investigated the role of endothelin-1 in T. cruzi acute infection in rats, using the orally active ETA receptor antagonist BSF-461314. Treatment with BSF-461314 ...
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Role of endothelin during experimental Trypanosoma cruzi infection in rats Elizabeth R. S. CAMARGOS*, Conceic: a4 o R. S. MACHADO*, Antonio L. TEIXEIRA, JR*, Lamara L. V. ROCHA*, Anderson J. FERREIRA†, Alvair P. ALMEIDA†, Matthias BARTON‡ and Mauro M. TEIXEIRA§ *Department of Morphology, Instituto de Cie# ncias Biolo! gicas, Federal University of Minas Gerais, Avenida Anto# nio Carlos 6627, Belo Horizonte, Minas Gerais, Brazil, †Department of Physiology and Biophysics, Instituto de Cie# ncias Biolo! gicas, Federal University of Minas Gerais, Avenida Anto# nio Carlos 6627, Belo Horizonte, Minas Gerais, Brazil, ‡Department of Medicine and Medical Policlinic, University Hospital Zurich, Zurich, Switzerland, and §Biochemistry and Immunology, Instituto de Cie# ncias Biolo! gicas, Federal University of Minas Gerais, Avenida Anto# nio Carlos 6627, Belo Horizonte, Minas Gerais, Brazil
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Chagas’ disease is caused by the intracellular protozoan Trypanosoma cruzi. Here we have investigated the role of endothelin-1 in T. cruzi acute infection in rats, using the orally active ETA receptor antagonist BSF-461314. Treatment with BSF-461314 markedly increased parasitaemia, but animals managed to control the infection by day 15. Histopathological analysis of heart tissue at the end of the acute phase showed greater numbers of parasite nests in BSF-461314-treated animals. The perfusion of isolated rat hearts from infected animals with bradykinin failed to induce an increase, and actually reduced, coronary blood flow. Pretreatment with BSF-461314 prevented changes in coronary flow induced by T. cruzi infection. Together these results demonstrate that endothelin-1, through ETA receptor activation, contributes to the protective immune response against acute T. cruzi infection. Moreover, these data suggest that endothelin-1 is a mediator of impaired endothelium-dependent vasomotion in the coronary microcirculation associated with acute T. cruzi infection.
INTRODUCTION Chagas’ disease is an endemic disease found in South and Central America that is caused by the protozoan parasite Trypanosoma cruzi and has important economic and social implications. It is estimated that around 16–18 million people are infected, 20–30 % of whom will develop severe chronic forms, especially the cardiac form, and will eventually die of Chagas’ disease [1]. Although infection is treatable in the first years after infection, no studies have provided irrefutable evidence that treatment is safe and effective after the second decade of life [2]. Thus understanding the basic mechanisms underlying the pathophysiology of Chagas’ diseases may aid in the development of novel strategies to control disease progression.
Recent developments using sensitive techniques (PCR and immunocytochemistry) have suggested that the association of chronic chagasic lesions, inflammation and parasite products (DNA and protein) occurs much more often than previously thought [3]. Indeed, there is now good evidence to suggest that parasitism of heart tissue is both necessary and sufficient for the induction of inflammation and tissue damage in Chagas’ disease [3]. In addition, it has been suggested that pathological and functional damage to the endothelium may be an important contributory factor to the cardiac injury observed during experimental T. cruzi infection [4]. Moreover, several studies have suggested that the release of endothelin-1 and its action on ETA receptors may underlie the altered vascular compromise characteristic of experimental T. cruzi infection in mice [4].
Key words : bradykinin, heart failure, Langerdorff, nitric oxide. Abbreviations : TNF-α, tumour necrosis factor-α. Correspondence : Dr Mauro Martins Teixeira (e-mail mmtex!icb.ufmg.br).
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Endothelin and experimental Chagas’ disease
In addition to having marked effects on vascular smooth muscle cells, there is now much evidence demonstrating that endothelins have the ability to activate leucocytes, and suggesting the participation of endogenous endothelins in several models of inflammation [5,6]. For example, chronic treatment with an ETA receptor antagonist inhibited the development of monocyte chemotactic protein-1-dependent atherosclerosis in apoliprotein E-deficient mice [7]. In the present study, we have investigated whether activation of the ETA receptor plays a role in the course of acute experimental T. cruzi infection in rats. To this end, T. cruzi-infected animals were treated with the ETA receptor antagonist BSF461314, and infectivity indices and coronary blood flow were assessed.
in 100 µl) was injected through a polyethylene tube inserted into a lateral branch of the perfusion cannula [9].
Histological processing Fragments of heart tissue were obtained after the perfusion experiments and fixed in 4 % buffered paraformaldehyde. The fragments were processed for paraffin embedding, and 7-µm-thick sections were stained with haematoxylin and eosin.
Statistical analysis Results are shown as meanspS.E.M.. Differences between groups were compared using Student’s t test (two sets of data) or ANOVA (three or more sets of data), followed by the Student–Newman–Keuls post hoc test. Differences were considered significant at P 0.05.
MATERIALS AND METHODS Infection model Male Holtzman rats aged 27–30 days were inoculated intraperitoneally with a single inoculum of 300 000 trypomastigotes of T. cruzi Y strain. Parasitaemia was estimated in 5 µl of peripheral blood sampled from the tail on alternate days from day 3 post-infection until the day of sacrifice. Control and infected animals were killed 20 days after inoculation, at the end of acute phase, as revealed by the virtual absence of parasitaemia and a drastic reduction of tissue parasitism [8]. Control and infected animals received drinking water with or without the orally active ETA receptor antagonist BSF-461314 (30 mg : kg−" : day−") from the day of inoculation. BSF461314 was kindly donated by Knoll AG (Ludwigshafen, Germany).
RESULTS Infection indices In untreated infected animals, there were small amounts of parasites until day 6 after infection. Parasitaemia peaked on day 9 and had subsided by day 15 (Figure 1). In contrast, in BSF-461314-treated animals, parasitaemia values increased until day 12, when the mean peak value was double that of the non-treated group (Figure 1). However, parasitaemia also subsided by day 15 in this group (Figure 1). In both groups, no circulating parasites could be detected on day 20. On day 20 after infection, histopathological analysis of the right ventricle free wall, the cardiac region that is most affected in this model, showed an inflammatory process in all infected animals (treated and non-treated),
Langendorff preparation Rats (90–145 g body weight) were anaesthetized with ether and the heart was carefully dissected from its connections. The heart was then perfused with Krebs– Ringer solution (composition in mM : NaCl 118.4 ; KCl 4.7 ; CaCl 2.5 ; NaHCO 26.2 ; glucose 11.6 ; KH PO # $ # % 1.2 ; MgSO 1.2) through a 1.0p0.3 cm aortic stump. The % perfusion fluid was kept at 37 mC, with a pressure of 88 cmH O and constant oxygenation (95 % O , 5 % CO ). # # # An isometric tensor transducer was attached through a heart clip to the apex of the ventricles in order to record contractile force (developed tension) using a data acquisition system (Lodas; Dataz Instruments Inc.). A diastolic tension of 0.5–1.0 g was applied to the heart. Electrical activity was recorded on an electrocardiograph (Nyhon Kohden, Tokyo, Japan) with the aid of two cotton wicks placed directly on the surface of the right atrium and the left ventricle (bipolar lead). Coronary flow was assessed by evaluating the total effluent every 1 min. After the preparation was stable, bradykinin (5 ng
Figure 1 Effects of treatment with the ETA receptor antagonist BSF-461314 on levels of blood parasites in T. cruzi infected rats
Male Holtzman rats aged 27–30 days were inoculated intraperitoneally with 300 000 trypomastigotes of the Y strain of T. cruzi. Blood parasitaemia was checked every 3 days after infection. BSF-4614314 at a dose of 30 mg : kg−1 : day−1 (treated) or vehicle (control) was given in the drinking water. Results are meanspS.E.M. of four untreated and six BSF-4614314-treated animals. *P 0.05 compared with control animals. # 2002 The Biochemical Society and the Medical Research Society
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Figure 2 Effects of treatment with the ETA receptor antagonist BSF-461314 on changes in coronary flow in T. cruzi infected rats
Male Holtzman rats aged 27–30 days were inoculated intraperitoneally with 30 0000 trypomastigotes of the Y strain of T. cruzi. BSF-4614314 at a dose of 30 mg : kg−1 : day−1 (treated) or vehicle (control) was given in the drinking water. Data from non-infected animals were pooled for ease of presentation. At 20 days after infection, animals were killed, and the hearts were isolated and perfused at a pressure of 88 cmH2O. Results are meanspS.E.M. of 4–6 animals in each group, and are shown as the changes in coronary flow obtained after a bolus injection of bradykinin (5 ng/100 µl). *P 0.05 when compared with noninfected and infected control animals. and apparently the groups behaved similarly. Regarding myocardial parasitism, only 33 % (n l 11) of the nontreated infected animals still displayed a few parasite nests. In contrast, amastigote nests were present in 64 % (n l 12) of the BSF-461314-treated animals, the nests being very numerous in 36 % of them.
Functional parameters In hearts from non-infected animals (untreated and BSF461314-treated), the bolus injection of bradykinin (5 ng in 100 µl) caused a marked increase in coronary flow, especially during the first 2 min after injection (Figure 2). In hearts from T. cruzi-infected animals, bradykinin failed to induce an increase, and actually significantly reduced coronary flow (Figure 2). The maximal decrease in flow was observed 4 min after bradykinin injection (Figure 2). Treatment of infected rats with BSF-461314 prevented most of the changes in coronary flow induced by T. cruzi infection (Figure 2).
DISCUSSION Data exist to support an important role for endothelin in the pathogenesis of the vasculopathy that accompanies experimental T. cruzi infection in mice [4]. Thus T. cruzi infection induces endothelin release from endothelial cells [10], and the released endothelin contracts vascular smooth muscle via activation of ETA receptors [11]. In addition, endothelin is expressed during T. cruzi infection # 2002 The Biochemical Society and the Medical Research Society
in mice [12], and high plasma levels of immunoreactive endothelin are found in patients with severe chagasic cardiomyopathy [13]. Overall, these data support a role for endothelin in Chagas’ disease. In addition to their vascular effects, it has become clear that the endothelins possess several pro-inflammatory effects in vitro and in vivo [5]. In our rat model of acute T. cruzi infection, blockade of ETA receptors with the orally active antagonist, BSF461314, was accompanied by greater levels of parasites in the blood and tissues of infected animals. This finding suggests an important role for endothelin and ETA receptor activation in the initial protective immune response against experimental T. cruzi infection in rats. However, it is clear that other mediators\mechanisms are operative, as essentially all animals were capable of clearing the infection. A study in mice using phosphoramidon, a non-selective inhibitor of endothelin-converting enzyme, showed lower levels of endothelin in treated animals, but failed to demonstrate differences in parasitaemia between control and treated mice [4]. Of note, lethality in the control infected group (60–70 %) was abolished by phosphoramidon treatment [4]. The differences compared with our present studies could lie in species peculiarities (rats versus mice) or the specificity of the strategy used, as phosphoramindon is not a specific inhibitor of endothelin release [4]. Despite the differences, both studies argue for a role for endothelin during acute experimental T. cruzi infections. The mechanisms by which endothelin controls T. cruzi infection were not investigated in the present work. However, endothelin acting mainly on ETA receptors can activate macrophages to release cytokines, such as tumour necrosis factor-α (TNF-α) [5]. As TNF-α-induced nitric oxide (NO) production by macrophages plays an important role in the acute phase of the infection [14], it is possible that the ability of endothelins to release TNF-α may account for the effects observed in our model. In this regard, we have shown recently that inflammatory mediators, such as platelet-activating factor and chemokines, which like endothelin act on seven-transmembrane G-protein-coupled receptors, activate macrophages to produce NO and kill T. cruzi [15,16]. The possibility that endothelin acts in a similar manner on macrophages to release NO and kill T. cruzi in a NO-dependent manner is under investigation in our laboratories. One interesting finding of our study was the contrasting effects of bradykinin on the isolated hearts obtained from non-infected and infected animals. In the former, bradykinin induced a significant and rapid increase in coronary flow, an effect that is dependent on the release of NO by endothelial cells [9]. In hearts from infected animals, the vasodilatory effects of bradykinin were lost, and a decrease in coronary flow was noticed throughout the 5-min observation period. If infected animals were treated with the ETA receptor antagonist,
Endothelin and experimental Chagas’ disease
the effects of bradykinin on the isolated hearts were similar to those in hearts from non-infected animals. One possible explanation of these results would be greater endothelin release and consequent coronary artery constriction upon bradykinin stimulation of the hearts from infected animals. In support of this hypothesis, T. cruzi has been shown to release endothelin from endothelial cells in vitro [10]. Of note, the ETA receptor antagonist was not present at the time bradykinin was administered to the isolated heart; thus a direct inhibition of endothelin receptors is unlikely in the in vitro Langendorff preparation. However, chronic ETA receptor blockade may decrease the inflammatory vasculopathy associated with experimental T. cruzi infection, with subsequent inhibition of endothelin release upon activation with bradykinin. In support of the latter hypothesis, chronic treatment with phosphoramidon decreased the vasculitis observed in T. cruzi-infected mice [4]. Overall, our data support an important role for endothelin via ETA receptor activation in the functional alterations observed in the coronary arteries of acutely T. cruzi-infected rats. In conclusion, the results presented here demonstrate that endothelin-1, through ETA receptor activation, contributes to the protective immune response against acute T. cruzi infection, and is a mediator of impaired endothelium-dependent vasomotion in the coronary microcirculation associated with the acute infection.
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ACKNOWLEDGMENTS This work was supported by FAPEMIG, CNPq and the Swiss National Foundation, and received financial assistance from the UNDP\World Bank\WHO Special Programme for Research and Training in Tropical Diseases (TDR 97 0728).
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