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Fibrosis and inflammatory cells in human chronic chagasic myocarditis: scanning electron microscopy and immunohistochemical observations
International Journal of Cardiology 66 (1998) 183–194
Fibrosis and inflammatory cells in human chronic chagasic myocarditis: scanning electron microscopy and immunohistochemical observations Marcos A. Rossi* ˜ Preto, University of Sao ˜ Paulo, 14049 -900 Ribeirao ˜ Preto, SP, Brazil Department of Pathology, Faculty of Medicine of Ribeirao Received 16 April 1998; received in revised form 2 July 1998; accepted 2 July 1998
Abstract The present study deals with both pathologic fibrosis and matrix connective tissue in chronic chagasic myocarditis. A total of 12 hearts were obtained at autopsy. Eight cases of chronic chagasic myocarditis were selected. Four cases without evidence of cardiac disease were used as controls. The diagnosis of chronic Chagas’ heart disease was based on previously established criteria. A cell-maceration method was utilized to evaluate the spatial organization of the fibrillar collagen accumulation after removal of the myocardial tissue non-fibrous elements. The relationship between inflammatory cells identified by monoclonal antibodies and interstitial fibrosis stained with picrosirius red was assessed. Striking structural alterations of the collagen matrix in the perimysium were detected: increase in number and thickness of tendon-like structures, and markedly thickened and aggregated collagen strands. Besides, a diffuse increase in the thickness of collagen fibers surrounding individual myocytes, consisting of the endomysial matrix, mainly adjacent to the perimysium, could be observed. The dense-weave endomysial meshwork was composed of fine collagen fibrils, and it was continuous with those of adjacent myocytes, obscuring the lateral struts. Sometimes, thicker struts tethering myocytes to myocytes could be seen. These changes were associated with scattered dense scar-like foci, probably reflecting repair fibrosis associated with myocyte necrosis. Furthermore, the present results clearly showed the colocalization of foci of myocyte necrosis and degeneration and associated fibrosed areas and fibroblasts with T lymphocytes and macrophages. The accumulation of interstitial collagen fibers in chronic chagasic myocarditis may be expected to decrease myocardial compliance and disrupt synchronous contraction of the ventricles during systole, contributing to a spectrum of ventricular dysfunction that involve either the diastolic or systolic phase of the cardiac cycle or both. Myocardial fibrosis can be also implicated in the genesis of malignant ventricular tachyarrhythmias, major causes of sudden death among chronic chagasic patients. The increase in myocardial fibrosis could be directly related to an inflammatory reaction mainly composed of T lymphocytes and macrophages. 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Chagas’ disease; Trypanosoma cruzi; Chagasic myocarditis; Connective tissue matrix; Immunohistochemistry; Myocardial fibrosis; Scanning electron microscopy
1. Introduction The most significant clinical manifestation of chronic Chagas’ heart disease include congestive heart failure, thromboembolic phenomena, severe arrhythmias, and sudden death. Hemodynamic studies of patients with the chronic form of Chagas’ heart *Tel. / Fax: 155 16 6023130. E-mail: [email protected]
disease have shown ventricular function abnormalities characterized by increased pulmonary artery and pulmonary wedge pressures, increased right and left ventricular volumes and reduced ventricular ejection fraction [1]. Sudden death, presumably related to severe arrhythmias, accounts for 30 to 44% of deaths among chagasic patients [2,3]. However, little is known with respect to the mechanism or mechanisms that underlie the progressive deteriora-
0167-5273 / 98 / $19.00 1998 Elsevier Science Ireland Ltd. All rights reserved. PII: S0167-5273( 98 )00208-3
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tion of right and left ventricular function in chronic chagasic patients. Considering that fibrosis is one of the most prominent features of chronic chagasic myocarditis, either in human or experimental animals [4,5], and the extracellular matrix has an important role in the structure and function of the myocardium [6–10], the progressive accumulation of interstitial collagen could well be the main factor responsible for the progressive impairment of the contractile performance of the myocardium and for the increase in arrhythmogenic risk in chronic Chagas’ heart. In previous work using picrosirius red stain, a collagen specific stain, used together with polarization microscopy, we could demonstrate the pattern of myocardial fibrosis in chronic chagasic cardiomyopathy at the light microscopic level characterized by diffuse increase in the thickness of collagen fibers surrounding muscle fiber bundles (perimysial matrix) and around intramyocardial coronary vessels combined with less pronounced increase in the collagen fibers of the endomysial matrix, and scattered dense stellate scars [4]. The objective here was to examine the fibrillar collagen accumulation in this pathological condition with scanning electron microscopy after removal of the myocardial tissue nonfibrous elements. Besides, since the expression of collagen by fibroblast can be controlled by colocalized inflammatory cells [11,12], the relationship between inflammatory cells and myocardial fibrosis was assessed. The cells responsible for collagen synthesis and the inflammatory cells were identified by monoclonal antibodies and the fibrosis deposits were stained with picrosirius red.
2. Methods
2.1. Human specimens A total of 12 hearts were obtained at autopsy. All hearts were obtained from the Autopsy Service of the Department of Pathology, University Hospital, Facul˜ Preto, University of Sao ˜ ty of Medicine of Ribeirao Paulo. Of the cases, four were without evidence of cardiac disease, these hearts coming from three men and one woman, ranging in age from 35 to 55 years, and weighing between 250 and 350 g. The cause of
death was noncardiac in all cases (two cases of sepsis, one case of pneumonia, and one case of cancer). Eight cases of Chagas’ heart disease were selected, six men and two women, ranging in age from 35 to 68 years, and hearts weighing between 420 and 655 g. The cause of death was cardiac in all cases: sudden cardiac death (three cases), congestive heart failure (three cases), and malignant arrhythmia (two cases). The diagnosis was based on previously established criteria: macroscopic features characterized by cardiomegaly with or without apical aneurysm, rosarybead type epicarditis, dilatation of the subpulmonary infundibulum and microscopically by chronic fibrosing myocarditis and clinical and laboratory findings (positive complement-fixation and / or immunofluorescence tests for Chagas’ disease done before death or in serous fluids at autopsy). Considering that the intensity of the inflammatory reaction varies from one case to another, we exclusively studied cases with moderate to severe chronic myocarditis.
2.2. Histology and morphometry Our routine procedure for examining hearts suspected to have Chagas’ heart disease is to fill the heart with buffered formalin after ligating the great vessels. After 48–72 h of fixation, the heart is opened by a single cut from apex to base. Small fragments were obtained in the basal and middle portion of the left ventricular free wall, immersed in formalin for additional 24 h, and embedded in paraffin. Sections (5 mm thick) were stained with hematoxylin–eosin and azan-Heidenhain. These areas were selected considering that the inflammatory reaction is more intensely and extensively found in the basal portions of the left ventricle and the basal portions of the right atrium. To study the matrix of fibrillar collagen, 5 mm sections were immersed in 0.2% phosphomolybdic acid for 1–5 min before staining for 90 min in a 0.1% solution of Sirius red F3BA dissolved in saturated aqueous picric acid at pH 2.0 [13]. The sections treated with picrosirius red were then examined in direct light and by polarization microscopy. When stained with picrosirius red, collagen is readily identified as red staining fibers with direct light or as yellow or yellow–red thick fibers or green thin fibers
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with polarization microscopy. The minor diameter of myocytes were measured in properly oriented crosssections in each 200 fibers from each heart (subepicardial and midmyocardial zones). Another method for the quantitative examination of the left ventricular myocardium was carried out at mediumpower light microscopic fields (3250): a 100-point ocular Integration eyepiece II (Carl Zeiss) was used to estimate the volume fraction (%) of fibrosis in picrosirius-red-stained sections. Twenty (20) fields of subepicardial and midmyocardial zones were analysed for each heart.
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broblasts, smooth muscle cells, and endothelial cells (mouse anti-swine vimentin, DAKO, Vimentin, V9), leukocyte common antigen (mouse anti-human leukocyte common antigen, DAKO, LCA), T lymphocytes (mouse anti-human T cells, DAKO, CD45RO, UCHL1), B lymphocytes (mouse antihuman B cells, DAKO, CD20, L26), macrophages (mouse anti-human macrophages, DAKO, CD68, clone KP-1), and desmin (mouse anti-human desmin, DAKO, desmin, D33) were used according to manufacturer’s directions.
2.5. Statistics 2.3. Scanning electron microscopy The specimens for scanning electron microscopy were processed according to a modification of the cell-maceration [14,15]. This method allows a good demonstration of the three-dimensional architecture of the collagen fibers of the myocardium, and is not affected by the storage procedure [16]. Adjacent small fragments 103533 mm in size were thoroughly rinsed in distilled water, immersed in 2.5% phosphate buffered glutaraldehyde, rinsed in distilled water, and immersed in a 10% NaOH solution for maceration and removal of the cellular elements for 6–10 days at room temperature. After, they were rinsed in distilled water until they became transparent. Then, they were immersed in 1% tannic acid for 4 h. Subsequently, the specimens were rinsed in distilled water overnight, post-fixed in 1% osmium tetroxide, rinsed, dehydrated in graded concentrations of ethanol, sectioned transversally or longitudinally in relation to the orientation of the myofibers with a very sharp and clean blade under dissecting microscope, and critical-point dried in liquid carbon dioxide. They were then glued to aluminum stubs, sputtercoated with gold and examined in a Zeiss 940-A scanning electron microscope.
2.4. Immunohistochemistry The fibroblasts and inflammatory cells population were identified on serial sections of formalin-fixed paraffin-embedded myocardial samples using the avidin–biotin–peroxidase complex method. Antibodies against intermediate filament vimentin to demonstrate mesenchimal cells in general, including fi-
All quantitative data are expressed as mean6standard deviation. Differences between means were calculated with Student’s t-test. Differences with a P value ,0.05 were considered significant.
3. Results In the myocardium from the hearts without disease, thick (appearing yellow or yellow–red at polarization microscopy) and thin (appearing green at polarization microscopy) fibers of collagen, often colocalized, were seen to form distinct perimysial sheaths which surrounded muscle fibers so as to group them together into bundles, and around intramyocardial coronary vessels. A fine endomysial sheath could be identified within the muscle bundles. The perimysial fibers were composed of strands orientated perpendicular to the long axis of the muscle (lateral connections), and of broad wavy longitudinal fibers. Most of the endo- and perimysial fibers were thick structures (Fig. 1A,B). These findings were in keeping with those reported in the literature [7]. In chronic Chagas’ heart disease the histologic examination of the myocardium showed a chronic fibrosing myocarditis characterized by diffuse foci of myocardial cell loss and degeneration with an inflammatory response composed predominantly of mononuclear cells, and striking interstitial fibrosis. Myofibers containing parasites were not observed. Diffuse interstitial fibrosis, present to variable degree but in all cases, was manifest by the finding of a diffuse increase in the thickness of collagen fibers
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Fig. 1. A, B. Light microscopy of control myocardium. Thick (appearing yellow at polarization microscopy) and thin (appearing green at polarization microscopy) colocalized collagen fibers form distinct perimysial sheaths surrounding muscle fiber bundles (Picrosirius red stain, direct (A) and polarization microscopy (B), 376). B,C, E, and F. Light microscopy of chronic chagasic heart. C. Chronic fibrosing myocarditis. Predominant perimysial and perivascular fibrosis combined with less pronounced endomysial fibrosis. (Movat pentachrome stain, 376). D. Focus of micronecrosis associated with an inflammatory response composed predominantly of lymphomononuclear cells. (Hematoxylin and eosin, 376). E, F. Predominant perimysial fibrosis combined with less pronounced increase in the thickness of endomysial collagen fibers. (Picrosirius red stain, direct and polarization microscopy, 376).
which surrounded the bundles of muscle fibers (the matrix of perymisial collagen), varying in intensity from one area to another, and around the intramural coronary vessels, combined with a less pronounced
increase in the thickness of collagen fibers which surrounded individual myocytes (the matrix of endomysial collagen). Scattered dense stellate scars, replacing areas of ‘dropout’ of myocytes, could also be
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seen. The volume fraction of fibrosis of chagasic hearts (30.768.8%) was markedly increased in comparison to that found in control hearts (6.561.6%). The areas of fibrosis were associated with marked infiltrates of inflammatory cells consisting of lymphomononuclear cells (predominantly macrophages and a few lymphocytes), together with myocytes showing degenerative changes. Most of the cells within the bundles of muscle fibers appeared hypertrophied. The minor diameter of myocytes of the chagasic group was 23.2566.28 mm as compared to 13.7467.88 mm of controls (Fig. 1C,D,E,F). The scanning electron microscopic study revealed that the organization of the connective tissue skeleton of human control hearts sectioned transversally is quite similar to a beehive. The perimysium envelopes groups of myocytes. The endomysium, as final arborization of the perimysium, supports and connects individual cells. The endomysial weave envelopes each individual myofiber and is connected to adjacent myocytes by lateral struts presenting branches of variable size and extension. The range of the length and diameter of these struts is very wide. Collagen struts also connect myocytes to interstitial microvessels (Fig. 2).
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To a variable degree, but present in all cases, interstitial and diffuse fibrosis could be observed in the chagasic hearts. The most striking feature was a diffuse increase of thick collagen fibers constituting broad bands and sheaths of collagen surrounding disorganized muscle bundles (perimysial matrix). Tendon-like collagen fibers increased in number and became much more thicker than those seen in normal hearts. The perimysial collagen bundles had a wavy appearance, most of them oriented parallel to the long axes of the myocytes. Thick collagen strands interconnected the endomysial collagen fibers in the perimysial collagen strands. This was associated with a less marked increase of the pericelllular collagen meshwork (endomysial matrix), mainly in the periphery of the muscle bundles, adjacent to the perimysium and markedly variable from an area to another. The myocytes were encased in a dense weave of collagen fibrils continuous with those of adjacent myocytes. The thickness of the meshwork of collagen fibrils was extremely variable from one cell to another, but the thickest endomysial weaves were the nearest to the perimysial collagen strands. The arrangement of collagen fibrils of the endomysium had a complicated criss-cross pattern. The endomysi-
Fig. 2. Scanning electron microscopy of control myocardium. The perimysium (p) envelopes groups of myocytes. The endomysium (e) supports and connects individual cells. The endomysial weave is connected to adjacent cells by lateral struts (arrow heads). Delicate collagen struts connect myocytes to interstitial microvessels and to the perimysial sheath (arrows). (A, bar520 mm; B, bar510 mm).
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al weave fibers are continuous with those of adjacent myocytes, obscuring the lateral connections. The dimension of the muscle fibers was markedly increased in chagasic heart, although extremely variable. Diffuse scar-like foci corresponding to areas of myocyte ‘dropout’, could be also seen. This disorganization of the connective tissue skeleton of the myocardium could be seen directly related to the intensity of the myocarditis and not in parallel to increase of heart weight. In addition, a remarkably developed perimysial and predominantly endomysial capillary network could be observed (Figs. 3 and 4). The immunoperoxidase findings were similar for all chagasic cases. The fibrotic areas contained connective tissue cells, mainly fibroblasts, stained with vimentin (V9), colocalized with inflammatory cells. Numerous inflammatory cells stained with the antibody against leukocyte common antigen (LCA), while the cardiac myocytes did not. The immunohistochemical staining for KP-1 (CD68), the macrophage marker, stained mononuclear cells with abundant cytoplasm. The mononuclear cells with scant cytoplasm stained strongly for UCHL-1 (CD45RO), demonstrating that the lymphocytes of the infiltrate are primarily T cells rather than B cells. Only a few of the mononuclear cells were positive for L26 (CD20), the B-cell marker. The desmin stain was positive throughout most of the myocytes, but negative within the inflammatory foci (Fig. 5).
4. Discussion The stroma of the heart maintains the structure of the myocardium, determining tissue tensile, strength and stiffness. Besides, it contributes to ventricular function through the transmission of myocyte-generated force to the atrial and ventricular chambers and to relengthening of myocytes in diastole [7–9,18,19]. The three-dimensional configuration of cardiac collagen in small laboratory animals has been determined by scanning electron microscopy through studies on whole fixed myocardial tissue without removal of its non-fibrous elements [6,19,20]. As previously reported, the evaluation of the collagen network of control hearts after removal of the myocardial tissue non-fibrous elements showed that the perimysium, which is an extension of the epi-
mysium, serves to enwrap groups of myocytes, and the endomysium, as final arborization of the perimysium, supports and connects individual cells. The endomysial weave envelopes each individual myocyte and is connected to adjacent myocytes and capillaries by lateral struts [15,17]. The present findings clearly demonstrate, for the first time, the three-dimensional architecture of collagen fibrils in chronic chagasic cardiomyopathy after digestion of the cellular elements. Striking structural alterations of the collagen matrix in the perimysium were detected: increase in number and thickness of tendon-like structures, and markedly thickened and aggregated collagen strands. Besides, a diffuse increase in the thickness of collagen fibers surrounding individual myocytes, consisting the endomysial matrix, mainly adjacent to the perimysium, could be observed. The dense-weave endomysial meshwork was composed of fine collagen fibrils, and it was continuous with those of adjacent myocytes, obscuring the lateral struts. Sometimes, thicker struts tethering myocytes to myocytes could be seen. These changes were associated with scattered dense scarlike foci, probably reflecting repair fibrosis associated with myocyte necrosis. The accumulation of interstitial collagen fibers in chronic chagasic myocarditis may be expected to decrease myocardial compliance and disrupt synchronous contraction of the ventricles during systole, contributing to a spectrum of ventricular dysfunction that involve either the diastolic or systolic phase of the cardiac cycle or both. Myocardial fibrosis can be also implicated in the genesis of malignant ventricular tachyarrhythmias (ventricular tachycardia and ventricular fibrillation), major causes of sudden death among patients with chronic Chagas’ heart disease. The collagen distribution could interfere with the electrical properties of the myocardium. Fibrosis can block the cardiac impulse which may recycle (reentry) through an alternative route and could slow conduction. In addition, the thick collagenous septa encompassing muscle fiber bundles could interfere with lateral impulse conduction, which would favor re-entry [21]. Furthermore, the present results clearly show the colocalization of foci of myocyte necrosis and degeneration and associated fibrosed areas and fibroblasts with T lymphocytes and macrophages. The
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Fig. 3. Scanning electron microscopy of chronic chagasic heart. A. Broad bands of perimysial collagen fibers (p) surround muscle fiber bundles. The increase of pericellular collagen matrix (endomysial matrix, e) occurs mainly in the periphery of the muscle bundles, adjacent to the perimysium (arrows) (Bar550 mm); B. Longitudinally-oriented section showing thick perimysial collagen bundles (p) with a wavy appearance, most of them oriented parallel to the long axes of the myocytes (Bar520 mm); C. Obliquely-sectioned myocardium showing tendon-like perimysial collagen fibers (arrows) and dense pericellular collagen meshworks (arrow heads) (Bar510 mm); D. Very thick coiled perimysial fibers (Bar510 mm).
inflammatory cells could act in coordination perhaps via cytokines and growth factors in fibrogenesis. In previous work we could demonstrate the pattern of myocardial fibrosis in idiopathic cardiomyopathies and chronic chagasic cardiopathy employing the
picrosirius staining technique and polarization microscopy [22]. The myocardium in both dilated and hypertrophic cardiomyopathies showed diffuse fibrosis varying from one to another, but present in all cases. The basic pattern was characterized by a
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Fig. 4. Scanning electron microscopy of chronic chagasic heart. A. Thickened perimysial sheath (p). The adjacent myocytes are encased in a dense weave of collagen fibers (e). Perivascular fibrosis (v). The endomysial weave fibers are continuous with those of adjacent myocytes, obscuring the lateral struts. Broad bands and struts of collagen connect muscle bundles to the perimysial sheath (arrow heads) (Bar520 mm); B. Longitudinal section showing the thick lateral connections tethering one myocyte to another (arrow heads) (Bar510 mm); C. Arrangement of the collage fibrils showing a complicated criss-cross pattern (Bar510 mm); D. Thickened endomysial weaves continuous to those of adjacent myocytes. Remarkably developed endomysial capillary network can be seen (arrows) (Bar520 mm).
diffuse increase in both endomysial and perimysial collagen thick fibers, particularly surrounding individual myocytes (endomysial sheath). The microscopic study of cases of endomyocardial fibrosis
showed a thickened fibrotic endocardium, consisting predominantly of thick collagen fibers extending as septum, corresponding to broad perimysial sheaths, into the myocardium. In the superficial muscle fiber
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Fig. 5. Immunohistochemistry of chronic chagasic heart. A. Numerous inflammatory cells stained with the antibody against leukocyte common antigen (LCA), while the cardiac myocytes did not. B. The immunohistochemical staining for KP-1 (CD68), the macrophage marker, stained mononuclear cells with abundant cytoplasm. C. The mononuclear cells with scant cytoplasm stained strongly for UCHL-1 (CD45RO), demonstrating that the lymphocytes of the infiltrate are primarily T cells rather than B cells. D. Only a few of the mononuclear cells were positive for L26 (CD20), the B-cell marker. E. The fibrotic areas contained connective tissue cells, mainly fibroblasts, stained with vimentin (V9), colocalized with inflammatory cells. F. The desmin stain was positive throughout most of the myocytes, but negative within the inflammatory foci. (3192).
bundles usually composed of atrophic fibers, there was an increase in thickness of collagen fibers in both the perimysial and endomysial connective tissue matrix. Interstitial and diffuse fibrosis could be
observed in the chronic myocarditis of Chagas’ disease. This was manifested by a diffuse increase in the amount of thick collagen fibers surrounding individual muscle fiber bundles (perimysial matrix),
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varying in intensity from one area to another, and around intramural coronary vessels, combined with less pronounced increase in the endomysial collagen matrix. A fundamental question raised is concerned with the potential mechanisms responsible for myocardial fibrosis. The pattern of myocardial fibrosis in chronic Chagas’ heart disease probably reflects the pathogenic mechanisms involved. Diffuse foci of myocardial myonecrosis may be the main etiology factor of the chronic expression in chronic chagasic myocarditis [23]. There is controversy surrounding theories concerning the pathogenesis of chronic chagasic cardiomyopathy. One theory attributes an autoimmune basis to the chronic myocarditis of Chagas’ disease [24–27], whereas another implicates a primary role for an impairment of the intrinsic and extrinsic cardiac autonomic nervous system [28,29]. A possible relationship between the presence of parasite antigens and the intensity of the inflammatory infiltrate has been observed during the chronic phase of the infection in both mice and man [30,31], suggesting therefore that the parasite may have a role in promoting a persistent antigenic stimulation throughout the chronic phase. However, more recently, clinical and experimental studies suggests that the consequences of infection converges on the microcirculation resulting in progressive and addictive focal cellular necrosis with associated inflammatory lymphomononuclear infiltrate, reactive and reparative fibrosis, and surrounding myocyte hypertrophy [26,32–37]. The present findings showing a welldeveloped capillary network in chagasic hearts are in accord with previous unpublished observations using laser confocal microscopy (Higuchi and Brito, personal communication), a probable cause of slow blood capillary flux, which could contribute to the hypoxic changes in chronic chagasic myocarditis [38]. The presence of infiltrates of lymphomononuclear cells is a consistent and prominent finding in the chronic chagasic cardiomyopathy. Our results clearly show the colocalization of the fibrosed areas and fibroblasts with T lymphocytes and macrophages. T lymphocytes have been demonstrated to play a role in the pathogenesis of fibrosis. Bleomycin-induced pul-
monary fibrosis in mice is attenuated by depletion of CD4 1 or CD8 1 T cells and completely abrogated by total T-cell depletion [39]. They may act directly on mesenchymal cells by means of cytokine production which leads to the proliferation of fibroblasts and the synthesis of collagen or indirectly by enhancing the activation of macrophages [39]. Macrophages, when activated by cytokines, have been shown to produce powerful inducers of fibrosis, such as transforming growth factors b (TGF-b) and platelet-derived growth factor (PDGF) [40,41]. Our study shows a predominance of T cells and macrophages concentration. Besides, since myocytes can produce growth factors such as fibroblast growth factor [42], the injured myocytes could potentially produce and release such factors, contributing to the fibrogenesis. In addition, increased production of endothelin-1 by cardiac myocytes correlates closely with the degree of hemodynamic and functional impairment [43,44], indicating that this peptide could also contribute to myocardial fibrosis through its collagen synthesisenhancing effect [45]. We have demonstrated striking changes in the three-dimensional architecture of the collagen fibers in formalin-fixed autopsied human hearts from patients with chronic Chagas’ heart disease. The increase in myocardial fibrosis could be directly related to an inflammatory reaction mainly composed of T lymphocytes and macrophages. These inflammatory cells could promote fibrosis by releasing mediators such as growth factors and cytokines which act on fibroblasts. Further studies to characterize these mediators are needed if possibilities of prevention and corrective forms of therapy are to be offered for the fibrotic process.
Acknowledgements This study was supported by a grant from the ˜ de Amparo a` Pesquisa do Estado de Sao ˜ Fundac¸ao Paulo (Processo FAPESP 97 / 07084-0). The author ´ thanks to Monica Abreu, Ana Abreu, Lıgia Santoro and Maria Elena Riul the excellent technical assistance. Professor Marcos Rossi is Senior Investigator (1A) of the Conselho Nacional de Desenvolvimento ´ ´ Cientıfico e Tecnologico (CNPq).
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[21] Spach MS, Dolber PC. Relating extracellular potentials and their derivatives to anisotropic propagation at a microscopical level in human cardiac muscle. Evidence for electrical uncoupling side-toside fiber connections with increasing age. Circ Res 1986;58:356– 71. [22] Rossi MA. Patterns of myocardial fibrosis in idiopathic cardiomyopathies and chronic Chagasic cardiopathy. Can J Cardiol 1991;7:287–94. [23] Rossi MA, Ramos SG. Pathogenesis of chronic Chagas’myocarditis: an overview. Cardiovasc Path 1996;5:197–202. [24] Ribeiro-dos-Santos R, Rossi MA. Imunopatologia: In: Canc¸ado JR, ´ Chuster M, editors. Cardiopatia chagasica. Belo Horizonte: Fun˜ Carlos Chagas, 1985:10–22. dac¸ao [25] Ribeiro-dos-Santos R, Rossi MA, Laus JL, Silva JS, Savino W, Mengel J. Anti-CD4 abrogates rejection and reestablishes long-term tolerance to syngeneic newborn hearts grafted in mice chronically infected with Trypanosoma cruzi. J Exp Med 1992;175:29–39. [26] Mengel J, Rossi MA. Chronic myocarditis pathogenesis: dependence on autoimmune and microvascular factors. Am Heart J 1992;124:1052–7. [27] Kalil J, Cunha-Neto E. Autoimmunity in Chagas’ disease cardiomyopathy: fulfilling the criteria at last? Parasitol Today 1996;12:396–9. [28] Oliveira JSM. A natural model of intrinsic heart system denervation: Chagas’ cardiopathy. Am Heart J 1985;110:1092–8. [29] Ramos SG, Matturri L, Rossi L, Rossi MA. Cardiac extrinsic neuropathy: lesions of mediastinal paraganglia in chronic chagasic cardiomyopathy. Cardiovasc Path 1996;5:227–31. ` [30] Ben Younes-Chennoufi A, Hontebeyrie-Joskowicz M, Tricottet V, Eisen H, Reynes M, Said G. Persistence of Trypanosoma cruzi antigens in the inflammatory lesion of chronically infected mice. Trans Roy Soc Trop Med Hyg 1988;82:77–83. [31] Higuchi ML, Brito T, Reis MM, et al. Correlation between Trypanosoma cruzi parasitism and myocardial inflammatory infiltrate in human chronic chagasic myocarditis: light microscopy and immunohistochemical findings. Cardiovasc Path 1993;2:101–6. [32] Rossi MA, Gonc¸alves S, Ribeiro-dos-Santos R. Experimental Trypanosoma cruzi myocarditis in BALB / c mice. The potential role of intravascular platelet aggregation in its genesis. Am J Pathol 1984;114:209–16. [33] Rossi MA. Microvascular changes as a cause of chronic cardiomyopathy in Chagas’ disease. Am Heart J 1990;120:233–6. [34] Morris SA, Tanowitz HB, Witner M, Bilezikian JP. Pathophysiological insights into the cardiomyopathy of Chagas’ disease. Circulation 1990;82:1900–9. [35] Tanowitz HB, Morris SA, Factor SM, Weiss LM, Wittner M. Parasitic diseases of the heart. I: Acute and chronic Chagas’ disease. Cardiovasc Path 1992;1:7–15. [36] Marin-Neto JA, Marzullo P, Marcassa C, et al. Myocardial perfusion abnormalities in chronic Chagas’ disease as detected by thalium-201 scintigraphy. Am J Cardiol 1992;69:780–4. [37] Torres FV, Acquatella H, Condado A, Dinsmore R, Palacios IF. Coronary vascular reactivity is abnormal in patients with Chagas’ disease. Am Heart J 1995;129:945–1001. [38] Rossi MA, Carobrez SG. Experimental Trypanosoma cruzi cardiomyopathy in BALB / c mice: histochemical evidence of hypoxic changes in the myocardium. Br J Exp Path 1985;66:155–66. [39] Piguet PF, Collart MA, Grau GE, Kapanci Y, Vassali P. Tumor necrosis factor / cachetin plays a key role in bleomycin-induced pneumopathy and fibrosis. J Exp Med 1989;170:655–63. [40] Kovacs EJ. Fibrogenic cytokines: the role of immune mediators in the development of scar tissue. Immunol Today 1991;12:17–23.
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[41] Lange LG, Schreiner GF. Immune mechanisms of cardiac disease. New Engl J Med 1994;330:1129–35. [42] Speir E, Yi-Fu Z, Lee M, Shrivastav S, Cassels W. Fibroblast growth factors are present in adult cardiac myocytes in vivo. Biochem Biophys Res Commun 1988;157:1336–40. [43] Wei CM, Lerman A, Rodeheffer RJ, et al. Endothelin in human congestive failure. Circulation 1994;89:1580–6.
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Fibrosis and inflammatory cells in human chronic chagasic myocarditis: scanning electron microscopy and immunohistochemical observations Marcos A. Rossi* ˜ Preto, University of Sao ˜ Paulo, 14049 -900 Ribeirao ˜ Preto, SP, Brazil Department of Pathology, Faculty of Medicine of Ribeirao Received 16 April 1998; received in revised form 2 July 1998; accepted 2 July 1998
Abstract The present study deals with both pathologic fibrosis and matrix connective tissue in chronic chagasic myocarditis. A total of 12 hearts were obtained at autopsy. Eight cases of chronic chagasic myocarditis were selected. Four cases without evidence of cardiac disease were used as controls. The diagnosis of chronic Chagas’ heart disease was based on previously established criteria. A cell-maceration method was utilized to evaluate the spatial organization of the fibrillar collagen accumulation after removal of the myocardial tissue non-fibrous elements. The relationship between inflammatory cells identified by monoclonal antibodies and interstitial fibrosis stained with picrosirius red was assessed. Striking structural alterations of the collagen matrix in the perimysium were detected: increase in number and thickness of tendon-like structures, and markedly thickened and aggregated collagen strands. Besides, a diffuse increase in the thickness of collagen fibers surrounding individual myocytes, consisting of the endomysial matrix, mainly adjacent to the perimysium, could be observed. The dense-weave endomysial meshwork was composed of fine collagen fibrils, and it was continuous with those of adjacent myocytes, obscuring the lateral struts. Sometimes, thicker struts tethering myocytes to myocytes could be seen. These changes were associated with scattered dense scar-like foci, probably reflecting repair fibrosis associated with myocyte necrosis. Furthermore, the present results clearly showed the colocalization of foci of myocyte necrosis and degeneration and associated fibrosed areas and fibroblasts with T lymphocytes and macrophages. The accumulation of interstitial collagen fibers in chronic chagasic myocarditis may be expected to decrease myocardial compliance and disrupt synchronous contraction of the ventricles during systole, contributing to a spectrum of ventricular dysfunction that involve either the diastolic or systolic phase of the cardiac cycle or both. Myocardial fibrosis can be also implicated in the genesis of malignant ventricular tachyarrhythmias, major causes of sudden death among chronic chagasic patients. The increase in myocardial fibrosis could be directly related to an inflammatory reaction mainly composed of T lymphocytes and macrophages. 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Chagas’ disease; Trypanosoma cruzi; Chagasic myocarditis; Connective tissue matrix; Immunohistochemistry; Myocardial fibrosis; Scanning electron microscopy
1. Introduction The most significant clinical manifestation of chronic Chagas’ heart disease include congestive heart failure, thromboembolic phenomena, severe arrhythmias, and sudden death. Hemodynamic studies of patients with the chronic form of Chagas’ heart *Tel. / Fax: 155 16 6023130. E-mail: [email protected]
disease have shown ventricular function abnormalities characterized by increased pulmonary artery and pulmonary wedge pressures, increased right and left ventricular volumes and reduced ventricular ejection fraction [1]. Sudden death, presumably related to severe arrhythmias, accounts for 30 to 44% of deaths among chagasic patients [2,3]. However, little is known with respect to the mechanism or mechanisms that underlie the progressive deteriora-
0167-5273 / 98 / $19.00 1998 Elsevier Science Ireland Ltd. All rights reserved. PII: S0167-5273( 98 )00208-3
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tion of right and left ventricular function in chronic chagasic patients. Considering that fibrosis is one of the most prominent features of chronic chagasic myocarditis, either in human or experimental animals [4,5], and the extracellular matrix has an important role in the structure and function of the myocardium [6–10], the progressive accumulation of interstitial collagen could well be the main factor responsible for the progressive impairment of the contractile performance of the myocardium and for the increase in arrhythmogenic risk in chronic Chagas’ heart. In previous work using picrosirius red stain, a collagen specific stain, used together with polarization microscopy, we could demonstrate the pattern of myocardial fibrosis in chronic chagasic cardiomyopathy at the light microscopic level characterized by diffuse increase in the thickness of collagen fibers surrounding muscle fiber bundles (perimysial matrix) and around intramyocardial coronary vessels combined with less pronounced increase in the collagen fibers of the endomysial matrix, and scattered dense stellate scars [4]. The objective here was to examine the fibrillar collagen accumulation in this pathological condition with scanning electron microscopy after removal of the myocardial tissue nonfibrous elements. Besides, since the expression of collagen by fibroblast can be controlled by colocalized inflammatory cells [11,12], the relationship between inflammatory cells and myocardial fibrosis was assessed. The cells responsible for collagen synthesis and the inflammatory cells were identified by monoclonal antibodies and the fibrosis deposits were stained with picrosirius red.
2. Methods
2.1. Human specimens A total of 12 hearts were obtained at autopsy. All hearts were obtained from the Autopsy Service of the Department of Pathology, University Hospital, Facul˜ Preto, University of Sao ˜ ty of Medicine of Ribeirao Paulo. Of the cases, four were without evidence of cardiac disease, these hearts coming from three men and one woman, ranging in age from 35 to 55 years, and weighing between 250 and 350 g. The cause of
death was noncardiac in all cases (two cases of sepsis, one case of pneumonia, and one case of cancer). Eight cases of Chagas’ heart disease were selected, six men and two women, ranging in age from 35 to 68 years, and hearts weighing between 420 and 655 g. The cause of death was cardiac in all cases: sudden cardiac death (three cases), congestive heart failure (three cases), and malignant arrhythmia (two cases). The diagnosis was based on previously established criteria: macroscopic features characterized by cardiomegaly with or without apical aneurysm, rosarybead type epicarditis, dilatation of the subpulmonary infundibulum and microscopically by chronic fibrosing myocarditis and clinical and laboratory findings (positive complement-fixation and / or immunofluorescence tests for Chagas’ disease done before death or in serous fluids at autopsy). Considering that the intensity of the inflammatory reaction varies from one case to another, we exclusively studied cases with moderate to severe chronic myocarditis.
2.2. Histology and morphometry Our routine procedure for examining hearts suspected to have Chagas’ heart disease is to fill the heart with buffered formalin after ligating the great vessels. After 48–72 h of fixation, the heart is opened by a single cut from apex to base. Small fragments were obtained in the basal and middle portion of the left ventricular free wall, immersed in formalin for additional 24 h, and embedded in paraffin. Sections (5 mm thick) were stained with hematoxylin–eosin and azan-Heidenhain. These areas were selected considering that the inflammatory reaction is more intensely and extensively found in the basal portions of the left ventricle and the basal portions of the right atrium. To study the matrix of fibrillar collagen, 5 mm sections were immersed in 0.2% phosphomolybdic acid for 1–5 min before staining for 90 min in a 0.1% solution of Sirius red F3BA dissolved in saturated aqueous picric acid at pH 2.0 [13]. The sections treated with picrosirius red were then examined in direct light and by polarization microscopy. When stained with picrosirius red, collagen is readily identified as red staining fibers with direct light or as yellow or yellow–red thick fibers or green thin fibers
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with polarization microscopy. The minor diameter of myocytes were measured in properly oriented crosssections in each 200 fibers from each heart (subepicardial and midmyocardial zones). Another method for the quantitative examination of the left ventricular myocardium was carried out at mediumpower light microscopic fields (3250): a 100-point ocular Integration eyepiece II (Carl Zeiss) was used to estimate the volume fraction (%) of fibrosis in picrosirius-red-stained sections. Twenty (20) fields of subepicardial and midmyocardial zones were analysed for each heart.
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broblasts, smooth muscle cells, and endothelial cells (mouse anti-swine vimentin, DAKO, Vimentin, V9), leukocyte common antigen (mouse anti-human leukocyte common antigen, DAKO, LCA), T lymphocytes (mouse anti-human T cells, DAKO, CD45RO, UCHL1), B lymphocytes (mouse antihuman B cells, DAKO, CD20, L26), macrophages (mouse anti-human macrophages, DAKO, CD68, clone KP-1), and desmin (mouse anti-human desmin, DAKO, desmin, D33) were used according to manufacturer’s directions.
2.5. Statistics 2.3. Scanning electron microscopy The specimens for scanning electron microscopy were processed according to a modification of the cell-maceration [14,15]. This method allows a good demonstration of the three-dimensional architecture of the collagen fibers of the myocardium, and is not affected by the storage procedure [16]. Adjacent small fragments 103533 mm in size were thoroughly rinsed in distilled water, immersed in 2.5% phosphate buffered glutaraldehyde, rinsed in distilled water, and immersed in a 10% NaOH solution for maceration and removal of the cellular elements for 6–10 days at room temperature. After, they were rinsed in distilled water until they became transparent. Then, they were immersed in 1% tannic acid for 4 h. Subsequently, the specimens were rinsed in distilled water overnight, post-fixed in 1% osmium tetroxide, rinsed, dehydrated in graded concentrations of ethanol, sectioned transversally or longitudinally in relation to the orientation of the myofibers with a very sharp and clean blade under dissecting microscope, and critical-point dried in liquid carbon dioxide. They were then glued to aluminum stubs, sputtercoated with gold and examined in a Zeiss 940-A scanning electron microscope.
2.4. Immunohistochemistry The fibroblasts and inflammatory cells population were identified on serial sections of formalin-fixed paraffin-embedded myocardial samples using the avidin–biotin–peroxidase complex method. Antibodies against intermediate filament vimentin to demonstrate mesenchimal cells in general, including fi-
All quantitative data are expressed as mean6standard deviation. Differences between means were calculated with Student’s t-test. Differences with a P value ,0.05 were considered significant.
3. Results In the myocardium from the hearts without disease, thick (appearing yellow or yellow–red at polarization microscopy) and thin (appearing green at polarization microscopy) fibers of collagen, often colocalized, were seen to form distinct perimysial sheaths which surrounded muscle fibers so as to group them together into bundles, and around intramyocardial coronary vessels. A fine endomysial sheath could be identified within the muscle bundles. The perimysial fibers were composed of strands orientated perpendicular to the long axis of the muscle (lateral connections), and of broad wavy longitudinal fibers. Most of the endo- and perimysial fibers were thick structures (Fig. 1A,B). These findings were in keeping with those reported in the literature [7]. In chronic Chagas’ heart disease the histologic examination of the myocardium showed a chronic fibrosing myocarditis characterized by diffuse foci of myocardial cell loss and degeneration with an inflammatory response composed predominantly of mononuclear cells, and striking interstitial fibrosis. Myofibers containing parasites were not observed. Diffuse interstitial fibrosis, present to variable degree but in all cases, was manifest by the finding of a diffuse increase in the thickness of collagen fibers
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Fig. 1. A, B. Light microscopy of control myocardium. Thick (appearing yellow at polarization microscopy) and thin (appearing green at polarization microscopy) colocalized collagen fibers form distinct perimysial sheaths surrounding muscle fiber bundles (Picrosirius red stain, direct (A) and polarization microscopy (B), 376). B,C, E, and F. Light microscopy of chronic chagasic heart. C. Chronic fibrosing myocarditis. Predominant perimysial and perivascular fibrosis combined with less pronounced endomysial fibrosis. (Movat pentachrome stain, 376). D. Focus of micronecrosis associated with an inflammatory response composed predominantly of lymphomononuclear cells. (Hematoxylin and eosin, 376). E, F. Predominant perimysial fibrosis combined with less pronounced increase in the thickness of endomysial collagen fibers. (Picrosirius red stain, direct and polarization microscopy, 376).
which surrounded the bundles of muscle fibers (the matrix of perymisial collagen), varying in intensity from one area to another, and around the intramural coronary vessels, combined with a less pronounced
increase in the thickness of collagen fibers which surrounded individual myocytes (the matrix of endomysial collagen). Scattered dense stellate scars, replacing areas of ‘dropout’ of myocytes, could also be
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seen. The volume fraction of fibrosis of chagasic hearts (30.768.8%) was markedly increased in comparison to that found in control hearts (6.561.6%). The areas of fibrosis were associated with marked infiltrates of inflammatory cells consisting of lymphomononuclear cells (predominantly macrophages and a few lymphocytes), together with myocytes showing degenerative changes. Most of the cells within the bundles of muscle fibers appeared hypertrophied. The minor diameter of myocytes of the chagasic group was 23.2566.28 mm as compared to 13.7467.88 mm of controls (Fig. 1C,D,E,F). The scanning electron microscopic study revealed that the organization of the connective tissue skeleton of human control hearts sectioned transversally is quite similar to a beehive. The perimysium envelopes groups of myocytes. The endomysium, as final arborization of the perimysium, supports and connects individual cells. The endomysial weave envelopes each individual myofiber and is connected to adjacent myocytes by lateral struts presenting branches of variable size and extension. The range of the length and diameter of these struts is very wide. Collagen struts also connect myocytes to interstitial microvessels (Fig. 2).
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To a variable degree, but present in all cases, interstitial and diffuse fibrosis could be observed in the chagasic hearts. The most striking feature was a diffuse increase of thick collagen fibers constituting broad bands and sheaths of collagen surrounding disorganized muscle bundles (perimysial matrix). Tendon-like collagen fibers increased in number and became much more thicker than those seen in normal hearts. The perimysial collagen bundles had a wavy appearance, most of them oriented parallel to the long axes of the myocytes. Thick collagen strands interconnected the endomysial collagen fibers in the perimysial collagen strands. This was associated with a less marked increase of the pericelllular collagen meshwork (endomysial matrix), mainly in the periphery of the muscle bundles, adjacent to the perimysium and markedly variable from an area to another. The myocytes were encased in a dense weave of collagen fibrils continuous with those of adjacent myocytes. The thickness of the meshwork of collagen fibrils was extremely variable from one cell to another, but the thickest endomysial weaves were the nearest to the perimysial collagen strands. The arrangement of collagen fibrils of the endomysium had a complicated criss-cross pattern. The endomysi-
Fig. 2. Scanning electron microscopy of control myocardium. The perimysium (p) envelopes groups of myocytes. The endomysium (e) supports and connects individual cells. The endomysial weave is connected to adjacent cells by lateral struts (arrow heads). Delicate collagen struts connect myocytes to interstitial microvessels and to the perimysial sheath (arrows). (A, bar520 mm; B, bar510 mm).
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al weave fibers are continuous with those of adjacent myocytes, obscuring the lateral connections. The dimension of the muscle fibers was markedly increased in chagasic heart, although extremely variable. Diffuse scar-like foci corresponding to areas of myocyte ‘dropout’, could be also seen. This disorganization of the connective tissue skeleton of the myocardium could be seen directly related to the intensity of the myocarditis and not in parallel to increase of heart weight. In addition, a remarkably developed perimysial and predominantly endomysial capillary network could be observed (Figs. 3 and 4). The immunoperoxidase findings were similar for all chagasic cases. The fibrotic areas contained connective tissue cells, mainly fibroblasts, stained with vimentin (V9), colocalized with inflammatory cells. Numerous inflammatory cells stained with the antibody against leukocyte common antigen (LCA), while the cardiac myocytes did not. The immunohistochemical staining for KP-1 (CD68), the macrophage marker, stained mononuclear cells with abundant cytoplasm. The mononuclear cells with scant cytoplasm stained strongly for UCHL-1 (CD45RO), demonstrating that the lymphocytes of the infiltrate are primarily T cells rather than B cells. Only a few of the mononuclear cells were positive for L26 (CD20), the B-cell marker. The desmin stain was positive throughout most of the myocytes, but negative within the inflammatory foci (Fig. 5).
4. Discussion The stroma of the heart maintains the structure of the myocardium, determining tissue tensile, strength and stiffness. Besides, it contributes to ventricular function through the transmission of myocyte-generated force to the atrial and ventricular chambers and to relengthening of myocytes in diastole [7–9,18,19]. The three-dimensional configuration of cardiac collagen in small laboratory animals has been determined by scanning electron microscopy through studies on whole fixed myocardial tissue without removal of its non-fibrous elements [6,19,20]. As previously reported, the evaluation of the collagen network of control hearts after removal of the myocardial tissue non-fibrous elements showed that the perimysium, which is an extension of the epi-
mysium, serves to enwrap groups of myocytes, and the endomysium, as final arborization of the perimysium, supports and connects individual cells. The endomysial weave envelopes each individual myocyte and is connected to adjacent myocytes and capillaries by lateral struts [15,17]. The present findings clearly demonstrate, for the first time, the three-dimensional architecture of collagen fibrils in chronic chagasic cardiomyopathy after digestion of the cellular elements. Striking structural alterations of the collagen matrix in the perimysium were detected: increase in number and thickness of tendon-like structures, and markedly thickened and aggregated collagen strands. Besides, a diffuse increase in the thickness of collagen fibers surrounding individual myocytes, consisting the endomysial matrix, mainly adjacent to the perimysium, could be observed. The dense-weave endomysial meshwork was composed of fine collagen fibrils, and it was continuous with those of adjacent myocytes, obscuring the lateral struts. Sometimes, thicker struts tethering myocytes to myocytes could be seen. These changes were associated with scattered dense scarlike foci, probably reflecting repair fibrosis associated with myocyte necrosis. The accumulation of interstitial collagen fibers in chronic chagasic myocarditis may be expected to decrease myocardial compliance and disrupt synchronous contraction of the ventricles during systole, contributing to a spectrum of ventricular dysfunction that involve either the diastolic or systolic phase of the cardiac cycle or both. Myocardial fibrosis can be also implicated in the genesis of malignant ventricular tachyarrhythmias (ventricular tachycardia and ventricular fibrillation), major causes of sudden death among patients with chronic Chagas’ heart disease. The collagen distribution could interfere with the electrical properties of the myocardium. Fibrosis can block the cardiac impulse which may recycle (reentry) through an alternative route and could slow conduction. In addition, the thick collagenous septa encompassing muscle fiber bundles could interfere with lateral impulse conduction, which would favor re-entry [21]. Furthermore, the present results clearly show the colocalization of foci of myocyte necrosis and degeneration and associated fibrosed areas and fibroblasts with T lymphocytes and macrophages. The
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Fig. 3. Scanning electron microscopy of chronic chagasic heart. A. Broad bands of perimysial collagen fibers (p) surround muscle fiber bundles. The increase of pericellular collagen matrix (endomysial matrix, e) occurs mainly in the periphery of the muscle bundles, adjacent to the perimysium (arrows) (Bar550 mm); B. Longitudinally-oriented section showing thick perimysial collagen bundles (p) with a wavy appearance, most of them oriented parallel to the long axes of the myocytes (Bar520 mm); C. Obliquely-sectioned myocardium showing tendon-like perimysial collagen fibers (arrows) and dense pericellular collagen meshworks (arrow heads) (Bar510 mm); D. Very thick coiled perimysial fibers (Bar510 mm).
inflammatory cells could act in coordination perhaps via cytokines and growth factors in fibrogenesis. In previous work we could demonstrate the pattern of myocardial fibrosis in idiopathic cardiomyopathies and chronic chagasic cardiopathy employing the
picrosirius staining technique and polarization microscopy [22]. The myocardium in both dilated and hypertrophic cardiomyopathies showed diffuse fibrosis varying from one to another, but present in all cases. The basic pattern was characterized by a
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Fig. 4. Scanning electron microscopy of chronic chagasic heart. A. Thickened perimysial sheath (p). The adjacent myocytes are encased in a dense weave of collagen fibers (e). Perivascular fibrosis (v). The endomysial weave fibers are continuous with those of adjacent myocytes, obscuring the lateral struts. Broad bands and struts of collagen connect muscle bundles to the perimysial sheath (arrow heads) (Bar520 mm); B. Longitudinal section showing the thick lateral connections tethering one myocyte to another (arrow heads) (Bar510 mm); C. Arrangement of the collage fibrils showing a complicated criss-cross pattern (Bar510 mm); D. Thickened endomysial weaves continuous to those of adjacent myocytes. Remarkably developed endomysial capillary network can be seen (arrows) (Bar520 mm).
diffuse increase in both endomysial and perimysial collagen thick fibers, particularly surrounding individual myocytes (endomysial sheath). The microscopic study of cases of endomyocardial fibrosis
showed a thickened fibrotic endocardium, consisting predominantly of thick collagen fibers extending as septum, corresponding to broad perimysial sheaths, into the myocardium. In the superficial muscle fiber
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Fig. 5. Immunohistochemistry of chronic chagasic heart. A. Numerous inflammatory cells stained with the antibody against leukocyte common antigen (LCA), while the cardiac myocytes did not. B. The immunohistochemical staining for KP-1 (CD68), the macrophage marker, stained mononuclear cells with abundant cytoplasm. C. The mononuclear cells with scant cytoplasm stained strongly for UCHL-1 (CD45RO), demonstrating that the lymphocytes of the infiltrate are primarily T cells rather than B cells. D. Only a few of the mononuclear cells were positive for L26 (CD20), the B-cell marker. E. The fibrotic areas contained connective tissue cells, mainly fibroblasts, stained with vimentin (V9), colocalized with inflammatory cells. F. The desmin stain was positive throughout most of the myocytes, but negative within the inflammatory foci. (3192).
bundles usually composed of atrophic fibers, there was an increase in thickness of collagen fibers in both the perimysial and endomysial connective tissue matrix. Interstitial and diffuse fibrosis could be
observed in the chronic myocarditis of Chagas’ disease. This was manifested by a diffuse increase in the amount of thick collagen fibers surrounding individual muscle fiber bundles (perimysial matrix),
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varying in intensity from one area to another, and around intramural coronary vessels, combined with less pronounced increase in the endomysial collagen matrix. A fundamental question raised is concerned with the potential mechanisms responsible for myocardial fibrosis. The pattern of myocardial fibrosis in chronic Chagas’ heart disease probably reflects the pathogenic mechanisms involved. Diffuse foci of myocardial myonecrosis may be the main etiology factor of the chronic expression in chronic chagasic myocarditis [23]. There is controversy surrounding theories concerning the pathogenesis of chronic chagasic cardiomyopathy. One theory attributes an autoimmune basis to the chronic myocarditis of Chagas’ disease [24–27], whereas another implicates a primary role for an impairment of the intrinsic and extrinsic cardiac autonomic nervous system [28,29]. A possible relationship between the presence of parasite antigens and the intensity of the inflammatory infiltrate has been observed during the chronic phase of the infection in both mice and man [30,31], suggesting therefore that the parasite may have a role in promoting a persistent antigenic stimulation throughout the chronic phase. However, more recently, clinical and experimental studies suggests that the consequences of infection converges on the microcirculation resulting in progressive and addictive focal cellular necrosis with associated inflammatory lymphomononuclear infiltrate, reactive and reparative fibrosis, and surrounding myocyte hypertrophy [26,32–37]. The present findings showing a welldeveloped capillary network in chagasic hearts are in accord with previous unpublished observations using laser confocal microscopy (Higuchi and Brito, personal communication), a probable cause of slow blood capillary flux, which could contribute to the hypoxic changes in chronic chagasic myocarditis [38]. The presence of infiltrates of lymphomononuclear cells is a consistent and prominent finding in the chronic chagasic cardiomyopathy. Our results clearly show the colocalization of the fibrosed areas and fibroblasts with T lymphocytes and macrophages. T lymphocytes have been demonstrated to play a role in the pathogenesis of fibrosis. Bleomycin-induced pul-
monary fibrosis in mice is attenuated by depletion of CD4 1 or CD8 1 T cells and completely abrogated by total T-cell depletion [39]. They may act directly on mesenchymal cells by means of cytokine production which leads to the proliferation of fibroblasts and the synthesis of collagen or indirectly by enhancing the activation of macrophages [39]. Macrophages, when activated by cytokines, have been shown to produce powerful inducers of fibrosis, such as transforming growth factors b (TGF-b) and platelet-derived growth factor (PDGF) [40,41]. Our study shows a predominance of T cells and macrophages concentration. Besides, since myocytes can produce growth factors such as fibroblast growth factor [42], the injured myocytes could potentially produce and release such factors, contributing to the fibrogenesis. In addition, increased production of endothelin-1 by cardiac myocytes correlates closely with the degree of hemodynamic and functional impairment [43,44], indicating that this peptide could also contribute to myocardial fibrosis through its collagen synthesisenhancing effect [45]. We have demonstrated striking changes in the three-dimensional architecture of the collagen fibers in formalin-fixed autopsied human hearts from patients with chronic Chagas’ heart disease. The increase in myocardial fibrosis could be directly related to an inflammatory reaction mainly composed of T lymphocytes and macrophages. These inflammatory cells could promote fibrosis by releasing mediators such as growth factors and cytokines which act on fibroblasts. Further studies to characterize these mediators are needed if possibilities of prevention and corrective forms of therapy are to be offered for the fibrotic process.
Acknowledgements This study was supported by a grant from the ˜ de Amparo a` Pesquisa do Estado de Sao ˜ Fundac¸ao Paulo (Processo FAPESP 97 / 07084-0). The author ´ thanks to Monica Abreu, Ana Abreu, Lıgia Santoro and Maria Elena Riul the excellent technical assistance. Professor Marcos Rossi is Senior Investigator (1A) of the Conselho Nacional de Desenvolvimento ´ ´ Cientıfico e Tecnologico (CNPq).
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