Diabetes-induced HDAC6-miR-34a pathway downregulates SIRT-1-miR-146a axis to promote stress-induced premature senescence and diabetic retinal microangiopathy

Abstract

Purpose :
We have previously shown that stress-induced premature senescence (SIPS) is a key pathogenic component of diabetic retinal vascular dysfunction. Here we have investigated the reciprocal role of histone deacetylase 6 (HDAC6) and sirtuin 1 (SIRT-1) in diabetes-induced SIPS. These two HDACs have been shown to play opposing roles in regulating SIPS, however their reciprocal interaction in this process was not investigated before.

Methods : We used streptozotocin-induced diabetic rats at 8 weeks of hyperglycemia (STZ-rats) and age-matched normoglycemic rats. Human retinal endothelial cells (HREC) were cultured for 48 hours in different glucose conditions (HG=25mM D-glucose). Senescence was determined by senescence-associated-beta-galactosidase activity assay. Extracellular vesicles (EVs), were isolated from HREC supernatants using ultracentrifugation. Inhibition of HDAC6 was achieved in STZ-rats by treatment with the specific inhibitor Tubastatin (TS;10mg/Kg/day) and in HREC by 5µM TS. HDAC6 expression in HuREC was also halted using human specific gRNA and compared to negative controls. MicroRNA arrays were conducted on HREC extracts and in EVs in response to the different treatments.

Results : Hyperglycemia stimulated HDAC6 while downregulating SIRT1 retinal expression and activity. TS prevented diabetes-induced loss of SIRT-1 in rats and significantly reduced senescence markers. In HuREC, HG treatment inhibited and TS treatment restored SIRT-1 while decreasing the number of senescent cells. MiRNA arrays of HREC extracts and EVs collected from the cells supernatants showed the up-regulation of miR-34a and down-regulation of miR-146a. Treatments of the cells with TS or transfection with HDAC6-specific gRNA, halted HG-induced cells senescence while restoring SIRT-1 expression and activity. Loss of HDAC6 (TS and gRNA) also down-regulated miR-34a expression while restoring miR-146a. Finally, transfection of HREC with miR-34a mimic overrode the effects of TS in blocking HG-induced loss of SIRT-1 and miR-146a.

Conclusions : We have identified in the HDCA6-miR-34a axis a key molecular event leading to diabetes-induced retinal vascular senescence and diabetic microangioapthy through loss of SIRT1 and of miR-146a. Overall, our results suggest the potential therapeutic use of HDAC6 inhibitors for diabetic retinopathy

This is a 2021 ARVO Annual Meeting abstract.