Abstract
Purpose :
Purpose: Retinal vascular senescence has emerged as an important pathogenic effect of diabetes and a potential contributor to diabetic retinal microangiopathy and retinopathy (DR). We have previously shown that the histone deacetylase HDAC6 plays a key role in this process, therefore pointing out to its interactome/acetylome as potential contributor to vascular senescence in the diabetic milieu. Here we have analyzed the effects of diabetes and high glucose on HDAC6 interactome in particular focusing on the inducible chaperone heat shock protein 90aa1 (HSP90a) because of its well-established role in vascular homeostasis.
Methods :
Methods: STRING analysis was used to identify HDAC6 interacting proteins. SILAC was conducted to assess changes in lysine acetylation of protein extracted from human retinal endothelial cells (HuREC) exposed to different glucose levels [5.5mM (NG), 25mM (HG) for 48hours] in presence or absence of the HDAC6 specific inhibitor Tubastatin A (TubA, 5mM). Immunoprecipitation and Western blotting analyses were used to determine HSP90a expression, acetylation and interaction with HDAC6 in HuREC exposed to different glucose levels or in retinas of streptozotocin-induced diabetic rats (STZ-rats) at 8 weeks of hyperglycemia and compared to age-matched normoglycemic rats.
Results :
Results: STRING analysis predicted structural and functional interaction between HDAC6 and HSP90a and SILAC analysis showed HSP90a deacetylation in HuREC exposed to HG, effect that was abrogated by the HDAC6 inhibitor TubA. Western blotting and immunoprecipitation analyses showed that the expression of HSP90a was not significantly changed in HuREC exposed to HG or in STZ-rat retinas. However, immunoprecipitation studies confirmed increased protein-protein interaction among HDAC6 and HSP90a with a significant decrease in HSP90a acetylation, that in HuREC was restored by treatments of the cells with TubA.
Conclusions :
Conclusions: We have identified HSP90a as a key downstream target of HDAC6 in the diabetic milieu and in retinal endothelial cells exposed to high glucose. Due to the crucial function of HSP90a in promoting vascular homeostasis by regulating nitric oxide production and endothelial cells survival and autophagy, these results warrant further studies to investigate the role of this molecular chaperone in retinal vascular senescence and DR.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.