RNA Sequencing Analysis Detection of a Novel Pathway of Endothelial Dysfunction in Pulmonary Arterial Hypertension

Abstract

Rationale: Pulmonary arterial hypertension is characterized by endothelial dysregulation, but global changes in gene expression have not been related to perturbations in function.Objectives: RNA sequencing was used to discriminate changes in transcriptomes of endothelial cells cultured from lungs of patients with idiopathic pulmonary arterial hypertension versus control subjects and to assess the functional significance of major differentially expressed transcripts.Methods: The endothelial transcriptomes from the lungs of seven control subjects and six patients with idiopathic pulmonary arterial hypertension were analyzed. Differentially expressed genes were related to bone morphogenetic protein type 2 receptor (BMPR2) signaling. Those down-regulated were assessed for function in cultured cells and in a transgenic mouse.Measurements and Main Results: Fold differences in 10 genes were significant (P , 0.05), four increased and six decreased in patients versus control subjects. No patient was mutant for BMPR2. However, knockdown of BMPR2 by siRNA in control pulmonary arterial endothelial cells recapitulated 6 of 10 patient-related gene changes, including decreased collagen IV (COL4A1, COL4A2) and ephrinA1 (EFNA1). Reduction of BMPR2-regulated transcripts was related to decreased b-catenin. Reducing COL4A1, COL4A2, and EFNA1 by siRNA inhibited pulmonary endothelial adhesion, migration, and tube formation. In mice null for the EFNA1 receptor, EphA2, versus control animals, vascular endothelial growth factor receptor blockade and hypoxia caused more severe pulmonary hypertension, judged by elevated right ventricular systolic pressure, right ventricular hypertrophy, and loss of small arteries. Conclusions:The novel relationship between BMPR2 dysfunction and reduced expression of endothelial COL4 and EFNA1 may underlie vulnerability to injury in pulmonary arterial hypertension.

Epigenetic mechanisms in pulmonary arterial hypertension: the need for global perspectives

“…The impact of epigenetic changes such as DNA methylation and histone modification has been better documented in the pathogenesis of other human diseases [33][34][35][36], while the application of genomic technologies in human PAH samples are relatively limited. To date, except for a few studies [37][38][39][40], the approaches employed to delineate the disease mechanisms in PAH have not provided a global perspective of the molecular network underlying the disease phenotype. To gain insight into the mechanisms leading to the development of PAH, a broad range of high-throughput techniques in genomics, proteomics and metabolomics can be employed to systematically dissect the disease-specific networks in isolated pulmonary vascular cells, peripheral blood mononuclear cells and pulmonary tissues collected from patients.…”

Amphetamines promote mitochondrial dysfunction and DNA damage in pulmonary hypertension

“…Total RNA was extracted and purified from cells (Zymo Research). The quantity and quality of RNA were determined by a spectrophotometer, and then RNA was reverse transcribed to cDNA (60). The primers are listed in the Supplemental Information.…”

Induced Pluripotent Stem Cell Model of Pulmonary Arterial Hypertension Reveals Novel Gene Expression and Patient Specificity

“…PAEC were obtained from explanted lungs through the Pulmonary Hypertension Breakthrough Initiative Network by scraping the endothelial layer of PAs (Tables 1 and 2) before culture in EC medium according to previous protocols (5). Procurement of the tissues from human subjects is approved by the Administrative Panel on Human Subjects in Medical Research at Stanford University (IRB #350, Panel 6).…”

“…Reduced BMPR2 is associated with impaired adhesion and migration of PA endothelial cells (PAEC) related to decreased expression of downstream targets collagen (COL) 4A1 and A2 and Ephrin A1 (5), heightened susceptibility to inflammation (7), and defects in cytoskeletal protein phosphorylation (8). In addition, decreased BMPR2 suppresses downstream activation of mothers against decapentaplegic homolog proteins (SMAD)1/5 and transcription of the early response gene inhibitor of DNA binding 1 (ID1) (9) that regulates angiogenesis (10), and SMAD1/5 independent signaling (3,9).…”