Eur Respir J 2013; 41: 1324–1330 DOI: 10.1183/09031936.0084112 CopyrightßERS 2013 Pulmonary arteriole gene expression signature in idiopathic pulmonary fibrosis Nina M. Patel*,#, Steven M. Kawut", Sanja Jelic*, Selim M. Arcasoy*,#,+, David J. Lederer*,#,+ and Alain C. Borczuk1 ABSTRACT: A third of patients with idiopathic pulmonary fibrosis (IPF) develop pulmonary AFFILIATIONS hypertension (PH-IPF), which is associated with increased mortality. Whether an altered gene *Division of Pulmonary, Allergy and Critical Care Medicine, Columbia expression profile in the pulmonary vasculature precedes the clinical onset of PH-IPF is unknown. University, New York, NY, We compared gene expression in the pulmonary vasculature of IPF patients with and without PH #Interstitial Lung Disease Program, with controls. New York Presbyterian Hospital, New Pulmonary arterioles were isolated using laser capture microdissection from 16 IPF patients: York, NY, "Dept of Medicine and the Center for eight with PH (PH-IPF) and eight with no PH (NPH-IPF), and seven controls. Probe was prepared Clinical Epidemiology and from extracted RNA, and hybridised to Affymetrix Hu133 2.0 Plus genechips. Biometric Research Biostatistics, Perelman School of Branch array tools and Ingenuity Pathway Analysis software were used for analysis of the Medicine, University of Pennsylvania, microarray data. Philadelphia, PA, +Lung Transplantation Program, New Univariate analysis revealed 255 genes that distinguished IPF arterioles from controls York Presbyterian Hospital, New York, (p,0.001). Mediators of vascular smooth muscle and endothelial cell proliferation, Wnt signalling NY, and and apoptosis were differentially expressed in IPF arterioles. Unsupervised and supervised 1Dept of Pathology and Cell Biology, clustering analyses revealed similar gene expression in PH-IPF and NPH-IPF arterioles. Columbia University, New York, NY, USA. The pulmonary arteriolar gene expression profile is similar in IPF patients with and without coexistent PH. Pathways involved in vascular proliferation and aberrant apoptosis, which may CORRESPONDENCE contribute to pulmonary vascular remodelling, are activated in IPF patients. N.M. Patel Division of Pulmonary, Allergy and Critical Care Medicine KEYWORDS: DNA microarray, laser capture microdissection, pulmonary arterial hypertension, Columbia University usual interstitial pneumonia 622 W. 168th Street PH 8 East diopathic pulmonary fibrosis (IPF) is a studies have utilised DNA microarrays to eval- Room 101 New York uate gene expression in idiopathic pulmonary debilitating and fatal diffuse parenchymal NY 10032 arterial hypertension (PAH) and PH-IPF. Genes I lung disease with a median survival of USA ,3 years. More than a third of patients with IPF associated with transforming growth factor E-mail: [email protected] already have pulmonary hypertension (PH-IPF) (TGF)-b, extracellular signal-related kinase (ERK)/ at the time of initial evaluation for lung trans- mitogen-activated protein kinase (MAPK), platelet- Received: plantation, with an overwhelming majority derived growth factor (PDGF) and cAMP signalling May 29 2012 developing clinically overt PH by the time of are differentially expressed in whole-lung homo- Accepted after revision: transplantation [1, 2]. It is unclear if the subset of genates from patients with PAH compared with Aug 29 2012 First published online: IPF patients with PH have a distinct vascular patients with PH-IPF [4]. Decreased expression of Sept 20 2012 molecular phenotype, or whether all patients angiogenic genes and upregulation of genes asso- with IPF have molecular differences in pulmon- ciated with vascular remodelling have been ary vascular gene expression compared to con- observed in whole-lung homogenates in PH-IPF trols. It is valuable to discern the mechanism of [5]. Considering that the pulmonary vasculature pulmonary vascular dysfunction in IPF, as PH- accounts for a relatively small portion of gene IPF is associated with decreased functional status expression in whole-lung homogenates, differences and an increased risk of death [1, 3]. in gene expression observed in these studies may predominantly reflect changes in the lung parench- Pulmonary arterioles from patients with PH-IPF yma rather than the pulmonary vasculature. exhibit marked intimal hypertrophy, medial Ascertaining directly whether gene expression is smooth muscle cell proliferation and adventitial altered in the pulmonary vasculature of IPF patients thickening due to fibroblast accumulation and with and without PH is clinically relevant as extracellular matrix deposition [1]. Previous differences between the two groups would imply European Respiratory Journal Print ISSN 0903-1936 This article has supplementary material available from www.erj.ersjournals.com Online ISSN 1399-3003 1324 VOLUME 41 NUMBER 6 EUROPEAN RESPIRATORY JOURNAL N.M. PATEL ET AL. IDIOPATHIC PULMONARY FIBROSIS distinct vascular phenotypes. Differences in vascular gene and biotinylate RNA for a minimum goal of 15 mg RNA for expression in IPF compared with controls would suggest specific hybridisation. Target cRNA was hybridised to Affymetrix pathologic processes in IPF, which might lead to pulmonary Hu133 2.0 Plus oligonucleotide arrays (Affymetrix, Santa vascular remodelling. Clara, CA, USA). Biometric Research Branch array tools software v3.7.2. (Richard Simon and Amy Lam at the Thus, we assessed the gene expression of laser capture National Cancer Institute, Bethesda, MA, USA) was used for microdissected (LCM) pulmonary arterioles from IPF patients normalisation, filtering and initial quality control assessment free of PH (NPH-IPF) and those with coexistent PH and of the genechip data. Supervised analyses of differential gene compared it with controls. We sought to identify genes and expression between class assignments was performed using biological pathways that could potentially mediate the devel- univariate analyses with p,0.001. The false discovery rate for opment of vascular remodelling in IPF. Some of the results all comparisons was set at q,10%. A permutation analysis was have previously been reported as an abstract [6]. also performed utilising 10 000 random permutations, with a global p,0.01. Gene lists were further categorised into METHODS biological pathways using Ingenuity Pathways Analysis (IPA; Study population Ingenuity Systems, Redwood City, CA, USA). Please see the online supplementary material for full details. We identified potentially eligible study subjects by querying Paraffinised tissue blocks of 10 IPF cases (five PH-IPF and five the Columbia University Tissue Bank database for the terms NPH-IPF) and five normal donor lungs were sectioned and ‘‘lung’’ and ‘‘usual interstitial pneumonia’’ (UIP) and/or stained for antibodies against secreted frizzled-related protein ‘‘pulmonary hypertension’’. We included IPF patients who (SFRP)-2 (monoclonal rabbit antihuman) (Sigma-Aldrich Corp. met American Thoracic Society criteria (previous and current) St Louis, MO, USA), SPARC-related modular calcium binding for a diagnosis of IPF [7, 8]. Patients with IPF were considered (SMOC)-2 (polyclonal rabbit antihuman) (Lifespan Biosciences in the PH-IPF group if they had a mean pulmonary arterial Inc., Seattle, WA, USA) and signal transducer and activator of pressure (mPAP) .25 mmHg with a pulmonary capillary transcription (STAT1) (monoclonal rabbit antihuman) (Sigma) wedge pressure f15 mmHg measured by right heart cathe- using the DAKO Envision Dual Link kit (Dako, Glostrup, terisation, meeting World Health Organization (WHO) group 3 Denmark). Examination for statistical significance was per- criteria [9, 10]. IPF patients were included in the NPH-IPF formed using Fisher’s exact test (SPSS, v16.0; IBM North group if they had mPAP f25 mmHg. America, New York, NY, USA). We excluded patients with clinical evidence of collagen vascular RESULTS disease, another known aetiology for pulmonary hypertension We included eight patients with PH-IPF, eight with NPH-IPF (e.g. chronic obstructive pulmonary disease, obstructive sleep and seven controls (table 1). IPF patients were older than the apnoea and thromboembolic disease), left ventricular dysfunc- controls and predominantly male. Patients with PH-IPF and tion or left-sided valvular disease, missing haemodynamic or NPH-IPF were similar with regards to age, sex, race, smoking pulmonary function data and age ,18 years. history and lung function. Pulmonary function data revealed the presence of a restrictive impairment and the absence of 15 IPF samples were obtained from lung explant and one from obstructive physiology in both groups. One patient had mild surgical lung biopsy. Specimens from four unused donor lung upper lobe emphysema by computed tomography (CT); no shavings and three normal lung sections obtained during patients had significant emphysema histologically or by chest resection of pulmonary carcinoid tumours were used as CT. Notably, the degree of PH present in the PH-IPF group controls. Approval and a waiver of consent for clinical data was mild, with mPAP of 29 mmHg. collection were obtained from the Columbia University Institutional Review Board (IRB-AAAC0695). The raw gene expression values for four commonly expressed endothelial and epithelial genes were examined to confirm Processing and LCM of tissue samples the isolation of pulmonary arterioles while minimising the All lung tissues were obtained peri-operatively, snap-frozen in inclusion of lung parenchyma or airways. Endothelial gene liquid nitrogen and