Idiopathic pulmonary arterial hypertension (IPAH), a subgroup of WHO grp 1 pulmonary arterial hypertension (PAH), is a rare disorder associated with severe morbidity and high mortality rates. There are no routine screening tests or validated markers of disease activity in IPAH, or the broader group of disease associated PAH (DaPAH). Therefore, patients usually present at advanced stages of disease. The pathogenesis of IPAH and DaPAH remain unclear. Current thinking focuses on a two-hit hypothesis: 1) genetic susceptibility, and 2) a triggering stimulus that initiates pulmonary vascular injury, resulting in endothelial cell (EC) dysfunction. Endothelial cells are normally shed into the circulation and are a valuable source of clinical material for studying diseases characterized by EC dysfunction. However, no clear methodology exists for isolating clinically relevant numbers of circulating ECs (CECs). In the bench phase of the project we were using flow cytometry to develop a methodology for isolating clinically relevant numbers of viable CECs from healthy volunteers and PAH patients. We hypothesized that CECs and/or peripheral blood mononuclear cells (PBMC) can be used to define a subset of differentially regulated biomarkers in IPAH and DaPAH that may lead to earlier diagnosis and better methods for measuring responses to therapy. We also hope to identify novel targets for future therapeutic interventions. In the clinical phase of the project, we recruited healthy volunteers and patients with IPAH and DaPAH. Peripheral blood specimens were obtained for CECs and PBMCs for microarrays; the remaining plasma was saved for future application to cultured microvascular cells. A subset of subjects underwent right heart catheterization to assess pulmonary pressures and to obtain pulmonary blood specimens. The protocol started actively enrolling in June 2006 and enrolled 31 subjects prior to closing to enrollment in 2009. Preliminary data suggested that there was no trend towards CEC enrichment in pulmonary vein blood compared to peripheral blood for both the healthy volunteers (4.4 CEC/ml vs. 4.8 CEC/ml) and the PAH patients (2.4 CEC/ml vs. 3.0 CEC/ml). There was a trend towards CEC enrichment in pulmonary artery blood compared to peripheral blood for both the healthy volunteers (13.8 CEC/ml vs. 4.8 CEC/ml) and the PAH patients (3.3 CEC/ml vs. 3.0 CEC/ml). In 2010 and 2011, total RNA was processed from PBMCs for genome-wide expression analysis. An abstract based on the PBMC differential gene expression patterns in PAH was presented at the 2011 Annual American Thoracic Society (ATS) Meeting. These patterns reflected both treatment related signatures and underlying disease pathophysiology. In addition to completing expression profiling of PBMCs, plasma samples from healthy volunteers and PAH patients were processed for application to cultured microvascular ECs. In 2011 to 2013, using PBMC expression profiles from 10 PAH subjects with 10 age, gender and race matched healthy control subjects we identified over 230 differentially regulated genes at a 20% false discovery rate (FDR). Ingenuity Pathway Analysis identified gene signatures for inflammation, cell-to-cell signaling and interaction, cytoskeletal rearrangement, cellular movement, hemostasis and cell death. In vitro data from our collaborating laboratory demonstrates that spironolactone suppresses phorbol 12-myristate 13-acetate-induced (PMA; an AP-1 activator) inflammatory gene transcription in primary human PAECs. In order to explore the effect of spironolactone on PAH-associated vascular inflammation we conducted a promoter level analysis of the up-regulated genes we identified in PAH subjects. Biobase ExPlain and Transfac bioinformatics software identified activator protein-1 (AP-1) as a key transcriptional regulator. Experiments using PBMCs isolated from healthy subjects and stimulated with PMA demonstrate that spironolactone suppresses these AP-1 inducible, PAH-associated genes in a dose-dependent manner. Similarly in PBMCs from healthy and PAH subjects, spironolactone strongly suppressed the basal expression of genes that had been up regulated in the PBMCs of PAH patients. In addition in 2011-12 we continued to develop a bioassay assessing global transcriptomic changes induced by plasma from PAH subjects compared to healthy controls using Affymetrix oligonucleotide microarrays. Exposure of human PAECs to plasma from 5 PAH subjects compared to 5 age, gender and race matched controls, identified over 300 differentially expressed transcripts at a 10% FDR. In additional work done in 2012-13, we explored the gene expression changes in cultured human PAECs induced by plasma from PAH subjects and found that 20% of this signature overlapped with the gene expression changes induced following bone morphogeneic protein receptor-2 (BMPR2) gene silencing in PAECs. Importantly, more than 90% of this overlap was directionally discordant, suggesting circulating factors may work to counter-regulate genotypic and phenotypic abnormalities that drive PAH. Future experiments will utilize stored plasma currently available from PAH patients and healthy controls to examine the effects of circulating mediators on gene expression in BMPR2-deficient PAECs. In 2013-14, we continued to actively investigate the mechanisms that mediate the anti-inflammatory effects of spironolactone using molecular techniques such as overexpression vectors and AP-1 promoter based reporter assays. Furthermore, in order to expand upon our expression findings in circulating mononuclear cells, we started to collect data from all of the other published human PAH PBMC genome-wide expression profiling studies for subsequent meta-analysis. In 2014-15 we completed the necessary steps of data collection, annotation and aggregation of all the published human PAH PBMC expression profiling studies, Meta-analysis of all studies comparing gene expression profiles from IPAH and DaPAH patients to healthy subjects identified 579 and 1186 differentially expressed transcripts, respectively, at a 1% FDR. Interestingly, comparing gene expression profiles from IPAH and DaPAH patients yielded zero differentially expressed transcripts at a 1% or 5% FDR. Sensitivity analyses are planned to determine the effects of any one study on the overall stringency of the gene list and on the bioinformatic pathway analyses. Defining a robust genomic signature across multiple studies of PBMCs may highlight previously unrecognized patterns of gene expression. Specifically we plan to focus on differentially expressed transcripts that overlap between the IPAH versus healthy and the DaPAH versus healthy comparisons. Hypotheses generated from this work will be tested in downstream in vitro experiments. In 2015-16, we added additional data from a recently published blood transcriptomic study conducted in patients with chronic respiratory disease (n=8 PAH patients) compared to healthy controls (n=28). With this updated data set, we completed further downstream analyses. Differentially expressed genes in PAH previously identified in the literature demonstrated only modest reproducibility, defined as a FDR 10% by meta-analysis. Different bioinformatic approaches consistently identified inflammatory signaling (MAPK, Toll-like receptors, and interferon responses) and regulators of cell proliferation (Myc) as overrepresented pathways among the shared genomic signature in IPAH and DaPAH. An abstract of this work was presented at the 2016 ATS International Conference (Meta-analysis of PBMC Genome-wide Expression Profiling in PAH. AJRCCM 193: A4619, 2016.). This protocol remains open to continue bioinformatic analyses of the gene expression data, completing downstream in vitro work as well as finishing a meta-analysis of the published PAH PBMC expression profilin
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