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DOI 10.1002/prca.201300047
RESEARCH ARTICLE
Human cystatin C: A new biomarker of idiopathic pulmonary fibrosis? Mariana Kasabova, Alix Joulin-Giet, Fabien Lecaille, Ahlame Saidi, Sylvain Marchand-Adam and Gilles Lalmanach ´ ´ ´ ´ ´ INSERM U1100, Pathologies Pulmonaires: Proteolyse et Aerosolth erapie, Equipe 2: Mecanismes Proteolytiques ´ dans l’Inflammation; Faculte´ de Medecine, Centre d’Etude des Pathologies Respiratoires (CEPR), Universite´ Franc¸ois Rabelais, Tours, France Purpose: Human idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disorder with a poor prognosis. The identification of a new and specific biomarker in bronchoalveolar lavage fluids (BALFs) may assist in the diagnosis of the disease. Experimental design: Characterization of cysteine Cats and their endogenous inhibitor, cystatin C, was conducted by immunochemical analysis and measurement of endopeptidase activity of control (n = 11) and IPF (n = 25) BALFs (normalized conditions, 20 g protein/assay). Results: Cathepsin (Cat) B was detected as proform and mature enzyme for both control and IPF samples, while Cats K, L, and S were found as zymogens with a strengthened staining in IPF BALFs. The overall endopeptidase activity related mainly to Cat B and did not vary significantly between control and IPF samples. Conversely a significant increase of immunoreactive cystatin C was measured in BALFs for each of three IPF grades. Conclusions and clinical relevance: An excessive deposition of extracellular matrix proteins is the hallmark of fibrotic disorders. Cats are potent collagenases and might be essential for lung homeostasis. Taken together, increase of cystatin C in IPF BALFs may reflect abnormal regulation of proteolytic activity of Cats in lung, which in turn can promote the development of fibrosis.
Received: June 19, 2013 Revised: September 4, 2013 Accepted: September 14, 2013
Keywords: Cathepsin / Cystatin C / Lung fibrosis / Protease / Protease inhibitor
1
Introduction
Idiopathic pulmonary fibrosis (IPF) is a chronic, irreversible lung interstitial disorder with poor prognosis since median survival is estimated at 3 years after diagnosis [1]. Despite an unknown etiology, cigarette smoking, viral (influenza A,
Correspondence: Professor Dr Gilles Lalmanach, INSERM, UMR ´ ´ ´ 1100 Pathologies Pulmonaires: Proteolyse et Aerosolth erapie ´ /CEPR, Faculte´ de Medecine, Centre d’Etude des Pathologies Respiratoires (CEPR), Universite´ Franc¸ois Rabelais 10 Boulevard ´ F – 37032 Tours cedex, France Tonnelle, E-mail: [email protected] Fax: +33-247366046 Abbreviations: AEBSF, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride; BALF, bronchoalveolar lavage fluid; Brij35, polyethylene glycol lauryl ether; CA-074, N-(L-3trans-propylcarbamoyloxirane-2-carbonyl)-L-isoleucyl-L-proline; Cats, cathepsins; E-64, L-3-carboxy-trans-2,3-epoxy-propionylleucylamide-(4-guanido)-butane; ECM, extracellular matrix; IPF, idiopathic pulmonary fibrosis; RT, room temperature C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
hepatitis C, Epstein-Barr, and other herpes viruses) infections, gastro-esophageal reflux disease as well inherited factors may increase the risk of IPF [2,3]. Apart a common shortness of breath (dyspnea), IPF remains usually asymptomatic and under diagnosed until onset of symptoms. About 100 000 people are affected in the United States, and 30 000–40 000 new cases are identified each year [4]. IPF leads to a progressive loss of lung function, which is accompanied by fibroblast proliferation and excessive extracellular matrix (ECM) deposition of collagens, and ultimately death. In 2011, the American Thoracic Society, the European Respiratory Society, the Japanese Respiratory Society, and the Latin-American Thoracic Society issued an official joint statement providing guidance for the diagnosis and management of IPF [5]. Some drugs (e.g. prednisonol, azathioprine, N-acetyl cysteine) have been used to treat IPF, either alone or in combination, but to date, no pharmacologic therapies have definitively been shown to improve survival of IPF patients. Other drugs such as pirfenidone could have a positive effect on pulmonary function decline in for patients with mild-to-moderate IPF [6]. It might be assumed that the efficiency of therapeutics could be www.clinical.proteomics-journal.com
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improved by an earlier diagnosis of IPF. Besides serum markers, the identification of new and sensitive biomarkers would be essential to allow an early and more specific diagnosis of the disease. Under these circumstances, identification of specific candidate molecules to facilitate IPF diagnosis remains a tricky and exciting challenge. Type 2 cystatins that belong to the family I25 are singlechain molecules (circa. 120 amino acids) exhibiting two disulfide bridges [7–9]. Cystatins are mainly extracellular, secreted proteins, which are synthesized with a signal peptide, allowing transport of the mature protein over the cell membrane to the extracellular space [10]. Cystatin C is the best wellcharacterized member of the type 2 family [7]. Cystatin C is a highly potent inhibitor of cysteine cathepsins (Cat) (family C1; MEROPS: the peptidase database: [9]) and is considered to be the major extracellular cysteine protease inhibitor [11, 12]. The lung expression of cysteine Cats and their putative functions have been reviewed elsewhere [13, 14]. Cystatin C that is expressed by a broad variety of human cells and tissues, has a widespread distribution, and is ubiquitously found in body fluids at significant concentrations, especially in milk, seminal plasma, and cerebrospinal fluid [11]. Serum creatinine is the most widely used marker of glomerular filtration rate to follow renal function and detect impairment and acutely changing kidney function [15]. Although less broadly used in the clinical routine, cystatin C has been also identified as a promising and valuable biomarker of glomerular filtration rate (The Lund Model) (for review see [16]). On the other hand increased cystatin C serum concentration was reported in some fibrotic diseases, such as multiple sclerosis [17], chronic hepatic fibrosis [18] as well during the progression of liver fibrosis [19]. A similar upregulation was reported for profibrogenic hepatic stellate cells [20]. Oral submucous fibrosis that is due to areca quid chewing is characterized by epithelial atrophy and progressive accumulation of collagen fibers in the lamina propria and submucosa. Consistently, cystatin C is dramatically upregulated during oral submucous fibrosis compared to normal buccal mucosa [21]. In a comparable way we have recently observed that level of cystatin C increased in primary myofibroblasts from IPF patients (Kasabova and Lalmanach, unpublished data). This led us to determine whether cystatin C could be specifically overexpressed in bronchoalveolar lavage fluids (BALFs) of fibrotic patients, raising the question of its potential use as a specific and new biomarker of IPF. In conjunction, the activity and expression of their biological partners, cysteine Cats, were also examined.
2
Materials and methods
2.1 Reagents Benzyloxycarbonyl-Phe-Arg-7-amino-4-methyl coumarin (ZPhe-Arg-AMC) was obtained from R&D Systems (Minneapolis, MN, USA). L-3-carboxy-trans-2,3-epoxy-propionyl C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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leucylamide-(4-guanido)-butane (E-64), N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-L-isoleucyl-L-proline (CA-074), 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (Pefabloc), pepstatin A, EDTA, S-methyl thiomethanesulfonate (MMTS) and BSA (bovine serum albumin) were purchased from Sigma–Aldrich (Saint-Quentin Fallavier, France). DTT was obtained from Calbiochem (VWR International, Pessac, France). Brij35 (polyethylene glycol lauryl ether) was from Pierce (Rockford, IL, USA). 2.2 Ethics statements and clinical specimens The Institutional Review Board of the French Learned Society for Respiratory Medicine (Soci´et´e de Pneumologie de Langue Franc¸aise) approved the study, and written informed consent was obtained from the participants to the study (Biocollection DC 2010-1216, CHU Tours). Clinical charts and functional records were collected on a standardized and anonymous collection form. Data are reported in Table 1 (non-IPF control patients: 7 males, 4 females; IPF patients: 18 males, 7 females). IPF patients were divided into three grades depending on the severity of the disease (stage I, mild, n = 11; stage II, moderate, n = 8; stage III, severe, n = 6) grades using the GAP index [22]. Serum creatinine concentration was measured by the routine clinical Jaffe’s reaction [23]. Absorbance was measured at 520 nm (Olympus spectrophotometer). Clearance was calculated by the Modification of Diet in Renal Disease method [24]. A broncho-alveolar lavage was required for an investigative purpose when a diagnosis of IPF was suspected with standard procedures [25]. Bronchoscopy was performed as described elsewhere [26]. BALFs were immediately buffered (final concentrations: 0.1 M sodium acetate, pH 5.5 plus the peptidase inhibitors: Pefabloc, 0.5 mM EDTA, 0.04 mM pepstatin A, and 0.5 mM MMTS) in order to stabilize and preserve Cats from irreversible pH inactivation and uncontrolled proteolysis. After centrifugation (1000 × g for 8 min at 4⬚C), the resulting supernatants were aliquoted and frozen at −80⬚C until use. Protein concentration was determined by a bicinchoninic acid assay (BCA protein assay kit, Interchim, Montluc¸on, France). 2.3 Immunoblotting Polyclonal rabbit anti-human Cat B (1:1000) and anti-Cat K (1:1000) antibodies were obtained from Calbiochem. Polyclonal goat antihuman Cat L (1:1000) and anti-Cat S (1:1000) antibodies were purchased from R&D Systems. The mouse antihuman cystatin C (1:500) was from R&D Systems. The goat antirabbit IgG-peroxidase conjugate, the goat antimouse IgG-peroxidase conjugate, and the rabbit antigoat IgG-peroxidase conjugate were from Sigma-Aldrich. Recombinant cystatin C (with a C-terminal 10-His tag) was obtained from R&D Systems. Prestained molecular masses (Precision Plus Protein Standards) came from Bio-Rad (Marnes-La-Coquette, France). Samples (20 g www.clinical.proteomics-journal.com
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Table 1. Biological and clinical characteristics and BALF data from control and IPF patients
Patients
Age
Cell number (×104 /mL)
Macrophages (%)
Lymphocytes (%)
PNN (%)
PNE (%)
Creatinine (mol/mL)
Clearance (mL/min)
Controls (n = 11) IPF (n = 25)
59 (±6) 77 (±2)
18 (±5.8) 19 (±4.3)
85 (±3) 80 (±4)
8 (±2) 5 (±4)
2 (±2) 6 (±2)
0 3 (±1)
89 (±10.1) 93 (±5.6)
75.5 (±6.1) 68 (±5.4)
Data are presented as mean ± SEM. PNN, polynuclear neutrophils; PNE, polynuclear eosinophils Non-IPF control patients, 11; IPF patients, 25 (stage I, mild, n = 11; stage II, moderate, n = 8; stage III, severe, n = 6, using the GAP index [22]). Serum creatinine concentration was measured by the routine clinical Jaffe’s reaction [23]. Clearance was calculated by the Modification of Diet in Renal Disease method [24]. No significant differences in clearance and serum creatinine levels, and in cell counting and distribution were observed.
protein/well) were diluted in Laemmli buffer under reducing conditions and boiled for 5 min at 95⬚C. Proteins were separated on 12% SDS-PAGE and blotted onto nitrocellulose membranes (Amersham Biosciences, Buckinghamshire, UK). Membranes were blocked with 5% nonfat dry milk in PBS, 0.1% Tween-20 (PBS-T) at room temperature (RT) for 1 h and incubated with the specific primary antibodies at 4⬚C overnight. After washing, peroxidase-conjugate secondary antibodies (1:5000) were added and incubated 1 h at RT. Revelation was performed using an ECL kit (Amersham). Band densities were quantified by densitometric analysis using the ImageJ software (NIH, Bethesda, MD, USA).
(10 M), the broad-spectrum cysteine protease inhibitor, or with CA-074 (10 M), a specific Cat B inhibitor. The experiment was repeated twice. 2.6 Statistical analysis All data were expressed as median (range) values (±SEM). Variations between control and IPF grades (stages I, II, and III) were determined using the Kruskal–Wallis test, and results were analyzed by using the nonparametric two ways Mann–Whitney test, using a software running Excel (AnaStats Scop, Rilly-sur-Vienne, France) (***p < 0.001; ** p < 0.05).
2.4 Cystatin C immunoassay The concentration of human cystatin C was determinated using the sandwich ELISA DuoSet kit from R&D Systems. Microtiter plates (Nunc A/S, Roskilde, Denmark) were coated with a murine monoclonal anticystatin C antibody and incubated overnight at RT. The plates were washed three times with PBS-T before blocking with 1% BSA in PBS for 1 h at RT. Standard concentrations of cystatin C used for the calibration curve and biological samples (BALFs) (duplicates), diluted in blocking solution, were then applied and incubated 2 h at RT. After washing step, biotinylated mouse antihuman cystatin C antibody was added for 2 h followed by streptavidin HRP conjugate for 20 min. After washing, samples were incubated with 0.04% v/v H2 O2 and orthophenylene diamine (0.4 mg/mL) in substrate buffer (50 mM phosphate, 20 mM citric acid, pH 5.5) in the dark. The reaction was stopped by adding 3M H2 SO4 . Absorbance was measured at 492 nm using a microplate reader (VersaMax, Molecular Devices, St Gr´egoire, France). The experiment was repeated twice. 2.5 Peptidase activity Cysteine Cats from BALF (20 L/assay, duplicates) were activated in the activity buffer (0.1 M sodium acetate, pH 5.5, 5 mM DTT, 2 mM EDTA, and 0.01% Brij35) for 3 min at 30⬚C. Enzymatic activity was measured using Z-Phe-Arg-AMC (50 M) as substrate (exc = 350 nm, em = 460 nm) using a Gemini spectrofluorimeter (Molecular Devices). Control experiments were made by pre-incubating BALFs with E-64 C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
3
Results and discussion
3.1 Characterization of BALFs cysteine Cats Clinical records (11 control patients, 25 IPF patients) were reported in Table 1. Differences in cell counting and distribution were not statistically significant. Following centrifugation, cell-free supernatants of BALFs were immediately buffered to preserve cysteine Cats from inactivation according to [26]. Median protein concentrations were not significantly different between control patients (0.16 ± 0.04 mg/mL) and IPF patients (0.21 ± 0.085 mg/mL). Constant amounts of samples (were analyzed by Western blot. Both immunoreactive mature Cat B and its zymogen were detected in BALFs (see representative samples: Fig. 1A), while staining was similar for control and IPF samples. Moreover mature Cat B was detected at ∼24/25 kDa supporting that the enzyme is mostly found as its double-chain form rather than ∼31 kDa single-chain Cat B. Cat L was mainly found as its proform while mature Cat L was weakly stained (or remained below the limits of immunodetection) (Fig. 1B). The full and/or partly processed proforms of both Cats K and S, but not mature enzymes, were also detected (Fig. 1C and D). In addition a more intense staining was observed for proforms of Cats K, L, and S in IPF BALFs. Similar immunoreactive outlines were observed for pro-Cats K and S in BALFs from patients suffering from sarcoidosis and alveolar proteinosis [26]. Conversely, enzyme profiles differed to that observed in silicotic BALFs [27] and in sputum from patients with cystic www.clinical.proteomics-journal.com
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Figure 1. Cysteine Cats in human IPF BALFs. Proteins (20 g/well) were separated by 12% SDSPAGE under reducing conditions, transferred to nitrocellulose membranes, and analyzed with polyclonal antibodies against human Cat B (A), Cat L (B), Cat K (C), and Cat S (D). Black arrows correspond to proforms of Cats and white arrows correspond to mature enzymes. (E) The peptidase activity related to cysteine Cats was measured toward the fluorogenic substrate Z-Phe-Arg-AMC, and expressed as arbitrary fluorescence unit per g of proteins. Median value is shown by solid line (n = 2). n.s, non significant; AUF, arbitrary units of fluorescence; C, BALFs from control patients; IPF, BALFs from IPF patients.
fibrosis where Cat S is predominantly found as a ∼25 kDa active protease [28,29]. In a previous work we proposed that procathepsins could represent a reservoir of activable enzymes in inflammatory BALFs [26]. Here, we failed to induce maturation of proenzymes under in vitro conditions, as reported for BALFs from patients with silicosis [27]. Regardless of cigarette smoking was reported to induce the release of Cats B, L, and S by macrophages [30–34], Western blot patterns did not depend on the fact that BALFs originated from smokers and nonsmokers (data not shown). Moreover, the Cat activity in BALFs was further measured using Z-FR-AMC as substrate. Despite a lesser (nearly twofold lower) peptidase activity for IPF versus control nonsmoker patients, this decrease appears to be not statistically significant (Fig. 1E, p > 0.1). Addition of CA-074 impaired over 85% of the overall activity (data not shown), strengthening that Cat B is the prevailing Cat in BALFs (for review see [13]). Present results are in good agreement with the fact that a similar amount of macrophages, which are the primary source of extracellular Cats in BALFs [13], were found for both control and IPF patients (see Table 1).
3.2 Immunodetection and quantitative analysis of cystatin C There is now a growing body of evidence that cysteine Cats take part in pulmonary homeostasis [14]. Dysregulation of the protease/antiprotease balance in favor to a reduction of the proteolytic activity by endogenous inhibitors may promote progression of the lung fibrosis. Recently we observed an increased protein level of cystatin C, the most potent circulating inhibitor of Cats in primary myofibroblasts from IPF patients (Kasabova & Lalmanach, unpublished data). This raises the issue whether cystatin C could be specifically over-
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expressed in BALFs of fibrotic patients, and questions its potential use as a specific and new biomarker of IPF. Recombinant human cystatin C used as control was detected to a higher molecular mass (circa. 17 kDa) as notified by the supplier, due to the presence of an additional C-terminal 10-His tag. Albeit variations of intensity, cystatin C was detected in all BALFs from IPF patients compared to control samples (Fig. 2A). In addition, pools of both control and IPF samples were prepared, analyzed by Western blotting and quantified by densitometry to give an overall drawing of the immunoreactivity levels of cystatin C in BALFs (Fig. 2B). ELISA assays confirmed that concentrations of cystatin C in IPF samples were highly significantly increased (p < 0.001) (median concentrations: Control, 14 ng/mL; IPF, 27.2 ng/mL) (Fig. 2C). Moreover, the increase of alveolar cystatin C level was significant for each of three IPF grades with a median concentration of 31.9 ng/mL (stage I, p < 0.001), 27 ng/mL (stage II, p < 0.001), and 24.7 ng/mL (stage III, p < 0.05), respectively (Fig. 2D). But there are no confident differences of concentration between the three IPF stages (p > 0.1), suggesting that increased levels of cystatin C are not statistically correlated with the severity of the disease. In routine clinical practice, the diagnosis and severity classification of acute kidney injury is based on a rise in serum creatinine levels. Likewise, concentration of cystatin C that has been also proposed as a promising biomarker during renal injury and clearance failure dramatically increases [35]. Remarkably, no significant variations of clearance (Control, 75.5 ± 6.1 mL/min; IPF, 68 ± 5.4 mL/min) and serum creatinine levels (Control, 89 ± 10.1 mol/mL; IPF, 93 ± 5.6 mol/mL) were observed in the present study (Table 1), supporting that the increased concentration of cystatin C in IPF BALFs did not rely on kidney damage. In addition, for each IPF grade (stages I-III), we did not detect a confident change in alveolar concentration of cystatin C by comparing the samples corresponding to a low
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clearance (60 mL/min) (p > 0.1). Since patients with chronic renal failure do not have greater risks to develop a pulmonary fibrosis, present data support that BALF cystatin C change relate directly to pathophysiological variations associated to lung fibrosis. We noted in the previous paragraph that despite the augmentation of procathepsins (Fig. 1B, C, and D) may correspond to an activatable reservoir of mature forms [26], no significant change of overall Cat-related activities was measured in IPF BALFs. That is most likely associated with the balancing increase in cystatin C in IPF samples. Consequently, we may presume that the ability of zymogens to be processed as mature Cats depends on the protease/antiprotease balance. A hallmark of fibrosis is the increase of ECM components deposition, including an excess of collagen fibers. On the other hand, Cats are potent collagenolytic enzymes and hydrolyze various ECM and basal membrane components (fibronectin, perlecan, nidogen) [36–38]. Accordingly these enzymes are essential for lung homeostasis by participating to ECM and basal membrane turnover and remodeling and their proteolytic potential tightly regulated [39, 40]. In a murine model of bleomycin-induced lung injury, Cat K deficiency exacerbated lung fibrosis, while increased levels of Cat K reduced excessive ECM deposition [41]. In a balanced manner, drug-induced overexpression of Cats K and L might be beneficial in the treatment of lung fibrosis in bleomycin-treated mice [42]. Alternatively, since cystatin C is a highly potent inhibitor of cysteine Cats (1:1 stoichiometric mechanism) that plays a regulatory role in numerous body fluids [7, 11, 12], it is reasonable to speculate that cystatin C may be involved in the pathogenesis of fibrosis.
4
Figure 2. Immunodetection and dosage of cystatin C in human BALFs. (A) Western blot analyses were conducted using a mouse monoclonal antibody against human cystatin C. CC, recombinant human cystatin C (R&D systems) that possesses an additional Cterminal 10 His-tag was used as control (apparent molecular mass of 17 kDa). Some representative samples are shown. (B) Control and IPF BALFs were pooled, respectively, separated by 12% SDSPAGE under reducing conditions and immunoblotted with the anticystatin C antibody. A densitometric analysis was used for quantification. (C) ELISA dosage of human cystatin C (non-IPF versus IPF patients) were performed in duplicates (***p < 0.001). The experiment was repeated twice. (D) ELISA assay for cystatin C according to the severity of the disease (i.e. IPF grades) (stage I, n = 11; stage II, n = 8; stage III, n = 6). Assays were performed in duplicates (***p < 0.001, **p < 0.05). The experiment was repeated twice.
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Concluding remarks
To our knowledge, this is the first attempt to evaluate the protein level of cystatin C in BALFs from IPF patients. We have demonstrated that cystatin C expression is critically increased in IPF BALFS compared to non-IPF BALFs (p < 0.001). Despite this augmentation occurs for the three IPF grades (stages I, II, and III) and apparently does not correlate directly with the severity of the disease, current results raise the question of the use of BALF cystatin C as a possible specific marker of lung fibrosis, beside the validated use of plasma cystatin C as biomarker of renal glomerular failure [11]. Present data suggest also that one of the molecular mechanisms involved in the pathogenesis of lung fibrosis might be the upregulation of cystatin C. Since ECM remodeling is the result of a subtle balance between synthesis and degradation, the rise of cystatin C might promote the development of lung fibrosis by impairing collagenolytic activity of cysteine Cats. More fundamental and mechanistic studies have to be undertaken right now to clarify the putative roles www.clinical.proteomics-journal.com
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Clinical Relevance Human idiopathic pulmonary fibrosis (IPF) that leads to a progressive loss of lung function is a chronic and lethal interstitial disorder. A hallmark of fibrosis is the excessive deposition of extracellular matrix proteins. Currently no therapies have been shown to improve survival, despite some molecules could have beneficial effects. It may be assumed that the efficiency of such drugs could be improved by an earlier diagnosis of IPF. Thus, the identification of new markers would be essential. Besides other proteases, cysteine cathepsins are potent collage-
of cysteine Cats and cystatin C during idiopathic pulmonary fibrosis at both in vivo and in vitro levels. We thank la R´egion Centre (France) for its financial assistance (FibroCat project). This work was also supported by institutional funding from the Institut National de la Sant´e et de la Recherche M´edicale (INSERM). M.K. holds a doctoral fellowship from MENRT (Minist`ere de l’Education Nationale, de la Recherche et de la Technologie, France). The authors have declared no conflict of interest.
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´ ´ [26] Serveau-Avesque, C., Martino, M. F.-D., Herve-Gr epinet, V., Hazouard, E. et al., Active cathepsins B, H, K, L and S in human inflammatory bronchoalveolar lavage fluids. Biol. Cell 2006, 98, 15–22.
[37] Reiser, J., Adair, B., Reinheckel, T., Specialized roles for cysteine cathepsins in health and disease. J. Clin. Invest. 2010, 120, 3421–3431.
[27] Perdereau, C., Godat, E., Maurel, M.-C., Hazouard, E. et al., Cysteine cathepsins in human silicotic bronchoalveolar lavage fluids. Biochim. Biophys. Acta 2006, 1762, 351–356. ´ [28] Naudin, C., Joulin-Giet, A., Couetdic, G., Plesiat, P. et al., Human cysteine cathepsins are not reliable markers of infection by Pseudomonas aeruginosa in cystic fibrosis. Plos One. 2011, 6, e25577. [29] Lecaille, F., Naudin, C., Sage, J., Joulin-Giet, A. et al., Specific cleavage of the lung surfactant protein A by human cathepsin S may impair its antibacterial properties. Int. J. Biochem. Cell Biol. 2013, 45, 1701–1709. [30] Chang, J. C., Lesser, M., Yoo, O. H., Orlowski, M., Increased cathepsin B-like activity in alveolar macrophages and bronchoalveolar lavage fluid from smokers. Am. Rev. Respir. Dis. 1986, 134, 538–541. [31] Ishii, Y., Hashizume, Y., Watanabe, T., Waguri, S. et al., Cysteine proteinases in bronchoalveolar epithelial cells and
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[38] Turk, V., Stoka, V., Vasiljeva, O., Renko, M. et al., Cysteine cathepsins: From structure, function and regulation to new frontiers. Biochim. Biophys. Acta 2012, 1824, 68–88. ¨ ¨ [39] Buhling, F., Rocken, C., Brasch, F., Hartig, R. et al., Pivotal role of cathepsin K in lung fibrosis. Am. J. Pathol. 2004, 164, 2203–2216. ¨ [40] Buhling, F., Waldburg, N., Reisenauer, A., Heimburg, A. et al., Lysosomal cysteine proteases in the lung: role in protein processing and immunoregulation. Eur. Respir. J. 2004, 23, 620–628. [41] Zhang, D., Leung, N., Weber, E., Saftig, P. et al., The effect of cathepsin K deficiency on airway development and TGF-1 degradation. Respir. Res. 2011, 12, 72. [42] Zhang, D., Huang, C., Yang, C., Liu, R. J. et al., Antifibrotic effects of curcumin are associated with overexpression of cathepsins K and L in bleomycin treated mice and human fibroblasts. Respir. Res. 2011, 12, 154.
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DOI 10.1002/prca.201300047
RESEARCH ARTICLE
Human cystatin C: A new biomarker of idiopathic pulmonary fibrosis? Mariana Kasabova, Alix Joulin-Giet, Fabien Lecaille, Ahlame Saidi, Sylvain Marchand-Adam and Gilles Lalmanach ´ ´ ´ ´ ´ INSERM U1100, Pathologies Pulmonaires: Proteolyse et Aerosolth erapie, Equipe 2: Mecanismes Proteolytiques ´ dans l’Inflammation; Faculte´ de Medecine, Centre d’Etude des Pathologies Respiratoires (CEPR), Universite´ Franc¸ois Rabelais, Tours, France Purpose: Human idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disorder with a poor prognosis. The identification of a new and specific biomarker in bronchoalveolar lavage fluids (BALFs) may assist in the diagnosis of the disease. Experimental design: Characterization of cysteine Cats and their endogenous inhibitor, cystatin C, was conducted by immunochemical analysis and measurement of endopeptidase activity of control (n = 11) and IPF (n = 25) BALFs (normalized conditions, 20 g protein/assay). Results: Cathepsin (Cat) B was detected as proform and mature enzyme for both control and IPF samples, while Cats K, L, and S were found as zymogens with a strengthened staining in IPF BALFs. The overall endopeptidase activity related mainly to Cat B and did not vary significantly between control and IPF samples. Conversely a significant increase of immunoreactive cystatin C was measured in BALFs for each of three IPF grades. Conclusions and clinical relevance: An excessive deposition of extracellular matrix proteins is the hallmark of fibrotic disorders. Cats are potent collagenases and might be essential for lung homeostasis. Taken together, increase of cystatin C in IPF BALFs may reflect abnormal regulation of proteolytic activity of Cats in lung, which in turn can promote the development of fibrosis.
Received: June 19, 2013 Revised: September 4, 2013 Accepted: September 14, 2013
Keywords: Cathepsin / Cystatin C / Lung fibrosis / Protease / Protease inhibitor
1
Introduction
Idiopathic pulmonary fibrosis (IPF) is a chronic, irreversible lung interstitial disorder with poor prognosis since median survival is estimated at 3 years after diagnosis [1]. Despite an unknown etiology, cigarette smoking, viral (influenza A,
Correspondence: Professor Dr Gilles Lalmanach, INSERM, UMR ´ ´ ´ 1100 Pathologies Pulmonaires: Proteolyse et Aerosolth erapie ´ /CEPR, Faculte´ de Medecine, Centre d’Etude des Pathologies Respiratoires (CEPR), Universite´ Franc¸ois Rabelais 10 Boulevard ´ F – 37032 Tours cedex, France Tonnelle, E-mail: [email protected] Fax: +33-247366046 Abbreviations: AEBSF, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride; BALF, bronchoalveolar lavage fluid; Brij35, polyethylene glycol lauryl ether; CA-074, N-(L-3trans-propylcarbamoyloxirane-2-carbonyl)-L-isoleucyl-L-proline; Cats, cathepsins; E-64, L-3-carboxy-trans-2,3-epoxy-propionylleucylamide-(4-guanido)-butane; ECM, extracellular matrix; IPF, idiopathic pulmonary fibrosis; RT, room temperature C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
hepatitis C, Epstein-Barr, and other herpes viruses) infections, gastro-esophageal reflux disease as well inherited factors may increase the risk of IPF [2,3]. Apart a common shortness of breath (dyspnea), IPF remains usually asymptomatic and under diagnosed until onset of symptoms. About 100 000 people are affected in the United States, and 30 000–40 000 new cases are identified each year [4]. IPF leads to a progressive loss of lung function, which is accompanied by fibroblast proliferation and excessive extracellular matrix (ECM) deposition of collagens, and ultimately death. In 2011, the American Thoracic Society, the European Respiratory Society, the Japanese Respiratory Society, and the Latin-American Thoracic Society issued an official joint statement providing guidance for the diagnosis and management of IPF [5]. Some drugs (e.g. prednisonol, azathioprine, N-acetyl cysteine) have been used to treat IPF, either alone or in combination, but to date, no pharmacologic therapies have definitively been shown to improve survival of IPF patients. Other drugs such as pirfenidone could have a positive effect on pulmonary function decline in for patients with mild-to-moderate IPF [6]. It might be assumed that the efficiency of therapeutics could be www.clinical.proteomics-journal.com
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improved by an earlier diagnosis of IPF. Besides serum markers, the identification of new and sensitive biomarkers would be essential to allow an early and more specific diagnosis of the disease. Under these circumstances, identification of specific candidate molecules to facilitate IPF diagnosis remains a tricky and exciting challenge. Type 2 cystatins that belong to the family I25 are singlechain molecules (circa. 120 amino acids) exhibiting two disulfide bridges [7–9]. Cystatins are mainly extracellular, secreted proteins, which are synthesized with a signal peptide, allowing transport of the mature protein over the cell membrane to the extracellular space [10]. Cystatin C is the best wellcharacterized member of the type 2 family [7]. Cystatin C is a highly potent inhibitor of cysteine cathepsins (Cat) (family C1; MEROPS: the peptidase database: [9]) and is considered to be the major extracellular cysteine protease inhibitor [11, 12]. The lung expression of cysteine Cats and their putative functions have been reviewed elsewhere [13, 14]. Cystatin C that is expressed by a broad variety of human cells and tissues, has a widespread distribution, and is ubiquitously found in body fluids at significant concentrations, especially in milk, seminal plasma, and cerebrospinal fluid [11]. Serum creatinine is the most widely used marker of glomerular filtration rate to follow renal function and detect impairment and acutely changing kidney function [15]. Although less broadly used in the clinical routine, cystatin C has been also identified as a promising and valuable biomarker of glomerular filtration rate (The Lund Model) (for review see [16]). On the other hand increased cystatin C serum concentration was reported in some fibrotic diseases, such as multiple sclerosis [17], chronic hepatic fibrosis [18] as well during the progression of liver fibrosis [19]. A similar upregulation was reported for profibrogenic hepatic stellate cells [20]. Oral submucous fibrosis that is due to areca quid chewing is characterized by epithelial atrophy and progressive accumulation of collagen fibers in the lamina propria and submucosa. Consistently, cystatin C is dramatically upregulated during oral submucous fibrosis compared to normal buccal mucosa [21]. In a comparable way we have recently observed that level of cystatin C increased in primary myofibroblasts from IPF patients (Kasabova and Lalmanach, unpublished data). This led us to determine whether cystatin C could be specifically overexpressed in bronchoalveolar lavage fluids (BALFs) of fibrotic patients, raising the question of its potential use as a specific and new biomarker of IPF. In conjunction, the activity and expression of their biological partners, cysteine Cats, were also examined.
2
Materials and methods
2.1 Reagents Benzyloxycarbonyl-Phe-Arg-7-amino-4-methyl coumarin (ZPhe-Arg-AMC) was obtained from R&D Systems (Minneapolis, MN, USA). L-3-carboxy-trans-2,3-epoxy-propionyl C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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leucylamide-(4-guanido)-butane (E-64), N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-L-isoleucyl-L-proline (CA-074), 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (Pefabloc), pepstatin A, EDTA, S-methyl thiomethanesulfonate (MMTS) and BSA (bovine serum albumin) were purchased from Sigma–Aldrich (Saint-Quentin Fallavier, France). DTT was obtained from Calbiochem (VWR International, Pessac, France). Brij35 (polyethylene glycol lauryl ether) was from Pierce (Rockford, IL, USA). 2.2 Ethics statements and clinical specimens The Institutional Review Board of the French Learned Society for Respiratory Medicine (Soci´et´e de Pneumologie de Langue Franc¸aise) approved the study, and written informed consent was obtained from the participants to the study (Biocollection DC 2010-1216, CHU Tours). Clinical charts and functional records were collected on a standardized and anonymous collection form. Data are reported in Table 1 (non-IPF control patients: 7 males, 4 females; IPF patients: 18 males, 7 females). IPF patients were divided into three grades depending on the severity of the disease (stage I, mild, n = 11; stage II, moderate, n = 8; stage III, severe, n = 6) grades using the GAP index [22]. Serum creatinine concentration was measured by the routine clinical Jaffe’s reaction [23]. Absorbance was measured at 520 nm (Olympus spectrophotometer). Clearance was calculated by the Modification of Diet in Renal Disease method [24]. A broncho-alveolar lavage was required for an investigative purpose when a diagnosis of IPF was suspected with standard procedures [25]. Bronchoscopy was performed as described elsewhere [26]. BALFs were immediately buffered (final concentrations: 0.1 M sodium acetate, pH 5.5 plus the peptidase inhibitors: Pefabloc, 0.5 mM EDTA, 0.04 mM pepstatin A, and 0.5 mM MMTS) in order to stabilize and preserve Cats from irreversible pH inactivation and uncontrolled proteolysis. After centrifugation (1000 × g for 8 min at 4⬚C), the resulting supernatants were aliquoted and frozen at −80⬚C until use. Protein concentration was determined by a bicinchoninic acid assay (BCA protein assay kit, Interchim, Montluc¸on, France). 2.3 Immunoblotting Polyclonal rabbit anti-human Cat B (1:1000) and anti-Cat K (1:1000) antibodies were obtained from Calbiochem. Polyclonal goat antihuman Cat L (1:1000) and anti-Cat S (1:1000) antibodies were purchased from R&D Systems. The mouse antihuman cystatin C (1:500) was from R&D Systems. The goat antirabbit IgG-peroxidase conjugate, the goat antimouse IgG-peroxidase conjugate, and the rabbit antigoat IgG-peroxidase conjugate were from Sigma-Aldrich. Recombinant cystatin C (with a C-terminal 10-His tag) was obtained from R&D Systems. Prestained molecular masses (Precision Plus Protein Standards) came from Bio-Rad (Marnes-La-Coquette, France). Samples (20 g www.clinical.proteomics-journal.com
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Table 1. Biological and clinical characteristics and BALF data from control and IPF patients
Patients
Age
Cell number (×104 /mL)
Macrophages (%)
Lymphocytes (%)
PNN (%)
PNE (%)
Creatinine (mol/mL)
Clearance (mL/min)
Controls (n = 11) IPF (n = 25)
59 (±6) 77 (±2)
18 (±5.8) 19 (±4.3)
85 (±3) 80 (±4)
8 (±2) 5 (±4)
2 (±2) 6 (±2)
0 3 (±1)
89 (±10.1) 93 (±5.6)
75.5 (±6.1) 68 (±5.4)
Data are presented as mean ± SEM. PNN, polynuclear neutrophils; PNE, polynuclear eosinophils Non-IPF control patients, 11; IPF patients, 25 (stage I, mild, n = 11; stage II, moderate, n = 8; stage III, severe, n = 6, using the GAP index [22]). Serum creatinine concentration was measured by the routine clinical Jaffe’s reaction [23]. Clearance was calculated by the Modification of Diet in Renal Disease method [24]. No significant differences in clearance and serum creatinine levels, and in cell counting and distribution were observed.
protein/well) were diluted in Laemmli buffer under reducing conditions and boiled for 5 min at 95⬚C. Proteins were separated on 12% SDS-PAGE and blotted onto nitrocellulose membranes (Amersham Biosciences, Buckinghamshire, UK). Membranes were blocked with 5% nonfat dry milk in PBS, 0.1% Tween-20 (PBS-T) at room temperature (RT) for 1 h and incubated with the specific primary antibodies at 4⬚C overnight. After washing, peroxidase-conjugate secondary antibodies (1:5000) were added and incubated 1 h at RT. Revelation was performed using an ECL kit (Amersham). Band densities were quantified by densitometric analysis using the ImageJ software (NIH, Bethesda, MD, USA).
(10 M), the broad-spectrum cysteine protease inhibitor, or with CA-074 (10 M), a specific Cat B inhibitor. The experiment was repeated twice. 2.6 Statistical analysis All data were expressed as median (range) values (±SEM). Variations between control and IPF grades (stages I, II, and III) were determined using the Kruskal–Wallis test, and results were analyzed by using the nonparametric two ways Mann–Whitney test, using a software running Excel (AnaStats Scop, Rilly-sur-Vienne, France) (***p < 0.001; ** p < 0.05).
2.4 Cystatin C immunoassay The concentration of human cystatin C was determinated using the sandwich ELISA DuoSet kit from R&D Systems. Microtiter plates (Nunc A/S, Roskilde, Denmark) were coated with a murine monoclonal anticystatin C antibody and incubated overnight at RT. The plates were washed three times with PBS-T before blocking with 1% BSA in PBS for 1 h at RT. Standard concentrations of cystatin C used for the calibration curve and biological samples (BALFs) (duplicates), diluted in blocking solution, were then applied and incubated 2 h at RT. After washing step, biotinylated mouse antihuman cystatin C antibody was added for 2 h followed by streptavidin HRP conjugate for 20 min. After washing, samples were incubated with 0.04% v/v H2 O2 and orthophenylene diamine (0.4 mg/mL) in substrate buffer (50 mM phosphate, 20 mM citric acid, pH 5.5) in the dark. The reaction was stopped by adding 3M H2 SO4 . Absorbance was measured at 492 nm using a microplate reader (VersaMax, Molecular Devices, St Gr´egoire, France). The experiment was repeated twice. 2.5 Peptidase activity Cysteine Cats from BALF (20 L/assay, duplicates) were activated in the activity buffer (0.1 M sodium acetate, pH 5.5, 5 mM DTT, 2 mM EDTA, and 0.01% Brij35) for 3 min at 30⬚C. Enzymatic activity was measured using Z-Phe-Arg-AMC (50 M) as substrate (exc = 350 nm, em = 460 nm) using a Gemini spectrofluorimeter (Molecular Devices). Control experiments were made by pre-incubating BALFs with E-64 C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
3
Results and discussion
3.1 Characterization of BALFs cysteine Cats Clinical records (11 control patients, 25 IPF patients) were reported in Table 1. Differences in cell counting and distribution were not statistically significant. Following centrifugation, cell-free supernatants of BALFs were immediately buffered to preserve cysteine Cats from inactivation according to [26]. Median protein concentrations were not significantly different between control patients (0.16 ± 0.04 mg/mL) and IPF patients (0.21 ± 0.085 mg/mL). Constant amounts of samples (were analyzed by Western blot. Both immunoreactive mature Cat B and its zymogen were detected in BALFs (see representative samples: Fig. 1A), while staining was similar for control and IPF samples. Moreover mature Cat B was detected at ∼24/25 kDa supporting that the enzyme is mostly found as its double-chain form rather than ∼31 kDa single-chain Cat B. Cat L was mainly found as its proform while mature Cat L was weakly stained (or remained below the limits of immunodetection) (Fig. 1B). The full and/or partly processed proforms of both Cats K and S, but not mature enzymes, were also detected (Fig. 1C and D). In addition a more intense staining was observed for proforms of Cats K, L, and S in IPF BALFs. Similar immunoreactive outlines were observed for pro-Cats K and S in BALFs from patients suffering from sarcoidosis and alveolar proteinosis [26]. Conversely, enzyme profiles differed to that observed in silicotic BALFs [27] and in sputum from patients with cystic www.clinical.proteomics-journal.com
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Figure 1. Cysteine Cats in human IPF BALFs. Proteins (20 g/well) were separated by 12% SDSPAGE under reducing conditions, transferred to nitrocellulose membranes, and analyzed with polyclonal antibodies against human Cat B (A), Cat L (B), Cat K (C), and Cat S (D). Black arrows correspond to proforms of Cats and white arrows correspond to mature enzymes. (E) The peptidase activity related to cysteine Cats was measured toward the fluorogenic substrate Z-Phe-Arg-AMC, and expressed as arbitrary fluorescence unit per g of proteins. Median value is shown by solid line (n = 2). n.s, non significant; AUF, arbitrary units of fluorescence; C, BALFs from control patients; IPF, BALFs from IPF patients.
fibrosis where Cat S is predominantly found as a ∼25 kDa active protease [28,29]. In a previous work we proposed that procathepsins could represent a reservoir of activable enzymes in inflammatory BALFs [26]. Here, we failed to induce maturation of proenzymes under in vitro conditions, as reported for BALFs from patients with silicosis [27]. Regardless of cigarette smoking was reported to induce the release of Cats B, L, and S by macrophages [30–34], Western blot patterns did not depend on the fact that BALFs originated from smokers and nonsmokers (data not shown). Moreover, the Cat activity in BALFs was further measured using Z-FR-AMC as substrate. Despite a lesser (nearly twofold lower) peptidase activity for IPF versus control nonsmoker patients, this decrease appears to be not statistically significant (Fig. 1E, p > 0.1). Addition of CA-074 impaired over 85% of the overall activity (data not shown), strengthening that Cat B is the prevailing Cat in BALFs (for review see [13]). Present results are in good agreement with the fact that a similar amount of macrophages, which are the primary source of extracellular Cats in BALFs [13], were found for both control and IPF patients (see Table 1).
3.2 Immunodetection and quantitative analysis of cystatin C There is now a growing body of evidence that cysteine Cats take part in pulmonary homeostasis [14]. Dysregulation of the protease/antiprotease balance in favor to a reduction of the proteolytic activity by endogenous inhibitors may promote progression of the lung fibrosis. Recently we observed an increased protein level of cystatin C, the most potent circulating inhibitor of Cats in primary myofibroblasts from IPF patients (Kasabova & Lalmanach, unpublished data). This raises the issue whether cystatin C could be specifically over-
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expressed in BALFs of fibrotic patients, and questions its potential use as a specific and new biomarker of IPF. Recombinant human cystatin C used as control was detected to a higher molecular mass (circa. 17 kDa) as notified by the supplier, due to the presence of an additional C-terminal 10-His tag. Albeit variations of intensity, cystatin C was detected in all BALFs from IPF patients compared to control samples (Fig. 2A). In addition, pools of both control and IPF samples were prepared, analyzed by Western blotting and quantified by densitometry to give an overall drawing of the immunoreactivity levels of cystatin C in BALFs (Fig. 2B). ELISA assays confirmed that concentrations of cystatin C in IPF samples were highly significantly increased (p < 0.001) (median concentrations: Control, 14 ng/mL; IPF, 27.2 ng/mL) (Fig. 2C). Moreover, the increase of alveolar cystatin C level was significant for each of three IPF grades with a median concentration of 31.9 ng/mL (stage I, p < 0.001), 27 ng/mL (stage II, p < 0.001), and 24.7 ng/mL (stage III, p < 0.05), respectively (Fig. 2D). But there are no confident differences of concentration between the three IPF stages (p > 0.1), suggesting that increased levels of cystatin C are not statistically correlated with the severity of the disease. In routine clinical practice, the diagnosis and severity classification of acute kidney injury is based on a rise in serum creatinine levels. Likewise, concentration of cystatin C that has been also proposed as a promising biomarker during renal injury and clearance failure dramatically increases [35]. Remarkably, no significant variations of clearance (Control, 75.5 ± 6.1 mL/min; IPF, 68 ± 5.4 mL/min) and serum creatinine levels (Control, 89 ± 10.1 mol/mL; IPF, 93 ± 5.6 mol/mL) were observed in the present study (Table 1), supporting that the increased concentration of cystatin C in IPF BALFs did not rely on kidney damage. In addition, for each IPF grade (stages I-III), we did not detect a confident change in alveolar concentration of cystatin C by comparing the samples corresponding to a low
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clearance (60 mL/min) (p > 0.1). Since patients with chronic renal failure do not have greater risks to develop a pulmonary fibrosis, present data support that BALF cystatin C change relate directly to pathophysiological variations associated to lung fibrosis. We noted in the previous paragraph that despite the augmentation of procathepsins (Fig. 1B, C, and D) may correspond to an activatable reservoir of mature forms [26], no significant change of overall Cat-related activities was measured in IPF BALFs. That is most likely associated with the balancing increase in cystatin C in IPF samples. Consequently, we may presume that the ability of zymogens to be processed as mature Cats depends on the protease/antiprotease balance. A hallmark of fibrosis is the increase of ECM components deposition, including an excess of collagen fibers. On the other hand, Cats are potent collagenolytic enzymes and hydrolyze various ECM and basal membrane components (fibronectin, perlecan, nidogen) [36–38]. Accordingly these enzymes are essential for lung homeostasis by participating to ECM and basal membrane turnover and remodeling and their proteolytic potential tightly regulated [39, 40]. In a murine model of bleomycin-induced lung injury, Cat K deficiency exacerbated lung fibrosis, while increased levels of Cat K reduced excessive ECM deposition [41]. In a balanced manner, drug-induced overexpression of Cats K and L might be beneficial in the treatment of lung fibrosis in bleomycin-treated mice [42]. Alternatively, since cystatin C is a highly potent inhibitor of cysteine Cats (1:1 stoichiometric mechanism) that plays a regulatory role in numerous body fluids [7, 11, 12], it is reasonable to speculate that cystatin C may be involved in the pathogenesis of fibrosis.
4
Figure 2. Immunodetection and dosage of cystatin C in human BALFs. (A) Western blot analyses were conducted using a mouse monoclonal antibody against human cystatin C. CC, recombinant human cystatin C (R&D systems) that possesses an additional Cterminal 10 His-tag was used as control (apparent molecular mass of 17 kDa). Some representative samples are shown. (B) Control and IPF BALFs were pooled, respectively, separated by 12% SDSPAGE under reducing conditions and immunoblotted with the anticystatin C antibody. A densitometric analysis was used for quantification. (C) ELISA dosage of human cystatin C (non-IPF versus IPF patients) were performed in duplicates (***p < 0.001). The experiment was repeated twice. (D) ELISA assay for cystatin C according to the severity of the disease (i.e. IPF grades) (stage I, n = 11; stage II, n = 8; stage III, n = 6). Assays were performed in duplicates (***p < 0.001, **p < 0.05). The experiment was repeated twice.
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Concluding remarks
To our knowledge, this is the first attempt to evaluate the protein level of cystatin C in BALFs from IPF patients. We have demonstrated that cystatin C expression is critically increased in IPF BALFS compared to non-IPF BALFs (p < 0.001). Despite this augmentation occurs for the three IPF grades (stages I, II, and III) and apparently does not correlate directly with the severity of the disease, current results raise the question of the use of BALF cystatin C as a possible specific marker of lung fibrosis, beside the validated use of plasma cystatin C as biomarker of renal glomerular failure [11]. Present data suggest also that one of the molecular mechanisms involved in the pathogenesis of lung fibrosis might be the upregulation of cystatin C. Since ECM remodeling is the result of a subtle balance between synthesis and degradation, the rise of cystatin C might promote the development of lung fibrosis by impairing collagenolytic activity of cysteine Cats. More fundamental and mechanistic studies have to be undertaken right now to clarify the putative roles www.clinical.proteomics-journal.com
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Clinical Relevance Human idiopathic pulmonary fibrosis (IPF) that leads to a progressive loss of lung function is a chronic and lethal interstitial disorder. A hallmark of fibrosis is the excessive deposition of extracellular matrix proteins. Currently no therapies have been shown to improve survival, despite some molecules could have beneficial effects. It may be assumed that the efficiency of such drugs could be improved by an earlier diagnosis of IPF. Thus, the identification of new markers would be essential. Besides other proteases, cysteine cathepsins are potent collage-
of cysteine Cats and cystatin C during idiopathic pulmonary fibrosis at both in vivo and in vitro levels. We thank la R´egion Centre (France) for its financial assistance (FibroCat project). This work was also supported by institutional funding from the Institut National de la Sant´e et de la Recherche M´edicale (INSERM). M.K. holds a doctoral fellowship from MENRT (Minist`ere de l’Education Nationale, de la Recherche et de la Technologie, France). The authors have declared no conflict of interest.
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nases that play a critical role in lung homeostasis. Cystatin C, their natural inhibitor, is a valuable serum biomarker of glomerular filtration rate. According to the level of cystatin C is also increased during multiple sclerosis and liver fibrosis, we investigated if cystatin C could be specifically overexpressed during IPF. Increase of cystatin C in IPF bronchoalveolar lavage fluids (p < 0.001) that may reflect dysregulation of proteolytic activity in lung, raise the question of its potential use as a specific and new marker of IPF.
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