Justyna Kiszałkiewicz

TGF-β and SMADs mRNA Expression in Pulmonary Sarcoidosis

Lung fibrosis is a complication of sarcoidosis, in which TGF-β/Smad pathway may play an important role. We evaluated gene expression of TGF-β1, SMAD2, 3 and 7 in bronchoalveolar lavage (BAL) cells and peripheral blood (PB) lymphocytes of sarcoidosis patients (n=94) to better understand the mechanisms of sarcoid inflammation. The relative gene expression was analyzed by qPCR method. Selected clinical/radiological features and biochemical markers were taken into account in the analysis. We found that TGF-β1 and SMAD3 expressions in PB lymphocytes were significantly higher in sarcoidosis patients. Up-regulation of SMAD7 (inhibitory Smad) and down-regulation of SMAD3 in BAL cells in all subgroups were found. The expression of TGF-β1 in PB lymphocytes was the highest in patients with lung parenchymal involvement and in the insidious onset phenotype. The expression of TGF-β1 in BAL cells was higher in patients with abnormal spirometry (p=0.012), and TGF-β1 and SMAD3 in patients with restrictive pattern (p=0.034 and 0.031, respectively). Several statistically significant negative correlations were found between the expression levels of SMAD2 and 3 in BAL cells and various LFT parameters. We conclude that TGF-β/Smad pathway is involved in the pathogenesis of pulmonary sarcoidosis. These biomarkers (especially TGF-β1, SMAD2 and 3) are of a negative prognostic value.

Altered miRNA expression in pulmonary sarcoidosis

BackgroundmiRNAs control important cellular functions including angiogenesis/angiostasis or fibrosis and reveal altered expression during pathological processes in the lung.MethodsThe aim of the study was to investigate the expression of selected miRNAs (miR-let7f, miR-15b, miR-16, miR-20a, miR-27b, miR-128a, miR-130a, miR-192 miR-221, miR-222) in patients with pulmonary sarcoidosis (n = 94) and controls (n = 50). The expression was assessed by q-PCR in BALF cells and peripheral blood lymphocytes (PB lymphocytes). For statistical analysis, the Kruskal–Wallis test, Mann–Whitney U- test, Neuman–Keuls’ multiple comparison test, and Spearman’s rank correlation were used.ResultsIn BALF cells, significantly higher expression of miR-192 and miR-221 and lower expression of miR-15b were found in patients than controls. MiR-27b, miR-192 and miR-221 expression was significantly higher in patients without parenchymal involvement (stages I) than those at stages II-IV. Patients with acute disease demonstrated significantly higher miR-27b, miR-192 and miR-221 expression than those with insidious onset. For PB lymphocytes, patients demonstrated significantly greater miR-15b, miR-27b, miR-192, miR-221 and miR-222 expression, but lower miR-let7f and miR-130a expression, than controls. Stage I patients demonstrated significantly higher miR-16 and miR-15b expression than those in stages II-IV, and patients with the acute form demonstrated higher miR-130a and miR-15b expression. In BALF cells, miR-16 and miR-20a expression was significantly higher in patients with lung volume restriction, and miR-let7f was higher in the PB lymphocytes in patients with obturation. Several correlations were observed between the pattern of miRNA expression, lung function parameters and selected laboratory markers.ConclusionThe obtained results suggest that the studied miRNAs play a role in the pathogenesis of sarcoidosis, and that some of them might have negative prognostic value.

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

BackgroundThe 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.MethodsWe 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.ResultsThe 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.ConclusionsTGF-β/Smad signalling pathway and VEGF-A participate in the pathogenesis of sarcoidosis. BAL TGF-β1, and Smad 2 in serum seem to be promising biomarkers with negative prognostic value, but further studies are required to confirmed our observations.

Decreased FAM107A Expression in Patients with Non-small Cell Lung Cancer

Lung cancer is the leading cause of cancer-related death in the world. Early detection, based on molecular markers, could decrease mortality from this disease. Tumor development is often associated with inactivation or loss of tumor suppressor genes (TSGs). The aim of the present study was to analyze the expression level of FAM107A gene, a TSG located in 3p21.1, in lung cancer tumors and in tumor adjacent normal lung samples. Promoter methylation status of FAM107A was evaluated as the potential mechanism of its epigenetic silencing. The relationship between gene mRNA expression and tumor staging, metastasis status, and non-small cell lung cancer (NSCLC) histopathological subtypes in 60 patients was analyzed. Total RNA was isolated from tissue samples and gene expression was assessed in qPCR assay. Gene promoter methylation status was evaluated in MSP reactions, using bisulfite converted DNA and two pairs of primers: methylated and unmethylated. We found that the expression of the gene was dramatically decreased in all NSCLC samples and was significantly lower than in tumor adjacent normal lung tissue. Promoter methylation of FAM107A gene was confirmed only in the minority of NSCLCs. The results highlight the importance of FAM107A in lung carcinogenesis, although indicate other than promoter hypermethylation mechanism of the gene decreased expression.

Expression analysis of three miRNAs, miR-26a, miR-29b and miR-519d, in relation to MMP-2 expression level in non-small cell lung cancer patients: a pilot study

Lung cancer is the most common cause of death in men and second only to breast cancer in women. MicroRNAs (miRNAs) are involved in tumorigenesis and function as oncogenes or tumor suppressor genes. Among other genes, miRNAs regulate matrix metalloproteinases (MMPs), the proteolytic enzymes playing a significant role in the degradation of extracellular matrix, enhancing tumor invasion and metastasis. The aim of the study was to evaluate the expression levels of selected miRNAs: miR-26a, miR-29b and miR-519d, and their target gene, matrix metalloproteinase-2 (MMP-2) in patients with non-small cell lung cancer (NSCLC). The results were correlated with tumor staging, NSCLC histopathological subtypes and patients’ demographical features to assess the possible diagnostic/prognostic value of the studied miRNAs and MMP-2. Total RNA was isolated from 38 NSCLC tissue samples, and the expression analysis was performed using TaqMan® probes in qPCR assay. The results indicated underexpression of selected miRNAs and overexpression of MMP-2. The decrease in miRNA-29b expression was statistically significant and differentiated NSCLC histopathological subtypes. Additionally, statistically significant negative correlation was found between MMP-2 expression and its regulatory miR-26a. There are very few studies reporting miRNA-MMPs analysis on mRNA level in lung cancer, and no similar reports are available from Polish population. The results of our pilot study indicated the diagnostic potential of miR-29b and MMP-2, an inverse association between miR-26a and MMP-2, and proved the role of MMP-2 and the studied miRNAs in lung carcinogenesis. Further studies are needed to verify their potential usefulness for the treatment of lung cancer.

Analysis of changes in expression of IL-4/IL-13/STAT6 pathway and correlation with the selected clinical parameters in patients with atopic asthma

Introduction: Asthma is associated with activation of interleukin-4 (IL-4)/interleukin-13 (IL-13)/signal transducer and activator of transcription factor-6(STAT6) inflammatory response via overexpression of all pathway components: IL-4, IL-13, and STAT6. Objectives: To evaluate the association of IL-4, IL-13, and STAT6 expression and immunoexpression with atopic asthma development. Patients and methods: Fifty patients with atopic asthma and 20 healthy controls were enrolled into the study. Relative gene expression was analyzed by qPCR method. Immunoexpression was assessed by ELISA method. Results: The expression levels of IL-4, IL-13, and STAT6 were higher in patients compared to the controls, but a statistically significant difference was observed only for IL-13 (P = 0.03). In immunoexpression analysis, a statistically significant difference between patients and controls was found for IgE (P = 0.03). Significant positive correlations in the patient group were found between IL-13 gene expression and total level of serum IgE (rho = 0.230, P = 0.033), STAT6 gene/STAT6 protein and total level of serum IgE (STAT6: rho = 0.077, P = 0.038; STAT6: rho = 0.049, P = 0.042), IL-4, and STAT6 expression (rho = 0.098, P = 0.048). Any significant correlations were found between expression/immunoexpression levels of the studied genes and clinical classification, clinical features, or lung function parameters. Conclusions: Our data support the role of Th2 cytokines (IL-4, IL-13) and STAT6 in Th1/Th2 imbalance and highlight the etiological relationship between IL-4/IL-13/STAT6 signaling and atopy and asthma.

Selected molecular events in the pathogenesis of sarcoidosis - recent advances.

Sarcoidosis is an orphan inflammatory disorder that can virtually affect any organ or system in the body, although the lungs and lymph nodes are most frequently involved. Sarcoidosis is believed to derive from an interaction between environmental and genetic agents. Many studies emphasize a strong association between certain human leukocyte antigen (HLA) alleles and sarcoidosis susceptibility. Several new insights have allowed the further evaluation of other candidate genes with a potential function in the immunopathogenesis of sarcoidosis. This review summarizes recent advances in the identification of novel molecular markers that may play a role in different stages of disease, such as the acute phase of inflammation, granuloma formation and fibrosis. Furthermore, this article elucidates the role of both TGF-b/Smad and (HIF)-1a-VEGF-ING-4 signaling pathways in the development of sarcoidosis. The potential epigenetic regulation of the processes occurring in sarcoidosis by miRNA is also discussed.

Expression of HIF-1A/VEGF/ING-4 Axis in Pulmonary Sarcoidosis

Angiogenesis/angiostasis regulated by hypoxia inducible factor-1A (HIF-1A)/vascular endothelial growth factor (VEGF)/inhibitor of growth protein 4 (ING-4) axis may be crucial for the course and outcome of sarcoidosis. Overexpression of angiogenic factors (activation of VEGF through HIF-1A) may predispose to chronic course and lung fibrosis, whereas immunoangiostasis (related to an overexpression of inhibitory ING-4) may be involved in granuloma formation in early sarcoid inflammation, or sustained or recurrent formation of granulomas. In this work we investigated gene expression of HIF-1A, VEGF and ING-4 in bronchoalveolar fluid (BALF) cells and in peripheral blood (PB) lymphocytes of sarcoidosis patients (n=94), to better understand mechanisms of the disease and to search for its biomarkers. The relative gene expression level (RQ value) was analyzed by qPCR. The results were evaluated according to the presence of lung parenchymal involvement (radiological stage I vs. II-IV), acute vs. insidious onset, lung function tests, calcium metabolism parameters, percentage of lymphocytes (BALL%) and BAL CD4+/CD8+ in BALF, age, and gender. In BALF cells, the ING-4 and VEGF RQ values were increased, while HIF-1A expression was decreased. In PB lymphocytes all studied genes were overexpressed. Higher expression of HIF-1A in PB lymphocytes of patients with abnormal spirometry, and in BALF cells of patients with lung volume restriction was found. VEGF gene expression in BALF cells was also higher in patients with abnormal spirometry. These findings were in line with previous data on the role of HIF-1A/VEGF/ING-4 axis in the pathogenesis of sarcoidosis. Up-regulated HIF-1A and VEGF genes are linked to acknowledged negative prognostics.

Signaling pathways and their miRNA regulators involved in the etiopathology of idiopathic pulmonary fibrosis (IPF) and hypersensitivity pneumonitis (HP).

Idiopathic pulmonary fibrosis (IPF) and hypersensitivity pneumonitis (HP) belong to heterogenic group of interstitial lung diseases (ILD). For the reason that this group of diseases present with complex clinical non-specific features, they represent a diagnostic and therapeutic challenge. In this review we focus on several crucial signaling pathways participating in inflammation, fibrosis and EMT processes, so important in the course of ILD: TNF-α/NFκβ, TGF-β/SMAD, Wnt-β-catenin and PI3K-Akt signaling. Moreover, this review summarizes the role of selected signaling pathways and some miRNAs which are their regulators during development and progression of IPF and HP. Recent advances indicate the potential role of miRNAs as a molecular markers differentiating clinical course of ILD.

The association between airway eosinophilic inflammation and IL-33 in stable non-atopic COPD

BackgroundInterleukin(IL)-33 is an epithelial alarmin important for eosinophil maturation, activation and survival. The aim of this study was to examine the association between IL-33, its receptor expression and airway eosinophilic inflammation in non-atopic COPD.MethodsIL-33 concentrations were measured in exhaled breath condensate (EBC) collected from healthy non-smokers, asthmatics and non-atopic COPD subjects using ELISA. Serum and sputum samples were collected from healthy non-smokers, healthy smokers and non-atopic COPD patients. Based on sputum eosinophil count, COPD subjects were divided into subgroups with airway eosinophilic inflammation (sputum eosinophils > 3%) or without (sputum eosinophils ≤3%). IL-33 and soluble form of IL-33 receptor (sST2) protein concentrations were measured in serum and sputum supernatants using ELISA. ST2 mRNA expression was measured in peripheral mononuclear cells and sputum cells by qPCR. Hemopoietic progenitor cells (HPC) expressing ST2 and intracellular IL-5 were enumerated in blood and induced sputum by means of flow cytometry.ResultsIL-33 levels in EBC were increased in COPD patients to a similar extent as in asthma and correlated with blood eosinophil count. Furthermore, serum and sputum IL-33 levels were higher in COPD subjects with sputum eosinophilia than in those with a sputum eosinophil count ≤3% (p < 0.001 for both). ST2 mRNA was overexpressed in sputum cells obtained from COPD patients with airway eosinophilic inflammation compared to those without sputum eosinophilia (p < 0.01). Similarly, ST2 + IL-5+ HPC numbers were increased in the sputum of COPD patients with airway eosinophilia (p < 0.001).ConclusionsOur results indicate that IL-33 is involved in the development of eosinophilic airway inflammation in non-atopic COPD patients.