Distinct differences in gene expression patterns in pulmonary arteries of patients with chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis with pulmonary hypertension.

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ORIGINAL ARTICLE

Distinct Differences in Gene Expression Patterns in Pulmonary Arteries of Patients with Chronic Obstructive Pulmonary Disease and Idiopathic Pulmonary Fibrosis with Pulmonary Hypertension Julia Hoffmann1*, Jochen Wilhelm2*, Leigh M. Marsh1, Bahil Ghanim1,3, Walter Klepetko3, Gabor Kovacs1,4, Horst Olschewski4, Andrea Olschewski1,5, and Grazyna Kwapiszewska1,5 1 Ludwig Boltzmann Institute for Lung Vascular Research, Graz, Austria; 2Department of Internal Medicine, Justus-Liebig-University Giessen, Universities of Giessen and Marburg Lung Center, German Center for Lung Research, Giessen, Germany; 3Division of Thoracic Surgery, Department of Surgery, Medical University of Vienna, Vienna, Austria; and 4Division of Pulmonology, Department of Internal Medicine, and 5Department of Anesthesiology and Intensive Care Medicine, Medical University of Graz, Graz, Austria

Abstract Rationale: The development of pulmonary hypertension (PH)

in patients with idiopathic pulmonary fibrosis (IPF) or chronic obstructive pulmonary disease (COPD) is associated with increased morbidity.

Objectives: To elucidate whether vascular remodeling in a wellcharacterized PH-COPD and PH-IPF patient cohort results from similar or divergent molecular changes. Methods: Vascular remodeling of donor, PH-COPD, and PH-IPF

pulmonary arteries was assessed. Laser capture microdissected pulmonary artery profiles in combination with whole genome microarrays were performed. Measurements and Main Results: Pulmonary arteries from patients with COPD and IPF with PH exhibited remodeling of vascular layers and reduction of lumen area. Pathway analyses comparing normalized gene expression profiles obtained from

Chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) are chronic and progressive lung diseases that lead to a decrease in lung function. Although exposure to smoke

patients with PH-IPF or PH-COPD revealed the retinol and extracellular matrix (ECM) receptor interaction to be the most perturbed processes. Within the ECM-receptor pathway, differential regulation of 5 out of the top 10 results (collagen, type III, a-1; tenascin C; collagen, type VI, a-3; thrombospondin 2; and von Willebrand factor) were verified by real-time polymerase chain reaction and immunohistochemical staining. Conclusions: Despite clinical and histologic vascular remodeling in

all patients with PH-COPD and PH-IPF, differential gene expression pattern was present in pulmonary artery profiles. Several genes involved in retinol metabolism and ECM receptor interaction enable discrimination of vascular remodeling in PH-IPF or PH-COPD. This suggests that pulmonary arterial remodeling in PH-COPD and PHIPF is caused by different molecular mechanisms and may require specific therapeutic options. Keywords: pulmonary hypertension; microarrays; laser capture

microdissection; remodeling

may be a common dominant risk factor for development of both COPD and IPF (1–3), the main pathologic characteristics of COPD and IPF are divergent. COPD is characterized by narrowing of small

conducting airways and chronic changes in lung parenchyma leading to emphysema. However, IPF is a progressive interstitial lung disease characterized by hyperplasia of resident

( Received in original form January 8, 2014; accepted in final form May 21, 2014 ) *Both authors contributed equally. Author Contributions: Conception and design, J.H., J.W., L.M.M., H.O., A.O., and G. Kwapiszewska. Analysis and interpretation, J.H., J.W., L.M.M., B.G., G. Kovacs, H.O., A.O., and G. Kwapiszewska. Drafting the manuscript for important intellectual content, J.H., J.W., L.M.M., B.G., W.K., G. Kovacs, H.O., A.O., and G. Kwapiszewska. Correspondence and requests for reprints should be addressed to Grazyna Kwapiszewska, Ph.D., Ludwig Boltzmann Institute for Lung Vascular Research, c/o ZMF, Stiftingtalstrasse 24, 8010 Graz, Austria. E-mail: [email protected] This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org Am J Respir Crit Care Med Vol 190, Iss 1, pp 98–111, Jul 1, 2014 Copyright © 2014 by the American Thoracic Society Originally Published in Press as DOI: 10.1164/rccm.201401-0037OC on June 11, 2014 Internet address: www.atsjournals.org

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American Journal of Respiratory and Critical Care Medicine Volume 190 Number 1 | July 1 2014

ORIGINAL ARTICLE cells, extracellular remodeling, honeycombing of the lung, and the pathologic pattern of usual interstitial pneumonia (UIP) (4). Development of pulmonary hypertension (PH), defined by an elevation of mean pulmonary arterial pressure (mPAP) greater than or equal to 25 mm Hg, is a frequent complication of advanced COPD (5, 6) and IPF (6, 7). PH leads to right ventricular dysfunction and is associated with poor prognosis in these patients (8). Remodeling of the small pulmonary arteries is the key pathologic hallmark of PH and includes among others increased stiffening of the elastic proximal pulmonary arteries and thickening of the intimal and/or medial layer of muscular arteries (9). Several key signaling pathways have been suggested to contribute to the remodeling process in pulmonary arterial smooth muscle cells and endothelial cells, such as endothelin1, serotonin, bone morphogenetic

At a Glance Commentary Scientific Knowledge on the Subject: Remodeling of pulmonary

arteries is present in patients with pulmonary hypertension caused by chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. Whether underlying gene expression patterns are similar or divergent is unknown. What This Study Adds to the Field: This study provides evidence

that vascular remodeling in pulmonary hypertension caused by chronic obstructive pulmonary disease or idiopathic pulmonary fibrosis might be driven by diverging molecular mechanisms and that different treatment options may be required.

proteins, Rho kinase, and hypoxiainducible factor 1 (10). To date it remains unclear whether PH associated with COPD or IPF and its features, such as arterial remodeling, share common or diverging pathomechanisms. Because remodeling ultimately results from changes in the transcriptome, we aimed to identify overlapping or diverging gene expression patterns or signaling pathways in the pulmonary artery walls of patients suffering from PH caused by lung diseases. Because expression of vascular-specific genes can be masked in lung homogenates because intrapulmonary arteries represent only a minor percentage of the lung tissue (11), we took advantage of compartmentspecific analysis via laser capture microdissection (LCM). Subsequently, we performed an unbiased whole genome screening on clinically well-characterized human lung samples with PH caused by IPF or COPD and compared the findings

Table 1. Clinical Characteristics of the Donors, Patients with PH-IPF, and Patients with PH-COPD

No. Sex 1 2 3 4 5 6 7 8 9* 10* 11 12 13 14 15 16 17 18 19* 20* 21 22 23 24 25 26 27 28 29* 30*

M F M M F F M F F M M M M F F M M M M M M F M F M M F F M F

Age at Ltx (yr)

Underlying Condition

mPAP (mm Hg)

Lung Function Test, Time before Ltx (mo)

FEV1 (%)

FVC (%)

FEV1/ FVC

Score Right Lung (%)

59 43 45 49 40 70 16 44 40 31 64 72 63 48 66 55 67 59 56 47 58 48 61 60 59 58 53 62 63 65

Donor Donor Donor Donor Donor Donor Donor Donor Donor Donor IPF 1 PH IPF 1 PH IPF 1 PH IPF 1 PH IPF 1 PH IPF 1 PH IPF 1 PH IPF 1 PH IPF 1 PH IPF 1 PH COPD 1 PH COPD 1 PH COPD 1 PH COPD 1 PH COPD 1 PH COPD 1 PH COPD 1 PH COPD 1 PH COPD 1 PH COPD 1 PH

NA NA NA NA NA NA NA NA NA NA 33 43 45 28 54 41 42 45 35 62 31 33 41 62 32 45 47 28 28 29

NA NA NA NA NA NA NA NA NA NA 1 0 16 5 2 6 10 2 5 16 10 2 12 15 5 12 1 9 2 0

NA NA NA NA NA NA NA NA NA NA 67.0 41.0 38.0 40.0 49.0 31.0 61.0 34.0 41.8 34.7 16.7 11.4 16.0 20.0 52.0 51.7 25.2 14.0 16.0 28.7



NA NA NA NA NA NA NA NA NA NA ,25 25–50 25–50 ,25 25–50 25–50 ,25 ,25 50–75 25–50 50–75 .75 50–75 50–75 NA 50–75 50–75 50–75 50–75 50–75

Score Left Lung (%) NA NA NA NA NA NA NA NA NA NA ,25 50–75 25–50 25–50 50–75 25–50 ,25 50–75 25–50 25–50 50–75 .75 50–75 50–75 NA 50–75 50–75 50–75 50–75 50–75

Definition of abbreviations: COPD = chronic obstructive pulmonary disease; IPF = idiopathic pulmonary fibrosis; Ltx = lung transplantation; mPAP = mean pulmonary arterial pressure; NA = not available; PH = pulmonary hypertension. *Samples additionally used for morphologic analyses.

Hoffmann, Wilhelm, Marsh, et al.: Gene Expression in Pulmonary Arteries in PH-COPD and PH-IPF

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ORIGINAL ARTICLE

Figure 1. Characterization of human donor, pulmonary hypertension (PH)-chronic obstructive pulmonary disease (COPD), and PH-idiopathic pulmonary fibrosis (IPF) lungs. ( A) Representative computed tomography images of PH-COPD and PH-IPF lungs. (B–D) Representative stainings of donor, COPD, and IPF lungs (n = 10 each) with (B) hematoxylin and eosin, (C) a-smooth muscle actin ( pink), and (D) von Willebrand factor (brown)/a-smooth muscle actin ( purple).

with results obtained from healthy control subjects. This approach allows the detection of novel pathways specifically involved in vascular remodeling. Our results may give rise to new target genes and specific therapeutic options for vascular remodeling in PH-IPF and PH-COPD. Some of these results have been presented in the form of an abstract (12).

characteristics including age, sex, mPAP, lung function parameters, and computer tomography (CT) lung scorings are reported in Table 1, and Table E1 in the online supplement. For microarray analyses, n = 8 samples per group were analyzed; for vascular remodeling quantification, n = 10 were analyzed. For COPD, n = 8 samples were available. For more information, see the online supplement.

Methods

Quantification of Vascular Remodeling

For full experimental details, see the online supplement.

Quantification of vascular remodeling was performed using Visiopharm integrated software VIS (Visiopharm, Hoersholm, Denmark) on trichrome-stained lung sections. More information is given in the online supplement.

Human Lungs

Human lung tissue samples were obtained from patients with PH-IPF (n = 10) and PH-COPD (n = 10) and patients with COPD without PH (n = 8) who underwent lung transplantation at the Department of Surgery, Division of Thoracic Surgery, Medical University of Vienna, Vienna, Austria. The protocol and tissue usage were approved by the institutional ethics committee (976/2010) and patient consent was obtained before lung transplantation. The patients’ 100

For information, see the online supplement.

amplified product (amplified DNA: aDNA) was Cy5-labeled using the SureTag DNA labeling kit (Agilent, Waldbronn, Germany) and hybridization to 60mer oligonucleotide spotted microarray slides (Human Whole Genome, SurePrint G3 Human GE v2 8 3 60K Microarray; Agilent Technologies, design ID 039494). Image analysis was performed with GenePix Pro 5.1 software, and calculated values for all spots were saved as GenePix results files. Log foldchanges in PH-IPF and in PH-COPD are changes relative to donors. Log foldchanges of the differential profiles PHIPF versus PH-COPD indicate the difference in expression in PH-IPF relative to PH-COPD. Here, “up- (down-) regulation” means that the ratio of expression in PH-IPF versus PH-COPD is larger (less) than 1. Logarithms were taken to the base 2. For further information, see the online supplement.

Genome-Wide Expression Profiling and Microarray Data Analysis

Real-Time Polymerase Chain Reaction

Purified total RNA (n = 8 per group) was amplified using the Ovation PicoSL WTA System V2 kit (NuGEN Technologies, Bemmel, Netherlands). Per sample, 2 mg

The polymerase chain reaction (PCR) reactions were set up using QuantiFast SYBR PCR kit (Qiagen, Hilden, Germany). The Ct values were normalized to internal

LCM and RNA Extraction


ORIGINAL ARTICLE

Figure 2. Quantification of vascular remodeling of donor, pulmonary hypertension (PH)-chronic obstructive pulmonary disease (COPD), and PH-idiopathic pulmonary fibrosis (IPF) pulmonary arteries. Mean percentage of adventitia, media, intima, and lumen for each patient presented for vessels (A) 50–200 mm and (B) 200–500 mm; n = 10 patients each, *P , 0.05. (C) Overlay of regression lines with 95% confidence bands (derived from Figure E2) showing the proportion of each respective compartment according to vessel diameter.

control b2-macroglobulin using the following formula: DCt = Ct reference gene-Ct gene of interest. For more information, see the online supplement.

Immunohistochemistry and Tissue Staining

For information, see the online supplement.

Statistical Analysis

Statistical analysis was performed in GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA) software. Mean responses by group were compared by Student t tests. Results for multiple comparisons were corrected by Tukey method. Comparisons with P values less than 0.05 are indicated by an asterisk in the diagrams. Results of

vascular remodeling are presented as individual patients’ (mean) and group means with 95% confidence interval. Proportions of vessel compartments were analyzed by a random effects logit model. Predictors were the disease group and the log vessel diameter, the random effect was the patient. Model assumptions were checked by a residual analysis. The analysis was performed with R


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ORIGINAL ARTICLE

Figure 3. Gene expression profiles of pulmonary hypertension (PH)-idiopathic pulmonary fibrosis (IPF) and PH-chronic obstructive pulmonary disease (COPD) patients compared with donors. (A) Heatmap donor versus PH-COPD versus PH-IPF: Unsupervised hierarchical clustering analysis of the union set of the 50 most significantly regulated genes. (B) Volcano plots for the comparisons of PH-IPF versus donor (top) and PH-COPD versus donor (bottom). Some genes at prominent coordinates are annotated. (C) Venn diagrams of regulated genes (5% false-discovery rate) in PH-COPD and PH-IPF. Top: Upregulated genes. Bottom: Down-regulated genes.

(3.1.0) (13). Mixed-effects models were calculated using the package lmerTest (2.0–6) (14). Real-time PCR results are presented as means with 95% confidence interval. For microarray analyses, statistical details are described in the online supplement.

Results Vascular Remodeling in PH-COPD and PH-IPF Lungs

Transplanted lungs of clinically wellcharacterized patients with PH-COPD and PH-IPF and nontumorous donors (n = 10 each) were enrolled in this study. Patient 102

information is presented in Table 1; age, sex, and mPAP pretransplant and most lung function parameters did not show significant differences between the two groups. Representative CT images of PH-COPD and PH-IPF lungs at the time of lung transplantation displayed characteristic features of each disease, such as emphysema in PH-COPD and honeycombing and pathologic UIP in the PH-IPF lungs (Figure 1A). In accordance with the severity of the disease CT scorings of right and left lungs were high (Table 1). Representative hematoxylin and eosin (Figure 1B), a-smooth muscle actin (SMA; Figure 1C),

and von Willebrand factor (vWF)/SMA (Figure 1D) stainings showed healthy unremodeled pulmonary arteries in donor lungs and remodeled arteries in PH-IPF and PH-COPD lungs. Quantification of remodeling in pulmonary vessels (50–200 mm and 200–500 mm) revealed a significant reduction in the vessel lumen in both PHCOPD and PH-IPF lungs. The most pronounced remodeling was observed in the intima followed by the media, with only minor contribution of the adventitia (Figures 2A and 2B). The distribution of each vessel component in all analyzed vessels according to vessel size is shown in Figure E2. In


ORIGINAL ARTICLE

Figure 4. Comparison of pulmonary hypertension (PH)-idiopathic pulmonary fibrosis (IPF) and PHchronic obstructive pulmonary disease (COPD) profiles. (A) Correlation of log2 fold-changes versus donor. Pearson coefficient of correlation: 0.42 6 0.1 (P , 0.001). Green and red areas indicate stronger induction (or less repression) in PH-COPD and PH-IPF, respectively. (B) Volcano plot for PHCOPD versus PH-IPF. Some genes at prominent coordinates are annotated. Green and red areas have the same meaning as in A.

PH-COPD and PH-IPF vessels increase in percentage media and percentage intima can be clearly observed by a shift in the regression line (Figure 2C; see Figure E2). Interestingly, smaller vessels in PH-COPD samples exhibited more pronounced lumen reduction than larger vessels (P = 0.008). In contrast, lumen reduction in PH-IPF samples was more uniform between larger and smaller vessels (P = 0.142) (Figure 2C). To investigate the possible PH effects, COPD samples with and without PH were compared. COPD patient characteristics are presented in Table E1. Of note, IPF patient samples without PH were not available at our collaborating transplant centrum. Representative CT image of a COPD lung is provided in Figure E3A. Remodeling was visible in COPD samples without PH as depicted in representative images in Figures E3B–E3D. Quantification of vascular remodeling revealed small changes in both the lumen and intima (see Figures E3E–E3H). In larger vessels no difference in any component was observed between COPD samples with and without PH; however, in smaller vessels an increase in the intima (P = 0.004) and a decrease in the lumen (P = 0.001) could be observed in PH-COPD compared with COPD (see Figure E3H). Gene Expression Profiles of LCM Vessels from PH-IPF and PH-COPD Samples Compared with Donors

LCM pulmonary artery profiles from PHIPF and PH-COPD lungs and healthy

donors were subjected to human whole genome microarrays and gene expression changes were identified. A heatmap of the 50 most significantly regulated genes in donor, PH-COPD, and PH-IPF depicts a clustered gene expression pattern between the three groups (Figure 3A). Volcano plots show significances (as negative log P values) versus means of differential expression for the comparisons of PH-IPF versus donor and for PH-COPD versus donor (Figure 3B). Based on a 5% falsediscovery rate, the numbers of genes classified as up- and down-regulated in both diseases as compared with donor were similar (IPF: 910/1024, COPD:855/850 [up/ down]). Slightly more genes were classified as regulated in PH-IPF versus donor (1,934) than in PH-COPD versus donor (1,705). For both up- and down-regulated genes, there was only a small overlap of 192 (13.9%) up- and 294 (22.9%) downregulated genes (Figure 3C). In accordance, most differentially expressed genes between normalized PH-COPD and normalized PH-IPF samples varied. Comparison of Expression Profiles from Intrapulmonary Arteries of PH-IPF and PH-COPD Lungs

The log fold-changes in both diseases versus donor show a positive correlation (Pearson coefficient of correlation, 0.42 6 0.1; P , 0.001) (Figure 4A). The significance of differential regulation between the diseases is shown in

Figure 4B. Genes in the green box display stronger induction (or lower repression) in PH-COPD, the red box stronger in PHIPF. At a false-discovery rate of 5%, only the two genes Family With Sequence Similarity 107, Member A (FAM107A), and B-cell translocation gene 2 (BTG2) can be classified as differentially regulated in vessels of PH-IPF versus PH-COPD. For a 10% false-discovery rate, 32 genes can be classified as differentially regulated (see Table E4). Genes highlighted that are classified as regulated either exclusively in one of the diseases or commonly regulated in both diseases are shown in Figure E4, Tables 2–4, and described in the online supplement. To identify possible gene expression differences between COPD with and without PH, 10 of the top up- and downregulated genes from donor versus PHCOPD (Table 2) were analyzed by real-time PCR. No significant differences in gene expression were observed between these groups (see Figure E5AB). We then sought to determine whether a PH signature can be detected. Here the influence of PH severity on the expression profiles was examined by subdividing all samples (PH-COPD, PHIPF) into two groups: moderate PH (,40 mm Hg) or severe PH (.40 mm Hg). Comparison of these samples revealed only minor gene regulations that can be attributed to a general mPAP-dependent regulation. Full details of these results are included in the online supplement. Gene Set Tests and Pathway Analysis in the Differential Profiles of PH-IPF versus PH-COPD Pulmonary Arteries

The top 10 results of the gene ontology analyses of the differential profiles between PH-COPD and PH-IPF are shown in Figure 5. Ontology nodes with the highest significances were cell adhesion (biologic process, GO:0007155, 1,563 gene products), integrin binding (molecular function, GO:0005178, 419 gene products), and extracellular space (cellular component, GO:0005615, 1,052 gene products). Pathway analyses using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway maps indicated retinol metabolism to be the most significantly differentially regulated pathway between PH-COPD and PH-IPF (Figure 6A). Color-coded expression of KEGG pathway genes for retinol metabolism (hsa00830) is provided in Figure E6A. The relative


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ORIGINAL ARTICLE Table 2. Top Regulated Genes: Distinctly Different Genes between PH-COPD and Donor Symbol Up-regulated FOSB BTG2 NPTXR MYOM1 ZNF776 RERGL EGR1 LOC572558 NTRK3 SIK1 Down-regulated UBE2C CMTM3 SNX24 CLDN4 NEDD4L LOC100287628 ZNF521 SLC34A3 SLC34A2 COL6A3 NCEH1 SERPINA1 HOPX DHCR24 IRX3 NKX2-1 SCNN1A TJP3 POSTN GGT5

Name

FBJ murine osteosarcoma viral oncogene homolog B BTG family, member 2 Neuronal pentraxin receptor Myomesin 1, 185 kD Zinc finger protein 776 RERG/RAS-like Early growth response 1 Uncharacterized LOC572558 Neurotrophic tyrosine kinase, receptor, type 3 Salt-inducible kinase 1 Ubiquitin-conjugating enzyme E2C CKLF-like MARVEL transmembrane domain containing 3 Sorting nexin 24 Claudin 4 Neural precursor cell expressed, developmentally down-regulated 4-like Uncharacterized LOC100287628 Zinc finger protein 521 Solute carrier family 34 (sodium phosphate), member 3 Solute carrier family 34 (sodium phosphate), member 2 Collagen, type VI, alpha 3 Neutral cholesterol ester hydrolase 1 Serpin peptidase inhibitor, clade A (a1-antiproteinase, antitrypsin), member 1 HOP homeobox 24-Dehydrocholesterol reductase Iroquois homeobox 3 NK2 homeobox 1 Sodium channel, nonvoltage-gated 1 alpha Tight junction protein 3 (zona occludens 3) Periostin, osteoblast specific factor g-Glutamyltransferase 5

A*

Log FC†

Unigene ID

RefSeq

Hs.590958

NM_006732

12.6

2.9 6 1.8

Hs.519162 Hs.91622 Hs.464469 Hs.720254 Hs.115497 Hs.326035 Hs.9015 Hs.410969

NM_006763 NM_014293 NM_003803 NM_173632 NM_024730 NM_001964 NR_015423 NM_001012338

9.8 5.5 6.3 5.8 9.0 12.9 5.7 7.5

2.7 2.3 2.3 2.2 2.1 2.1 2.0 2.0

Hs.282113

NM_173354

Hs.93002 Hs.298198

6 6 6 6 6 6 6 6

P Value‡

0.012

0.9 1.3 1.3 1.2 2.1 1.3 1.7 1.4

,0.001 0.009 0.010 0.009 0.060 0.012 0.031 0.017

6.8

2.0 6 1.3

0.012

NM_181803 NM_144601

6.3 8.3

22.0 6 1.4 22.0 6 1.3

0.013 0.012

Hs.483200 Hs.647036 Hs.185677

NM_014035 NM_001305 NM_015277

6.8 7.9 8.1

22.0 6 1.3 22.0 6 1.8 22.1 6 1.6

0.012 0.039 0.022

Hs.720980 Hs.116935 Hs.432442

XR_109254 NM_015461 NM_001177317

6.6 7.6 8.8

22.1 6 1.5 22.2 6 1.6 22.2 6 1.8

0.017 0.017 0.034

Hs.479372

NM_006424

8.0

22.2 6 2.1

0.059

Hs.233240 Hs.444099

NM_004369 NM_020792

7.0 8.9

22.2 6 1.3 22.3 6 1.3

0.009 0.010

Hs.525557

NM_001002236

11.3

22.4 6 1.5

0.012

Hs.619396 Hs.498727 Hs.499205 Hs.94367 Hs.591047

NM_139211 NM_014762 NM_024336 NM_003317 NM_001038

7.6 6.8 7.7 7.6 6.5

22.4 22.5 22.5 22.5 22.7

1.6 1.7 1.8 1.9 1.7

0.012 0.013 0.017 0.022 0.012

Hs.25527

NM_014428

6.5

23.0 6 1.4

0.003

Hs.136348 Hs.437156

NM_006475 NM_001099781

8.5 8.2

23.1 6 1.7 23.2 6 1.2

0.009 ,0.001

6 6 6 6 6

*Average log2 spot-intensity, a measure of the overall expression intensity (,5, background; 16, saturation). † Log2 fold-change (differential expression in the respective contrast), mean 6 95% confidence interval. ‡ P value for differential expression, Benjamini-Hochberg adjusted (controlling the false-discovery rate).

expression and differential regulation of the pathway genes is shown in Figure E6B and in the volcano plot in Figure 6B. The genes belonging to the retinol metabolism pathway are marked by squares, whereas the filled squares indicate top 10 (log fold-change) genes from this pathway. Annotation details, log2 fold-change, and significance for these genes are provided in Table 5. The second most significantly regulated KEGG pathway was extracellular matrix (ECM)-receptor interaction (hsa04512; see Figure E6C). This pathway

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was also overrepresented in GO analysis with the highest number of genes being differentially regulated. The relative expression and differential regulation of the pathway genes is shown in Figure E6D. The volcano plot in Figure 6C shows the significances of their differential regulation. The genes belonging to the ECM-receptor interaction pathway are marked by squares. Filled squares indicate top 10 (log-fold change) genes from this pathway. Details for these genes are summarized in Table 6.

Verification of Regulation of ECM-related Genes

Differential regulations between donor versus PH-IPF and donor versus PH-COPD of ECM genes (collagen a-1[III] chain [COL3A1], tenascin C [TNC], collagen a-3 [VI] chain [COL6A3], thrombospondin-2 [THBS2], and vWF) were verified by real-time PCR analyses. COL3A1, TNC, COL6A3, and THBS2 were up-regulated in lung vessels from patients with PH-IPF, whereas vWF was down-regulated in PH-IPF as compared with PH-COPD


ORIGINAL ARTICLE Table 3. Top Regulated Genes: Distinctly Different Genes between PH-IPF and Donor Symbol Up-regulated S100A2 MMP1 SCGB1A1 MMP7 C20orf85 BCAS1 CXCL14 KRT17 WDR38 TRIM29 SIX1 BDKRB2 GPX2 CLIC6 TUBB3 HMGB3 SCGB3A1 CLCA2 C6orf165 SOX2 CBX8 STK33 SAA2 MUC4 FANK1 Down-regulated TMEM88 PDE1B MMRN2 PLA1A C1orf115 PTPRR IDO1 CLDN5 CLEC14A PNMT PCYT2 SDPR HBA2 ADRB2

Log FC†

P Value‡

Name

Unigene ID

RefSeq

A*

S100 calcium binding protein A2 Matrix metallopeptidase 1 (interstitial collagenase) Secretoglobin, family 1A, member 1 (uteroglobin) Matrix metallopeptidase 7 (matrilysin, uterine) Chromosome 20 open reading frame 85 Breast carcinoma amplified sequence 1 Chemokine (C-X-C motif) ligand 14 Keratin 17 WD repeat domain 38 Tripartite motif containing 29 SIX homeobox 1 Bradykinin receptor B2 Glutathione peroxidase 2 (gastrointestinal) Chloride intracellular channel 6 Tubulin, beta 3 class III High mobility group box 3 Secretoglobin, family 3A, member 1 Chloride channel accessory 2 Chromosome 6 open reading frame 165 SRY (sex determining region Y)-box 2 Chromobox homolog 8 Serine/threonine kinase 33 Serum amyloid A2 Mucin 4, cell surface associated Fibronectin type III and ankyrin repeat domains 1

Hs.516484 Hs.83169 Hs.523732 Hs.2256 Hs.43977 Hs.400556 Hs.483444 Hs.2785 Hs.343383 Hs.504115 Hs.633506 Hs.726620 Hs.2704 Hs.473695 Hs.511743 Hs.19114 Hs.62492 Hs.241551 Hs.82921 Hs.518438 Hs.387258 Hs.501833 Hs.1955 Hs.369646 Hs.352591

NM_005978 NM_002421 NM_003357 NM_002423 NM_178456 NM_003657 NM_004887 NM_000422 NM_001045476 NM_012101 NM_005982 NM_000623 NM_002083 NM_053277 NM_006086 NM_005342 NM_052863 NM_006536 NM_001031743 NM_003106 NM_020649 NM_030906 NM_030754 NM_018406 NM_145235

7.7 5.4 9.6 4.9 5.9 5.0 5.1 4.4 5.5 4.6 4.9 5.6 5.3 4.5 5.8 5.3 12.4 4.4 4.7 4.0 6.0 5.1 4.2 12.6 5.6

5.5 4.2 4.2 3.6 3.6 3.1 3.0 3.0 2.7 2.6 2.5 2.5 2.5 2.5 2.4 2.4 2.4 2.2 2.2 2.1 2.1 2.1 2.1 2.0 2.0

6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6

2.2 1.9 2.4 2.0 1.6 1.5 1.5 1.4 1.4 1.3 1.5 1.7 1.7 1.4 1.5 1.4 1.4 1.6 1.7 1.1 1.5 1.5 1.7 1.5 1.6

,0.001 0.002 0.005 0.004 0.002 0.003 0.003 0.002 0.003 0.002 0.006 0.012 0.010 0.004 0.007 0.004 0.005 0.014 0.019 0.004 0.013 0.012 0.024 0.015 0.020

Transmembrane protein 88 Phosphodiesterase 1B, calmodulin-dependent Multimerin 2 Phospholipase A1 member A Chromosome 1 open reading frame 115 Protein tyrosine phosphatase, receptor type, R Indoleamine 2,3-dioxygenase 1 Claudin 5 C-type lectin domain family 14, member A Phenylethanolamine N-methyltransferase Phosphate cytidylyltransferase 2, ethanolamine Serum deprivation response Hemoglobin, alpha 2 Adrenergic, b2-receptor, surface

Hs.389669 Hs.530871 Hs.524479 Hs.437451 Hs.519839 Hs.506076 Hs.840 Hs.505337 Hs.525307 Hs.1892 Hs.569843 Hs.26530 Hs.654744 Hs.2551

NM_203411 NM_000924 NM_024756 NM_015900 NM_024709 NM_002849 NM_002164 NM_001130861 NM_175060 NM_002686 NM_001184917 NM_004657 NM_000517 NM_000024

6.0 7.5 7.9 5.2 5.8 5.8 8.4 9.2 8.3 5.7 6.3 6.5 12.0 8.3

22.0 22.1 22.1 22.1 22.1 22.2 22.3 22.3 22.3 22.4 22.5 22.6 22.7 23.4

6 6 6 6 6 6 6 6 6 6 6 6 6 6

2.0 1.2 1.3 1.2 1.1 1.4 1.5 1.3 1.5 2.0 1.3 1.2 1.6 1.2

0.056 0.004 0.007 0.005 0.003 0.007 0.010 0.004 0.007 0.027 0.003 0.002 0.006 ,0.001


pulmonary arteries (Figure 7A). No significant difference was detectable between COPD with and without PH (see Figure E5C). When comparing IPF intima/ media with IPF parenchyma, COL6A3, TNC, and THBS2 were not differentially expressed. However, COL3A1 was more prominently expressed and vWF less prominently in the lung parenchyma (see Figure E7). In a next step, we performed immunohistochemical staining for one up- and one down-regulated gene, TNC, and vWF, respectively, to identify their subcellular localization within the vessel wall. TNC expression was localized to the intima and media of the vessel wall

(Figure 7B). Expression of vWF was exclusively localized to the endothelium of the pulmonary vessels (Figure 7C).

Discussion In the present study we first analyzed the degree of vascular remodeling in PH-COPD and PH-IPF patient lungs and subsequently used RNA isolated from LCM pulmonary vessel profiles to specifically identify genes involved in vascular remodeling underlying both diseases. To our knowledge, this is the first study comparing gene expression patterns in LCM lung vessel profiles of two

distinct lung diseases, both of which are associated with vascular remodeling. Additionally, all our control samples were nontumorous healthy lung tissue resected from lungs used for lung transplantation. To identify possible PH effects we included a third group, COPD without PH. A limitation of this study is the lack of an IPF group without PH, because all end-stage IPF lungs from the transplant centrum displayed moderate to severe PH as defined by an elevated mPAP shortly pretransplant. Detailed analysis of our patient cohort revealed that COPD, PH-COPD, and PH-IPF pulmonary arteries displayed various degrees of vascular remodeling. To


105

ORIGINAL ARTICLE Table 4. Top Regulated Genes: Commonly Regulated Genes for PH-COPD and PH-IPF

Symbol

Name

Up-regulated PHKA1 Phosphorylase kinase, a1 (muscle) CXCL1 Chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating activity-a) KCTD11 Potassium channel tetramerization domain containing 11 CACNG6 Calcium channel, voltage-dependent, gamma subunit 6 COL9A2 Collagen, type IX, a2 CSAD Cysteine sulfinic acid decarboxylase Down-regulated KCNAB2 Potassium voltage-gated channel, shaker-related subfamily, beta member 2 BTNL9 Butyrophilin-like 9 GGTLC1 g-Glutamyltransferase light chain 1 ANKRD1 Ankyrin repeat domain 1 (cardiac muscle) CLDN18 Claudin 18 GLT25D2 Glycosyltransferase 25 domain containing 2 DAPK2 Death-associated protein kinase 2 AGER Advanced glycosylation end product–specific receptor SFTPA1 Surfactant protein A1 PLA2G1B Phospholipase A2, group IB (pancreas) MME Membrane metalloendopeptidase TMEM97 Transmembrane protein 97 PCSK9 Proprotein convertase subtilisin/kexin type 9 HHIP Hedgehog interacting protein SFTA2 Surfactant associated 2 FCN3 Ficolin (collagen/fibrinogen domain containing) 3 (Hakata antigen) IL1RL1 Interleukin 1 receptor-like 1 LAMP3 Lysosomal-associated membrane protein 3

PH-COPD vs. Donor P A* Log FC† Value‡

PH-IPF vs. Donor P Log FC† Value‡

Unigene ID

RefSeq

Hs.201379

NM_002637

6.9

3.3 6 1.4

0.002

2.4 6 1.4

0.005

Hs.789

NM_001511

7.9

2.6 6 1.6

0.011

3.0 6 1.6

0.003

Hs.592112

NM_001002914

7.1

2.4 6 1.6

0.016

2.7 6 1.6

0.005

Hs.631560

NM_145814

6.0

2.3 6 2.0

0.036

2.2 6 1.9

0.035

Hs.418012 Hs.279815

NM_001852 NM_015989

6.2 6.7

2.2 6 1.7 2.1 6 1.1

0.021 0.004

2.9 6 1.6 2.4 6 1.0

0.003 0.001

Hs.440497

NM_003636

5.6 22.0 6 1.5

0.019

22.3 6 1.4

0.008

Hs.546502 Hs.355394 Hs.448589

NM_152547 NM_178311 NM_014391

7.0 22.0 6 1.5 7.8 22.0 6 1.2 5.1 22.0 6 1.4

0.019 0.006 0.016

22.4 6 1.5 22.1 6 1.1 22.9 6 1.3

0.006 0.003 0.002

Hs.655324 Hs.387995

NM_016369 NM_015101

8.6 22.1 6 2.6 6.1 22.1 6 1.6

0.131 0.023

23.6 6 2.6 23.1 6 1.6

0.015 0.003

Hs.237886

NM_014326

5.6 22.1 6 1.0

0.004

22.0 6 1.0

0.003

Hs.534342

NM_001136

11.4 22.1 6 1.8

0.033

23.3 6 1.7

0.003

Hs.535295 Hs.992

NM_005411 NM_000928

13.0 22.2 6 2.2 6.2 22.4 6 1.7

0.070 0.016

22.3 6 2.2 22.3 6 1.6

0.048 0.015

Hs.307734 Hs.199695 Hs.18844

NM_007289 NM_014573 NM_174936

7.2 22.5 6 1.5 6.0 22.5 6 1.6 5.4 22.6 6 1.8

0.006 0.011 0.017

23.1 6 1.4 22.1 6 1.5 22.2 6 1.8

0.002 0.016 0.027

Hs.49265 Hs.211267 Hs.333383

XR_109840 NM_205854 NM_003665

7.7 22.8 6 1.6 8.0 22.9 6 1.6 8.2 22.9 6 2.1

0.006 0.006 0.019

22.4 6 1.5 22.1 6 1.5 22.8 6 2.1

0.008 0.016 0.016

Hs.66 Hs.518448

NM_016232 NM_014398

5.8 23.2 6 1.3 8.6 23.5 6 1.9

0.001 0.006

22.9 6 1.2 22.1 6 1.9

0.001 0.036


ensure robust, unbiased data we have analyzed 4 sections of 10 (eight for COPD) patients per group. In all diseases the vessel lumen was reduced accompanied by a thickening of media and especially intima layers. Although IPF is a fibrotic disease with broaden septa, one could expect that the neighboring adventitia would also be prone to remodeling; however, in our patient cohort percentage of adventitia was only significantly elevated in small vessels in PHCOPD. The lack of prominent thickening of 106

perivascular tissue (adventitia) has previously been described by Stacher and coworkers (9) in idiopathic PAH pulmonary arteries. UIP patterns as present in our end-stage PH-IPF lungs have previously been associated with thickening of the smooth muscle cell layer and proliferative intimal lesions (15, 16). The structural changes in the pulmonary vasculature of PH-COPD have been controversially reported. Our results and that of other studies have shown significant

intimal hyperplasia and media thickening of small pulmonary arteries in patients with mild or severe COPD (17) and even in smokers with normal lung function (18, 19), whereas others did not identify pronounced vascular remodeling, even in patients with severe PH-COPD (17, 20). To identify differences in gene expression underlying vascular remodeling we performed an unbiased screening approach. To date, several studies have performed microarray analyses in lung


ORIGINAL ARTICLE

Figure 5. Top 10 significantly perturbed Gene Ontology (GO) nodes in pulmonary hypertension (PH)-idiopathic pulmonary fibrosis (IPF) versus PH-chronic obstructive pulmonary disease (COPD). Only nodes with more than 10 genes were considered. Left, statistical significance of the perturbation as determined from a gene set test; right, percentages of genes in the respective pathways that are down-regulated (gray bars, to the left) and up-regulated (black bars, to the right), controlling for 20% false-discovery rate.

homogenates of PAH or PH caused by lung diseases (21–25). However, to our knowledge only three studies have so far investigated compartment-specific gene expression patterns in pulmonary arteries from human PH lung samples (26–28). In pulmonary arteries of patients with IPAH compared with healthy control subjects, planar cell polarity pathway has been demonstrated to play a decisive role in vascular remodeling underlying IPAH (27). Jonigk and colleagues (26) applied real-time PCR and revealed distinct cellular composition of plexiform lesions as compared with the adjacent arteries from which they sprout. Recently, gene expression profiles of pulmonary arteries of patients with IPF with either mild or without PH were compared with control subjects. In this study resection material

from lung tissue next to pulmonary carcinoid tumors was used as control tissue. The gene signature appeared similar in patients with IPF with and without coexisting mild PH. Of note, genes that promote cellular proliferation were upregulated in IPF pulmonary arterioles regardless of the presence of coexistent PH (28). Similarly, in our study we did not detect pronounced differences between COPD with and without PH regarding vascular remodeling or gene expression of selected genes. In the present microarray analyses, the small overlap of significantly differentially regulated genes between PH-COPD and PH-IPF pulmonary arteries suggests different mechanisms and pathways to be involved in the underlying vascular remodeling. Retinol metabolism was the top

significantly regulated KEGG pathway between PH-IPF and PH-COPD. Within this pathway several genes, such as cytochromes and aldehyde dehydrogenases, have been implicated in cardiovascular remodeling (29). In this regard, retinoic acid has been shown to inhibit airway SMC migration (30), vascular SMC proliferation (31), and adhesion (32), and regulate endothelial cell proliferation (33). The top regulated gene from the retinol metabolism pathway was uridine diphosphoglucuronosyltransferases 1A6, which is the only UGT1A isoform expressed in lung tissue and is responsible for the detoxification of benezoapyrene from cigarette smoke (34). The observed down-regulation in PH-COPD as compared with PH-IPF might account for less detoxification of benezoapyrene from


107

ORIGINAL ARTICLE

Figure 6. Pathway analysis of pulmonary hypertension (PH)-idiopathic pulmonary fibrosis (IPF) versus PH-chronic obstructive pulmonary disease (COPD). (A) Ten most significantly perturbed Kyoto Encyclopedia of Genes and Genomes pathways. Left, statistical significance of the perturbation as determined from a gene set test; right, percentages of genes in the respective pathways that are down-regulated (gray bars, to the left) and up-regulated (black bars, to the right), controlling for 20% false-discovery rate. (B and C) Volcano plots for the comparison of PH-IPF versus PH-COPD profiles. Genes belonging to (B) the retinol metabolism pathway or (C) the ECM-receptor interaction pathway are highlighted by squares. Black squares indicate the 10 pathway genes with highest differential regulation.

cigarette smoke. Oppositely, the most significantly down-regulated gene in vessels of PH-IPF versus PH-COPD from the retinol metabolism pathway was cytochrome P-450 (CYP) 26B1, which is expressed in endothelial, smooth muscle cells and in atherosclerotic lesions (35). Inhibition of CYP26B1 by profibrotic transforming growth factor-b has recently been reported (36). Furthermore, blockage of CYP26B1 has been described to be a therapeutic approach in vascular proliferative disorders (37), suggesting potential therapeutic use in the treatment of vascular remodeling in PH caused by IPF. These results indicate that genes related to retinol metabolism might play an important role in differentiating processes involved in vascular remodeling of PH caused by different lung diseases. Another significantly regulated pathway between the two diseases was ECM-receptor interaction. Interestingly, retinol metabolism has been linked to ECM 108

receptor interaction pathway in the lung (38–40). The GO nodes for molecular function and cellular compartment identified ECM to be among the top 10 regulated. Furthermore, several top regulated genes out of the KEGG ECMreceptor interaction pathway have recently been identified in the context of vascular remodeling or in the pathophysiology of PH. This implies that ECM might represent a tool to distinguish between vascular remodeling processes of pulmonary arteries of patients with PH-IPF and PH-COPD. One set of ECM-related genes with elevated expression in PH-IPF were profibrotic collagens, COL3A1 and COL6A3 and TNC. Elevated levels of COL3A1 have been identified in a model of flow-induced pulmonary vascular remodeling (41) and COL6A3 is a target of transforming growth factor-b (42). TNC has been shown to be expressed in human sera from patients with SSc-associated pulmonary fibrosis (43) and in the early

phase of pulmonary fibrosis in a rat model of bleomycin-induced pulmonary injury (44). Functionally, TNC is related to increased smooth muscle cell proliferation (45). However, our study is the first to implicate TNC in vascular remodeling in human patients with IPF. Furthermore, THBS2 modulates collagen fibrillogenesis and angiogenesis (46) and could exert this role also in the development of PH caused by IPF, which could explain the higher expression in PH-IPF pulmonary arteries. In contrast to the previous genes, vWF was significantly higher expressed in pulmonary arteries of PH-COPD than in PH-IPF. In previous studies, plasma vWF has been shown to be increased in serum of COPD smokers (47), to be a marker of endothelial dysfunction with coexisting COPD exacerbation (48), and to be a useful tool in identifying individuals at increased risk of accelerated decline in FEV1 (49). In PH endothelial production of vWF is increased and associated with abnormal interaction


ORIGINAL ARTICLE Table 5. Ten Genes of the Retinol Metabolism Pathway with Strongest Differential Regulation between PH-IPF and PH-COPD Symbol Up-regulated UGT1A6 ALDH1A1 CYP2A7

Name

UDP glucuronosyltransferase 1 family, polypeptide A6 Aldehyde dehydrogenase 1 family, member A1 Cytochrome P-450, family 2, subfamily A, polypeptide 7 Retinol dehydrogenase 10 (all-trans)

RDH10 Down-regulated CYP4A11 Cytochrome P-450, family 4, subfamily A, polypeptide 11 CYP2C19 Cytochrome P-450, family 2, subfamily C, polypeptide 19 ADH5 Alcohol dehydrogenase 5 (class III), chi polypeptide PNPLA4 Patatin-like phospholipase domain containing 4 ALDH1A2 Aldehyde dehydrogenase 1 family, member A2 CYP26B1 Cytochrome P-450, family 26, subfamily B, polypeptide 1

Unigene ID

RefSeq

A*

Log FC†

P Value‡

Hs.554822

NM_001072

4.8

4.1 6 1.8

0.003

Hs.76392

NM_000689

7.9

1.6 6 1.0

0.037

Hs.719890

NM_000764

4.6

0.9 6 1.4

0.492

Hs.244940

NM_172037

4.9

0.6 6 1.1

0.660

Hs.1645

NM_000778

4.3

20.5 6 0.7

0.452

Hs.282409

NM_000769

3.4

20.7 6 0.8

0.452

Hs.78989

NM_000671

5.3

20.8 6 1.0

0.452

Hs.264

NM_004650

3.6

20.9 6 0.6

0.037

Hs.643455

NM_170697

5.0

21.4 6 1.4

0.361

Hs.91546

NM_019885

5.1

22.3 6 1.5

0.037


with platelets, which leads to worse prognosis (50). Our results indicate that proteins involved in ECM receptor interaction might be a useful tool for the early detection of PH caused by COPD or IPF in plasma because they displayed marked differential regulation in the pulmonary vessel walls of end-stage lungs. However, further studies are needed to provide evidence for the usefulness of ECM proteins as diagnostic markers.

Taken together, this study provides for the first time an overview of compartmentspecific gene expression analyses of PHCOPD versus PH-IPF pulmonary arteries using clinically well-characterized lung samples. Even though both lung diseases display pulmonary vascular remodeling, albeit to different degrees, gene expression patterns and identified pathways underlying the pathophysiologic development of vascular remodeling in these two disorders

are distinctly different. This study implies that vascular remodeling, the hallmark of PH, is based on differential mechanisms in PH-COPD and PH-IPF. n Author disclosures are available with the text of this article at www.atsjournals.org. Acknowledgment: The authors thank Lisa Oberreiter, Susanna Ziegler, Julia Schittl, and Ida Niklasson for excellent technical support and LBI colleagues for fruitful discussions.

Table 6. Ten Genes of the ECM Receptor Interaction Pathway with Strongest Differential Regulation between PH-IPF and PH-COPD Symbol Up-regulated LAMC2 SDC1 LAMB3 COL3A1 COL6A3 TNC ITGB6 THBS2 Down-regulated VWF ITGA8

Name

Unigene ID

RefSeq

A*

Laminin, gamma 2 Syndecan 1 Laminin, beta 3 Collagen, type III, alpha 1 Collagen, type VI, alpha 3 Tenascin C Integrin, beta 6 Thrombospondin 2

Hs.591484 Hs.224607 Hs.497636 Hs.443625 Hs.233240 Hs.143250 Hs.470399 Hs.371147

NM_018891 NM_00100694 NM_00101740 NM_000090 NM_004369 NM_002160 NM_000888 NM_003247

7.7 6.6 6.5 11.0 7.0 9.0 5.7 9.8

Von Willebrand factor Integrin, alpha 8

Hs.440848 Hs.171311

NM_000552 NM_003638

10.8 9.8

Log FC†

3.7 2.5 2.3 2.0 2.0 1.9 1.9 1.6

6 6 6 6 6 6 6 6

P Value‡

1.4 1.2 1.6 1.3 1.3 1.8 1.3 1.1

0.003 0.016 0.086 0.086 0.086 0.203 0.086 0.086

21.7 6 1.2 21.8 6 1.6

0.086 0.203



109

ORIGINAL ARTICLE

Figure 7. Verification of differential gene expression of extracellular matrix (ECM) receptor interaction pathway genes. (A) Real-time polymerase chain reaction, data are normalized to b2-macroglobulin (b2M) and presented as mean with confidence interval, *P < 0.05. (B and C) Immunohistochemistry against tenascin C (TNC) (B) and von Willebrand factor (vWF) (C) on pulmonary hypertension (PH)–chronic obstructive pulmonary disease (COPD) and PH–idiopathic pulmonary fibrosis (IPF) lungs; arrows depict positive staining.

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Right Atrium Volume Index in Patients with Secondary Pulmonary Hypertension Due to Chronic Obstructive Pulmonary Disease.

Incidence and clinical characteristics of pulmonary hypertension in patients with idiopathic pulmonary fibrosis.

Integrated Genomics Reveals Convergent Transcriptomic Networks Underlying Chronic Obstructive Pulmonary Disease and Idiopathic Pulmonary Fibrosis.

24-Hour Hypoxia and Pulmonary Hypertension in Patients with Idiopathic Pulmonary Fibrosis.

Non-invasive screening for pulmonary hypertension in idiopathic pulmonary fibrosis.

Role of vasoactive intestinal peptide in chronic obstructive pulmonary disease with pulmonary hypertension.

Expression variations of connective tissue growth factor in pulmonary arteries from smokers with and without chronic obstructive pulmonary disease.

Acute and chronic effects of nitrendipine in patients with precapillary pulmonary hypertension due to pulmonary fibrosis.

Pulmonary thromboendarterectomy in 106 patients with chronic thromboembolic pulmonary hypertension.

Pulmonary Hypertension in Patients with Chronic Fibrosing Idiopathic Interstitial Pneumonias.

High Frequency of Pulmonary Hypertension-Causing Gene Mutation in Chinese Patients with Chronic Thromboembolic Pulmonary Hypertension.

Bosentan for pulmonary hypertension secondary to idiopathic pulmonary fibrosis.

Relationship between coronary artery disease and pulmonary arterial pressure in patients with chronic obstructive pulmonary disease.

Assessment of pulmonary rehabilitation efficacy in chronic obstructive pulmonary disease patients using the chronic obstructive pulmonary disease assessment test.

Differences in pulmonary function and exercise capacity in patients with idiopathic dilated cardiomyopathy and idiopathic pulmonary arterial hypertension.