Long non-coding RNA PANDA drives diabetic vascular dysfunction by promoting endothelial senescence and oxidative damage
Authors: Mongelli A , Mohammed S A , Gorica E , Mengozzi A , Telesca M , Matter C M , Paneni F , Ruschitzka F , Costantino S 
Published: May 2024

Abstract

Funding Acknowledgements
None.

Background
ROS and inflammation are major features of diabetic vasculopathy, yet the underlying mechanisms remain elusive. Long non-coding RNAs (lncRNAs) are emerging as important players in the pathogenesis of cardiovascular disease. Recent work has shown that PANDA, a newly identified lncRNA, is a key regulator of cellular senescence and apoptosis.

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

Methods
Human aortic endothelial cells (HAECs) were exposed to normal (NG, 5 mmol/L) and high glucose concentrations (HG, 25 mmol/L). PANDA depletion in HG-treated HAECs was obtained by siRNA transfection. Expression of PANDA was assessed by real time PCR. RNAs sequencing (RNA-seq) and bioinformatic analysis (network perturbation amplitude, NPA) were leveraged to unveil transcriptional changes upon PANDA depletion. PANDA RNA immuno-precipitation (RIP) was performed to check its binding to relevant transcriptional factor. Western Blots was used to investigate on NRF2 protein and its products. Beta-galactosidase staining was used to detect endothelial senescence while, migration and tube formation were employed to evaluate angiogenic properties of HAECs. ROS production and NRF2 localization were investigated by fluorescence staining. The ex vivo effect of PANDA siRNA on endothelial function was assessed in aortic rings from diabetic mice.

Results
PANDA expression was significantly increased in HAECs exposed to HG as compared to NG. Transcriptomic analysis revealed dysregulation of several genes upon PANDA silencing with the antioxidant gene heme oxygenase-1 as the top-ranking transcript in HG-treated cells (Fig). NPA analysis showed a strong involvement of PANDA in senescence, DNA damage, NRF2 signaling, hypoxic stress response and proliferation. Under HG conditions PANDA perturbed the pathway of the transcription factor NRF2, thus inhibiting the expression of NRF2-dependent pro-survival and anti-aging gene. Indeed, silencing of PANDA in HG-treated HAECs reduced the apotosis and senescence (Figure 1). PANDA depletion in HG improved endothelial migration and tube formation, while ROS production was reduced. In HG, there was an increasing of PANDA-NRF2 binding (Fig). Interesting, in HG NRF2 was more present in the cytosol, while PANDA siRNA allowed its translocation into the where it can promote the transcription of HMOX1. Of interest, PANDA levels were increased in aortas from diabetic mice while depletion of PANDA rescued endothelial dysfunction (Figure 2).

Conclusions
The hyperglycemia induced the upregulation of PANDA driving endothelial senescence and impairing angiogenic properties. PANDA depletion in HG-treated HAECs rescued maladaptive transcriptional changes enhancing NRF2 pathway. Of note, targeting PANDA in the diabetic vasculature was able to rescue endothelial dysfunction. These results indicate PANDA as a novel molecular target in the setting of diabetic vascular disease.