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The systemic inflammatory response in heart failure
Progress in Pediatric Cardiology 11 Ž2000. 219᎐230
The systemic inflammatory response in heart failure Michael R. Anderson Department of Pediatrics, Case Western Reser¨ e Uni¨ ersity School of Medicine, Di¨ ision of Pediatric Pharmacology and Critical Care, Rainbow Babies and Childrens’ Hospital, 11100 Euclid A¨ enue, Cle¨ eland, OH 44106, USA
Abstract The physiologic diagnosis of heart failure has changed very little over the past several decades: heart failure is the inability of the cardiac output to meet the metabolic demands of the organism. The clinical definition of heart failure Žalso relatively unchanged. describes it as ventricular dysfunction that is accompanied by reduced exercise tolerance. Our understanding of the true pathophysiologic processes involved in heart failure have, however, changed. We have moved from thinking of heart failure as primarily a circulatory phenomenon to seeing it as a pathophysiologic state under the control of multiple complex systems. Over the past several years the dramatic explosion of research in the fields of immunology and immunopathology have added an additional piece to the puzzle that defines heart failure and have lead to an understanding of heart failure, at least in some part, as an ‘inflammatory disease’. In this review we will examine several of the key inflammatory mediators as they relate to heart failure while at the same time attempting to define the sourceŽs. of these mediators. We will examine key elements of the inflammatory cascade as they relate to heart failure such as: cytokines, ‘proximal mediators’ Že.g. NF-B., and distal mediators Že.g. nitric oxide.. We will end with a discussion of the potential therapeutic role of anti-inflammatory strategies in the future treatment of heart failure. Also, throughout the review we will examine the potential pitfalls encountered in applying bench discoveries to the bedside as have been learned in the field of septic shock research. 䊚 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Pediatric heart failure; Cytokines; Inflammation; NF-B, Nitric oxide; Tumor necrosis factor; Systemic inflammatory response syndrome
1. Introduction The physiologic diagnosis of heart failure has changed very little over the past several decades: heart failure is the inability of the cardiac output to meet the metabolic demands of the organism w1x. The clinical diagnosis of heart failure defines it as ventricular dysfunction that is accompanied by reduced exercise tolerance. Our understanding of the true pathophysiologic processes involved in heart failure have, however, changed. We have moved from thinking of heart failure as primarily a circulatory phenomenon to seeing it as a pathophysiologic state under the
E-mail address: [email protected] ŽM.R. Anderson..
control of refined neuro-hormonal influences w1x. Furthermore, clinicians and researchers have not only attempted to define heart failure more clearly but also to develop a more thorough understanding of the mechanisms by which heart failure progresses. Over the past several years the dramatic explosion of research in the fields of immunology and immunopathology have added additional complexity to our understanding of heart failure and may ultimately lead to a more complete picture of this syndrome. Here enters the concept of the systemic inflammatory response syndrome ŽSIRS.. The inflammatory response has been studied for many years. Inflammation is a well-choreographed, complex series of events wherein the organism attempts to respond to a variety of external and internal
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stimuli so as to return to homeostasis. When the system works well, invading organisms are killed, tissues are repaired, and vascular integrity is maintained. When the inflammatory machinery goes amuck, either through excessive production of pro-inflammatory molecules, the Under-production of antiinflammatory molecules or from some other yet unknown mechanism, the consequences can be devastating: tissues are damaged, capillaries leak, effective circulating volume diminishes and patients die, all as a result of an unchecked inflammatory response. There is growing evidence that this same cascade, well studied in diseases such as septic shock and autoimmune states, may play a central role in heart failure. The central regulators of SIRS are cytokines w2,3x. These molecules are produced by a variety of cells and can have dramatic effects in both a paracrine and endocrine fashion. ‘Pro-inflammatory’ cytokines Ži.e. TNF-␣, IL-1, IFN-gamma. can activate other immune cells, depress myocyte activity, as well as suppress anti-inflammatory molecule production. Anti-inflammatory cytokines Ži.e. IL-4, IL-5, IL-10. can stabilize membranes, down-regulate the production of pro-inflammatory molecules, and may play a ‘protective’ role in certain disease states w4x. It is thought that several clinical conditions such as septic shock result from an unchecked, pro-inflammatory response to an external stimulus, i.e. an infectious organism. The same immune system derangements that perpetuate disease states such as sepsis may also play a central role in the pathophysiology and progression of heart failure. For example, the cytokine TNF-␣, a classic ‘pro-inflammatory’ cytokine implicated in numerous disease states, appears to play a major role in both the pathophysiology and progression of heart failure. While much work has focused on defining the pathophysiologic derangements of the inflammatory cascade that occur in a variety of clinical heart failure types, the true role of cytokines in heart failure and the implications for future therapy remain to be resolved. While an understanding of heart failure as an ‘inflammatory’ process may lead to a better overall understanding of this disease process, it is still just one piece of the puzzle of heart failure. Only through both an understanding of the role of inflammation in heart failure and an appreciation of how inflammation figures in to the entire pathophysiology of heart failure can we truly begin to outline effective strategies for combating this important disease. In this review we examine several of the key inflammatory mediators as they relate to heart failure while at the same time attempting to define the sourceŽs. of these mediators. Finally, we review the potential therapeutic role of inflammatory manipulation in heart failure. Fig. 1 portrays a simplified
Fig. 1. A schematic representation of some of the areas of the systemic inflammatory response that may be involved in heart failure. A variety of stimuli can activate local monocytes to produce cytokines, influence cardiac myocyte activity and vascular endothelial cell function. Each of these areas will be reviewed in this paper.
schematic of the specific inflammatory components we will examine as they relate to heart failure: cytokines such as TNF and IL-1, ‘proximal mediators’ such as NF-B, and distal mediators such as nitric oxide. Obviously the entire inflammatory cascade is much more complex than this, but an understanding of these basic components will give the reader a better appreciation for the fundamental mediators of the SIRS and a context in which to examine the role of inflammation in heart failure.
2. Specific cytokine networks in heart failure 2.1. Tumor necrosis factor Tumor necrosis factor ŽTNF-␣r. appears to play a pivotal role in a myriad of human diseases including septic shock and autoimmune diseases such as rheumatoid arthritis. TNF normally exists as a 25-kDa transmembrane protein that is proteolytically cleaved from the surface of cells. The 17-kDa fragment then circulates as a stable 51-kDa trimer w5x. Like many of the mediators we will examine, much of the information on TNF comes from investigations of sepsis. At low concentrations TNF acts as a paracrine and autocrine molecule, upregulating vascular adhesion molecules, activating neutrophils and stimulating monocytes to secrete IL-1, IL-6 and TNF w5,6x. At higher concentrations, TNF acts in an endocrine fashion and as a pyrogen, activating the clotting cascade and suppressing bone marrow stem cell development. At even higher serum concentrations TNF acts as a myocardial depressant and induces nitric oxide ŽNO. synthesis leading to decreased vascular tone. TNF-␣ is produced mainly by cells of the monocytermacrophage lineage, although glial cells in the brain, Kuppfer cells in the liver, and T and B cells
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also produce TNF w7x. TNF appears to exert its effects by binding to one of two receptors, TNF-R1 Ž55 kDa. and TNF-R2 Ž75 kDa.. While both receptors have similar affinities for TNF, they appear to exert different intracellular effects once bound. Also both receptors can be cleaved, releasing soluble TNF receptors into the circulation. Serum and urine levels of these soluble receptors have been studied and found to be elevated in patients with sepsis and cancer as well as others with febrile illnesses w8x. The first reports implicating TNF as a factor in heart failure appeared in 1990 when Levine showed increased levels of TNF in patients with advanced heart failure w9x. The authors measured TNF levels in 33 adults with chronic heart failure and in 33 agematched controls. The patients with heart failure had significantly increased TNF levels Ž115 " 3 unitsrml. as compared with controls Ž9 " 3 unitsrml.. Furthermore, patients with the most cachexia, defined as less than 82% of ideal body weight, had the highest circulating TNF levels. Nine patients with renal failure had levels similar to those of controls, suggesting that higher TNF levels in patients with heart failure were not simply due to decreased renal perfusion. The complete story is not quite that simple, however. Out of the 33 patients with heart failure, 14 had levels less than 2 S.D. above the control group; thus, while TNF concentration appears to be related to cachexia in heart failure, there is obviously a large variation in serum TNF levels in heart failure patients. Furthermore, although quite practical, simply measuring serum cytokine levels is fraught with pitfalls, Ži.e. does a serum concentration actually reflect the tissuespecific concentration of interest?. Other authors have also demonstrated a relationship between TNF concentration and ischemic changes in the myocardium, measuring TNF at the site of most interest, the myocardium. Meldrum et al. measured TNF levels in myocardial biopsies taken from patients undergoing cardiopulmonary bypass w10x. Right atrial appendage biopsies were obtained before and after cardiopulmonary bypass in four patients undergoing coronary artery bypass grafting. TNF was measured both by ELISA analysis of myocardial homogenate and by immunohistochemical staining of the biopsies. As seen in Fig. 2, bypass exposure was associated with increased levels of myocardial TNF. These authors also demonstrated that TNF, which was primarily localized to the cardiac interstitium in myocardial samples obtained before bypass, was now localized to the myocytes themselves following bypass-induced ischemia. Thus, it appears that in addition to the monocytermacrophage system, the myocardium itself may also produce TNF.
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Fig. 2. Human myocardial tissue TNF-␣ levels before and after global ischemia in vivo, as determined by ELISA Ža. or by cytotoxicity assay Žb.. Mean levels " S.E.M. before and after ischemia for all patients are shown w1x. U Ps 0.001 vs. before ischemia and †Ps 0.0032 vs. before ischemia. ŽFrom: Meldrum et al. w10x with permission..
2.2. TNF-receptors
As outlined above, TNF appears to exert its effect by binding to the TNF receptors, TNF-R1 and TNFR2, found on a variety of tissues. The cleaved Žor soluble. forms of these receptors, sTNF-R1 and sTNF-R2, appear to modulate TNF activity. At higher concentrations, sTNF-R may compete for and neutralize free TNF, while at lower concentrations, the free receptor may stabilize the trimeric structure of TNF w7x. Several authors have attempted to correlate TNF-R1 and R2 concentrations with heart failure disease severity.
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Ferrari et al. examined levels of sTNF-R and found that patients with severe congestive heart failure ŽNew York Heart Association Class IV. had increased levels of both sTNF-R1 Ž4.43" 2.14 ngrml vs. 1.17" 0.43. and sTNF-R2 Ž7.55" 2.28 vs. 2.2" 0.44. when compared to age-matched, healthy controls. w11x The sTNF-R2 levels also appeared to correlate with mortality, as patients who died within 1 month of study entry had the highest levels of sTNF-R2 Ž8.18" 1.92 ngrml. of all subjects studied. The exact source of the soluble TNF receptors remains to be fully elucidated. Multiple tissues could serve as a source for increased TNF-R1 and R2 cleavage during heart failure. Recently Nozaki et al. examined the role of the mononuclear cell as a source for the sTNF-R shed by patients with heart failure w12x. These investigators isolated blood mononuclear cells from patients with heart failure ŽNYHA classification II᎐IV. and examined serum sTNF-R2 levels by ELISA and TNF-R2 cell-surface expression by FACS analysis. In a rather heterogeneous population of adults with heart failure, patients had significantly higher levels of circulating sTNF-R2 and enhanced expression of sTNF-R2 on the surface of mononuclear cells examined by FACS than did healthy controls. The mononuclear cells also demonstrated increased shedding of TNF-R2 upon stimulation with mitogen compared with control monocytes. The authors concluded that patients with heart failure show an increased expression of TNF-R2 both on the mononuclear cell surface as well as in the circulation. Whether the increased release of sTNF-R2 from stimulated monocytes represents purely an increased pool of protein available for cleavage or a difference in the response of white cells from heart failure patients remains to be determined. While it is clear that in patients with heart failure there is an increase in both TNF and TNF-regulating proteins such as sTNF-Rs, the exact pathologic effect of TNF on the patient with heart failure remains unclear. Studies on isolated myocytes have shown that exposure to a medium rich in TNF and IL-1 renders these cells less responsive to -adrenergic increases in contractility and in intracellular cAMP accumulation w13x. There is also clear evidence that TNF acts as a direct myocardial depressant in studies of whole animals. Using an experimental septic shock model, Natanson et al. showed that infusion of TNF in dogs produced left ventricular dilation and decreased cardiac output, similar to the effects of direct endotoxin infusion w14x. When analyzing studies outlined above it is interesting to question whether or not the amounts of cytokine administered truly reflect the in vivo quantities that are encountered in heart failure. While the administration of exogenous TNF certainly has dele-
terious effects in the whole animal model, one must still question whether the amounts used are relevant when compared with the levels of circulating TNF found in heart failure patients. One fascinating study that has addressed this pivotal issue was conducted by Bozkurt et al. w15x. These authors implanted osmotic pumps in rats and titrated the amount of TNF infused to the end point of serum TNF concentrations of 80᎐100 unitsrml, similar to the levels found in patients with heart failure. Rats were serially assessed by transthoracic echocardiography. Fifteen days of TNF treatment lead to a progressive depression in left ventricular function, impaired cardiac myocyte shortening, and increased left ventricular dilation ŽFig. 3.. Not all data points to TNF as a villain, however. As mentioned above, TNF can act in a autocriner paracrine manner when released in small quantities. There is some evidence that TNF may actually play a protective role in the stressed myocardium. Nakano and co-workers demonstrated that pre-incubation of feline myocytes with TNF rendered the cells less prone to hypoxic stress w16x. Cells incubated with TNF in paracrine concentrations were less susceptible to hypoxia-induced cell damage as measured by LDH release, calcium uptake and MTT metabolism. The pathway for this protective response is not known. TNF appears, therefore, to be an important mediator in human heart failure. The central question re-
Fig. 3. Effects of continuous TNF-␣ infusion on LV structure in vivo. LV dimensions were studied for 15 days in rats that underwent implantation of an intraperitoneal osmotic pump infusion that contained either diluent Ž n s 20. or TNF-␣ Ž n s 38.. After 15 days osmotic pumps were removed and animals allowed to recover. LV dimensions were serially assessed at baseline and every 5 days for a total of 30 days. ŽFrom: Bozkurt et al. w15x, with permission..
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mains: exactly what role does TNF play in the physiological imbalance known as heart failure. That is, does TNF simply represent a marker of an organism that is in a homeostatic imbalance such as severe heart failure or does it, indeed, represent a central and key mediator of progressive heart failure. Furthermore, while TNF has the ability to protect myocytes at one concentration, it can also be deleterious at a different concentration. A better understanding of the pathophysiology of heart failure is crucial, but will our new knowledge lead to improved treatment of this complex disease? If the studies of sepsis are a guide, medicine has a long way to go before it is able to interpret fully the role of TNF in heart failure and to utilize TNF-modulating therapies. 2.3. Anti-TNF strategies Fueled by the knowledge that TNF appears to also play a central role in sepsis, several trials of anti-TNF therapies have been conducted in septic patients. Vincent et al. found that a murine anti-TNF antibody administered to adults with sepsis improved left ventricular performance as measured by the left ventricular stroke work index ŽLVSWI. w17x. Other authors, however, have met with less favorable results when analyzing mortality in septic patients following administration of either anti-TNF antibodies or soluble TNF receptors Žs-TNF-Rs.. The TNF-␣ MAb Sepsis Study Group administered anti-TNF antibody to over 900 patients with sepsis as defined by objective signs of infection and at least three of five signs of SIRS: temperature instability, tachycardia, tachypnea, abnormal white blood cell count and altered end-
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organ perfusion. These authors found no difference in 28-day total mortality, although there was a trend towards decreased mortality in patients presenting with severe shock w18x. A similar trial of s-TNF-R administration to 498 patients with sepsis also found no decrease in overall 28-day mortality when compared with placebo-treated patients w19x. However, when patients with severe sepsis, Ži.e. patients with sufficient hypotension to require inotropic support. were analyzed, a significant decrease in mortality was observed in patients treated with the p55 TNF receptor protein compared with placebo-treated patients. No large trials of TNF-modulating therapy have been conducted in human heart failure patients. If the septic shock trials are any indication, we will need much more data before we can precisely use anti-TNF therapy in the treatment of heart failure.
2.4. Interleukin-1
While TNF may be one of the most widely studied cytokines in inflammation, a myriad of other cytokines have been studied in heart failure in an attempt to define more precisely the cytokine networks activated in this complex disease. Interleukin-1 ŽIL-1. is a pro-inflammatory cytokine primarily produced by monocytes and macrophages. Infusion of IL-1 reproduces many of the physiologic changes seen in septic shock such as hypotension and fever w20x. IL-1 may also cause skeletal muscle proteolysis. In a rabbit model of IL-1-induced septic shock, IL-1 has been shown to induce hypotension and to be synergistic with TNF in creating a shock state w21x. The role of
Fig. 4. Quantitative analysis of TNF-␣, IL-1 and IL-6. The value at each point in time represents the normalized mean " SEM for four rats. U P- 0.05 compared with sham operation group. 噛P- 0.05 compared with infarcted region. ŽFrom: Ono et al. w22x, with permission..
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IL-1 in heart failure has been studied both in animal and human models. In an experimental model of ischemic heart disease ŽIHD., Ono et al. studied the effects of a large myocardial infarction on IL-1, IL-6 and TNF levels as measured by PCR from myocardium in infarcted and non-infarcted tissue w22x. As seen in Fig. 4, IL-1 levels rose dramatically in the infarcted areas of the rat heart 1 week after myocardial infarction, whereas infarcted areas demonstrated an increase in IL-1 over non-infarcted areas at 8 and 20 weeks following the MI. Interestingly, these cytokine levels in non-infarcted areas correlated with left ventricular end-diastolic diameter as measured by echocardiography. Also, IL-1 levels correlated with collagen deposition in the non-infarcted tissue, implicating this cytokine in the re-modeling seen after an ischemic injury. Likewise, TNF levels increased in infarcted areas shortly after the ischemic event and increased in non-infarcted areas 5 months after ischemia. In pressure overloaded hearts other authors have shown a correlation between IL-1 and ventricular remodeling. Shioi et al. demonstrated that IL-1 RNA levels correlated with the development of left ventricular hypertrophy in hypertensive Dahl salt-sensitive rats as measured by PCR w23x. In both ischemia and pressure overload models of CHF, therefore, IL-1 has been shown to play some role in ventricular remodeling. The human data on the role of IL-1 in heart failure is just beginning to be elucidated. Francis and colleagues demonstrated that IL-1 levels were increased in human hearts with DCM as compared with hearts from patients with ischemic heart disease ŽIHD. w24x. These investigators compared the total IL-1 mRNA from explanted hearts of patients with DCM Žmean left ventricular ejection fraction of 28.5%. with that of hearts from patients transplanted for IHD ŽNYHA class III or IV.. The authors also examined frozen sections of myocardium for IL-1 by immunohistochemical staining. IL-1 mRNA bands were detected in all patients with DCM and only weakly in patients with IHD. Likewise, IL-1 was increased in eight of eight ventricles from patients with DCM as compared with three of nine patients with IHD as determined by Northern blot analysis. Immuno-staining revealed that IL-1 was present in endothelial cells of small blood vessels, in the myocytes themselves, and in the interstitium of the myocardium in patients with DCM. The distribution of IL-1 in IHD samples was not commented on. The authors concluded that IL-1 may play a role in human heart failure, at least in a more ‘inflammatory’ disease such as DCM. The lack of ‘normal’ controls, however, makes the data difficult to interpret.
It appears, then, that IL-1 may play a role in the pathogenesis of heart failure, particularly in the ‘inflammatory type’ of cardiac disease. As with many of the cytokines covered in this review, however, how this data is used in the diagnosis and management of patients will be the pivotal question as this research progresses. 2.5. Anti-IL-1 strategies Similar to studies performed using anti-TNF antibodies in septic patients, several centers have attempted to treat SIRS patients with anti-IL1 monoclonal antibodies, and like the anti-TNF trials, discovered that new found data concerning the liberation of a cytokine does not always lead to positive clinical results. In a follow-up to a smaller study w25x, Fisher and colleagues administered IL-1 receptor antagonist to over 890 adults with the sepsis syndrome w26x. While 28-day mortality was not statistically different between placebo and IL-1RA-treated patients, a trend towards decreased mortality was found in those IL-1RA treated patients with one or more organ system failures present at study entry. When designing anti-IL1 therapies or any anti-cytokine therapy for heart failure, obvious questions revolve around pharmacokinetics. If we believe that blocking IL-1 activity may be useful in heart failure, what dose of which antibody delivered to which site should be used to achieve the desired effect. Obviously we must look to the laboratory bench for many more clues to the role of IL-1 in heart failure before any such therapy can be designed.
3. Proximal mediators of inflammation 3.1. NF- B While cytokines have been studied extensively in sepsis and now in heart failure, a key process proximal to the release of cytokines is the activation of regulatory molecules such as COX-2 and NF-B, which in turn upregulate cytokine transcription and release. These important mediators may also play a role in the pathogenesis of heart failure and in the future may be a target for therapy. NF-B is a heterodimer made up of two subunits, p65 and p50, although other subunits may be present in the activated form of the protein. It is likely that different NF-B forms Ži.e. different conglomerations of subunits. may affect different regulatory genes, and thus NF-B appears to have a broad range of effects when activated w27x. When not activated, NF-B is bound
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with I B-␣ and I B-, which prevents its influx into the nucleus. When activated, NF-B is liberated from these regulatory proteins and enters the nucleus. A variety of stimuli can activate NF-B, including cytokines, lipopolysaccharide, viral infection and hypoxia. NF-B in turn controls a plethora of biological responses and regulates immune and inflammatory responses. Production of pro-inflammatory cytokines ŽIL-2, IL-1, TNF-␣ ., cell adhesion molecules, and immunoglobulins are all under the control of NF-B regulation w28x; thus, when one looks at heart failure in the context of the systemic inflammatory response, NF-B is a logical and perhaps pivotal molecule to investigate. A great deal of the work on NF-B regulation of heart failure has taken place in the context of a decidedly non-pediatric disease, IHD from coronary artery occlusion. Ritche recently analyzed NF-B activity in white blood cells isolated from patients with unstable angina pectoris or other cardiac conditions Ži.e. valvular disease, heart failure, or atypical chest pain. who were undergoing cardiac catheterization w29x. In 17 of the 19 patients with unstable angina pectoris, marked activation of NF-B was found as measured by electromobility shift assay ŽEMSA.. As demonstrated in Fig. 5, NF-B elevation correlated with the presence of unstable angina. The authors also measured NF-B levels in patients with ‘inflammatory or infectious’ diseases and found no significant elevations of NF-B. However, the specific data was not presented in the paper. In a similar paper, Wong et al. examined NF-B and COX-2 Žan additional pro-inflammatory nuclear regulator. by immunohistochemical staining of myocardium isolated either at the time of cardiac transplantation in patients with IHD or DCM or at autopsy in the case of two patients with sepsis w30x. These investigators demonstrated intense activation of NF-B in the myocardium of patients with IHD with less intense staining found in patients with DCM. Intense staining for NF-B was also demonstrated in hearts isolated from septic patients the time of autopsy. Once again it appears that the inflammatory cascade is indeed activated in patients with heart failure from a variety of causes. Likewise, it appears that one of the known ‘proximal’ activators of the inflammatory cascade is involved in the pathogenesis of heart failure.
4. Distal mediators of inflammation Just as dissection of the events proximal to the secretion of cytokine may shed some light on the pathophysiology of heart failure, an examination of the events ‘distal’ to cytokine secretion may bring us
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Fig. 5. NF-B levels of activation of patients with stable angina pectoris ŽSAP. or unstable angina pectoris ŽUSA. extrapolated from NF-B binding availability derived from EMSAs. NF-B levels were quantified by scanning laser densitometer and are shown as arbitrary units ŽAU.. Each dot represents an individual patient. ŽFrom: Ritchie w29x, with permission..
closer to understanding the inflammatory processes. Perhaps no mediator has been more widely studied both in inflammation as well as in heart failure as nitric oxide ŽNO.. Once thought of merely as an environmental polluting gas, NO is now known to be a major regulator of a variety of physiological processes. NO is produced during the conversion of L-arginine to L-citrulline by the nitric oxide synthases ŽNOS., of which there are two basic types-constitutive NOS Žc-NOS. and inducible NOS Ži-NOS. w31x. NOS nomenclature has changed over the past several years as a more thorough understanding of the synthesis of NO has emerged. c-NOS has two sub-types, NOS1 Žalso known as neuronal c-NOS. and NOS3 Žalso known as endothelial c-NOS. w32x. i-NOS is now also known as NOS2. While c-NOS appears to regulate basal NO production and plays an important role in myocardial regulation, i-NOS appears to modulate harmful upregulation of NO production which may have deleterious effects in the human heart, including decreased contractility and blunting of -adrenergic responsiveness w33x. Hypoxia and increased levels of inflammatory cytokines appear to be important triggers for increased i-NOS activity, which then leads to increased NO production in a variety of cells including endothelial cells, monocytes and cardiac myocytes w32x. Regulation of NO has been studied in animal models of heart failure as well as in humans with a variety of cardiac diseases. Iwasaki and colleagues recently used a murine model of viral myocarditis to examine the role of a new phosphodiesterase inhibitor, pimobendan, in disease survival and in the liberation of
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Fig. 6. Effects of pimobendan on intracardiac production of TNF-␣, IL-1 and IL-6 measured on day seven after EMC virus inoculation. U P- 0.001 vs. control; UU P - 0.01 vs. control. ŽFrom: Iwasaki et al. w34x, with permission..
cardiac cytokines and i-NOS activity w34x. These investigators found that in untreated infected animals, levels of pro-inflammatory cytokines TNF-␣ and IL-1 were increased as compared with the groups of animals treated with pimobendan ŽFig. 6.. Likewise, levels of intracardiac NO Žas measured by total nitrite concentration as well as i-NOS levels by PCR. were decreased in the treated animals as compared with the infected, untreated animals. While this data would appear to implicate increased production of NO and pro-inflammatory cytokines in experimental myocarditis, the lack of an uninfected control group makes the data slightly more difficult to utilize. Nevertheless, the potential for a drug that both increases myocardial contractility through phosphodiesterase inhibition and decreases inflammation is intriguing. Human studies have also begun to shed light on the role of NO in human heart failure. In autopsies performed within 6 h of death, Haywood et al. examined myocardial specimens from hearts of victims of sudden non-cardiac deaths as well as from patients under-going heart transplantation for congestive heart failure Žboth donor and recipient hearts were biopsied. w35x. i-NOS was quantified by RT-PCR as well as localized by immunohistochemical staining of the myocardial tissue. Interestingly, while no control hearts demonstrated i-NOS activity, the majority of donor hearts and an even greater percentage of diseased hearts showed i-NOS RNA by PCR ŽFig. 7.. i-NOS expression appeared to be inversely related to the severity of heart failure, since patients with the mildest symptoms ŽNYH class II. had the highest percentage of i-NOS expression compared with more severely affected patients. When analyzed by im-
munostaining, they found that all hearts with congestive heart failure showed i-NOS staining while no donor hearts demonstrated any significant i-NOS staining; thus, i-NOS expression may be a non-specific inflammatory finding in the ‘stressed’ myocardium Ži.e. organ donation patients., while patients with heart failure may produce quantitatively more NO and have more localized myocardial production of i-NOS. The relatively small numbers of patients, the study of post-mortem samples, and the use of donor hearts from brain dead patients, however, make the interpretation of this data problematic.
Fig. 7. Frequency of i-NOS and atrial natriuretic peptide ŽANP. mRNA expression in ventricular myocardium detected by RT-PCR. DCM Židiopathic dilated cardiomyopathy.; IHD Žischemic heart disease.; and valvular Žvalvular heart disease.. ŽFrom: Haywood et al. w35x, with permission..
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In a similar series of experiments investigators analyzed human left ventricular myocardium from patients with IHD and DCM who were undergoing cardiac transplantation w36x. i-NOS RNA was detected in all of the hearts and no differences between IHD and DCM patients could be found. Furthermore, all biopsies demonstrated i-NOS in vascular endothelium and vascular smooth muscle cells and some biopsies also demonstrated direct staining of cardiac myocytes for i-NOS. It is important to note, however, that no control tissues were used in the experiments. Drexler et al. took this experimental model one step further w37x. These authors isolated strips of myocardium from explanted hearts at the time of cardiac transplantation and demonstrated increased i-NOS RNA content in failing hearts compared with ‘healthy’ donor hearts ŽFig. 8.. They also demonstrated that when examining isometric contractions of cardiac muscles strips isolated from these patients, increased i-NOS activity was associated with shortening of relaxation and a blunted response to inotropic stimulation. Likewise, the inhibition of i-NOS by the agent L-NMMA improved the -agonist-induced contractile responses in the failing heart. Not all investigators have been able to demonstrate a direct correlation between NO modulation and improved cardiac myocyte function. Harding et al. isolated single ventricular myocytes from explanted failing human hearts and ‘healthy’ donor hearts w38x. Myocyte contractility was measured in response to electrical stimulation at 0.2 Hz or 1 Hz. Non-failing myocytes showed a marked increase in contraction amplitude between 0.2 and 1 Hz, while failing myocytes showed little change. The authors concluded that tonic production of NO was not responsible for
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the depressed contractility, as incubation of the cells with the NOS inhibitor L-NMMA improved neither their contractility nor their -adrenergic responsiveness. A major problem with these types of studies, however, is the lack of an ideal control. As demonstrated by the donor heart group in Haywood’s work, even ‘healthy’ donor hearts subjected to stress Ži.e. explantation, critical illness, brain death, etc.. appear to increase i-NOS expression. How to elucidate the role of i-NOS in healthy vs. diseased myocardium remains to be determined.
5. Potential anti-inflammatory treatments of heart failure A logical extension of the SIRS research in heart failure reviewed here is to the field of therapeutics. Can we extend our relatively limited knowledge of cytokine networks to the clinical arena to treat heart failure more precisely and effectively? As we have seen from our review of anti-cytokine therapies, much work remains to be done. Some of the most interesting work in the field of inflammatory modulation of heart failure has come out of the discovery of the anti-inflammatory effects of agents already used in the treatment of heart failure, particularly the phosphodiesterase inhibitors. Matsumori et al. have examined the effects of vesnarinone, a quinolone and phosphodiesterase inhibitor, on patients with heart failure. In previous human studies, investigators had found that while the mortality rate was improved in patients treated with vesnarinone, a small percentage of them developed profound neutropenia w39x, leading
Fig. 8. Quantification of c-NOS and i-NOS gene expression by competitive RT-PCR in non-failing ŽNF; n s 5. and failing hearts ŽCHF; n s 24.. The number of transcripts Žrepresenting RNA molecules per microgram total RNA. is depicted. ŽFrom: Drexler et al. w37x, with permission..
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the Matsumori group to examine the anti-inflammatory effects of the drug w40x. White cells were isolated from patients with heart failure and the production of cytokines in response to lipopolysaccharide ŽLPS. was examined in the presence or absence of the drug. Patients with heart failure had marked increases in TNF-␣ production in response to LPS stimulation, and vesnarinone inhibited the production of TNF-␣ and IFN-gamma in both healthy and diseased patients. These investigators expanded their earlier experiments by examining the effects of several other inotropes on WBC cytokine production w41x. They found that amrinone, pimobendan, and vesnarinone all inhibited TNF-␣ production of white blood cells as measured by ELISA. Furthermore, amrinone and pimobendan enhanced IL-1 production whereas vesnarinone had no effect on production of this cytokine. While there was no direct study of in-vivo cytokine production and no measurement of heart failure patients’ cytokine production, the authors concluded that the differences in the clinical effects of phosphodiesterase inhibitors in patients with heart failure may be related to differences in their anti-inflammatory effects. In similar studies, other investigators have also demonstrated that phosphodiesterase inhibitors can dampen myocyte TNF-␣ production in response to LPS in a rat model of heart failure w42x. In vivo data concerning the role of ‘anti-inflammatory’ therapy in heart failure are few. Matsumori’s group compared the anti-inflammatory role of vesnarinone vs. amrinone in a murine model of acute viral myocarditis w43,44x. Mice were infected with encephalomyocarditis virus ŽECMV., and their survival, histopathological scoring, NK cellular activity and
TNF-␣ production were analyzed. A dramatic improvement in survival was detected in the vesnarinone-treated group as compared with the control group Ž60% vs. 20%., whereas no difference in mortality was found between controls and amrinonetreated animals ŽFig. 9.. Vesnarinone-treated animals also displayed less myocardial necrosis, decreased natural killer cell activity, and less TNF-␣ production compared to untreated, ECMV-infected mice. The authors concluded that one of the beneficial effects of vesnarinone therapy may be related to its anti-inflammatory actions in addition to its phosphodiesterase inhibition. While experimental data indicating that a drug with both inotropic and anti-inflammatory properties may benefit patients with heart failure may seem compelling, caution should be exercised. A larger trial of vesnarinone was recently published in abstract form and reported an increased mortality in the vesnarinone-treated group w45x. A large human trial of antiinflammatory modulation in heart failure has yet to be published.
6. Conclusions We have reviewed the role of the inflammatory cascade in heart failure from several different perspectives. As mentioned at the beginning of the article, this is only the ‘tip of the iceberg’ as far as the breadth and depth of the inflammatory processes and others involved in the pathogenesis of heart failure. When examining the data en mass one must con-
Fig. 9. Effect of vesnarinone and amrinone on survival rate after EMCV inoculation. Four-week-old DBAr2 mice were inoculated intraperitoneally with 10 pfu of EMCV. Ža. Two groups were treated with vesnarinone ŽVN. at doses of 10 and 50 mgrkg by mouth daily. Survival of the group treated with vesnarinone at 10 mgrkg was relatively improved compared with the control group but was not significantly different. Treatment with 50 mgrkg of vesnarinone significantly reduced mortality at a very early stage ŽU P- 0.01 vs. control.. Žb. Mice were treated with amrinone ŽAM. at doses of 5 and 25 mgrkg by mouth daily. Survival was not improved compared to control mice. ŽFrom: Matsui S et al. w43x, with permission..
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clude, however, that inflammatory mediators play an important role in both the pathogenesis and progression of heart failure. What then are we to make of the new understanding of heart failure as, at least in some part, an inflammatory disease? What parallels can we draw between sepsis research and the study of inflammation in heart failure? On the one hand, we know log-fold more today than we did even 5 years ago about the individual mediators of both sepsis and heart failure. TNF-␣, NO, IL-1 and the like are now part of the language of medicine. We now know a great deal about each of these mediators, the individual pieces of the puzzle. But the real challenge for the next several years and beyond is to piece together the whole puzzle of heart failure as best we can. We need to know more about the entire ‘cytokine network’ and the interplay between individual mediators. We need to define more precisely the pharmacokinetics and pharmacodynamics of the substances we are studying. Further, we need to define more fully the instigating factors behind inflammatory mediator release and determine which factors propagate the continued release of these mediators in the failing cardiovascular system. Likewise, we need to search for relevant animal and non-animal models in which to study the inflammatory network as a whole. We also need to define how we will use this newfound data. Do we use our knowledge simply as a new insight into the pathophysiology of an age-old disease. Or do we look to a future in which precise modulation of this inflammatory cascade will benefit patients with heart failure. Many more questions will need to be answered before this dream becomes a reality, but more effective treatment and improved outcome in heart failure is still the main motivation behind this important area of research.
Acknowledgements Supported in part by National Institutes of Health Grant AI01578. References w1x De Keulenaer GW, Brutsaert DL. Pathogenesis of heart failure. Changing conceptual paradigms. Acta Cardiol 1998;53:131. w2x Bone RC. Toward a theory regarding the pathogenesis of the systemic inflammatory response syndrome: what we do and do not know about cytokine regulation. Crit Care Med 1996;24:163. w3x Glauser MP. The inflammatory cytokines. New developments in the pathophysiology and treatment of septic shock. Drugs 1996;52ŽSuppl 2.:9᎐17. w4x Schafer R, Sheil JM. Superantigens and their role in infectious disease. Adv Pediatr Infect Dis 1995;369:10᎐90.
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w5x Tracey KJ, Cerami A. Tumor necrosis factor in septic shock. In: Neugebauer EA, Holaday JA, editors. Handbook of mediators in septic shock. Boca Raton: CRC Press, 1993:291᎐308. w6x Anderson MR, Blumer JL. Advances in the therapy for sepsis in children. Pediatr Clin North Am 1997;44:179. w7x Tracey KJ, Cerami A. Tumor necrosis factor: an updated review of its biology. Crit Care Med 1993;21:S415. w8x Shapiro L, Clark BD, Orencole SF, Poutsiaka DD, Granowitz EV, Dinarello CA. Detection of tumor necrosis factor soluble receptor p55 in blood samples from healthy and endotoxemic humans. J Infect Dis 1993;167:1344. w9x Levine B, Kalman J, Mayer L, Fillit HM, Packer M. Elevated circulating levels of tumor necrosis factor in severe chronic heart failure. N Engl J Med 1990;323:236. w10x Meldrum DR, Meng X, Dinarello CA et al. Human myocardial tissue TNFalpha expression following acute global ischemia in vivo. J Mol Cell Cardiol 1998;30:1683. w11x Ferrari R, Bachetti T, Confortini R et al. Tumor necrosis factor soluble receptors in patients with various degrees of congestive heart failure wsee commentsx. Circulation 1995; 92:1479. w12x Nozaki N, Yamaguchi S, Yamaoka M, Okuyama M, Nakamura H, Tomoike H. Enhanced expression and shedding of tumor necrosis factor ŽTNF. receptors from mononuclear leukocytes in human heart failure. J Mol Cell Cardiol 1998;30:2003. w13x Gulick T, Chung MK, Pieper SJ, Lange LG, Schreiner GF. Interleukin 1 and tumor necrosis factor inhibit cardiac myocyte beta-adrenergic responsiveness. Proc Natl Acad Sci USA 1989;86:6753. w14x Natanson C, Eichenholz PW, Danner RL et al. Endotoxin and tumor necrosis factor challenges in dogs simulate the cardiovascular profile of human septic shock. J Exp Med 1989;169:823. w15x Bozkurt B, Kribbs SB, Clubb FJJ et al. Pathophysiologically relevant concentrations of tumor necrosis factor-alpha promote progressive left ventricular dysfunction and remodeling in rats. Circulation 1998;97:1382. w16x Nakano M, Knowlton AA, Dibbs Z, Mann DL. Tumor necrosis factor-alpha confers resistance to hypoxic injury in the adult mammalian cardiac myocyte. Circulation 1998;97:1392. w17x Vincent JL, Bakker J, Marecaux G, Schandene L, Kahn RJ, Dupont E. Administration of anti-TNF antibody improves left ventricular function in septic shock patients. Results of a pilot study. Chest 1992;101:810. w18x Abraham E, Wunderink R, Silverman H et al. Efficacy and safety of monoclonal antibody to human tumor necrosis factor alpha in patients with sepsis syndrome. A randomized, controlled, double-blind, multicenter clinical trial. TNF-alpha MAb Sepsis Study Group. J Am Med Assoc 1995;273:934. w19x Abraham E, Glauser MP, Butler T et al. p55 Tumor necrosis factor receptor fusion protein in the treatment of patients with severe sepsis and septic shock. A randomized controlled multicenter trial. Ro 45-2081 Study Group. J Am Med Assoc 1997;277:1531. w20x Spitzer JA. Interleukins in sepsis. In: Neugebauer EA, Holaday JA, editors. Handbook of mediators in septic shock. Boca Raton: CRC Press, 1993:279᎐289. w21x Okusawa S, Gelfand JA, Ikejima T, Connolly RJ, Dinarello CA. Interleukin 1 induces a shock-like state in rabbits. Synergism with tumor necrosis factor and the effect of cyclooxygenase inhibition. J Clin Invest 1988;81:1162. w22x Ono K, Matsumori A, Shioi T, Furukawa Y, Sasayama S. Cytokine gene expression after myocardial infarction in rat hearts: possible implication in left ventricular remodeling. Circulation 1998;98:149. w23x Shioi T, Matsumori A, Kihara Y et al. Increased expression
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M.R. Anderson r Progress in Pediatric Cardiology 11 (2000) 219᎐230 of interleukin-1 beta and monocyte chemotactic and activating factorrmonocyte chemoattractant protein-1 in the hypertrophied and failing heart with pressure overload. Circ Res 1997;81:664. Francis SE, Holden H, Holt CM, Duff GW. Interleukin-1 in myocardium and coronary arteries of patients with dilated cardiomyopathy. J Mol Cell Cardiol 1998;30:215. Fisher CJJ, Slotman GJ, Opal SM et al. Initial evaluation of human recombinant interleukin-1 receptor antagonist in the treatment of sepsis syndrome: a randomized, open- label, placebo-controlled multicenter trial. The IL-1RA Sepsis Syndrome Study Group wsee commentsx. Crit Care Med 1994;22:12. Fisher CJJ, Dhainaut JF, Opal SM et al. Recombinant human interleukin 1 receptor antagonist in the treatment of patients with sepsis syndrome. Results from a randomized, doubleblind, placebo-controlled trial. Phase III rhIL-1ra Sepsis Syndrome Study Group wsee commentsx. J Am Med Assoc 1994;271:1836. Lee JI, Burckart GJ. Nuclear factor kappa B: important transcription factor and therapeutic target. J Clin Pharmacol 1998;38:981. Barnes PJ, Karin M. Nuclear factor-kappaB: a pivotal transcription factor in chronic inflammatory diseases. N Engl J Med 1997;336:1066. Ritchie ME. Nuclear factor-kappaB is selectively and markedly activated in humans with unstable angina pectoris. Circulation 1998;98:1707. Wong SC, Fukuchi M, Melnyk P, Rodger I, Giaid A. Induction of cyclooxygenase-2 and activation of nuclear factor-kappaB in myocardium of patients with congestive heart failure wsee commentsx. Circulation 1998;98:100. Moncada S, Higgs A. The L-arginine-nitric oxide pathway. N Engl J Med 1993;329:2002. Sawyer DB, Colucci WS. Nitric oxide in the failing myocardium. Cardiol Clin 1998;16:657. Hare JM, Loh E, Creager MA, Colucci WS. Nitric oxide inhibits the positive inotropic response to beta-adrenergic stimulation in humans with left ventricular dysfunction. Circulation 1995;92:2198. Iwasaki A et al. Pimobendan inhibits the production of proinflammatory cytokines and gene expression of inducible nitric oxide synthase in a murine model of viral myocarditis win process citationx. J Am Coll Cardiol 1999;33:1400.
w35x Haywood GA, Tsao PS, von der L et al. Expression of inducible nitric oxide synthase in human heart failure wsee commentsx. Circulation 1996;93:1087. w36x Vejlstrup NG, Bouloumie A, Boesgaard S et al. Inducible nitric oxide synthase ŽiNOS. in the human heart: expression and localization in congestive heart failure. J Mol Cell Cardiol 1998;30:1215. w37x Drexler H, Kastner S, Strobel A, Studer R, Brodde OE, Hasenfuss G. Expression, activity and functional significance of inducible nitric oxide synthase in the failing human heart. J Am Coll Cardiol 1998;32:955. w38x Harding SE, Davies CH, Money-Kyrle AM, Poole-Wilson PA. An inhibitor of nitric oxide synthase does not increase contraction or beta-adrenoceptor sensitivity of ventricular myocytes from failing human heart. Cardiovasc Res 1998;40:523. w39x Feldman AM, Bristow MR, Parmley WW et al. Effects of vesnarinone on morbidity and mortality in patients with heart failure. Vesnarinone Study Group wsee commentsx. N Engl J Med 1993;329:149. w40x Matsumori A, Shioi T, Yamada T, Matsui S, Sasayama S. Vesnarinone, a new inotropic agent, inhibits cytokine production by stimulated human blood from patients with heart failure. Circulation 1994;89:955. w41x Matsumori A, Ono K, Sato Y, Shioi T, Nose Y, Sasayama S. Differential modulation of cytokine production by drugs: implications for therapy in heart failure. J Mol Cell Cardiol 1996;28:2491. w42x Bergman MR, Holycross BJ. Pharmacological modulation of myocardial tumor necrosis factor alpha production by phosphodiesterase inhibitors wpublished erratum appears in J Pharmacol Exp Ther 1997;280Ž1.:520x. J Pharmacol Exp Ther 1996;279:247. w43x Matsui S, Matsumori A, Matoba Y, Uchida A, Sasayama S. Treatment of virus-induced myocardial injury with a novel immunomodulating agent, vesnarinone. Suppression of natural killer cell activity and tumor necrosis factor-alpha production. J Clin Invest 1994;94:1212. w44x Matsumori A, Sasayama S. Immunomodulating agents for the management of heart failure with myocarditis and cardiomyopathy ᎏ lessons from animal experiments. Eur Heart J ŽSuppl O. 1995;16:140᎐143. w45x Feldman AM, Young J, Bourge R et al. Mechanism of increased mortality from vesnarinone in the Severe Heart Trial ŽVesT. wAbstractx, J Am Coll Cardiol 1997;29.
The systemic inflammatory response in heart failure Michael R. Anderson Department of Pediatrics, Case Western Reser¨ e Uni¨ ersity School of Medicine, Di¨ ision of Pediatric Pharmacology and Critical Care, Rainbow Babies and Childrens’ Hospital, 11100 Euclid A¨ enue, Cle¨ eland, OH 44106, USA
Abstract The physiologic diagnosis of heart failure has changed very little over the past several decades: heart failure is the inability of the cardiac output to meet the metabolic demands of the organism. The clinical definition of heart failure Žalso relatively unchanged. describes it as ventricular dysfunction that is accompanied by reduced exercise tolerance. Our understanding of the true pathophysiologic processes involved in heart failure have, however, changed. We have moved from thinking of heart failure as primarily a circulatory phenomenon to seeing it as a pathophysiologic state under the control of multiple complex systems. Over the past several years the dramatic explosion of research in the fields of immunology and immunopathology have added an additional piece to the puzzle that defines heart failure and have lead to an understanding of heart failure, at least in some part, as an ‘inflammatory disease’. In this review we will examine several of the key inflammatory mediators as they relate to heart failure while at the same time attempting to define the sourceŽs. of these mediators. We will examine key elements of the inflammatory cascade as they relate to heart failure such as: cytokines, ‘proximal mediators’ Že.g. NF-B., and distal mediators Že.g. nitric oxide.. We will end with a discussion of the potential therapeutic role of anti-inflammatory strategies in the future treatment of heart failure. Also, throughout the review we will examine the potential pitfalls encountered in applying bench discoveries to the bedside as have been learned in the field of septic shock research. 䊚 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Pediatric heart failure; Cytokines; Inflammation; NF-B, Nitric oxide; Tumor necrosis factor; Systemic inflammatory response syndrome
1. Introduction The physiologic diagnosis of heart failure has changed very little over the past several decades: heart failure is the inability of the cardiac output to meet the metabolic demands of the organism w1x. The clinical diagnosis of heart failure defines it as ventricular dysfunction that is accompanied by reduced exercise tolerance. Our understanding of the true pathophysiologic processes involved in heart failure have, however, changed. We have moved from thinking of heart failure as primarily a circulatory phenomenon to seeing it as a pathophysiologic state under the
E-mail address: [email protected] ŽM.R. Anderson..
control of refined neuro-hormonal influences w1x. Furthermore, clinicians and researchers have not only attempted to define heart failure more clearly but also to develop a more thorough understanding of the mechanisms by which heart failure progresses. Over the past several years the dramatic explosion of research in the fields of immunology and immunopathology have added additional complexity to our understanding of heart failure and may ultimately lead to a more complete picture of this syndrome. Here enters the concept of the systemic inflammatory response syndrome ŽSIRS.. The inflammatory response has been studied for many years. Inflammation is a well-choreographed, complex series of events wherein the organism attempts to respond to a variety of external and internal
1058-9813r00r$ - see front matter 䊚 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 1 0 5 8 - 9 8 1 3 Ž 0 0 . 0 0 0 5 3 - 9
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stimuli so as to return to homeostasis. When the system works well, invading organisms are killed, tissues are repaired, and vascular integrity is maintained. When the inflammatory machinery goes amuck, either through excessive production of pro-inflammatory molecules, the Under-production of antiinflammatory molecules or from some other yet unknown mechanism, the consequences can be devastating: tissues are damaged, capillaries leak, effective circulating volume diminishes and patients die, all as a result of an unchecked inflammatory response. There is growing evidence that this same cascade, well studied in diseases such as septic shock and autoimmune states, may play a central role in heart failure. The central regulators of SIRS are cytokines w2,3x. These molecules are produced by a variety of cells and can have dramatic effects in both a paracrine and endocrine fashion. ‘Pro-inflammatory’ cytokines Ži.e. TNF-␣, IL-1, IFN-gamma. can activate other immune cells, depress myocyte activity, as well as suppress anti-inflammatory molecule production. Anti-inflammatory cytokines Ži.e. IL-4, IL-5, IL-10. can stabilize membranes, down-regulate the production of pro-inflammatory molecules, and may play a ‘protective’ role in certain disease states w4x. It is thought that several clinical conditions such as septic shock result from an unchecked, pro-inflammatory response to an external stimulus, i.e. an infectious organism. The same immune system derangements that perpetuate disease states such as sepsis may also play a central role in the pathophysiology and progression of heart failure. For example, the cytokine TNF-␣, a classic ‘pro-inflammatory’ cytokine implicated in numerous disease states, appears to play a major role in both the pathophysiology and progression of heart failure. While much work has focused on defining the pathophysiologic derangements of the inflammatory cascade that occur in a variety of clinical heart failure types, the true role of cytokines in heart failure and the implications for future therapy remain to be resolved. While an understanding of heart failure as an ‘inflammatory’ process may lead to a better overall understanding of this disease process, it is still just one piece of the puzzle of heart failure. Only through both an understanding of the role of inflammation in heart failure and an appreciation of how inflammation figures in to the entire pathophysiology of heart failure can we truly begin to outline effective strategies for combating this important disease. In this review we examine several of the key inflammatory mediators as they relate to heart failure while at the same time attempting to define the sourceŽs. of these mediators. Finally, we review the potential therapeutic role of inflammatory manipulation in heart failure. Fig. 1 portrays a simplified
Fig. 1. A schematic representation of some of the areas of the systemic inflammatory response that may be involved in heart failure. A variety of stimuli can activate local monocytes to produce cytokines, influence cardiac myocyte activity and vascular endothelial cell function. Each of these areas will be reviewed in this paper.
schematic of the specific inflammatory components we will examine as they relate to heart failure: cytokines such as TNF and IL-1, ‘proximal mediators’ such as NF-B, and distal mediators such as nitric oxide. Obviously the entire inflammatory cascade is much more complex than this, but an understanding of these basic components will give the reader a better appreciation for the fundamental mediators of the SIRS and a context in which to examine the role of inflammation in heart failure.
2. Specific cytokine networks in heart failure 2.1. Tumor necrosis factor Tumor necrosis factor ŽTNF-␣r. appears to play a pivotal role in a myriad of human diseases including septic shock and autoimmune diseases such as rheumatoid arthritis. TNF normally exists as a 25-kDa transmembrane protein that is proteolytically cleaved from the surface of cells. The 17-kDa fragment then circulates as a stable 51-kDa trimer w5x. Like many of the mediators we will examine, much of the information on TNF comes from investigations of sepsis. At low concentrations TNF acts as a paracrine and autocrine molecule, upregulating vascular adhesion molecules, activating neutrophils and stimulating monocytes to secrete IL-1, IL-6 and TNF w5,6x. At higher concentrations, TNF acts in an endocrine fashion and as a pyrogen, activating the clotting cascade and suppressing bone marrow stem cell development. At even higher serum concentrations TNF acts as a myocardial depressant and induces nitric oxide ŽNO. synthesis leading to decreased vascular tone. TNF-␣ is produced mainly by cells of the monocytermacrophage lineage, although glial cells in the brain, Kuppfer cells in the liver, and T and B cells
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also produce TNF w7x. TNF appears to exert its effects by binding to one of two receptors, TNF-R1 Ž55 kDa. and TNF-R2 Ž75 kDa.. While both receptors have similar affinities for TNF, they appear to exert different intracellular effects once bound. Also both receptors can be cleaved, releasing soluble TNF receptors into the circulation. Serum and urine levels of these soluble receptors have been studied and found to be elevated in patients with sepsis and cancer as well as others with febrile illnesses w8x. The first reports implicating TNF as a factor in heart failure appeared in 1990 when Levine showed increased levels of TNF in patients with advanced heart failure w9x. The authors measured TNF levels in 33 adults with chronic heart failure and in 33 agematched controls. The patients with heart failure had significantly increased TNF levels Ž115 " 3 unitsrml. as compared with controls Ž9 " 3 unitsrml.. Furthermore, patients with the most cachexia, defined as less than 82% of ideal body weight, had the highest circulating TNF levels. Nine patients with renal failure had levels similar to those of controls, suggesting that higher TNF levels in patients with heart failure were not simply due to decreased renal perfusion. The complete story is not quite that simple, however. Out of the 33 patients with heart failure, 14 had levels less than 2 S.D. above the control group; thus, while TNF concentration appears to be related to cachexia in heart failure, there is obviously a large variation in serum TNF levels in heart failure patients. Furthermore, although quite practical, simply measuring serum cytokine levels is fraught with pitfalls, Ži.e. does a serum concentration actually reflect the tissuespecific concentration of interest?. Other authors have also demonstrated a relationship between TNF concentration and ischemic changes in the myocardium, measuring TNF at the site of most interest, the myocardium. Meldrum et al. measured TNF levels in myocardial biopsies taken from patients undergoing cardiopulmonary bypass w10x. Right atrial appendage biopsies were obtained before and after cardiopulmonary bypass in four patients undergoing coronary artery bypass grafting. TNF was measured both by ELISA analysis of myocardial homogenate and by immunohistochemical staining of the biopsies. As seen in Fig. 2, bypass exposure was associated with increased levels of myocardial TNF. These authors also demonstrated that TNF, which was primarily localized to the cardiac interstitium in myocardial samples obtained before bypass, was now localized to the myocytes themselves following bypass-induced ischemia. Thus, it appears that in addition to the monocytermacrophage system, the myocardium itself may also produce TNF.
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Fig. 2. Human myocardial tissue TNF-␣ levels before and after global ischemia in vivo, as determined by ELISA Ža. or by cytotoxicity assay Žb.. Mean levels " S.E.M. before and after ischemia for all patients are shown w1x. U Ps 0.001 vs. before ischemia and †Ps 0.0032 vs. before ischemia. ŽFrom: Meldrum et al. w10x with permission..
2.2. TNF-receptors
As outlined above, TNF appears to exert its effect by binding to the TNF receptors, TNF-R1 and TNFR2, found on a variety of tissues. The cleaved Žor soluble. forms of these receptors, sTNF-R1 and sTNF-R2, appear to modulate TNF activity. At higher concentrations, sTNF-R may compete for and neutralize free TNF, while at lower concentrations, the free receptor may stabilize the trimeric structure of TNF w7x. Several authors have attempted to correlate TNF-R1 and R2 concentrations with heart failure disease severity.
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Ferrari et al. examined levels of sTNF-R and found that patients with severe congestive heart failure ŽNew York Heart Association Class IV. had increased levels of both sTNF-R1 Ž4.43" 2.14 ngrml vs. 1.17" 0.43. and sTNF-R2 Ž7.55" 2.28 vs. 2.2" 0.44. when compared to age-matched, healthy controls. w11x The sTNF-R2 levels also appeared to correlate with mortality, as patients who died within 1 month of study entry had the highest levels of sTNF-R2 Ž8.18" 1.92 ngrml. of all subjects studied. The exact source of the soluble TNF receptors remains to be fully elucidated. Multiple tissues could serve as a source for increased TNF-R1 and R2 cleavage during heart failure. Recently Nozaki et al. examined the role of the mononuclear cell as a source for the sTNF-R shed by patients with heart failure w12x. These investigators isolated blood mononuclear cells from patients with heart failure ŽNYHA classification II᎐IV. and examined serum sTNF-R2 levels by ELISA and TNF-R2 cell-surface expression by FACS analysis. In a rather heterogeneous population of adults with heart failure, patients had significantly higher levels of circulating sTNF-R2 and enhanced expression of sTNF-R2 on the surface of mononuclear cells examined by FACS than did healthy controls. The mononuclear cells also demonstrated increased shedding of TNF-R2 upon stimulation with mitogen compared with control monocytes. The authors concluded that patients with heart failure show an increased expression of TNF-R2 both on the mononuclear cell surface as well as in the circulation. Whether the increased release of sTNF-R2 from stimulated monocytes represents purely an increased pool of protein available for cleavage or a difference in the response of white cells from heart failure patients remains to be determined. While it is clear that in patients with heart failure there is an increase in both TNF and TNF-regulating proteins such as sTNF-Rs, the exact pathologic effect of TNF on the patient with heart failure remains unclear. Studies on isolated myocytes have shown that exposure to a medium rich in TNF and IL-1 renders these cells less responsive to -adrenergic increases in contractility and in intracellular cAMP accumulation w13x. There is also clear evidence that TNF acts as a direct myocardial depressant in studies of whole animals. Using an experimental septic shock model, Natanson et al. showed that infusion of TNF in dogs produced left ventricular dilation and decreased cardiac output, similar to the effects of direct endotoxin infusion w14x. When analyzing studies outlined above it is interesting to question whether or not the amounts of cytokine administered truly reflect the in vivo quantities that are encountered in heart failure. While the administration of exogenous TNF certainly has dele-
terious effects in the whole animal model, one must still question whether the amounts used are relevant when compared with the levels of circulating TNF found in heart failure patients. One fascinating study that has addressed this pivotal issue was conducted by Bozkurt et al. w15x. These authors implanted osmotic pumps in rats and titrated the amount of TNF infused to the end point of serum TNF concentrations of 80᎐100 unitsrml, similar to the levels found in patients with heart failure. Rats were serially assessed by transthoracic echocardiography. Fifteen days of TNF treatment lead to a progressive depression in left ventricular function, impaired cardiac myocyte shortening, and increased left ventricular dilation ŽFig. 3.. Not all data points to TNF as a villain, however. As mentioned above, TNF can act in a autocriner paracrine manner when released in small quantities. There is some evidence that TNF may actually play a protective role in the stressed myocardium. Nakano and co-workers demonstrated that pre-incubation of feline myocytes with TNF rendered the cells less prone to hypoxic stress w16x. Cells incubated with TNF in paracrine concentrations were less susceptible to hypoxia-induced cell damage as measured by LDH release, calcium uptake and MTT metabolism. The pathway for this protective response is not known. TNF appears, therefore, to be an important mediator in human heart failure. The central question re-
Fig. 3. Effects of continuous TNF-␣ infusion on LV structure in vivo. LV dimensions were studied for 15 days in rats that underwent implantation of an intraperitoneal osmotic pump infusion that contained either diluent Ž n s 20. or TNF-␣ Ž n s 38.. After 15 days osmotic pumps were removed and animals allowed to recover. LV dimensions were serially assessed at baseline and every 5 days for a total of 30 days. ŽFrom: Bozkurt et al. w15x, with permission..
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mains: exactly what role does TNF play in the physiological imbalance known as heart failure. That is, does TNF simply represent a marker of an organism that is in a homeostatic imbalance such as severe heart failure or does it, indeed, represent a central and key mediator of progressive heart failure. Furthermore, while TNF has the ability to protect myocytes at one concentration, it can also be deleterious at a different concentration. A better understanding of the pathophysiology of heart failure is crucial, but will our new knowledge lead to improved treatment of this complex disease? If the studies of sepsis are a guide, medicine has a long way to go before it is able to interpret fully the role of TNF in heart failure and to utilize TNF-modulating therapies. 2.3. Anti-TNF strategies Fueled by the knowledge that TNF appears to also play a central role in sepsis, several trials of anti-TNF therapies have been conducted in septic patients. Vincent et al. found that a murine anti-TNF antibody administered to adults with sepsis improved left ventricular performance as measured by the left ventricular stroke work index ŽLVSWI. w17x. Other authors, however, have met with less favorable results when analyzing mortality in septic patients following administration of either anti-TNF antibodies or soluble TNF receptors Žs-TNF-Rs.. The TNF-␣ MAb Sepsis Study Group administered anti-TNF antibody to over 900 patients with sepsis as defined by objective signs of infection and at least three of five signs of SIRS: temperature instability, tachycardia, tachypnea, abnormal white blood cell count and altered end-
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organ perfusion. These authors found no difference in 28-day total mortality, although there was a trend towards decreased mortality in patients presenting with severe shock w18x. A similar trial of s-TNF-R administration to 498 patients with sepsis also found no decrease in overall 28-day mortality when compared with placebo-treated patients w19x. However, when patients with severe sepsis, Ži.e. patients with sufficient hypotension to require inotropic support. were analyzed, a significant decrease in mortality was observed in patients treated with the p55 TNF receptor protein compared with placebo-treated patients. No large trials of TNF-modulating therapy have been conducted in human heart failure patients. If the septic shock trials are any indication, we will need much more data before we can precisely use anti-TNF therapy in the treatment of heart failure.
2.4. Interleukin-1
While TNF may be one of the most widely studied cytokines in inflammation, a myriad of other cytokines have been studied in heart failure in an attempt to define more precisely the cytokine networks activated in this complex disease. Interleukin-1 ŽIL-1. is a pro-inflammatory cytokine primarily produced by monocytes and macrophages. Infusion of IL-1 reproduces many of the physiologic changes seen in septic shock such as hypotension and fever w20x. IL-1 may also cause skeletal muscle proteolysis. In a rabbit model of IL-1-induced septic shock, IL-1 has been shown to induce hypotension and to be synergistic with TNF in creating a shock state w21x. The role of
Fig. 4. Quantitative analysis of TNF-␣, IL-1 and IL-6. The value at each point in time represents the normalized mean " SEM for four rats. U P- 0.05 compared with sham operation group. 噛P- 0.05 compared with infarcted region. ŽFrom: Ono et al. w22x, with permission..
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IL-1 in heart failure has been studied both in animal and human models. In an experimental model of ischemic heart disease ŽIHD., Ono et al. studied the effects of a large myocardial infarction on IL-1, IL-6 and TNF levels as measured by PCR from myocardium in infarcted and non-infarcted tissue w22x. As seen in Fig. 4, IL-1 levels rose dramatically in the infarcted areas of the rat heart 1 week after myocardial infarction, whereas infarcted areas demonstrated an increase in IL-1 over non-infarcted areas at 8 and 20 weeks following the MI. Interestingly, these cytokine levels in non-infarcted areas correlated with left ventricular end-diastolic diameter as measured by echocardiography. Also, IL-1 levels correlated with collagen deposition in the non-infarcted tissue, implicating this cytokine in the re-modeling seen after an ischemic injury. Likewise, TNF levels increased in infarcted areas shortly after the ischemic event and increased in non-infarcted areas 5 months after ischemia. In pressure overloaded hearts other authors have shown a correlation between IL-1 and ventricular remodeling. Shioi et al. demonstrated that IL-1 RNA levels correlated with the development of left ventricular hypertrophy in hypertensive Dahl salt-sensitive rats as measured by PCR w23x. In both ischemia and pressure overload models of CHF, therefore, IL-1 has been shown to play some role in ventricular remodeling. The human data on the role of IL-1 in heart failure is just beginning to be elucidated. Francis and colleagues demonstrated that IL-1 levels were increased in human hearts with DCM as compared with hearts from patients with ischemic heart disease ŽIHD. w24x. These investigators compared the total IL-1 mRNA from explanted hearts of patients with DCM Žmean left ventricular ejection fraction of 28.5%. with that of hearts from patients transplanted for IHD ŽNYHA class III or IV.. The authors also examined frozen sections of myocardium for IL-1 by immunohistochemical staining. IL-1 mRNA bands were detected in all patients with DCM and only weakly in patients with IHD. Likewise, IL-1 was increased in eight of eight ventricles from patients with DCM as compared with three of nine patients with IHD as determined by Northern blot analysis. Immuno-staining revealed that IL-1 was present in endothelial cells of small blood vessels, in the myocytes themselves, and in the interstitium of the myocardium in patients with DCM. The distribution of IL-1 in IHD samples was not commented on. The authors concluded that IL-1 may play a role in human heart failure, at least in a more ‘inflammatory’ disease such as DCM. The lack of ‘normal’ controls, however, makes the data difficult to interpret.
It appears, then, that IL-1 may play a role in the pathogenesis of heart failure, particularly in the ‘inflammatory type’ of cardiac disease. As with many of the cytokines covered in this review, however, how this data is used in the diagnosis and management of patients will be the pivotal question as this research progresses. 2.5. Anti-IL-1 strategies Similar to studies performed using anti-TNF antibodies in septic patients, several centers have attempted to treat SIRS patients with anti-IL1 monoclonal antibodies, and like the anti-TNF trials, discovered that new found data concerning the liberation of a cytokine does not always lead to positive clinical results. In a follow-up to a smaller study w25x, Fisher and colleagues administered IL-1 receptor antagonist to over 890 adults with the sepsis syndrome w26x. While 28-day mortality was not statistically different between placebo and IL-1RA-treated patients, a trend towards decreased mortality was found in those IL-1RA treated patients with one or more organ system failures present at study entry. When designing anti-IL1 therapies or any anti-cytokine therapy for heart failure, obvious questions revolve around pharmacokinetics. If we believe that blocking IL-1 activity may be useful in heart failure, what dose of which antibody delivered to which site should be used to achieve the desired effect. Obviously we must look to the laboratory bench for many more clues to the role of IL-1 in heart failure before any such therapy can be designed.
3. Proximal mediators of inflammation 3.1. NF- B While cytokines have been studied extensively in sepsis and now in heart failure, a key process proximal to the release of cytokines is the activation of regulatory molecules such as COX-2 and NF-B, which in turn upregulate cytokine transcription and release. These important mediators may also play a role in the pathogenesis of heart failure and in the future may be a target for therapy. NF-B is a heterodimer made up of two subunits, p65 and p50, although other subunits may be present in the activated form of the protein. It is likely that different NF-B forms Ži.e. different conglomerations of subunits. may affect different regulatory genes, and thus NF-B appears to have a broad range of effects when activated w27x. When not activated, NF-B is bound
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with I B-␣ and I B-, which prevents its influx into the nucleus. When activated, NF-B is liberated from these regulatory proteins and enters the nucleus. A variety of stimuli can activate NF-B, including cytokines, lipopolysaccharide, viral infection and hypoxia. NF-B in turn controls a plethora of biological responses and regulates immune and inflammatory responses. Production of pro-inflammatory cytokines ŽIL-2, IL-1, TNF-␣ ., cell adhesion molecules, and immunoglobulins are all under the control of NF-B regulation w28x; thus, when one looks at heart failure in the context of the systemic inflammatory response, NF-B is a logical and perhaps pivotal molecule to investigate. A great deal of the work on NF-B regulation of heart failure has taken place in the context of a decidedly non-pediatric disease, IHD from coronary artery occlusion. Ritche recently analyzed NF-B activity in white blood cells isolated from patients with unstable angina pectoris or other cardiac conditions Ži.e. valvular disease, heart failure, or atypical chest pain. who were undergoing cardiac catheterization w29x. In 17 of the 19 patients with unstable angina pectoris, marked activation of NF-B was found as measured by electromobility shift assay ŽEMSA.. As demonstrated in Fig. 5, NF-B elevation correlated with the presence of unstable angina. The authors also measured NF-B levels in patients with ‘inflammatory or infectious’ diseases and found no significant elevations of NF-B. However, the specific data was not presented in the paper. In a similar paper, Wong et al. examined NF-B and COX-2 Žan additional pro-inflammatory nuclear regulator. by immunohistochemical staining of myocardium isolated either at the time of cardiac transplantation in patients with IHD or DCM or at autopsy in the case of two patients with sepsis w30x. These investigators demonstrated intense activation of NF-B in the myocardium of patients with IHD with less intense staining found in patients with DCM. Intense staining for NF-B was also demonstrated in hearts isolated from septic patients the time of autopsy. Once again it appears that the inflammatory cascade is indeed activated in patients with heart failure from a variety of causes. Likewise, it appears that one of the known ‘proximal’ activators of the inflammatory cascade is involved in the pathogenesis of heart failure.
4. Distal mediators of inflammation Just as dissection of the events proximal to the secretion of cytokine may shed some light on the pathophysiology of heart failure, an examination of the events ‘distal’ to cytokine secretion may bring us
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Fig. 5. NF-B levels of activation of patients with stable angina pectoris ŽSAP. or unstable angina pectoris ŽUSA. extrapolated from NF-B binding availability derived from EMSAs. NF-B levels were quantified by scanning laser densitometer and are shown as arbitrary units ŽAU.. Each dot represents an individual patient. ŽFrom: Ritchie w29x, with permission..
closer to understanding the inflammatory processes. Perhaps no mediator has been more widely studied both in inflammation as well as in heart failure as nitric oxide ŽNO.. Once thought of merely as an environmental polluting gas, NO is now known to be a major regulator of a variety of physiological processes. NO is produced during the conversion of L-arginine to L-citrulline by the nitric oxide synthases ŽNOS., of which there are two basic types-constitutive NOS Žc-NOS. and inducible NOS Ži-NOS. w31x. NOS nomenclature has changed over the past several years as a more thorough understanding of the synthesis of NO has emerged. c-NOS has two sub-types, NOS1 Žalso known as neuronal c-NOS. and NOS3 Žalso known as endothelial c-NOS. w32x. i-NOS is now also known as NOS2. While c-NOS appears to regulate basal NO production and plays an important role in myocardial regulation, i-NOS appears to modulate harmful upregulation of NO production which may have deleterious effects in the human heart, including decreased contractility and blunting of -adrenergic responsiveness w33x. Hypoxia and increased levels of inflammatory cytokines appear to be important triggers for increased i-NOS activity, which then leads to increased NO production in a variety of cells including endothelial cells, monocytes and cardiac myocytes w32x. Regulation of NO has been studied in animal models of heart failure as well as in humans with a variety of cardiac diseases. Iwasaki and colleagues recently used a murine model of viral myocarditis to examine the role of a new phosphodiesterase inhibitor, pimobendan, in disease survival and in the liberation of
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Fig. 6. Effects of pimobendan on intracardiac production of TNF-␣, IL-1 and IL-6 measured on day seven after EMC virus inoculation. U P- 0.001 vs. control; UU P - 0.01 vs. control. ŽFrom: Iwasaki et al. w34x, with permission..
cardiac cytokines and i-NOS activity w34x. These investigators found that in untreated infected animals, levels of pro-inflammatory cytokines TNF-␣ and IL-1 were increased as compared with the groups of animals treated with pimobendan ŽFig. 6.. Likewise, levels of intracardiac NO Žas measured by total nitrite concentration as well as i-NOS levels by PCR. were decreased in the treated animals as compared with the infected, untreated animals. While this data would appear to implicate increased production of NO and pro-inflammatory cytokines in experimental myocarditis, the lack of an uninfected control group makes the data slightly more difficult to utilize. Nevertheless, the potential for a drug that both increases myocardial contractility through phosphodiesterase inhibition and decreases inflammation is intriguing. Human studies have also begun to shed light on the role of NO in human heart failure. In autopsies performed within 6 h of death, Haywood et al. examined myocardial specimens from hearts of victims of sudden non-cardiac deaths as well as from patients under-going heart transplantation for congestive heart failure Žboth donor and recipient hearts were biopsied. w35x. i-NOS was quantified by RT-PCR as well as localized by immunohistochemical staining of the myocardial tissue. Interestingly, while no control hearts demonstrated i-NOS activity, the majority of donor hearts and an even greater percentage of diseased hearts showed i-NOS RNA by PCR ŽFig. 7.. i-NOS expression appeared to be inversely related to the severity of heart failure, since patients with the mildest symptoms ŽNYH class II. had the highest percentage of i-NOS expression compared with more severely affected patients. When analyzed by im-
munostaining, they found that all hearts with congestive heart failure showed i-NOS staining while no donor hearts demonstrated any significant i-NOS staining; thus, i-NOS expression may be a non-specific inflammatory finding in the ‘stressed’ myocardium Ži.e. organ donation patients., while patients with heart failure may produce quantitatively more NO and have more localized myocardial production of i-NOS. The relatively small numbers of patients, the study of post-mortem samples, and the use of donor hearts from brain dead patients, however, make the interpretation of this data problematic.
Fig. 7. Frequency of i-NOS and atrial natriuretic peptide ŽANP. mRNA expression in ventricular myocardium detected by RT-PCR. DCM Židiopathic dilated cardiomyopathy.; IHD Žischemic heart disease.; and valvular Žvalvular heart disease.. ŽFrom: Haywood et al. w35x, with permission..
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In a similar series of experiments investigators analyzed human left ventricular myocardium from patients with IHD and DCM who were undergoing cardiac transplantation w36x. i-NOS RNA was detected in all of the hearts and no differences between IHD and DCM patients could be found. Furthermore, all biopsies demonstrated i-NOS in vascular endothelium and vascular smooth muscle cells and some biopsies also demonstrated direct staining of cardiac myocytes for i-NOS. It is important to note, however, that no control tissues were used in the experiments. Drexler et al. took this experimental model one step further w37x. These authors isolated strips of myocardium from explanted hearts at the time of cardiac transplantation and demonstrated increased i-NOS RNA content in failing hearts compared with ‘healthy’ donor hearts ŽFig. 8.. They also demonstrated that when examining isometric contractions of cardiac muscles strips isolated from these patients, increased i-NOS activity was associated with shortening of relaxation and a blunted response to inotropic stimulation. Likewise, the inhibition of i-NOS by the agent L-NMMA improved the -agonist-induced contractile responses in the failing heart. Not all investigators have been able to demonstrate a direct correlation between NO modulation and improved cardiac myocyte function. Harding et al. isolated single ventricular myocytes from explanted failing human hearts and ‘healthy’ donor hearts w38x. Myocyte contractility was measured in response to electrical stimulation at 0.2 Hz or 1 Hz. Non-failing myocytes showed a marked increase in contraction amplitude between 0.2 and 1 Hz, while failing myocytes showed little change. The authors concluded that tonic production of NO was not responsible for
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the depressed contractility, as incubation of the cells with the NOS inhibitor L-NMMA improved neither their contractility nor their -adrenergic responsiveness. A major problem with these types of studies, however, is the lack of an ideal control. As demonstrated by the donor heart group in Haywood’s work, even ‘healthy’ donor hearts subjected to stress Ži.e. explantation, critical illness, brain death, etc.. appear to increase i-NOS expression. How to elucidate the role of i-NOS in healthy vs. diseased myocardium remains to be determined.
5. Potential anti-inflammatory treatments of heart failure A logical extension of the SIRS research in heart failure reviewed here is to the field of therapeutics. Can we extend our relatively limited knowledge of cytokine networks to the clinical arena to treat heart failure more precisely and effectively? As we have seen from our review of anti-cytokine therapies, much work remains to be done. Some of the most interesting work in the field of inflammatory modulation of heart failure has come out of the discovery of the anti-inflammatory effects of agents already used in the treatment of heart failure, particularly the phosphodiesterase inhibitors. Matsumori et al. have examined the effects of vesnarinone, a quinolone and phosphodiesterase inhibitor, on patients with heart failure. In previous human studies, investigators had found that while the mortality rate was improved in patients treated with vesnarinone, a small percentage of them developed profound neutropenia w39x, leading
Fig. 8. Quantification of c-NOS and i-NOS gene expression by competitive RT-PCR in non-failing ŽNF; n s 5. and failing hearts ŽCHF; n s 24.. The number of transcripts Žrepresenting RNA molecules per microgram total RNA. is depicted. ŽFrom: Drexler et al. w37x, with permission..
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the Matsumori group to examine the anti-inflammatory effects of the drug w40x. White cells were isolated from patients with heart failure and the production of cytokines in response to lipopolysaccharide ŽLPS. was examined in the presence or absence of the drug. Patients with heart failure had marked increases in TNF-␣ production in response to LPS stimulation, and vesnarinone inhibited the production of TNF-␣ and IFN-gamma in both healthy and diseased patients. These investigators expanded their earlier experiments by examining the effects of several other inotropes on WBC cytokine production w41x. They found that amrinone, pimobendan, and vesnarinone all inhibited TNF-␣ production of white blood cells as measured by ELISA. Furthermore, amrinone and pimobendan enhanced IL-1 production whereas vesnarinone had no effect on production of this cytokine. While there was no direct study of in-vivo cytokine production and no measurement of heart failure patients’ cytokine production, the authors concluded that the differences in the clinical effects of phosphodiesterase inhibitors in patients with heart failure may be related to differences in their anti-inflammatory effects. In similar studies, other investigators have also demonstrated that phosphodiesterase inhibitors can dampen myocyte TNF-␣ production in response to LPS in a rat model of heart failure w42x. In vivo data concerning the role of ‘anti-inflammatory’ therapy in heart failure are few. Matsumori’s group compared the anti-inflammatory role of vesnarinone vs. amrinone in a murine model of acute viral myocarditis w43,44x. Mice were infected with encephalomyocarditis virus ŽECMV., and their survival, histopathological scoring, NK cellular activity and
TNF-␣ production were analyzed. A dramatic improvement in survival was detected in the vesnarinone-treated group as compared with the control group Ž60% vs. 20%., whereas no difference in mortality was found between controls and amrinonetreated animals ŽFig. 9.. Vesnarinone-treated animals also displayed less myocardial necrosis, decreased natural killer cell activity, and less TNF-␣ production compared to untreated, ECMV-infected mice. The authors concluded that one of the beneficial effects of vesnarinone therapy may be related to its anti-inflammatory actions in addition to its phosphodiesterase inhibition. While experimental data indicating that a drug with both inotropic and anti-inflammatory properties may benefit patients with heart failure may seem compelling, caution should be exercised. A larger trial of vesnarinone was recently published in abstract form and reported an increased mortality in the vesnarinone-treated group w45x. A large human trial of antiinflammatory modulation in heart failure has yet to be published.
6. Conclusions We have reviewed the role of the inflammatory cascade in heart failure from several different perspectives. As mentioned at the beginning of the article, this is only the ‘tip of the iceberg’ as far as the breadth and depth of the inflammatory processes and others involved in the pathogenesis of heart failure. When examining the data en mass one must con-
Fig. 9. Effect of vesnarinone and amrinone on survival rate after EMCV inoculation. Four-week-old DBAr2 mice were inoculated intraperitoneally with 10 pfu of EMCV. Ža. Two groups were treated with vesnarinone ŽVN. at doses of 10 and 50 mgrkg by mouth daily. Survival of the group treated with vesnarinone at 10 mgrkg was relatively improved compared with the control group but was not significantly different. Treatment with 50 mgrkg of vesnarinone significantly reduced mortality at a very early stage ŽU P- 0.01 vs. control.. Žb. Mice were treated with amrinone ŽAM. at doses of 5 and 25 mgrkg by mouth daily. Survival was not improved compared to control mice. ŽFrom: Matsui S et al. w43x, with permission..
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clude, however, that inflammatory mediators play an important role in both the pathogenesis and progression of heart failure. What then are we to make of the new understanding of heart failure as, at least in some part, an inflammatory disease? What parallels can we draw between sepsis research and the study of inflammation in heart failure? On the one hand, we know log-fold more today than we did even 5 years ago about the individual mediators of both sepsis and heart failure. TNF-␣, NO, IL-1 and the like are now part of the language of medicine. We now know a great deal about each of these mediators, the individual pieces of the puzzle. But the real challenge for the next several years and beyond is to piece together the whole puzzle of heart failure as best we can. We need to know more about the entire ‘cytokine network’ and the interplay between individual mediators. We need to define more precisely the pharmacokinetics and pharmacodynamics of the substances we are studying. Further, we need to define more fully the instigating factors behind inflammatory mediator release and determine which factors propagate the continued release of these mediators in the failing cardiovascular system. Likewise, we need to search for relevant animal and non-animal models in which to study the inflammatory network as a whole. We also need to define how we will use this newfound data. Do we use our knowledge simply as a new insight into the pathophysiology of an age-old disease. Or do we look to a future in which precise modulation of this inflammatory cascade will benefit patients with heart failure. Many more questions will need to be answered before this dream becomes a reality, but more effective treatment and improved outcome in heart failure is still the main motivation behind this important area of research.
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