Severe Fibrosis and Increased Expression of Fibrogenic Cytokines in the Gastric Wall of Systemic Sclerosis Patients

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1 ARTHRITIS & RHEUMATISM Vol. 56, No. 10, October 2007, pp DOI /art , American College of Rheumatology Severe Fibrosis and Increased Expression of Fibrogenic Cytokines in the Gastric Wall of Systemic Sclerosis Patients Mirko Manetti, 1 Elena Neumann, 2 Anna Franca Milia, 3 Ingo H. Tarner, 2 Paolo Bechi, 3 Marco Matucci-Cerinic, 3 Lidia Ibba-Manneschi, 3 and Ulf Müller-Ladner 2 Objective. Systemic sclerosis (SSc) is a connective tissue disorder characterized by fibrosis of the skin and internal organs. Although the esophagus is the most frequently affected part of the gastrointestinal tract, all other segments can be involved. The present study was undertaken to evaluate the fibrotic process and the expression of fibrogenic cytokines in the gastric wall of SSc patients with gastroesophageal involvement. Methods. Full-thickness surgical and endoscopic gastric biopsy samples were obtained from 14 SSc patients and 10 controls. Tissue sections were either stained with Masson s trichrome or by immunohistochemistry and analyzed for the expression of types I, III, and IV collagen, -smooth muscle actin ( -SMA), transforming growth factor (TGF ), connective tissue growth factor (CTGF), and endothelin 1 (ET-1). Results. In the gastric wall of SSc patients, Masson s trichrome staining and immunohistochemistry for types I and III collagen revealed a high amount of collagen in the lamina propria that increased toward the Supported by grants from the Ministero Italiano dell Università e della Ricerca, the Associazione per lo Studio della Sclerosi Sistemica e delle Malattie Fibrosanti, and the German Ministry of Education and Research/German Network for Systemic Sclerosis. 1 Mirko Manetti, PhD: University of Florence, Florence, Italy, Justus-Liebig-University of Giessen, Giessen, Germany, and Kerckhoff-Klinik, Bad Nauheim, Germany; 2 Elena Neumann, PhD, Ingo H. Tarner, MD, Ulf Müller-Ladner, MD: Justus-Liebig- University of Giessen, Giessen, Germany, and Kerckhoff-Klinik, Bad Nauheim, Germany; 3 Anna Franca Milia, PhD, Paolo Bechi, MD, Marco Matucci-Cerinic, MD, Lidia Ibba-Manneschi, MD: University of Florence, Florence, Italy. Address correspondence and reprint requests to Lidia Ibba- Manneschi, MD, University of Florence, Department of Anatomy, Histology, and Forensic Medicine, Viale G. B. Morgagni 85, Florence, Italy ( ibba@unifi.it); or to Ulf Müller-Ladner, MD, Justus-Liebig-University of Giessen, Department of Internal Medicine and Rheumatology, Division of Rheumatology and Clinical Immunology, Kerckhoff-Klinik, Benekestrasse 2-8, D Bad Nauheim, Germany ( u.mueller-ladner@kerckhoff-klinik.de). Submitted for publication April 19, 2007; accepted in revised form June 29, muscularis mucosae. In addition, muscle layers showed features of atrophy, with wide areas of focal fibrosis surrounding smooth muscle cells. Type IV collagen was present around glands and small vessels, suggesting a thickening of the basal lamina. The expression of the fibrogenic cytokines TGF and CTGF, ET-1, and the myofibroblast marker -SMA was stronger in SSc patients than in controls. Conclusion. A pronounced deposition of collagen, the presence of myofibroblasts, and increased expression of several profibrotic factors are important hallmarks in the stomach of patients with SSc. The fibrotic involvement of the gastric wall may account for muscle atrophy leading to stomach hypomotility in SSc. Systemic sclerosis (SSc; scleroderma) is a chronic connective tissue disorder of unknown etiology that leads to fibrosis of the skin and various internal organs, including the heart, lung, kidney, and gastrointestinal (GI) tract (1). Perivascular inflammatory infiltration and reduced capillary density are the early histopathologic hallmarks of SSc. In the later stages of the disease, the excessive synthesis and deposition of extracellular matrix (ECM) components lead to dysfunction of the affected organs, often resulting in death (1,2). SSc fibroblasts are activated and produce high amounts of types I, III, VI, and VII collagen, fibronectins, and proteoglycans (2,3). Myofibroblasts, which are characterized by the expression of -smooth muscle actin ( -SMA), are believed to be mainly responsible for the increased ECM production in the affected tissues (3). Profibrotic cytokines, such as transforming growth factor (TGF ) and connective tissue growth factor (CTGF), play a crucial role in the pathologic remodeling of connective tissues and, therefore, in the pathogenesis of SSc (4). GI tract dysfunction is one of the most common features of SSc patients, both in those with the diffuse 3442

2 FIBROSIS AND FIBROGENIC FACTORS IN SSC GASTRIC WALL 3443 cutaneous (dcssc) and in those with the limited cutaneous (lcssc) form of the disease (5). Among the different parts of the GI tract, the esophagus and the anorectal segment are most frequently affected, even though all other segments can be involved (5,6). Several studies in SSc patients have addressed the GI manifestations, but only a few histologic studies of the GI tract have been performed (7,8), and in particular, only 1 ultrastructural study of the muscle coat of the gastric wall has been performed (9). This prompted us to investigate the key molecules involved in the fibrotic process of the gastric wall. In the present study, we analyzed endoscopic and surgical gastric samples from SSc patients with gastroesophageal involvement to evaluate the extent of collagen deposition, the presence of myofibroblasts, and the expression of key fibrogenic cytokines (TGF and CTGF), as well as of endothelin 1 (ET-1), a mediator of fibroblast proliferation and ECM synthesis, whose role in the pathogenesis of SSc is the subject of worldwide interest (2,10). PATIENTS AND METHODS Patients. Fourteen SSc patients (12 women and 2 men; mean SD age years) were enrolled after giving their written informed consent to participate in the study. The protocol was approved by the ethics committees of the hospitals. SSc was classified as dcssc (8 patients) or lcssc (6 patients) according to the criteria of LeRoy et al (1). The mean SD disease duration was years. GI involvement was defined symptomatically according to the recent guidelines (11). Patients with gastroesophageal symptoms were selected and underwent esophagogastroscopy. Three of the 14 patients also underwent esophageal surgery. One of them, a 52-year-old woman with longstanding lcssc (disease duration 19 years) and severe involvement of the upper digestive tract, did not respond to treatment with ranitidine and, later, with omeprazole and cisapride. This patient therefore underwent a Nissen-Rossetti laparoscopic fundoplication, and later, a total gastrectomy with a Roux-en-Y esophagojejunal anastomosis was performed. The other 2 patients, both of whom were women with dcssc (disease duration 7 years and 11 years, respectively), had severe and refractory gastroesophageal reflux, gastroparesis, and bleeding. They underwent antrectomy with Roux-en-Y anastomosis. Gastric biopsy samples. Biopsy samples were obtained during esophagogastroscopy, from the fundus, corpus, and antrum of each SSc patient. Samples were embedded in OCT medium (TissueTek OCT; VWR Scientific Products, San Diego, CA) and immediately snap-frozen in liquid nitrogen. All cryoblocks were stored at 80 C until used. Full-thickness samples of the gastric anterior wall, near the greater curvature, were obtained from the 3 gastric areas of the lcssc patient and from the antrum of the dcssc patients after gastrectomy. The specimens were fixed in 10% buffered formalin, dehydrated in a graded alcohol series, and embedded in paraffin. As control gastric tissue, we used endoscopic biopsy samples from 7 age- and sex-matched healthy subjects and full-thickness surgical samples from 3 patients who underwent total gastrectomy because of adenocarcinoma of the stomach. We carefully selected samples that appeared to have no inflammatory or neoplastic infiltration (at least 8 cm from the margins of the cancers) according to macroscopic observation. Two fragments were obtained from each biopsy sample. The first fragment was processed for hematoxylin and eosin staining to exclude the presence of inflammatory and neoplastic infiltration. The second fragment was used as a control in our study. Histologic and immunohistochemical analyses. Paraffin sections and ice-cold acetone-fixed cryosections (5 m thick) were stained either with hematoxylin and eosin or with Masson s trichrome to evaluate histopathologic changes and tissue fibrosis. Immunohistochemistry was performed on serial sections according to established methods. When necessary, antigen unmasking on paraffin sections was performed by microwave heating for 20 minutes at 350W in 10 mm citrate buffer (ph 6.0) and 0.05% Tween 20. To block endogenous peroxidase activity, sections were treated with 0.3% H 2 O 2 in methanol for 15 minutes at room temperature. After blocking nonspecific site binding for 30 minutes with normal horse serum, sections were incubated overnight at 4 C with the primary antibody. The primary antibodies used were mouse monoclonal antibody (mab) anti type I collagen (1:500 dilution; Sigma, St. Louis, MO), mouse mab anti type III collagen (1:500 dilution; Sigma), mouse mab anti type IV collagen (1:50 dilution; DakoCytomation, Hamburg, Germany), mouse mab anti -SMA (1:50 dilution; Abcam, Cambridge, UK), mouse mab anti-tgf (1:100 dilution; Abcam), rabbit polyclonal anti-ctgf (1:400 dilution; Abcam), and mouse mab anti ET-1 (1:100 dilution; Abcam). The immunoreactivity was detected using biotinylated secondary antibodies and avidin biotin peroxidase complex (UltraVision Detection System; LabVision, Fremont, CA), followed by color development using 3-amino-9-ethylcarbazole substrate (AEC kit; Vector, Burlingame, CA) or 3,3 diaminobenzidine tetrahydrochloride substrate (Sigma). Some sections were counterstained with hematoxylin and then mounted with Crystal-Mount (Biomeda, Foster City, CA). Negative controls were obtained by omitting the primary antibodies or by incubating the tissue sections with irrelevant isotype-matched normal IgG (Sigma). The sections were examined using a light microscope (Eclipse E400; Nikon, Tokyo, Japan) and photographed with a digital camera (Coolpix 2500; Nikon). Three fields (20 objective magnification) from 2 random sections of each gastric biopsy sample were analyzed by 2 blinded observers (MM and AFM). We performed a semiquantitative analysis of the expression of types I and IV collagen, TGF, CTGF, and ET-1. Histologic grading was based on the intensity of the staining, as follows: no staining, / weak staining, moderate staining, and intense staining. The final result was the median of the 2 different observations for each sample.

3 3444 MANETTI ET AL RESULTS Findings of histochemical staining. Masson s trichrome staining was performed to analyze tissue fibrosis in gastric biopsy samples from SSc patients and Figure 2. Immunohistochemical detection of myofibroblasts in gastric biopsy samples from systemic sclerosis (SSc) patients and controls. A and B, Immunostaining of the gastric wall for -smooth muscle actin ( -SMA). In the mucosa of the SSc patient and control samples, glandular myoepithelial cells and vascular pericytes show immunopositivity for -SMA (A). Strong immunostaining for -SMA is evident close to the vessels of the lamina propria and in the muscularis mucosae in the SSc patient sample (A), and at higher magnification, strongly immunopositive spindle-shaped cells and vascular pericytes are seen (inset). The muscle layers show higher -SMA positivity in the SSc patient sample than in the control sample (B). (Original magnification 20; 40 in inset.) Figure 1. Histochemical and immunohistochemical analyses of extracellular matrix components in gastric biopsy samples from systemic sclerosis (SSc) patients and controls. A and B, Masson s trichrome staining. In the gastric wall of the SSc patient sample, relevant fibrosis in the lamina propria and muscularis mucosae is evident as compared with the control sample (note the intense and diffuse green staining) (A). In the muscle layers of the SSc patient sample, there are wide areas of focal fibrosis (B), and at higher magnification, smooth muscle cells surrounded by fibrotic tissue are seen (inset). C and D, Immunostaining of endoscopic biopsy samples for type I and type IV collagen. In the lamina propria of the SSc patient sample, there is increased expression of type I collagen, particularly among the glands (C), and at higher magnification, immunopositive dense collagen bundles are seen (inset). Type IV collagen around the glands (arrowheads) and vessel walls (arrows) is more pronounced in the SSc patient sample than in the control sample (D). (Original magnification 10 in A and B; 20 in C and D; 40 in insets.) controls. In the gastric wall of patients with SSc, Masson s trichrome staining revealed an accumulation of ECM components in the lamina propria that became more severe in the muscularis mucosae (Figure 1A). In the circular and longitudinal muscle layers, wide areas of marked focal fibrosis surrounded smooth muscle cells (SMCs), thereby widening the intercellular spaces (Figure 1B and inset). Moreover, the involvement of the muscle layers showed a patchy distribution, with areas of either atrophic or normal smooth muscle fibers in the same tissue section. No disease subset- or durationrelated differences in the extent of fibrosis of the analyzed tissue samples were observed. Immunohistochemical findings. In gastric tissues from patients with SSc, immunohistochemistry showed an increased accumulation of both types I and III collagen, mirroring the findings with the Masson s trichrome staining. Type I collagen deposition was prominent in the lamina propria of the SSc gastric wall, particularly among the glands (Figure 1C and inset). More intense immunopositivity for type IV collagen in SSc samples than in control samples was observed

4 FIBROSIS AND FIBROGENIC FACTORS IN SSC GASTRIC WALL 3445 around the glands and small blood vessels, suggesting a thickening of the basal lamina (Figure 1D). The presence and distribution of myofibroblasts in the gastric wall were investigated using an antibody against -SMA, a marker expressed by myofibroblasts, SMCs, and pericytes, but not by stromal fibroblasts. In the gastric mucosa, immunopositivity for -SMA was detected in myoepithelial cells around the glands and in the vascular pericytes, both in SSc patients and controls (Figure 2A). Interestingly, in SSc samples, a diffuse -SMA immunostaining was frequently observed also in the stroma close to the vessels (Figure 2A and inset). The muscularis mucosae and the muscle layers showed more -SMA positive cells in SSc samples as compared with the controls, with a patchy distribution (Figures 2A and B). This pattern was similar to the wide areas of marked focal fibrosis and smooth muscle atrophy identified by Masson s trichrome staining. In samples from patients with SSc, strong expression of CTGF was evident in all components of the gastric wall (Figures 3A and B). Diffuse immunostaining was observed in stromal cells of the lamina propria and muscularis mucosae (Figure 3A). Endothelial cells (ECs) and pericytes of the small vessels showed intense immunopositivity for CTGF, whereas larger vessels were weakly immunostained (Figure 3A and inset). Moreover, in SSc stomach samples, CTGF was focally distributed in wide areas of the circular and longitudinal muscle layers, with many strongly immunopositive small vessels and spindle-shaped cells (Figure 3B). In contrast, only a few CTGF-positive cells were detected in the muscularis mucosae and muscle layers of control tissues. In addition, vascular ECs and pericytes showed weaker expression of CTGF in control gastric wall samples than in SSc gastric wall samples (Figures 3A and B). Similar to the findings for CTGF, the expression of TGF was strongly increased in SSc samples as compared with controls, particularly in the muscularis mucosae and muscle layers (Figure 3C). Immunostaining for ET-1 was evident in the epithelial cells and vessels in SSc gastric mucosa, and it was focally distributed in wide muscle areas, where many spindle-shaped cells displayed very strong positivity (Figure 3D and inset). In control samples, weak positivity or no positivity was detected in the muscle layers (Figure 3D). The immunohistochemistry results are summarized in Table 1. In particular, no considerable differences were observed between dcssc and lcssc patients with regard to the various parameters assessed (Table 1). Figure 3. Expression of fibrogenic cytokines in the gastric wall of systemic sclerosis (SSc) patients and controls. A and B, Immunostaining for connective tissue growth factor (CTGF). In the lamina propria and muscularis mucosae of the gastric wall in the SSc patient sample, many stromal cells and vessels show strong immunopositivity as compared with the sample from the control (A), and at higher magnification, small vessels with strongly immunopositive endothelial cells and pericytes are seen (inset). In the muscle layers of the SSc patient sample, many cells and small vessels positive for CTGF are evident in wide areas (B). In the gastric wall of the control samples, only a few CTGF-positive cells and weakly stained vessels are seen (A and B). C, Immunostaining for transforming growth factor (TGF ). Expression of TGF is higher in the muscle layers of the SSc gastric wall than in the control. D, Immunostaining for endothelin 1 (ET-1). Expression of ET-1 is more intense in wide muscle areas of the SSc gastric wall than in the control (D), and in the mucosa, glandular epithelial cells with diffuse immunopositivity for ET-1 are seen (inset). (Original magnification 20 in A, B, and D and inset in D; 40 in C and inset in A.) DISCUSSION This study provides evidence that profibrotic mechanisms may play a role in determining fibrosis

5 3446 MANETTI ET AL Table 1. Immunohistochemical findings in gastric wall biopsy tissues from SSc patients and control subjects* Type I collagen Type IV collagen TGF CTGF ET-1 Patients with lcssc / 5 / 6 Patients with dcssc / Control subjects 1 / / / / 2 / / 3 / / / / / 4 / / / / 5 / / 6 / / 7 / / / / 8 / / / 9 / / / 10 / / / / / * Semiquantitative scoring of immunostained gastric wall sections was performed independently by 2 blinded observers, as follows: no staining, / weak staining, moderate staining, and intense staining. SSc systemic sclerosis; TGF transforming growth factor ; CTGF connective tissue growth factor; ET-1 endothelin 1; lcssc limited cutaneous SSc; dcssc diffuse cutaneous SSc. within the gastric wall of patients with SSc. In particular, we analyzed the extent of collagen deposition and the expression of different fibrogenic cytokines, which are known to play a key role in the pathogenesis of SSc. Organ failure secondary to fibrosis is the main cause of morbidity and death in patients with SSc (1). GI dysfunction occurs in up to 90% of SSc patients, both in dcssc and in lcssc, causing high morbidity and, in some cases, mortality (5,12). The esophagus and the anorectal segment are the most commonly involved areas, although any part of the GI tract can be affected (12). GI tract dysmotility is a major visceral manifestation in SSc, clinically ranging from an asymptomatic form to severe paresis (6,12). Different theories support vascular damage, with concomitant or subsequent neurogenic and immunologic abnormalities, or primary autonomic dysfunction as initiating event in the pathogenesis of GI disease in SSc (6,12,13). Nevertheless, there is substantial evidence that with disease progression, generalized GI dysmotility is mainly caused by abnormal collagen deposition and fibrosis (6,12,13). Several clinical studies have assessed GI manifestations, but only a few morphologic analyses have been performed on the GI tract of SSc patients, and these were mainly of the esophageal, colonic, and rectal wall (7,13). To our knowledge, only 1 ultrastructural study has been performed on the stomach of patients with SSc (9). The main limitation to performing such studies is the poor availability of GI tissue samples from SSc patients. Furthermore, in the few surveys performed on large numbers of subjects at autopsy, the authors state that immunohistochemical evaluation was possible only in a small number of samples because of the scarce tissue preservation (8). Our present observations on samples obtained surgically and endoscopically revealed the presence of generalized fibrosis and increased accumulation of both type I and type III collagen in the gastric wall of SSc patients. In particular, the lamina propria, muscularis mucosae, and muscle coat were all affected, with more severe changes in the muscularis mucosae and muscle layers. Involvement of muscle layers occurred in a patchy

6 FIBROSIS AND FIBROGENIC FACTORS IN SSC GASTRIC WALL 3447 distribution, with wide areas of marked focal fibrosis surrounding SMCs. These findings are consistent with those of our previous ultrastructural study (9) and with other morphologic studies of the GI tract of patients with SSc (6,13). The prominent fibrosis enveloping SMCs might account for the impaired cellular contraction and its propagation from cell to cell, which together with autonomic dysfunctions, contribute to gastric dysmotility and poor gastric emptying. Moreover, we found an increased deposition of type IV collagen around the glands and small vessels in the mucosa, suggesting a thickening of the basement membrane, which is likely involved in secretory alterations and vascular tone dysfunction. Myofibroblasts, which can differentiate from fibroblasts or vascular pericytes, are believed to drive the development of fibrosis, and their presence in the skin of patients with SSc has been extensively studied (3). Our results provide the first evidence that -SMA expressing cells with a myofibroblast phenotype are also present in the stomach of SSc patients and might account for the increased ECM synthesis and deposition. The increased -SMA expressing cellularity in the muscle coat of tissues from SSc patients, which mirrored the areas of focal fibrosis, suggests a notable presence of myofibroblasts together with the normally resident SMCs, which are probably active in the fibrotic process. TGF appears to play a crucial role in the pathogenesis of skin and lung fibrosis in SSc, activating fibroblasts and causing a potent stimulation of CTGF synthesis in fibroblasts, vascular SMCs, and ECs (3,4). ET-1 is a potent activator of fibroblast proliferation and ECM synthesis, and its enhanced production has been well described in SSc (3,10). We found a marked overexpression of all these key profibrotic factors in the stomach of patients with SSc, mostly in the small vessel wall and focally in the fibrotic areas of muscle layers, suggesting their active role in inducing and perpetuating the fibrotic response. In conclusion, this study is the first to show that a severe fibrosis with increased deposition of different collagen types, myofibroblast differentiation, and strong expression of several profibrotic factors are important hallmarks in the gastric wall of patients with SSc. Such alterations likely account for the muscle atrophy that leads to the hypomotility of the stomach that is commonly observed in SSc patients. ACKNOWLEDGMENT The authors would like to thank Dr. Luca Manneschi for collecting the biopsy samples that were used as controls in the study. AUTHOR CONTRIBUTIONS Drs. Ibba-Manneschi and Müller-Ladner had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study design. Manetti, Matucci-Cerinic, Ibba-Manneschi, Müller- Ladner. Acquisition of data. Manetti, Neumann, Milia, Tarner, Bechi. Analysis and interpretation of data. Manetti, Milia, Ibba-Manneschi, Müller-Ladner. Manuscript preparation. Manetti, Matucci-Cerinic, Ibba-Manneschi, Müller-Ladner. REFERENCES 1. LeRoy EC, Black C, Fleischmajer R, Jablonska S, Krieg T, Medsger TA Jr, et al. Scleroderma (systemic sclerosis): classification, subsets, and pathogenesis. J Rheumatol 1988;15: Jimenez SA, Derk CT. Following the molecular pathways toward an understanding of the pathogenesis of systemic sclerosis. Ann Intern Med 2004;140: Abraham DJ, Varga J. Scleroderma: from cell and molecular mechanisms to disease models. Trends Immunol 2005;26: Denton CP, Abraham DJ. 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