PFKFB3 nuclear translocation improves diabetic retinopathy by attenuating endothelial cell senescence through inhibition of USP7-p53 axis.

Single-cell RNA sequencing identified endothelial cell subclusters in diabetic retinas that are enriched for senescence transcripts and simultaneously depleted for barrier and tight-junction pathways.

• Single-cell RNA sequencing was used to profile EC senescence signatures and barrier/tight-junction programs in diabetic retinas.
• Distinct EC subclusters were identified showing concurrent upregulation of senescence-associated transcripts and downregulation of barrier/tight-junction pathway genes.
• This analysis linked EC senescence to retinal vascular dysfunction at the transcriptomic level in the diabetic context.

Hyperglycemia reduced PFKFB3 expression and impaired its nuclear entry, leading to prominent cellular senescence both in vitro and in vivo.

• PFKFB3 abundance and nuclear localization were assessed by Western blotting, immunofluorescence, and related assays under hyperglycemic conditions.
• Senescence was quantified using SA-β-gal staining, immunofluorescence, and cell-cycle flow cytometry.
• Restoration of PFKFB3 effectively reversed the hyperglycemia-induced senescence phenotype in both cell culture and animal models.

Anti-senescent activity of PFKFB3 required its nuclear localization, as demonstrated by stable endothelial cell lines expressing compartment-restricted PFKFB3 mutants.

• Stable endothelial cell lines were established expressing PFKFB3 only in the nucleus (NLS mutant) or only in the cytoplasm (K472Q mutant).
• The nuclear-localized NLS mutant retained anti-senescent activity, whereas the cytoplasm-restricted K472Q mutant did not.
• This compartment-specific approach directly distinguished the nuclear function of PFKFB3 from its cytoplasmic glycolytic role.

Nuclear-localized PFKFB3 physically interacted with USP7 and regulated the USP7-p53 axis by constraining their coupling, thereby promoting proteasomal degradation of p53.

• PFKFB3-USP7 interaction was examined by co-immunoprecipitation, mass spectrometry, and nuclear colocalization studies.
• Nuclear PFKFB3 constrained the coupling between USP7 and p53, reducing USP7-mediated deubiquitination and stabilization of p53.
• Reduced USP7-p53 coupling led to increased proteasomal degradation of p53.
• USP7 is described as 'a critical modulator of the p53 pathway.'

USP7 abrogated the protective effect of PFKFB3, and its inhibition attenuated hyperglycemia-induced senescence and mitigated retinal vascular dysfunction.

• USP7 acted as a downstream effector of PFKFB3 in the senescence pathway.
• Pharmacological or genetic inhibition of USP7 was sufficient to attenuate hyperglycemia-induced EC senescence.
• USP7 inhibition also mitigated retinal vascular dysfunction in the diabetic setting.
• Retinal vascular dysfunction was quantified by Evans blue leakage and PAS-stained retinal trypsin digests.

PFKFB3 is recognized for its role in EC glycolysis and angiogenesis, but this study reveals a previously uncharacterized nuclear function in suppressing endothelial senescence in diabetic retinopathy.

• Prior literature established PFKFB3 as a regulator of EC glycolysis and angiogenesis.
• The contribution of PFKFB3 to endothelial senescence in DR had not previously been elucidated.
• The study identifies nuclear translocation of PFKFB3 as a distinct, non-metabolic mechanism relevant to DR pathogenesis.