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Secreted Frizzled-related Protein 5 Diminishes Cardiac Inflammation and Protects the Heart from Ischemia/Reperfusion Injury*

  • Kazuto Nakamura
    Affiliations
    From the Whitaker Cardiovascular Institute, Boston University Medical Campus, Boston, Massachusetts 02118 and
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  • Soichi Sano
    Affiliations
    From the Whitaker Cardiovascular Institute, Boston University Medical Campus, Boston, Massachusetts 02118 and
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  • José J. Fuster
    Affiliations
    From the Whitaker Cardiovascular Institute, Boston University Medical Campus, Boston, Massachusetts 02118 and
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  • Ryosuke Kikuchi
    Affiliations
    From the Whitaker Cardiovascular Institute, Boston University Medical Campus, Boston, Massachusetts 02118 and
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  • Ippei Shimizu
    Affiliations
    From the Whitaker Cardiovascular Institute, Boston University Medical Campus, Boston, Massachusetts 02118 and
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  • Kousei Ohshima
    Affiliations
    From the Whitaker Cardiovascular Institute, Boston University Medical Campus, Boston, Massachusetts 02118 and
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  • Yasufumi Katanasaka
    Affiliations
    From the Whitaker Cardiovascular Institute, Boston University Medical Campus, Boston, Massachusetts 02118 and
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  • Noriyuki Ouchi
    Affiliations
    From the Whitaker Cardiovascular Institute, Boston University Medical Campus, Boston, Massachusetts 02118 and

    the Department of Molecular Cardiovascular Medicine, Nagoya University Graduate School of Medicine, Nagoya, Aichi 466-8550, Japan
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  • Kenneth Walsh
    Correspondence
    To whom correspondence should be addressed: Molecular Cardiology/Whitaker Cardiovascular Institute, Boston University Medical Campus, 700 Albany St., W611, Boston, MA 02118. Tel.: 617-414-2390; Fax: 617-414-2391; E-mail:
    Affiliations
    From the Whitaker Cardiovascular Institute, Boston University Medical Campus, Boston, Massachusetts 02118 and
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  • Author Footnotes
    * This work was supported by National Institutes of Health Grants HL081587, HL116591, HL126141, and HL120160 (to K. W.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the author and does not necessarily represent the official views of the National Institutes of Health.
    ♦ This article was selected as a Paper of the Week.
Open AccessPublished:December 02, 2015DOI:https://doi.org/10.1074/jbc.M115.693937
      Wnt signaling has diverse actions in cardiovascular development and disease processes. Secreted frizzled-related protein 5 (Sfrp5) has been shown to function as an extracellular inhibitor of non-canonical Wnt signaling that is expressed at relatively high levels in white adipose tissue. The aim of this study was to investigate the role of Sfrp5 in the heart under ischemic stress. Sfrp5 KO and WT mice were subjected to ischemia/reperfusion (I/R). Although Sfrp5-KO mice exhibited no detectable phenotype when compared with WT control at baseline, they displayed larger infarct sizes, enhanced cardiac myocyte apoptosis, and diminished cardiac function following I/R. The ischemic lesions of Sfrp5-KO mice had greater infiltration of Wnt5a-positive macrophages and greater inflammatory cytokine and chemokine gene expression when compared with WT mice. In bone marrow-derived macrophages, Wnt5a promoted JNK activation and increased inflammatory gene expression, whereas treatment with Sfrp5 blocked these effects. These results indicate that Sfrp5 functions to antagonize inflammatory responses after I/R in the heart, possibly through a mechanism involving non-canonical Wnt5a/JNK signaling.

      Introduction

      Inflammation is widely recognized to be involved in the pathogenesis, severity, and outcome of ischemic heart disease (
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      ). Obesity is thought to contribute to cardiovascular disorders, at least in part, through the systemic release of pro-inflammatory adipokines by dysfunctional white adipose tissue (WAT)
      The abbreviations used are: WAT, white adipose tissue; I/R, ischemia/reperfusion; Sfrp, secreted frizzled-related protein; BMDM, bone marrow-derived macrophage; NRVM, neonatal rat ventricular myocyte; AAR, area at risk; IA, infarction area; LV, left ventricular; LVEDD, LV end diastolic diameter; LVESD, LV end systolic diameter; %FS, percentage of fractional shortening; qRT-PCR, quantitative real-time PCR; GSK-3β, glycogen synthase kinase 3β.
      (
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      At the cellular level, inflammatory processes are tightly orchestrated by secreted signaling molecules that bind to specific cell surface receptors and activate intracellular signaling pathways. Signaling by Wnt ligands is a major regulator of several biological processes, but its roles in modulating inflammatory responses are relatively understudied. The 19 Wnt family proteins contain cysteine-rich domains and activate signaling by binding to one or more of the 10 frizzled family receptors (
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      ). Wnt signaling can be classified as canonical or a non-canonical (
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      ,
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      ). Canonical Wnt signaling involves activation of the β-catenin signaling pathway. Non-canonical Wnt signaling involves other pathways including planar cell polarity and Ca2+ pathways (
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      ). Wnt3a is the prototypical ligand that induces canonical signaling, whereas Wnt5a has mostly been associated with non-canonical signaling. Canonical and non-canonical Wnt signaling pathways are generally thought to functionally oppose each other. It is reported that both canonical and non-canonical Wnt signaling pathways play an important role in cardiac development and myocyte differentiation (
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      ), and in the adult, have been reported to be involved in maladaptive hypertrophy (
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      ).
      Both canonical and non-canonical Wnt signaling pathways can be regulated by several endogenous antagonists, including Wnt inhibitory factor (WIF), Cerberus (CER), and Dickkopf (Dkk) (
      • Kawano Y.
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      Secreted antagonists of the Wnt signalling pathway.
      ). Secreted frizzled-related proteins (Sfrps) represent a family of decoy receptors that negatively modulate Wnt signaling (
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      Fritz: a secreted frizzled-related protein that inhibits Wnt activity.
      • Bovolenta P.
      • Esteve P.
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      • Lopez-Rios J.
      Beyond Wnt inhibition: new functions of secreted Frizzled-related proteins in development and disease.
      ). Sfrps have cysteine-rich domain domains (
      • Bovolenta P.
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      • Lopez-Rios J.
      Beyond Wnt inhibition: new functions of secreted Frizzled-related proteins in development and disease.
      ,
      • Rattner A.
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      • Copeland N.G.
      • Jenkins N.A.
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      A family of secreted proteins contains homology to the cysteine-rich ligand-binding domain of frizzled receptors.
      ), and the antagonistic effect of Sfrps on signaling is thought to be mediated by the ability to bind either Wnt ligands and/or the frizzled receptors (
      • Kikuchi A.
      • Yamamoto H.
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      Multiplicity of the interactions of Wnt proteins and their receptors.
      ,
      • Kawano Y.
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      Secreted antagonists of the Wnt signalling pathway.
      ,
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      ).
      Accumulating evidence implicates the participation of Sfrps in ischemic cardiovascular diseases. It is reported that Sfrp2 activates mesenchymal stem cells and promotes cardiac repair following myocardial infarction via inhibition of bone morphogenic protein (BMP) and canonical Wnt signaling (
      • Alfaro M.P.
      • Vincent A.
      • Saraswati S.
      • Thorne C.A.
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      • Lee E.
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      sFRP2 suppression of bone morphogenic protein (BMP) and Wnt signaling mediates mesenchymal stem cell (MSC) self-renewal promoting engraftment and myocardial repair.
      ,
      • He W.
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      • Mao L.
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      • Dzau V.J.
      Exogenously administered secreted frizzled related protein 2 (Sfrp2) reduces fibrosis and improves cardiac function in a rat model of myocardial infarction.
      ). Sfrp1 has also been shown to have a protective role in post-infarct left ventricular (LV) remodeling (
      • Barandon L.
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      • Leroux L.
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      • Allières C.
      • Lamazière J.M.
      • Dufourcq P.
      • Couffinhal T.
      • Duplàa C.
      Secreted frizzled-related protein-1 improves postinfarction scar formation through a modulation of inflammatory response.
      ). Furthermore, the combined blockade of Wnt/frizzled signaling by Wnt3a/Wnt5a homologues suppresses development of heart failure following myocardial infarction (
      • Laeremans H.
      • Hackeng T.M.
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      • Thijssen V.L.
      • Janssen B.J.
      • Ottenheijm H.C.
      • Smits J.F.
      • Blankesteijn W.M.
      Blocking of frizzled signaling with a homologous peptide fragment of Wnt3a/Wnt5a reduces infarct expansion and prevents the development of heart failure after myocardial infarction.
      ). Although these studies provide compelling evidence for the importance of Sfrp/Wnt signaling in cardiac development and disease, the role of this signaling pathway in the context of inflammatory cardio-metabolic disease has not been investigated.
      We and other groups have reported that Sfrp5 is highly expressed in WAT and is involved in obesity and metabolic function (
      • Ouchi N.
      • Higuchi A.
      • Ohashi K.
      • Oshima Y.
      • Gokce N.
      • Shibata R.
      • Akasaki Y.
      • Shimono A.
      • Walsh K.
      Sfrp5 is an anti-inflammatory adipokine that modulates metabolic dysfunction in obesity.
      • Schulte D.M.
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      • Güdelhöfer H.
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      • Krone W.
      • Laudes M.
      Pro-inflammatory wnt5a and anti-inflammatory sFRP5 are differentially regulated by nutritional factors in obese human subjects.
      ,
      • Jura M.
      • Jaroslawska J.
      • Chu D.T.
      • Kozak L.P.
      Mest and Sfrp5 are biomarkers for healthy adipose tissue.
      ,
      • Flehmig G.
      • Scholz M.
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      • Fasshauer M.
      • Tönjes A.
      • Stumvoll M.
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      • Blüher M.
      Identification of adipokine clusters related to parameters of fat mass, insulin sensitivity and inflammation.
      ,
      • Hu W.
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      • Luo X.
      • Ran W.
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      • Xiong Z.
      • Liu H.
      • Yang G.
      Circulating Sfrp5 is a signature of obesity-related metabolic disorders and is regulated by glucose and liraglutide in humans.
      • Prats-Puig A.
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      • Ibáñez L.
      • Bassols J.
      • López-Bermejo A.
      Balanced duo of anti-inflammatory SFRP5 and proinflammatory WNT5A in children.
      ). Most of these reports are consistent with the interpretation that Sfrp5 has an anti-inflammatory effect and that it exerts these effects through the suppression of non-canonical Wnt5a/JNK signaling pathway. To date, nothing has been reported about the role of Sfrp5 in ischemic heart disease. Here, we investigate whether Sfrp5 affects the inflammatory response of the heart to acute ischemic injury. Specifically, we tested the effects of Sfrp5 on myocardial infarct size, apoptotic activity, and inflammatory responses following ischemia reperfusion (I/R) injury. We also tested the effects of Sfrp5 on mouse bone marrow-derived macrophage (BMDM) phenotype in vitro. Our observations indicate that Sfrp5 is cardio-protective in the context of I/R injury. This protection is associated with the inhibition of Wnt5a/JNK-dependent anti-inflammatory actions of Sfrp5.

      Discussion

      The data from this study show, for the first time, that Sfrp5 confers resistance to damage caused by acute myocardial ischemia. The genetic deficiency of Sfrp5 in mice led to a greater myocardial infarct size following I/R injury, and this was accompanied by a greater amount of apoptotic cell death of cardiac myocytes and a greater degree of inflammation in the infarct zone. These data indicate a new mechanism by which obesity contributes to heart disease. A growing body of evidence suggests that obesity contributes to cardiovascular disease through unbalanced adipokine secretion that contributes to a chronic low-grade inflammatory state (
      • Ouchi N.
      • Parker J.L.
      • Lugus J.J.
      • Walsh K.
      Adipokines in inflammation and metabolic disease.
      ). In this regard, we identified Sfrp5 as an anti-inflammatory adipokine that antagonizes the pro-inflammatory activity of Wnt5a, a regulator of non-canonical Wnt signaling (
      • Ouchi N.
      • Higuchi A.
      • Ohashi K.
      • Oshima Y.
      • Gokce N.
      • Shibata R.
      • Akasaki Y.
      • Shimono A.
      • Walsh K.
      Sfrp5 is an anti-inflammatory adipokine that modulates metabolic dysfunction in obesity.
      ). Utilizing genetic models, it was shown that Sfrp5/Wnt5a signaling is a regulator of adipose tissue inflammation and systemic metabolic health (
      • Ouchi N.
      • Higuchi A.
      • Ohashi K.
      • Oshima Y.
      • Gokce N.
      • Shibata R.
      • Akasaki Y.
      • Shimono A.
      • Walsh K.
      Sfrp5 is an anti-inflammatory adipokine that modulates metabolic dysfunction in obesity.
      ,
      • Fuster J.J.
      • Zuriaga M.A.
      • Ngo D.T.
      • Farb M.G.
      • Aprahamian T.
      • Yamaguchi T.P.
      • Gokce N.
      • Walsh K.
      Noncanonical Wnt signaling promotes obesity-induced adipose tissue inflammation and metabolic dysfunction independent of adipose tissue expansion.
      ). Here we show that genetic ablation of Sfrp5, a condition that is mimicked by severe metabolic dysfunction, is sufficient to promote adverse effects on the ischemic heart that are associated with elevated inflammation in the injured myocardium. Notably, the effects of Sfrp5 deficiency can be observed in mice fed a normal chow diet, which have normal systemic metabolic function (
      • Ouchi N.
      • Higuchi A.
      • Ohashi K.
      • Oshima Y.
      • Gokce N.
      • Shibata R.
      • Akasaki Y.
      • Shimono A.
      • Walsh K.
      Sfrp5 is an anti-inflammatory adipokine that modulates metabolic dysfunction in obesity.
      ), suggesting a direct effect on the injured myocardium. Furthermore, these data in metabolically normal mice document that the modulation of Sfrp5 expression is sufficient to confer changes in cardiovascular function independent of any potential confounding metabolic actions.
      Mechanistically, the infarcted hearts of the Sfrp5-deficient mice displayed enhanced inflammation and greater levels of myocyte death. A loss of Sfrp5 led to greater cytokine and chemokine production in the infarct area, and this was accompanied by the greater influx of Wnt5a-positive macrophages. In cell culture studies, Wnt5a promotes the expression of pro-inflammatory mediators, and this activity is completely blocked by co-incubation with Sfrp5. Although Wnt signaling has been extensively studied in the context of development and differentiation, it has more recently been appreciated that Wnt5a can function as a modulator of innate immunity in adult organisms (
      • Fuster J.J.
      • Zuriaga M.A.
      • Ngo D.T.
      • Farb M.G.
      • Aprahamian T.
      • Yamaguchi T.P.
      • Gokce N.
      • Walsh K.
      Noncanonical Wnt signaling promotes obesity-induced adipose tissue inflammation and metabolic dysfunction independent of adipose tissue expansion.
      ,
      • Pereira C.
      • Schaer D.J.
      • Bachli E.B.
      • Kurrer M.O.
      • Schoedon G.
      Wnt5A/CaMKII signaling contributes to the inflammatory response of macrophages and is a target for the antiinflammatory action of activated protein C and interleukin-10.
      ,
      • Naskar D.
      • Maiti G.
      • Chakraborty A.
      • Roy A.
      • Chattopadhyay D.
      • Sen M.
      Wnt5a-Rac1-NF-κB homeostatic circuitry sustains innate immune functions in macrophages.
      • Pereira C.P.
      • Bachli E.B.
      • Schoedon G.
      The wnt pathway: a macrophage effector molecule that triggers inflammation.
      ). Wnt5a is recognized as the prototypical regulator of non-canonical Wnt signaling, partly through its ability to activate the planar cell polarity pathway. JNK is a key component of the planar cell polarity pathway, and this study found that JNK is activated to a greater extent in the infarcted myocardium of the Sfrp5-deficient mice and that Wnt5a activates JNK in cultured macrophages in an Sfrp5-repressible manner. JNK signaling participates in inflammatory responses and is central in the process of macrophage-driven metabolic dysfunction in obesity (
      • Han M.S.
      • Jung D.Y.
      • Morel C.
      • Lakhani S.A.
      • Kim J.K.
      • Flavell R.A.
      • Davis R.J.
      JNK expression by macrophages promotes obesity-induced insulin resistance and inflammation.
      ). Consistent with these data, we recently provided evidence that macrophage Wnt5a expression is associated with greater inflammation and impaired revascularization in a murine model of peripheral artery disease (
      • Kikuchi R.
      • Nakamura K.
      • MacLauchlan S.
      • Ngo D.T.
      • Shimizu I.
      • Fuster J.J.
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      • Yoshida S.
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      • Matsushita T.
      • Murohara T.
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      • Bates D.O.
      • Hamburg N.M.
      • Walsh K.
      An antiangiogenic isoform of VEGF-A contributes to impaired vascularization in peripheral artery disease.
      ).
      There is a high degree of complexity in the Wnt signaling system (
      • Dawson K.
      • Aflaki M.
      • Nattel S.
      Role of the Wnt-Frizzled system in cardiac pathophysiology: a rapidly developing, poorly understood area with enormous potential.
      ,
      • Deb A.
      Cell-cell interaction in the heart via Wnt/β-catenin pathway after cardiac injury.
      ). There are 19 Wnt ligands, and it is possible that other non-canonical Wnts, in addition to Wnt5a, participate in the suppression of JNK and the anti-apoptotic and anti-inflammatory actions of Sfrp5. Other Wnts that activate non-canonical signaling include Wnt5b, Wnt11, and possibly Wnt4. However, transcripts that encode at least two of these candidates are expressed at low or undetectable levels in the heart (Wnt5b and Wnt4). Although additional experiments will be required to document a causal link between Sfrp5 and Wnt5a with mouse genetic models, multiple lines of evidence support the notion that Sfrp5 acts via Wnt5a in the ischemic heart. These data include the observations that Sfrp5 deficiency leads to the greater influx of Wnt5a-positive macrophages into the infarct zone and cell culture studies showing that Sfrp5 can suppress the pro-inflammatory and pro-apoptotic actions of Wnt5a on macrophages and cardiac myocytes, respectively.
      It has also been reported that Wnt5a stimulates hypertrophy in cultured myocytes (
      • Hagenmueller M.
      • Riffel J.H.
      • Bernhold E.
      • Fan J.
      • Katus H.A.
      • Hardt S.E.
      Dapper-1 is essential for Wnt5a induced cardiomyocyte hypertrophy by regulating the Wnt/PCP pathway.
      ). Cardiac hypertrophy was not examined in our study because it is not a significant end point in the acute I/R model. However, the pro-hypertrophic effect of Wnt5a would be expected to be greater under conditions where Sfrp5 levels are limiting, such as severe metabolic dysfunction, suggesting that an imbalance in Sfrp5/Wnt5a expression could also contribute to worsened post-myocardial infarction remodeling at later time points or in the development of left ventricle hypertrophy, which is often associated with obesity-related metabolic dysfunction.
      Sfrp5 expression is highly enriched in adipocytes, and it is now appreciated to be a marker of adipose tissue (
      • Jura M.
      • Jaroslawska J.
      • Chu D.T.
      • Kozak L.P.
      Mest and Sfrp5 are biomarkers for healthy adipose tissue.
      ,
      • Flehmig G.
      • Scholz M.
      • Klöting N.
      • Fasshauer M.
      • Tönjes A.
      • Stumvoll M.
      • Youn B.S.
      • Blüher M.
      Identification of adipokine clusters related to parameters of fat mass, insulin sensitivity and inflammation.
      ,
      • Koza R.A.
      • Nikonova L.
      • Hogan J.
      • Rim J.S.
      • Mendoza T.
      • Faulk C.
      • Skaf J.
      • Kozak L.P.
      Changes in gene expression foreshadow diet-induced obesity in genetically identical mice.
      ,
      • Anunciado-Koza R.P.
      • Higgins D.C.
      • Koza R.A.
      Adipose tissue Mest and Sfrp5 are concomitant with variations of adiposity among inbred mouse strains fed a non-obesogenic diet.
      ,
      • Wang R.
      • Hong J.
      • Liu R.
      • Chen M.
      • Xu M.
      • Gu W.
      • Zhang Y.
      • Ma Q.
      • Wang F.
      • Shi J.
      • Wang J.
      • Wang W.
      • Ning G.
      SFRP5 acts as a mature adipocyte marker but not as a regulator in adipogenesis.
      ) and to function as an adipokine (
      • Ouchi N.
      • Higuchi A.
      • Ohashi K.
      • Oshima Y.
      • Gokce N.
      • Shibata R.
      • Akasaki Y.
      • Shimono A.
      • Walsh K.
      Sfrp5 is an anti-inflammatory adipokine that modulates metabolic dysfunction in obesity.
      ). Sfrp5 shows a biphasic pattern of regulation in adipose tissue upon diet-induced obesity. Sfrp5 is transiently up-regulated in the early stages of murine obesity, but its expression declines at later time points (
      • Ouchi N.
      • Higuchi A.
      • Ohashi K.
      • Oshima Y.
      • Gokce N.
      • Shibata R.
      • Akasaki Y.
      • Shimono A.
      • Walsh K.
      Sfrp5 is an anti-inflammatory adipokine that modulates metabolic dysfunction in obesity.
      ). A similar pattern of Sfrp5 expression has been reported where it was noted that the decrease in Sfrp5 expression coincides with the plateau in adipose tissue expansion, suggesting that Sfrp5 is a marker of “healthy” fat (
      • Jura M.
      • Jaroslawska J.
      • Chu D.T.
      • Kozak L.P.
      Mest and Sfrp5 are biomarkers for healthy adipose tissue.
      ). Here, we document another facet of Sfrp5 regulation, that ischemic injury in a remote tissue will lead to a reduction in Sfrp5 expression in pericardial and epididymal adipose tissue depots. Similarly, levels of other anti-inflammatory adipokines, adiponectin and CTRP9, were down-regulated in pericardial adipose tissue following myocardial I/R injury, and CTRP9 was also down-regulated in epididymal adipose tissue under these conditions. Relatively little is known about the mechanisms of Sfrp5 regulation. However, it has features in common with adiponectin, including marked up-regulation during adipocyte differentiation and suppression by agents that promote adipocyte dysfunction including endoplasmic reticulum stress, oxidant stress, and inflammation. With regard to the regulation of Sfrp5 expression, the acute production of pro-inflammatory cytokines following myocardial I/R may contribute to its down-regulation, as has been proposed for the down-regulation of adiponectin under these conditions (
      • Shibata R.
      • Sato K.
      • Kumada M.
      • Izumiya Y.
      • Sonoda M.
      • Kihara S.
      • Ouchi N.
      • Walsh K.
      Adiponectin accumulates in myocardial tissue that has been damaged by ischemia-reperfusion injury via leakage from the vascular compartment.
      ). Furthermore, because adiponectin, Sfrp5, and potentially CTRP9 represent markers of functional adipose tissue, their down-regulation following myocardial I/R is suggestive of a mechanism of heart-to-fat crosstalk that could contribute to metabolic dysfunction under conditions of cardiovascular disease.
      In summary, we show that Sfrp5 functions to limit infarct size in the heart following ischemia-reperfusion injury. Because excessive inflammation contributes to infarct expansion, we propose that the myocardium-sparing properties of Sfrp5 are mediated, at least in part, by its abilities to suppress pro-inflammatory Wnt5a/JNK signaling within the macrophages that infiltrate the infarct and pro-apoptotic Wnt5a/JNK signaling within myocytes. Because Sfrp5 is a secreted factor that is highly expressed by adipose tissue, we proposed further that it functions as a cardio-protective adipokine, and that its reduced expression could contribute to the increased prevalence of myocardial infarction in obese individuals.

      Author Contributions

      K. W. and K. N. designed the study. K. N., S. S., and J. J. F. performed the experiments, analyzed the data, and wrote the paper. R. K., I. S., K. O., Y. K., and N. O. provided technical assistance and contributed to the preparation of the figures. All authors analyzed the results and approved the final version of the manuscript.

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