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Simultaneous Transforming Growth Factor β-Tumor Necrosis Factor Activation and Cross-talk Cause Aberrant Remodeling Response and Myocardial Fibrosis in Timp3-deficient Heart*

Open AccessPublished:July 22, 2009DOI:https://doi.org/10.1074/jbc.M109.028449
      The pleiotropic cytokines, transforming growth factor β1 (TGFβ1), and tumor necrosis factor (TNF) play critical roles in tissue homeostasis in response to injury and are implicated in multiple human diseases and cancer. We reported that the loss of Timp3 (tissue inhibitor of metalloproteinase 3) leads to abnormal TNF signaling and cardiovascular function. Here we show that parallel deregulation of TGFβ1 and TNF signaling in Timp3−/− mice amplifies their cross-talk at the onset of cardiac response to mechanical stress (pressure overload), resulting in fibrosis and early heart failure. Microarray analysis showed a distinct gene expression profile in Timp3−/− hearts, highlighting activation of TGFβ1 signaling as a potential mechanism underlying fibrosis. Neonatal cardiomyocyte-cardiofibroblast co-cultures were established to measure fibrogenic response to agonists known to be induced following mechanical stress in vivo. A stronger response occurred in neonatal Timp3−/− co-cultures, as determined by increased Smad signaling and collagen expression, due to increased TNF processing and precocious proteolytic maturation of TGFβ1 to its active form. The relationship between TGFβ1 and TNF was dissected using genetic and pharmacological manipulations. Timp3−/−/Tnf−/− mice had lower TGFβ1 than Timp3−/−, and anti-TGFβ1 antibody (1D11) negated the abnormal TNF response, indicating their reciprocal stimulatory effects, with each manipulation abolishing fibrosis and improving heart function. Thus, TIMP3 is a common innate regulator of TGFβ1 and TNF in tissue response to injury. The matrix-bound TIMP3 balances the anti-inflammatory and proinflammatory processes toward constructive tissue remodeling.
      Tissue repair requires the coordinated response between cellular and stromal compartments, involving regulated cytokine release, inflammation, cellular turnover, and structural remodeling, in order to restore organ function. The consequence of an inadequate remodeling program is reflected as necrosis, hyperplasia, and fibrosis, which are the hallmarks of multiple human diseases. Tissue fibrosis is the outcome of excessive and disorganized deposition of extracellular matrix (ECM)
      The abbreviations used are: ECM
      extracellular matrix
      Ang II
      angiotensin II
      PE
      phenylephrine
      TGFβ
      transforming growth factor β
      TNF
      tumor necrosis factor
      MMP
      matrix metalloproteinase
      AB
      aortic banding
      WT
      wild type
      PSR
      Picro-Sirius Red
      ELISA
      enzyme-linked immunosorbent assay
      CT
      cycle threshold
      qPCR
      quantitative PCR.
      3The abbreviations used are: ECM
      extracellular matrix
      Ang II
      angiotensin II
      PE
      phenylephrine
      TGFβ
      transforming growth factor β
      TNF
      tumor necrosis factor
      MMP
      matrix metalloproteinase
      AB
      aortic banding
      WT
      wild type
      PSR
      Picro-Sirius Red
      ELISA
      enzyme-linked immunosorbent assay
      CT
      cycle threshold
      qPCR
      quantitative PCR.
      proteins, resulting in disruption of normal tissue architecture and homeostasis that contributes to organ dysfunction. Myocardial fibrosis is the underlying cause of diastolic heart failure and a complicating factor in multiple heart disorders (
      • Zile M.R.
      • Baicu C.F.
      • Gaasch W.H.
      ,
      • Katz A.M.
      • Zile M.R.
      ,
      • Herpel E.
      • Pritsch M.
      • Koch A.
      • Dengler T.J.
      • Schirmacher P.
      • Schnabel P.A.
      ). Although cell surface-bound and soluble matrix metalloproteinases (MMPs) along with their natural tissue inhibitors (tissue inhibitors of metalloproteinase) constitute an important system for regulating ECM turnover (
      • Spinale F.G.
      ), inflammation is emerging as an important co-contributor to fibrosis (
      • Stramer B.M.
      • Mori R.
      • Martin P.
      ,
      • Sugimoto H.
      • Grahovac G.
      • Zeisberg M.
      • Kalluri R.
      ). Recent studies have linked specific subsets of metalloproteinases to inflammatory processes through their ability to cleave a wide variety of ECM-bound and cell surface cytokines (
      • Yu Q.
      • Stamenkovic I.
      ,
      • Murphy G.
      • Murthy A.
      • Khokha R.
      ). Common regulators of inflammatory cytokines and fibrogenic ligands may be the key to building an adequate tissue response, and their identification can lead to developing new strategies against tissue fibrosis.
      TGFβ1 is considered the major regulator of fibroblast response during normal ECM homeostasis as well as during the pathogenesis of fibrosis (
      • Gauldie J.
      • Bonniaud P.
      • Sime P.
      • Ask K.
      • Kolb M.
      ). TGFβ1-activated fibroblasts, characterized by the expression of α-smooth muscle actin, are the main source of collagen biosynthesis in the myocardium (
      • Stawowy P.
      • Kallisch H.
      • Veinot J.P.
      • Kilimnik A.
      • Prichett W.
      • Goetze S.
      • Seidah N.G.
      • Chrétien M.
      • Fleck E.
      • Graf K.
      ). TGFβ1 can trigger the differentiation of cardiac fibroblasts to activated myofibroblasts that synthesize collagen types I and III (
      • Petrov V.V.
      • Fagard R.H.
      • Lijnen P.J.
      ,
      • Asano Y.
      • Ihn H.
      • Yamane K.
      • Kubo M.
      • Tamaki K.
      ). It also regulates the proteolytic systems responsible for ECM turnover (
      • Overall C.M.
      • Wrana J.L.
      • Sodek J.
      ). Secreted TGFβ1 is sequestered and concentrated in the ECM by specific binding proteins, which render it biologically inactive (
      • Hyytiäinen M.
      • Penttinen C.
      • Keski-Oja J.
      ). Enhanced TGFβ1 signaling can result from defective matrix-binding due to fibrillin mutations as observed in Marfan syndrome or by increased release from the ECM through excessive proteolysis (
      • Yu Q.
      • Stamenkovic I.
      ,
      • Mu D.
      • Cambier S.
      • Fjellbirkeland L.
      • Baron J.L.
      • Munger J.S.
      • Kawakatsu H.
      • Sheppard D.
      • Broaddus V.C.
      • Nishimura S.L.
      ,
      • Jenkins G.
      ). As such, MMP2, -9, and -14 have been suggested to activate latent TGFβ1 directly, via cleavage and release of latency-associated peptide (
      • Yu Q.
      • Stamenkovic I.
      ,
      • Mu D.
      • Cambier S.
      • Fjellbirkeland L.
      • Baron J.L.
      • Munger J.S.
      • Kawakatsu H.
      • Sheppard D.
      • Broaddus V.C.
      • Nishimura S.L.
      ). MMPs and TGFβ1 also play a role in fibroblast migration (
      • Stawowy P.
      • Margeta C.
      • Kallisch H.
      • Seidah N.G.
      • Chrétien M.
      • Fleck E.
      • Graf K.
      ). Altogether, protease activity and TGFβ1 may cooperate at several levels to generate tissue fibrosis.
      TGFβ1 and TNF are two multifunctional cytokines, and each inhibits the signal transduction pathways activated by the other cytokine (
      • Inagaki Y.
      • Truter S.
      • Tanaka S.
      • Di Liberto M.
      • Ramirez F.
      ,
      • Bitzer M.
      • von Gersdorff G.
      • Liang D.
      • Dominguez-Rosales A.
      • Beg A.A.
      • Rojkind M.
      • Böttinger E.P.
      ). During fibrosis, both compete for p300, a transcriptional cofactor that can positively or negatively modulate collagen synthesis to orient the fibrotic response (
      • Ghosh A.K.
      • Varga J.
      ). TNF has been shown to negatively regulate TGFβ1 signaling through its central effector molecule NF-κB, which activates the inhibitory Smad7 (
      • Roman-Blas J.A.
      • Stokes D.G.
      • Jimenez S.A.
      ,
      • Goldberg M.T.
      • Han Y.P.
      • Yan C.
      • Shaw M.C.
      • Garner W.L.
      ), providing another means of cross-talk between these two cytokines. Both TGFβ1 and TNF exist as proproteins anchored to ECM and cell surface, respectively (
      • Murphy G.
      • Murthy A.
      • Khokha R.
      ,
      • Hyytiäinen M.
      • Penttinen C.
      • Keski-Oja J.
      ). Therefore, another level of control involves the proteolytic cleavage of TNF on the cell surface and of TGFβ1 anchored to the ECM, to become soluble effector proteins. Overall, an adequate interplay of these opposing pathways is critical for a coordinated cellular stress response, which depends in part on their extracellular bioavailability.
      Timp3−/− mice have an abnormal inflammatory response due to excessive TNF signaling (
      • Mohammed F.F.
      • Smookler D.S.
      • Taylor S.E.
      • Fingleton B.
      • Kassiri Z.
      • Sanchez O.H.
      • English J.L.
      • Matrisian L.M.
      • Au B.
      • Yeh W.C.
      • Khokha R.
      ,
      • Smookler D.S.
      • Mohammed F.F.
      • Kassiri Z.
      • Duncan G.S.
      • Mak T.W.
      • Khokha R.
      ). Timp3−/− hearts are more susceptible to mechanical stress induced by constriction of the aorta (aortic banding (AB)) and exhibit accelerated dilated cardiomyopathy with an early onset of heart failure, due to increased ADAM17 (a disintegrin and metalloproteinase 17)-mediated TNF processing and enhanced MMP-mediated ECM degradation (
      • Kassiri Z.
      • Oudit G.Y.
      • Sanchez O.
      • Dawood F.
      • Mohammed F.F.
      • Nuttall R.K.
      • Edwards D.R.
      • Liu P.P.
      • Backx P.H.
      • Khokha R.
      ). Here we show that after aortic banding, Timp3−/− as well as Timp3+/− mice develop marked myocardial fibrosis offering a model to study the role of deregulated cytokine signaling in fibrosis. Using microarrays, a primary neonatal cardiomyocyte-cardiofibroblast co-culture system, and genetic and pharmacological manipulations in mice, we demonstrate the importance of TGFβ-TNF co-activation and cross-talk. Our study highlights the unique ability of TIMP3 to co-regulate these cytokines in the heart.

      DISCUSSION

      Aortic constriction is a well established experimental model to generate cardiac pressure overload (mechanical stress), representing the condition in patients with hypertension or aortic stenosis. Such tissue perturbation must invoke a coordinated response between cellular and stromal compartments through the interplay of cytokines that are regulated both in time and space. We show that TIMP3 is a common stromal regulator of critical proinflammatory (TNF) and anti-inflammatory (TGFβ1) cytokines involved in this process, and its loss initiates a distinct gene expression program after aortic banding. The concurrent increase of these two cytokines occurs at the levels of RNA expression and protein maturation involving proteolytic cleavage, leading to their concomitant bioavailability as seen through the activation of their signal transduction pathways in TIMP3 null mice. Their reciprocal induction generates an aberrant cross-talk responsible for the unconstructive tissue remodeling, which culminates in myocardial fibrosis, dysfunction, and heart failure.
      Microarray analyses to map changes in global gene expression suggested an early involvement of TGFβ1 in Timp3−/− mice after aortic banding. To test directly the hypothesis that TGFβ1 is responsible for fibrosis in TIMP3-deficient mice, we established a well controlled in vitro system of primary neonatal murine cardiomyocytes and cardiofibroblasts that were cultured separately or in combination. This system mimics the cellular environment and in vivo responses for expression of cytokines, MMPs, and matrix molecules. We demonstrate that the cardiomyocyte-cardiofibroblast interaction is required for a strong fibrogenic response and that Timp3−/− co-cultures have the capacity to generate a more potent fibrogenic response compared with WT co-cultures in response to agonists. Using rat neonatal co-cultures, another group has shown the importance of fibroblast-myocyte interaction for Ang II-induced collagen production (
      • Pathak M.
      • Sarkar S.
      • Vellaichamy E.
      • Sen S.
      ). Furthermore, we show that co-cultures are far more efficient at TGFβ production and release. The molecular mechanism underlying the severe phenotype associated with TIMP3 deficiency includes increased transcription as well as proteolytic processing of TGFβ1 and TNF, heightened Smad2/3 phosphorylation, and up-regulation of specific metalloproteinases capable of cleaving interstitial collagens. Additionally, the co-culture setting reveals base-line perturbations in expression of collagen I, MMPs, and cytokines, indicating altered propensity toward matrix remodeling upon loss of TIMP3.
      Since a neonatal culture system bears limitations, we confirmed the role of TGFβ in the severe myocardial fibrosis in Timp3−/− hearts in vivo. The removal of TNF through the use of Tnf−/− mice or blocking TGFβ1 by 1D11 antibody allowed us to dissect the importance of each cytokine. TGFβ1 was still somewhat induced in Timp3−/−/Tnf−/− mice, whereas TGFβ1-neutralizing antibody profoundly suppressed TNF induction after aortic banding, indicating that the TGFβ1 effects may supersede those of TNF. Although TNF levels rise at later points in these mice, the MMP expressions remain suppressed, with the cardiomyopathy remaining significantly improved at 3 and 6 weeks post-AB. This could suggest that the initial rise in TNF (in the presence of TGFβ) in Timp3−/− heart is the key event in triggering the disease initiation and progression and involves MMP elevation. These data collectively show that severity of the cardiac phenotype resulting from the loss of TIMP3 is due to its simultaneous impact on two opposing cytokines, one anti-inflammatory and the other proinflammatory. It disrupts their time of action, the magnitude of increase, and co-regulation, resulting in maladaptive tissue remodeling that leads to fibrosis.
      TGFβ1 is a key regulator of inflammation and fibrosis and is itself regulated at several levels. TGFβ1 null mice develop a multifocal inflammatory disorder and die shortly after weaning (
      • Shull M.M.
      • Ormsby I.
      • Kier A.B.
      • Pawlowski S.
      • Diebold R.J.
      • Yin M.
      • Allen R.
      • Sidman C.
      • Proetzel G.
      • Calvin D.
      ,
      • Kulkarni A.B.
      • Huh C.G.
      • Becker D.
      • Geiser A.
      • Lyght M.
      • Flanders K.C.
      • Roberts A.B.
      • Sporn M.B.
      • Ward J.M.
      • Karlsson S.
      ), whereas TGFβ1 overexpression results in kidney, liver, and lung fibrosis (
      • Kopp J.B.
      • Factor V.M.
      • Mozes M.
      • Nagy P.
      • Sanderson N.
      • Böttinger E.P.
      • Klotman P.E.
      • Thorgeirsson S.S.
      ,
      • Sanderson N.
      • Factor V.
      • Nagy P.
      • Kopp J.
      • Kondaiah P.
      • Wakefield L.
      • Roberts A.B.
      • Sporn M.B.
      • Thorgeirsson S.S.
      ). In humans, TGFβ levels are increased in patients with fibrotic kidney disease, idiopathic pulmonary fibrosis, and hepatic fibrosis (
      • Blobe G.C.
      • Schiemann W.P.
      • Lodish H.F.
      ). Plasma levels of TGFβ1 are twice as high in patients who have developed idiopathic dilated cardiomyopathy compared with controls (
      • Kühl U.
      • Noutsias M.
      • Schultheiss H.P.
      ). TGFβ1 polymorphisms affect its level of expression and can play a role in predisposition to fibrotic disease (
      • Blobe G.C.
      • Schiemann W.P.
      • Lodish H.F.
      ,
      • Li B.
      • Khanna A.
      • Sharma V.
      • Singh T.
      • Suthanthiran M.
      • August P.
      ). For instance, a polymorphism that increases TGFβ1 production has been linked to the development of fibrotic lung disease (
      • Awad M.R.
      • El-Gamel A.
      • Hasleton P.
      • Turner D.M.
      • Sinnott P.J.
      • Hutchinson I.V.
      ), and one that correlates with elevated circulating levels of TGFβ1 is associated with dilated cardiomyopathy (
      • Holweg C.T.
      • Baan C.C.
      • Niesters H.G.
      • Vantrimpont P.J.
      • Mulder P.G.
      • Maat A.P.
      • Weimar W.
      • Balk A.H.
      ,
      • Yamada Y.
      • Miyauchi A.
      • Goto J.
      • Takagi Y.
      • Okuizumi H.
      • Kanematsu M.
      • Hase M.
      • Takai H.
      • Harada A.
      • Ikeda K.
      ). The current study reveals yet another level of regulation for the ECM-bound TGFβ1. Its bioavailability is controlled by the proteolytic release mediated by TIMP3-sensitive metalloproteinase(s), with the loss of TIMP3 resulting in myocardial fibrosis, diastolic dysfunction, and dilated cardiomyopathy. Interruption of the TGFβ pathway by an anti-TGFβ antibody (1D11) prevented myocardial fibrosis. Echocardiographic assessment of diastolic function mirrored the changes in systolic function; the diastolic filling parameters were more affected in Timp3−/−-AB mice but exhibited a marked improvement following TGFβ blockade. The improvement in diastolic dysfunction in these mice could be partly due to improved systolic function. These results are consistent with a pivotal role of maladaptive cytokine signaling in mediating systolic and diastolic dysfunctions.
      Myocardial fibrosis causes reduced compliance, compromised LV filling, and diastolic heart failure (
      • Mandinov L.
      • Eberli F.R.
      • Seiler C.
      • Hess O.M.
      ,
      • Senni M.
      • Redfield M.M.
      ,
      • Hogg K.
      • Swedberg K.
      • McMurray J.
      ). Fibrosis is a characteristic of hypertensive, hypertrophic, and dilated cardiomyopathy. A recent study reported the occurrence of interstitial fibrosis in dilated left ventricles even when no evidence of ischemic heart disease was observed (
      • Brooks A.
      • Schinde V.
      • Bateman A.C.
      • Gallagher P.J.
      ). Further, TIMP3 levels are significantly reduced in patients with dilated cardiomyopathy and heart failure (
      • Li Y.Y.
      • Feldman A.M.
      • Sun Y.
      • McTiernan C.F.
      ), and as we report here, even the loss of a single TIMP3 allele in mice leads to myocardial fibrosis. Thus, TIMP3 can be a powerful therapeutic candidate in strategies aimed at blocking myocardial fibrosis at early stages of heart disease.

      Acknowledgments

      We thank Aditya Murthy and Dr. Paul Waterhouse for critical review of the manuscript.

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