Long non-coding RNA PANDA promotes endothelial senescence and oxidative damage through the interaction with NRF2
Authors: Mongelli A , Mohammed S , Gorica E , Mengozzi A , Telesca M , Matter C M , Paneni F , Ruschitzka F , Costantino S 
Published: October 2024

Abstract

Background
Accumulation of reactive oxygen species and inflammation are major features of diabetic vasculopathy, yet the underlying mechanisms remain elusive. LncRNAs are emerging as important players in the pathogenesis of cardiovascular disease. PANDA, a newly identified lncRNA, is a key regulator of cellular senescence, apoptosis and oxidative stress.

Purpose
To investigate the role and molecular mechanism of PANDA in diabetic vascular disease.

Methods
RNAseq was performed to aorta specimen from diabetic and no diabetic patients as well to HAECs exposed to 5 mM and 25 mM concentrations (Normal -NG- and High -HG-, respectively). PANDA depletion in HG-treated HAECs was obtained by siRNA transfection while a scrambled siRNA wass used as a negative control. RNAseq and network perturbation analysis (NPA) of treated HAECs unveiled transcriptional changes upon PANDA depletion. Expression of PANDA was assessed by real time PCR. PANDA RNA-IP was performed to check its binding to relevant transcriptional factor (such as NRF2) as well Ch-IP was performed to check the NRF2 binding on oxidative gene promoters. Cellular localization of NRF2 was investigated by Immunofluorescence. Beta-galactosidase and superoxide staining was used to detect endothelial senescence and ROS formation; while, migration and tube formation were employed to evaluate angiogenic properties of HAECs.

Results
PANDA expression was significantly increased in diabetic human aorta as well in HG-treated HAECs (Figure1 A-B). Transcriptomic analysis revealed dysregulation of several genes upon PANDA silencing with the antioxidant gene heme oxygenase-1 (HMOX1) as the top-ranking transcript in HG cells (Figure1 C-D). Western blot analysis reveals the restored level of HMOX1 and TFRC1 upon PANDA depletion (Figure1 E-G). NPA analysis showed a strong involvement of PANDA in senescence, DNA damage, NRF2 signaling, hypoxic stress response and proliferation. In HG, NFR2 is downregulated while PANDA silencing restores its level (Figure1 I-J). We found that in HG condition PANDA binds the transcription factor NRF2 and blocks its nuclear translocation thus impeding its binding on HMOX1 and TFRC promoters (Figure1 K-M).
Silencing of PANDA in HG-treated HAECs reduced genes and cellular features of senescence (Figure2 A-B), restored the expression of anti-apoptotic genes and decreases caspase 3 activity (Figure2 C-D), improved endothelial migration and tube formation (Figure2 E and G), and reduced ROS formation (Figure2 F).

Conclusions
In hyperglycemia PANDA upregulation drives endothelial senescence while impairing angiogenic properties. On the other hand, 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. The results indicate PANDA as a putative novel molecular target in the setting of diabetic vascular disease (Figure2 H).