Serial mRNA profiling of peripheral blood monocytes from PAH patients treated with phosphodiesterase 5 inhibitors (PDE5i) and PDE5i+macitentan
Purpose: We performed single cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) from non-diseased control individuals (N=3) as well as from individuals with PAH, naïve to treatment (N=4), 3 months after phosphodiesterase 5 inhibitor (PDE5i) treatment (N=5), and 3 months after treatment with PDE5i plus an endothelin receptor antagonist, macitentan (N=5). We performed complementary analyses of differential gene expression and differential gene network dependency. Method: Blood samples collected in EDTA purple top vacutainer were centrifuged at 2000 RPM for 15 minutes and plasma was removed for banking. Buffy coats were collected and used for Ficoll gradient to isolate PBMC which are used for the single cell RNA sequencing analysis. RNA libraries were prepared using Chromium Single Cell 3' Reagent Kits V2 per manufacturer's guidelines (10x Genomics manual CG00052, cat# PN-12237). Single cell RNA-sequencing was performed using the GemCodeTM Single Cell Instrument (Chromium, 10X Genomics) which included at least 800 cells (~50K reads per cell) per N=3 non-PAH control samples, N=4 PAH no treatment samples, N=8 PDE5i treated samples, and N=6 PDE5i+Macitentan treated samples. Overall design: B cells, CD8+ T cells, CD4+ T cells, and NK cell RNA profile from non-PAH control, PAH no treatment, PDE5i treated, and PDE5i+macitentan treated samples