Volume 141, Issue 3, Pages 661-673 (March 2012) Gene Expression Profiling in the Lungs of Patients With Pulmonary Hypertension Associated With Pulmonary Fibrosis  Marco Mura, MD, PhD, Masaki Anraku, MD, Zhihong Yun, BSc, Karen McRae, MD, Mingyao Liu, MD, Thomas K. Waddell, MD, PhD, Lianne G. Singer, MD, John T. Granton, MD, Shaf Keshavjee, MD, Marc de Perrot, MD  CHEST  Volume 141, Issue 3, Pages 661-673 (March 2012) DOI: 10.1378/chest.11-0449 Copyright © 2012 The American College of Chest Physicians Terms and Conditions

Figure 1 Design of the study. Patients in the development set were divided based on their mean pulmonary arterial pressures. The criteria for SAM were a q value < 5% and a fold change ≥ 1.5. A 222-gene signature was found, and the differentially expressed genes were entered into the pathways/networks discovery analysis and the gene set enrichment analysis. This characterized the two different gene signatures found as “proproliferative” in the severe PH group and “proinflammatory” in the NoPH group. Next, patients in the intermediate PH group and in the validation set were clustered based on the gene signatures found, and the outcomes of the newly selected groups were analyzed. IPAH = idiopathic pulmonary arterial hypertension; PH = pulmonary hypertension; RT-PCR = real-time polymerase chain reaction. SAM = significance analysis of microarray CHEST 2012 141, 661-673DOI: (10.1378/chest.11-0449) Copyright © 2012 The American College of Chest Physicians Terms and Conditions

Figure 2 Representative histology slides from a patient with severe pulmonary hypertension and pulmonary fibrosis. A, Pulmonary artery branch in an area of dense fibrosis, showing medial thickening and significant intimal proliferation (hematoxylin and eosin stain, original magnification × 10). B, Pulmonary artery branch in a relatively fibrosis-spared area, showing similar changes (hematoxylin and eosin stain; original magnification × 10). CHEST 2012 141, 661-673DOI: (10.1378/chest.11-0449) Copyright © 2012 The American College of Chest Physicians Terms and Conditions

Figure 3 Ingenuity network analysis, networks with highest scores. Networks were scored based on the number of network eligible molecules they contained. Network eligible molecules with relatively increased expression in the PH group are shown in red, whereas molecules with relatively increased expression in the NoPH group are shown in green. The intensity of the color is proportional to the fold change. Fold change and q value are indicated under each network eligible molecule. Noncolored nodes represent genes added by Ingenuity pathway analysis based on its network algorithm but not upregulated in the actual microarray data. A, Respiratory disease, antigen presentation, cell-mediated immune response (Network 1, score 20). B, Cellular movement, hematologic system development and function, immune cell trafficking (Network 2, score 15). C, Antigen presentation, cell-mediated immune response, humoral immune response(Network 3, score 11). D, Cell death, cancer, cell cycle (Network 4, score 10). See Figure 1 legend for expansion of abbreviations. CHEST 2012 141, 661-673DOI: (10.1378/chest.11-0449) Copyright © 2012 The American College of Chest Physicians Terms and Conditions

Figure 3 Ingenuity network analysis, networks with highest scores. Networks were scored based on the number of network eligible molecules they contained. Network eligible molecules with relatively increased expression in the PH group are shown in red, whereas molecules with relatively increased expression in the NoPH group are shown in green. The intensity of the color is proportional to the fold change. Fold change and q value are indicated under each network eligible molecule. Noncolored nodes represent genes added by Ingenuity pathway analysis based on its network algorithm but not upregulated in the actual microarray data. A, Respiratory disease, antigen presentation, cell-mediated immune response (Network 1, score 20). B, Cellular movement, hematologic system development and function, immune cell trafficking (Network 2, score 15). C, Antigen presentation, cell-mediated immune response, humoral immune response(Network 3, score 11). D, Cell death, cancer, cell cycle (Network 4, score 10). See Figure 1 legend for expansion of abbreviations. CHEST 2012 141, 661-673DOI: (10.1378/chest.11-0449) Copyright © 2012 The American College of Chest Physicians Terms and Conditions

Figure 3 Ingenuity network analysis, networks with highest scores. Networks were scored based on the number of network eligible molecules they contained. Network eligible molecules with relatively increased expression in the PH group are shown in red, whereas molecules with relatively increased expression in the NoPH group are shown in green. The intensity of the color is proportional to the fold change. Fold change and q value are indicated under each network eligible molecule. Noncolored nodes represent genes added by Ingenuity pathway analysis based on its network algorithm but not upregulated in the actual microarray data. A, Respiratory disease, antigen presentation, cell-mediated immune response (Network 1, score 20). B, Cellular movement, hematologic system development and function, immune cell trafficking (Network 2, score 15). C, Antigen presentation, cell-mediated immune response, humoral immune response(Network 3, score 11). D, Cell death, cancer, cell cycle (Network 4, score 10). See Figure 1 legend for expansion of abbreviations. CHEST 2012 141, 661-673DOI: (10.1378/chest.11-0449) Copyright © 2012 The American College of Chest Physicians Terms and Conditions

Figure 3 Ingenuity network analysis, networks with highest scores. Networks were scored based on the number of network eligible molecules they contained. Network eligible molecules with relatively increased expression in the PH group are shown in red, whereas molecules with relatively increased expression in the NoPH group are shown in green. The intensity of the color is proportional to the fold change. Fold change and q value are indicated under each network eligible molecule. Noncolored nodes represent genes added by Ingenuity pathway analysis based on its network algorithm but not upregulated in the actual microarray data. A, Respiratory disease, antigen presentation, cell-mediated immune response (Network 1, score 20). B, Cellular movement, hematologic system development and function, immune cell trafficking (Network 2, score 15). C, Antigen presentation, cell-mediated immune response, humoral immune response(Network 3, score 11). D, Cell death, cancer, cell cycle (Network 4, score 10). See Figure 1 legend for expansion of abbreviations. CHEST 2012 141, 661-673DOI: (10.1378/chest.11-0449) Copyright © 2012 The American College of Chest Physicians Terms and Conditions

Figure 4 Hierarchic clustering. A, Hierarchic clustering based on 222 differentially expressed genes (PH vs NoPH groups) in the severe PH (n = 17), NoPH (n = 22) and, below, the idiopathic PH (n = 7) groups of patients. Each row corresponds to an individual sample, and each column corresponds to an individual gene. Each square on the matrix represents the expression level of an individual gene in each sample, with red and green indicating gene expression levels above or below, respectively, compared with each other. The patients clustering in the subgroup with relatively increased expression of genes seen in the PH group are identified as PH phenotype, whereas the patients clustering in the subgroup with relatively increased expression of genes seen in the NoPH group are identified as NoPH phenotype. B, Hierarchic clustering based on 222 differentially expressed genes (PH vs NoPH groups) in the intermediate PH group of patients (n = 45). C, Hierarchic clustering based on 222 differentially expressed genes (PH vs NoPH groups) in the development set (n = 84) and in the validation set of patients (n = 32). See Figure 1 legend for expansion of abbreviations. CHEST 2012 141, 661-673DOI: (10.1378/chest.11-0449) Copyright © 2012 The American College of Chest Physicians Terms and Conditions

Figure 5 Quantitative RT-PCR confirmation of selected genes. Relative gene expression levels of six selected genes by quantitative RT-PCR comparing patients in the severe PH and NoPH groups. For each gene, the ratio with the expression the housekeeping gene RPLP0 is presented. Data are presented as mean ± SD. A, SPP1. B, TP63. C, MMP1. D, EDNRB. E, PTX3. F, S100A8. EDNRB = endothelin receptor type B; MMP1 = metalloproteinase 1; PTX3 = long pentraxin 3; RPLP0 = ribosomal protein large P0; S100A8 = S100 calcium-binding protein A8; SPP1 = osteopontin. See Figure 1 legend for expansion of other abbreviations. CHEST 2012 141, 661-673DOI: (10.1378/chest.11-0449) Copyright © 2012 The American College of Chest Physicians Terms and Conditions