- Email: [email protected]
The study of fibulin-1 as a novel biomarker in bronchial asthma and its association with disease severity
Egyptian Journal of Chest Diseases and Tuberculosis xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Egyptian Journal of Chest Diseases and Tuberculosis journal homepage: www.sciencedirect.com
The study of fibulin-1 as a novel biomarker in bronchial asthma and its association with disease severity Dalal El Giziry b, Nermine HossamEldin Zakaria b, Abeer Hassan Kassem a,⇑, Mona Mustafa Abdellatif a a b
Chest Department, Faculty of Medicine, Alex University, Egypt Clinical Pathology Department, Faculty of Medicine, Alex University, Egypt
a r t i c l e
i n f o
Article history: Received 27 October 2016 Accepted 7 December 2016 Available online xxxx Keywords: Fibulin-1 Mediator Bronchial asthma
a b s t r a c t A key feature of asthmatic airways is remodeling which involves thickening of the airway wall, altered deposition of extracellular matrix (ECM) proteins and increased airway smooth muscle (ASM) mass. Fibulin-1 (FBLN-1) assists in stabilizing the ECM which maintains airway function and structure by providing mechanical support in addition to constituting a dynamic and complex network that influences cellular function. The present study aimed at investigation of possible association between the fibulin1 levels in asthmatic patients and its relation to asthma severity. Subjects: The study was carried out on forty five asthmatic patients and thirty control normal subjects age and sex matched. Methods: All subjects included in the present study were subjected to: full history taking, complete clinical examination, laboratory investigation and chest X-ray. Pulmonary function test and reversibility test. Serum samples from all studied patients and controls were taken for estimation of level of fibulin-1 Enzyme-linked immune-sorbent assay (ELISA). All asthmatic patients will be examined with fibroptic bronchoscope and bronchoalveolar lavage were taken for estimation of level of fibulin-1. Results: The mean level of serum fibulin-1 in asthmatic patients was 244.10 ± 98.28 pg/ml in mild group, 217.97 ± 121.16 pg/ml in moderate group, 172.20 ± 53.85 pg/ml in severe group compared to a mean level of 187.23 ± 67.97 pg/ml in control group. It was found that fibulin-1 increased in serum of asthmatic patients than in controls with statistical significant difference (p < 0.05) but there was no significant relation to asthma severity. Estimation of BAL fibulin-1 in the three asthmatic groups, the mean level of BAL fibulin-1 was 507.0 ± 152.27 pg/ml in mild group, 692.81 ± 207.14 pg/ml in moderate group, 702.0– 127.67 pg/ml in severe group. It was found that fibulin-1 was increased in BAL of severe degree bronchial asthma than in mild and moderate degrees with statistical significant difference (p < 0.05). Conclusion: This may highlights the potential role of fibulin-1 in airway wall remodeling. Ó 2016 The Egyptian Society of Chest Diseases and Tuberculosis. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-ncnd/4.0/).
Introduction Asthma is a chronic inflammatory disorder of the airways characterized by variable and reversible airflow obstruction and airway hyper-responsiveness (AHR). A key feature of asthmatic airways is
Abbreviations: FBLN-1, fibulin-1; ECM, extracellular matrix; AHR, airway hyperresponsiveness; ASM, airway smooth muscle; FN, fibronectin; TGF-b, transforming growth factor b; BAL, bronchoalveolar lavage; ELISA, Enzyme-linked immunesorbent assay. Peer review under responsibility of The Egyptian Society of Chest Diseases and Tuberculosis. ⇑ Corresponding author. E-mail address: [email protected] (A.H. Kassem).
remodeling which involves thickening of the airway wall, altered deposition of extracellular matrix (ECM) proteins [1,2] and increased airway smooth muscle (ASM) mass. These structural changes may result from an aberrant repair process in the lung, which includes increased proliferation of the ASM cells [3,4]. Whilst current treatments control the symptoms of asthma, they are unable to fully prevent or reverse airway remodeling. The ECM maintains airway function and structure by providing mechanical support in addition to constituting a dynamic and complex network that influences cellular function [5]. The ECM deposited by asthma derived ASM cells is altered such that increased amounts of collagen I and laminin [6–8], as well as
http://dx.doi.org/10.1016/j.ejcdt.2016.12.003 0422-7638/Ó 2016 The Egyptian Society of Chest Diseases and Tuberculosis. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: D.E. Giziry et al., The study of fibulin-1 as a novel biomarker in bronchial asthma and its association with disease severity, Egypt. J. Chest Dis. Tuberc. (2016), http://dx.doi.org/10.1016/j.ejcdt.2016.12.003
2
D.E. Giziry et al. / Egyptian Journal of Chest Diseases and Tuberculosis xxx (2016) xxx–xxx
fibronectin (FN) are produced which mediate a range of cellular interactions including migration, growth and differentiation. Levels of the profibrotic cytokine transforming growth factor b (TGF-b) are elevated in the bronchoalveolar lavage (BAL) in asthma [9], and are increased in bronchial tissue [10]. TGF-bstimulated FN deposition is also enhanced in asthma derived bronchial epithelium and fibroblasts [11,12]. Fibulin-1 (FBLN-1), a secreted glycoprotein, assists in stabilizing the ECM. It associates with FN and a variety of other ECM proteins including laminin and fibrinogen [13]. FBLN-1 expression has been reported in human lung tissue using microarray technology, however, FBLN-1 was not verified by PCR or at the protein level, nor were functional studies carried out [14–16]. In other study reported reduction of FBLN-1D expression in asthma derived bronchial biopsies compared with those derived from non-asthmatics [14]. However, the function of FBLN-1 in the lungs and its role in asthma is unknown. The present study aimed at investigation of possible association between the fibulin-1 levels in asthmatic patients and its relation to asthma severity.
FBLN1 in the samples is then determined by comparing the optical density (O.D) of the samples to the standard curve. Calculation: The standard curve was drawn on graph paper with the standard density as the horizontal, the OD value for the vertical. The corresponding density according to the sample OD value had been found out by the Sample curve, multiplied by the dilution multiple. Assay range 20 varied from 20 pg/ml to 800 pg/ml.
Subjects The study was carried out on forty-five asthmatic patients were classified into three groups [17] group I: Fifteen asthmatic patients (mild stage(ﻭgroup II: Fifteen asthmatic patients (moderate stage (and group III: Fifteen asthmatic patients (severe stage) and thirty age and sex- matched normal control subjects with no history of asthma or other lung disease and normal spirometry recruited from the clinical and chemical pathology department and chest diseases department of the Alexandria Main University Hospital. An informed consent was taken from all subjects prior to the onset of the study. Inclusion criteria: asthmatic patients in different degrees of disease (mild, moderate and severe degrees). Exclusion criteria: renal, hepatic, immunological diseases, connective tissue diseases and smoking. Methods All subjects included in the present study were subjected to: full history taking, complete clinical examination, laboratory investigation and chest X-ray with Pulmonary function and reversibility tests. Serum samples from all studied patients and controls were taken for estimation of level of fibulin-1 by Enzyme-linked immune-sorbent assay (ELISA). All asthmatic patients were examined with fibroptic bronchoscope and bronchoalveolar lavage was taken for estimation of level of fibulin-1. Bronchoalvoelar lavage (BAL) [18] was obtained (from patients only)by flexible fibreoptic bronchoscopy. Specimens were collected via normal saline lavage of the segmental airways and alveolar spaces in sterile containers, centrifuged for 20 min at speed of 2000–3000 r.p.m. to remove mucus and cells. Supernatant was removed, aliquot and stored at 20 °C till later use. Samples were centrifuged again after thawing before they were processed. Enzyme-linked immune-sorbent assay (ELISA) [19–22]. The assay measure Human FBLN1 level in the sample using Sandwich High Sensitivity ELISA kit for Quantitative Detection of Human Fibulin-3/EFEMP1. The sensitivity is up to <10 pg/ml It use Purified Human FBLN1 antibody to coat microtiter plate wells, Combined FBLN1 antibody which with enzyme labeled, become antibody - antigen - enzyme-antibody complex. After washing completely, add substrate. Substrate becomes blue color at HRP enzyme-catalyzed. Reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm. The concentration of
Results Table 1 shows comparison between the four studied groups according to demographic data. The control group included 3 males (10%) and 27females (90%) with a mean age of 40.66 ± 9.33 years (ranging from 1962 years). The asthmatic patients include 2 male (13.3%) and 13 females (86.78%) with a mean age of 46.20 ± 10.42 (ranging from 26–62 years) in mild stage, 1males (6.7%) and 14 females (93.3%) with a mean age of 45.53 ± 14.6 (ranging from 26–72 years) in moderate stage, 2 males (13.3%) and 13 females (86.7%) with a mean age of 42.33 ± 10.24 (ranging from 22–56 years) in severe stage. Table 2 shows estimation of serum levels fibulin-1 in both asthmatic patients and control group. The mean level of Serum fibulin1 in asthmatic patients was 244.10 ± 98.28 pg/ml (ranging from 170.50–577.0 pg/ml) in mild group, 217.97 ± 121.16 pg/ml (ranging from 45.50–454.50 pg/ml) in moderate group, 172.20 ± 53.85 pg/ml (ranging from 101.50–265 pg/ml) in severe group compared to a mean level of 187.23 ± 67.97 pg/ml (ranging from 49.50–426.50 pg/ml) in control group. It was found that fibulin-1 increased in serum of asthmatic patients but without relation to asthma severity. Table 3 shows estimation of BAL fibulin-1 in the three asthmatic groups. The mean level of BAL fibulin-1 was 507.0 ± 152.27 pg/ml (ranging from 290.50–750.0 pg/ml) in mild group, 692.81 ± 207.14 pg/ml (ranging from 260.50–960.0 pg/ml) in moderate group, 702.0–127.67 pg/ml (ranging from 550.0–1050.0 pg/ ml) in severe group. It was found that fibulin-1 increased in BAL of severe degree bronchial asthma than in mild and moderate degrees with statistical significant difference (p < 0.05).
Discussion Asthma is a chronic inflammatory disorder of the airways characterized by variable and reversible airflow obstruction and airway hyper-responsiveness (AHR). It is thought to be caused by a combination of genetic and environmental factors [23]. Its diagnosis is usually made based on the pattern of symptoms and/or response to therapy over time [24].
Please cite this article in press as: D.E. Giziry et al., The study of fibulin-1 as a novel biomarker in bronchial asthma and its association with disease severity, Egypt. J. Chest Dis. Tuberc. (2016), http://dx.doi.org/10.1016/j.ejcdt.2016.12.003
3
D.E. Giziry et al. / Egyptian Journal of Chest Diseases and Tuberculosis xxx (2016) xxx–xxx Table 1 Comparison between the four studied groups according to demographic data. Mild (n = 15) No. Age (years) <20 20–40 40–60 >60 Min.–Max. Mean ± SD. Median p1 p2 p3 Sex Male Female
Moderate (n = 15) %
No.
%
Severe (n = 15)
Control (n = 30)
No.
No.
%
%
0 0.0 5 33.3 9 60.0 1 6.7 26.0–62.0 46.20 ± 10.42 47.0 <0.001* 0.235 0.766
0 0.0 6 40.0 7 46.7 2 13.3 26.0–72.0 45.53 ± 14.60 50.0 <0.001* 0.372
0 00 8 53.3 7 46.7 0 00 22.0–56.0 42.33 ± 10.24 40.0 <0.001*
1 16 10 3 19.0–62.0 40.66 ± 9.33 39.0
3.3 53.3 33.3 10.0
2 13
1 14
2 13
3 27
10.0 90.0
13.3 86.78
6.7 93.3
13.3 86.7
Test of sig.
p
v2 = 3.62*
MC
F = 1.95
0.166
v2 = 0.982
MC
p 0.109
p = 0.366
P: p value for comparing between the four studied groups. p1: p value for Post Hoc test (LSD) for comparing between control with each other group. p2: p value for Post Hoc test (LSD) for comparing between sever with each other group. p3: p value for Post Hoc test (LSD) for comparing between mild and moderate. F: F test (ANOVA). v2: Chi square test. MC: Monte Carlo test. * Statistically significant at p 6 0.05.
Table 2 Comparison between the four studied groups according to serum fibulin-1 in pg/ml.
Serum fibulin-1 in pg/ml Min.–Max. Mean ± SD. Median p1 p2 p3
Mild (n = 15)
Moderate (n = 15)
Severe (n = 15)
Control (n = 30)
KWv2
P
170.50–577.0 244.10 ± 98.28 237.0 0.002* 0.010* 0.383
45.50–454.50 217.97 ± 121.16 202.50 0.492 0.418
101.50–265.0 172.20 ± 53.84 151.50 0.433
49.50–426.50 187.23 ± 67.97 178.0
9.498*
0.023*
p: p value for comparing between the four studied groups. p1: p value for Mann Whitney test for comparing between control with each other group. p2: p value for Mann Whitney test for comparing between sever with each other group. p3: p value for Mann Whitney test for comparing between mild and moderate. KWv2: Chi square for Kruskal Wallis test. * Statistically significant at p 6 0.05.
Table 3 Comparison between the three asthmatic groups according to BAL fibulin-1 in pg/ml.
BAL fibulin-1 in pg/ml. Min.–Max. Mean ± SD. Median p1 p2
Mild (n = 15)
Moderate (n = 15)
Severe (n = 15)
F
P
290.50–750.0 507.0 ± 152.27 500.0
260.50–960.0 692.81 ± 207.14 750.0 0.004* 0.880
550.0–1050.0 702.0–127.67 665.0 0.002*
6.612*
0.003*
F: F test (ANOVA). p1: p value for Post Hoc test (LSD) for comparing between mild with each other group. p2: p value for Post Hoc test (LSD) for comparing between moderate and severe. * Statistically significant at p 6 0.05.
A key feature of asthmatic airways is remodeling which involves thickening of the airway wall, altered deposition of extracellular matrix (ECM) proteins [2,3] and increased airway smooth muscle (ASM) mass. These structural changes may result from an aberrant repair process in the lung, which includes increased proliferation of the ASM cells [4,5]. The ECM maintains airway function and structure by providing mechanical support in addition to constituting a dynamic and complex network that influences cellular function [6]. The ECM deposited by asthma derived ASM cells is altered such that increased amounts of collagen I and laminin [7–9], as well as fibronectin (FN) are produced which mediate a range of cellular interactions including migration, growth and differentiation. Fibulin-1 (FBLN-1), a secreted glycoprotein, assists in
stabilizing the ECM. It associates with FN and a variety of other ECM proteins including laminin and fibrinogen [25]. Mice deficient in FN and FBLN-1 die perinatally due to abnormal lung development [14]. FBLN-1 expression has been reported in human lung tissue using microarray technology [15,16]. The present study aimed at investigation of possible association between the fibulin-1 levels in asthmatic patients and its relation to asthma severity. The study was carried out on forty-five asthmatic patients with different degrees of asthma severity and thirty age and sexmatched normal control subjects. According to our results, we found that fibulin-1 increased in serum of asthmatic patients without relation to asthma severity
Please cite this article in press as: D.E. Giziry et al., The study of fibulin-1 as a novel biomarker in bronchial asthma and its association with disease severity, Egypt. J. Chest Dis. Tuberc. (2016), http://dx.doi.org/10.1016/j.ejcdt.2016.12.003
4
D.E. Giziry et al. / Egyptian Journal of Chest Diseases and Tuberculosis xxx (2016) xxx–xxx
and highly increased in BAL of sever degree asthmatic patients than in mild and moderate degrees. So, fibulin-1 in BAL can be used as a marker in diagnosis of severe degrees of bronchial asthma. Transforming growth factor beta 1 (TGF-b1) is a pro-fibrotic cytokine which is increased in several forms of acute and chronic adult lung diseases such as asthma [15], COPD [26,27], and pulmonary fibrosis [28,29]. It is considered to play a crucial role in the pathogenesis of tissue fibrosis, stimulating the production of various collagens and ECM proteins [30,31,6]. Levels of the pro-fibrotic cytokine TGF-b1 are elevated in the BAL fluid in asthma [22], and are increased in bronchial tissue [15]. TGF-b1 increased the expression of FBLN-1 in ASM cells. The increased FBLN-1 resulted in exaggerated proliferation and wound repair in asthma derived ASM cells. TGF-b1 not only increased FBLN-1 in asthma derived ASM cells, but also enhanced its deposition in the asthmatic ECM. The architecture of the asthmatic airway often undergoes prominent and permanent structural changes, including alterations of the molecular composition of the ECM. In particular, ECM protein deposition is increased in the lamina reticularis [32] resulting in basement membrane thickening [33]. Given that TGF-b1 levels may alter the ECM assembly process, it is likely that the raised TGF-b1 levels in the asthmatic airway may contribute to an increase in the ECM thereby augmenting the effects of airway remodeling. AHR is a hallmark feature of asthma that has been linked to the pathological events associated with remodeling of the airway wall. TGFb-1 has also been linked to the mechanism underpinning AHR in sthma. In particular, TGF-b1 administration to the airways in mice induces peribronchial fibrosis that results in the development of AHR [34]. When antisense oligomer targeting FBLN-1 (AOs) was used in mice treatment, TGF-b1 induced AHR was inhibited demonstrating a critical role for FBLN-1 in the mechanism of TGF-b1 induced AHR. The regulated expression of FBLN-1 downstream of inflammation and its regulation of the remodeling/AHR axis identify the therapeutic potential of targeting this glycoprotein. While FBLN1 is normally found in the blood, there is not much known about how the body regulates FBLN1 levels or where the molecule comes from. TGF-b1 causes increased release of soluble FBLN1 from airway epithelial cells, which may contribute to the increased levels found in the serum and BAL of people with asthma. Lau JY, et al. [35] found that FBLN-1 is increased in the serum and BAL of people with asthma, and shown that FBLN-1 regulates airway smooth muscle (ASM) cell proliferation, therefore highlighting the potential role of FBLN-1 in airway wall remodeling. They also found that there was no correlation between the FBLN-1 level detected in the serum or BAL from the asthmatic volunteers and their FEV1/FVC ratio. In contrast, Chen L, et al. [36] found that TGF-b1 decreased FBLN-1 mRNA, and that FBLN-1 protein production was controlled. Therefore it was hypothesized that the increased ECM FBLN-1 following TGF-b1 stimulation was due to sequestration of soluble FBLN-1 and not de novo synthesis. Conclusion It was found that fibulin-1 was increased in BAL of severe degree bronchial asthma than in mild and moderate degrees with statistical significant difference (p < 0.05). This may highlights the potential role of fibulin-1 in airway wall remodeling. References [1] M. Ebina, T. Takahashi, T. Chiba, M. Motomiya, Cellular hypertrophy and hyperplasia of airway smooth muscles underlying bronchial asthma. A 3-D morphometric study, Am. Rev. Respir. Dis. 148 (1993) 720–726.
[2] P.G. Woodruff, G.M. Dolganov, R.E. Ferrando, S. Donnelly, S.R. Hays, Hyperplasia of smooth muscle in mild to moderate asthma without changes in cell size or gene expression, Am. J. Respir. Crit. Care Med. 169 (2004) 1001– 1006. [3] P.R. Johnson, M. Roth, M. Tamm, M. Hughes, Q. Ge, Airway smooth muscle cell proliferation is increased in asthma, Am. J. Respir. Crit. Care Med. 164 (2001) 474–477. [4] T. Trian, G. Benard, H. Begueret, R. Rossignol, P.O. Girodet, D. Ghosh, et al., Bronchial smooth muscle remodeling involves calcium-dependent enhanced mitochondrial biogenesis in asthma, J. Exp. Med. 204 (2007) 3173–3181. [5] S.K. Akiyama, S.S. Yamada, W.T. Chen, K.M. Yamada, Analysis of fibronectin receptor function with monoclonal antibodies: roles in cell adhesion, migration, matrix assembly, and cytoskeletal organization, J. Cell Biol. 109 (1989) 863–875. [6] P.R. Johnson, J.K. Burgess, P.A. Underwood, W. Au, M.H. Poniris, M. Tamm, et al., Extracellular matrix proteins modulate asthmatic airway smooth muscle cell proliferation via an autocrine mechanism, J. Allergy Clin. Immunol. 113 (2004) 690–696. [7] L.A. Laitinen, A. Laitinen, Inhaled corticosteroid treatment and extracellular matrix in the airways in asthma, Int. Arch. Allergy Immunol. 107 (1995) 215– 216. [8] K. Parameswaran, K. Radford, J. Zuo, L.J. Janssen, P.M. O’Byrne, P.G. Cox, Extracellular matrix regulates human airway smooth muscle cell migration, Eur. Respir. J. 24 (4) (2004) 545–551. [9] A.E. Redington, J. Madden, A.J. Frew, R. Djukanovic, W.R. Roche, S.T. Holgate, et al., Transforming growth factor-beta 1 in asthma. Measurement in bronchoalveolar lavage fluid, Am. J. Respir. Crit. Care Med. 156 (2 Pt 1) (1997) 642–647. [10] A.M. Vignola, P. Chanez, G. Chiappara, A. Merendino, E. Pace, Transforming growth factor-beta expression in mucosal biopsies in asthma and chronic bronchitis, Am. J. Respir. Crit. Care Med. 156 (1997) 591–599. [11] D.J. Romberger, J.D. Beckmann, L. Claassen, R.F. Ertl, S.I. Rennard, Modulation of fibronectin production of bovine bronchial epithelial cells by transforming growth factor-beta, Am. J. Respir. Cell Mol. Biol. 7 (1992) 149–155. [12] G. Westergren-Thorsson, J. Chakir, M.J. Lafreniere-Allard, L.P. Boulet, G.M. Tremblay, Correlation between airway responsiveness and proteoglycan production by bronchial fibroblasts from normal and asthmatic subjects, Int. J. Biochem. Cell Biol. 34 (2002) 1256–1267. [13] H. Tran, W.J. VanDusen, W.S. Argraves, The self-association and fibronectinbinding sites of fibulin-1 map to calcium-binding epidermal growth factor-like domains, J. Biochem. 272 (1997) 22600–22606. [14] C. Laprise, R. Sladek, A. Ponton, M.C. Bernier, T.J. Hudson, M. Laviolette, Functional classes of bronchial mucosa genes that are differentially expressed in asthma, BMC Genomics 5 (2004) 21. [15] F. Syed, R.A. Panettieri Jr., O. Tliba, C. Huang, K. Li, M. Bracht, et al., The effect of IL-13 and IL-13R130Q, a naturally occurring IL-13 polymorphism, on the gene expression of human airway smooth muscle cells, Respir. Res. 6 (2005) 9. [16] N. Zimmermann, N.E. King, J. Laporte, M. Yang, A. Mishra, Dissection of experimental asthma with DNA microarray analysis identifies arginase in asthma pathogenesis, J. Clin. Invest 111 (2003) 1863–1874. [17] Global Initiative for Asthma. Global strategy for asthma management and prevention. Issued January 1995 (NIH Publication No. 02–3659); updated 2015. [18] S.E. Wenzel, S.J. Szefler, D.Y.M. Leung, S.I. Sloan, M.D. Rex, Bronchoscopic evaluation of severe asthma, Am. J. Respir. Crit. Care Med. 156 (1997) 737–743, 214. [19] T. Musso, I. Espinosa-Delgado, K. Pulkki, G.L. Gusella, D.L. Longo, L. Vareso, Transforming growth factor-ß1 downregulates interleukin-1 (IL-1)-induced IL-6 production by human monocytes, Blood 76 (1990) 2466–2469. [20] E. Engvall, P. Perlmann, Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G, Immunochemistry 8 (9) (1971) 871–874. [21] E. Engvall, P. Perlmann, Enzyme-linked immunosorbent assay, Elisa. 3. Quantitation of specific antibodies by enzyme-labeled anti-immunoglobulin in antigen-coated tubes, J. Immunol. 109 (1) (1972) 129–135. [22] B.K. Van Weemen, A.H. Schuurs, Immunoassay using antigen enzyme conjugates, FEBS Lett. 15 (1971) 232. [23] F.D. Martinez, Genes, environments, development and asthma: a reappraisal, Eur. Respir. J. 29 (1) (2007) 179–184. [24] R.F. Lemanske, W.W. Busse, Asthma: clinical expression and molecular mechanisms, J. Allergy Clin. Immunol. 125 (2 Suppl 2) (2010) S95–S102. [25] G. Kostka, R. Giltay, W. Bloch, K. Addicks, R. Timpl, R. Fassler, et al., Perinatal lethality and endothelial cell abnormalities in several vessel compartments of fibulin-1-deficient mice, Mol. Cell. Biol. 21 (20) (2001) 7025–7034. [26] W.I. de Boer, A. van Schadewijk, J.K. Sont, H.S. Sharma, J. Stolk, H.S. Sharma, et al., Transforming growth factor beta1 and recruitment of macrophages and mast cells in airways in chronic obstructive pulmonary disease, Am. J. Respir. Crit. Care Med. 158 (1998) 1951–1957. [27] J.C. Mak, M.M. Chan-Yeung, S.P. Ho, K.S. Chan, K. Choo, Elevated plasma TGFbeta1 levels in patients with chronic obstructive pulmonary disease, Respir. Med. 103 (2009) 1083–1089. [28] N. Khalil, R.N. O’Connor, H.W. Unruh, P.W. Warren, K.C. Flanders, A. Kemp, et al., Increased production and immunohistochemical localization of transforming growth factor-beta in idiopathic pulmonary fibrosis, Am. J. Respir. Cell Mol. Biol. 5 (1991) 155–162.
Please cite this article in press as: D.E. Giziry et al., The study of fibulin-1 as a novel biomarker in bronchial asthma and its association with disease severity, Egypt. J. Chest Dis. Tuberc. (2016), http://dx.doi.org/10.1016/j.ejcdt.2016.12.003
D.E. Giziry et al. / Egyptian Journal of Chest Diseases and Tuberculosis xxx (2016) xxx–xxx [29] K. Zhang, K.C. Flanders, S.H. Phan, Cellular localization of transforming growth factor-beta expression in bleomycin-induced pulmonary fibrosis, Am. J. Pathol. 147 (1995) 352–361. [30] L.M. Moir, J.K. Burgess, J.L. Black, Transforming growth factor beta 1 increases fibronectin deposition through integrin receptor alpha 5 beta 1 on human airway smooth muscle, J. Allergy Clin. Immunol. 121 (2008) 1034–1039. [31] J. Rosenbloom, S.V. Castro, S.A. Jimenez, Narrative review: fibrotic diseases: cellular and molecular mechanisms and novel therapies, Ann. Intern. Med. 152 (2010) 159–166. [32] S.J. Hirst, C.H. Twort, T.H. Lee, Differential effects of extracellular matrix proteins on human airway smooth muscle cell proliferation and phenotype, Am. J. Respir. Cell Mol. Biol. 23 (2000) 335–344.
5
[33] P.K. Jeffery, Pathology of asthma, Br. Med. Bull. 48 (1992) 23–39. [34] N.J. Kenyon, R.W. Ward, G. McGrew, J.A. Last, TGF-ß1 causes airway fibrosis and increased collagen I and III mRNA in mice, Thorax 58 (2003) 772–777. [35] J.Y. Lau, B.G. Oliver, M. Baraket, E.L. Beckett, N.G. Hansbro, Fibulin-1 is increased in asthma – a novel mediator of airway remodeling?, PLoS One 5 (10) (2010) e13360 [36] L. Chen, Q. Ge, J.L. Black, L. Deng, J.K. Burgess, B.G. Oliver, Differential regulation of extracellular matrix and soluble fibulin-1 levels by TGF-ß1 in airway smooth muscle cells, PLoS One 8 (6) (2013) e65544.
Please cite this article in press as: D.E. Giziry et al., The study of fibulin-1 as a novel biomarker in bronchial asthma and its association with disease severity, Egypt. J. Chest Dis. Tuberc. (2016), http://dx.doi.org/10.1016/j.ejcdt.2016.12.003
Contents lists available at ScienceDirect
Egyptian Journal of Chest Diseases and Tuberculosis journal homepage: www.sciencedirect.com
The study of fibulin-1 as a novel biomarker in bronchial asthma and its association with disease severity Dalal El Giziry b, Nermine HossamEldin Zakaria b, Abeer Hassan Kassem a,⇑, Mona Mustafa Abdellatif a a b
Chest Department, Faculty of Medicine, Alex University, Egypt Clinical Pathology Department, Faculty of Medicine, Alex University, Egypt
a r t i c l e
i n f o
Article history: Received 27 October 2016 Accepted 7 December 2016 Available online xxxx Keywords: Fibulin-1 Mediator Bronchial asthma
a b s t r a c t A key feature of asthmatic airways is remodeling which involves thickening of the airway wall, altered deposition of extracellular matrix (ECM) proteins and increased airway smooth muscle (ASM) mass. Fibulin-1 (FBLN-1) assists in stabilizing the ECM which maintains airway function and structure by providing mechanical support in addition to constituting a dynamic and complex network that influences cellular function. The present study aimed at investigation of possible association between the fibulin1 levels in asthmatic patients and its relation to asthma severity. Subjects: The study was carried out on forty five asthmatic patients and thirty control normal subjects age and sex matched. Methods: All subjects included in the present study were subjected to: full history taking, complete clinical examination, laboratory investigation and chest X-ray. Pulmonary function test and reversibility test. Serum samples from all studied patients and controls were taken for estimation of level of fibulin-1 Enzyme-linked immune-sorbent assay (ELISA). All asthmatic patients will be examined with fibroptic bronchoscope and bronchoalveolar lavage were taken for estimation of level of fibulin-1. Results: The mean level of serum fibulin-1 in asthmatic patients was 244.10 ± 98.28 pg/ml in mild group, 217.97 ± 121.16 pg/ml in moderate group, 172.20 ± 53.85 pg/ml in severe group compared to a mean level of 187.23 ± 67.97 pg/ml in control group. It was found that fibulin-1 increased in serum of asthmatic patients than in controls with statistical significant difference (p < 0.05) but there was no significant relation to asthma severity. Estimation of BAL fibulin-1 in the three asthmatic groups, the mean level of BAL fibulin-1 was 507.0 ± 152.27 pg/ml in mild group, 692.81 ± 207.14 pg/ml in moderate group, 702.0– 127.67 pg/ml in severe group. It was found that fibulin-1 was increased in BAL of severe degree bronchial asthma than in mild and moderate degrees with statistical significant difference (p < 0.05). Conclusion: This may highlights the potential role of fibulin-1 in airway wall remodeling. Ó 2016 The Egyptian Society of Chest Diseases and Tuberculosis. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-ncnd/4.0/).
Introduction Asthma is a chronic inflammatory disorder of the airways characterized by variable and reversible airflow obstruction and airway hyper-responsiveness (AHR). A key feature of asthmatic airways is
Abbreviations: FBLN-1, fibulin-1; ECM, extracellular matrix; AHR, airway hyperresponsiveness; ASM, airway smooth muscle; FN, fibronectin; TGF-b, transforming growth factor b; BAL, bronchoalveolar lavage; ELISA, Enzyme-linked immunesorbent assay. Peer review under responsibility of The Egyptian Society of Chest Diseases and Tuberculosis. ⇑ Corresponding author. E-mail address: [email protected] (A.H. Kassem).
remodeling which involves thickening of the airway wall, altered deposition of extracellular matrix (ECM) proteins [1,2] and increased airway smooth muscle (ASM) mass. These structural changes may result from an aberrant repair process in the lung, which includes increased proliferation of the ASM cells [3,4]. Whilst current treatments control the symptoms of asthma, they are unable to fully prevent or reverse airway remodeling. The ECM maintains airway function and structure by providing mechanical support in addition to constituting a dynamic and complex network that influences cellular function [5]. The ECM deposited by asthma derived ASM cells is altered such that increased amounts of collagen I and laminin [6–8], as well as
http://dx.doi.org/10.1016/j.ejcdt.2016.12.003 0422-7638/Ó 2016 The Egyptian Society of Chest Diseases and Tuberculosis. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article in press as: D.E. Giziry et al., The study of fibulin-1 as a novel biomarker in bronchial asthma and its association with disease severity, Egypt. J. Chest Dis. Tuberc. (2016), http://dx.doi.org/10.1016/j.ejcdt.2016.12.003
2
D.E. Giziry et al. / Egyptian Journal of Chest Diseases and Tuberculosis xxx (2016) xxx–xxx
fibronectin (FN) are produced which mediate a range of cellular interactions including migration, growth and differentiation. Levels of the profibrotic cytokine transforming growth factor b (TGF-b) are elevated in the bronchoalveolar lavage (BAL) in asthma [9], and are increased in bronchial tissue [10]. TGF-bstimulated FN deposition is also enhanced in asthma derived bronchial epithelium and fibroblasts [11,12]. Fibulin-1 (FBLN-1), a secreted glycoprotein, assists in stabilizing the ECM. It associates with FN and a variety of other ECM proteins including laminin and fibrinogen [13]. FBLN-1 expression has been reported in human lung tissue using microarray technology, however, FBLN-1 was not verified by PCR or at the protein level, nor were functional studies carried out [14–16]. In other study reported reduction of FBLN-1D expression in asthma derived bronchial biopsies compared with those derived from non-asthmatics [14]. However, the function of FBLN-1 in the lungs and its role in asthma is unknown. The present study aimed at investigation of possible association between the fibulin-1 levels in asthmatic patients and its relation to asthma severity.
FBLN1 in the samples is then determined by comparing the optical density (O.D) of the samples to the standard curve. Calculation: The standard curve was drawn on graph paper with the standard density as the horizontal, the OD value for the vertical. The corresponding density according to the sample OD value had been found out by the Sample curve, multiplied by the dilution multiple. Assay range 20 varied from 20 pg/ml to 800 pg/ml.
Subjects The study was carried out on forty-five asthmatic patients were classified into three groups [17] group I: Fifteen asthmatic patients (mild stage(ﻭgroup II: Fifteen asthmatic patients (moderate stage (and group III: Fifteen asthmatic patients (severe stage) and thirty age and sex- matched normal control subjects with no history of asthma or other lung disease and normal spirometry recruited from the clinical and chemical pathology department and chest diseases department of the Alexandria Main University Hospital. An informed consent was taken from all subjects prior to the onset of the study. Inclusion criteria: asthmatic patients in different degrees of disease (mild, moderate and severe degrees). Exclusion criteria: renal, hepatic, immunological diseases, connective tissue diseases and smoking. Methods All subjects included in the present study were subjected to: full history taking, complete clinical examination, laboratory investigation and chest X-ray with Pulmonary function and reversibility tests. Serum samples from all studied patients and controls were taken for estimation of level of fibulin-1 by Enzyme-linked immune-sorbent assay (ELISA). All asthmatic patients were examined with fibroptic bronchoscope and bronchoalveolar lavage was taken for estimation of level of fibulin-1. Bronchoalvoelar lavage (BAL) [18] was obtained (from patients only)by flexible fibreoptic bronchoscopy. Specimens were collected via normal saline lavage of the segmental airways and alveolar spaces in sterile containers, centrifuged for 20 min at speed of 2000–3000 r.p.m. to remove mucus and cells. Supernatant was removed, aliquot and stored at 20 °C till later use. Samples were centrifuged again after thawing before they were processed. Enzyme-linked immune-sorbent assay (ELISA) [19–22]. The assay measure Human FBLN1 level in the sample using Sandwich High Sensitivity ELISA kit for Quantitative Detection of Human Fibulin-3/EFEMP1. The sensitivity is up to <10 pg/ml It use Purified Human FBLN1 antibody to coat microtiter plate wells, Combined FBLN1 antibody which with enzyme labeled, become antibody - antigen - enzyme-antibody complex. After washing completely, add substrate. Substrate becomes blue color at HRP enzyme-catalyzed. Reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm. The concentration of
Results Table 1 shows comparison between the four studied groups according to demographic data. The control group included 3 males (10%) and 27females (90%) with a mean age of 40.66 ± 9.33 years (ranging from 1962 years). The asthmatic patients include 2 male (13.3%) and 13 females (86.78%) with a mean age of 46.20 ± 10.42 (ranging from 26–62 years) in mild stage, 1males (6.7%) and 14 females (93.3%) with a mean age of 45.53 ± 14.6 (ranging from 26–72 years) in moderate stage, 2 males (13.3%) and 13 females (86.7%) with a mean age of 42.33 ± 10.24 (ranging from 22–56 years) in severe stage. Table 2 shows estimation of serum levels fibulin-1 in both asthmatic patients and control group. The mean level of Serum fibulin1 in asthmatic patients was 244.10 ± 98.28 pg/ml (ranging from 170.50–577.0 pg/ml) in mild group, 217.97 ± 121.16 pg/ml (ranging from 45.50–454.50 pg/ml) in moderate group, 172.20 ± 53.85 pg/ml (ranging from 101.50–265 pg/ml) in severe group compared to a mean level of 187.23 ± 67.97 pg/ml (ranging from 49.50–426.50 pg/ml) in control group. It was found that fibulin-1 increased in serum of asthmatic patients but without relation to asthma severity. Table 3 shows estimation of BAL fibulin-1 in the three asthmatic groups. The mean level of BAL fibulin-1 was 507.0 ± 152.27 pg/ml (ranging from 290.50–750.0 pg/ml) in mild group, 692.81 ± 207.14 pg/ml (ranging from 260.50–960.0 pg/ml) in moderate group, 702.0–127.67 pg/ml (ranging from 550.0–1050.0 pg/ ml) in severe group. It was found that fibulin-1 increased in BAL of severe degree bronchial asthma than in mild and moderate degrees with statistical significant difference (p < 0.05).
Discussion Asthma is a chronic inflammatory disorder of the airways characterized by variable and reversible airflow obstruction and airway hyper-responsiveness (AHR). It is thought to be caused by a combination of genetic and environmental factors [23]. Its diagnosis is usually made based on the pattern of symptoms and/or response to therapy over time [24].
Please cite this article in press as: D.E. Giziry et al., The study of fibulin-1 as a novel biomarker in bronchial asthma and its association with disease severity, Egypt. J. Chest Dis. Tuberc. (2016), http://dx.doi.org/10.1016/j.ejcdt.2016.12.003
3
D.E. Giziry et al. / Egyptian Journal of Chest Diseases and Tuberculosis xxx (2016) xxx–xxx Table 1 Comparison between the four studied groups according to demographic data. Mild (n = 15) No. Age (years) <20 20–40 40–60 >60 Min.–Max. Mean ± SD. Median p1 p2 p3 Sex Male Female
Moderate (n = 15) %
No.
%
Severe (n = 15)
Control (n = 30)
No.
No.
%
%
0 0.0 5 33.3 9 60.0 1 6.7 26.0–62.0 46.20 ± 10.42 47.0 <0.001* 0.235 0.766
0 0.0 6 40.0 7 46.7 2 13.3 26.0–72.0 45.53 ± 14.60 50.0 <0.001* 0.372
0 00 8 53.3 7 46.7 0 00 22.0–56.0 42.33 ± 10.24 40.0 <0.001*
1 16 10 3 19.0–62.0 40.66 ± 9.33 39.0
3.3 53.3 33.3 10.0
2 13
1 14
2 13
3 27
10.0 90.0
13.3 86.78
6.7 93.3
13.3 86.7
Test of sig.
p
v2 = 3.62*
MC
F = 1.95
0.166
v2 = 0.982
MC
p 0.109
p = 0.366
P: p value for comparing between the four studied groups. p1: p value for Post Hoc test (LSD) for comparing between control with each other group. p2: p value for Post Hoc test (LSD) for comparing between sever with each other group. p3: p value for Post Hoc test (LSD) for comparing between mild and moderate. F: F test (ANOVA). v2: Chi square test. MC: Monte Carlo test. * Statistically significant at p 6 0.05.
Table 2 Comparison between the four studied groups according to serum fibulin-1 in pg/ml.
Serum fibulin-1 in pg/ml Min.–Max. Mean ± SD. Median p1 p2 p3
Mild (n = 15)
Moderate (n = 15)
Severe (n = 15)
Control (n = 30)
KWv2
P
170.50–577.0 244.10 ± 98.28 237.0 0.002* 0.010* 0.383
45.50–454.50 217.97 ± 121.16 202.50 0.492 0.418
101.50–265.0 172.20 ± 53.84 151.50 0.433
49.50–426.50 187.23 ± 67.97 178.0
9.498*
0.023*
p: p value for comparing between the four studied groups. p1: p value for Mann Whitney test for comparing between control with each other group. p2: p value for Mann Whitney test for comparing between sever with each other group. p3: p value for Mann Whitney test for comparing between mild and moderate. KWv2: Chi square for Kruskal Wallis test. * Statistically significant at p 6 0.05.
Table 3 Comparison between the three asthmatic groups according to BAL fibulin-1 in pg/ml.
BAL fibulin-1 in pg/ml. Min.–Max. Mean ± SD. Median p1 p2
Mild (n = 15)
Moderate (n = 15)
Severe (n = 15)
F
P
290.50–750.0 507.0 ± 152.27 500.0
260.50–960.0 692.81 ± 207.14 750.0 0.004* 0.880
550.0–1050.0 702.0–127.67 665.0 0.002*
6.612*
0.003*
F: F test (ANOVA). p1: p value for Post Hoc test (LSD) for comparing between mild with each other group. p2: p value for Post Hoc test (LSD) for comparing between moderate and severe. * Statistically significant at p 6 0.05.
A key feature of asthmatic airways is remodeling which involves thickening of the airway wall, altered deposition of extracellular matrix (ECM) proteins [2,3] and increased airway smooth muscle (ASM) mass. These structural changes may result from an aberrant repair process in the lung, which includes increased proliferation of the ASM cells [4,5]. The ECM maintains airway function and structure by providing mechanical support in addition to constituting a dynamic and complex network that influences cellular function [6]. The ECM deposited by asthma derived ASM cells is altered such that increased amounts of collagen I and laminin [7–9], as well as fibronectin (FN) are produced which mediate a range of cellular interactions including migration, growth and differentiation. Fibulin-1 (FBLN-1), a secreted glycoprotein, assists in
stabilizing the ECM. It associates with FN and a variety of other ECM proteins including laminin and fibrinogen [25]. Mice deficient in FN and FBLN-1 die perinatally due to abnormal lung development [14]. FBLN-1 expression has been reported in human lung tissue using microarray technology [15,16]. The present study aimed at investigation of possible association between the fibulin-1 levels in asthmatic patients and its relation to asthma severity. The study was carried out on forty-five asthmatic patients with different degrees of asthma severity and thirty age and sexmatched normal control subjects. According to our results, we found that fibulin-1 increased in serum of asthmatic patients without relation to asthma severity
Please cite this article in press as: D.E. Giziry et al., The study of fibulin-1 as a novel biomarker in bronchial asthma and its association with disease severity, Egypt. J. Chest Dis. Tuberc. (2016), http://dx.doi.org/10.1016/j.ejcdt.2016.12.003
4
D.E. Giziry et al. / Egyptian Journal of Chest Diseases and Tuberculosis xxx (2016) xxx–xxx
and highly increased in BAL of sever degree asthmatic patients than in mild and moderate degrees. So, fibulin-1 in BAL can be used as a marker in diagnosis of severe degrees of bronchial asthma. Transforming growth factor beta 1 (TGF-b1) is a pro-fibrotic cytokine which is increased in several forms of acute and chronic adult lung diseases such as asthma [15], COPD [26,27], and pulmonary fibrosis [28,29]. It is considered to play a crucial role in the pathogenesis of tissue fibrosis, stimulating the production of various collagens and ECM proteins [30,31,6]. Levels of the pro-fibrotic cytokine TGF-b1 are elevated in the BAL fluid in asthma [22], and are increased in bronchial tissue [15]. TGF-b1 increased the expression of FBLN-1 in ASM cells. The increased FBLN-1 resulted in exaggerated proliferation and wound repair in asthma derived ASM cells. TGF-b1 not only increased FBLN-1 in asthma derived ASM cells, but also enhanced its deposition in the asthmatic ECM. The architecture of the asthmatic airway often undergoes prominent and permanent structural changes, including alterations of the molecular composition of the ECM. In particular, ECM protein deposition is increased in the lamina reticularis [32] resulting in basement membrane thickening [33]. Given that TGF-b1 levels may alter the ECM assembly process, it is likely that the raised TGF-b1 levels in the asthmatic airway may contribute to an increase in the ECM thereby augmenting the effects of airway remodeling. AHR is a hallmark feature of asthma that has been linked to the pathological events associated with remodeling of the airway wall. TGFb-1 has also been linked to the mechanism underpinning AHR in sthma. In particular, TGF-b1 administration to the airways in mice induces peribronchial fibrosis that results in the development of AHR [34]. When antisense oligomer targeting FBLN-1 (AOs) was used in mice treatment, TGF-b1 induced AHR was inhibited demonstrating a critical role for FBLN-1 in the mechanism of TGF-b1 induced AHR. The regulated expression of FBLN-1 downstream of inflammation and its regulation of the remodeling/AHR axis identify the therapeutic potential of targeting this glycoprotein. While FBLN1 is normally found in the blood, there is not much known about how the body regulates FBLN1 levels or where the molecule comes from. TGF-b1 causes increased release of soluble FBLN1 from airway epithelial cells, which may contribute to the increased levels found in the serum and BAL of people with asthma. Lau JY, et al. [35] found that FBLN-1 is increased in the serum and BAL of people with asthma, and shown that FBLN-1 regulates airway smooth muscle (ASM) cell proliferation, therefore highlighting the potential role of FBLN-1 in airway wall remodeling. They also found that there was no correlation between the FBLN-1 level detected in the serum or BAL from the asthmatic volunteers and their FEV1/FVC ratio. In contrast, Chen L, et al. [36] found that TGF-b1 decreased FBLN-1 mRNA, and that FBLN-1 protein production was controlled. Therefore it was hypothesized that the increased ECM FBLN-1 following TGF-b1 stimulation was due to sequestration of soluble FBLN-1 and not de novo synthesis. Conclusion It was found that fibulin-1 was increased in BAL of severe degree bronchial asthma than in mild and moderate degrees with statistical significant difference (p < 0.05). This may highlights the potential role of fibulin-1 in airway wall remodeling. References [1] M. Ebina, T. Takahashi, T. Chiba, M. Motomiya, Cellular hypertrophy and hyperplasia of airway smooth muscles underlying bronchial asthma. A 3-D morphometric study, Am. Rev. Respir. Dis. 148 (1993) 720–726.
[2] P.G. Woodruff, G.M. Dolganov, R.E. Ferrando, S. Donnelly, S.R. Hays, Hyperplasia of smooth muscle in mild to moderate asthma without changes in cell size or gene expression, Am. J. Respir. Crit. Care Med. 169 (2004) 1001– 1006. [3] P.R. Johnson, M. Roth, M. Tamm, M. Hughes, Q. Ge, Airway smooth muscle cell proliferation is increased in asthma, Am. J. Respir. Crit. Care Med. 164 (2001) 474–477. [4] T. Trian, G. Benard, H. Begueret, R. Rossignol, P.O. Girodet, D. Ghosh, et al., Bronchial smooth muscle remodeling involves calcium-dependent enhanced mitochondrial biogenesis in asthma, J. Exp. Med. 204 (2007) 3173–3181. [5] S.K. Akiyama, S.S. Yamada, W.T. Chen, K.M. Yamada, Analysis of fibronectin receptor function with monoclonal antibodies: roles in cell adhesion, migration, matrix assembly, and cytoskeletal organization, J. Cell Biol. 109 (1989) 863–875. [6] P.R. Johnson, J.K. Burgess, P.A. Underwood, W. Au, M.H. Poniris, M. Tamm, et al., Extracellular matrix proteins modulate asthmatic airway smooth muscle cell proliferation via an autocrine mechanism, J. Allergy Clin. Immunol. 113 (2004) 690–696. [7] L.A. Laitinen, A. Laitinen, Inhaled corticosteroid treatment and extracellular matrix in the airways in asthma, Int. Arch. Allergy Immunol. 107 (1995) 215– 216. [8] K. Parameswaran, K. Radford, J. Zuo, L.J. Janssen, P.M. O’Byrne, P.G. Cox, Extracellular matrix regulates human airway smooth muscle cell migration, Eur. Respir. J. 24 (4) (2004) 545–551. [9] A.E. Redington, J. Madden, A.J. Frew, R. Djukanovic, W.R. Roche, S.T. Holgate, et al., Transforming growth factor-beta 1 in asthma. Measurement in bronchoalveolar lavage fluid, Am. J. Respir. Crit. Care Med. 156 (2 Pt 1) (1997) 642–647. [10] A.M. Vignola, P. Chanez, G. Chiappara, A. Merendino, E. Pace, Transforming growth factor-beta expression in mucosal biopsies in asthma and chronic bronchitis, Am. J. Respir. Crit. Care Med. 156 (1997) 591–599. [11] D.J. Romberger, J.D. Beckmann, L. Claassen, R.F. Ertl, S.I. Rennard, Modulation of fibronectin production of bovine bronchial epithelial cells by transforming growth factor-beta, Am. J. Respir. Cell Mol. Biol. 7 (1992) 149–155. [12] G. Westergren-Thorsson, J. Chakir, M.J. Lafreniere-Allard, L.P. Boulet, G.M. Tremblay, Correlation between airway responsiveness and proteoglycan production by bronchial fibroblasts from normal and asthmatic subjects, Int. J. Biochem. Cell Biol. 34 (2002) 1256–1267. [13] H. Tran, W.J. VanDusen, W.S. Argraves, The self-association and fibronectinbinding sites of fibulin-1 map to calcium-binding epidermal growth factor-like domains, J. Biochem. 272 (1997) 22600–22606. [14] C. Laprise, R. Sladek, A. Ponton, M.C. Bernier, T.J. Hudson, M. Laviolette, Functional classes of bronchial mucosa genes that are differentially expressed in asthma, BMC Genomics 5 (2004) 21. [15] F. Syed, R.A. Panettieri Jr., O. Tliba, C. Huang, K. Li, M. Bracht, et al., The effect of IL-13 and IL-13R130Q, a naturally occurring IL-13 polymorphism, on the gene expression of human airway smooth muscle cells, Respir. Res. 6 (2005) 9. [16] N. Zimmermann, N.E. King, J. Laporte, M. Yang, A. Mishra, Dissection of experimental asthma with DNA microarray analysis identifies arginase in asthma pathogenesis, J. Clin. Invest 111 (2003) 1863–1874. [17] Global Initiative for Asthma. Global strategy for asthma management and prevention. Issued January 1995 (NIH Publication No. 02–3659); updated 2015. [18] S.E. Wenzel, S.J. Szefler, D.Y.M. Leung, S.I. Sloan, M.D. Rex, Bronchoscopic evaluation of severe asthma, Am. J. Respir. Crit. Care Med. 156 (1997) 737–743, 214. [19] T. Musso, I. Espinosa-Delgado, K. Pulkki, G.L. Gusella, D.L. Longo, L. Vareso, Transforming growth factor-ß1 downregulates interleukin-1 (IL-1)-induced IL-6 production by human monocytes, Blood 76 (1990) 2466–2469. [20] E. Engvall, P. Perlmann, Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G, Immunochemistry 8 (9) (1971) 871–874. [21] E. Engvall, P. Perlmann, Enzyme-linked immunosorbent assay, Elisa. 3. Quantitation of specific antibodies by enzyme-labeled anti-immunoglobulin in antigen-coated tubes, J. Immunol. 109 (1) (1972) 129–135. [22] B.K. Van Weemen, A.H. Schuurs, Immunoassay using antigen enzyme conjugates, FEBS Lett. 15 (1971) 232. [23] F.D. Martinez, Genes, environments, development and asthma: a reappraisal, Eur. Respir. J. 29 (1) (2007) 179–184. [24] R.F. Lemanske, W.W. Busse, Asthma: clinical expression and molecular mechanisms, J. Allergy Clin. Immunol. 125 (2 Suppl 2) (2010) S95–S102. [25] G. Kostka, R. Giltay, W. Bloch, K. Addicks, R. Timpl, R. Fassler, et al., Perinatal lethality and endothelial cell abnormalities in several vessel compartments of fibulin-1-deficient mice, Mol. Cell. Biol. 21 (20) (2001) 7025–7034. [26] W.I. de Boer, A. van Schadewijk, J.K. Sont, H.S. Sharma, J. Stolk, H.S. Sharma, et al., Transforming growth factor beta1 and recruitment of macrophages and mast cells in airways in chronic obstructive pulmonary disease, Am. J. Respir. Crit. Care Med. 158 (1998) 1951–1957. [27] J.C. Mak, M.M. Chan-Yeung, S.P. Ho, K.S. Chan, K. Choo, Elevated plasma TGFbeta1 levels in patients with chronic obstructive pulmonary disease, Respir. Med. 103 (2009) 1083–1089. [28] N. Khalil, R.N. O’Connor, H.W. Unruh, P.W. Warren, K.C. Flanders, A. Kemp, et al., Increased production and immunohistochemical localization of transforming growth factor-beta in idiopathic pulmonary fibrosis, Am. J. Respir. Cell Mol. Biol. 5 (1991) 155–162.
Please cite this article in press as: D.E. Giziry et al., The study of fibulin-1 as a novel biomarker in bronchial asthma and its association with disease severity, Egypt. J. Chest Dis. Tuberc. (2016), http://dx.doi.org/10.1016/j.ejcdt.2016.12.003
D.E. Giziry et al. / Egyptian Journal of Chest Diseases and Tuberculosis xxx (2016) xxx–xxx [29] K. Zhang, K.C. Flanders, S.H. Phan, Cellular localization of transforming growth factor-beta expression in bleomycin-induced pulmonary fibrosis, Am. J. Pathol. 147 (1995) 352–361. [30] L.M. Moir, J.K. Burgess, J.L. Black, Transforming growth factor beta 1 increases fibronectin deposition through integrin receptor alpha 5 beta 1 on human airway smooth muscle, J. Allergy Clin. Immunol. 121 (2008) 1034–1039. [31] J. Rosenbloom, S.V. Castro, S.A. Jimenez, Narrative review: fibrotic diseases: cellular and molecular mechanisms and novel therapies, Ann. Intern. Med. 152 (2010) 159–166. [32] S.J. Hirst, C.H. Twort, T.H. Lee, Differential effects of extracellular matrix proteins on human airway smooth muscle cell proliferation and phenotype, Am. J. Respir. Cell Mol. Biol. 23 (2000) 335–344.
5
[33] P.K. Jeffery, Pathology of asthma, Br. Med. Bull. 48 (1992) 23–39. [34] N.J. Kenyon, R.W. Ward, G. McGrew, J.A. Last, TGF-ß1 causes airway fibrosis and increased collagen I and III mRNA in mice, Thorax 58 (2003) 772–777. [35] J.Y. Lau, B.G. Oliver, M. Baraket, E.L. Beckett, N.G. Hansbro, Fibulin-1 is increased in asthma – a novel mediator of airway remodeling?, PLoS One 5 (10) (2010) e13360 [36] L. Chen, Q. Ge, J.L. Black, L. Deng, J.K. Burgess, B.G. Oliver, Differential regulation of extracellular matrix and soluble fibulin-1 levels by TGF-ß1 in airway smooth muscle cells, PLoS One 8 (6) (2013) e65544.
Please cite this article in press as: D.E. Giziry et al., The study of fibulin-1 as a novel biomarker in bronchial asthma and its association with disease severity, Egypt. J. Chest Dis. Tuberc. (2016), http://dx.doi.org/10.1016/j.ejcdt.2016.12.003