Project description:Rationale: Pulmonary arterial hypertension is a common and potentially fatal complication of scleroderma that may involve inflammatory and autoimmune mechanisms. Alterations in the gene expression of peripheral blood mononuclear cells have been previously described in patients with pulmonary arterial hypertension. The ability to identify patients at risk for developing pulmonary hypertension would be clinically beneficial. Objective: To identify genes that are differentially expressed in peripheral blood mononuclear cells in scleroderma patients with and without pulmonary hypertension which could be used as biomarkers of disease for early diagnosis and provide insight into pathogenesis of pulmonary hypertension in at-risk populations. Methods and Results: Gene expression analysis was performed on a carefully characterized Microarray Cohort of scleroderma patients with (n=10) and without (n=10) pulmonary hypertension. Differentially expressed genes were confirmed in the Microarray Cohort and validated in a separate Validation Cohort of scleroderma patients with (n=15) and without (n=19) pulmonary hypertension by RT-qPCR. We identified inflammatory and immune-related genes including interleukin-7 receptor (IL-7R) and chemokine receptor 7 (CCR7) as differentially expressed in patients with scleroderma-associated pulmonary hypertension. Flow cytometry confirmed decreased expression of IL-7R on circulating CD4+ T cells from scleroderma patients with pulmonary hypertension. Conclusions: Differences exist in the expression of inflammatory and immune-related genes in peripheral blood cells derived from patients with scleroderma-related pulmonary hypertension compared to those with normal pulmonary artery pressures. These findings may have implications as biomarkers to screen at-risk populations to facilitate early diagnosis and provide insight into inflammatory and autoimmune mechanisms of scleroderma-related pulmonary hypertension. Gene expression analysis was performed on a carefully characterized Microarray Cohort of scleroderma patients with (n=10) and without (n=10) pulmonary hypertension. Differentially expressed genes were confirmed in the Microarray Cohort by RT-qPCR.

Project description:Despite recent improvements in management of idiopathic pulmonary arterial hypertension, mortality remains high. Understanding the alterations in the transcriptome–phenotype of the key lung cells involved could provide insight into the drivers of pathogenesis. In this study, we examined differential gene expression of cell types implicated in idiopathic pulmonary arterial hypertension from lung explants of patients with idiopathic pulmonary arterial hypertension compared to control lungs. After tissue digestion, we analyzed all cells from three idiopathic pulmonary arterial hypertension and six control lungs using droplet-based single cell RNA-sequencing. After dimensional reduction by t-stochastic neighbor embedding, we compared the transcriptomes of endothelial cells, pericyte/smooth muscle cells, fibroblasts, and macrophage clusters, examining differential gene expression and pathways implicated by analysis of Gene Ontology Enrichment. We found that endothelial cells and pericyte/smooth muscle cells had the most differentially expressed gene profile compared to other cell types. Top differentially upregulated genes in endothelial cells included novel genes: ROBO4, APCDD1, NDST1, MMRN2, NOTCH4, and DOCK6, as well as previously reported genes: ENG, ORAI2, TFDP1, KDR, AMOTL2, PDGFB, FGFR1, EDN1, and NOTCH1. Several transcription factors were also found to be upregulated in idiopathic pulmonary arterial hypertension endothelial cells including SOX18, STRA13, LYL1, and ELK, which have known roles in regulating endothelial cell phenotype. In particular, SOX18 was implicated through bioinformatics analyses in regulating the idiopathic pulmonary arterial hypertension endothelial cell transcriptome. Furthermore, idiopathic pulmonary arterial hypertension endothelial cells upregulated expression of FAM60A and HDAC7, potentially affecting epigenetic changes in idiopathic pulmonary arterial hypertension endothelial cells. Pericyte/smooth muscle cells expressed genes implicated in regulation of cellular apoptosis and extracellular matrix organization, and several ligands for genes showing increased expression in endothelial cells. In conclusion, our study represents the first detailed look at the transcriptomic landscape across idiopathic pulmonary arterial hypertension lung cells and provides robust insight into alterations that occur in vivo in idiopathic pulmonary arterial hypertension lungs.

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