Wt

Fig. 2. LV remodeling in transgenic mouse model of TNF overexpression. Magnetic resonance images of the heart were obtained from 24-wk-old transgenic (TG) mice with targeted TNF overexpression (A—C) and an age-matched control (WT) mouse (D—F). As shown, there was significant LV dilation in the animal harboring the TNF transgene in the cardiac compartment. Bars = 2.5 mm. (Reproduced from ref. 48 with permission of the American Heart Association ©1997.)

model, following TNF infusion, LV end diastolic dimension increased by >25% when compared to time-matched controls (Fig. 1B). Moreover, in a transgenic mouse model, myocardial TNF-a overexpression results in increased systolic and diastolic LV volumes, a dilated and globoid LV phenotype (Fig. 2) (48).

Important determinants of myocardial wall remodeling include changes in myocyte size and number, alignment of myocytes in the myocardial wall, and changes in myocardial extracellular matrix (ECM). Increased levels of these inflammatory mediators have been demonstrated to cause changes in each of these determinants of remodeling and can result in myocyte hypertrophy (53), alterations in fetal gene expression (48), and progressive myocyte loss through apoptosis (54). In addition to these effects, there are several lines of evidence suggesting that TNF may promote left ventricular remodeling through alterations in the ECM. Increased TNF in animal models has been demonstrated to cause significant changes in the ECM (3,55). For example, chronic TNF-a infusion in rats reduces fibrillar collagen by 50% when compared with controls (3). Scanning electron micrographs of myocardial sections from TNF-a-infused animals reveal disruptions in the myocardial extracellular fibrillar collagen network (Fig. 3). Transgenic mice overexpressing myocardial TNF-a have increased soluble myocardial collagen, suggestive of reduced collagen crosslinking (55). Similar findings have also been reported by others (56,57) who observed LV dysfunction and LV dilation in transgenic mice with targeted overexpression of TNF in the heart.

Thus, in animal models of increased TNF-a expression, defects in extracellular fibrillar collagen structure and composition are known to occur. With respect to the mechanisms that are involved in TNF-induced LV dilation, it has been suggested that TNF-induced activation of matrix metalloproteinases is responsible for this effect (55,57). The current hypothesis regarding the role of TNF in LV remodeling proposes that without altering

Fig. 2. LV remodeling in transgenic mouse model of TNF overexpression. Magnetic resonance images of the heart were obtained from 24-wk-old transgenic (TG) mice with targeted TNF overexpression (A—C) and an age-matched control (WT) mouse (D—F). As shown, there was significant LV dilation in the animal harboring the TNF transgene in the cardiac compartment. Bars = 2.5 mm. (Reproduced from ref. 48 with permission of the American Heart Association ©1997.)

Fig. 3. Rats were infused with diluent or TNF-a for 15 d. Representative scanning electron micrographs of LV myocardial sections were taken. In myocardial samples from diluent-treated animals (control), a fine weave of collagen around the myocytes was observed. In myocardial samples from TNF-a-infused animals (TNF-a), the collagen weave appeared to be significantly disrupted. (Reproduced from ref. 3 with permission of the American Heart Association ©1998.)

Fig. 3. Rats were infused with diluent or TNF-a for 15 d. Representative scanning electron micrographs of LV myocardial sections were taken. In myocardial samples from diluent-treated animals (control), a fine weave of collagen around the myocytes was observed. In myocardial samples from TNF-a-infused animals (TNF-a), the collagen weave appeared to be significantly disrupted. (Reproduced from ref. 3 with permission of the American Heart Association ©1998.)

preload or afterload, TNF induces LV dilation, perhaps as a result of ECM degradation, allowing rearrangement ("slippage") of bundles or groups of cardiac myocytes. These alterations in LV geometry are further accompanied by systolic and diastolic dysfunction (3,58). Thus, excessive activation of TNF, and possibly of other proinflammatory cytokines, may contribute to the LV remodeling observed in heart failure via mechanisms that involve both myocyte and nonmyocyte elements of the myocardium.

Interleukin-6

Originally identified as T-cell-derived cytokine, IL-6 has been recognized as a multifunctional cytokine produced by several cell types of nonimmunological origin. IL-6 can produce myocyte hypertrophy, myocardial dysfunction, and muscle wasting (59). In advanced heart failure, cardiac expression of IL-6 and IL-6 receptor mRNA is increased (60). IL-6 is thought to be released in direct response to TNF-a, and a linear correlation between the two cytokine levels has been described (59,61). IL-6 has two different types of receptors: a private ligand-binding receptor (called membrane a receptor [R]) and a membrane P receptor (called transmembrane glycoprotein [gp] 130) (62,63). Interestingly, it is the small transmembrane glycoprotein, gp130, but not IL-6 soluble receptor (IL-6R) itself, that renders cells susceptible to IL-6 (63). Indeed, IL-6 can act on cells lacking the expression of IL-6R after complex formation with soluble IL-6R. Both gp130 and IL-6R are always required for signaling. The soluble form of gp130 inactivates the solu-

Fig. 4. TNF-a levels in patients with class I—IV heart failure. Compared with age-matched control subjects (open bar), there was a progressive increase in serum TNF-a levels in direct relation to decreasing functional heart failure classification. The solid bars denote values for patients enrolled in Studies ofLeft Ventricular Dysfunction (SOLVD) (8) ; the shaded bar denotes values for NYHA class IV patients who were undergoing cardiac transplantation (9). *Significantly different from normal. (Reproduced from ref. 59 with permission of Churchill Livingstone ©1996.)

Fig. 4. TNF-a levels in patients with class I—IV heart failure. Compared with age-matched control subjects (open bar), there was a progressive increase in serum TNF-a levels in direct relation to decreasing functional heart failure classification. The solid bars denote values for patients enrolled in Studies ofLeft Ventricular Dysfunction (SOLVD) (8) ; the shaded bar denotes values for NYHA class IV patients who were undergoing cardiac transplantation (9). *Significantly different from normal. (Reproduced from ref. 59 with permission of Churchill Livingstone ©1996.)

ble IL-6/IL-6R complex. However, both the concentration of gp130 and the overall level of bioactivity of IL-6 are increased in heart failure (62).

Interleukin-1

Another important cytokine in the setting of heart failure is IL-1. This cytokine and TNF-a are generally thought of as the prototypical proinflammatory cytokines. IL-1 has been demonstrated in the myocardium of patients with idiopathic dilated cardiomyopathy (64). Experimentally, it is known to depress myocardial contractility in a dose-dependent fashion (65). The alterations in gene expression seen in response to IL-1 resemble, in many ways, the phenotype of the failing heart (66). These effects are synergistic with those of TNF-a. Additional studies have shown that IL-1 is also involved in myocardial apoptosis, hypertrophy, and arrhythmogenesis (66,67).

Proinflammatory Cytokine Levels Are Elevated and Correlate With Severity of Disease in Heart Failure

Circulating levels of TNF and IL-6 are elevated in patients with heart failure. Because they were initially identified in patients with cardiac cachexia (17,68), and edematous decompensation (69), these cytokines were thought to be expressed only in patients with end-stage heart failure. However, as reported in a number of studies (1,8,18-20,24,31, 61,70), proinflammatory molecules are activated starting at earlier phases ofheart failure (i.e., New York Heart Association [NYHA] class II [8] or asymptomatic left ventricular dysfunction [70]) and continue to rise in direct relation to worsening NYHA functional classification (8,31,59,71) (Fig. 4) regardless ofthe etiology ofthe heart failure (8,24,25,61).

In addition to the inflammatory cytokines, circulating levels of cytokine receptors are elevated in heart failure. These include the soluble TNFR (22) and soluble transmembrane

Fig. 5. Kaplan-Meier survival analysis. The circulating levels of (A) TNF, (B) IL-6, (C) sTNFRl, and (D) sTNFR2 were examined in relation to patient survival during follow-up (mean duration: 55 wk; maximum duration: 78 wk). For this analysis, the circulating levels of cytokines and cytokine receptors were arbitrarily divided into quartiles. (Reproduced from ref. 31 with permission of the American Heart Association ©2001.)

Fig. 5. Kaplan-Meier survival analysis. The circulating levels of (A) TNF, (B) IL-6, (C) sTNFRl, and (D) sTNFR2 were examined in relation to patient survival during follow-up (mean duration: 55 wk; maximum duration: 78 wk). For this analysis, the circulating levels of cytokines and cytokine receptors were arbitrarily divided into quartiles. (Reproduced from ref. 31 with permission of the American Heart Association ©2001.)

gp130 (one ofthe receptors for the IL-6 family), which are increased in patients with heart failure in close relation to functional class (61). Of note, even though IL-6 and gp130 levels are elevated, IL-6R levels are not increased in patients with heart failure (61,72).

Furthermore, increased myocardial TNF-a-converting enzyme expression, which results in the production of a functional enzyme that has precursor TNF-a in the mature form (73), is associated with the degree of left ventricular systolic dysfunction in patients with dilated cardiomyopathy (73).

Proinflammatory Cytokines Predict Poor Prognosis in Heart Failure

As well as correlating with severity of disease (i.e., with worsening functional class), elevated blood levels of proinflammatory cytokines are associated with increased mortality in patients with heart failure. Increased circulating levels of TNF-a (31,74), IL-6 (28,71, 75-77), and sTNFRl and sTNFR2 (31,74) have been reported to predict poorer survival. As shown in Fig. 5A, among patients participating in the multicenter Vesnarinone Trial (VEST), survival gradually declined as a function of increasing TNF levels, with the worst survival in patients with TNF levels >75th percentile (31). Similarly, circulating levels of IL-6 (Fig. 5B) and sTNFRl and sTNFR2 were associated with outcome (Fig. 5C,D) (31). Of interest, in a study of 37 patients with heart failure and 26 age-matched control subjects (22), the circulating level of sTNFR2 appeared to be the most powerful predictor of mortality. In a larger study of152 patients with heart failure, Rauchhaus et al. (74), however, reported that sTNFR1 was the strongest and most accurate prognosticator; the receiver operating characteristic area under the curve for sTNFR1 was greater than for sTNFR2 at 6, 12, and 18 mo (all p < 0.05). When examined in VEST with Cox regression modeling adjusting for age, sex, etiology of heart failure, NYHA class, ejection fraction, and serum sodium, TNF, IL-6, sTNFRl, and sTNFR2 remained significant independent predictors of mortality, along with NYHA class and ejection fraction (31).

Although these clinical studies cannot address whether the findings represent an epiphe-nomenon that is associated with, but not causally related to, worsening severity of disease and outcomes, the preponderance of data support that the proinflammatory cytokines TNF-a and IL-6 contribute further to progression of heart failure and worsening outcomes in heart failure.

Inflammatory Cytokines as Markers for Monitoring Response to Therapy in Heart Failure

Several studies have examined the changes in levels of inflammatory cytokines during standard therapy for heart failure. Some of these changes can be attributed to direct interaction of the medications used, such as the interaction between the neurohormonal antagonists and the proinflammatory cytokines (78,79). Clinical studies have shown that treatment with angiotensin receptor antagonists can lead to significant reductions in circulating levels of TNF and/or cell adhesion molecules in patients with heart failure (80). ^-Adrenergic blockade also prevents the expression of inflammatory mediators in postinfarction animal models (81) and reduces proinflammatory cytokine levels in patients with heart failure (82-87). Compared to angiotensin receptor and ^-blockers, the effect of angiotensin-converting enzyme (ACE) inhibitors on inflammatory cytokines is not as clear. In an animal infarct model, the use of ACE inhibitors over 28 d resulted in a reduction in cardiac cytokine expression (79). By contrast, in a clinical study by Gullestad et al. (88), treatment with ACE inhibitors over 34 wk was associated with a rise in the peripheral levels of chemokines, cell adhesion molecules, and proinflammatory cytokines except for IL-6. In another study, treatment with the long-acting dihydropyridine calcium antagonist, amlodipine, over 26 wk lowered plasma IL-6 levels in patients with heart failure (28). Similarly, optimization of background standard therapy of heart failure with diuretics, ACE inhibitors, ^-blockers, and digoxin can result in significant reductions in circulating levels of TNF-a and IL-6 (75). Histochemical sections of failing human myocardium have shown that after several weeks of mechanical circulatory support with a ventricular assist device, myocardial expression of TNF-a was markedly reduced in patients with heart failure (89).

These studies suggest that there are important interactions between the renin-angio-tensin, adrenergic systems and proinflammatory cytokines, and many of the conventional therapies for heart failure may work, at least in part, through the modulation of proinflammatory cytokines. Nevertheless, it should be noted that despite these temporal parallel changes in the levels of cytokines with optimal heart failure therapy, currently there are no data from large-scale studies of the relationship between changes in inflammatory biomarkers over time and morbidity and mortality in patients with heart failure. Furthermore, the sensitivity, specificity, and negative and positive predictive values ofinflamma-tory biomarkers for predicting adverse outcomes in the setting ofheart failure are not well defined, and any clinical value ofinflammatory markers over and above established parameters remains to be proven (72).

Proinflammatory Cytokines as Therapeutic Targets in Heart Failure

Clinical studies have been carried out to selectively antagonize inflammatory cyto-kines, specifically TNF-a, in patients with heart failure using recombinant human TNFRs that act as "decoys" to bind TNF, thereby preventing TNF from binding to TNFRs on cell-

surface membranes of target cells, or monoclonal antibodies (MAbs) from binding and neutralizing circulating cytokines.

Early preclinical studies ofTNF antagonism using the TNFR etanercept (Enbrel) showed that etanercept could reverse the deleterious negative inotropic effects of TNF in vitro (90) and in vivo (3). Subsequently, a series ofphase 1 and phase 2 clinical studies was performed in patients with moderate to advanced heart failure and showed improvements in quality of life, 6-min walking distance, and left ventricular ejection performance after treatment with etanercept for up to 3 mo (45,91). Two multicenter clinical trials using etanercept were then completed in patients with NYHA class II-IV heart failure. The trial in North America, entitled Randomized Etanercept North American Strategy to Study Antagonism of Cytokines (RENAISSANCE) (n = 900), and the trial in Europe and Australia, entitled Research into Etanercept Cytokine Antagonism in Ventricular Dysfunction (RECOVER) (n = 900), were both quality-of-life trials that used a clinical composite as the primary end point. A third trial that used the pooled data from the RENAISSANCE and RECOVER trials, entitled Randomized Etanercept Worldwide Evaluation (RENEWAL) (n = 1500), had a primary end point of all-cause mortality and hospitalization for heart failure. The trials were stopped early after the Data Monitoring Safety Board deemed that it is was unlikely that the trials would show benefit on the primary end points ifthe two trials were allowed to go to completion (12). In a post hoc analysis, patients taking the etanercept treatment over a longer period of time had increased risk of death/heart failure hospitalization compared with patients receiving placebo (5,12).

The second targeted anticytokine approach in clinical heart failure trials was the use of MAbs directed against TNF-a. The Anti-TNF-a Therapy Against CHF was a phase 2 study in 150 patients with moderate to advanced heart failure using infliximab (Remicade), a chimeric MAb consisting of a genetically engineered murine Fab fragment (that binds human TNF) fused to a human FC portion of human IgG1. The study showed a dose-related increase in death and heart failure hospitalizations with infliximab compared with placebo (13). In the aftermath of these clinical studies, it was questioned whether the biological agents that were used in the trials had intrinsic toxicity, and/or whether TNF antagonism had untoward effects in the setting of heart failure. Infliximab may be directly cytotoxic to cells expressing TNF on the membrane by inducing complement fixation in the heart, which may lead to sustained myocarditis as well as cardiac myocyte lysis (5). Etanercept, on the other hand, may have stabilized TNF and resulted in the accumulation of high concentrations of immunoreactive TNF in the peripheral circulation (5). This potential increase in the circulating levels of biologically active TNF patients with heart failure might actually worsen the heart failure status.

Proinflammatory Cytokines as Predictors of Development of Heart Failure in Asymptomatic Patients

Elevated levels of IL-6 (92) and TNF (8) have been reported in patients with LV dysfunction in the absence of the clinical symptoms of heart failure. In a subgroup of 732 elderly Framingham study subjects without prior MI and heart failure, Vasan et al. (70) reported that baseline levels of IL-6 and spontaneous production of TNF by peripheral blood mononuclear cells (PBMCs) were predictive of the development of heart failure in the next 5 yr. After adjusting for established risk factors, including the occurrence of MI during follow-up, the investigators found that the risk of developing heart failure increased ~1.6- to 1.7-fold per tertile increment in PBMC TNF and IL-6 levels, respectively. Sub jects with increased levels of IL-6, PBMC TNF, and CRP ©5 mg/dL had a 4.1-fold risk of developing heart failure. The study population consisted of predominantly elderly, Caucasian subjects (67% female) with a high prevalence of hypertension (~70%), atrial fibrillation (~7%), and preexisting cardiovascular disease without prior documented MI. It is important to point out that in this study there was no assessment of LV function at baseline. Elevated inflammatory markers in this study may have identified patients with vascular disease at risk of MI (93,94) or patients with preexisting subclinical LV dysfunction (92). As pointed out by Murray et al. (95), without a baseline assessment of ventricular function, the study cannot conclude whether IL-6, TNF, and CRP predict the de novo development of cardiomyopathy vs the transition between subclinical LV dysfunction and overt heart failure.

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