Abstract 13600: RNAseq Analysis of Pulmonary Arterial Endothelial Cells from Patients with Idiopathic Pulmonary Arterial Hypertension Reveals Dysregulation of Angiogenic Factors
Pulmonary arterial (PA) endothelial cell (EC) dysfunction is a pathological feature in idiopathic PA hypertension (IPAH) reflected both in vulnerability to apoptosis and dysregulation of angiogenesis, which may be in part due to defective BMPR2 signalling. To better understand and target this abnormality, we analyzed gene expression in PAEC using RNA-Seq, a sequencing-based method that provides comprehensive analysis of the transcriptome. Using 2 independent sets of PAECs cultured from (n=3, 4) unused donor and (n=3, 3) IPAH lungs, we identified consistently altered expression levels of 23 genes (>50% average change in both sets). These include ephrin-A1, IQSEC1, ISG20 and collagen IV alpha-1 and -2 chains (down in IPAH) and endothelial nitric oxide synthase (eNOS, up in IPAH), all p<0.05. Fold-changes with p<0.1 detected by RNAseq were validated by qPCR analysis. BMPR2 knockdown in control PAECs recapitulated some of the IPAH-related gene expression changes, including downregulation of ephrinA1 mRNA and upregulation of eNOS mRNA levels (both p=0.02), further implicating dysregulation of these genes in PAH. Ephrin-A1 stimulates systemic EC migration, adhesion and tube formation and regulates basement membrane collagen IV deposition. IQSEC1 and ISG20 have also been implicated in the regulation and promotion of systemic angiogenesis. Western blotting confirmed downregulation of collagen IV chains at the protein level (p<0.05). Moreover ephrin-A1-Fc constructs stimulated migration of PAECs. Future studies will address whether loss of ephrin-A1 abrogates migration and tube formation in controls and whether supplementation with ephrin-A1-Fc rescues function in patients. In conclusion, RNAseq reveals novel changes in gene expression related to aberrant angiogenesis that may explain failure to regenerate normal distal microvessels and obliterative remodeling of proximal vessels in patients with PAH.
- © 2012 by American Heart Association, Inc.