Role of the long non-coding RNA DSCR9 in pulmonary artery endothelial cells

Pulmonary arterial hypertension (PAH) is a rare and life-threatening disease. PAH has a multifactorial aetiology and several genetic alterations have been implicated in its development, of which mutations in bone morphogenic protein receptor 2 (BMPR2) are the most prevalent ones. A defective nitric oxide (NO) production in PAH patients’ pulmonary endothelium is considered one of the main drivers of PAH pathology, leading either to a loss of pulmonary vascular homeostasis or pulmonary vascular remodelling. Despite several studies investigating this alteration, the molecular factors leading to NO deficiency in PAH are still only partially known. To better understand the mechanism causing NO deficiency in PAH, commercial primary human Pulmonary Artery Endothelial Cells (hPAEC) were incubated for 24 hours at atmospheric or 40 mmHg-higher pressure in a dedicated chamber. NO production was evaluated using diamino-fluorescein diacetate-FM. Cell viability and proliferation were assessed with MTS assay, flow cytometry, cell cycle analysis and scratch assay, while endothelial nitric oxide synthase (eNOS) phosphorylation and abundance were evaluated by western blot. Changes in the RNA profile were explored with next-generation sequencing (NGS) and differentially expressed genes (DEGs) analysed with ShinyGO vo.6.1. DEGs were considered significant when displaying a p.value < 0.05 and falling within the 90th centile. Following transcriptomic data validation, key DEGs were analysed with RT-PCR, also in primary hPAEC of PAH patients (PAH-hPAEC) and control (control-hPAEC), and endothelial cells (ECs) derived from induced pluripotent stem cells (iPSC-EC) from patients with a PAH-causing mutation in BMPR2. Finally, the effects of lentiviral DSCR9 overexpression were assessed in primary hPAEC. Exposure to high pressure resulted in a decrease in NO production in commercial hPAEC, not due to a decrease in cell viability or proliferation. Furthermore, NO deficiency was not reverted by the addition of exogenous L-Arginine. Despite an increase in total eNOS expression, there was a decrease in phosphorylated eNOS under high-pressure conditions, as revealed by Western Blot. Among 11,486 DEGs, the long non-coding RNA Down Syndrome Critical Region 9 (DSCR9) was the most upregulated upon incubation of commercial hPAEC at high pressure, and in silico analysis revealed its possible involvement in the eNOS pathway. DSCR9 was enriched in Gene Expression Omnibus microarray datasets from patients with PAH (GSE90943, GSE151971, GSE117261) and RT-qPCR confirmed its upregulation. DSCR9 upregulation was also confirmed in PAH-hPAECs and in BMPR2-mutated iPSC-ECs. Furthermore, overexpression of DSCR9 in PAH-hPAEC recreated the imbalance in the eNOS pathway observed in commercial hPAEC exposed to high pressure, with increased total, but decreased phosphorylated protein levels. These results suggest that DSCR9 is involved in PAH pathobiology by modulating eNOS and NO synthesis.