Long non-coding RNA PANDA drives diabetic vascular dysfunction by promoting endothelial senescence and oxidative stress

Background Long non-coding RNAs (lncRNAs) are emerging as pivotal players in the pathogenesis of cardiovascular disease. Recent work has shown that PANDA, a newly identified lncRNA, is a key regulator of cellular senescence, apoptosis and oxidative stress acting as decoy lncRNA. Purpose To investigate the role and molecular mechanism of lncRNA PANDA in diabetic vascular disease. Methods RNAseq was performed to aorta specimen from diabetic and no diabetic patients as well in human aortic endothelial cells (HAECs) exposed to normal (NG, 5 mM) and high glucose concentrations (HG, 25 mM). PANDA modulations were performed by siRNA transfection and by the lipid transfection of PANDA carrying plasmid. RNAs sequencing (RNA-seq) and bioinformatic analysis (network perturbation amplitude, NPA) of treated HAECs unveiled transcriptional changes upon PANDA depletion. Expression of PANDA was assessed by real time PCR. PANDA RNA immuno-precipitation (RIP) was performed to check its binding to relevant transcriptional factor (such as NRF2) as well chromatin immunoprecipitation (ChIP) was performed to check the NRF2 binding on oxidative gene promoters. Cellular localization of NRF2 was investigated by Immunofluorescence. Beta-galactosidase, superoxide staining and lipid peroxidation assay were used to detect endothelial senescence and ROS formation while migration and tube formation were employed to evaluate angiogenic properties of HAECs. Results In diabetic human aorta as well in HAECs exposed to HG, PANDA levels were significantly increased. Transcriptomic analysis revealed the upregulation of antioxidant genes (HMOX1, TFRC1) upon PANDA removal and the data was also confirmed at protein level. NPA analysis showed a strong involvement of PANDA in senescence, DNA damage as well as NRF2 signaling, which is the main transcription factor of antioxidant genes. In HG, NFR2 is downregulated while PANDA silencing restores its level. Under HG condition PANDA binds the transcription factor NRF2 and blocks its nuclear translocation thus impeding its binding on HMOX1 and TFRC promoters. Silencing of PANDA in HG-treated HAECs prevents cellular senescence, restores the expression of anti-apoptotic genes, and reduces ROS formation and lipid peroxidation, a marker of ferroptosis. Interestingly, overexpression of PANDA in NG-treated cells leads to increased oxidative stress and lipid peroxidation, thus mimicking the effect observed under HG conditions. Conclusions In hyperglycemia PANDA upregulation drives endothelial senescence while impairing angiogenic properties. Accordingly, PANDA depletion in HG-treated HAECs rescues maladaptive transcriptional changes through the release of NRF2 that in turn translocate into the nucleus enhancing the expression of the antioxidant gene HMOX1 and TFRC. The results indicate PANDA as a putative novel molecular target in the setting of diabetic vascular disease.