Idiopathic pulmonary arterial hypertension (IPAH), a subgroup of WHO grp 1 PAH, is a rare disorder with severe morbidity and high mortality. There are no routine screening tests or validated markers of disease activity in IPAH, or the broader group of disease associated PAH (DaPAH). Patients can 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. We hypothesized that circulating ECs (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, better methods for assessing responses to therapy, and potentially identify novel targets for future therapeutic interventions. Endothelial cells shed into the circulation 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 CECs. This project used flow cytometry to develop a methodology for isolating relevant numbers of viable CECs from healthy and PAH subjects. In addition, peripheral blood (PB) specimens were obtained for CECs and PBMCs for microarrays from healthy volunteers and IPAH and DaPAH patients. Plasma was also saved for application to cultured microvascular cells. A subset of subjects had a right heart catheterization to assess pulmonary pressures and to obtain pulmonary blood specimens. The protocol started actively enrolling in June 2006 and closed to enrollment in 2009 (n=31). Available data suggested there was no trend towards CEC enrichment in pulmonary vein blood compared to PB for both healthy volunteers (4.4 CEC/ml vs. 4.8 CEC/ml) and PAH patients (2.4 CEC/ml vs. 3.0 CEC/ml). There was a trend towards CEC enrichment in pulmonary artery blood compared to PB for both healthy volunteers (13.8 CEC/ml vs. 4.8 CEC/ml) and PAH patients (3.3 CEC/ml vs. 3.0 CEC/ml). In 2010-11, total RNA was processed from PBMCs for genome-wide expression analysis. An abstract on PBMC differential gene expression patterns in PAH was presented at the American Thoracic Society (ATS) Meeting (AJRCCM 183:A5511, 2011). The patterns reflected both treatment related signatures and underlying disease pathophysiology. In 2011-2013, using PBMC expression profiles from 10 PAH subjects with 10 age, gender and race matched healthy 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 lab showed that spironolactone suppresses phorbol 12-myristate 13-acetate-induced (PMA; an AP-1 activator) inflammatory gene transcription in primary human PAECs. 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 upregulated in PBMCs of PAH patients. In 2011-12 we also continued to develop a bioassay assessing global transcriptomic changes induced by plasma from PAH compared to healthy subjects using Affymetrix oligonucleotide microarrays. Exposure of human PAECs to plasma from 5 PAH subjects compared to 5 matched controls, identified over 300 differentially expressed transcripts at a 10% FDR. We also explored (2012-13) the gene expression changes in cultured human PAECs induced by plasma from PAH subjects and found that 20% of the signature overlapped with the gene expression changes induced following bone morphogeneic protein receptor-2 gene silencing in PAECs. Importantly, over 90% of the overlap was directionally discordant, suggesting circulating factors may work to counter-regulate genotypic and phenotypic abnormalities that drive PAH. In 2013-14, we expanded our PBMC expression findings, by starting to collect data from all 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 no differentially expressed transcripts at a 1% or 5% FDR. Defining a robust genomic signature across multiple PBMC studies may highlight previously unrecognized gene expression patterns. In 2015-16, we added data from a recently published blood transcriptomic study conducted in chronic respiratory disease patients (n=8 PAH patients) compared to healthy controls (n=28). With the 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 and regulators of cell proliferation as overrepresented pathways among the shared genomic signature in IPAH and DaPAH. An abstract was presented at ATS 2016 (AJRCCM 193: A4619, 2016). In 2016-17, over 30 differentially expressed genes in the combined PAH cohort were selected for validation using samples from this protocol. Importantly, over 900 genes identified by meta-analysis were not previously reported in the 7 published studies. Functional analysis of the 1269 differentially expressed genes derived from the combined PAH cohort identified several inflammatory signaling pathways as significantly overrepresented. Among these EIF2 and mTOR signaling have recently been linked to PAH pathogenesis demonstrating the validity of our meta-analytic approach. We also found that in circulating immune cells interferon plays a prominent role in the inflammatory component of PAH pathobiology. Next we performed a gene set analysis using data from all 22753 genes included in the meta-analysis without using arbitrary gene selection criteria. The analysis further supported the prominence of interferon signaling in circulating immune cells of PAH patients. Lastly we conducted a promoter-level analysis to determine if there was enrichment for specific transcription factor binding sites in the upregulated genes from the combined PAH cohort. The upregulated genes were compared to a group of genes that were not differentially regulated between PAH and healthy subjects. The results show highly significant enrichment of binding sites for the interferon regulatory factor family of transcription factors among the upregulated PAH genes. Project data was presented at the 2017 American College of Cardiology Annual Scientific Session as an invited talk entitled Genomics: Meta-Analysis of Blood Expression Profiling Studies in PAH. The protocol remains open to continue bioinformatic analyses of gene expression data, complete downstream in vitro work and finish the meta-analysis of the PAH PBMC expression profiling.
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