Plasma apolipoprotein H levels are different between aspirin induced respiratory diseases and aspirin tolerant asthma

Pulmonary Pharmacology & Therapeutics 27 (2014) 184e189

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Plasma apolipoprotein H levels are different between aspirin induced respiratory diseases and aspirin tolerant asthma Hee-Jeong Kim, Jong-Sook Park, Jeong-Seok Heo, Kuk-Young Moon, Choon-Sik Park* Genome Research Center and Division for Allergy and Respiratory Diseases, Soonchunhyang University Bucheon Hospital, 1174 Jung-dong, Wonmi-gu, Bucheon, Gyeonggi-do 420-767, South Korea

a r t i c l e i n f o

a b s t r a c t

Article history: Received 16 May 2013 Received in revised form 30 September 2013 Accepted 2 October 2013

Aspirin-exacerbated respiratory disease (AERD) has attracted a great deal of attention because of its association with increased asthma severity. To identify plasma biomarkers for the prediction of AERD, the six most abundant plasma proteins (albumin, IgG, antitrypsin, IgA, transferrin, and haptoglobin) in pooled plasma samples were removed using a multiple affinity removal system column. Twodimensional gel electrophoresis (2DE) was used for differential display proteomic analysis of the pooled plasma. Proteins were identified by matrix assisted laser desorption ionization time-of-flight (MALDI-TOF)/TOF. Enzyme-linked immunosorbent assay (ELISA) was performed to identify and quantify apolipoprotein H (Apo H) in plasma from subjects with AERD and aspirin-tolerant asthma (ATA). Eight protein spots showed differences in relative intensity between pooled plasma from subjects with AERD (n ¼ 8) and those with ATA (n ¼ 8). MALDI-TOF/TOF analysis showed decreases in the levels of alpha-fibrinogen precursor, Apo H, fibrin beta, and proapolipoprotein in AERD as compared with ATA, and increases in chain A human complement component C3, 90-kDa heat shock protein, complement component C4a, and kininogen-1 isoform 2. Apo H concentrations were significantly increased in plasma from subjects with ATA than those with AERD and normal controls, as measured by ELISA (P < 0.01). AERD is characterized by changes in the levels of proteins involved in the coagulation and complement pathways. In addition, Apo H is up-regulated in ATA compared to AERD and normal controls, suggesting that Apo H may be involved in different pathogenesis of ATA from AERD. Ó 2013 Published by Elsevier Ltd.

Keywords: Acetylsalicylic acid Asthma Apolipoprotein H Blood plasma Two-dimensional gel electrophoresis

1. Introduction Aspirin-exacerbated respiratory disease (AERD) refers to the development of bronchoconstriction and nasal and ocular manifestations in individuals with asthma following the ingestion of aspirin or other non-steroidal anti-inflammatory drugs (NSAIDs) [23]. Recently, aspirin hypersensitivity has attracted a great deal of attention because of its association with increased asthma severity and possible remodeling of both the upper and lower airways [14]. Out of asthma patients who require emergency mechanical ventilation, 25% are aspirin intolerant [13,20]. However, AERD remains widely underdiagnosed in patients with asthma due to insufficient awareness of the relationship between ASA ingestion and asthma exacerbation. In a multicenter study of 500 patients in 10 European

* Corresponding author. Division of Allergy and Respiratory Medicine, Department of Internal Medicine, Soonchunhyang University Bucheon Hospital, 1174 Jung Dong, Wonmi-Gu, Bucheon, Gyeonggi Do 420-021, South Korea. Tel.: þ82 32 621 5105; fax: þ82 32 621 5023. E-mail addresses: [email protected], [email protected] (C.-S. Park). 1094-5539/$ e see front matter Ó 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.pupt.2013.10.001

countries, 15% of patients were unaware of intolerance to aspirin before aspirin provocation tests were performed [25]. In Korea, the sensitivity and specificity of the history of aspirin hypersensitivity for prediction of AERD were 64.7% and 92.0%, respectively [3]. Thus, the identification of aspirin hypersensitivity, especially hidden cases, is essential for avoiding serious complications. Diagnosis can only be established with certainty by provocation tests using increasing doses of ASA. Oral aspirin challenge (OAC) is the gold standard for confirming the diagnosis [24]. However, OAC is a time-consuming procedure and causes serious complications in some cases. Thus, the development of non-invasive methods for easy diagnosis is necessary to prevent the unexpected complications of aspirin use in susceptible patients. We previously found that plasma levels of human complement components C3a and C4a were elevated in AERD compared to aspirin-tolerant asthma (ATA) at baseline and following aspirin challenge [12]. In addition, we found changes in apolipoprotein A-1, modified albumin, and unknown proteins. However, the presence of highly abundant plasma proteins was a technical limitation of the previous study, impairing the ability to identify proteins whose

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spots in Two-dimensional gel electrophoresis (2DE) images overlapped with those for these highly abundant proteins. In the present study, we removed highly abundant proteins from the plasma before performing 2DE analysis and compared the intensities of protein spots between AERD and ATA. 2. Materials and methods 2.1. Subjects Plasma from subjects with AERD (n ¼ 53) and ATA (n ¼ 53) and normal controls (n ¼ 24) were obtained from a biobank at Soonchunhyang University Hospital, Bucheon, Korea, after a protocol approved by the local ethics committee of Soonchunhyang University Hospital (approval No. Schbc-biobank-2011-007). Normal controls were recruited from spouses of the patients and members of the general population who responded negatively to a screening questionnaire for respiratory symptoms and had an FEV1 (forced expiratory volume in 1 s) value of >75% of the predicted value, a PC20 (concentration causing a 20% fall in FEV1) for methacholine of >10 mg/ml, and normal findings on a simple chest radiograph. All study subjects were Korean and gave informed consent to donate their blood to a biobank in the hospital. All patients were diagnosed by physicians and met the criteria for asthma, as defined by the Global Initiative for Asthma (GINA) guidelines and Online Supplement [1]. The patients with asthma had experienced no exacerbation of asthma or respiratory tract infection in the 6 weeks preceding OAC. OAC was performed with increasing doses of aspirin [9,16] as detailed in Online Supplements. Aspirin-induced bronchospasm, reflected by a decline (%) in FEV1, was calculated as pre-challenge FEV1 minus post-challenge FEV1 divided by pre-challenge FEV1. OAC reactions were categorized into two groups as follows: (1) 15% decrease in FEV1 or nasal reactions (AERD); or (2) <15% decrease in FEV1 without naso-ocular or cutaneous reactions (ATA). Plasma was obtained from peripheral venous blood before OAC. 2.2. Preparation of plasma and 2-dimensional gel electrophoresis (2DE) A 1.6 ml pooled plasma sample was prepared from 200 ml of plasma from each of eight subjects with AERD, and a similar pooled plasma sample was prepared from plasma of eight subjects with ATA. The pooled plasma was diluted 4-fold with equilibration buffer and filtered through a 0.22-mm microcentrifuge filter tube (Agilent Technologies, DE, USA). The six most abundant plasma proteins (albumin, IgG, antitrypsin, IgA, transferrin, and haptoglobin) were removed using a multiple affinity removal system (MARS) high-performance liquid chromatography (HPLC) column (4.6  100 mm; Agilent Technologies) [18]. Following depletion, the flow-through fractions were concentrated by centrifugation in Amicon Ultra-4 tubes (5-kDa molecular weight cut-off, Millipore, MA, USA). The concentrated samples were pooled and stored in aliquots at 70  C for future use. 2DE was performed as previously described [12] as detailed in online supplements. Coomassie-stained spots on the gels were quantified on the basis of their normalized volumes using the following formula: relative spot intensity ¼ spot volume/total of all spot volumes.

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MALDI-TOF [12,22]. MALDI-TOF mass spectrometry (MS) analysis was performed [12] as detailed in Online Supplements. 2.4. Measurement of plasma Apo H levels by ELISA Plasma Apo H levels were measured using a quantitative sandwich enzyme immunoassay kit (Abnova; Teipeh, Taiwan) according to the manufacturer’s instructions. The lower and upper limits of detection ranges were 0.625e40 ng/ml. The plasma was diluted 2105 times with Phosphate buffer saline before assay. Values below the lower limit were assumed to be the lower limit for the purposes of statistical analysis. 2.5. Statistical analysis Statistical analyses were performed with SPSS 10.0. Differences between independent groups and samples were compared using the non-parametric KruskaleWallis H test for continuous data. If differences were found to be statistically significant, ManneWhitney U test was used to analyze differences between pairs of groups. Receiver operating characteristic (ROC) curves were analyzed. All data are expressed as the mean  standard error of mean. Statistical significance was defined as P < 0.05. 3. Results 3.1. Characteristics of patients participating in the study The 106 patients with asthma were classified into two groups (AERD and ATA) according to the results of the OAC (Table 1). Eight subjects with AERD and eight with ATA were selected for 2DE analysis. There were no significant differences in terms of age, onset of age, sex, FVC%, FEV1%, blood eosinophil %, total IgE, atopy frequency, or smoking history between the AERD and ATA patients. 3.2. 2-D electrophoretic analysis of patient plasma samples To examine the differential expression of proteins in ATA and AERD patients, pooled plasma from eight subjects in each group was analyzed by high-resolution 2DE after removal of highabundance proteins. No significant difference in the number of spots was observed among the groups (AERD, 585; ATA, 590) (Fig. 1). By applying the cut-off value of a >3-fold difference in plasma protein expression, as measured by relative intensity on 2DE, we identified eight protein spots that showed differences between the two groups (Fig. 1, Table 2). The relative intensities of the spots numbered 1, 2, 3, and 4 were significantly lower in the AERD group than in the ATA group. In contrast, the intensities of spots 5, 6, 7 and 8 were significantly higher in the AERD group as compared with the ATA group (Fig. 1, Table 2). 3.3. Identification of proteins by MALDI-TOF MS The proteins were identified by MALDI-TOF MS (Table 2). Spots 1 and 3 corresponded to alpha-fibrinogen precursor and fibrin beta, respectively. Spots 2 and 4 were identified as apolipoprotein H and proapolipoprotein, respectively. Spots 5 and 7 were identified as complement C3 and C4a components, respectively. The other two proteins were identified as a 90-kDa heat shock protein and kininogen-1 isoform 2.

2.3. Intra-gel digestion and mass spectrometric analysis

3.4. Concentration of apolipoprotein H in plasma

Differentially expressed protein spots were excised from gels, cut into smaller pieces, and digested with trypsin (Promega) and

In order to validate changes in Apo H protein levels in AERD, a caseecontrol study was conducted. The concentrations of Apo H in

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Table 1 Clinical characteristics of the study subjects. Clinical profile

AERD

ATA

AERD

ATA

NC

Number Age [year (range)] Onset of age [year (range)] Sex (male, %) Atopy (%) Current smoker plus ExSmoker (%) Total IgE (IU/ml) Blood eosinophils (%) FVC%, predicted FEV1%, predicted Decline of FEV1% after OAC

8 47 (33e65) 32 (18e61) 37.5 62.5 37.5 275.6304.8 3.032.7 82.618.7 87.021.4 28.524.1

8 52 (32e64) 45 (24e57) 50.0 62.5 0 492.7179.5 8.75.9 92.38.6 92.613.7 2.94.4*

53 53 (28e80) 42(10e68) 33.9 41.5 30.1 539.0150.8 7.70.8 89.01.9 89.32.5 37.42.3

53 51 (28e84) 38(4e70) 50.9 45.2 35.8 256.752.3 6.550.9 84.92.1 84.62.4 4.80.70*

24 54 (31e79) 44(11e77) 20.8 8.3 20.8 120.444.6 2.50.7 97.72.3 109.32.6 3.80.9

AERD: Aspirin Exacerbated Respiratory Disease; ATA: Aspirin Tolerant Asthma; NC: Normal Control; OAC: Oral aspirin challenge. P-values are obtained using T test or X2 test between AERD and ATA. *P < 0.01 for difference between AERD and ATA.

plasma from AERD patients (n ¼ 53), ATA patients (n ¼ 53), and normal controls (n ¼ 23) are shown in Fig. 2. The plasma concentrations of Apo H were significantly lower in AERD patients as compared with ATA patients (300.00  8.34 mg/ml vs. 348.98  9.18 mg/ml, respectively; P ¼ 0.0001). When compared with plasma Apo H concentrations in normal controls (288.59  17.72 mg/ml), ATA patients had significantly higher levels of Apo H (p ¼ 0.002), while Apo H levels in AERD patients were comparable to those in normal controls. There was a significant inverse correlation between Apo H concentration and the decline of FEV1% after OAC in patients with asthma (r2 ¼ 0.246, P ¼ 0.005) (Fig. 3). 3.5. Receiver operating curve (ROC) analysis of Apo H as a predictor of aspirin-induced respiratory diseases ROC was created for Apo H in AERD and ATA (Fig. 2B). ROC analysis of Apo H gave an area under the curve of 0.717 (Fig. 2B). A cut-off of 315.00 mg/ml for Apo H predicts AERD with a sensitivity of 71.6% and a specificity of 56.6%. 4. Discussion Although it is reasonable to attribute the effects of aspirin to liberation of the 5-lipoxygenase (5-LO) pathway from suppression by endogenous PGE2, we were primarily concerned with other factors that contribute to differences between AERD and ATA. To better understand the molecular basis of this pathology, we

adopted a proteomic approach. In our previous study, among more than 500 protein spots on a 2DE gel, fewer than 10 spots, which included complement components, apolipoproteins AI and AII, and human serum albumin in a complex with myristic acid, were different between aspirin-intolerant asthma (AIA) and ATA [12]. As the initial processing step in the present study, we removed from high-abundance proteins from plasma, including albumin, IgG, antitrypsin, IgA, transferrin, and haptoglobin, because these proteins can mask other candidate proteins in 2DE images. As a result, eight proteins with >3-fold differences in relative intensities on 2DE gels between the two groups were identified. Among them, five were identified as new candidate proteins for AERD (the other threedtwo complement components and one a proapolipoproteindwere identified in our previous study) [12]. These proteins are alpha-fibrinogen precursor, fibrin beta, kininogen, and apolipoprotein H, all of which function in the coagulation pathway, and 90-kDa heat shock protein. These data indicate that an alteration in the coagulation pathway may contribute to the development of aspirin hypersensitivity in asthma. Few coagulation-related changes have been reported in subjects with aspirin hypersensitivity. Subjects with AERD had a significant decrease in euglobulin lysis time during aspirin-induced bronchospasm and an increase in the plasma recalcification time [10], suggesting that activation of fibrinolysis may contribute to the pathogenesis of aspirin intolerance through generalized activation of plasma proteolytic activity. The protein spot for Apo H on the 2DE gel was about 50 kDa with a pI of 6.3. These values are compatible with beta-2-glycoprotein I (B2GPI), a 50-kDa phospholipid-binding protein [2,27]. In the

Fig. 1. Two-dimensional electrophoresis analysis of plasma samples from subjects with aspirin-induced respiratory disease (AERD) (n ¼ 8) and aspirin-tolerant asthma (ATA) (n ¼ 8). Plasma proteins (1 mg) were focused on a pH 3e10 immobilized pH gradient strip, separated by 7.5e20% gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and visualized (as described in the Materials and Methods). Protein spots identified by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)/ TOF (arrows) are labeled with numbers. Spot #13 is shown at higher magnification in the small boxes.

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Table 2 List of differentially expressed proteins identified by MALDI-TOF/TOF. No.

Protein name

Accession no.

Determined sequence

MW(kD)/Pi

Relative intensity

Fold change

Function

14.692 3.093 3.065 3.032 10.554 9.961 9.726 3.308

Coagulation Coagulation Coagulation HDL deficiencies Complement system Signal transduction Complement system Coagulation

AERD ATA 1 2 3 4 5 6 7 8

Alpha-fibrinogen precursor Beta-2-glycoprotein I (apolipoprotein H) Fibrin beta Proapolipoprotein Chain A complement component C3 90 kDa heat shock protein Complement component C4A Kininogen-1 isoform 2

182424 28810 223002 178775 78101267 306891 443671 4504893

K.DSHSLTTNIMEILR.G K.EHSSLAFWK.T K.HGTDDGVVWMNWK.G K.LLDNWDSVTSTFSK.L K.GLEVTITAR.F R.ALLFIPR.R K.AEFQDALEKLNMGITDLQGLR.L K.YFIDFVAR.E

present study, beta-2-glycoprotein I (apolipoprotein H) is down regulated in plasma from subjects with AERD compared to plasma from subjects with ATA. However, ATA patients had significantly higher levels of Apo H (p ¼ 0.002) when compared with plasma Apo H concentrations in normal controls, while Apo H levels in AERD patients were comparable to those in normal controls. This data suggest that functional alteration in the coagulation pathway may be present in aspirin tolerant asthma, and may be normalized in AERD. Evidence supports the alteration of coagulation pathway in asthma. B2GPI is regarded as a natural anticoagulant that inhibits prothrombinase activity on platelets and factor XII activation, and modulates the adenosine diphosphate (ADP)-dependent activation of platelets [15]. An interaction between B2GPI dimers and apolipoprotein E receptor 2 on platelets results in increased platelet adhesion to collagen, probably as a result of the induction of thromboxane synthesis [19]. The main role of TXA2 in asthma is as a potent inducer of bronchospasmodic activity, and an inducer of platelet activation [8]. Recently, platelets were implicated in airway remodeling [21]. In AIA, compelling evidence indicates that excessive arachidonate production precedes exposure to aspirin/

69.767/8.26 50.287/6.34 51.358/7.95 28.944/5.45 71.146/6.82 83.242/4.97 195.195/6.79 48.936/6.29

0.007 0.240 0.081 0.023 0.211 0.088 0.889 0.179

< < < < > > > >

0.107 0.743 0.248 0.069 0.020 0.009 0.091 0.054

NSAIDs. Even under baseline conditions, before aspirin challenge, levels of TXB2 and its metabolites are increased in the urine and airways [11,17]. After aspirin challenge, urine and bronchoalveolar lavage (BAL) fluids from both AIA and ATA patients were reported to contain reduced concentrations of TXB2 [26]. These data suggest that Apo H may contribute to the development of asthma via altered generation of thromboxane. The kinin-kallikrein system generates bradykinin (BK) through proteolytic cleavage of its kininogen precursor, high-molecularweight kininogen, by the enzyme kallikrein [5]. Elevated amounts of tissue kallikrein and kinin are present in the bronchoalveolar spaces of subjects with asthma. Kinin generation may directly contribute to the asthmatic response through edema formation and smooth muscle contraction, and by augmenting release and/or production of preformed mediators (e.g., histamine) and secondary mediators such as leukotrienes and platelet-activating factor [4]. The lectin pathway of the complement system causes oxidative stress in endothelial cells, thereby activating the serine proteases MASP-1 and MASP-2. MASP-1 is able to cleave high-molecular weight kininogen (HK), resulting in BK production [5]. Aspirin interferes the formation of kallikrein, that is essential enzyme for

Fig. 2. Comparison of Apolipoprotein H (Apo H) expression in ATA and AERD subjects. (A) Plasma levels of Apo H were significantly lower in subjects with AERD than in those with ATA (ATA, 348.98  9.18 mg/ml; AERD; 300.00  8.34 mg/ml; P ¼ 0.0001). (B) ROC analysis of Apo H under baseline conditions in AERD and ATA patients gave an area under the curve of 0.717. A cut-off of 315.00 mg/ml for Apo H in AERD under baseline conditions gave a sensitivity of 71.6% and a specificity of 56.6%.

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Acknowledgments Plasma samples were generously provided by the Soonchunhyang University Bucheon Hospital Biobank, a member of the National Biobank of Korea, supported by the Ministry of Health, Welfare and Family Affairs, Republic of Korea. This study was supported by a research grant of Soonchunhyang University and the Ministry of Health, Welfare and Family Affairs, Republic of Korea (HI13C0319). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.pupt.2013.10.001. References

Fig. 3. Correlation of Apolipoprotein H (Apo H) expression and FEV1 declines after aspirin challenge in asthmatics including AERD and ATA. There was a significant inverse correlation between Apo H concentration and the decline of FEV1% after OAC in patients with asthma including AERD and ATA.

kallenogen, thus inhibits the activation of the mediator. Aspirin has been reported to be an activator of the complement system in vitro and in vitro [6,7]. Based on these data, coagulation pathway activation leading to enhanced kinin production may, in concert with complement activation, be associated with development of AERD. To screen differentially expressed proteins on 2DE gels, the cut-off level between the two conditions was set to a 20% fall in FEV1 induced by aspirin challenge. However, the percentage fall in FEV1 induced by aspirin challenge is a continuous value. Therefore, we had to select study subjects with a percentage fall in FEV1 of <5% of that in ATA and those with a percentage fall in FEV1 of >25% of that in AERD to properly discriminate between these two phenotypes. We pooled plasma samples from eight subjects from each group and analyzed them by 2DE. In our opinion, analyzing only eight cases from each group is not optimal, but it is sufficient to look for differences between the two groups in an initial screening study. However, pooling plasma from eight subjects per group may a limitation of the present study. A Korean study [28] demonstrated the three allelic variants of Apo H: APOH*1 (10%), APOH*2 (91.3%) and APOH*3 (7.3%). These genetic variants potentially lead to different levels and functions of Apo H. However, any association studies have not been performed in asthma and aspirin hypersensitivity. DNA genotyping may be a solution for relation of genetic variants and different levels of Apo H in the AERD and ATA subjects. 5. Conclusion Our study is the first to show differences in the plasma expression of proteins involved in coagulation pathways between patients with AERD and those with ATA. We identified four proteinsdalpha-fibrinogen precursor, fibrin beta, kininogen, and Apo Hdthat were significantly up- or down-regulated in AERD patients. Plasma levels of apolipoprotein H were validated in a caseecontrol study, the results of which showed significantly increased levels of Apo H in the plasma of patients with ATA compared with those with AERD and normal controls. The Apo H values in asthma significantly correlated with declines in FEV1 following aspirin challenge. These results suggest that Apo H, and possibly other coagulation factors and complement proteins, contribute to the pathogenesis of ATA as well as AERD.

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