. Légaré J.F. Early fibroblast progenitor cell migration to the AngII-exposed myocardium is not CXCL12 or CCL2 dependent as previously thought.Am J Pathol. 2013; 183: 459-469Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 22Rosin N.L. Falkenham A. Sopel M.J. Lee T.D. Legare J.F. Regulation and role of connective tissue growth factor in AngII-induced myocardial fibrosis.Am J Pathol. 2013; 182: 714-726Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 23Rosin N.L. Sopel M. Falkenham A. Myers T.L. Legare J.F. Myocardial migration by fibroblast progenitor cells is blood pressure dependent in a model of angII myocardial fibrosis.Hypertens Res. 2012; 35: 449-456Crossref PubMed Scopus (13) Google Scholar The AngII model is characterized by significant macrophage infiltrate, a profibrotic milieu, and significant ECM deposition without the loss of healthy tissue as seen in ischemic injury.15Sopel M.J. Rosin N.L. Lee T.D. Légaré J.F. Myocardial fibrosis in response to Angiotensin II is preceded by the recruitment of mesenchymal progenitor cells.Lab Invest. 2011; 91: 565-578Crossref PubMed Scopus (54) Google Scholar, 16Sopel M. Falkenham A. Oxner A. Ma I. Lee T.D. Legare J.F. Fibroblast progenitor cells are recruited into the myocardium prior to the development of myocardial fibrosis.Int J Exp Pathol. 2012; 93: 115-124Crossref PubMed Scopus (29) Google Scholar, 24Sugino H. Ozono R. Kurisu S. Matsuura H. Ishida M. Oshima T. Kambe M. Teranishi Y. Masaki H. Matsubara H. Apoptosis is not increased in myocardium overexpressing type 2 angiotensin II receptor in transgenic mice.Hypertension. 2001; 37: 1394-1398Crossref PubMed Scopus (50) Google Scholar Furthermore, our work with Ccr2−/− mice in this model suggests that the nonclassical Cx3cr1 pathway may be important to nonischemic myocardial healing.21Falkenham A. Sopel M. Rosin N. Lee T.D. Issekutz T. Légaré J.F. Early fibroblast progenitor cell migration to the AngII-exposed myocardium is not CXCL12 or CCL2 dependent as previously thought.Am J Pathol. 2013; 183: 459-469Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar This model offers the unique opportunity to study myocardial fibrosis without conflicting ischemia that is seen after myocardial infarction and to focus the depletion to the fibrotic stage of the response. Here, we have characterized early macrophage accumulation and the effects of monocyte depletion on the development of AngII-mediated myocardial fibrosis. Specifically, we examined the effects of liposome monocyte depletion on cellular infiltrate, the profibrotic environment, and the development of myocardial fibrosis. In addition, we are the first group to characterize myocardial healing in the absence of Cx3cr1 signaling, the outcomes of which could have important therapeutic implications. Animal experiments were performed in accordance with the Canadian Council on Animal Care and approved by the Dalhousie University Committee on Laboratory Animals. Male C57BL/6 mice [8 to 10 weeks old; wild-type (WT)] were purchased from The Jackson Laboratory (Bar Harbor, ME), and male Cx3cr1−/− mice (8 to 10 weeks old) on a C57BL/6 background were acquired through a material transfer agreement with Taconic Farms (Germantown, NY).25Combadiere C. Potteaux S. Gao J.L. Esposito B. Casanova S. Lee E.J. Debre P. Tedgui A. Murphy P.M. Mallat Z. Decreased atherosclerotic lesion formation in CX3CR1/apolipoprotein E double knockout mice.Circulation. 2003; 107: 1009-1016Crossref PubMed Scopus (407) Google Scholar Mice were housed in the Carleton Animal Care Facility at Dalhousie University and were provided food and water ad libitum for 1 week before beginning experimentation. Osmotic minipumps (Alzet, Palo Alto, CA) were implanted as previously described.21Falkenham A. Sopel M. Rosin N. Lee T.D. Issekutz T. Légaré J.F. Early fibroblast progenitor cell migration to the AngII-exposed myocardium is not CXCL12 or CCL2 dependent as previously thought.Am J Pathol. 2013; 183: 459-469Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar Mice were randomly assigned treatments of vehicle (saline) or 2.8 mg/kg per day AngII (Sigma-Aldrich, Oakville, ON, Canada). The pumps remained in for 3 days, during which time, the mice were observed for morbidity. Mice were weighed, and blood pressure measurements were taken with the Coda2 noninvasive tail cuff system (Kent Scientific, Torrington, CT) for a minimum of five measurements per mouse. At the time of harvest, hearts were extracted, flushed with saline, and weighed. Liposomes were generated as previously described, with modification.26van Rooijen N. Hendrikx E. Liposomes for specific depletion of macrophages from organs and tissues.Methods Mol Biol. 2010; 605: 189-203Crossref PubMed Scopus (262) Google Scholar In brief, egg phosphotidyl choline and cholesterol (82.4:15.6% mol/L ratio, respectively) in chloroform were added to sterile pear-shaped flasks and placed under rotary evaporation until excess chloroform was removed to dryness, and a uniform film developed. Lipid-soluble DiO was added for liposome binding and uptake studies. The film was subsequently hydrated with either 300 mg/mL clodronate (Sigma-Aldrich) or saline, and liposomes were left to swell for 2 hours at room temperature. Liposomes were then sonicated (4 minutes at 55 Hz) and washed of free clodronate by centrifuging four to five times at 24,000 × g for 5 minutes at 10°C. Before injection, liposomes were passed through a 3.0-μm pore filter (Millipore, Billerica, MA). The final concentration of lipid in the liposomes was 33.07 mmol/L. Liposomes were injected in 100-μL doses via tail vein every 24 hours, beginning at −1 day before AngII infusion. At 3 days, mice were sacrificed, and blood was collected for smears. Smears were stained with Giemsa (GS500; Sigma-Aldrich) for nuclear morphology, and leukocyte populations were counted by a blind observer (A.F.). A total of 350 cells were characterized per slide. Hearts were processed in 10% buffered formalin overnight and paraffin-embedded. Serial sections (5 μm) were stained for histologic analysis. Basic myocardial histology and cellular infiltrate were assessed with heart sections stained with hematoxylin and eosin. The area of the heart affected was calculated as previously described.21Falkenham A. Sopel M. Rosin N. Lee T.D. Issekutz T. Légaré J.F. Early fibroblast progenitor cell migration to the AngII-exposed myocardium is not CXCL12 or CCL2 dependent as previously thought.Am J Pathol. 2013; 183: 459-469Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 22Rosin N.L. Falkenham A. Sopel M.J. Lee T.D. Legare J.F. Regulation and role of connective tissue growth factor in AngII-induced myocardial fibrosis.Am J Pathol. 2013; 182: 714-726Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 23Rosin N.L. Sopel M. Falkenham A. Myers T.L. Legare J.F. Myocardial migration by fibroblast progenitor cells is blood pressure dependent in a model of angII myocardial fibrosis.Hypertens Res. 2012; 35: 449-456Crossref PubMed Scopus (13) Google Scholar Fibrotic deposition was examined with heart sections stained with Picosirius Red (SR) and the counterstain Fast Green (FG). Collagen content was semiquantified by photographing representative SR/FG whole heart sections at ×5 magnification. With the use of Photoshop CS6 (Adobe Systems, San Jose, CA), red pixels were positively selected and summed for a total number of red (collagen) pixels. Subsequently, nonbackground pixels were summed for the total heart pixels. Collagen pixels were divided by the total heart pixels to provide a semiquantitative measurement of the percentage of collagen content in the heart. All tissues were processed simultaneously for SR/FG, and the same red color palette was used to select red pixels. For frozen sections, anesthetized mice were perfused with saline, followed by 4% paraformaldehyde. Hearts were extracted and placed in 4% paraformaldehyde overnight. The hearts were transferred to 30% sucrose, and 10-μm sections were cut on a cryostat. Immunofluorescence was performed on both frozen heart sections and fixed cocultures for CD11b (AbD Serotec, Raleigh, NC), collagen type-I (Rockland, Gilbertsville, PA), F4/80 (Abcam, Cambridge, MA), α-smooth muscle cell actin (α-SMA; Sigma-Aldrich), CD206 (AbD Serotec), CX3CR1 (Abcam), and CD107b (AbD Serotec). In brief, sections were permeabilized with 0.1% Triton X-100/phosphate-buffered saline (PBS), blocked with 5% bovine serum albumin/PBS, and then incubated with primary antibodies overnight at 4°C. Sections were then incubated with Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 647, or biotin-conjugated secondary antibodies. Biotin-conjugated antibodies were linked to avidin and developed with diaminobenzidine. In fluorescence, nuclei were stained with DAPI. Slides were visualized with Axiovert 200M (Carl Zeiss Inc., Thornwood, NY) and photographed with an Orca R2 camera (Hamamatsu Corporation, Bridgewater, NJ). The macrophage-fibroblast coculture was conducted as previously described with modification.5Ma F. Li Y. Jia L. Han Y. Cheng J. Li H. Qi Y. Du J. Macrophage-stimulated cardiac fibroblast production of IL-6 is essential for TGF β/Smad activation and cardiac fibrosis induced by angiotensin II.PLoS One. 2012; 7: e35144Crossref PubMed Scopus (202) Google Scholar, 27Holt D.J. Chamberlain L.M. Grainger D.W. Cell-cell signaling in co-cultures of macrophages and fibroblasts.Biomaterials. 2010; 31: 9382-9394Crossref PubMed Scopus (95) Google Scholar Bone marrow-derived macrophages (BMDMs) were generated as previously described.28Weischenfeldt J. Porse B. Bone marrow-derived macrophages (BMM): isolation and applications.CSH Protoc. 2008; (doi: 10.1101/pdb.prot5080)PubMed Google Scholar In brief, mice were anesthetized and then sacrificed, and the femurs and tibias were isolated. Cells were flushed from the marrow by using a 30-gauge needle attached to a syringe that contained DMEM (Dulbecco's Modified Eagle Media) complete (DMEM-C: DMEM, l-glutamine, Pen Strep, 10% fetal bovine serum). After washes and cell straining, BMDMs were resuspended in DMEM-C with 10% L929 conditioned medium and plated in T75 flasks. Media were replaced on day 5. On day 7, the BMDMs were lifted with 0.25% trypsin, washed with DMEM-C, counted, and replated-plated in 12-well plates at a density of 2 × 105 per well. Additional macrophages were screened for CD11b and Ly6C expression by using flow cytometry as described in Immunohistochemistry and Immunofluorescence. NIH/3T3 cells (ATCC, Manassas, VA) served as fibroblasts in monocultures and cocultures. NIH/3T3 cells were maintained in DMEM-C until lifted with 0.25% trypsin, washed with DMEM-C, counted, and replated at 2 × 105 per well. Monocultures and cocultures were allowed to sit down overnight in DMEM-C before being serum-starved for 24 hours. The next day, the media were replaced with DMEM-C, and cells were incubated for 48 hours. At 48 hours, the supernatant fluid was removed, and the cells were washed with PBS, then fixed in 4% paraformaldehyde. The cells were immunofluorescently labeled as described in Immunohistochemistry and Immunofluorescence, and wells were read for fluorescence by using a Tecan infinite M200 Pro (Tecan, Männedorf, Germany) plate reader. α-SMA expression was standardized to DAPI fluorescence intensity for cocultures and fibroblast monocultures. The coculture expression was then calculated relative to fibroblast monoculture expression. Immunofluorescence and flow cytometry were used to quantify cell purity. Hearts from WT and knockout mice infused with AngII for 3 days were harvested under sterile conditions and were used for cell isolation as previously described with modification.16Sopel M. Falkenham A. Oxner A. Ma I. Lee T.D. Legare J.F. Fibroblast progenitor cells are recruited into the myocardium prior to the development of myocardial fibrosis.Int J Exp Pathol. 2012; 93: 115-124Crossref PubMed Scopus (29) Google Scholar, 21Falkenham A. Sopel M. Rosin N. Lee T.D. Issekutz T. Légaré J.F. Early fibroblast progenitor cell migration to the AngII-exposed myocardium is not CXCL12 or CCL2 dependent as previously thought.Am J Pathol. 2013; 183: 459-469Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar In brief, hearts were mechanically and enzymatically digested in a collagenase solution (0.652 mg/mL collagenase II; Cedarlane Laboratories, Ltd., Burlington, ON, Canada) in DMEM-C at 37°C, with agitation for 45 minutes. Cell isolates were twice washed in DMEM-C and purified over a Percoll gradient (30% and 70%). Cells were then counted and used for flow cytometric analysis. Cells were washed with fluorescence-activated cell sorting buffer (Dulbecco PBS, 1% bovine serum albumin, 0.1% NaN3) and incubated with antibodies anti-CD11b-allophycocyanin (eBioscience, San Diego, CA), anti-F4/80–peridinin chlorophyll protein complex cyanine 5.5 (Biolegend, San Diego, CA), and anti-Ly6C-phycoerythrin (Biolegend). After incubation, cells were washed twice with fluorescence-activated cell sorting buffer and fixed with 1% formalin/ fluorescence-activated cell sorting buffer. Isotype control (Santa Cruz Biotechnology, Santa Cruz, CA) was used for negative gating. Data were acquired with a BD FACSCalibur (BD Biosciences, San Jose, CA) and were analyzed with WinList 3D (Verity Software, Topsham, ME). Macrophage gating was performed by gating on cells with the use of forward and side scatter (SSC), which was then applied to a CD11b × F4/80 dot plot. The F4/80+ events were gated and applied to a CD11b × Ly6C dot plot for the characterization Ly6C mean fluorescent index of mature and immature macrophage populations in the myocardium. When F4/80 labeling was not performed, CD11b × SSC was used to distinguish polymorphonuclear cells (SSChigh) and monocyte-derived cells (SSClow−mid). Before echocardiography, mice were anesthetized, and the fur was removed with hair removal cream (Nair; Church & Dwight Co., Inc., Ewing, NJ). Images and recordings were acquired on a Vivid 7 Ultrasound (GE Healthcare, Chalfont St. Giles, UK). Short-axis M-mode measurements for left ventricular posterior wall and intraventricular septal wall thicknesses were performed in Photoshop CS6 (Adobe Systems). RNA isolation, cDNA generation, and quantitative real-time PCR were performed as previously described.15Sopel M.J. Rosin N.L. Lee T.D. Légaré J.F. Myocardial fibrosis in response to Angiotensin II is preceded by the recruitment of mesenchymal progenitor cells.Lab Invest. 2011; 91: 565-578Crossref PubMed Scopus (54) Google Scholar, 21Falkenham A. Sopel M. Rosin N. Lee T.D. Issekutz T. Légaré J.F. Early fibroblast progenitor cell migration to the AngII-exposed myocardium is not CXCL12 or CCL2 dependent as previously thought.Am J Pathol. 2013; 183: 459-469Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 22Rosin N.L. Falkenham A. Sopel M.J. Lee T.D. Legare J.F. Regulation and role of connective tissue growth factor in AngII-induced myocardial fibrosis.Am J Pathol. 2013; 182: 714-726Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar Primers were designed against mRNA sequences of chemokine (C-X3-C motif) ligand 1 (CX3CL1; forward, 5′-GCAACCCCACCCCTTATCAA-3′; reverse, 5′-CTTGCCAGCCCTCAGAATCA-3′), TGF-β (forward, 5′-CTCTTGAGTCCCTCGCATCC-3′; reverse, 5′-GG
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