Different Angiogenic Activity in Pulmonary Sarcoidosis and Idiopathic Pulmonary Fibrosis

Original Research INTERSTITIAL LUNG DISEASE

Different Angiogenic Activity in Pulmonary Sarcoidosis and Idiopathic Pulmonary Fibrosis* Katerina M. Antoniou, MD, PhD; Argyris Tzouvelekis, MD; Michael G. Alexandrakis, MD, PhD; Katerina Sfiridaki, MD; Ioanna Tsiligianni, MD, PhD; George Rachiotis, MD, PhD; Nikolaos Tzanakis, MD, PhD; Demosthenes Bouros, MD, PhD, FCCP; Joseph Milic-Emili, MD, PhD; and Nikolaos M. Siafakas, MD, PhD, FCCP

Background: Recent evidence has shown that several chemokines—including those involved in angiogenesis— have been implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF) and sarcoidosis. We speculated that these differences could be attributed to distinct angiogenic and angiostatic profiles. This hypothesis was investigated by estimating the levels of three angiogenic chemokines (growth-related gene [GRO]-␣, epithelial neutrophil-activating protein [ENA]-78, and interleukin [IL]-8), and three angiostatic chemokines (monokine induced by interferon (IFN)-␥ [MIG], IFN-␥–inducible protein [IP]-10, and IFN-␥–inducible T-cell ␣ chemoattractant) in serum and BAL fluid (BALF). Methods: We studied prospectively 20 patients with sarcoidosis (median age, 46 years; range, 25 to 65 years), 20 patients with IPF (median age, 68 years; range, 40 to 75 years), and 10 normal subjects (median age, 39 years; range, 26 to 60 years). Results: The GRO-a serum and BALF levels of IPF patients were found significantly increased in comparison with healthy subjects (799 pg/mL vs 294 pg/mL [p ⴝ 0.022] and 1,827 pg/mL vs 94 pg/mL [p < 0.001], respectively) and sarcoidosis patients (799 pg/mL vs 44 pg/mL [p < 0.001] and 1,827 pg/mL vs 214 pg/mL [p < 0.001], respectively). Moreover, ENA-78 and IL-8 BALF levels in IPF patients were significantly higher compared with sarcoidosis patients (191 pg/mL vs 30 pg/mL [p < 0.001] and 640 pg/mL vs 94 pg/mL [p ⴝ 0.03], respectively). MIG serum levels in IPF patients were found significantly upregulated in comparison with sarcoidosis patients and healthy control subjects. However, MIG and IP-10 BALF levels (1,136 pg/mL vs 66 pg/mL [p < 0.001] and 112 pg/mL vs 56 pg/mL [p ⴝ 0.037], respectively) were significantly higher in sarcoidosis patients compared with IPF patients. Conclusions: Our data suggest distinct angiogenic profiles between IPF and sarcoidosis, indicating a potential different role of CXC chemokines in the local immunologic response in IPF and pulmonary sarcoidosis. (CHEST 2006; 130:982–988) Key words: angiogenesis; angiostasis; CXC chemokines; growth-related gene-␣; interferon-␥–inducible protein-10; interferon-␥–inducible T-cell ␣ chemoattractant; monokine induced by interferon-␥ Abbreviations: ACE ⫽ angiotensin-converting enzyme; BALF ⫽ BAL fluid; CXCL ⫽ CXC ligand; ELISA ⫽ enzymelinked immunosorbent assay; ELR ⫽ three amino acid (glutamic acid-leucine-arginine); ENA ⫽ epithelial neutrophilactivating protein; GRO ⫽ growth-related gene; IFN ⫽ interferon; IL ⫽ interleukin; IP ⫽ interferon-␥–inducible protein; IPF ⫽ idiopathic pulmonary fibrosis; ITAC ⫽ interferon-␥–inducible T-cell ␣ chemoattractant; MIG ⫽ monokine induced by interferon-␥; NS ⫽ not significant; Th1 ⫽ T-helper 1; Th2 ⫽ T-helper 2

lung diseases are a heterogeneous T hegroupinterstitial of diffuse parenchymal lung diseases including sarcoidosis and idiopathic pulmonary fibrosis (IPF). IPF is characterized by tissue damage and exuberant repair with an aberrant wound-healing response leading to severe disruption of the pulmo982

nary architecture.1 Sarcoidosis, however, is a multisystem inflammatory disease of unknown etiology that is characterized by noncaseating epithelioid cell granulomas and the accumulation of CD4⫹ T cells and macrophages at sites of inflammation.2 Recent evidence3 lends support to the notion that several Original Research

cytokines and chemokines—including those involved with angiogenesis— have been implicated in their pathogenesis. Angiogenesis, defined as the process of new blood vessel growth, plays a pivotal role in wound healing and may contribute to the fibroproliferation and extracellular matrix deposition observed in IPF and in advanced stages of sarcoidosis. Neovascularization in fibroproliferative disorders is regulated by an opposing balance between angiogenic and angiostatic factors. Molecules that originally promote angiogenesis include members of the CXC chemokine family that contain a three amino-acid (glutamic acid-leucine-arginine) [ELR] motif such as interleukin (IL)-8/CXC ligand (CXCL)-8, epithelial neutrophil-activating protein (ENA)-78/CXCL-5, and growth-related genes (GROs)-␣, -␤, -␥/CXCL-1, -2, -3.3 By contrast, other members of this unique chemokine family that do not contain the angiogenic ELR motif behave as potent inhibitors of angiogenesis. These are interferon (IFN)-␥–inducible monokine induced by IFN-␥ (MIG)/CXCL-9, IFN-␥– inducible protein (IP)-10/CXCL-10, and IFN-␥inducible T-cell ␣ chemoattractant (ITAC)/CXCL11.3 Several studies4 – 8 have implicated aberrant vascular remodeling in the etiopathogenesis of pulmonary fibrosis. Seminal observations implicating angiogenic activity as an important aspect of progressive fibrosis were originally made by Turner-Warwick4 in 1963. In the last decade, several studies9 –15 suggested a potential role of neovascularization in the etiopathogenesis of IPF. However, the contribution of aberrant vascular remodeling in IPF is still controversial. Immunologic advances in sarcoidosis, however, have revealed a T-helper 1 (Th1) and T-helper 2 (Th2) paradigm with predominance of the Th1 response in its immunopathogenesis.16,17 The last years have seen the emergence of Th1 mediators *From the Departments of Thoracic Medicine (Drs. Antoniou, Tsiligianni, Tzanakis, and Siafakas) and Hematology (Dr. Alexandrakis), University of Crete, Heraklion, Greece; Department of Pneumonology (Drs. Tzouvelekis and Bouros), Democritus University of Thrace, Alexandroupolis, Greece; Department of Hematology (Dr. Sfiridaki), Venizeleion General Hospital, Heraklion, Greece; Department of Experimental Physiology (Dr. Rachiotis), University of Athens, Greece; and Department of Pneumonology (Dr. Milic-Emili), McGill University, Montreal, QC, Canada. No financial or other potential conflicts of interest exist. Manuscript received January 23, 2006; revision accepted March 23, 2006. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (www.chestjournal. org/misc/reprints.shtml). Correspondence to: Nikolaos M. Siafakas, MD, PhD, FCCP, Professor of Thoracic Medicine, Department of Thoracic Medicine, University Hospital, Medical School, University of Crete, Heraklion 71110 Crete, Greece; e-mail: [email protected] DOI: 10.1378/chest.130.4.982 www.chestjournal.org

with pleiotropic properties including the IFN-␥– regulated CXC chemokines that lack the ELR motif (ELR⫺) at the NH2 terminus. So far, there are only few studies in the literature18 –22 that have implicated angiogenesis in the immunomodulatory cascade of sarcoidosis, correlating its immunopathogenesis with members of the angiostatic group of CXC chemokines. While IPF and sarcoidosis are both members of the same disease group, they present with notable differences in terms of immunopathogenesis, clinical course, prognosis, and response to steroid treatment. Based on the hypothesis that these differences may be related to distinct angiogenic and angiostatic profiles and that expression of angiogenic cytokines is associated with the development of pulmonary fibrosis, we assessed levels of three angiogenic (GRO-a, ENA-78, and IL-8) and three angiostatic (MIG, IP-10, and ITAC) chemokines in serum and BAL fluid (BALF) in IPF patients and patients with pulmonary sarcoidosis. The results of the present study support the above hypothesis. Materials and Methods Subjects Twenty consecutive sarcoidosis patients from the sarcoidosis clinic of the University Hospital of Heraklion were enrolled in the study (8 men and 12 women; median age, 46 years; range, 25 to 65 years). Two of them were smokers, and 18 were nonsmokers. The American Thoracic Society/European Respiratory Society/ World Association of Sarcoidosis and Granulomatous Disorders statement2 on sarcoidosis was used for the diagnosis, based on history, clinical symptoms, standard chest radiography, CT, and laboratory tests (serum angiotensin-converting enzyme [ACE]). All patients underwent transbronchial or open-lung biopsy with histopathologic evidence of noncaseating epithelioid cell granulomas. According to chest radiographic classification of sarcoidosis, eight patients had stage I disease (lymphadenopathy alone), seven patients had stage II disease (lymphadenopathy and parenchymal opacities), and five patients had stage III disease (only parenchymal opacities). From January 2000 to 2003, we recruited 20 patients with newly diagnosed IPF (14 men and 6 women; 16 ex-smokers and 4 nonsmokers; median, age 68 years; range, 40 to 75 years). The diagnosis of IPF was based on accepted clinical and imaging criteria1 and was confirmed histologically. Lung biopsy samples were obtained using video-assisted thoracoscopic surgery. The histologic diagnosis was usual interstitial pneumonia in all patients.1 The control group included 10 healthy subjects (5 men and 5 women; median age, 39 years; range, 26 to 60 years). Patients and control subjects with acute respiratory infection during the 6 weeks prior to the study were excluded. The ethics committee of our hospital approved the protocol, and all patients and control subjects gave consent. Spirometry Spirometry was performed using standard protocols (MasterLab 2.12; Jaeger; Wu¨rzburg, Germany).23 CHEST / 130 / 4 / OCTOBER, 2006

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BAL The BAL procedure was well tolerated by all subjects without any adverse events. Fiberoptic bronchoscopy with BAL was performed as part of routine clinical management according to recommended guidelines.24

CXCL-5, IL-8/CXCL-8, MIG/CXCL-9, IP10/CXCL-10, and ITAC/ CXCL-11 levels and pulmonary function tests (FEV1, FVC, total lung capacity, and carbon monoxide transfer coefficient) and serum ACE levels. A p value ⬍ 0.05 was considered statistically significant. Statistical analysis was performed using statistical software (version 10.0; SPSS; Chicago, IL).

BAL Fluid Processing

Results

Recovered BAL fluid (BALF) was filtered through sterile gauze and centrifuged at 400g for 15 min at 4°C. Total cell counts were determined using an improved Neubauer counting chamber, and were expressed as the total number of cells per milliliter of aspirated fluid. The pellet was washed three times with cold phosphate-buffered saline-Dulbecco solution, and the cells were adjusted to a final concentration of 106/mL with RPMI 1640 plus 2% fetal calf serum. The slide preparation was performed as previously reported.24 Assay of Chemokine Levels Using Specific Enzyme-Linked Immunosorbent Assay Measurements of GRO-a/CXCL-1, ENA-78/CXCL-5, IL-8/ CXCL-8, MIG/CXCL-9, IP-10/CXCL-10, and ITAC/CXCL-11 were performed using specific enzyme-linked immunosorbent assays (ELISAs). The ELISA capture and detection antibodies for assaying angiogenic and angiostatic chemokines were selected paired reagents optimized for ELISA performance (Quantikine; R&D Systems; Minneapolis MN). The detection limits for the assays were as follows: 10 pg/mL for GRO-a/CXCL-1, 15 pg/mL for ENA-78/CXCL-5, 7.5 pg/mL for IL-8/CXCL-8, 3.9 pg/mL for MIG/CXCL-9, 1.7 pg/mL for IP-10/CXCL-10 and 13.9 pg/mL for ITAC/CXCL-11. Angiogenic and angiostatic chemokines were quantitated according to the protocols provided by the manufacturer. Although BAL procedures generally have a dilutional effect on the recovery of chemokines, a good correlation was observed between the original values and standardized values by albumin concentrations in BALF (data not shown). Thus, the original values of chemokine levels were reported in this study rather than corrected values by albumin concentrations. Statistical Analysis Results are expressed as mean ⫾ SD and/or median (range). The Mann-Whitney U test was used to compare the levels of GRO-a/ CXCL-1, ENA-78/CXCL-5, IL-8/CXCL-8, MIG/CXCL9, IP-10/ CXCL-10 and ITAC/CXCL-11between patients and control subjects, as well as between IPF and sarcoidosis patients, in both biological fluids. A Spearman correlation coefficient was used to evaluate the correlation between GRO-a/CXCL-1, ENA-78/

The demographic and spirometric data of normal control subjects, patients with IPF, and patients with sarcoidosis are shown in Table 1. The GRO-a serum and BALF levels of IPF patients were found significantly increased in comparison with healthy subjects (799 pg/mL vs 294 pg/mL [p ⫽ 0.022] and 1,827 pg/mL vs 94 pg/mL [p ⬍ 0.001], respectively) and patients with sarcoidosis (799 pg/mL vs 44 pg/mL [p ⬍ 0.001] and 1,827 pg/mL vs 214 pg/mL [p ⬍ 0.001], respectively) [Table 2; Fig 1, top, A; Fig 2, top, A]. Moreover, ENA-78 BALF levels in IPF patients were significantly higher compared with healthy control subjects and sarcoidosis patients (191 pg/mL vs 30 pg/mL [p ⬍ 0.001] and 191 pg/mL vs 41 pg/mL [p ⬍ 0.001], respectively), whereas no significant differences were found between sarcoidosis patients and healthy control subjects (277 pg/mL vs 460 pg/mL and 41 pg/mL vs 30 pg/mL) [Table 2; Fig 1, top, A; Fig 2, top, A]. IL-8 BALF levels were significantly higher in IPF patients than in sarcoidosis patients (640 pg/mL vs 93.5 pg/mL, p ⫽ 0.03), whereas differences were not significant (NS) between serum and BALF levels in IPF patients and control subjects (Table 2; Fig 1, top, A; Fig 2, top, A). Regarding angiostatic mediators, MIG serum levels of IPF patients were significantly higher compared with sarcoidosis patients and healthy control subjects (261 pg/mL vs 55 pg/mL [p ⫽ 0.013] and 261 pg/mL vs 58 pg/mL [p ⫽ 0.043], respectively). However, MIG BALF levels were elevated in sarcoidosis patients in comparison with control subjects and IPF patients (1,136 pg/mL vs 236 pg/mL [p ⫽ 0.017] and 1,136 pg/mL vs 66 pg/mL [p ⬍ 0.001], respectively). IP-10 serum and BALF levels of sarcoidosis patients were found significantly increased in comparison with

Table 1—Demographic and Spirometric Characteristics of the Study Population* Characteristics

Sarcoidosis

IPF

Normal Subjects

Subjects, No. Male/female gender, No. Age, yr Smokers/nonsmokers, No. FVC, % of predicted FEV1, % of predicted Kco, % of predicted ACE serum levels (U/L)

20 12/8 46 (25–65) 2/18 92 ⫾ 7 91 ⫾ 6 83 ⫾ 8 42 ⫾ 7

20 14/6 68 (40–75) 16/4 79 ⫾ 3† 86 ⫾ 4† 67 ⫾ 4†

10 5/5 39 (26–60) 0/10 103 ⫾ 14 101 ⫾ 19 96 ⫾ 6 13 ⫾ 5†

*Data are presented as mean ⫾ SD or median (range) unless otherwise indicated. Kco ⫽ carbon monoxide transfer coefficient. †Statistical significance between sarcoidosis and IPF patients and healthy control subjects (p ⬍ 0.05). 984

Original Research

Table 2—Median Values of Angiogenic and Angiostatic Chemokines in Serum and BALF in the Study Population* Normal Subjects

p Value Sarcoidosis

IPF

Serum

BALF

Variables

Serum

BALF

Serum

BALF

Serum

BALF

C-IPF

C-SARC

IPF-SARC

C-IPF

C-SARC

IPF-SARC

GRO-␣/CXCL-1 ENA-78/CXCL-5 IL-8/CXCL-8 MIG/CXCL-9 IP-10/CXCL-10 ITAC/CXCL-11

294 460 294 58 13 86

93.5 30 94 236 258 126

44 277 44 54.5 163 117

214 41 214 1,136 112 54

799 480 90 261 97 104

1,827 191 640 66 56 43

0.022 NS NS 0.043 0.003 NS

0.034 NS NS NS 0.001 0.009

⬍ 0.001 NS NS 0.013 0.002 NS

0.001 ⬍ 0.001 0.03 NS 0.006 0.015

NS NS NS 0.017 0.043 0.02

⬍ 0.001 ⬍ 0.001 NS ⬍ 0.001 0.037 NS

*C ⫽ control; SARC ⫽ sarcoidosis.

IPF patients (163 pg/mL vs 97 pg/mL [p ⫽ 0.002] and 112 pg/mL vs 56 pg/mL [p ⫽ 0.037], respectively); whereas in comparison with healthy subjects, serum and BALF levels were increased (163 pg/mL vs 13 pg/mL [p ⬍ 0.001]) and decreased (112 pg/mL vs 258 pg/mL [p ⫽ 0.043]), respectively. Finally, differences were NS between IPF and sarcoidosis patients regarding ITAC serum (104 pg/mL vs 117 pg/mL) and BALF (43 pg/mL vs 54 pg/mL) levels. Nevertheless, ITAC BALF levels in both patient groups were significantly downregulated in comparison with healthy control subjects (43 pg/mL vs 126 pg/mL [p ⫽ 0.015] and 54 pg/mL vs 126 pg/mL [p ⫽ 0.02]) [Table 2; Fig 1, top, A; Fig 2, bottom, B]. Discussion To the best of our knowledge, this is the first study to investigate both the local and systemic expression of the CXC chemokines (GRO-a, ENA-78, IL-8, MIG, IP-10, and ITAC) in patients with IPF and pulmonary sarcoidosis without pulmonary fibrosis. Our major finding was the presence of a distinct local and systemic angiogenic profile of CXC chemokines in IPF patients compared to sarcoidosis patients. In particular, BALF (GRO-a and ENA-78) and serum (GRO-a) levels of angiogenic CXC chemokines were found significantly elevated in IPF patients in comparison with sarcoidosis patients. In addition, BALF levels of angiostatic mediators (MIG and IP-10) of the same family were found significantly reduced in IPF compared to sarcoidosis patients. An opposing balance of angiogenic and angiostatic factors regulates angiogenesis.19 Keane et al demonstrated increased angiogenic activity in lung specimens of IPF and experimental fibrosis,5– 6 and described an opposing balance of angiogenic (IL-8, ENA-78) and angiostatic factors (IP-10) that favors angiogenesis.5–7 Earlier data demonstrated that increased production of IL-8 in BAL immune cells is characteristic for IPF and sarcoid patients who show progressive disease25 or chronic disease.26 It has www.chestjournal.org

been also demonstrated that the degree of neutrophilic alveolitis in IPF is reflected by increased serum levels of IL-8, suggesting that the serologic assessment of IL-8 may provide a useful parameter for clinicians in monitoring patients with IPF.27 Moreover, the same group of investigators28 showed that increased percentages of neutrophils in BALF from patients with newly diagnosed pulmonary sarcoidosis were associated with progressive disease and need for treatment. A significant correlation of elevated serial plasma levels of angiogenic mediators with clinical parameters of disease severity in a heterogeneous group of patients with idiopathic interstitial pneumonias has also been reported.29 Additionally, differential profiles of CXC chemokines in patients with IPF and nonspecific interstitial pneumonia have recently been described.30 In line with these findings, our study group demonstrated a distinct local and systemic angiogenic profile in patients with IPF compared to sarcoidosis patients. The latter evidence implicates angiogenesis in the fibrotic (Th2) pathway of interstitial lung diseases and highlights novel noninvasive biomarkers to identify patients who are likely to acquire progressive disease, allowing anti-inflammatory and other treatments to be evaluated or eventually modified before they have failed.31 Despite these data suggesting a potential role of the chemokine molecules in the pathogenesis of the fibrotic process via regulation of angiogenesis, doubt has been cast on the perception that vascular remodeling and angiogenesis play such pivotal role in IPF. In fact, there are several reports that question the role of angiogenesis: vascular density was decreased in fibrotic areas of IPF8 –10 and fibroblastic foci, present at the leading edge of fibrosis and linked to disease progression,11 showed almost a complete lack of capillaries.10,12–14 Decreased levels of vascular endothelial growth factor have been reported in BALF,13 and increased levels of potent angiostatic factors in tissue biopsy homogenates of IPF were shown by Cosgrove et al.15 Moreover, substantial CHEST / 130 / 4 / OCTOBER, 2006

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Figure 1. Top, A: Serum GRO-a, ENA-78, and IL-8 levels in normal control subjects (CTRLS), patients with IPF, and patients with sarcoidosis (SARCO). Values are expressed as median. Bottom, B: Serum MIG, IP-10, and ITAC levels in normal control subjects, patients with IPF, and patients with sarcoidosis.

neovascularization occurs in the fibromyxoid lesions of organizing pneumonia, a disorder that does not usually progress to irreversible fibrosis.15 The concept of disparate activity of the IFN-␥– induced CXC chemokines in the context of Th1-like immune disorders, such as sarcoidosis, was originally raised by Agostini et al,20 who documented an enhanced expression of IP-10 in sarcoid tissues and a positive relationship of BALF IP-10 levels and the degree of T-cell alveolitis, suggesting its pivotal role in ruling the migration of T cells to sites of ongoing inflammation. In addition, Miotto et al21 described a specific for Th1-mediated response upregulation of IP-10 BALF levels, further implicating angiostatic CXC chemokines in the inflammatory cascade of 986

sarcoidosis. Recently, Katoh et al22 reported elevated BALF concentrations of IP-10 and MIG in patients with sarcoidosis and chronic eosinophilic pneumonia. Our study group has recently showed a shift vs local Th1 immunologic response in sarcoidosis32 expressed by upregulation of two major Th1 cytokines: IL-12 and IL-18.33 The results of the current study are in agreement with these early findings. The present study further substantiates the assertion that IFN-␥–induced CXC chemokines are strongly involved in the immunomodulatory cascade of sarcoidosis implicating angiostasis with Th1 immune response. Taken together, our study group revealed that while IPF and sarcoidosis are members of the same Original Research

Figure 2. Top, A: BALF GRO-a, ENA-78, and IL-8 levels in normal control subjects, patients with IPF, and patients with sarcoidosis. Values are expressed as median. Bottom, B: BAL MIG, IP-10, and ITAC levels in normal control subjects, patients with IPF, and patients with sarcoidosis. See Figure 1 legend for expansion of abbreviations.

disease group, they present with distinct local and systemic angiogenic and/or angiostatic profiles. The latter observation could provide scientific basis on the conception that these two disease entities present with significant differences in terms of immunopathogenesis, clinical course, prognosis, and response to treatment. In addition, our findings implicate angiogenesis with the Th2 and angiostasis with the Th1 immune response, suggesting that neovascularization is actively involved in the development of the fibrogenic process and is associated with detrimental prognosis and clinical course.3 Moreover, the latter statement could explain the worst clinical prognosis in patients with sarcoidosis www.chestjournal.org

with pulmonary fibrosis. Therefore it is tempting to speculate that aberrant angiogenesis is responsible for the switch of the immune response from Th1 to Th2 in these patients. Further studies in well-defined subgroups of patients with sarcoidosis with and without pulmonary fibrosis are warranted to elucidate the role of angiogenesis during this pathogenetic process. Although this study exhibited a number of limitations, including major discrepancies regarding the BALF and serum levels of angiogenic and angiostatic chemokines in both populations, the lack of serial measurements, and their relationships with clinical and radiologic parameters of disease severity, these findings demonstrate for the first time CHEST / 130 / 4 / OCTOBER, 2006

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a differential angiogenic profile between patients with IPF and sarcoidosis, indicating a crucial role of impaired angiogenesis in Th1 and Th2 immune paradigms. In conclusion, we showed distinct profiles of angiogenic and angiostatic CXC chemokines in IPF and sarcoidosis patients. Our findings suggest an instrumental role of aberrant angiogenesis in the Th1 and Th2 immune response and may explain differences between these two disease entities in terms of pathogenesis, clinical course, prognosis, and response to treatment. Future studies in large-scale populations are needed to substantiate these observations and elucidate whether an opposing balance between angiogenic and angiostatic mediators that favors angiogenesis shifts the critical balance of Th1 to Th2 toward the Th2 response. References 1 American Thoracic Society/European Respiratory Society International Multidisciplinary Consensus Classification of the Idiopathic Interstitial Pneumonias. Am J Respir Crit Care Med 2002; 165: 277–304 2 Hunninghake GW, Costabel U, Ando M, et al. ATS/ERS/ WASOG statement on sarcoidosis. American Thoracic Society/European Respiratory Society/World Association of Sarcoidosis and Granulomatous Disorders. Sarcoidosis Vasc Diffuse Lung Dis 1999; 16:149 –173 3 Strieter RM, Burdick MD, Gomperts BN, et al. CXC chemokines in angiogenesis. Cytokine Growth Factor Rev 2005; 16:593– 609 4 Turner-Warwick M. Precapillary systemic-pulmonary anastomoses. Thorax 1963; 18:225–237 5 Keane MP, Arenberg DA, Lynch JP III, et al. The CXC chemokines, IL-8 and IP-10, regulate angiogenic activity in idiopathic pulmonary fibrosis. J Immunol 1997; 159:1437–1443 6 Keane MP, Belperio JA, Burdick MD, et al. ENA-78 is an important angiogenic factor in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 2001; 164:2239 –2242 7 Keane MP, Belperio JA, Arenberg DA, et al. IFN-␥-inducible protein-10 attenuates bleomycin-induced pulmonary fibrosis via inhibition of angiogenesis. J Immunol 1999; 163:5686 –5692 8 Cassan SM, Divertie MB, Brown AL Jr. Fine structural morphometry on biopsy specimens of human lung: 2. Diffuse idiopathic pulmonary fibrosis. Chest 1974; 65:275–278 9 Gracey DR, Divertie MB, Brown AL Jr. Alveolar-capillary membrane in idiopathic interstitial pulmonary fibrosis: electron microscopic study of 14 cases. Am Rev Respir Dis 1968; 98:16 –21 10 Renzoni EA, Walsh DA, Salmon M, et al. Interstitial vascularity in fibrosing alveolitis. Am J Respir Crit Care Med 2003; 167:438 – 443 11 Keane MP. Angiogenesis and pulmonary fibrosis: feast or famine? Am J Respir Crit Care Med 2004; 170:207–209 12 Renzoni EA. Neovascularization in idiopathic pulmonary fibrosis: too much or too little? Am J Respir Crit Care Med 2004; 169:1179 –1180 13 Ebina M, Shimizukawa M, Shibata N, et al. Heterogeneous increase in CD34-positive alveolar capillaries in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 2004; 169: 1203–1208 14 Meyer KC, Cardoni A, Xiang ZZ. Vascular endothelial growth 988

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