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Impact of angiopoietin-1 and -2 on clinical course of idiopathic pulmonary fibrosis
Respiratory Medicine 114 (2016) 18e26
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Impact of angiopoietin-1 and -2 on clinical course of idiopathic pulmonary fibrosis Masahiro Uehara a, Noriyuki Enomoto a, b, *, Masashi Mikamo a, Yoshiyuki Oyama a, Masato Kono a, Tomoyuki Fujisawa a, Naoki Inui c, Yutaro Nakamura a, Takafumi Suda a a b c
Second Division, Department of Internal Medicine, Hamamatsu University School of Medicine, Hamamatsu, Japan Health Administration Center, Hamamatsu University School of Medicine, Hamamatsu, Japan Department of Clinical Pharmacology and Therapeutics, Hamamatsu University School of Medicine, Hamamatsu, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 September 2015 Received in revised form 17 January 2016 Accepted 3 March 2016 Available online 5 March 2016
Background: Angiopoietin (Ang) -1 and -2 are glycoproteins that play roles in vascular development, angiogenesis, and lung vascular permeability. Although the serum concentrations of Ang-1 and -2 have been evaluated in patients with sepsis, those in patients with idiopathic pulmonary fibrosis (IPF) have received less attention. Objective: To elucidate the clinical significance of Ang-1 and -2 in patients with IPF. Methods: Seventy-five patients with IPF were retrospectively studied. Serum concentrations of Ang-1 and -2 at diagnosis of IPF were measured by enzyme-linked immunosorbent assay. The relationships of the Ang-1 and -2 concentrations with pulmonary function test results, high-resolution computed tomography findings, histologic findings, occurrence of acute exacerbation of IPF (AE-IPF), and prognosis were evaluated. Results: The median patient age was 68 year-old and the median observation period was 44 months. IPF patients with high Ang-2 concentrations showed a significantly lower forced vital capacity (FVC) (2.28 vs. 2.69 L, respectively; p ¼ 0.047) and lower percent diffusion lung capacity for carbon monoxide (%DLCO) (61.4 vs. 81.4%, respectively; p ¼ 0.015) than patients with low Ang-2 concentrations. Serum Ang-2 concentrations were negatively correlated with %DLCO (r ¼ 0.375, p ¼ 0.021) and the change in % FVC in 12 months (r ¼ 0.348, p ¼ 0.043). The Ang-2 concentration was significantly higher in several patients with AE-IPF than in patients in stable condition (p ¼ 0.011). Finally, patients with high Ang-2 concentrations showed a significantly worse prognosis than those with low Ang-2 concentrations (log-rank p ¼ 0.039). Multivariate analysis showed that the serum Ang-2 concentration, but not the Ang1 concentration, was a significant prognostic factor in patients with IPF (hazard ratio 1.439, p ¼ 0.028). Conclusion: Increases in the serum Ang-2 concentration were associated with disease progression and poor prognosis in patients with IPF. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Angiopoietin Angiogenesis Usual interstitial pneumonia Idiopathic pulmonary fibrosis
1. Introduction Idiopathic pulmonary fibrosis (IPF) is a progressive and lifethreatening interstitial lung disease with an extremely poor prognosis [1e3]. The clinical course of IPF is not uniform and may be rapidly progressive, slowly progressive, or mixed [2]. In addition, acute exacerbation of IPF (AE-IPF) sometimes occurs and has a great
* Corresponding author. Health Administration Center, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu 431-3192, Japan. E-mail address: [email protected] (N. Enomoto). http://dx.doi.org/10.1016/j.rmed.2016.03.001 0954-6111/© 2016 Elsevier Ltd. All rights reserved.
impact on the prognosis [4e6]. However, the mechanisms that determine the clinical course of IPF and occurrence of AE-IPF have not been fully elucidated. The existence of angiogenesis in the lungs is heterogeneous, and the role of angiogenesis in the pathogenesis of IPF is also still controversial [7,8]. Ebina et al. reported that compared with control lungs, the vascular density was higher in those with mild fibrosis and lower in those with the most extensively fibrotic lesions [9]. Therefore, mildly fibrotic areas contain more microvascular structures, and may facilitate the extension of fibrosis in IPF-affected lungs. Moreover, microvascular injury may be the key to the development and pathogenesis of AE-IPF [6,10].
M. Uehara et al. / Respiratory Medicine 114 (2016) 18e26
Several angiogenic factors, including vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF), and several angiostatic factors, including pigment epithelium-derived factor and endostatin, are reportedly related to the development of lung lesion in patients with IPF [8]. Angiopoietin-1 and -2 (Ang-1 and -2) are glycoproteins that play a role in vascular development, angiogenesis, and vascular permeability through the tunica internal endothelial cell kinase-2 (Tie-2) receptor, which is primarily located on endothelial cells [11e13]. Although the changes in serum Ang-1 and -2 concentrations were reported in patients with acute respiratory distress syndrome (ARDS) [14e16], such changes in patients with chronic interstitial pneumonia are not well known. In addition, the relationships between the serum angiopoietin concentration and clinical parameters or prognosis have not been fully elucidated in patients with IPF. In this study, to determine the significance of angiopoietins in the clinical course of IPF, we measured serum Ang-1 and -2 concentrations and examined whether they are related to the clinical, radiological, and pathological findings and outcomes in patients with IPF.
2. Methods 2.1. Study population Seventy-nine patients who were diagnosed with IPF at our hospital from 1995 to 2014 were enrolled. Four patients with AEIPF at their first visit were excluded from the study. Thus, 75 patients with IPF were retrospectively studied. All patients met the 2011 IPF consensus criteria of the American Thoracic Society (ATS)/ European Respiratory Society (ERS)/Japanese Respiratory Society (JRS)/Latin American Thoracic Association (ALAT) [1]. Patients who met the criteria for any connective tissue disorders were excluded from this study. Diagnosis of Lung-dominant connective tissue disorder was based on the original criteria [17]. AE-IPF was diagnosed according to the modified diagnostic criteria described by Collard et al., in 2007 [5]. Briefly, the criteria for a diagnosis of AEIPF were: 1) previous or concurrent diagnosis of IPF, 2) unexplained worsening or development of dyspnea within 30 days, 3) highresolution computed tomography (HRCT) findings of new bilateral ground-glass abnormality and/or consolidation superimposed on a background reticular or honeycomb pattern, 4) no evidence of pulmonary infection, and 5) exclusion of alternative causes including left heart failure, pulmonary embolism, or an identifiable cause of acute lung injury. The study protocol was approved by the Ethical Committee of Hamamatsu University School of Medicine (approval number E14-362, E14-109).
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2.3. Evaluation of pathological findings in surgical lung biopsy specimens The amount of fibroblastic foci was evaluated in surgical lung biopsy (SLB) specimens. Severity of each finding was scored on a scale from 0 to 3: 0, none; 1, mild; 2, moderate; and 3, severe. The specimens were reviewed by two observers; when the score differed between the observers, a consensus was reached after discussion. 2.4. Evaluation of HRCT findings The extent of lung fibrosis on HRCT was scored by the same procedure as that in our previous paper [20]. Briefly, the extent of lung fibrosis was measured using HRCT on slices taken at the bifurcation of the trachea, the base of the lower lobes, and the midpoint. The extent of fibrosis in each lobe was scored using following system: 0, none; 1, 1%e10%; 2, 11%e25%; 3, 26%e50%; 4, 51%e75%; and 5, 76%e100%. The sum of the scores from five lobes (0e25) was used to express the extent of fibrosis throughout each patient's lungs. Usual interstitial pneumonia (UIP) patterns on HRCT were classified into three patterns: UIP-pattern, possible UIPpattern, and inconsistent with UIP-pattern as described in the 2011 IPF consensus criteria of the ATS/ERS/JRS/ALAT [1]. All HRCT scans were reviewed by two observers; when the evaluation differed between the observers, a consensus was reached after discussion. 2.5. Measurement of serum angiopoietins Blood samples were collected at the time of IPF diagnosis. The serum concentrations of Ang-1 and -2 were measured using an enzyme-linked immunosorbent assay (R&D Systems, Inc., Minneapolis, MN, USA). These angiopoietin concentrations were compared with those of age-matched healthy controls. 2.6. Immunohistochemical staining of angiopoietins Angiopoietins were immunohistochemically stained in SLB specimens. Briefly, deparaffinized sections were steeped in 0.3% hydrogen peroxide to inactivate endogenous peroxidase activity, then blocked with 10% normal goat serum. The sections were incubated with rabbit polyclonal antibody against angiopoietins (anti-Ang-1 antibody: anti-ANGPT1, or anti-Ang-2 antibody: antiANGPT2 (C-term), Sigma-Aldrich, Saint Louis, MO, USA). After rinsing with PBS, the sections were incubated with biotinconjugated goat anti-rabbit IgG polyclonal antibody (Nichirey, Tokyo, Japan). The sections were then incubated with streptoavidin-peroxidase complex (Nichirey, Tokyo, Japan). The antigen-antibody complex was visualized with 3, 30 -diaminobenzidine (DAB; Nichirey, Tokyo, Japan) and counterstained with hematoxylin.
2.2. Data collection 2.7. Statistical analysis Clinical data on sex, age, smoking-history, symptoms, treatments, and survival were obtained from the medical records. Laboratory and pulmonary function test results and bronchoalveolar lavage data at the time of IPF diagnosis were also recorded. Disease severity of IPF was assessed using the gender, age, and physiology (GAP) staging system [18] or the JRS severity staging system. The JRS severity staging includes the arterial partial pressure of oxygen (PaO2) at rest and minimum arterial blood hemoglobin saturation (SpO2) during the 6-min walk test (6MWT) [19]. Evaluation of pulmonary hypertension (PH) was performed with echocardiography and/or measurement of brain natriuretic peptide (BNP) in peripheral blood at the diagnosis of IPF.
Statistical analyses were performed using StatView J-4.5 (SAS Institute Inc; Cary, NC, US). Continuous data were compared using the unpaired t-test. Relationships between the serum angiopoietin concentration and serial data were analyzed using Pearson's correlation coefficient. Continuous data in the same patient were compared using the paired t-test. The overall survival of each group was estimated by Kaplan-Meier curves. The log-rank test was used to compare survival between two groups. The effects of variables on the risk of death were modeled using Cox proportional hazards regression. All tests were two-sided and statistical significance was defined as p < 0.05.
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with IPF tended to be lower compared to that in healthy controls (median, 1.9 vs. 2.2 ng/mL, respectively; p ¼ 0.066) (Fig. 1B).
3. Results 3.1. Clinical characteristics and laboratory and physiological findings The clinical characteristics and laboratory and physiological data of the patients are summarized in Table 1. The median age was 68 year-old and the median observation period was 44 months. The median period from the onset of respiratory symptoms such as dyspnea and dry cough was 12 months. Of 75 patients with IPF, 37 were diagnosed by SLB. The median percent forced vital capacity (% FVC) and percent diffusion lung capacity for carbon monoxide (% DLCO) were slightly impaired (76.1% and 70.8%, respectively). Although PaO2 at rest was preserved (median 81.2 mmHg), the minimum SpO2 was low during 6MWT (median, 87%). The serum concentrations of Krebs von den Lungen-6 (KL-6) and surfactant protein D (SP-D) were increased (median, 828 U/mL and 229 ng/mL, respectively). Most patients was classified in stage I, the mildest stage in the severity grading system of both JRS [19] and GAP [18]. Therapeutic interventions were performed in 54 participants (72.0%). Four received corticosteroids alone, 19 pirfenidone alone, 1 N-acetylcysteine alone, 1 intravenous immunoglobulin (IVIG) alone, 17 corticosteroids þ immunosuppressive agents (ISA), 1 corticosteroids þ pirfenidone, 1 pirfenidone þ IVIG, 1 pirfenidone þ sildenafil, 7 corticosteroids þ ISA þ pirfenidone, 1 corticosteroids þ ISA þ N-acetylcysteine, and 1 corticosteroids þ ISA þ pirfenidone þ IVIG. In addition, 19 (25.3%) received long-term oxygen therapy. During the observation period, AE-IPF was diagnosed in 24 patients (32%). Thirty-six patients (48%) died. Among them, 30 (83.3%) died of respiratory insufficiency, including 17 (47.2%) of AE-IPF.
3.3. Comparison between patients with low versus high angiopoietins Patients with IPF were divided into two groups based on the median serum Ang-1 concentration: The Ang-1 high group (18.5 ng/mL) and the Ang-1 low group (<18.5 ng/mL) (Table 2). The Ang-1 high group included all four female patients in the study population, all of whom had a significantly higher KL-6 concentration (p ¼ 0.025) than the patients in the Ang-1 low group. Other physiologic parameters and severity grading stages were not significantly different between the two groups. Next, patients with IPF were divided into two groups based on the median serum Ang2 concentration: Ang-2 high group (1.9 ng/mL) and Ang-2 low group (<1.9 ng/mL) (Table 3). Patients in the Ang-2 high group were significantly older (71.0 vs. 66.5 year-old, respectively; p ¼ 0.013), had a lower FVC (2.28 L vs. 2.69 L, respectively; p ¼ 0.047), had a lower %DLCO (61.4% vs. 81.4%, respectively; p ¼ 0.015), and had a shorter 6MWT distance (417 m vs. 455 m, respectively; p ¼ 0.013) than those in the Ang-2 low group. In addition, the Ang-2 high group included significantly more patients with severe disease based on the JRS severity grading system (p ¼ 0.008). Forty-three patients were assessed with echocardiography and/or measurement of peripheral blood BNP, and 2 (4.7% of 43 patients screened; 2.7% of all patients) were suspected of having mild PH although right heart catheterization was not done. These two patients without left heart disease showed relatively high Ang-2 concentrations (4.3 and 4.5 ng/mL). There was no significant difference in the number of patients who received treatments between the high and low angiopoietins groups (Ang-1, p ¼ 0.225; Ang-2, p ¼ 0.071).
3.2. Evaluation of serum angiopoietins The serum concentrations of Ang-1 and -2 in the 75 patients with IPF were measured at diagnosis of IPF and were compared with those of 52 age-matched healthy controls (Fig. 1). The serum Ang-1 concentration in patients with IPF was significantly lower than that in healthy controls (median, 18.5 vs. 29.0 ng/mL, p ¼ 0.001) (Fig. 1A). The serum Ang-2 concentration in patients
3.4. Relationships between angiopoietin concentrations at diagnosis and physiologic parameters The relationships between the serum Ang-2 concentration and several physiological parameters were evaluated (Fig. 2). The Ang-2 concentration was inversely correlated with the %DLCO (r ¼ 0.375, p ¼ 0.021) (Fig. 2A). The serum Ang-2 concentration in
Table 1 Clinical characteristics, laboratory, and physiologic data of all patients with IPF. n ¼ 75, median (range) Age, year Sex, male/female Smoking, never/ex/current Pack-year of smoking Diagnosis, surgical lung biopsy/clinical, n Period from symptom onset, m Observation period, m %FVC, % %FEV1, % %DLCO, % PaO2 at rest, Torr Distance in 6MWT, m Minimum SpO2 in 6MWT, % Serum KL-6, U/mL Serum SP-D, ng/mL Severity grade of interstitial pneumonia in JRS, Ⅰ/Ⅱ/Ⅲ/Ⅳ/unknown The GAP staging system, Ⅰ/Ⅱ/Ⅲ/unknown Therapeutic intervention, n (%) Acute exacerbation, n (%) Death during the study period, n (%)
68 (47, 90) 71/4 9/48/18 37 (0, 192) 37/38 12 (0, 1364) 44 (1, 217) 76.1 (41.0, 128.1) 81.3 (44.9, 102.4) 70.8 (27.9, 133.6) 81.2 (54.0, 105.7) 427 (100, 580) 87 (68, 98) 828 (312, 3290) 229 (55, 821) 39/5/13/15/3 23/11/2/39 54 (72) 24 (32) 36 (48)
Definition of abbreviations: FVC: forced vital capacity, FEV1: forced expiratory volume in 1 s, DLCO: diffusion lung capacity for carbon monoxide, 6MWT: 6-min walk test, KL-6: Krebs von den Lungen-6, SP-D: surfactant protein D, JRS: Japanese Respiratory Society, GAP: gender, age, and physiology.
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Fig. 1. Serum concentrations of Ang-1 and -2 in patients with IPF. The serum Ang-1 (A) and Ang-2 (B) concentrations at the time of IPF diagnosis were measured using enzymelinked immunosorbent assay in patients, and these angiopoietin concentrations were compared with those of age-matched healthy controls. The serum concentrations of Ang-1 (A) and Ang-2 (B) in 75 patients with IPF and in 52 healthy controls are shown. The horizontal bar represents the median value.
Table 2 Comparison between patients with low and high angiopoietin-1 concentrations.
Age, year Sex, male/female Smoking, never/ex/current Pack-year of smoking FVC, L %FVC, % pred. %DLCO, % pred. PaO2 at rest, Torr Distance in 6MWT, m Minimum SpO2 in 6MWT, % Serum KL-6, U/mL Serum SP-D, ng/mL Severity grade of interstitial pneumonia in JRS, Ⅰ/Ⅱ/Ⅲ/Ⅳ/unknown The GAP staging, Ⅰ/Ⅱ/Ⅲ/unknown HRCT Extent scores on HRCT (full score: 25) UIP-pattern on HRCT, definite/possible/inconsistent Surgical lung biopsy specimens Fibroblastic focus score, 0/1/2/3 BAL Lymphocytes Neutrophils Eosinophils Acute exacerbation, þ/-
Ang-1 low (n ¼ 38, median (range))
Ang-1 high (n ¼ 37, median (range))
p value
68.5 (51, 82) 38/0 4/26/8 32.5 (0, 100) 2.50 (1.26, 3.88) 80.4 (44.0, 108.8) 70.6 (27.9, 133.6) 83.0 (62.0, 105.7) 436 (100, 550) 87 (72, 96) 719 (312, 3030) 203 (55, 821) 22/4/6/5/1 13/4/1/20
68 (47, 90) 33/4 5/22/10 39.5 (0, 192) 2.65 (1.06, 4.45) 72.2 (41.0, 128.1) 71.0 (28.1, 125.9) 79.4 (54.0, 102.0) 420 (124, 580) 85 (68, 98) 989 (354, 3290) 238 (115, 561) 17/1/7/10/2 10/7/1/19
0.319 0.037 0.721 0.227 0.941 0.859 0.652 0.097 0.590 0.331 0.025 0.832 0.248 0.546
9 (3, 18) 27/11/0
10 (3, 19) 21/13/1
0.406 0.407
0/5/4/4
0/8/6/5
0.960
4.4 (0, 27.4) 0.8 (0, 8.4) 0.8 (0, 5.2) 11/27
4.0 (0.6, 36) 0.9 (0, 53.4) 0.5 (0, 3.8) 13/24
0.742 0.194 0.220 0.566
Definition of abbreviations: Ang: angiopoietin, FVC: forced vital capacity, FEV1: forced expiratory volume in 1 s, DLCO: diffusion lung capacity for carbon monoxide, 6MWT: 6min walk test, KL-6: Krebs von den Lungen-6, SP-D: surfactant protein D, JRS: Japanese Respiratory Society, GAP: gender, age, and physiology, UIP: usual interstitial pneumonia, HRCT: high-resolution computed tomography, BAL: broncho-alveolar lavage.
patients with advanced IPF and a %DLCO 70% was significantly higher than that in age-matched healthy controls (median, 2.5 vs. 2.2 ng/mL, respectively; p ¼ 0.031) (Supplementary Figure A.1). Furthermore, the Ang-2 concentration was inversely correlated with the changes in %FVC during the 12 months after measurement of serum Ang-2 (r ¼ 0.348, p ¼ 0.043) (Fig. 2B). Unlike Ang-2, the serum Ang-1 concentration was not associated with physiological parameters.
3.5. Expressions of Ang-1 and -2 in SLB specimens Expression of Ang-1 and -2 was confirmed with immunohistochemical staining of SLB specimens (Fig. 3). In one patient with IPF (case No.55) with a high serum Ang-1 concentration (65.0 ng/mL) and high serum Ang-2 concentration (2.23 ng/mL), Ang-1 was found mainly on vascular smooth muscle cells (Fig. 3A: 100 magnification; 3B: 400 magnification). In addition, Ang-2 was
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Table 3 Comparison between patients with low and high angiopoietin-2 concentrations.
Age, year Sex, male/female Smoking, never/ex/current Pack-year of smoking FVC, L %FVC, % pred. %DLCO, % pred. PaO2 at rest, Torr Distance in 6MWT, m Minimum SpO2 in 6MWT, % Serum KL-6, U/mL Serum SP-D, ng/mL Severity grade of interstitial pneumonia in JRS, Ⅰ/Ⅱ/Ⅲ/Ⅳ/unknown The GAP staging, Ⅰ/Ⅱ/Ⅲ/unknown HRCT Extent scores on HRCT (full score: 25) UIP-pattern on HRCT, definite/possible/inconsistent Surgical lung biopsy specimens Fibroblastic focus score, 0/1/2/3 BAL Lymphocytes Neutrophils Eosinophils Acute exacerbation, þ/-
Ang-2 low (n ¼ 38, median (range))
Ang-2 high (n ¼ 37, median (range))
p value
66.5 (47, 82) 38/0 3/26/9 30.3 (0, 177) 2.69 (1.35, 4.45) 80.9 (44.0, 128.1) 81.4 (33.5, 133.6) 84.3 (54.0, 99.2) 455 (260, 580) 86 (79, 94) 813 (312, 3290) 259 (87, 561) 24/3/1/8/2 12/4/2/20
71 (54, 90) 33/4 6/22/9 50 (0, 192) 2.28 (1.06, 3.89) 75.0 (41.0, 108.8) 61.4 (27.9, 102.0) 79.2 (58.0, 105.7) 417 (100, 516) 87 (68, 98) 868 (359, 3070) 203 (55, 821) 15/2/12/7/1 11/7/0/19
0.013 0.037 0.517 0.118 0.047 0.261 0.015 0.445 0.013 0.899 0.645 0.630 0.008 0.239
9 (3, 18) 21/15/0
11 (3, 19) 27/9/1
0.512 0.198
0/8/6/7
0/5/4/2
0.662
5.5 (0, 36) 0.4 (0, 53.4) 0.8 (0, 5.2) 16/22
3.9 (0, 36) 1.2 (0, 18.8) 0.8 (0, 5.2) 8/29
0.216 0.899 0.473 0.057
Definition of abbreviations: Ang: angiopoietin, FVC: forced vital capacity, FEV1: forced expiratory volume in 1 s, DLCO: diffusion lung capacity for carbon monoxide, 6MWT: 6min walk test, KL-6: Krebs von den Lungen-6, SP-D: surfactant protein D, JRS: Japanese Respiratory Society, GAP: gender, age, and physiology, UIP: usual interstitial pneumonia, HRCT: high-resolution computed tomography, BAL: broncho-alveolar lavage.
Fig. 2. Relationships between the serum Ang-2 concentration and physiologic parameters. The serum Ang-2 concentration was measured at the time of IPF diagnosis. Ang-2 levels were inversely correlated with %DLCO (r ¼ 0.375, p ¼ 0.021) (A). The Ang-2 concentration was also inversely correlated with the changes in %FVC during the 12 months after the measurement of serum Ang-2 (r ¼ 0.348, p ¼ 0.043) (B).
found on endothelial cells of small vessels in the fibrotic lung area (Fig. 3C: 100 magnification; 3D: 400 magnification), although it was not found on endothelial cells of larger arteries or veins. No expression of Ang-1 or -2 was found on fibroblastic foci or normal lung areas.
3.6. Increased serum angiopoietin concentrations at first AE-IPF Serum angiopoietin concentrations at diagnosis and first AE-IPF were serially evaluated in eight patients with IPF. The Ang-1 concentration was higher at first AE-IPF than at diagnosis of IPF in five of eight patients (p ¼ 0.037) (Fig. 4A). The Ang-2 concentration was clearly increased at first AE-IPF (p ¼ 0.011) (Fig. 4B). In fact, the Ang2 concentration was elevated at AE-IPF in seven of eight patients.
3.7. Correlation between angiopoietin concentrations and prognosis in patients with IPF Patients with IPF were divided into two groups based on the median serum Ang-2 concentration: the Ang-2 high group (1.9 ng/mL) and the Ang-2 low group (<1.9 ng/mL). The KaplanMeier plot of survival probability from the time of measurement of Ang-2 is shown in Fig. 5. Patients with a high Ang-2 concentration had significantly lower survival than those with a low Ang-2 concentration (log-rank p ¼ 0.039) (Fig. 5). The 5-year survival rate was 36.1% in the Ang-2 high group and 50.9% in the Ang-2 low group. Survival rates were not different between the Ang-1 high and low groups (log-rank p ¼ 0.351). Univariate Cox proportional hazards models were used to identify factors that predict a poor prognosis in patients with IPF
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Fig. 3. Expressions of angiopoietins in SLB specimens. Expression of Ang-1 and -2 was confirmed by immunohistochemical staining of SLB specimens. In one patient with IPF who showed a high serum Ang-1 concentration (65.0 ng/mL) and a high serum Ang-2 concentration (2.23 ng/mL), expression of Ang-1 was found mainly on vascular smooth muscle cells (A, 100 magnification; B, 400 magnification). Expression of Ang-2 was found on endothelial cells of small vessels in fibrotic lung areas (C, 100 magnification; D, 400 magnification). Representative micrographs are shown.
Fig. 4. Increased serum angiopoietin concentrations at first AE-IPF. The serum concentrations of Ang-1 and -2 at diagnosis and first AE-IPF were serially evaluated in eight patients with IPF. The serum Ang-1 was higher at first AE-IPF than at diagnosis of IPF in five of eight patients (p ¼ 0.037) (A). Ang-2 clearly increased at first AE-IPF in seven of eight patients (p ¼ 0.011) (B). The horizontal bar represents the median value.
(Table 4). The serum Ang-2 concentration, but not the Ang-1 concentration, was a significant prognostic factor (p < 0.001 and p ¼ 0.969, respectively) (Table 4). In addition, age, %FVC, %DLCO, resting PaO2, minimum SpO2 during the 6MWT, serum SP-D concentration, extent score on HRCT, and JRS severity grade also had significant prognostic value (Table 4). Age- and sex-adjusted
multivariate Cox proportional hazards models showed that the serum Ang-2 concentration, but not the Ang-1 concentration, continued to have a significant influence (p ¼ 0.028 and p ¼ 0.633, respectively) (Table 5). Furthermore, %FVC, %DLCO, resting PaO2, extent score on HRCT, JRS severity grade, and serum SP-D concentration still had significant prognostic value.
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Fig. 5. Survival analysis of all patients with IPF. Patients with IPF were divided into two groups based on the median serum Ang-2 concentration at the diagnosis of IPF: the Ang-2 high and Ang-2 low groups. The Kaplan-Meier plot of survival probability from the time of measurement of Ang-2 is shown. Patients with high Ang-2 concentration had significantly lower survival than those with low Ang-2 concentration (log-rank, p ¼ 0.039). The 5-year survival rate was 36.1% in the Ang-2 high group and 50.9% in the Ang-2 low group.
4. Discussion This study was conducted to elucidate the role of angiopoietins in the pathogenesis of IPF, and determine their clinical significance in IPF. We found that the serum Ang-1 concentration was lower in patients with IPF than in healthy controls, and patients with a high Ang-2 concentration had a significantly lower FVC, lower %DLCO, and shorter 6MWT walking distance than those with a low Ang-2 concentration. The serum Ang-2 concentration was negatively
correlated with %DLCO and the change in %FVC during 12 months. Patients with a high Ang-2 concentration had a significantly worse prognosis than those with a low Ang-2 concentration. Finally, the Ang-2 concentration, but not the Ang-1 concentration, was a significantly worse prognostic factor in patients with IPF. Heterogeneous angiogenesis has been reported in patients with IPF; however, whether the degree of angiogenesis is increased in the lungs of these patients remains unknown [8]. Ebina et al. reported that more CD34-positive alveolar capillaries were found in IPF lesions with mild fibrosis, while the vascular density gradually decreased as the degree of IPF-related lung fibrosis increased [9]. These results led us to the hypothesis that angiogenesis in relatively early fibrotic lesion may facilitate the extension of fibrosis in the lungs of patients with IPF. Ang-1 and -2 are glycoproteins that are related to vascular development, angiogenesis, and vascular permeability through the Tie-2 receptor on endothelial cells [11e13]. In addition, Ang-1 and -2 maintain vascular homeostasis [11e13]. Ang-1 is produced by pericytes, smooth muscle cells, and fibroblast, and Ang-1/Tie-2 binding promotes vascular integrity and survival of endothelial cells [21,22]. On the other hand, Ang-2 is stored in Weibel-Palade bodies in endothelial cells and is rapidly released upon endothelial cell activation and damage [23]. Furthermore, Ang-2 is expressed only at sites of vascular remodeling [24] and Ang-2/Tie-2 binding promotes vascular permeability [23,25,26]. Whereas the role of Ang-1 and -2 in patients with ARDS has been evaluated in several studies [14e16], only a few papers have reported the value of Ang-1 and -2 in patients with chronic interstitial pneumonia; the role of these glycoproteins in such patients thus remains unknown. Takahashi et al. reported that the serum Ang-2 concentration was increased in patients with systemic sclerosis with supervening interstitial pneumonia, and that the Ang-2 concentration was correlated with the disease activity of interstitial pneumonia [27]. In addition, Margaritopoulos et al. reported a lower concentration of Ang-1, but not Ang-2, in the broncho-alveolar lavage fluid of patients with IPF than in that of control subjects [28]. In their report, however, the relationships between the concentration of
Table 4 Univariate Cox proportional hazards models of survival. Variable
Age, yr Sex, female Pack-year of smoking Symptom onset, mo FVC, % pred. DLCO, % pred. PaO2 at rest, Torr Distance in 6MWT Minimum SpO2 in 6MWT Serum LDH, IU/L Serum KL-6, U/mL Serum SP-D, ng/mL Extent score on HRCT Existence of honeycombing on HRCT, þ Fibroblastic focus score on SLB Severity grade of interstitial pneumonia in JRS The GAP staging system Lung-dominant CTD, þ Treatments for IPF, þ Acute exacerbation of IPF, þ Serum Ang-1, ng/mL Serum Ang-2, ng/mL
Hazard ratio
1.059 1.970 0.991 1.000 0.960 0.971 0.963 0.998 0.836 1.000 1.000 1.004 1.265 0.790 1.005 1.778 1.682 0.716 0.617 1.238 1.000 1.619
95% CI
p Value
Lower
Upper
1.016 0.468 0.979 0.999 0.941 0.952 0.927 0.995 0.761 0.993 1.000 1.002 1.141 0.405 0.597 1.343 0.869 0.170 0.255 0.626 0.982 1.220
1.103 8.299 1.003 1.001 0.979 0.990 1.000 1.002 0.918 1.001 1.001 1.007 1.402 1.541 1.866 2.353 3.256 3.012 1.495 2.448 1.018 2.149
0.007 0.355 0.155 0.999 <0.001 0.003 0.048 0.383 <0.001 0.712 0.232 <0.001 <0.001 0.488 0.853 <0.001 0.122 0.649 0.285 0.540 0.969 <0.001
Definition of abbreviations: FVC: forced vital capacity, DLCO: diffusion lung capacity for carbon monoxide, 6MWT: 6-min walk test, KL-6: Krebs von den Lungen-6, SP-D: surfactant protein D, HRCT: high-resolution computed tomography, SLB: surgical lung biopsy, JRS: Japanese Respiratory Society, GAP: gender, age, and physiology, CTD: connective tissue disease, Ang: angiopoietin.
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Table 5 Multivariate Cox proportional hazards models of survival adjusted for age and sex. Variable
FVC, % pred. DLCO, % pred. PaO2 at rest, Torr Extent score on HRCT Severity grade of interstitial pneumonia in JRS Serum SP-D, ng/mL Acute exacerbation, þ Serum Ang-1, ng/mL Serum Ang-2, ng/mL
Hazard ratio
95% CI
0.960 0.972 0.950 1.266 1.659 1.004 2.094 1.005 1.439
p Value
Lower
Upper
0.941 0.950 0.914 1.132 1.228 1.002 0.981 0.985 1.039
0.980 0.995 0.988 1.417 2.242 1.007 4.468 1.026 1.992
<0.001 0.016 0.010 <0.001 0.001 <0.001 0.055 0.633 0.028
Definition of abbreviations: FVC: forced vital capacity, DLCO: diffusion lung capacity for carbon monoxide, HRCT: high-resolution computed tomography, JRS: Japanese Respiratory Society, SP-D: surfactant protein D, 6MWT: 6-min walk test, Ang: angiopoietin.
these angiopoietins and clinical parameters or prognosis were not shown in patients with IPF. Consistent with their study, we found significantly lower serum concentrations of serum Ang-1, but not Ang-2, in patients with IPF than in healthy controls. Furthermore, although the serum Ang-2 concentration was not higher in patients with IPF than in healthy controls, patients with high Ang-2 concentrations showed impaired lung functions, more pronounced decrease in %FVC in 12 months, and a worse prognosis than did patients with low Ang-2 concentrations. Therefore, it is likely that Ang-2 is not a diagnostic biomarker, but a prognostic biomarker [29] that may reflect microvascular damage and disease progression in patients with IPF. VEGF, basic FGF, and hypoxia increase the Ang-2 mRNA level in vascular endothelial cells [30]. In addition, activated neutrophil-endothelial interactions contribute to Ang-2 release from endothelial cells in acute lung injury [12]. In fact, VEGF and FGF are reportedly related to the development of lung lesions in patients with IPF [8]. Furthermore, high Ang-2 concentrations were reported in patients with idiopathic pulmonary arterial hypertension [31]. In previous studies, prevalence of PH in advanced IPF was 31.6% [32] while that in mild-moderate IPF was 14% [33]. In our series of patients, whereas only a small number of patients (4.7% of 43 patients screened; 2.7% of all patients) were suspected of having mild PH, these patients showed relatively high Ang-2 concentrations. Thus, these factors may be related to increased Ang-2 release from activated endothelial cells in patients with IPF. The Ang-2 concentration was significantly higher in patients with AE-IPF than in patients in stable condition. AE-IPF has a great impact on the prognosis of IPF [4e6]. However, the mechanisms that determine the occurrence of AE-IPF are unknown. Unusually increased vascular permeability, so called “leaky” vessels, may play a role in the development of AE-IPF [34]. Ang-2/Tie-2 binding reportedly promotes vascular permeability [23,25,26]. On the other hand, Ang-1/Tie-2 binding on endothelial cells promotes vascular integrity and leads to an angiostatic “non-leaky” condition [21,22], competing with Ang-2. Therefore, the balance between Ang-1 and Ang-2 may be important to the development of AE-IPF. In fact, in seven of eight patients of the present study, the serum Ang-2 concentration was 2- to 7-fold higher in patients with AE-IPF than in those in stable condition. We recently reported that the increased Ang-2 concentration in patients with AE-IPF was significantly reduced by treatments with corticosteroids and direct hemoperfusion with a polymyxin B-immobilized fiber column (PMX-DHP) [35]. The interaction between activated neutrophils and endothelial cells induces the release of Ang-2 from endothelial cells in acute lung injury [12]. Thus, Ang-2, which facilitates lung vascular permeability, may also play a role in AE-IPF, and monitoring Ang-2 as a disease activity marker [29] may be beneficial for predicting AE-IPF. Although the Ang-1 concentration also increased
in patients with AE-IPF, this increase may have been a self-limiting response against Ang-2. Our study has a number of limitations. First, the number of participants with IPF was relatively small. Second, this study was retrospectively conducted. Therefore, the severity grade according to the GAP staging system was not sufficiently evaluated due to the lack of physiologic data in several cases. Furthermore, secondary PH was not precisely screened in all cases. Finally, the serum concentrations of angiopoietins were evaluated only at the time of IPF diagnosis in many cases. A larger, prospectively enrolled cohort study would be the ideal study design to confirm our observations. In conclusion, we found that patients with IPF with high Ang-2 concentrations had more severely impaired pulmonary function, a greater decrease in %FVC in 12 months, and a significantly worse prognosis than patients with low Ang-2 concentrations. Ang-2, but not Ang-1, was a significantly worse prognostic factor in patients with IPF. Taken together, our data indicate that Ang-2 may not be a diagnostic biomarker, but a prognostic biomarker and a disease activity biomarker that reflects lung microvascular damage and predicts the progression of IPF. It seems that monitoring the concentrations of these angiopoietins, especially Ang-2, is beneficial for the treatment and management of IPF. Ang-2 may thus become a novel therapeutic target for the treatment of IPF. VEGF and FGF increase the Ang-2 concentration in endothelial cells [30]; therefore, nintedanib, which is an intracellular inhibitor of multiple tyrosine kinases such as VEGF and FGF receptors [36], may be effective for the suppression of Ang-2 in patients with IPF. Further study is needed to elucidate the significance of angiopoietins in IPF in the near future. Conflict of interest statements None. Acknowledgments This study was supported by the Study Group on Diffuse Lung Disease, Scientific Research/Research on Intractable Diseases in the Ministry of Health, Labour and Welfare, Japan. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.rmed.2016.03.001. References [1] G. Raghu, H.R. Collard, J.J. Egan, et al., An official ATS/ERS/JRS/ALAT statement: idiopathic pulmonary fibrosis: evidence-based guidelines for diagnosis and
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management, Am. J. Respir. Crit. care Med. 183 (2011) 788e824. [2] B. Ley, H.R. Collard, T.E. King Jr., Clinical course and prediction of survival in idiopathic pulmonary fibrosis, Am. J. Respir. Crit. Care Med. 183 (2011) 431e440. [3] T.E. King Jr., J.A. Tooze, M.I. Schwarz, et al., Predicting survival in idiopathic pulmonary fibrosis: scoring system and survival model, Am. J. Respir. Crit. care Med. 164 (2001) 1171e1181. [4] Y. Kondoh, H. Taniguchi, Y. Kawabata, et al., Acute exacerbation in idiopathic pulmonary fibrosis. Analysis of clinical and pathologic findings in three cases, Chest 103 (1993) 1808e1812. [5] H.R. Collard, B.B. Moore, K.R. Flaherty, et al., Acute exacerbations of idiopathic pulmonary fibrosis, Am. J. Respir. Crit. care Med. 176 (2007) 636e643. [6] Y. Kondoh, H. Taniguchi, T. Katsuta, et al., Risk factors of acute exacerbation of idiopathic pulmonary fibrosis, Sarcoidosis Vasc. Diffuse Lung Dis. 27 (2010) 103e110. [7] M. Selman, T.E. King, A. Pardo, et al., Idiopathic pulmonary fibrosis: prevailing and evolving hypotheses about its pathogenesis and implications for therapy, Ann. Intern Med. 134 (2001) 136e151. [8] C. Hanumegowda, L. Farkas, M. Kolb, Angiogenesis in pulmonary fibrosis: too much or not enough? Chest 142 (2012) 200e207. [9] M. Ebina, M. Shimizukawa, N. Shibata, et al., Heterogeneous increase in CD34positive alveolar capillaries in idiopathic pulmonary fibrosis, Am. J. Respir. Crit. Care Med. 169 (2004) 1203e1208. [10] K. Kataoka, H. Taniguchi, Y. Kondoh, et al., Recombinant human thrombomodulin in acute exacerbation of idiopathic pulmonary fibrosis, Chest 148 (2015) 436e443. [11] T. Mammoto, A. Jiang, E. Jiang, A. Mammoto, Platelet-rich plasma extract prevents pulmonary edema through angiopoietin-Tie2 signaling, Am. J. Respir. Cell Mol. Biol. 52 (2015) 56e64. [12] J. Lomas-Neira, F. Venet, C.S. Chung, et al., Neutrophil-endothelial interactions mediate angiopoietin-2-associated pulmonary endothelial cell dysfunction in indirect acute lung injury in mice, Am. J. Respir. Cell Mol. Biol. 50 (2014) 193e200. [13] M. van der Heijden, G.P. van Nieuw Amerongen, J. van Bezu, et al., Opposing effects of the angiopoietins on the thrombin-induced permeability of human pulmonary microvascular endothelial cells, PLoS One 6 (2011) e23448. [14] M. van der Heijden, G.P. van Nieuw Amerongen, P. Koolwijk, et al., Angiopoietin-2, permeability oedema, occurrence and severity of ALI/ARDS in septic and non-septic critically ill patients, Thorax 63 (2008) 903e909. [15] I. Ebihara, K. Hirayama, K. Nagai, et al., Angiopoietin balance in septic shock patients treated by direct hemoperfusion with polymyxin b-immobilized fiber, Ther. Apher. dialysis official peer rev. J. Int. Soc. Apher. Jpn. Soc. Apher. Jpn. Soc. Dialysis Ther. 13 (2009) 520e527. [16] I. Ebihara, K. Hirayama, M. Nagai, et al., Angiopoietin balance in septic shock patients with acute lung injury: effect of direct hemoperfusion with polymyxin B-immobilized fiber, Ther. Apher. dialysis official peer rev. J. Int. Soc. Apher. Jpn. Soc. Apher. Jpn. Soc. Dialysis Ther. 15 (2011) 349e354. [17] A. Fischer, S.G. West, J.J. Swigris, et al., Connective tissue disease-associated interstitial lung disease: a call for clarification, Chest 138 (2010) 251e256. [18] B. Ley, C.J. Ryerson, E. Vittinghoff, et al., A multidimensional index and staging system for idiopathic pulmonary fibrosis, Ann. Intern. Med. 156 (2012)
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Impact of angiopoietin-1 and -2 on clinical course of idiopathic pulmonary fibrosis Masahiro Uehara a, Noriyuki Enomoto a, b, *, Masashi Mikamo a, Yoshiyuki Oyama a, Masato Kono a, Tomoyuki Fujisawa a, Naoki Inui c, Yutaro Nakamura a, Takafumi Suda a a b c
Second Division, Department of Internal Medicine, Hamamatsu University School of Medicine, Hamamatsu, Japan Health Administration Center, Hamamatsu University School of Medicine, Hamamatsu, Japan Department of Clinical Pharmacology and Therapeutics, Hamamatsu University School of Medicine, Hamamatsu, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 September 2015 Received in revised form 17 January 2016 Accepted 3 March 2016 Available online 5 March 2016
Background: Angiopoietin (Ang) -1 and -2 are glycoproteins that play roles in vascular development, angiogenesis, and lung vascular permeability. Although the serum concentrations of Ang-1 and -2 have been evaluated in patients with sepsis, those in patients with idiopathic pulmonary fibrosis (IPF) have received less attention. Objective: To elucidate the clinical significance of Ang-1 and -2 in patients with IPF. Methods: Seventy-five patients with IPF were retrospectively studied. Serum concentrations of Ang-1 and -2 at diagnosis of IPF were measured by enzyme-linked immunosorbent assay. The relationships of the Ang-1 and -2 concentrations with pulmonary function test results, high-resolution computed tomography findings, histologic findings, occurrence of acute exacerbation of IPF (AE-IPF), and prognosis were evaluated. Results: The median patient age was 68 year-old and the median observation period was 44 months. IPF patients with high Ang-2 concentrations showed a significantly lower forced vital capacity (FVC) (2.28 vs. 2.69 L, respectively; p ¼ 0.047) and lower percent diffusion lung capacity for carbon monoxide (%DLCO) (61.4 vs. 81.4%, respectively; p ¼ 0.015) than patients with low Ang-2 concentrations. Serum Ang-2 concentrations were negatively correlated with %DLCO (r ¼ 0.375, p ¼ 0.021) and the change in % FVC in 12 months (r ¼ 0.348, p ¼ 0.043). The Ang-2 concentration was significantly higher in several patients with AE-IPF than in patients in stable condition (p ¼ 0.011). Finally, patients with high Ang-2 concentrations showed a significantly worse prognosis than those with low Ang-2 concentrations (log-rank p ¼ 0.039). Multivariate analysis showed that the serum Ang-2 concentration, but not the Ang1 concentration, was a significant prognostic factor in patients with IPF (hazard ratio 1.439, p ¼ 0.028). Conclusion: Increases in the serum Ang-2 concentration were associated with disease progression and poor prognosis in patients with IPF. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Angiopoietin Angiogenesis Usual interstitial pneumonia Idiopathic pulmonary fibrosis
1. Introduction Idiopathic pulmonary fibrosis (IPF) is a progressive and lifethreatening interstitial lung disease with an extremely poor prognosis [1e3]. The clinical course of IPF is not uniform and may be rapidly progressive, slowly progressive, or mixed [2]. In addition, acute exacerbation of IPF (AE-IPF) sometimes occurs and has a great
* Corresponding author. Health Administration Center, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu 431-3192, Japan. E-mail address: [email protected] (N. Enomoto). http://dx.doi.org/10.1016/j.rmed.2016.03.001 0954-6111/© 2016 Elsevier Ltd. All rights reserved.
impact on the prognosis [4e6]. However, the mechanisms that determine the clinical course of IPF and occurrence of AE-IPF have not been fully elucidated. The existence of angiogenesis in the lungs is heterogeneous, and the role of angiogenesis in the pathogenesis of IPF is also still controversial [7,8]. Ebina et al. reported that compared with control lungs, the vascular density was higher in those with mild fibrosis and lower in those with the most extensively fibrotic lesions [9]. Therefore, mildly fibrotic areas contain more microvascular structures, and may facilitate the extension of fibrosis in IPF-affected lungs. Moreover, microvascular injury may be the key to the development and pathogenesis of AE-IPF [6,10].
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Several angiogenic factors, including vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF), and several angiostatic factors, including pigment epithelium-derived factor and endostatin, are reportedly related to the development of lung lesion in patients with IPF [8]. Angiopoietin-1 and -2 (Ang-1 and -2) are glycoproteins that play a role in vascular development, angiogenesis, and vascular permeability through the tunica internal endothelial cell kinase-2 (Tie-2) receptor, which is primarily located on endothelial cells [11e13]. Although the changes in serum Ang-1 and -2 concentrations were reported in patients with acute respiratory distress syndrome (ARDS) [14e16], such changes in patients with chronic interstitial pneumonia are not well known. In addition, the relationships between the serum angiopoietin concentration and clinical parameters or prognosis have not been fully elucidated in patients with IPF. In this study, to determine the significance of angiopoietins in the clinical course of IPF, we measured serum Ang-1 and -2 concentrations and examined whether they are related to the clinical, radiological, and pathological findings and outcomes in patients with IPF.
2. Methods 2.1. Study population Seventy-nine patients who were diagnosed with IPF at our hospital from 1995 to 2014 were enrolled. Four patients with AEIPF at their first visit were excluded from the study. Thus, 75 patients with IPF were retrospectively studied. All patients met the 2011 IPF consensus criteria of the American Thoracic Society (ATS)/ European Respiratory Society (ERS)/Japanese Respiratory Society (JRS)/Latin American Thoracic Association (ALAT) [1]. Patients who met the criteria for any connective tissue disorders were excluded from this study. Diagnosis of Lung-dominant connective tissue disorder was based on the original criteria [17]. AE-IPF was diagnosed according to the modified diagnostic criteria described by Collard et al., in 2007 [5]. Briefly, the criteria for a diagnosis of AEIPF were: 1) previous or concurrent diagnosis of IPF, 2) unexplained worsening or development of dyspnea within 30 days, 3) highresolution computed tomography (HRCT) findings of new bilateral ground-glass abnormality and/or consolidation superimposed on a background reticular or honeycomb pattern, 4) no evidence of pulmonary infection, and 5) exclusion of alternative causes including left heart failure, pulmonary embolism, or an identifiable cause of acute lung injury. The study protocol was approved by the Ethical Committee of Hamamatsu University School of Medicine (approval number E14-362, E14-109).
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2.3. Evaluation of pathological findings in surgical lung biopsy specimens The amount of fibroblastic foci was evaluated in surgical lung biopsy (SLB) specimens. Severity of each finding was scored on a scale from 0 to 3: 0, none; 1, mild; 2, moderate; and 3, severe. The specimens were reviewed by two observers; when the score differed between the observers, a consensus was reached after discussion. 2.4. Evaluation of HRCT findings The extent of lung fibrosis on HRCT was scored by the same procedure as that in our previous paper [20]. Briefly, the extent of lung fibrosis was measured using HRCT on slices taken at the bifurcation of the trachea, the base of the lower lobes, and the midpoint. The extent of fibrosis in each lobe was scored using following system: 0, none; 1, 1%e10%; 2, 11%e25%; 3, 26%e50%; 4, 51%e75%; and 5, 76%e100%. The sum of the scores from five lobes (0e25) was used to express the extent of fibrosis throughout each patient's lungs. Usual interstitial pneumonia (UIP) patterns on HRCT were classified into three patterns: UIP-pattern, possible UIPpattern, and inconsistent with UIP-pattern as described in the 2011 IPF consensus criteria of the ATS/ERS/JRS/ALAT [1]. All HRCT scans were reviewed by two observers; when the evaluation differed between the observers, a consensus was reached after discussion. 2.5. Measurement of serum angiopoietins Blood samples were collected at the time of IPF diagnosis. The serum concentrations of Ang-1 and -2 were measured using an enzyme-linked immunosorbent assay (R&D Systems, Inc., Minneapolis, MN, USA). These angiopoietin concentrations were compared with those of age-matched healthy controls. 2.6. Immunohistochemical staining of angiopoietins Angiopoietins were immunohistochemically stained in SLB specimens. Briefly, deparaffinized sections were steeped in 0.3% hydrogen peroxide to inactivate endogenous peroxidase activity, then blocked with 10% normal goat serum. The sections were incubated with rabbit polyclonal antibody against angiopoietins (anti-Ang-1 antibody: anti-ANGPT1, or anti-Ang-2 antibody: antiANGPT2 (C-term), Sigma-Aldrich, Saint Louis, MO, USA). After rinsing with PBS, the sections were incubated with biotinconjugated goat anti-rabbit IgG polyclonal antibody (Nichirey, Tokyo, Japan). The sections were then incubated with streptoavidin-peroxidase complex (Nichirey, Tokyo, Japan). The antigen-antibody complex was visualized with 3, 30 -diaminobenzidine (DAB; Nichirey, Tokyo, Japan) and counterstained with hematoxylin.
2.2. Data collection 2.7. Statistical analysis Clinical data on sex, age, smoking-history, symptoms, treatments, and survival were obtained from the medical records. Laboratory and pulmonary function test results and bronchoalveolar lavage data at the time of IPF diagnosis were also recorded. Disease severity of IPF was assessed using the gender, age, and physiology (GAP) staging system [18] or the JRS severity staging system. The JRS severity staging includes the arterial partial pressure of oxygen (PaO2) at rest and minimum arterial blood hemoglobin saturation (SpO2) during the 6-min walk test (6MWT) [19]. Evaluation of pulmonary hypertension (PH) was performed with echocardiography and/or measurement of brain natriuretic peptide (BNP) in peripheral blood at the diagnosis of IPF.
Statistical analyses were performed using StatView J-4.5 (SAS Institute Inc; Cary, NC, US). Continuous data were compared using the unpaired t-test. Relationships between the serum angiopoietin concentration and serial data were analyzed using Pearson's correlation coefficient. Continuous data in the same patient were compared using the paired t-test. The overall survival of each group was estimated by Kaplan-Meier curves. The log-rank test was used to compare survival between two groups. The effects of variables on the risk of death were modeled using Cox proportional hazards regression. All tests were two-sided and statistical significance was defined as p < 0.05.
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with IPF tended to be lower compared to that in healthy controls (median, 1.9 vs. 2.2 ng/mL, respectively; p ¼ 0.066) (Fig. 1B).
3. Results 3.1. Clinical characteristics and laboratory and physiological findings The clinical characteristics and laboratory and physiological data of the patients are summarized in Table 1. The median age was 68 year-old and the median observation period was 44 months. The median period from the onset of respiratory symptoms such as dyspnea and dry cough was 12 months. Of 75 patients with IPF, 37 were diagnosed by SLB. The median percent forced vital capacity (% FVC) and percent diffusion lung capacity for carbon monoxide (% DLCO) were slightly impaired (76.1% and 70.8%, respectively). Although PaO2 at rest was preserved (median 81.2 mmHg), the minimum SpO2 was low during 6MWT (median, 87%). The serum concentrations of Krebs von den Lungen-6 (KL-6) and surfactant protein D (SP-D) were increased (median, 828 U/mL and 229 ng/mL, respectively). Most patients was classified in stage I, the mildest stage in the severity grading system of both JRS [19] and GAP [18]. Therapeutic interventions were performed in 54 participants (72.0%). Four received corticosteroids alone, 19 pirfenidone alone, 1 N-acetylcysteine alone, 1 intravenous immunoglobulin (IVIG) alone, 17 corticosteroids þ immunosuppressive agents (ISA), 1 corticosteroids þ pirfenidone, 1 pirfenidone þ IVIG, 1 pirfenidone þ sildenafil, 7 corticosteroids þ ISA þ pirfenidone, 1 corticosteroids þ ISA þ N-acetylcysteine, and 1 corticosteroids þ ISA þ pirfenidone þ IVIG. In addition, 19 (25.3%) received long-term oxygen therapy. During the observation period, AE-IPF was diagnosed in 24 patients (32%). Thirty-six patients (48%) died. Among them, 30 (83.3%) died of respiratory insufficiency, including 17 (47.2%) of AE-IPF.
3.3. Comparison between patients with low versus high angiopoietins Patients with IPF were divided into two groups based on the median serum Ang-1 concentration: The Ang-1 high group (18.5 ng/mL) and the Ang-1 low group (<18.5 ng/mL) (Table 2). The Ang-1 high group included all four female patients in the study population, all of whom had a significantly higher KL-6 concentration (p ¼ 0.025) than the patients in the Ang-1 low group. Other physiologic parameters and severity grading stages were not significantly different between the two groups. Next, patients with IPF were divided into two groups based on the median serum Ang2 concentration: Ang-2 high group (1.9 ng/mL) and Ang-2 low group (<1.9 ng/mL) (Table 3). Patients in the Ang-2 high group were significantly older (71.0 vs. 66.5 year-old, respectively; p ¼ 0.013), had a lower FVC (2.28 L vs. 2.69 L, respectively; p ¼ 0.047), had a lower %DLCO (61.4% vs. 81.4%, respectively; p ¼ 0.015), and had a shorter 6MWT distance (417 m vs. 455 m, respectively; p ¼ 0.013) than those in the Ang-2 low group. In addition, the Ang-2 high group included significantly more patients with severe disease based on the JRS severity grading system (p ¼ 0.008). Forty-three patients were assessed with echocardiography and/or measurement of peripheral blood BNP, and 2 (4.7% of 43 patients screened; 2.7% of all patients) were suspected of having mild PH although right heart catheterization was not done. These two patients without left heart disease showed relatively high Ang-2 concentrations (4.3 and 4.5 ng/mL). There was no significant difference in the number of patients who received treatments between the high and low angiopoietins groups (Ang-1, p ¼ 0.225; Ang-2, p ¼ 0.071).
3.2. Evaluation of serum angiopoietins The serum concentrations of Ang-1 and -2 in the 75 patients with IPF were measured at diagnosis of IPF and were compared with those of 52 age-matched healthy controls (Fig. 1). The serum Ang-1 concentration in patients with IPF was significantly lower than that in healthy controls (median, 18.5 vs. 29.0 ng/mL, p ¼ 0.001) (Fig. 1A). The serum Ang-2 concentration in patients
3.4. Relationships between angiopoietin concentrations at diagnosis and physiologic parameters The relationships between the serum Ang-2 concentration and several physiological parameters were evaluated (Fig. 2). The Ang-2 concentration was inversely correlated with the %DLCO (r ¼ 0.375, p ¼ 0.021) (Fig. 2A). The serum Ang-2 concentration in
Table 1 Clinical characteristics, laboratory, and physiologic data of all patients with IPF. n ¼ 75, median (range) Age, year Sex, male/female Smoking, never/ex/current Pack-year of smoking Diagnosis, surgical lung biopsy/clinical, n Period from symptom onset, m Observation period, m %FVC, % %FEV1, % %DLCO, % PaO2 at rest, Torr Distance in 6MWT, m Minimum SpO2 in 6MWT, % Serum KL-6, U/mL Serum SP-D, ng/mL Severity grade of interstitial pneumonia in JRS, Ⅰ/Ⅱ/Ⅲ/Ⅳ/unknown The GAP staging system, Ⅰ/Ⅱ/Ⅲ/unknown Therapeutic intervention, n (%) Acute exacerbation, n (%) Death during the study period, n (%)
68 (47, 90) 71/4 9/48/18 37 (0, 192) 37/38 12 (0, 1364) 44 (1, 217) 76.1 (41.0, 128.1) 81.3 (44.9, 102.4) 70.8 (27.9, 133.6) 81.2 (54.0, 105.7) 427 (100, 580) 87 (68, 98) 828 (312, 3290) 229 (55, 821) 39/5/13/15/3 23/11/2/39 54 (72) 24 (32) 36 (48)
Definition of abbreviations: FVC: forced vital capacity, FEV1: forced expiratory volume in 1 s, DLCO: diffusion lung capacity for carbon monoxide, 6MWT: 6-min walk test, KL-6: Krebs von den Lungen-6, SP-D: surfactant protein D, JRS: Japanese Respiratory Society, GAP: gender, age, and physiology.
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Fig. 1. Serum concentrations of Ang-1 and -2 in patients with IPF. The serum Ang-1 (A) and Ang-2 (B) concentrations at the time of IPF diagnosis were measured using enzymelinked immunosorbent assay in patients, and these angiopoietin concentrations were compared with those of age-matched healthy controls. The serum concentrations of Ang-1 (A) and Ang-2 (B) in 75 patients with IPF and in 52 healthy controls are shown. The horizontal bar represents the median value.
Table 2 Comparison between patients with low and high angiopoietin-1 concentrations.
Age, year Sex, male/female Smoking, never/ex/current Pack-year of smoking FVC, L %FVC, % pred. %DLCO, % pred. PaO2 at rest, Torr Distance in 6MWT, m Minimum SpO2 in 6MWT, % Serum KL-6, U/mL Serum SP-D, ng/mL Severity grade of interstitial pneumonia in JRS, Ⅰ/Ⅱ/Ⅲ/Ⅳ/unknown The GAP staging, Ⅰ/Ⅱ/Ⅲ/unknown HRCT Extent scores on HRCT (full score: 25) UIP-pattern on HRCT, definite/possible/inconsistent Surgical lung biopsy specimens Fibroblastic focus score, 0/1/2/3 BAL Lymphocytes Neutrophils Eosinophils Acute exacerbation, þ/-
Ang-1 low (n ¼ 38, median (range))
Ang-1 high (n ¼ 37, median (range))
p value
68.5 (51, 82) 38/0 4/26/8 32.5 (0, 100) 2.50 (1.26, 3.88) 80.4 (44.0, 108.8) 70.6 (27.9, 133.6) 83.0 (62.0, 105.7) 436 (100, 550) 87 (72, 96) 719 (312, 3030) 203 (55, 821) 22/4/6/5/1 13/4/1/20
68 (47, 90) 33/4 5/22/10 39.5 (0, 192) 2.65 (1.06, 4.45) 72.2 (41.0, 128.1) 71.0 (28.1, 125.9) 79.4 (54.0, 102.0) 420 (124, 580) 85 (68, 98) 989 (354, 3290) 238 (115, 561) 17/1/7/10/2 10/7/1/19
0.319 0.037 0.721 0.227 0.941 0.859 0.652 0.097 0.590 0.331 0.025 0.832 0.248 0.546
9 (3, 18) 27/11/0
10 (3, 19) 21/13/1
0.406 0.407
0/5/4/4
0/8/6/5
0.960
4.4 (0, 27.4) 0.8 (0, 8.4) 0.8 (0, 5.2) 11/27
4.0 (0.6, 36) 0.9 (0, 53.4) 0.5 (0, 3.8) 13/24
0.742 0.194 0.220 0.566
Definition of abbreviations: Ang: angiopoietin, FVC: forced vital capacity, FEV1: forced expiratory volume in 1 s, DLCO: diffusion lung capacity for carbon monoxide, 6MWT: 6min walk test, KL-6: Krebs von den Lungen-6, SP-D: surfactant protein D, JRS: Japanese Respiratory Society, GAP: gender, age, and physiology, UIP: usual interstitial pneumonia, HRCT: high-resolution computed tomography, BAL: broncho-alveolar lavage.
patients with advanced IPF and a %DLCO 70% was significantly higher than that in age-matched healthy controls (median, 2.5 vs. 2.2 ng/mL, respectively; p ¼ 0.031) (Supplementary Figure A.1). Furthermore, the Ang-2 concentration was inversely correlated with the changes in %FVC during the 12 months after measurement of serum Ang-2 (r ¼ 0.348, p ¼ 0.043) (Fig. 2B). Unlike Ang-2, the serum Ang-1 concentration was not associated with physiological parameters.
3.5. Expressions of Ang-1 and -2 in SLB specimens Expression of Ang-1 and -2 was confirmed with immunohistochemical staining of SLB specimens (Fig. 3). In one patient with IPF (case No.55) with a high serum Ang-1 concentration (65.0 ng/mL) and high serum Ang-2 concentration (2.23 ng/mL), Ang-1 was found mainly on vascular smooth muscle cells (Fig. 3A: 100 magnification; 3B: 400 magnification). In addition, Ang-2 was
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Table 3 Comparison between patients with low and high angiopoietin-2 concentrations.
Age, year Sex, male/female Smoking, never/ex/current Pack-year of smoking FVC, L %FVC, % pred. %DLCO, % pred. PaO2 at rest, Torr Distance in 6MWT, m Minimum SpO2 in 6MWT, % Serum KL-6, U/mL Serum SP-D, ng/mL Severity grade of interstitial pneumonia in JRS, Ⅰ/Ⅱ/Ⅲ/Ⅳ/unknown The GAP staging, Ⅰ/Ⅱ/Ⅲ/unknown HRCT Extent scores on HRCT (full score: 25) UIP-pattern on HRCT, definite/possible/inconsistent Surgical lung biopsy specimens Fibroblastic focus score, 0/1/2/3 BAL Lymphocytes Neutrophils Eosinophils Acute exacerbation, þ/-
Ang-2 low (n ¼ 38, median (range))
Ang-2 high (n ¼ 37, median (range))
p value
66.5 (47, 82) 38/0 3/26/9 30.3 (0, 177) 2.69 (1.35, 4.45) 80.9 (44.0, 128.1) 81.4 (33.5, 133.6) 84.3 (54.0, 99.2) 455 (260, 580) 86 (79, 94) 813 (312, 3290) 259 (87, 561) 24/3/1/8/2 12/4/2/20
71 (54, 90) 33/4 6/22/9 50 (0, 192) 2.28 (1.06, 3.89) 75.0 (41.0, 108.8) 61.4 (27.9, 102.0) 79.2 (58.0, 105.7) 417 (100, 516) 87 (68, 98) 868 (359, 3070) 203 (55, 821) 15/2/12/7/1 11/7/0/19
0.013 0.037 0.517 0.118 0.047 0.261 0.015 0.445 0.013 0.899 0.645 0.630 0.008 0.239
9 (3, 18) 21/15/0
11 (3, 19) 27/9/1
0.512 0.198
0/8/6/7
0/5/4/2
0.662
5.5 (0, 36) 0.4 (0, 53.4) 0.8 (0, 5.2) 16/22
3.9 (0, 36) 1.2 (0, 18.8) 0.8 (0, 5.2) 8/29
0.216 0.899 0.473 0.057
Definition of abbreviations: Ang: angiopoietin, FVC: forced vital capacity, FEV1: forced expiratory volume in 1 s, DLCO: diffusion lung capacity for carbon monoxide, 6MWT: 6min walk test, KL-6: Krebs von den Lungen-6, SP-D: surfactant protein D, JRS: Japanese Respiratory Society, GAP: gender, age, and physiology, UIP: usual interstitial pneumonia, HRCT: high-resolution computed tomography, BAL: broncho-alveolar lavage.
Fig. 2. Relationships between the serum Ang-2 concentration and physiologic parameters. The serum Ang-2 concentration was measured at the time of IPF diagnosis. Ang-2 levels were inversely correlated with %DLCO (r ¼ 0.375, p ¼ 0.021) (A). The Ang-2 concentration was also inversely correlated with the changes in %FVC during the 12 months after the measurement of serum Ang-2 (r ¼ 0.348, p ¼ 0.043) (B).
found on endothelial cells of small vessels in the fibrotic lung area (Fig. 3C: 100 magnification; 3D: 400 magnification), although it was not found on endothelial cells of larger arteries or veins. No expression of Ang-1 or -2 was found on fibroblastic foci or normal lung areas.
3.6. Increased serum angiopoietin concentrations at first AE-IPF Serum angiopoietin concentrations at diagnosis and first AE-IPF were serially evaluated in eight patients with IPF. The Ang-1 concentration was higher at first AE-IPF than at diagnosis of IPF in five of eight patients (p ¼ 0.037) (Fig. 4A). The Ang-2 concentration was clearly increased at first AE-IPF (p ¼ 0.011) (Fig. 4B). In fact, the Ang2 concentration was elevated at AE-IPF in seven of eight patients.
3.7. Correlation between angiopoietin concentrations and prognosis in patients with IPF Patients with IPF were divided into two groups based on the median serum Ang-2 concentration: the Ang-2 high group (1.9 ng/mL) and the Ang-2 low group (<1.9 ng/mL). The KaplanMeier plot of survival probability from the time of measurement of Ang-2 is shown in Fig. 5. Patients with a high Ang-2 concentration had significantly lower survival than those with a low Ang-2 concentration (log-rank p ¼ 0.039) (Fig. 5). The 5-year survival rate was 36.1% in the Ang-2 high group and 50.9% in the Ang-2 low group. Survival rates were not different between the Ang-1 high and low groups (log-rank p ¼ 0.351). Univariate Cox proportional hazards models were used to identify factors that predict a poor prognosis in patients with IPF
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Fig. 3. Expressions of angiopoietins in SLB specimens. Expression of Ang-1 and -2 was confirmed by immunohistochemical staining of SLB specimens. In one patient with IPF who showed a high serum Ang-1 concentration (65.0 ng/mL) and a high serum Ang-2 concentration (2.23 ng/mL), expression of Ang-1 was found mainly on vascular smooth muscle cells (A, 100 magnification; B, 400 magnification). Expression of Ang-2 was found on endothelial cells of small vessels in fibrotic lung areas (C, 100 magnification; D, 400 magnification). Representative micrographs are shown.
Fig. 4. Increased serum angiopoietin concentrations at first AE-IPF. The serum concentrations of Ang-1 and -2 at diagnosis and first AE-IPF were serially evaluated in eight patients with IPF. The serum Ang-1 was higher at first AE-IPF than at diagnosis of IPF in five of eight patients (p ¼ 0.037) (A). Ang-2 clearly increased at first AE-IPF in seven of eight patients (p ¼ 0.011) (B). The horizontal bar represents the median value.
(Table 4). The serum Ang-2 concentration, but not the Ang-1 concentration, was a significant prognostic factor (p < 0.001 and p ¼ 0.969, respectively) (Table 4). In addition, age, %FVC, %DLCO, resting PaO2, minimum SpO2 during the 6MWT, serum SP-D concentration, extent score on HRCT, and JRS severity grade also had significant prognostic value (Table 4). Age- and sex-adjusted
multivariate Cox proportional hazards models showed that the serum Ang-2 concentration, but not the Ang-1 concentration, continued to have a significant influence (p ¼ 0.028 and p ¼ 0.633, respectively) (Table 5). Furthermore, %FVC, %DLCO, resting PaO2, extent score on HRCT, JRS severity grade, and serum SP-D concentration still had significant prognostic value.
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Fig. 5. Survival analysis of all patients with IPF. Patients with IPF were divided into two groups based on the median serum Ang-2 concentration at the diagnosis of IPF: the Ang-2 high and Ang-2 low groups. The Kaplan-Meier plot of survival probability from the time of measurement of Ang-2 is shown. Patients with high Ang-2 concentration had significantly lower survival than those with low Ang-2 concentration (log-rank, p ¼ 0.039). The 5-year survival rate was 36.1% in the Ang-2 high group and 50.9% in the Ang-2 low group.
4. Discussion This study was conducted to elucidate the role of angiopoietins in the pathogenesis of IPF, and determine their clinical significance in IPF. We found that the serum Ang-1 concentration was lower in patients with IPF than in healthy controls, and patients with a high Ang-2 concentration had a significantly lower FVC, lower %DLCO, and shorter 6MWT walking distance than those with a low Ang-2 concentration. The serum Ang-2 concentration was negatively
correlated with %DLCO and the change in %FVC during 12 months. Patients with a high Ang-2 concentration had a significantly worse prognosis than those with a low Ang-2 concentration. Finally, the Ang-2 concentration, but not the Ang-1 concentration, was a significantly worse prognostic factor in patients with IPF. Heterogeneous angiogenesis has been reported in patients with IPF; however, whether the degree of angiogenesis is increased in the lungs of these patients remains unknown [8]. Ebina et al. reported that more CD34-positive alveolar capillaries were found in IPF lesions with mild fibrosis, while the vascular density gradually decreased as the degree of IPF-related lung fibrosis increased [9]. These results led us to the hypothesis that angiogenesis in relatively early fibrotic lesion may facilitate the extension of fibrosis in the lungs of patients with IPF. Ang-1 and -2 are glycoproteins that are related to vascular development, angiogenesis, and vascular permeability through the Tie-2 receptor on endothelial cells [11e13]. In addition, Ang-1 and -2 maintain vascular homeostasis [11e13]. Ang-1 is produced by pericytes, smooth muscle cells, and fibroblast, and Ang-1/Tie-2 binding promotes vascular integrity and survival of endothelial cells [21,22]. On the other hand, Ang-2 is stored in Weibel-Palade bodies in endothelial cells and is rapidly released upon endothelial cell activation and damage [23]. Furthermore, Ang-2 is expressed only at sites of vascular remodeling [24] and Ang-2/Tie-2 binding promotes vascular permeability [23,25,26]. Whereas the role of Ang-1 and -2 in patients with ARDS has been evaluated in several studies [14e16], only a few papers have reported the value of Ang-1 and -2 in patients with chronic interstitial pneumonia; the role of these glycoproteins in such patients thus remains unknown. Takahashi et al. reported that the serum Ang-2 concentration was increased in patients with systemic sclerosis with supervening interstitial pneumonia, and that the Ang-2 concentration was correlated with the disease activity of interstitial pneumonia [27]. In addition, Margaritopoulos et al. reported a lower concentration of Ang-1, but not Ang-2, in the broncho-alveolar lavage fluid of patients with IPF than in that of control subjects [28]. In their report, however, the relationships between the concentration of
Table 4 Univariate Cox proportional hazards models of survival. Variable
Age, yr Sex, female Pack-year of smoking Symptom onset, mo FVC, % pred. DLCO, % pred. PaO2 at rest, Torr Distance in 6MWT Minimum SpO2 in 6MWT Serum LDH, IU/L Serum KL-6, U/mL Serum SP-D, ng/mL Extent score on HRCT Existence of honeycombing on HRCT, þ Fibroblastic focus score on SLB Severity grade of interstitial pneumonia in JRS The GAP staging system Lung-dominant CTD, þ Treatments for IPF, þ Acute exacerbation of IPF, þ Serum Ang-1, ng/mL Serum Ang-2, ng/mL
Hazard ratio
1.059 1.970 0.991 1.000 0.960 0.971 0.963 0.998 0.836 1.000 1.000 1.004 1.265 0.790 1.005 1.778 1.682 0.716 0.617 1.238 1.000 1.619
95% CI
p Value
Lower
Upper
1.016 0.468 0.979 0.999 0.941 0.952 0.927 0.995 0.761 0.993 1.000 1.002 1.141 0.405 0.597 1.343 0.869 0.170 0.255 0.626 0.982 1.220
1.103 8.299 1.003 1.001 0.979 0.990 1.000 1.002 0.918 1.001 1.001 1.007 1.402 1.541 1.866 2.353 3.256 3.012 1.495 2.448 1.018 2.149
0.007 0.355 0.155 0.999 <0.001 0.003 0.048 0.383 <0.001 0.712 0.232 <0.001 <0.001 0.488 0.853 <0.001 0.122 0.649 0.285 0.540 0.969 <0.001
Definition of abbreviations: FVC: forced vital capacity, DLCO: diffusion lung capacity for carbon monoxide, 6MWT: 6-min walk test, KL-6: Krebs von den Lungen-6, SP-D: surfactant protein D, HRCT: high-resolution computed tomography, SLB: surgical lung biopsy, JRS: Japanese Respiratory Society, GAP: gender, age, and physiology, CTD: connective tissue disease, Ang: angiopoietin.
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Table 5 Multivariate Cox proportional hazards models of survival adjusted for age and sex. Variable
FVC, % pred. DLCO, % pred. PaO2 at rest, Torr Extent score on HRCT Severity grade of interstitial pneumonia in JRS Serum SP-D, ng/mL Acute exacerbation, þ Serum Ang-1, ng/mL Serum Ang-2, ng/mL
Hazard ratio
95% CI
0.960 0.972 0.950 1.266 1.659 1.004 2.094 1.005 1.439
p Value
Lower
Upper
0.941 0.950 0.914 1.132 1.228 1.002 0.981 0.985 1.039
0.980 0.995 0.988 1.417 2.242 1.007 4.468 1.026 1.992
<0.001 0.016 0.010 <0.001 0.001 <0.001 0.055 0.633 0.028
Definition of abbreviations: FVC: forced vital capacity, DLCO: diffusion lung capacity for carbon monoxide, HRCT: high-resolution computed tomography, JRS: Japanese Respiratory Society, SP-D: surfactant protein D, 6MWT: 6-min walk test, Ang: angiopoietin.
these angiopoietins and clinical parameters or prognosis were not shown in patients with IPF. Consistent with their study, we found significantly lower serum concentrations of serum Ang-1, but not Ang-2, in patients with IPF than in healthy controls. Furthermore, although the serum Ang-2 concentration was not higher in patients with IPF than in healthy controls, patients with high Ang-2 concentrations showed impaired lung functions, more pronounced decrease in %FVC in 12 months, and a worse prognosis than did patients with low Ang-2 concentrations. Therefore, it is likely that Ang-2 is not a diagnostic biomarker, but a prognostic biomarker [29] that may reflect microvascular damage and disease progression in patients with IPF. VEGF, basic FGF, and hypoxia increase the Ang-2 mRNA level in vascular endothelial cells [30]. In addition, activated neutrophil-endothelial interactions contribute to Ang-2 release from endothelial cells in acute lung injury [12]. In fact, VEGF and FGF are reportedly related to the development of lung lesions in patients with IPF [8]. Furthermore, high Ang-2 concentrations were reported in patients with idiopathic pulmonary arterial hypertension [31]. In previous studies, prevalence of PH in advanced IPF was 31.6% [32] while that in mild-moderate IPF was 14% [33]. In our series of patients, whereas only a small number of patients (4.7% of 43 patients screened; 2.7% of all patients) were suspected of having mild PH, these patients showed relatively high Ang-2 concentrations. Thus, these factors may be related to increased Ang-2 release from activated endothelial cells in patients with IPF. The Ang-2 concentration was significantly higher in patients with AE-IPF than in patients in stable condition. AE-IPF has a great impact on the prognosis of IPF [4e6]. However, the mechanisms that determine the occurrence of AE-IPF are unknown. Unusually increased vascular permeability, so called “leaky” vessels, may play a role in the development of AE-IPF [34]. Ang-2/Tie-2 binding reportedly promotes vascular permeability [23,25,26]. On the other hand, Ang-1/Tie-2 binding on endothelial cells promotes vascular integrity and leads to an angiostatic “non-leaky” condition [21,22], competing with Ang-2. Therefore, the balance between Ang-1 and Ang-2 may be important to the development of AE-IPF. In fact, in seven of eight patients of the present study, the serum Ang-2 concentration was 2- to 7-fold higher in patients with AE-IPF than in those in stable condition. We recently reported that the increased Ang-2 concentration in patients with AE-IPF was significantly reduced by treatments with corticosteroids and direct hemoperfusion with a polymyxin B-immobilized fiber column (PMX-DHP) [35]. The interaction between activated neutrophils and endothelial cells induces the release of Ang-2 from endothelial cells in acute lung injury [12]. Thus, Ang-2, which facilitates lung vascular permeability, may also play a role in AE-IPF, and monitoring Ang-2 as a disease activity marker [29] may be beneficial for predicting AE-IPF. Although the Ang-1 concentration also increased
in patients with AE-IPF, this increase may have been a self-limiting response against Ang-2. Our study has a number of limitations. First, the number of participants with IPF was relatively small. Second, this study was retrospectively conducted. Therefore, the severity grade according to the GAP staging system was not sufficiently evaluated due to the lack of physiologic data in several cases. Furthermore, secondary PH was not precisely screened in all cases. Finally, the serum concentrations of angiopoietins were evaluated only at the time of IPF diagnosis in many cases. A larger, prospectively enrolled cohort study would be the ideal study design to confirm our observations. In conclusion, we found that patients with IPF with high Ang-2 concentrations had more severely impaired pulmonary function, a greater decrease in %FVC in 12 months, and a significantly worse prognosis than patients with low Ang-2 concentrations. Ang-2, but not Ang-1, was a significantly worse prognostic factor in patients with IPF. Taken together, our data indicate that Ang-2 may not be a diagnostic biomarker, but a prognostic biomarker and a disease activity biomarker that reflects lung microvascular damage and predicts the progression of IPF. It seems that monitoring the concentrations of these angiopoietins, especially Ang-2, is beneficial for the treatment and management of IPF. Ang-2 may thus become a novel therapeutic target for the treatment of IPF. VEGF and FGF increase the Ang-2 concentration in endothelial cells [30]; therefore, nintedanib, which is an intracellular inhibitor of multiple tyrosine kinases such as VEGF and FGF receptors [36], may be effective for the suppression of Ang-2 in patients with IPF. Further study is needed to elucidate the significance of angiopoietins in IPF in the near future. Conflict of interest statements None. Acknowledgments This study was supported by the Study Group on Diffuse Lung Disease, Scientific Research/Research on Intractable Diseases in the Ministry of Health, Labour and Welfare, Japan. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.rmed.2016.03.001. References [1] G. Raghu, H.R. Collard, J.J. Egan, et al., An official ATS/ERS/JRS/ALAT statement: idiopathic pulmonary fibrosis: evidence-based guidelines for diagnosis and
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