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Protective role of ETA endothelin receptors during the acute phase of Trypanosoma cruzi infection in rats
Microbes and Infection 6 (2004) 650–656 www.elsevier.com/locate/micinf
Original article
Protective role of ETA endothelin receptors during the acute phase of Trypanosoma cruzi infection in rats Elizabeth R.S. Camargos a, Lamara L.V. Rocha a,1, Milene A. Rachid a, Alvair P. Almeida b, Anderson J. Ferreira b, Antonio L. Teixeira-Jr a, Egler Chiari c, Matthias Barton d, Mauro M. Teixeira e, Conceição R.S. Machado a,* a
Departamento de Morfologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais (UFMG), Av. Antonio Carlos 6627, P.O. Box 486, CEP: 31270-901 Belo Horizonte, MG, Brazil b Departamento de Fisiologia/Biofísica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais (UFMG), 31270901 Belo Horizonte, MG, Brazil c Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais (UFMG), 31270901 Belo Horizonte, MG, Brazil d Medical Policlinic, Department of Medicine, University Hospital Zurich, Switzerland e Departamento de Bioquímica/Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais (UFMG), 31270901 Belo Horizonte, MG, Brazil Received 14 November 2003; accepted 2 March 2004 Available online 16 April 2004
Abstract Chagas’ disease, caused by Trypanosoma cruzi, has an acute phase characterized by blood-circulating trypomastigotes and amastigote proliferation in several cell types, especially muscle cells. In the chronic phase, around 70% of infected people are asymptomatic (latent form). The remainder develop chagasic cardiomyopathy and/or digestive syndromes. There is evidence for aggravation of the chronic cardiac pathology by endothelin-mediated vasoconstriction. Holtzman rats have proven to be a good model for Chagas’ disease acute phase and latent chronic phase. Now, we investigate the effects of prolonged treatment with an endothelin ETA receptor antagonist, BSF 461314, during the acute phase on parasitemia, coronary flow, tissue parasitism and the inflammatory process. Using isolated heart in Langendorff’s preparation, endothelial dysfunction was observed only in non-treated infected animals. Histoquantitative analyses carried out in heart and diaphragm showed higher tissue parasitism and/or inflammatory process in BSF 461314-treated animals. Our data indicate that endothelin ETA receptors contribute to the initial mechanisms of parasite control. Impairment of the endothelium-dependent vasodilatation favors hazardous effects. However, blocking endothelin ETA receptors can prevent the latter. © 2004 Elsevier SAS. All rights reserved. Keywords: Chagas’ disease; Endothelin; Parasite clearance; Inflammation; Coronary flow
1. Introduction Although it is progressively becoming controlled, Chagas’ disease (American trypanosomiasis) still affects about 16 million people in South and Central America. Its etiological agent, the protozoan Trypanosoma cruzi, is highly pleomorphic, exhibiting distinct forms during its life cycle. In * Corresponding author. Tel.: +55-31-3499-2796; fax: +55-31-3499-2810. E-mail address: [email protected] (C.R.S. Machado). 1 Present address: Departamento de Ciências Biológicas, Fundação Educacional de Caratinga, 35300-049 Caratinga, MG, Brazil. © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.micinf.2004.03.002
mammalian hosts, the trypomastigote infective forms circulate in the blood, and amastigotes proliferate inside several cell types, mainly cardiac, skeletal and smooth muscle cells. The life-long chronic phase has three forms: latent or indeterminate, cardiac (chagasic cardiomyopathy) and digestive (megacolon and/or megaesophagus). Around 70% of infected people stay in the indeterminate form characterized by absence of symptoms, and presence of sparse inflammatory foci and rare amastigote nests in autopsied tissues [1,2]. Muscular pain and weakness are frequent symptoms in chagasic patients [1]. In experimental models of the disease, parasitism of skeletal muscle fibers and myositis occur during both acute and chronic phases [3]. In our rat model, the
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diaphragm is the most affected skeletal muscle during the acute phase [4]. Mechanisms responsible for the chronic phase manifestations are not completely elucidated but most studies favor the involvement of autoimmunity and/or parasite persistence [5,6]. However, microvascular compromise and the resulting ischemia are suggested to play an important role in the chronic myocardial damage [7–9]. Endothelins comprise a family of vasoactive peptides secreted mainly by the endothelium. Among the wide variety of cell types able to produce endothelin are cardiomyocytes, vascular smooth muscle, macrophages, mast cells [10] and cardiac fibroblast [11]. Two types of receptors, ETA and ETB, mediate endothelin effects [10]. Endothelin-1 (ET-1) is a potent vasoconstrictor [12] with an important role in vascular tone modulation. Vasoconstriction is mediated mainly by vascular smooth muscle ETA receptors. Vascular muscle ETB receptors also mediate vasoconstriction, but those located on endothelial cells have vasodilator action through the release of nitric oxide [13] and prostacyclin. This explains the transient endothelium-dependent vasodilatation followed by marked vasoconstriction provoked by ET-1, as reviewed [14]. Patients with Chagas’ cardiomyopathy exhibit high levels of plasma ET-1 [15]. This finding is an important basis for the microvascular hypothesis of the genesis of chronic Chagas’ heart disease. Moreover, human endothelial cells release ET-1 after infection with T. cruzi [16]. Studies on murine models of Chagas’ disease also showed generalized vasculitis in several vascular beds. Among the vascular disorders are vasospasm, focal ischemia, platelet thrombi, and elevated plasma levels of ET-1 and thromboxane A2 breakdown products [17–20]. In several previous studies, we demonstrated that the juvenile rat is a good model for the acute phase and the indeterminate form of the chronic disease as discussed elsewhere [4,21]. In the present paper, this rat model of Chagas’ disease was used to investigate the effects of chronic treatment with BSF 461314, an antagonist of endothelin ETA receptors, on coronary flow, parasitemia, tissue parasitism and the acute inflammatory process. BSF 461314 is a highly selective ETA receptor antagonist (more than 700-fold selectivity over ETB), and it was obtained by modifying the heterocyclic moiety of darusentan (LU135252), an ETA receptor antagonist [22]. Because severe acute myocarditis is accompanied by a drastic reduction in autonomic nerve terminals [21,23,25], sympathetic efferent innervation of the heart was analyzed.
2. Material and methods 2.1. Infection and groups of animals Juvenile male Holtzman rats aged 27–29 days were inoculated intraperitoneally with 1 ml of T. cruzi Y strain-infected
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mouse blood (300,000 trypomastigotes). BSF 461314 was kindly provided by Dr Klaus Muenter (Knoll AG, Ludwigshafen, Germany). The treatment (30 mg kg–1 per day in drinking water) started 1 day before trypomastigote inoculation, and was maintained until the day rats were sacrificed. The drug was dissolved in 0.023% NaOH, and the pH was adjusted to 7.5. The volume of water rats drank was measured daily and the body weight was taken every 3 days for dosage adjustment. Non-treated animals (control and infected) received tap water. Fifty-seven T. cruzi-infected and age-matched control rats composed four groups (control, control-treated, infected, and infected-treated) to be killed under ether anesthesia at days 13 and 20 postinoculation. Parasitemia was estimated by Brener’s method [24], in 5 µl of blood obtained from the tail from day 3–18 postinoculation, using 34 infected rats (BSF 461314-treated and untreated) in four experiments. All experimental protocols were performed in accordance with the guidelines of our Institute for laboratory use of animals. 2.2. Langendorff’s technique Before anesthesia, the animals received heparin (400 IU) intraperitoneally. About 5–10 min later, the hearts were carefully dissected and mounted in Langendorff’s preparation. The heart was perfused through a 1.0 ± 0.3-cm aortic stump with Krebs-Ringer solution (KRS) containing (in mM) the following: NaCl (118.4), KCl (4.7), KH2PO4 (1.2), MgSO4.7 H2O (1.2), CaCl2 2 H2O (2.5), glucose (11.7), NaHCO3 (26.5). The perfusion fluid was maintained at 37 ± 1 °C, with a pressure of 65 mm Hg and constant oxygenation (5% CO2 and 95% O2). Coronary flow was measured by collecting the perfusate over a period of 1 min at regular intervals. After an equilibration period of 20–30 min, the basal coronary flow of each heart was measured (ml/min). The doses of bradykinin (Bk, 5.0 ng) and sodium nitroprusside (SNP, 10 µg) were applied as a bolus via a polyethylene tube inserted into a lateral branch of the perfusion cannula. The volume injected was 0.1 ml. After Bk administration, the cannula was washed with KRS for 10–15 min and then SNP was tested. 2.3. Histochemical demonstration of sympathetic innervation Cardiac sympathetic efferent innervation was analyzed in treated and non-treated uninfected and infected animals (n = 5 in each group) at day 20 of infection. In 30-µm-thick cryostat sections of right ventricle tissues, a glyoxylic acidinduced fluorescence method [26] allowed visualization of the noradrenergic nerve fibers. Briefly, sections obtained at –30 °C were immersed in the sucrose-phosphate bufferglyoxylic acid solution for 30 s, dried, covered with mineral oil and heated at 60 °C for 30 min, then coverslipped. Fluo-
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rescence was examined under a Zeiss Axioplan 2 microscope equipped with an HBO 100 mercury lamp. 2.4. Histological and histoquantitative methods Heart base and diaphragm fragments were fixed in 4% phosphate-buffered paraformaldehyde for 24 h and routinely processed for Paraplast (Oxford Labware) embedding. The hearts were processed after ex vivo functional evaluation in Langendorff’s preparation. At least four sections of each organ (7-µm thick) stained with hematoxylin and eosin were analyzed at 70-µm intervals. The volumetric proportion of myocyte amastigote nests and inflammation were assessed in the right ventricle with the help of a Zeiss (Oberkochen, Germany) Kpl integrating eyepiece with 100 hits, as in our earlier paper [25]. At a final magnification of 400×, tissue components coinciding with each hit were counted up until a total of 5000 hits for each organ. In both heart and diaphragm, the tissue components were counted as (1) amastigote nests in myocytes, (2) myocytes without nests, (3) normal connective tissue and vessels, (4) inflammatory process, and (5) artifacts. The values were expressed as mean percentages of the tissue components. In diaphragm, the inflammatory cells inside myocytes were also counted as inflammatory process, and degenerating or regenerating myocytes as myocytes. At day 13, histometric analyses were made in organs of four non-treated and four treated control animals, and three non-treated and five treated infected rats. At day 20, seven control (four treated) and 12 infected (seven treated) animals were used. 2.5. Statistical analysis Data are reported as mean ± S.D., except for the parasitemia data, which are mean ± S.E.M. SigmaStat software (St Louis, MO) were used for all analyses with significance at P < 0.05. For parasitemia, differences between two groups were assessed by Student’s t-test. Non-parametric methods tested the other parameters: Mann–Whitney test for coronary flow and Student-Newman-Keuls method for histometric results.
3. Results 3.1. Mortality and parasitemia Until day 20, mortality was about the same in the two infected groups. Including animals found dead and those dying, we lost six untreated and eight BSF 461314-treated infected rats. The parasitemia was followed in 16 infected and 18 infected treated animals (Fig. 1). Mean peak values occurred at days 11 or 12, respectively, for untreated and
Fig. 1. Mean parasitemia for BSF 461314-treated (n = 18) and non-treated (n = 16) T. cruzi-infected rats obtained in four independent experiments. * P < 0.05 at day 3 and 12.
treated rats. In both infected groups, the expected abrupt fall occurred by day 15, despite significant differences at day 3 and 12. Dying animals were excluded from all other analyses. 3.2. Coronary flow Bk-induced vasodilatation is endothelium-dependent, and a paradoxical response, i.e. vasoconstriction instead of vasodilatation, indicates endothelial dysfunction. In the hearts of untreated infected animals, Bk demonstrated endothelial dysfunction at day 20 (Fig. 2A). In the same hearts, the vasodilator response to SNP-derived nitric oxide was maintained throughout the course of acute infection (Fig. 2B). At day 20, chronic treatment with BSF 461314 prevented the paradoxical response to Bk (Fig. 2A), but had no effect on the SNP response (Fig. 2B). At day 13, the vasodilator response to Bk or SNP was partially abrogated by BSF 461314 chronic treatment in both uninfected and infected rats (Fig. 2). 3.3. Sympathetic innervation The four experimental groups were compared by doubleblind procedure. All uninfected animals (BSF 461314treated and untreated) exhibited a rich myocardial and vascular innervation. All infected animals (treated and untreated) were grouped together because of the virtual absence or drastic reduction of the fluorescent varicose nerve terminals around arterial vessels and in the myocardium. Nerve preterminals and trunks along blood vessels were preserved as expected [25]. Thus, BSF 461314 treatment did not interfere with the severe sympathetic denervation provoked by T. cruzi infection at day 20 postinoculation. 3.4. Histopathological findings The right ventricle was more affected than the left ventricle during the acute phase of T. cruzi infection. Thus,
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Fig. 3. Effect of treatment with the endothelin ETA receptor antagonist on the volumetric proportions (mean ± S.D.) of inflamed stroma in hearts of rats at days 13 (three untreated and five treated) and 20 (five untreated and seven treated) postinoculation with T. cruzi. The percentage of inflammation was higher in infected treated animals in comparison to the untreated group only at day 13 of infection. Uninfected groups had four untreated and five treated rats. * P < 0.05.
Fig. 2. Coronary flow in response to bradykinin (A) and nitroprusside (B) stimuli in isolated hearts of control and T. cruzi-infected rats subjected or not to BSF 461314 chronic treatment. The values are mean percentage changes in perfusion flow-rate (PFR) in relation to the basal flow taken after stabilization of each Langendorff-perfused heart. After stimulation, the flow was measured at 1 min. (A) Impaired endothelial dependent-vasodilatation occurred at day 20 of infection in non-treated rats. (B) Preserved endotheliumindependent vasodilatation in all groups. At day 13, n = 4 untreated and five treated infected animals. At day 20, n = 5 untreated and eight treated rats. For uninfected rats, n = 6 per group, at day 20. * P < 0.05 and #<0.001 vs. respective untreated group.
comparisons among groups were made in the right ventricles. All infected animals showed amastigote nests in cardiomyocytes at day 13, and the myocarditis was intense and diffuse in the epicardial third of the ventricular free wall, confirming previous studies in infected untreated animals [21,25]. Scattered perivascular infiltrating cells were observed in both treated and untreated infected animals mainly around arterial vessels, but no detectable abnormalities were found in the vascular wall. At day 20, 20% of infected and 33% of treated /infected animals exhibited amastigote nests. In the diaphragm, amastigote nests in myocytes and focal myositis were observed in all infected rats at day 13. At day 20, amastigote nests were observed in 60% of untreated and in all BSF 461314-treated animals. A full description of the T. cruzi-induced myositis can be found elsewhere [4]. In the heart, our histoquantitative analysis showed similar volumetric proportions of amastigote nests in BSF 461314treated (0.52 ± 0.28) and untreated (0.71 ± 0.53) animals at day 13. At day 20, a significant reduction in amastigote nest
proportion occurred in both treated (0.03 ± 0.06) and untreated animals (0.02 ± 0.04). Regarding the volumetric proportions of inflammatory process, the mean values were significantly higher in treated infected animals at day 13 (Fig. 3). In the diaphragm, the amount of amastigote nests was higher in treated animals at day 13 (Fig. 4A). Regarding the inflammatory process, the treated group showed significantly higher values at both infection periods (Fig. 4B). Interestingly, only in treated animals was there a significant difference between 13-day and 20-day mean values for inflammation. In the heart, the mean value was higher at day 13 (29.0 ± 1.7) than at day 20 (21.0 ± 5.6). In diaphragm, the highest mean value occurred at day 20 (30.9 ± 7 vs. 21.6 ± 2.4 at day 13).
4. Discussion Endothelin is the most potent endothelium-derived vasoconstrictor, and NO is the main endothelial vasodilator [10]. The latter is quickly elicited by factors such as acetylcholine and bradykinin. Individual vascular beds vary considerably in their sensitivity to ET-1. Within coronary circulation, the resistive vessels are more sensitive than conductive vessels [27]. In the current paper, isolated heart in Langendorff’s preparation was used to test coronary flow in a T. cruzi infection model. The vascular ability to relax was evaluated by the response to Bk and SNP. At the end of the patent parasitemic period, impaired endothelial-dependent vasodilatation (Bk) with preserved endothelium-independent vasodilatation (SNP) was demonstrated. This endothelial dys-
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drome X [30], in which attenuation of the endotheliumdependent vasodilatation occurs. One possible mechanism to explain this endothelial dysfunction is endothelin stimulation of an endogenous NO-synthase inhibitor, the asymmetric dimethylarginine (ADMA), as indicated by studies on heart failure [31] and cardiac syndrome X [30]. Why endothelin receptor activation occurs during the acute phase of a parasitic infection is a very intriguing question. In juvenile rats, T. cruzi Y provokes high parasitemia with peak values around day 11 postinoculation. The patent parasitemic period ends around day 20 [25], reflecting the marked reduction in parasite proliferation in tissues, mainly in the heart [21,25]. Nest rupture provokes the inflammatory response. Heart parasitism is known to become significantly higher from day 6 to 15, but a significant reduction in the inflammatory process occurs only after day 20 [21]. In skeletal muscles, the amount of amastigote nests remains elevated until day 20 [4]. The current work confirmed a faster parasitism control in the heart, as parasite nests became rare at day 20 only in the heart. Treatment with the endothelin ETA receptor antagonist caused higher parasite proliferation in the diaphragm at day 13, a fact not observed in the heart. However, in treated rats, the inflammatory process was more severe at day 13 in both heart and diaphragm. At day 20, significantly higher inflammation values occurred only in the diaphragm of the treated animals. Altogether, our histological and histoquantitative findings point to a precocious role for endothelins in the early stages of parasitism control.
Fig. 4. Effect of treatment with the endothelin ETA receptor antagonist on volumetric proportions (mean ± S.D.) of amastigote nests (A) and inflammatory cells (B) in diaphragm of rats at days 13 and 20 of the T. cruzi infection. (A) Higher percentage of parasitized cardiomyocytes occurred in treated rats at day 13. The inflammatory process was higher in treated infected animals than in the non-treated group at both time periods analyzed. * P < 0.05. Number of animals as in Fig. 3.
function was blocked by chronic treatment with the ETA receptor antagonist, indicating that it was provoked by ET-1. In addition to this blockade at day 20, pretreatment with BSF 461314 (control and infected animals) induced an overall decrease in the vasodilator response to Bk and SNP at day 13. The explanation for this result is not immediately apparent but could be related to the ability of chronic ETA receptor blockade to modify basal vascular tonus in coronary vessels, reinforcing the involvement of ET-1. BSF 461314 treatment did not interfere with sympathetic denervation of the heart induced by the acute infection [21,23,25]. Thus, differences among treated and untreated infected groups cannot be attributed to noradrenaline-mediated mechanisms. Improvement of endothelial dysfunction mediated by ETA blockade has been described in other pathologies characterized by enhanced ET-1 levels [28]. Plasma ET-1 levels are increased in several cardiovascular pathologies, such as heart failure [14], Raynaud’s phenomenon [29] and cardiac syn-
In our rat model, gradual recovery from both myocarditis and myositis takes place after the acute phase halting. As occurs in infected humans with the asymptomatic form of the chronic phase, few amastigote nests and sparse focal inflammatory foci remain without provoking appreciable tissue damage or fibrosis [4,21]. Probably, endothelin rise is restricted to the acute phase and could have beneficial effects by partaking in the initial control of parasitism. Endothelin production can be stimulated either in vitro or in vivo by bacterial lipopolysaccharide and by several cytokines, growth factors, and humoral inflammatory mediators. On the other hand, endothelins can also induce the release of inflammatory mediators such as tumor necrosis factor-a (TNF-a), interleukin-1 (IL-1), IL6, IL-8, monocyte chemotatic protein-1 and granulocyte/macrophage colony-stimulating factor as well as phospholipase A2 activation, as reviewed in [32]. In addition, ET-1 promotes leukocyte adhesion and emigration, and endothelin ETA receptor antagonism is able to protect against leukocyte-induced cardiac injury during ischemia/reperfusion [33]. Also, endothelin receptor antagonism reduces the degree of leukocyte activation in a rat model of colitis [33], and modulates the lymphocyte recruitment and cytokine production in a model of allergic inflammation [34]. It is tempting to hypothesize that T. cruzi-derived molecules provoke overexpression of ET-1. Elevated levels could promote a faster or more efficient inflammatory response. In accordance with this view, ET-1 is overexpressed in the
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myocardium and vascular endothelium of T. cruzi-infected mice during a short period of the acute phase [20]. Moreover, T. cruzi-mediated production of endothelin by cultured human endothelial cells [16] strongly favors this hypothesis. There is no study on endothelin levels during the indeterminate chronic phase of human Chagas’ disease, from which the rat chronic phase works as a model. In patients with chagasic cardiomyopathy, the plasmatic ET-1 level is enhanced [15]. However, this finding could be related to heart failure. Indeed, in heart failure caused by different pathologies, there is ET-1 overproduction, as reviewed [14,20], in parallel with ADMA stimulation [31,35]. Studies on endothelin levels in patients with the indeterminate form as well as in those with different degrees of heart failure would help to clarify this point, contributing to understanding the progression from the indeterminate form to fatal chronic chagasic cardiomyopathy. In conclusion, ET-1 action on ETA receptors could play a role in the vascular functional changes observed in the acute phase of T. cruzi infection. It is unlikely that ETA receptor activation occurs to cause vascular dysfunction; more likely, the ETA receptor activation participates in parasite clearance. ETA receptor activation contributes to the vascular dysfunction seen early after the infection and is part of the cascade of events that results in the control of parasitism.
Acknowledgements Supported by grants 470516/01-1, 500357/03-0 and 551783/2002-8 from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil, and 32.58421.99 and 3254426.99 from the Swiss National Science Foundation.
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[30] P.M. Piatti, G. Fragasso, L.D. Monti, E. Setola, P. Lucotti, I. Fermo, R. Paroni, E. Galluccio, G. Pozza, S. Chierchia, A. Margonato, Acute intravenous L-arginine decreases endothelin-1 levels and improves endothelial function in patients with angina pectoris and normal coronary arteriograms. Correlation with asymmetric dimethylarginina levels, Circulation 107 (2003) 429–436. [31] M. Ohnishi, A. Wada, T. Tsutamoto, M. Fujii, T. Matsumoto, T. Yamamoto, T. Takayama, X. Wang, M. Kinoshita, Endothelin stimulates and endogenous nitric oxide synthase inhibitor, asymmetric dimethylarginin, in experimental heart failure, Clin. Sci. 103 (Suppl. 48) (2002) 241S–244S. [32] G.A. Rae, M.G.M.O. Henriques, Endothelins in inflammation, in: S.M. Said (Ed.), Proinflammatory and Antiinflammatory peptides, Marcel Decker, New York, 1998, pp. 163–202. [33] M. Wanecek, E. Weitzberg, A. Rudehill, A. Oldner, The endothelin system in septic and endotoxin shock, Eur. J. Pharmacol. 407 (2000) 1–15. [34] A.L. Sampaio, G.A. Rae, M.M. Henriques, Role of endothelins on lymphocyte accumulation in allergic pleurisy, J. Leukoc. Biol. 67 (2000) 189–195. [35] M. Usui, H. Matsuoka, H. Miyazaki, S. Ueda, S. Okuda, T. Imaizumi, Increased endogenous nitric oxide synthase inhibitor in patients with congestive heart failure, Life Sci. 62 (1998) 3425–3430.
Original article
Protective role of ETA endothelin receptors during the acute phase of Trypanosoma cruzi infection in rats Elizabeth R.S. Camargos a, Lamara L.V. Rocha a,1, Milene A. Rachid a, Alvair P. Almeida b, Anderson J. Ferreira b, Antonio L. Teixeira-Jr a, Egler Chiari c, Matthias Barton d, Mauro M. Teixeira e, Conceição R.S. Machado a,* a
Departamento de Morfologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais (UFMG), Av. Antonio Carlos 6627, P.O. Box 486, CEP: 31270-901 Belo Horizonte, MG, Brazil b Departamento de Fisiologia/Biofísica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais (UFMG), 31270901 Belo Horizonte, MG, Brazil c Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais (UFMG), 31270901 Belo Horizonte, MG, Brazil d Medical Policlinic, Department of Medicine, University Hospital Zurich, Switzerland e Departamento de Bioquímica/Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais (UFMG), 31270901 Belo Horizonte, MG, Brazil Received 14 November 2003; accepted 2 March 2004 Available online 16 April 2004
Abstract Chagas’ disease, caused by Trypanosoma cruzi, has an acute phase characterized by blood-circulating trypomastigotes and amastigote proliferation in several cell types, especially muscle cells. In the chronic phase, around 70% of infected people are asymptomatic (latent form). The remainder develop chagasic cardiomyopathy and/or digestive syndromes. There is evidence for aggravation of the chronic cardiac pathology by endothelin-mediated vasoconstriction. Holtzman rats have proven to be a good model for Chagas’ disease acute phase and latent chronic phase. Now, we investigate the effects of prolonged treatment with an endothelin ETA receptor antagonist, BSF 461314, during the acute phase on parasitemia, coronary flow, tissue parasitism and the inflammatory process. Using isolated heart in Langendorff’s preparation, endothelial dysfunction was observed only in non-treated infected animals. Histoquantitative analyses carried out in heart and diaphragm showed higher tissue parasitism and/or inflammatory process in BSF 461314-treated animals. Our data indicate that endothelin ETA receptors contribute to the initial mechanisms of parasite control. Impairment of the endothelium-dependent vasodilatation favors hazardous effects. However, blocking endothelin ETA receptors can prevent the latter. © 2004 Elsevier SAS. All rights reserved. Keywords: Chagas’ disease; Endothelin; Parasite clearance; Inflammation; Coronary flow
1. Introduction Although it is progressively becoming controlled, Chagas’ disease (American trypanosomiasis) still affects about 16 million people in South and Central America. Its etiological agent, the protozoan Trypanosoma cruzi, is highly pleomorphic, exhibiting distinct forms during its life cycle. In * Corresponding author. Tel.: +55-31-3499-2796; fax: +55-31-3499-2810. E-mail address: [email protected] (C.R.S. Machado). 1 Present address: Departamento de Ciências Biológicas, Fundação Educacional de Caratinga, 35300-049 Caratinga, MG, Brazil. © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.micinf.2004.03.002
mammalian hosts, the trypomastigote infective forms circulate in the blood, and amastigotes proliferate inside several cell types, mainly cardiac, skeletal and smooth muscle cells. The life-long chronic phase has three forms: latent or indeterminate, cardiac (chagasic cardiomyopathy) and digestive (megacolon and/or megaesophagus). Around 70% of infected people stay in the indeterminate form characterized by absence of symptoms, and presence of sparse inflammatory foci and rare amastigote nests in autopsied tissues [1,2]. Muscular pain and weakness are frequent symptoms in chagasic patients [1]. In experimental models of the disease, parasitism of skeletal muscle fibers and myositis occur during both acute and chronic phases [3]. In our rat model, the
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diaphragm is the most affected skeletal muscle during the acute phase [4]. Mechanisms responsible for the chronic phase manifestations are not completely elucidated but most studies favor the involvement of autoimmunity and/or parasite persistence [5,6]. However, microvascular compromise and the resulting ischemia are suggested to play an important role in the chronic myocardial damage [7–9]. Endothelins comprise a family of vasoactive peptides secreted mainly by the endothelium. Among the wide variety of cell types able to produce endothelin are cardiomyocytes, vascular smooth muscle, macrophages, mast cells [10] and cardiac fibroblast [11]. Two types of receptors, ETA and ETB, mediate endothelin effects [10]. Endothelin-1 (ET-1) is a potent vasoconstrictor [12] with an important role in vascular tone modulation. Vasoconstriction is mediated mainly by vascular smooth muscle ETA receptors. Vascular muscle ETB receptors also mediate vasoconstriction, but those located on endothelial cells have vasodilator action through the release of nitric oxide [13] and prostacyclin. This explains the transient endothelium-dependent vasodilatation followed by marked vasoconstriction provoked by ET-1, as reviewed [14]. Patients with Chagas’ cardiomyopathy exhibit high levels of plasma ET-1 [15]. This finding is an important basis for the microvascular hypothesis of the genesis of chronic Chagas’ heart disease. Moreover, human endothelial cells release ET-1 after infection with T. cruzi [16]. Studies on murine models of Chagas’ disease also showed generalized vasculitis in several vascular beds. Among the vascular disorders are vasospasm, focal ischemia, platelet thrombi, and elevated plasma levels of ET-1 and thromboxane A2 breakdown products [17–20]. In several previous studies, we demonstrated that the juvenile rat is a good model for the acute phase and the indeterminate form of the chronic disease as discussed elsewhere [4,21]. In the present paper, this rat model of Chagas’ disease was used to investigate the effects of chronic treatment with BSF 461314, an antagonist of endothelin ETA receptors, on coronary flow, parasitemia, tissue parasitism and the acute inflammatory process. BSF 461314 is a highly selective ETA receptor antagonist (more than 700-fold selectivity over ETB), and it was obtained by modifying the heterocyclic moiety of darusentan (LU135252), an ETA receptor antagonist [22]. Because severe acute myocarditis is accompanied by a drastic reduction in autonomic nerve terminals [21,23,25], sympathetic efferent innervation of the heart was analyzed.
2. Material and methods 2.1. Infection and groups of animals Juvenile male Holtzman rats aged 27–29 days were inoculated intraperitoneally with 1 ml of T. cruzi Y strain-infected
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mouse blood (300,000 trypomastigotes). BSF 461314 was kindly provided by Dr Klaus Muenter (Knoll AG, Ludwigshafen, Germany). The treatment (30 mg kg–1 per day in drinking water) started 1 day before trypomastigote inoculation, and was maintained until the day rats were sacrificed. The drug was dissolved in 0.023% NaOH, and the pH was adjusted to 7.5. The volume of water rats drank was measured daily and the body weight was taken every 3 days for dosage adjustment. Non-treated animals (control and infected) received tap water. Fifty-seven T. cruzi-infected and age-matched control rats composed four groups (control, control-treated, infected, and infected-treated) to be killed under ether anesthesia at days 13 and 20 postinoculation. Parasitemia was estimated by Brener’s method [24], in 5 µl of blood obtained from the tail from day 3–18 postinoculation, using 34 infected rats (BSF 461314-treated and untreated) in four experiments. All experimental protocols were performed in accordance with the guidelines of our Institute for laboratory use of animals. 2.2. Langendorff’s technique Before anesthesia, the animals received heparin (400 IU) intraperitoneally. About 5–10 min later, the hearts were carefully dissected and mounted in Langendorff’s preparation. The heart was perfused through a 1.0 ± 0.3-cm aortic stump with Krebs-Ringer solution (KRS) containing (in mM) the following: NaCl (118.4), KCl (4.7), KH2PO4 (1.2), MgSO4.7 H2O (1.2), CaCl2 2 H2O (2.5), glucose (11.7), NaHCO3 (26.5). The perfusion fluid was maintained at 37 ± 1 °C, with a pressure of 65 mm Hg and constant oxygenation (5% CO2 and 95% O2). Coronary flow was measured by collecting the perfusate over a period of 1 min at regular intervals. After an equilibration period of 20–30 min, the basal coronary flow of each heart was measured (ml/min). The doses of bradykinin (Bk, 5.0 ng) and sodium nitroprusside (SNP, 10 µg) were applied as a bolus via a polyethylene tube inserted into a lateral branch of the perfusion cannula. The volume injected was 0.1 ml. After Bk administration, the cannula was washed with KRS for 10–15 min and then SNP was tested. 2.3. Histochemical demonstration of sympathetic innervation Cardiac sympathetic efferent innervation was analyzed in treated and non-treated uninfected and infected animals (n = 5 in each group) at day 20 of infection. In 30-µm-thick cryostat sections of right ventricle tissues, a glyoxylic acidinduced fluorescence method [26] allowed visualization of the noradrenergic nerve fibers. Briefly, sections obtained at –30 °C were immersed in the sucrose-phosphate bufferglyoxylic acid solution for 30 s, dried, covered with mineral oil and heated at 60 °C for 30 min, then coverslipped. Fluo-
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rescence was examined under a Zeiss Axioplan 2 microscope equipped with an HBO 100 mercury lamp. 2.4. Histological and histoquantitative methods Heart base and diaphragm fragments were fixed in 4% phosphate-buffered paraformaldehyde for 24 h and routinely processed for Paraplast (Oxford Labware) embedding. The hearts were processed after ex vivo functional evaluation in Langendorff’s preparation. At least four sections of each organ (7-µm thick) stained with hematoxylin and eosin were analyzed at 70-µm intervals. The volumetric proportion of myocyte amastigote nests and inflammation were assessed in the right ventricle with the help of a Zeiss (Oberkochen, Germany) Kpl integrating eyepiece with 100 hits, as in our earlier paper [25]. At a final magnification of 400×, tissue components coinciding with each hit were counted up until a total of 5000 hits for each organ. In both heart and diaphragm, the tissue components were counted as (1) amastigote nests in myocytes, (2) myocytes without nests, (3) normal connective tissue and vessels, (4) inflammatory process, and (5) artifacts. The values were expressed as mean percentages of the tissue components. In diaphragm, the inflammatory cells inside myocytes were also counted as inflammatory process, and degenerating or regenerating myocytes as myocytes. At day 13, histometric analyses were made in organs of four non-treated and four treated control animals, and three non-treated and five treated infected rats. At day 20, seven control (four treated) and 12 infected (seven treated) animals were used. 2.5. Statistical analysis Data are reported as mean ± S.D., except for the parasitemia data, which are mean ± S.E.M. SigmaStat software (St Louis, MO) were used for all analyses with significance at P < 0.05. For parasitemia, differences between two groups were assessed by Student’s t-test. Non-parametric methods tested the other parameters: Mann–Whitney test for coronary flow and Student-Newman-Keuls method for histometric results.
3. Results 3.1. Mortality and parasitemia Until day 20, mortality was about the same in the two infected groups. Including animals found dead and those dying, we lost six untreated and eight BSF 461314-treated infected rats. The parasitemia was followed in 16 infected and 18 infected treated animals (Fig. 1). Mean peak values occurred at days 11 or 12, respectively, for untreated and
Fig. 1. Mean parasitemia for BSF 461314-treated (n = 18) and non-treated (n = 16) T. cruzi-infected rats obtained in four independent experiments. * P < 0.05 at day 3 and 12.
treated rats. In both infected groups, the expected abrupt fall occurred by day 15, despite significant differences at day 3 and 12. Dying animals were excluded from all other analyses. 3.2. Coronary flow Bk-induced vasodilatation is endothelium-dependent, and a paradoxical response, i.e. vasoconstriction instead of vasodilatation, indicates endothelial dysfunction. In the hearts of untreated infected animals, Bk demonstrated endothelial dysfunction at day 20 (Fig. 2A). In the same hearts, the vasodilator response to SNP-derived nitric oxide was maintained throughout the course of acute infection (Fig. 2B). At day 20, chronic treatment with BSF 461314 prevented the paradoxical response to Bk (Fig. 2A), but had no effect on the SNP response (Fig. 2B). At day 13, the vasodilator response to Bk or SNP was partially abrogated by BSF 461314 chronic treatment in both uninfected and infected rats (Fig. 2). 3.3. Sympathetic innervation The four experimental groups were compared by doubleblind procedure. All uninfected animals (BSF 461314treated and untreated) exhibited a rich myocardial and vascular innervation. All infected animals (treated and untreated) were grouped together because of the virtual absence or drastic reduction of the fluorescent varicose nerve terminals around arterial vessels and in the myocardium. Nerve preterminals and trunks along blood vessels were preserved as expected [25]. Thus, BSF 461314 treatment did not interfere with the severe sympathetic denervation provoked by T. cruzi infection at day 20 postinoculation. 3.4. Histopathological findings The right ventricle was more affected than the left ventricle during the acute phase of T. cruzi infection. Thus,
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Fig. 3. Effect of treatment with the endothelin ETA receptor antagonist on the volumetric proportions (mean ± S.D.) of inflamed stroma in hearts of rats at days 13 (three untreated and five treated) and 20 (five untreated and seven treated) postinoculation with T. cruzi. The percentage of inflammation was higher in infected treated animals in comparison to the untreated group only at day 13 of infection. Uninfected groups had four untreated and five treated rats. * P < 0.05.
Fig. 2. Coronary flow in response to bradykinin (A) and nitroprusside (B) stimuli in isolated hearts of control and T. cruzi-infected rats subjected or not to BSF 461314 chronic treatment. The values are mean percentage changes in perfusion flow-rate (PFR) in relation to the basal flow taken after stabilization of each Langendorff-perfused heart. After stimulation, the flow was measured at 1 min. (A) Impaired endothelial dependent-vasodilatation occurred at day 20 of infection in non-treated rats. (B) Preserved endotheliumindependent vasodilatation in all groups. At day 13, n = 4 untreated and five treated infected animals. At day 20, n = 5 untreated and eight treated rats. For uninfected rats, n = 6 per group, at day 20. * P < 0.05 and #<0.001 vs. respective untreated group.
comparisons among groups were made in the right ventricles. All infected animals showed amastigote nests in cardiomyocytes at day 13, and the myocarditis was intense and diffuse in the epicardial third of the ventricular free wall, confirming previous studies in infected untreated animals [21,25]. Scattered perivascular infiltrating cells were observed in both treated and untreated infected animals mainly around arterial vessels, but no detectable abnormalities were found in the vascular wall. At day 20, 20% of infected and 33% of treated /infected animals exhibited amastigote nests. In the diaphragm, amastigote nests in myocytes and focal myositis were observed in all infected rats at day 13. At day 20, amastigote nests were observed in 60% of untreated and in all BSF 461314-treated animals. A full description of the T. cruzi-induced myositis can be found elsewhere [4]. In the heart, our histoquantitative analysis showed similar volumetric proportions of amastigote nests in BSF 461314treated (0.52 ± 0.28) and untreated (0.71 ± 0.53) animals at day 13. At day 20, a significant reduction in amastigote nest
proportion occurred in both treated (0.03 ± 0.06) and untreated animals (0.02 ± 0.04). Regarding the volumetric proportions of inflammatory process, the mean values were significantly higher in treated infected animals at day 13 (Fig. 3). In the diaphragm, the amount of amastigote nests was higher in treated animals at day 13 (Fig. 4A). Regarding the inflammatory process, the treated group showed significantly higher values at both infection periods (Fig. 4B). Interestingly, only in treated animals was there a significant difference between 13-day and 20-day mean values for inflammation. In the heart, the mean value was higher at day 13 (29.0 ± 1.7) than at day 20 (21.0 ± 5.6). In diaphragm, the highest mean value occurred at day 20 (30.9 ± 7 vs. 21.6 ± 2.4 at day 13).
4. Discussion Endothelin is the most potent endothelium-derived vasoconstrictor, and NO is the main endothelial vasodilator [10]. The latter is quickly elicited by factors such as acetylcholine and bradykinin. Individual vascular beds vary considerably in their sensitivity to ET-1. Within coronary circulation, the resistive vessels are more sensitive than conductive vessels [27]. In the current paper, isolated heart in Langendorff’s preparation was used to test coronary flow in a T. cruzi infection model. The vascular ability to relax was evaluated by the response to Bk and SNP. At the end of the patent parasitemic period, impaired endothelial-dependent vasodilatation (Bk) with preserved endothelium-independent vasodilatation (SNP) was demonstrated. This endothelial dys-
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drome X [30], in which attenuation of the endotheliumdependent vasodilatation occurs. One possible mechanism to explain this endothelial dysfunction is endothelin stimulation of an endogenous NO-synthase inhibitor, the asymmetric dimethylarginine (ADMA), as indicated by studies on heart failure [31] and cardiac syndrome X [30]. Why endothelin receptor activation occurs during the acute phase of a parasitic infection is a very intriguing question. In juvenile rats, T. cruzi Y provokes high parasitemia with peak values around day 11 postinoculation. The patent parasitemic period ends around day 20 [25], reflecting the marked reduction in parasite proliferation in tissues, mainly in the heart [21,25]. Nest rupture provokes the inflammatory response. Heart parasitism is known to become significantly higher from day 6 to 15, but a significant reduction in the inflammatory process occurs only after day 20 [21]. In skeletal muscles, the amount of amastigote nests remains elevated until day 20 [4]. The current work confirmed a faster parasitism control in the heart, as parasite nests became rare at day 20 only in the heart. Treatment with the endothelin ETA receptor antagonist caused higher parasite proliferation in the diaphragm at day 13, a fact not observed in the heart. However, in treated rats, the inflammatory process was more severe at day 13 in both heart and diaphragm. At day 20, significantly higher inflammation values occurred only in the diaphragm of the treated animals. Altogether, our histological and histoquantitative findings point to a precocious role for endothelins in the early stages of parasitism control.
Fig. 4. Effect of treatment with the endothelin ETA receptor antagonist on volumetric proportions (mean ± S.D.) of amastigote nests (A) and inflammatory cells (B) in diaphragm of rats at days 13 and 20 of the T. cruzi infection. (A) Higher percentage of parasitized cardiomyocytes occurred in treated rats at day 13. The inflammatory process was higher in treated infected animals than in the non-treated group at both time periods analyzed. * P < 0.05. Number of animals as in Fig. 3.
function was blocked by chronic treatment with the ETA receptor antagonist, indicating that it was provoked by ET-1. In addition to this blockade at day 20, pretreatment with BSF 461314 (control and infected animals) induced an overall decrease in the vasodilator response to Bk and SNP at day 13. The explanation for this result is not immediately apparent but could be related to the ability of chronic ETA receptor blockade to modify basal vascular tonus in coronary vessels, reinforcing the involvement of ET-1. BSF 461314 treatment did not interfere with sympathetic denervation of the heart induced by the acute infection [21,23,25]. Thus, differences among treated and untreated infected groups cannot be attributed to noradrenaline-mediated mechanisms. Improvement of endothelial dysfunction mediated by ETA blockade has been described in other pathologies characterized by enhanced ET-1 levels [28]. Plasma ET-1 levels are increased in several cardiovascular pathologies, such as heart failure [14], Raynaud’s phenomenon [29] and cardiac syn-
In our rat model, gradual recovery from both myocarditis and myositis takes place after the acute phase halting. As occurs in infected humans with the asymptomatic form of the chronic phase, few amastigote nests and sparse focal inflammatory foci remain without provoking appreciable tissue damage or fibrosis [4,21]. Probably, endothelin rise is restricted to the acute phase and could have beneficial effects by partaking in the initial control of parasitism. Endothelin production can be stimulated either in vitro or in vivo by bacterial lipopolysaccharide and by several cytokines, growth factors, and humoral inflammatory mediators. On the other hand, endothelins can also induce the release of inflammatory mediators such as tumor necrosis factor-a (TNF-a), interleukin-1 (IL-1), IL6, IL-8, monocyte chemotatic protein-1 and granulocyte/macrophage colony-stimulating factor as well as phospholipase A2 activation, as reviewed in [32]. In addition, ET-1 promotes leukocyte adhesion and emigration, and endothelin ETA receptor antagonism is able to protect against leukocyte-induced cardiac injury during ischemia/reperfusion [33]. Also, endothelin receptor antagonism reduces the degree of leukocyte activation in a rat model of colitis [33], and modulates the lymphocyte recruitment and cytokine production in a model of allergic inflammation [34]. It is tempting to hypothesize that T. cruzi-derived molecules provoke overexpression of ET-1. Elevated levels could promote a faster or more efficient inflammatory response. In accordance with this view, ET-1 is overexpressed in the
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myocardium and vascular endothelium of T. cruzi-infected mice during a short period of the acute phase [20]. Moreover, T. cruzi-mediated production of endothelin by cultured human endothelial cells [16] strongly favors this hypothesis. There is no study on endothelin levels during the indeterminate chronic phase of human Chagas’ disease, from which the rat chronic phase works as a model. In patients with chagasic cardiomyopathy, the plasmatic ET-1 level is enhanced [15]. However, this finding could be related to heart failure. Indeed, in heart failure caused by different pathologies, there is ET-1 overproduction, as reviewed [14,20], in parallel with ADMA stimulation [31,35]. Studies on endothelin levels in patients with the indeterminate form as well as in those with different degrees of heart failure would help to clarify this point, contributing to understanding the progression from the indeterminate form to fatal chronic chagasic cardiomyopathy. In conclusion, ET-1 action on ETA receptors could play a role in the vascular functional changes observed in the acute phase of T. cruzi infection. It is unlikely that ETA receptor activation occurs to cause vascular dysfunction; more likely, the ETA receptor activation participates in parasite clearance. ETA receptor activation contributes to the vascular dysfunction seen early after the infection and is part of the cascade of events that results in the control of parasitism.
Acknowledgements Supported by grants 470516/01-1, 500357/03-0 and 551783/2002-8 from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil, and 32.58421.99 and 3254426.99 from the Swiss National Science Foundation.
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