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Current Pharmaceutical Design, 2003, 9, ???-??? 1

1381-6128/03 $35.00+.00 © 2003 Bentham Science Publishers Ltd.

Angiotensin Converting Enzyme Inhibitors and Angiotensin II ReceptorAntagonists in Experimental Myocarditis

Lisa M. Godsel, Juan S. Leon and David M. Engman*

Departments of Pathology and Microbiology-Immunology, Northwestern University FeinbergSchool of Medicine, Chicago, IL 60611, USA

Abstract: Myocarditis is a disease whose pathogenesis is not completely understoodand whose prevalence is likely underestimated. Individuals afflicted with this conditionmay be treated with agents that relieve symptoms arising from inflammation andconcurrent cellular damage. One class of drugs commonly used in the treatment ofmyocarditis includes the angiotensin converting enzyme inhibitors, such as captopril,enalapril and lisinopril, and the angiotensin ΙΙ receptor antagonists, such as L-158,809and losartan. The effects of these drugs on cardiomyopathy have been studied using avariety of animal models of heart failure and hypertension. However, less research hasbeen done in the area of animal models of frank myocarditis. Here we review the use of angiotensin convertingenzyme inhibitors and angiotensin ΙΙ receptor antagonists in animal models of myocarditis. We extend theimplications of that published work by correlation with results from studies of other disease models and invitro experiments that highlight the immunomodulatory potential of these compounds. The literature stronglysuggests that aggressive therapy employing angiotensin converting enzyme inhibition and/or blockade ofangiotensin ΙΙ receptors is beneficial. Treatment is useful not only for reducing complications associated withmyocarditis, but also for downregulating the potential autoimmune component of disease—withoutincreasing the levels of the infectious agent that may initiate the myocarditis.

Key words: angiotensin converting enzyme, angiotensin II, renin-angiotensin system, immune modulation, myocarditis,autoimmunity.

MYOCARDITIS

Myocarditis, or inflammation of the heart, is formallydefined as mononuclear cell infiltration of cardiac tissue,with or without myocyte necrosis, degeneration or fibrosis[1]. There are many potential etiologies of myocarditis andthe prevalence of this group of diseases is probablyunderestimated. For example, in a study of 176 individualswith clinically suspected dilated cardiomyopathy, 14 weregiven a clinical diagnosis of borderline myocarditis, while67 were found to have cardiac mononuclear cell infiltrationupon biopsy [2,3]. A number of different cell types maycomprise the tissue infiltrate, including CD4+ and CD8+ Tlymphocytes, B lymphocytes, natural killer cells,neutrophils and macrophages [4-6] and there are distincttypes of myocarditis in which one cell type is particularlyabundant, including those predominated by lymphocytes,eosinophils, and giant cells [1]. It is estimated that 2,500individuals develop myocarditis each year in the UnitedStates, the majority being children [7], and myocarditis is amajor cause of sudden death in young persons [5]. There area number of agents that may cause myocarditis, includingbacteria, viruses and parasites, drugs and toxins amongmany others [1,3,5,8]. Viral infection is the most commoncause of myocarditis in North America and Europe while

*Address correspondence to this author at the Department of Pathology,Ward 6-175, 303 E. Chicago Ave., Chicago, IL 60611; Tel: 312-503-1288;Fax: 312-503-1265; E-mail: [email protected]

Trypanosoma cruzi infection is a major cause of myocarditisin Central and South America [9].

Treatments for myocarditis mainly involve managementof the associated disease complications, such as congestiveheart failure, cardiogenic shock, conduction abnormalities,dysrhythmias and thromboembolism [10]. These typicallyinvolve the administration of diuretics, digitalis, betablockers and vasodilators such as nitroglycerin, inotropicdrugs and angiotensin converting enzyme (ACE) inhibitors[11]. In cases of diagnosed myocarditis, immunosuppressivetherapy may be warranted. However, the effectiveness of thiscourse of treatment is unpredictable and may be contraindi-cated, for example, in the setting of infection [5,12-20].There is a wealth of literature comparing the relative efficacyof ACE inhibition and angiotensin ΙΙ receptor (AIIR)antagonism for the treatment of heart failure andhypertension [21]. For this reason, we will restrict ourdiscussion to the comparatively small number of studiesinvolving animal models of myocarditis. Interestingly, thesereports suggest that ACE inhibitors and AIIR antagonistsmay be helpful in treating disease even in the presence ofacute viral infection and, despite being potentially immuno-suppressive, these agents do not increase the viral load.

ACE AND ACE INHIBITORS

ACE is a bivalent dipeptidyl carboxyl metallopeptidasepresent in soluble form in bodily fluids and as a membrane

2 Current Pharmaceutical Design, 2003, Vol. 9, No. 3 Engman et al.

bound form in endothelial, epithelial or neuroepithelial cellsof several organs—the heart being the focus of thisdiscussion. ACE is a key component of the renin-angiotensin system in which angiotensinogen, synthesizedby the liver and released into the blood, is cleaved by reninto generate the decapeptide angiotensin Ι (AI). ACE binds toAΙ and cleaves its C-terminal dipeptide, His-Leu, creatingangiotensin ΙΙ (AΙΙ ). ACE also cleaves the C-terminaldipeptide Phe-Arg from bradykinin, thus regulating thebalance between the renin-angiotensin and kallikrein-kininsystems [22]. Generation of the octapeptide AΙΙ initiates anumber of physiologic events associated with the renin-angiotensin system, such as vasoconstriction. Although therenin-angiotensin system is endocrine, a number of tissues,including the heart, actually contain and synthesizecomponents of the system. An outstanding review by Dostaland Baker discusses the evidence suggesting the presence ofa functional cardiac-specific renin-angiotensin system [23].Recently, a second ACE (ACE2) was identified in thevascular endothelial cells of the kidney and heart [24, 25].ACE2 is hypothesized to regulate blood pressure but is morelikely involved in heart function. Interestingly, ACE2 isinsensitive to classical ACE inhibitors, including captopril.

ACE inhibitors are drugs that inhibit the formation ofAΙΙ from AΙ , as well as the breakdown of bradykinin, bybinding to ACE via its peptide-binding pocket. A largenumber of ACE inhibitors is commonly prescribed today,including but not limited to captopril, enalaprilat, enalapril,lisinopril, quinapril, ramipril, trandolapril and zofenopril.AΙΙ is a potent vasoconstrictor, but may also be an importantimmunumodulator in its own right. Some of theimmunomodulatory properties of AΙΙ are also brieflydiscussed below in the context of ACE inhibition. ACEinhibitors are commonly prescribed both as a first course ofand as continued treatment for myocarditis, includingautoimmune and infectious forms of the disease. Whilemany experimental animal studies have explored theusefulness of modulating the renin-angiotensin system inheart failure and hypertension [20], little has been done tolook at the effectiveness of the treatments in animal modelsof myocarditis. The few studies published suggest that ACEinhibitors are immunomodulatory drugs and their use inmyocarditis may aid in reducing disease morbidity, even inthe presence of viral or protozoan agents. These studies aredetailed in the sections below, together with a summary ofthe research done to characterize the immunomodulatorymechanisms of these commonly prescribed vasodilators.

AIIR AND THEIR ANTAGONISTS

There are two subtypes of AΙΙ R in humans, AT1R andAT2R [26] and, in rats, AT1R has two subtypes, AT1ARand AT1BR [27]. AIIR are seven-transmembrane G protein-coupled receptors which mediate the physiologic actions ofAΙΙ , such as vasoconstriction, hypertophy, proliferation ofcardiac fibroblasts and production of extracellular matrixcomponents [28]. Both AT1R and AT2R are expressed in theheart; however, AT1R is the receptor through which AΙΙexerts most of its effects by activating phospholipase C orinactivating adenylate cyclase [29]. AΙΙR have been reportedto be increased in myocardial infarction [30], and models of

cardiomyopathy [31-34], and hypertrophy [35-38]. Receptorantagonists, such as losartan and PD123319, interact withthe transmembrane region of the receptor and inhibit thebinding of AΙΙ to AT1R or AT2R, respectively.

EXPERIMENTAL INFECTIOUS AND AUTO-IMMUNE MYOCARDITIS MODELS

The most extensively studied models of myocarditis areviral models utilizing the cardiotropic encephalomyocarditisvirus (EMCV) or coxsackievirus B3 (CB3). The diseaseshave three phases—acute, subacute, and chronic. The acutephase occurs within the first few days, during which deathdue to viral infection can occur without the presence of frankcardiac inflammation. The subacute phase occurs 4-14 dayspost infection and, during this time, heart failure isconcurrent with inflammation and cardiac fibrosis.Inflammatory cells observed in cardiac lesions first includenatural killer cells. By 7 days post infection macrophagesand lymphocytes are observed. The presence of thelymphocytes correlates with the most severe cardiac damage.In the chronic stage of the disease, virus cannot be culturedfrom tissue; however, persistent cardiac damage is observed.A variety of immunosuppressive agents have shown nofavorable effects and, in fact, led to increased damageresulting from an increased viral titer [5,39].

Experimental autoimmune myocarditis (EAM) is inducedin susceptible strains of mice upon immunization with thecardiac myosin α heavy chain [40]. This disease model isuseful in that anti-cardiac autoimmune responses can bestudied separately from those directed against an infectiousagent. Interestingly, immune responses against cardiacantigens, cardiac myosin in particular, have been observed inhuman inflammatory heart disease [41-47], making myosin arelevant and effective antigen for disease induction in themouse model. Recent studies utilizing EAM have shownthat myosin-specific immunosuppression is possible and anavenue worth pursuing [48,49]. EAM is histologicallysimilar to human myocarditis, with myocyte swelling andnecrosis accompanied by mononuclear cell infiltration andfibrosis. Studies in animals have shown that EAM is a Tcell mediated disease, requiring both CD4+ and CD8+subsets [50-54]. B cells are not vital for antigen presentationin EAM and autoantibodies are not necessary for theprogression of myocarditis [55].

Another model of infectious myocarditis is that inducedin mice upon infection with the protozoan Trypanosomacruzi. T. cruzi is the agent of human Chagas heart disease, achronic, progressive, and fibrosing myocarditis of variabledegree. Upon infection and entry into the bloodstream, T.cruzi invades cardiac myocytes, replicates in the cytoplasm,and is released into the interstitium upon myocyte lysis. Theacute phase of Chagas heart disease is characterized by afocal inflammation in the myocardium composed ofmononuclear cells (lymphocytes, macrophages, and plasmacells), mast cells, and granulocytes. Intense tissue parasitosisis typically found in both cardiac and skeletal muscle. Theacute myocarditis resolves after several weeks. Entry into thechronic phase is characterized by development of a globoseheart with a rounded apex due to biventricular hypertrophy

ACE and Myocarditis Current Pharmaceutical Design, 2003, Vol. 9, No. 3 3

Table 1. Use of ACE Inhibitors and AIIR Antagonists in Myocarditis Models

AgentInoculation and Treatment

Protocols

'ViralTiter orParasit-

emia

Heart Wtor HeartWt:BodyWt Ratio

LV WallThickness

and CavityDiameter

Myofib-rillar

DiameterInflamm-

ation NecrosisCalcifi-cation Fibrosis Ref

CB3 Captopril treatment 1-6 days postinoculation

nc ↓ n d n d ↓ ↓ ↓ n d [58]

Captopril treatment starting 10days post inoculation

nc ↓ n d n d nc nc nc n d [58]

Captopril treatment 3 days postinoculation

n d ↓ n d n d nc ↓ ↓ n d [59]

Captopril treatment 10-30 dayspost inoculation

n d ↓ nc ↓ ↓ ↓ nc ↓ [64]


n d nc nc nc nc nc nc nc [64]

EMCV Captopril treatment from day ofinoculation

n d ↓ n d n d ↓ ↓ ↓ n d [60]

TCV116 (3 mg/kg dosage) nc ↓ n d n d ↓ ↓ ↓ n d [66]

Captopril n d ↓ ↓ n d ↓ ↓ n d n d [62]

Enalapril n d nc nc n d nc ↓ n d n d [62]

Losartan n d ↓ ↓ n d nc nc n d n d [62]

Captopril treatment from 4-16weeks post inoculation

n d ↓ ↓ ↓ n d n d n d ↓ [63]

Losartan treatment from 4-16weeks post inoculation

n d ↓ ↓ ↓ n d n d n d nc [63]


n d ↓ n d ↓ ↓ ↓ n d n d [65]

Enalapril treatment 7-21 dayspost inoculation

n d ↓ n d ↓ nc nc n d n d [65]

L-158,809 treatment 7-21 dayspost inoculation

n d ↓ n d ↓ ↓ ↓ n d n d [65]

Myosin inCFA

Temocapril treatment from day ofimmunization

na ↓ n d n d ↓ n d n d n d [57]

Myosin inCFA

Temocapril treatment 15-21 dayspost immunization

Na nc n d n d nc nc n d n d [57]

Myosin inCFA

Captopril treatment from day ofimmunization

na ↓ n d n d ↓ ↓ n d ↓ thisreview

T. cruzi Captopril treatment from day ofinoculation

nc nc n d n d ↓ ↓ n d ↓ thisreview

na, not applicable; nc, no change from controls; nd, not determined; ↓ reduction compared to controls.

and chamber dilatation. The exudate in chronic chagasicmyocarditis is mainly composed of lymphocytes and, to alesser degree, by macrophages, eosinophils, plasma cells,neutrophils, and mast cells. T lymphocytes predominate,with CD8+ T cells outnumbering CD4+ cells 3 to 1(reviewed in [56]). In contrast to what is found in the acutephase, parasites are often undetectable in the hearts duringthe chronic phase. There is a variable degree of myofibrillardestruction and replacement by connective tissue, leading toprogressive fibrosis.

ACE INHIBITORS AND AIIR ANTAGONISTS INEXPERIMENTAL MYOCARDITIS

Captopril, enalapril and temocapril are the only ACEinhibitors that have been studied in animal models ofmyocarditis. The earliest work focused on CB3 and EMCVmyocarditides and is summarized in Table 1. Recent studies

from our laboratory and others [57] have addressed theeffects of captopril in a non-infectious, purely autoimmunemurine model of the disease. The earliest studies focused onthe efficacy of captopril in treating an animal model of viralmyocarditis. Rezkalla et al. published work on the effect ofcaptopril on both early (6 days of treatment) and late (20 or30 days of treatment) models of CB3 myocarditis [58]. Inthe early treatment protocol, mice received captopril fromday 1 through day 6, while in the late model they did notreceive captopril until 10 days post infection. Mice treated inboth the early and late protocols had significant reduction inheart weight and heart weight/body weight ratio compared tocontrol animals. Viral titers were the same in the captopriltreated and control animals. This indicates that anyimmunosuppressive effect of captopril did not affect theantiviral immune response. Hearts from animals given theearly protocol had less necrosis and calcification anddecreased inflammation; however, hearts from the lateprotocol with treatment at either 20 or 30 days post infection


were not statistically different in any of these three criteria.The effects of captopril on virus-specific immunity were notstudied.

The same authors published on another short-term modelof CB3 myocarditis (9 days of infection) [59]. In thatmodel, captopril treatment was initiated 3 days afterinfection and continued until the completion of theexperiment. The heart weights of the captopril treatedanimals were different from those of the non-treated controls;however, this difference was not addressed carefully bydetermining the heart weight/body weight ratio. Myocytenecrosis and calcification were also decreased upon captopriltreatment, although the degree of inflammation wasunchanged. These studies suggest that captopril treatmentcan be beneficial in decreasing cardiac damage. Based on thefinding that captopril may decrease inflammation, it wouldbe useful to know whether the viral titer increased in thecaptopril treated animals. Taken together, the results of thesepapers seem to indicate that there is a narrow window inwhich captopril treatment should be initiated early to havethe most beneficial effect on the inflammatory process,although later treatment initiation is still of some utility.

Captopril and some other ACE inhibitors containsulfhydryl groups which have been hypothesized to beinvolved in the drug’s effectiveness [60,61]. The EMCV-induced myocarditis model was used to compare the effectsof captopril and N ,2-mercapto-propionyl glycine, asulfhydryl-containing amino acid derivative without ACEinhibiting properties [60]. In this paper the authors reportedthat both drug treatments significantly increased survival anddecreased the amount of cellular infiltration, myocytenecrosis and calcification in the hearts of the animals. Bodyweights of the treated animals were significantly higher thanthose of controls, as were the heart weight/body weightratios. The authors made the comparison between these twodrugs in an attempt to explore the effectiveness of thesulfhydryl group in mediating captopril’s protective effectsbeyond ACE inhibition. In autoimmune myocarditis,administration of temocapril to myosin-immunized mice atday 0, but not 21, post immunization, decreased heartweight/body weight ratios, pericardial effusion, andmacroscopic and microscopic histologic scores [57]. Theauthors suggested that this effect may be due to thioredoxin,a protein involved in protecting cells from oxidative stress.This result may support the findings of other groups thatsuggest that the sulfhydryl group of ACE inhibitorsameliorate heart function through a redox mechanism.

Araki et al. then studied the effects of captopril andenalapril in treating EMCV-induced myocarditis andcompared their effects to those of losartan, an AΙΙ Rantagonist [62]. Heart weights and heart weight/body weightratios were reduced and left ventricular wall thickness andcavity dimension were both decreased by captopril andlosartan treatment, but not by enalapril. Captopril reducedcardiac inflammation and myocyte necrosis, while enalaprilonly decreased myocyte necrosis. Losartan did not have anyeffect on these two parameters, even though several doseswere tested. Therefore, these studies suggest that, while theeffect of captopril and enalapril on the renin-angiotensinsystem is the same, there are some differences in the

immune system regulation. These changes in the immunesystem resulting from captopril treatment, at least in theEMCV model, are not simply due to the lack of circulatingAΙΙ in the animals. In another study, losartan and captopriltreatment were compared in a long term model of EMCVmyocarditis [63]. Mice received the drugs from 4 weeks afterviral inoculation to the termination of the experiment at 16weeks post infection. Losartan and captopril had similareffects on all parameters tested—heart weight, left ventricularthickness, left ventricular cavity dimension and myocardialfiber diameter. The two drugs differed in their effect onfibrosis. Only captopril lowered the degree of fibrosis, anactivity of this drug that has been explored by others inmyocarditis and other disease models.

Takada et al. used CB3 induced myocarditis as a modelto study the effect of captopril on fibrosis [64]. The authorslooked at both the inflammatory phase, with captopril beingadministered on days 10 through 30 post infection, or thefibrotic phase, with the drug administered on days 30through 60 post infection. In the inflammatory phase,survival was higher in the captopril treated group andinflammation, necrosis and fibrin deposition were alldecreased. Furthermore, connective tissue architecture wasmaintained, myocyte hypertrophy was decreased and theshift of myosin isoforms from α to β was prevented. In thiscase, calcification was not statistically different from that ofcontrols. The fact that inflammation is decreased contrastswith results of Rezkalla et al., who observed decreasedinflammation and calcification only when captopril wasadministered at the time of infection, and not when the drugwas administered 10 days post infection [58]. The disparitymay be explained by differences in the specific experimentalprotocols, the mouse and/or virus strain, the number ofplaque-forming units of virus administered or the dosage ofdrug. In the fibrotic phase of disease the only significantdifference in the results of Rezkalla et al. was that interstitialreticulin fibers showed decreased thickening in treatedanimals compared to controls. This indicated that captoprilafforded protection from virally mediated damage, even iftreatment was begun 10 days after the infection wasinitiated.

Baba et al. published a study which addressed the effectsof the ACE inhibitors captopril and enalapril and the AΙΙRantagonist L-158,809 on cardiac damage after EMCVinfection [65]. Mice were given the drugs for 14 daysstarting 7 days post inoculation of virus. Heart weight/bodyweight ratios and myofibrillar hypertrophy were significantlydecreased in the animals treated with any of the three agentscompared to untreated controls. Myocardial necrosis andinflammation were reduced in the captopril and the L-158,809 treated animals, but not in the enalapril treatedmice. The AΙΙ R antagonist, but not either ACE inhibitor,also reduced the amount of another potent vasoconstrictor,endothelin-1. These findings illustrate that ACE inhibitionand AΙΙ R blockade are not entirely interchangeableapproaches and that treatment regimens should beindividualized.

There are only four reports in the literature describingAΙΙR blockade in myocarditis and three involving L-158,809and losartan, which were mentioned above. Mice were


Fig. (1). Captopril reduces myocardial inflammation and fibrosis in A/J mice immunized with cardiac myosin or phosphate-bufferedsaline (PBS) in complete Freund’s adjuvant. Representative cardiac sections of captopril treated (75 mg/L in the drinking waterbeginning at first immunization) and untreated mice at 28 days post treatment. Sections are at 40X and are stained with Masson’strichrome or hemotoxylin and eosin.

treated with a variety of concentrations of the AIIR blockerTCV-116 one day before or two days (1 or 10 mg/kg and0.3 or 3 mg/kg, respectively) after EMCV inoculation [66].3 mg/kg treatment resulted in a significantly lower heartweight, myocyte necrosis, calcification and cellularinfiltration than in untreated control animals. However, thedrug at this concentration did not have any effect on viral1sp titer. While the findings in this particular paper areintriguing, it is difficult to compare the groups because ofthe very different amount of the drug used. These datacorrelate well with those in the EMCV model addressing theeffects of L-158,809 [65], but do differ from those forlosartan, in which there was no effect on inflammation ornecrosis [62]. As mentioned above for the CB3 papers, thedifferent results obtained in studies of AIIR blockade couldbe due to variations in the experimental methods andreagents used in each study.

It is noteworthy that, in the above experiments, treatmentwith ACE inhibitors or AΙΙR antagonists did not lead to anincrease in viral load, as might be expected if the agents areimmunosuppressive. However, it is possible that the agentshave antiviral activity. The direct effects of the treatments onthe virus have not been examined in any detail. One in vitrostudy examined the effects of captopril or losartan treatmenton herpes simplex virus type 2 infection of cardiac neonatalmyocytes. Losartan reduced cellular damage, as measured bylactate dehydrogenase release into the medium, and viral

release. However, the rate of viral replication was notaffected. Captopril had no effect on the same parameters, sowhile this drug may not be as efficacious as losartan, it doesnot increase the damage or viral load as compared to controls[67].

Work from our laboratory strongly suggests thatcaptopril is useful for the prevention of autoimmunemyocarditis, as well as for myocarditis resulting frominfection with the cardiopathogenic protozoan parasiteTrypanosoma cruzi. A/J and BALB/cJ strains of miceimmunized with syngeneic cardiac myosin develop a severe,diffuse inflammatory disease specifically localized to thecardiac tissue and absent from skeletal muscle and smoothmuscle. Captopril prevented development of increased heartweight, heart weight/body weight ratio, cardiacinflammation and cardiac fibrosis (Fig. (1)). The captopriltreated animals also failed to develop myosin-specificcellular immunity in response to myosin-immunization asmeasured by delayed-type hypersensitivity. Furtherexperiments indicate that this immunosuppression was notcardiac specific, since ovalbumin-specific cellular immuneresponses were also decreased in ovalbumin-immunizedanimals treated with captopril (data not shown).

Therefore, it appears that both ACE inhibitors and AIIRantagonists may be beneficial when myocarditis issuspected, since they seem to suppress the complications


Table 2. Use of ACE Inhibitors and AIIR Antagonists in Clinical Chagas Disease

Ace Inhibitor Type Size Dose mg/day Length Effects/Comments Ref

Captopril Single blind,cross-over

18 150 12 weeks (6weeks

treatment)

↓Heart rate, ↓Ventricular couplets (ns), ↓Nonventriculartachycardia (ns),↑Plasma renin, ↓Urinary, norepinephrine,

Systolic/Arterial blood pressure (nc)

[70]

Enalapril/Captopril

ProspectiveCohort

56 “conventionaldoses”

2 years No association with mortality (ns) [139]

Enalapril Intervention 13 5 96 hours ↓Functional Class, ↓Weight, ↓Plasma Norepinephrine,↓Aldosterone, ↓Renin, Systolic/Arterial blood pressure

(ns)

[72]

Enalapril Intervention 20 5-10 8 weeks ↓ E/A relationship (improved diastolic function) [140]nc, no change from controls; ns, not significant; ↓ reduction compared to controls.

associated with myocarditis without increasing diseaseseverity. They have the added advantage of decreasingcardiac-specific inflammation without promoting increasedreplication of the infectious agent. From the work cited inthis section, it is clear that critical information on the use ofthese agents in myocarditis is lacking and that there aremany aspects of both ACE inhibition and AIIR blockadethat should be investigated more extensively. The remainderof this review will focus on studies of theimmunomodulatory effects of both ACE inhibitors and AIIRantagonists on immune cell function and correlate thosefindings with the results described above.

ACE INHIBITORS IN CHAGAS HEART DISEASE

ACE inhibitors are also used to treat another prevelantcardiomyopathy, Chagas heart disease (CHD), caused byinfection with the parasite Trypanosoma cruzi. CHD is acommon cause of cardiac death in Latin America and maymanifest as congestive heart failure, cardiac dysrhythmia orthromboembolism. Once heart failure develops in CHDpatients, life expectancy is reduced to a few years and suddendeath occurs in approximately 40% of patients [68].Management of clinical CHD is based on the treatment ofother cardiomyopathies. CHD patients with congestive heartfailure respond to routine management, including sodiumrestriction and treatment with diuretics, digitalis, and ACEinhibitors.

Despite the routine administration of ACE inhibitors topatients with CHD, only a few pilot studies have beenconducted to test their potential benefits. These studies aresummarized in Table 2. Captopril and enalapril, two ACEinhibitors, seem to improve cardiac function and are welltolerated by Chagas patients; at least 90% of patients do notdevelop side effects [69,70]. Patients with cardiomyopathy,including CHD patients, were given captopril (75 mg/day)for 12 weeks and these individuals exhibited a significantdecrease in heart rate and objective improvement of cardiacfunction in 98 (85.2%) patients [69]. However, noconclusions can be drawn about the CHD patients since theresults of their treatment were not presented separately.

ACE inhibitors may improve cardiac function in CHDpatients by interfering with the renin-angiotensin system.While ACE is probably the main target of these inhibitors,

ACE activity levels were not measured in any of thesestudies. ACE inhibitors can also affect renin levels, thoughthey are believed to operate downstream of the reninbiosynthetic pathway. Renin levels have been observed to behigher in CHD patients with heart failure than in those withasymptomatic CHD [71]. One study of CHD patientsreported an increase in plasma renin levels uponadministration of captopril for 6 weeks [70], while a secondstudy reported decreased renin levels upon administration ofenalapril over 96 hours [72]. The change in renin levelsinduced by ACE inhibitors probably influences AΙΙ levelsand cardiac function. Interestingly, one study reported thatthe ACE inhibitor captopril enhanced T. cruzi invasion ofhost cells in vitro and therefore may affect cardiac functionin the host [73]. Captopril blocks the degradation ofbradykinin, which was shown to increase infection of hostcells by T. cruzi. However, there are no published reports ofACE inhibitors affecting the parasite burden of CHDpatients. Lastly, these ACE inhibitors may affect cardiacfunction through the parasympathetic and sympatheticsystems, since the levels of neurohormones such asnorepinephrine were affected.

In conclusion, preliminary reports suggest that cardiacfunction in individuals with CHD may be improved byACE inhibitor therapy. The mechanisms by which ACEinhibitors may provide this benefit are unknown but mayinclude modulation of the renin-angiotensin system and theparasympathetic/sympathetic systems. Large scale controlledstudies on morbidity and long term survival of CHDpatients treated with ACE inhibitors are necessary to confirmthe benefits of ACE inhibitors in CHD and to identifyspecific populations best suited to receive such therapy.

CAPTOPRIL IN EXPERIMENTAL CHAGAS HEARTDISEASE

Preliminary evidence in our laboratory suggests thatcaptopril treatment also reduced myocarditis in anexperimental model of acute Chagas heart disease. Captopriltreatment of A/J mice infected with T. cruzi resulted in adecrease in cardiac inflammation and cardiac fibrosis (Fig.(2)). However, the reduction in inflammation and fibrosiswas not associated with a decrease in heart weight or heartweight/body weight ratio compared to infected controls thatdid not receive captopril. Interestingly, infected mice treated


Fig. (2). Captopril reduces myocardial inflammation and fibrosis in A/J mice acutely infected with T. cruzi. Representative cardiacsections of captopril treated (75 mg/L in the drinking water beginning at infection) and untreated mice at 28 days post treatment.Sections are at 40X and are stained with Masson’s trichrome or hemotoxylin and eosin. Arrowheads indicate T. cruzi pseudocysts.

with increasing amounts of captopril had higher mortality.In addition, captopril treated mice exhibited reduced anti-T.cruzi cellular immune responses as measured by delayedtype hypersensitivity, similar to the immunosuppresiveeffect observed in captopril-treated, myosin-immunizedmice. These results suggest that, while captopril treatmentreduces inflammation in T. cruzi induced myocarditis,captopril may also enhance T. cruzi - induced mortality.

ACE INHIBITORS IN OTHER AUTOIMMUNE ANDINFECTIOUS DISEASE MODELS

A number of other disease models, both infectious andautoimmune, have yielded more information regarding theeffects of ACE inhibitors on the immune response. In ananimal model of multiple sclerosis, experimentalautoimmune encephalomyelitis, captopril treatment resultedin decreased clinical disease severity and a suppressed andshortened duration of the disease. In vitro studies showedthat lymphocytes from treated animals had a diminishedresponse to myelin basic protein and to the mitogenconcanavalin A (Con A) [74].

Several papers describe the use of captopril in a numberof granulomatous inflammation models. The drug reduced

the size of liver granulomas in murine schistosomiasis [75],but not the size of hypersensitivity granulomas induced bybovine serum albumin-coated sepharose beads. Since thelatter granulomas contained no ACE, the paper suggestedthat the immunomodulatory effects of captopril may be due,at least in part, to the inhibition of ACE. In another study,captopril was used to decrease lung inflammation andsplenomegaly in Bacille Calmette Guerin-induced granuloma[76]. In vitro the drug did not affect the macrophagemigration nor did it change the chemotactic activity ofisolated granulomas. In yet another paper the granulomatousresponse to Histoplasma capsulatum was modulated byadministration of captopril with different findings from anyof those reported above. With ACE inhibition present duringthe early stages of the infection, the clinical severity ofinfection and the histopathologic changes in mice weresignificantly worsened and the growth of the yeast in liversand spleens was increased [77]. If captopril was firstadministered later during the resolution phase of theinfection, there was no effect on the healing of thegranulomas, but the drug did not cause a relapse ofinfection. Some in vitro experiments demonstrated thatcaptopril treatment of normal animals did not result insuppression of splenocyte responses to the mitogens Con Aor phytohemagglutinin in vitro or delayed typehypersensitivity. The somewhat contradictory results of


these in vitro experiments may be due to differences in thespecific ACE inhibitor employed, or to differences in thedosage and treatment regimen used both in the animals andin the culture dish. The presence of AΙΙ during the course ofinfection may therefore affect the outcome, depending on thetype of infection involved, and a decrease in AΙΙ may be themethod through which ACE inhibition decreases the size ofthe granulomas.

The protective effects of AΙΙ against infection arepresented in a paper studying bacterial peritonitis in whichtreatment of rats treated with AII inhibitors resulted in adecreased abscess size and an increase in host resistance [78].These data were correlated with in vitro experimentsshowing that AΙΙ treatment increased respiratory burst of ratperitoneal macrophages and increased phagocytosis of ratmacrophages and human peripheral blood mononuclear cells.Thus, the presence of AΙΙ can increase the activation of theimmune response against an infectious agent, while theinhibition of AΙΙ production via ACE inhibition or AΙΙ Rantagonism may decrease the activation of immune cells.Information is available regarding the effects of captopril andother ACE inhibitors and AΙΙ R antagonists that differenough from each other to suggest that not all of the effectsmediated by ACE inhibitors can be explained simply by alack of AΙΙ in the circulation or resident in tissues. Thesection below strives to summarize the research on a varietyof indicators of normal inflammatory cell function.

IMMUNOMODULATORY EFFECTS OF ACEINHIBITORS AND AIIR ANTAGONISTS

There are a number of ways that ACE inhibitors andA ΙΙ R antagonists may modulate the immune response.Several of the processes that may be affected includechemotaxis, motility and adhesion, differentiation,activation and cytokine/chemokine production. There is alsoa large base of research demonstrating an involvement of AΙΙin basic immunity, suggesting potential utility for ACEinhibitors and AΙ Ι R antagonists in modulatinginflammation. Much of the research has focused on cellularadhesion and chemotaxis and how ACE inhibitors and AΙΙRantagonists modulate these processes. For example, bothtypes of agent may inhibit mononuclear cell infiltration,either because of drug-induced decrease in expression ofadhesion molecules on the mononuclear, endothelial orinterstitial cells or because of decreased chemokineproduction.

A variety of in vivo and in vitro studies havedemonstrated that AII is involved in the chemotaxis andadhesion of monocytes and macrophages. Angiotensinknockout animals have decreased production of MCP-1 [79]AIII is an AII processing product that also increased MCP-1[80]. ACE inhibition resulted in decreased MCP-1 levels ina model of atherosclerosis [81,82] as well as in patientssuffering myocardial infarction, which correlated with adecreased accumulation of macrophages in heart tissue [83].In addition, AΙΙ has been shown to induce monocytemigration [84], leukocyte rolling, adhesion and extravasationwhile AΙΙ R antagonists inhibit these processes [85]. AΙΙinduced leukocyte-endothelial cell interactions in vivo [86].

AΙΙR antagonists and ACE inhibitors reduced the amountof neutrophil [87,88] and macrophage [89] recruitment anddecreased neutrophil [90], polymorphonuclear cell [91,92]and leukocyte [93] adhesion. Enalapril decreased ICAM-1,VCAM-1, MCP-1 levels in circulating adhesion moleculesin serum of patients [94]. In some of the studies, differencesamong the results of AΙΙR antagonism and ACE inhibitionwere highlighted. For example, study of the effects ofcaptopril on neutrophils showed that treatment did not affectneutrophil adherence [90] and, in fact, actually mightenhance neutrophil migration [95] and activation [96], incontrast to the results detailed above. In another study,treatment decreased the activated circulating andendothelium-adherent monocytes [97]. AIIR antagonistsprevented increased expression of ICAM-1 and VCAM-1,which correlated with a decrease in macrophage infiltration[98,99]. Other studies did not support this conclusion,however, possibly because a different agent was employed[100,101].

A ΙΙ therefore may upregulate chemotactic factorsimportant for attracting immune cells to the affected tissue,and may induced expression of molecules that enable thecells to adhere to endothelium. Administration of ACEinhibitors or AΙΙ R antagonists decreases the amount ofcirculating and tissue AΙΙ and may decrease inflammation inthis manner. Another way that ACE inhibitors and AΙΙ Rantagonists may modulate the immune response is via theireffects on immune cell proliferation and differentiation. AΙΙhas been shown to act through AT1R to stimulate theproliferation of splenic lymphocytes [102,103].Interestingly, ACE activity, AΙΙ release, and AT1R andAT2R expression all increased when the human leukemiacell line THP-1 is induced to differentiate into a macrophage[104].

The majority of the ACE inhibition studies that focus oncell proliferation and differentiation utilized captopril, whilea small number utilized enalapril and enalaprilat.Unfortunately, many of the results are contradictory.Captopril inhibited the proliferation of hematopoietic stemcells and progenitors [105] and also reduced proliferation ofthe stem cells in vivo [106]. In contrast, the drug stimulatedthe proliferation of both human [107] and rat [108]lymphocytes in vitro, but proliferation of lymphocytesderived from captopril treated rats was inhibited. In a mousemodel of lupus, captopril was used to inhibit T cellstimulation, but enalapril did not have this effect [109].However, in another study, captopril increased Con A-stimulated T cell proliferation while enalapril and enalaprilatinhibited the proliferation [110]. Finally, T cells werestimulated when cocultured with a low concentration ofserum from patients treated with either captopril or enalapril,but were inhibited when the serum concentration was high[111]. Because AΙΙ stimulates lymphocyte proliferation, itfollows that ACE inhibitors would decrease proliferation ofimmune cells through reduction of AΙΙ . There is asuggestion that all these effects are directed at least in part atmacrophages and there may be some effects of the drugs thatare independent of AΙΙ .

Another way that ACE inhibitors and AT1R and AT2Rantagonists might modulate the immune response is via


alteration of cytokine release. AΙΙ induces transcriptionalactivation of a number of cytokine genes. ACE inhibitorsand AT1R and AT2R antagonists might therefore inhibitcytokine secretion by decreasing the amount or antagonizingthe effects of AΙΙ . AΙΙ increases the transcription of IL-6 inmacrophages, which can be inhibited by AT1R antagonists[112,113]. This decrease in IL-6 production has also beenobserved in vivo in a model of chronic heart failure andmyocardial infarction [114,115]. Quinapril treatment in amodel of myocardial infarction reduced expression of IL-1β,IL-5, IL-6, and TNF-α. Captopril suppressed IL-1β inducedproduction of IL1-α and TNF-α in peripheral blood [116].Interestingly, captopril even increased production of IL-1Rα[113]. ACE inhibition was also shown to decrease theamount of IL-2, IL-12 and IFN-γ produced by T cells [117,118]. In contrast, murine T cells secreted higher levels of IL-2 upon captopril treatment after Con A stimulation [119].Peripheral blood monocytes release TNF-α in response toAII treatment—an effect blocked by AT1R antagonism andACE inhibition [113,116,120,121]. However, not everyACE inhibitor was able to block TNF-α expression by thesame cell populations [116]. ACE inhibitors were alsoshown to be more efficient at decreasing the plasmaconcentration of TNF-α than are AΙΙ R antagonists [122].These results raise the point that the activities of ACEinhibitors are not all directly related to their effects on AΙΙ .Caution must be taken when analyzing the effects of theseagents. In some cases, a large amount of drug is needed toobtain a decrease in cytokine production and the observed invitro responses may therefore not be physiologically relevant[113,116,123]. TGF-β is a cytokine that is regulated by AΙΙand is decreased by ACE inhibition and AIIR antagonism[124-127]. While cytokines are markers of cellularactivation, there are other important markers of activationthat have also been analyzed. For example, AΙΙ has beenshown to increase macrophage phagocytosis [128] and theadherence of peripheral blood monocytes to endothelial cellmonolayers [120]. AΙΙ increased cytosolic calcium inperipheral blood mononuclear cells, which could be blockedby the AT1R antagonist losartan [130]. Along these lines,treatment of hypertensive rats with the ACE inhibitorcaptopril significantly lowered the intracellular calcium inthymocytes, which correlated with a decrease in bloodpressure [130].

The studies mentioned above support the hypothesis thatACE inhibition and AΙΙ R antagonism are involved in thedownregulation of the immune response. Thisdownregulation may be mediated in part by the simple factthat AΙΙ is not produced, as in the case of ACE inhibition,or its activity is inhibited, as in the case with receptorantagonism. However, there are differences in the way thatACE inhibitors function compared to receptor antagonistsand even how drugs within the same class effect immunecell function. Some papers even showed that the activities ofsome types of immune cells are actually enhanced bytreatment with ACE inhibitors. ACE inhibitors have alsobeen shown to increase the activation of neutrophils andmast cells as demonstrated by myeloperoxidase, lysozymeand histamine release [96,131,132]. These results indicatethat the actions of these drugs are more complex than simplyto reduce AΙΙ production and suggest that each drug needs tobe analyzed individually. Certainly one must be aware that

an effect observed for one inhibitor or antagonist cannot beassumed to be the same for other agents.

Underlying all of the data in the above reports is thepossibility that ACE inhibitors and the receptor antagonistsmodify the immune response via cell signaling pathways.Certainly there are results suggesting that AΙΙ is involved incell signaling and, in the most obvious scenario, ACEinhibitors and AT1R and AT2R antagonists could modulatethe immune response by decreasing the concentration of AΙΙavailable for signaling. AΙΙ -mediated degradation of IκB andactivation of NFκB increased transcription and translation ofVCAM-1 and ICAM-1, which are important for recruitmentof leukocytes to inflamed tissues [1-3]. This upregulationmay be due, at least in part, to the AΙΙ -induced production ofendothelin-1 [133-135]. Angiotensin III, a product of AΙΙcleavage, increases the production of MCP-1 via NFκBactivation [80]. AΙΙ has also been shown to augment tyrosinekinase activity [136]. Interestingly, captopril treatment hasbeen shown to inhibit pp60c-src tyrosine phosphorylation inhuman mesanglial cells in vitro [137]. Studies using ACEinhibitors and an AT1R antagonist make the case for cellsignaling even stronger as NFκB activation of macrophagesand vascular smooth muscle cells is decreased upontreatment with these agents [81,82]. ACE inhibition withcaptopril, idapril, fosinopril, and losartan all reduced theexpression of tissue factor in monocytes via the inhibition ofNFκB translocation to the tissue factor promoter [138].

CONCLUSIONS

ACE inhibitors and AΙΙ R antagonists are extremelyuseful in the management of heart disease. However, littlehas been done to study the effects of these drugs inmyocarditis specifically. There is a concern that use of theseagents will decrease the immune response against aninfectious agent that has induced the heart damage and/orthat has precipitated the inflammation against self proteins(autoimmunity). There is cause for concern, sincecontradictory information regarding the effects of AΙΙ and itsinhibitors on the immune response continues to begenerated. However, the general theme arising from thepapers on this topic suggests that these drugs may notincrease the proliferation of an infectious agent and candecrease the morbidity of the inflammatory heart disease.

The amount and type of ACE inhibitor or AΙΙ Rantagonist used may make a difference in the effectsobserved on the immune system. Certainly there aredifferences between the ACE inhibitors and the AΙΙ receptorantagonists and between drugs within either category,suggesting that each has its own specific effects on theimmune system beyond inhibition of AΙΙ activity bydecreasing the amount of AΙΙ or by blocking its effects.Some of the effects include chemotaxis and motility, theproduction of cytokines important in activation andproliferation, the accumulation of bradykinin andprostaglandin production and the deposition of extracellularmatrix. With so many effects attributed to such widelyadministered medications, further research is necessary tofully understand the impact of these agents.


ACKNOWLEDGEMENTS

Research in our laboratory was supported by grants andfellowships from the National Institutes of Health and theAmerican Heart Association. We thank Drs. William Ward,Agostino Molteni and Carl Waltenbaugh for helpful adviceand assistance.

ABBREVIATIONS

ACE = Angiotensin converting enzyme

AI = Angiotensin I

AII = Angiotensin II

AIII = Angiotensin III

AIIR = Angiotensin II receptor

AT1R = Angiotensin II receptor 1

AT2R = Angiotensin II receptor 2

CHD = Chagas heart disease

CHF = Congestive heart failure

CB3 = Coxsackievirus B3

Con A = Concanavalin A

EAM = Experimental autoimmune myocarditis

EMCV = Encephalomyocarditis virus

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