Immunoexpression of TGF-β/Smad and VEGF-A proteins in serum and BAL fluid of sarcoidosis patients

Piotrowski et al. BMC Immunology (2015) 16:58 DOI 10.1186/s12865-015-0123-y RESEARCH ARTICLE Open Access Immunoexpression of TGF-β/Smad and VEGF-A proteins in serum and BAL fluid of sarcoidosis patients Wojciech J. Piotrowski1, Justyna Kiszałkiewicz2, Paweł Górski1, Adam Antczak3, Witold Górski1, Dorota Pastuszak-Lewandoska2, Monika Migdalska-Sęk2, Daria Domańska-Senderowska2, Ewa Nawrot2, Karolina H. Czarnecka2, Zofia Kurmanowska2 and Ewa Brzeziańska-Lasota2* Abstract Background: The chronic course of pulmonary sarcoidosis can lead to lung dysfunction due to fibrosis, in which the signalling pathways TGF-β/Smad and VEGF-A may play a key role. Methods: We evaluated immunoexpression of TGF-β1, Smad2, 3, and 7, and VEGF-A in serum and bronchoalveolar lavage (BAL) fluid of patients (n = 57) classified according to the presence of lung parenchymal involvement (radiological stage I vs. II-III), acute vs. insidious onset, lung function test (LFT) results, calcium metabolism parameters, percentage of BAL lymphocytes (BAL-L%), BAL CD4+/CD8+ ratio, age, and gender. Immunoexpression analysis of proteins was performed by ELISA. Results: The immunoexpression of all studied proteins were higher in serum than in BAL fluid of patients (p >0.05). The serum levels of TGF-β1 (p = 0.03), Smad2 (p = 0.01), and VEGF-A (p = 0.0002) were significantly higher in sarcoidosis patients compared to healthy controls. There were no differences within the sarcoidosis group between patients with vs. without parenchymal involvement, acute vs. insidious onset, or patients with normal vs. abnormal spirometry results. In patients with abnormal spirometry results a negative correlation was found between forced vital capacity (FVC) % predicted value and TGF-β1 immunoexpression in BAL fluid, and positive correlations were observed between the intensity of lung parenchymal changes estimated by high-resolution computed tomography (HRCT scores) and Smad 2 level in serum. Conclusions: TGF-β/Smad signalling pathway and VEGF-A participate in the pathogenesis of sarcoidosis. BAL TGFβ1, and Smad 2 in serum seem to be promising biomarkers with negative prognostic value, but further studies are required to confirmed our observations. Keywords: Sarcoidosis, TGF-β1, VEGF-A, Prognosis, Growth factors, Angiogenesis Background Sarcoidosis is a chronic inflammatory disorder of unknown aetiology. The diagnosis is made based on a clinical and radiological picture, and is usually confirmed by the presence of non-caseating granulomas in involved organs. In about 90 % of patients granulomas are present in intrathoracic lymph nodes and/or lung parenchyma, but extrapulmonary presentations are frequent [1, 2]. * Correspondence: 2 Department of Molecular Bases of Medicine, 1st Chair of Internal Medicine, Medical University of Lodz, 251 Pomorska St., 92-213 Lodz, Poland Full list of author information is available at the end of the article The prognosis is relatively good; in about 60 % of patients with a plethora of phenotypes the disease disappears without any clinically significant remains. However, in other patients the course may be chronic, sometimes progressive, or recurrent. The most severe complication is lung fibrosis, occurring in 10–15 % of patients and leading to severe functional impairment, disability, and sometimes to death. Among different negative prognostic factors—lung interstitial disease, lung function test abnormality (of both restrictive and obstructive patterns), and severe impairment of calcium homeostasis may be listed as examples, whereas acute © 2015 Piotrowski et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Piotrowski et al. BMC Immunology (2015) 16:58 disease onset and isolated intrathoracic lymph node enlargement (radiological stage I) are considered good prognostic markers [3]. A tremendous research effort has been made to find a reliable biomarker that would be useful to predict long-term prognosis in sarcoidosis patients. Unfortunately, the results have been inconclusive, and it may be difficult at the disease onset to anticipate which patients would be free of disease and which would develop the progressive and fibrotic form in future. The role of TGFβ and TGFβ signalling pathway elements (SMADs) have been extensively studied in animal models of lung fibrosis and in idiopathic lung fibrosis (IPF), and this particular pathway seems to be critical in wound healing, scarring, and fibrosis in different organs and different diseases [4–6]. VEGF is a major contributor to angiogenesis and regulates several cell functions via its receptors (VEGFRs). The angiostaticangiogenic axis (HIF-1a—VEGF—ING-4) may play a role in the pathogenesis of experimental lung fibrosis and IPF [7, 8]. Moreover, it was shown recently that these two molecular pathways are closely interrelated. For instance, in cultured human umbilical vein endothelial cells (HUVEC) physiological concentrations of VEGF attenuated TGF-β-related phosphorylation of Smad2/3 [9]. TGF-β1 has been shown to stimulate VEGF-A expression in human lung fibroblast via the Smad3 signalling pathway, but it downregulates VEGF-D expression through TGF-β receptor and JNK signalling pathway [10]. Interestingly, the same authors found decreased expression of VEGF-D in lung tissue of IPF patients [10]. In a rat model of lung fibrosis treatment with adenoviral delivery of VEGF resulted in reduced endothelial apoptosis, increased vascularisation, and decreased pulmonary hypertension due to reduced remodelling, but significantly worsened pulmonary fibrosis [7]. Therefore, the net effect of VEGF on lung fibrosis may depend on the isoform predominance, as well as the extent to which it is embedded in a cytokine network. In sarcoidosis data are scarce and even more ambiguous. TGF-β1 concentration was increased in BAL fluid of sarcoidosis patients, but only in those with impaired lung function [11]. Contrary to this, polymorphic alleles of TGF-β1, implicated in lower levels of protein production, were associated with more severe disease presentation [12]. Other genetic studies indicate the role of polymorphic variants of TGF-β3 (with presumed modulating role on TGF-β1 activity) in sarcoidosis-related fibrotic lung disease [13], and a protective role of TGF-β2 SNP [14]. Smad proteins have not been studied in sarcoidosis so far. Data on the role of VEGF in the pathogenesis of sarcoidosis are also inconclusive. VEGF BAL concentra (...truncated)