Journal of the American College of Cardiology Vol. 35, No. 3, 2000 © 2000 by the American College of Cardiology ISSN 0735-1097/00/$20.00 Published by Elsevier Science Inc. PII S0735-1097(99)00600-2 REVIEW ARTICLES The Role of Tumor Necrosis Factor in the Pathophysiology of Failure Arthur M. Feldman, MD, PHD, FACC, Alain Combes, MD, Daniel Wagner, MD, Toshiaki Kadakomi, MD, Toru Kubota, MD, PHD, Yun You Li, PHD, Charles McTiernan, PHD Pittsburgh, Pennsylvania

Recent studies have focused their attention on the role of the proinflammatory cytokine tumor necrosis factor (TNF) in the development of . First recognized as an endotoxin- induced serum factor that caused necrosis of tumors and cachexia, it is now recognized that TNF participates in the pathophysiology of a group of inflammatory diseases including rheumatoid arthritis and Crohn’s disease. The normal heart does not express TNF; however, the failing heart produces robust quantities. Furthermore, there is a direct relationship between the level of TNF expression and the severity of disease. In addition, both in vivo and in vitro studies demonstrate that TNF effects cellular and biochemical changes that mirror those seen in patients with congestive heart failure. Furthermore, in animal models, the development of the heart failure phenotype can be abrogated at least in part by anticytokine therapy. Based on information from experimental studies, investigators are now evaluating the clinical efficacy of novel anticytokine and anti-TNF strategies in patients with heart failure; one such strategy is the use of a recombinantly produced chimeric TNF alpha soluble receptor. Thus, in view of the emerging importance of proinflammatory cytokines in the pathogenesis of heart disease, we review the biology of TNF, its role in inflammatory diseases, the effects of TNF on the physiology of the heart and the development of clinical strategies that target the cytokine pathways. (J Am Coll Cardiol 2000;35:537–44) © 2000 by the American College of Cardiology

Dilated cardiomyopathy is a disease of epidemic proportion The proinflammatory cytokine TNF. In 1975, Carswell in the U.S. Although more prevalent in the elderly, it affects et al. (1) first identified tumor necrosis factor (TNF), an patients of all ages, races and genders. Heart failure is endotoxin-induced serum factor that caused necrosis of generally believed to begin with myocyte damage secondary tumors. A decade later, investigators isolated a protein from to a variety of insults including ischemia, toxins such as endotoxin-treated cells that was named cachectin because of alcohol or immune mechanisms. However, in many cases its presumed role in the molecular basis of cachexia (2,3). the etiology remains undefined. Initially the heart compen- The subsequent cloning of the genes encoding cachectin sates by dilatation and cellular hypertrophy; however, over a and TNF alpha confirmed that these two molecules were period of time, the heart eventually decompensates and identical (4,5). Produced as a prohormone of 233 amino patients present with signs and symptoms of heart failure. It acids, TNF alpha is anchored in the cell membrane and then is the transition from compensation to decompensation that processed to a 157 residue mature protein by cleavage of a has been the focus of clinical and basic research over the past 76 residue signal peptide. In response to a wide variety of two decades and the role of the neurohormones, norepi- infectious or inflammatory stimuli (e.g., lipopolysaccharide, nephrine and angiotensin II, in the development of symp- viruses, fungal or parasitic , interleukin-1 [IL-1], tomatic heart failure. However, investigators have recently TNF alpha), both transcription and translation of TNF focused their attention on the proinflammatory cytokine, precursor is increased, and large amounts of mature protein tumor necrosis factor (TNF). This manuscript reviews the are rapidly released into the circulation. However, not all of investigational data supporting the role of TNF in the the TNF alpha is released as some remains cell-associated in pathogenesis of heart failure and its usefulness as a thera- a transmembrane form (6). Tumor necrosis factor regulates peutic target. the expression of a variety of peptide regulatory factors including IL-1, IL-6, platelet derived growth factor, trans- forming growth factor-beta, as well as a group of eico- From the Cardiovascular Institute of the UPMC Health System, Pittsburgh, sanoids and hormones including platelet-activating factor Pennsylvania. This study was supported, in part, by NIH grant 1 P50 HL and adrenaline (7). In addition, TNF alpha (and IL-1beta) 6348A5-01. Ј Manuscript received July 2, 1999; revised manuscript received October 5, 1999, contains a 33 nucleotide 3 -untranslated sequence that accepted November 18, 1999. shortens messenger RNA (mRNA) half-life and, in so 538 Feldman et al. JACC Vol. 35, No. 3, 2000 Tumor Necrosis Factor in Heart Failure March 1, 2000:537–44

activity (18) and promotes leucostasis by inducing increased Abbreviations and Acronyms expression of intracellular adhesion molecules (ICAMS) alpha-MHC ϭ alpha myosin heavy chain and endothelial leukocyte adhesion molecules (ELAMS) at AMP ϭ adenosine monophosphate sites of inflammation. At higher concentrations, TNF alpha ϭ beta-MHC beta myosin heavy chain production exceeds the number of TNF alpha receptors CHF ϭ congestive heart failure ELAMS ϭ endothelial leukocyte adhesion molecules located on the cell surface with excess TNF alpha being ICAMS ϭ intracellular adhesion molecules released into the circulation. Once released, TNF alpha IL-1 ϭ interleukin-1 exerts endocrine or exocrine effects including initiation of LPS ϭ lipopolysaccharide metabolic wasting, microvascular coagulation, hypotension ϭ MCP monocyte chemoattractant protein and fever (19,20). Paradoxically, TNF alpha also appears to mRNA ϭ messenger RNA NYHA ϭ New York Heart Association modulate both tissue destruction and rebuilding. Tumor ϩ SERCA ϭ sarcoplasmic reticulum Ca2 ATPase necrosis factor alpha stimulates fibroblast and mesenchymal TNF ϭ tumor necrosis factor cell proliferation directly and induces the biosynthesis of ϭ TNFR soluble tumor necrosis factor receptor other growth factors. By contrast, it is directly cytotoxic to endothelial cells and can induce the biosynthesis of collag- enases, proteases, reactive oxygen intermediates and arachi- donic acid metabolites (7). Tumor necrosis factor alpha has doing, limits the production of large quantities of this also been shown to have an important role in cell death potent peptide (4). through a variety of mechanisms including second messen- TNF receptors. Once released from the cell, TNF alpha ger pathways, arachidonate metabolism, protein kinases, interacts with one of two TNF alpha receptors—a 55 kd oxygen free radicals, nitric oxide, transcription of a variety of high affinity receptor, soluble tumor necrosis factor receptor cytotoxic genes, regulation of nuclear regulatory factors, 1 (TNFR-1), and a 75 kd low affinity receptor (TNFR-2) ADP-ribosylation and, potentially, DNA fragmentation (8,9). Intracellular signaling occurs as a result of TNF (21). Under normal situations, these cytotoxic effects are alpha-induced cross-linking of the receptors. The extracel- important in host defense by initiating antitumor activity lular portions of these two proteins form a receptor family (1,22) and modulating cell growth and differentiation. containing four characteristic domains with regularly spaced However, the role of TNF alpha in the pathogenesis of cystine residues; however, no significant homology exists malignancies remains controversial. While therapy with between the intracytoplasmic portions of the two TNF TNF improved survival in tumor-bearing mice, it also alpha receptors (10). Thus, investigators have proposed that promoted tumor cell adhesion, nodule development (23) these receptors may be coupled to distinct signaling path- and metastasis (24). In addition, a direct relationship exists ways (11). In the presence of noxious stimulants including between the level of TNF expression and tumor grade in TNF alpha, lipopolysaccharide (LPS), okadaic acid and ovarian cancer (25). By contrast, excessive activation of phorbol esters, TNF alpha receptors are “shed” into the TNF alpha or nontissue specific expression leads to tissue circulation (12–14). These TNFR receptor proteins appear necrosis and apoptosis. Indeed, recent studies have demon- to be truncated fragments of the extracellular regions of the strated a causative role for TNF alpha in a group of diseases type 1 and type 2 membrane-bound receptors for TNF including: septic shock, rheumatoid arthritis (26), pre- alpha (13,15). Although TNF receptors do not possess eclampsia (27), hemolytic uremic syndrome (28), allograft intrinsic protein kinase activity, within minutes of exposure rejection (29) and regional enteritis (30). to TNF alpha, phosphorylation of several distinct proteins Proinflammatory cytokines and the heart. The first rec- occurs, which is likely due to activation of cellular kinases ognition that TNF alpha might participate in the develop- (16). The soluble receptors are able to bind ligand and ment of congestive heart failure (CHF) came in 1990 when inhibit the cytotoxic activities of TNF alpha (15). Thus, the Levine et al. (31) demonstrated that circulating levels of shedding of soluble binding proteins might serve as a TNF alpha were elevated in patients with end stage heart “biological buffer” that can rapidly neutralize the activities of failure and cachexia. Subsequent studies demonstrated com- TNF alpha. parable elevations in IL-6 and IL-1beta (32) and a direct Pleiotropic effects of TNF alpha. The cellular effects of relationship between TNF alpha levels and functional heart TNF alpha are highly pleiotropic. At low concentrations, failure classification (Fig. 1 [33]). Furthermore, direct rela- TNF alpha effects paracrine or autocrine regulation of tionships were identified between circulating levels of TNF leukocytes and endothelial cells and, thus, serves as an alpha and neurohumoral activation and the degree of important regulator of the inflammatory response. Indeed, anemia; however, there was no relationship between cyto- mice lacking the p55 TNF alpha receptor are highly kine levels and the degree of cachexia (34,35). Cytokine sensitive to some types of bacterial infections (17). Tumor levels were also elevated in patients with heart failure due to necrosis factor alpha enhances chemotaxis of macrophages (36). That the observed increases were of and , increases their phagocytic and cytotoxic physiologic significance was demonstrated by studies in JACC Vol. 35, No. 3, 2000 Feldman et al. 539 March 1, 2000:537–44 Tumor Necrosis Factor in Heart Failure

Figure 3. Tumor necrosis factor alpha acutely depresses contractile Figure 1. Circulating levels of tumor necrosis factor alpha (TNF activity and calcium transients of isolated adult rat cardiomyocytes. alpha) in normal subjects and patients with New York Heart Cells were loaded with the calcium sensitive dye, fura-2, and Association (NYHA) functional class I to III heart failure. exposed to 200 U/ml rat TNF alpha or diluent (control) for Adapted after Torre-Amione et al. (33) with permission of the 30 min. Cells were then electrically stimulated to contract (1 HZ) authors. and a video edge-detection system used to record diastolic cell length and contraction to determine fractional shortening. Cells were alternately illuminated at 340- and 380-nm light and emis- which injections of endotoxin into humans resulted in TNF sion of the fura-2 measured at 520-nm. The ratio of light emission alpha elevations, depressed left ventricular function and from the two illuminating wavelengths was used to follow intra- cellular free calcium transients. (A) Fractional shortening (n ϭ 30 decreases in mean arterial pressure (37). Parenthetically, it each group). (B) Mean calcium transient amplitude (difference should be noted that the different assay techniques, for between peak systolic and peak diastolic 340/380 nm ratio; n ϭ 40 example bioassays or ELISA measurements, used by various per group). (Wagner, Combes, Janczewski, McTiernan and Feld- investigators provide varying values, thus obfuscating the man; unpublished data). Open square ϭ control; solid square ϭ literature to some extent. TNF alpha 200 U/ml. The increase in circulating TNF alpha in patients with CHF was associated with a substantial decrease in TNF alpha levels in patients with CHF (Fig. 3). First, it was myocardial TNF alpha receptors and an increase in soluble demonstrated that the negative inotropic effects of TNF alpha receptors; however, the stoichiometry favored TNF alpha are virtually immediate (40,41) and appear to be free TNF alpha (38). Perhaps of greatest importance was completely reversible upon removal of the cytokine. How- the demonstration by Mann and colleagues (38) in 1996 ever, not only does TNF alpha have immediate negative that the nonfailing human heart does not express TNF inotropic properties, but it can recapitulate the cellular and alpha, whereas the end stage failing human heart reexpresses biochemical abnormalities that characterize the failing hu- robust amounts of protein (Fig. 2). man heart. For example, IL-1beta induces a down regula- 2ϩ Biologic effects of TNF alpha on the myocardium. tion of the expression of sarcoplasmic reticulum Ca Beginning with the observation that TNF alpha could ATPase (SERCA) and phospholamban at both the mRNA and protein level in neonatal myocytes (42), which is inhibit contractility of isolated hamster papillary muscles in 2ϩ a concentration-dependent and reversible manner (39), a associated with a depression and prolongation of the Ca series of in vivo and in vitro basic science studies were transient, effects that may be initiated by TNF alpha (40). In performed, which paralleled the clinical studies assessing addition, TNF alpha effectively uncouples the beta- adrenergic receptors from adenylyl cyclase via an effect on the G inhibitory protein (43,44). Furthermore, TNF alpha activates metalloproteinases and inhibits the expression of inhibitors of metalloproteinases in vivo—effects that would be expected to activate extracellular matrix remodeling (45). Although initial studies suggested that the negative inotrop- ic properties of TNF alpha were attributable to the ability of cytokines to induce nitric oxide synthase, subsequent inves- tigations demonstrated that the induction of inducible nitric oxide synthase could not in and of itself induce contractile dysfunction in cardiac myocytes and that alterations in calcium homeostasis played an important role (40). Tumor necrosis factor alpha also provokes a hypertrophic growth response in cardiac myocytes, which may be an adaptive Figure 2. Immunodetectable TNF alpha (picograms of TNF alpha per g of cystolic protein) in nonfailing and failing myocardium. response to hemodynamic or environmental stress (46). Adapted after Torre-Amione et al. (38) with permission of the Furthermore, a recent study suggested that the immediate authors. negative inotropic effects of TNF alpha may be mediated by 540 Feldman et al. JACC Vol. 35, No. 3, 2000 Tumor Necrosis Factor in Heart Failure March 1, 2000:537–44

to overexpression of TNF alpha has recently been confirmed by a second laboratory (51). Taken together, the existing TNF alpha transgenic studies suggest that there is a direct relationship between transgene copy number and the degree of inflammation (52). Recent studies suggest that mortality in these animals is due, in part, to an arrhythmogenic event since continuous holter monitoring of transgenic mice demonstrates high grade ventricular ectopy with frequent runs of nonsustained ventricular tachycardia (53). At the cellular and molecular level, the TNF alpha transgenics are characterized by: 1) reexpression of the fetal gene program (e.g., atrial natriuretic factor and beta-MHC), 2) reexpres- sion of downstream TNF alpha responsive cytokines and chemokines including IL-1beta, monocyte chemoattractant protein (MCP), and regulated upon activation, normal T-cell expressed and secreted, 3) increased activity of the metalloproteinases and diminished expression of inhibitors of metalloproteinases (54), 4) activation of both pro- and antiapoptotic pathways (55), 5) down-regulation of phos- pholamban and SERCA expression, and 6) a decrease in the alpha- and beta-myosin heavy chain mRNA ratio (56,57). Furthermore, the transgenic mice demonstrated reduced ejection fractions and fractional shortening and diminished ventricular compliance. The TNF alpha transgenic also Figure 4. Transgenic mice with cardiac-specific overexpression of demonstrated a substantial amount of apoptosis; however, TNF alpha (TNF1.6) develop dilated cardiomyopathy. Represen- tative magnetic resonance image (MRI, coronal view) from 24 this was largely isolated to the interstitium, and myocyte week-old wild type (A) and TNF1.6 transgenic mice (B) at apoptosis was rare (55). Thus, the TNF alpha overexpress- equivalent stage of cardiac cycle. (C) Transgenic animals demon- ing transgenic recapitulates both the pathologic, cellular and strate significant cardiac hypertrophy with chamber dilation and molecular phenotype of the human failing heart and, there- decreased ejection fraction as determined from MRI data (short- axis images). Adapted after Kubota et al. (50) with author’s fore, provides an ideal model in which to study the patho- permission. BW ϭ body weight; EDV ϭ end-diastolic volume; biology of progressive CHF and to test therapeutic strate- ESV ϭ end-systolic volume; HW ϭ heart weight. Open gies. square ϭ wild type; solid square ϭ TNF1.6. *p Ͻ 0.05. “Rescuing” the TNF alpha transgenic with TNFR. That there was a direct relationship between TNF alpha expres- sphingosine (47). Finally, chronic infusion of TNF alpha sion and negative inotropic effects was first shown by produces a reversible dilated cardiomyopathy in a rat mod- Kapadia et al. (58) using soluble TNF alpha binding el—without evidence of inflammatory infiltrate or myocyte proteins. However, to “prove” that overexpression of TNF necrosis (48). alpha is a critical step in the development of end stage CHF (52), transgenic and wild type mice were injected with an TNF alpha transgenic mice—a model of dilated cardio- adenoviral vector encoding a chimeric soluble TNF alpha myopathy. To pursue further studies of the role of cyto- protein consisting of two moieties of the human 55-kDa kines in the development of CHF and to provide additional TNF alpha receptor extracellular domain fused to a mouse support for the “cytokine hypothesis,” a series of mice that IgG heavy chain (59). Inoculation of mice with 109 plaques harbored a transgene effecting cardiac-specific overexpres- forming units resulted in inhibitor production and release by sion of TNF alpha were generated using the cardiac-specific hepatocytes and marked elevations in serum and myocardial alpha myosin heavy chain (alpha-MHC) promoter. Robust TNF alpha receptor levels for as long as six weeks (57). overexpression proved lethal due to (49) fulminant myocar- These levels of soluble receptor were several orders of ditis; however, lower levels of TNF alpha expression allowed magnitude greater than the levels of myocardial TNF alpha, the production of viable founders (50). These mice recapit- suggesting a favorable stoichiometry for TNF alpha inhibi- ulate heart failure in humans as they demonstrate: 1) tion. After both two and six weeks of treatment, TNFR four-chamber dilatation, 2) myocyte hypertrophy, 3) inter- completely abrogated the presence of interstitial infiltrates stitial infiltrates, 4) extracellular matrix remodeling with and normalized the expression of alpha-MHC, SERCA, fibrosis, 5) diminished beta-adrenergic responsiveness; and and phospholamban. In addition, the reexpression of down- 6) premature death with a six-month mortality of 25% (Fig. stream cytokines and chemokines including IL-1beta, 4). The development of a heart failure phenotype secondary MCP-1 and rantes was completely inhibited by TNFR JACC Vol. 35, No. 3, 2000 Feldman et al. 541 March 1, 2000:537–44 Tumor Necrosis Factor in Heart Failure therapy. By contrast, there was continued activation of mononuclear cells to adrenergic agonists inhibited LPS- beta-MHC expression, which was consistent with the induced production of TNF alpha (76). Phosphodiesterase finding of persistent ventricular hypertrophy in these TNFR inhibitors can also serve as potent inhibitors to TNF alpha treated mice. Finally, there was normalization of matrix production (77,78). Indeed, the phosphodiesterase inhibi- metalloproteinase and tissue inhibitor of metalloproteinase tor, pentoxyfilline, demonstrates salutorius effects on heart expression and stabilization of the degree of interstitial failure signs and symptoms while substantially lowering fibrosis, suggesting that extracellular matrix remodeling had circulating TNF alpha levels (79,80). However, other stud- abated. Thus, these studies suggest that some, although not ies have failed to show an effect of cyclic AMP, cyclic AMP all, of the phenotypic characteristics of CHF can be abated derivatives or beta-adrenergic agonists on TNF alpha pro- by anticytokine strategies. However, both hypertrophy and duction (81). Other inhibitors of cytokine expression in- fibrosis persist despite therapeutic intervention, suggesting clude: amiodarone (82), ouabain (83) and thalidomide (84). early, but not late, therapy using anticytokine approaches In addition, a recent report suggests that estrogen may might benefit patients with CHF. constitutively down-regulate TNF alpha expression (85). Recently, studies have demonstrated that adenosine is a The molecular genetics of TNF alpha. Genetic polymor- potent inhibitor of TNF alpha expression by both neonatal phisms in the TNF locus have been associated with higher myocytes, adult myocytes, rodent papillary muscle prepara- levels of TNF alpha production (60,61) and are known to be tions and adult human heart via activation of the adenosine related to several autoimmune, infectious and neoplastic A receptor (86,87). This effect appears to be partially diseases (62,63). These polymorphisms include a G to A 2 selective for the heart as adenosine’s anticytokine effects in transition at position Ϫ308 in the promoter region of the white cells is mediated via the A3 adenosine receptor (88). TNF alpha gene (TNF alpha2) (64) and a G to A transition That adenosine may have an important role in regulating at position ϩ252 in the first intron of the TNFbeta gene myocardial cytokine expression was further supported by a (65). The frequency of the TNF alpha2 allele, the allele recent study by Loh et al. (89) demonstrating improved associated with increased TNF expression, is increased in survival in heart failure patients having a common mutation patients with rheumatoid arthritis and systemic lupus ery- in at least one allele of the AMP deaminase gene. In the thematosus (66). Similarly, people homozygous for the peripheral muscle, and presumably in the heart, patients TNF alpha2 allele have a higher risk for death due to with this mutation have diminished AMP deaminase activ- cerebral malaria (67) while homozygous for the TNFbeta2 ity resulting in decreased metabolism of AMP to inosine allele have a higher mortality with sepsis (61). To date, and enhanced production of adenosine, leading us to hy- investigators have been unable to demonstrate an associa- pothesize that adenosine agonists might have therapeutic tion between either the TNF alpha or TNFbeta polymor- utility in the management of patients with CHF (69). phisms and the presence of CHF (68); however, these Effective blockade of TNF alpha activity has also been studies had an inherent bias, i.e., that patients with a effected using passive immunization. Monoclonal potentially unfavorable polymorphism might have died prior against TNF alpha have proved successful in ameliorating to receiving medical attention (69). Therefore, further the effects of sepsis (in animal models) (90,91), cancer (92), evaluation of this possibility is warranted. An additionally synovial inflammation (93), inflammatory bowel disease important, but as yet unanswered, question is how myocar- (94,95) and chronic bacterial infection (96,97). While im- dial TNF alpha expression is regulated at the molecular munotherapy is effective for investigation studies in animal level. However, interactions of nuclear proteins with an models, it has limitations in long-term disease therapy as Ap-1/CRE-like (cis-acting regulatory site) promoter se- the antibodies serve as immunogens and, therefore, can only quence in the human TNF alpha gene (70) and the marked be used for a limited period of time. To abrogate the sensitivity of TNF alpha expression to corticosteroids (and immunogenicity of the , several groups have pro- possibly cyclic adenosine monophosphate [AMP]) suggest posed using monoclonal antibodies in combination with an the importance of transcriptional regulation in modifying immunosuppressive such as methotrexate. However, the TNF alpha expression (71–73). rationale for using a potent immunosuppressive in a patient Clinical strategies for anticytokine therapy. Based on the with a chronic disease such as heart failure remains ques- basic science and clinical observations regarding the role of tionable. proinflammatory cytokines in the development of CHF, Perhaps the most exciting strategy that has recently been efforts have been directed towards developing anticytokine developed for inhibiting the effects of myocardial TNF strategies that might be useful in the therapy of patients. alpha production is the use of a recombinantly produced Yoshimura et al. (74) suggested that beta-adrenergic ago- chimeric TNF alpha soluble receptor (Etanercept; Immu- nists could inhibit the production of both TNF alpha and nex, Seattle, Washington) consisting of the p75 receptor IL-1beta by elevating intracellular cyclic AMP levels. Sim- linked to the Fc portion of human IgG (TNFR:Fc). Unlike ilarly, pretreatment with isoproterenol blunted the ability of synthetic pharmacologic agents that can have multiple LPS to induce TNF alpha production in conscious mice cellular effects, recombinant proteins have the theoretical (75), whereas short-term, but not long-term preexposure of benefit of being highly specific. First used in the treatment 542 Feldman et al. JACC Vol. 35, No. 3, 2000 Tumor Necrosis Factor in Heart Failure March 1, 2000:537–44 of rheumatoid arthritis, Etanercept was associated with 2. Beutler B, Mahoney J, Le Trang N, Pekala P, Cerami A. Purification dose-related reductions in disease activity without dose- of cachectin, a lipoprotein lipase-suppressing hormone, secreted by endotoxin-induced RAW 264.7 cells. J Exp Med 1985;161:984–95. limiting toxicity or the development of antibodies to the 3. Evans RD, Argiles JM, Williamson DH. Metabolic effects of tumor recombinant receptor protein (98). 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