**4. Discussion**

The increased incidence of diabetes and DR, urges the realization of early interventional therapeutic strategies. In this study, we investigated the role of the histone deacetylase HDAC6 in the early events characterizing the progression of an experimental model of DR. Dysregulation of the processes of acetylation/deacetylation of nuclear and cellular proteins has been shown to be associated with different pathologic conditions including diabetes [12–18,23]. Previous findings linked the activity of several histone deacetylases to the pathogenesis of DR [23,24]. A specific contribution of HDAC1, 2, and 8 to global acetylation of retinal histones in the diabetic retina was found to be involved not only to progression of DR but also to the metabolic memory phenomenon [23]. When compared to the other members of the large family of the histone deacetylases HDAC6 presents unique structural and functional properties, including a double catalytic domain and cytosolic and nuclear intracellular localization and sites of action [9]. Among other unique functions, HDAC6 can influence the redox state of the cells through deacetylation of endogenous antioxidants and modulation of oxidase activities [16].

Our results show that HDAC6 expression and activity are upregulated in the diabetic retinas of STZ-rats and, most importantly, in postmortem retinas of diabetic donors. Using an interventional approach, we assessed the effects of pharmacological inhibition of HDAC6 by administration of the specific inhibitor TS in an experimental model of Type 1 diabetes (STZ-rats). Pharmacological inhibition of HDAC6 by TS lessened DR pathology in STZ-rats, as evidenced by maintenance of total retinal thickness and amelioration of vascular hyperpermeability, a key feature of DR microangiopathy [2,3]. The observed effects of TS on BRB stabilization in STZ-rats, are in agreemen<sup>t</sup> with previous studies that have underscored the role of HDAC6 activity in altering endothelial and epithelial cells permeability and the stability of intercellular junctions [25–27].

While the specific role of HDAC6 in the diabetic retina has not been studied before, HDAC6 inhibition has been shown to alleviate myocardial ischemia-reperfusion injury in diabetic rats [16] and to ameliorate diabetic kidney disease [15], predominantly through antioxidant effects. Interestingly, in the diabetic retina, downregulation of HDAC6 was linked to the effects of exogenous GLP-1 in alleviating oxidative stress-induced apoptosis and autophagy of retinal cells [24].

The results of our studies show that HDAC6 inhibition by TS decreased the appearance of the senescence marker γH2AX and significantly augmented the expression of the redox homeostatic histone deacetylase SIRT1, of which expression levels are inversely correlated with the induction of endothelial cell senescence [4,7]. A contrasting balance between HDAC6 and SIRT1 could significantly impact the redox status of a number of cells including endothelial and other retinal cells [28].

Previous studies have shown that increased HDAC6 activity results in oxidative stress due to mitochondrial dysfunction [16,29] and to altered endogenous antioxidant function [11]. Increased oxidative/nitrative stress has been shown to play a key contributing role to the pathogenesis of DR due to its effects in promoting retinal chronic inflammation, microvascular injury, and accelerated endothelial cell senescence [4]. Inhibition of HDAC6 by TS, significantly reduced superoxide formation, diminished the levels of oxidative and nitrative stress markers (4-HNE and NT, respectively) and rescued the activity of endogenous antioxidants such as Nrf2 and Trx-1. Taken together, these antioxidant properties of TS further confirm the pro-oxidative capacity of overactive HDAC6 in the diabetic retina.

The results of our experiments in vitro, specifically addressing HDCA6 role in HuREC, largely recapitulated the results obtained in the in vivo experiments as TS prevented high glucose-induced HuREC senescence (SA-β-Gal activity assay) and halted high glucose effects in downregulating SIRT1 expression. These protective effects correlated with significant reduction of superoxide production from cellular and mitochondrial sources as well as with the rescuing of the activity of the endogenous antioxidant Trx-1. Consistent with our results, blocking of HDAC6 activity was shown to be protective against high glucose-induced oxidative stress in RPE via mechanisms involving inhibition NF-κB and NLRP3 inflammasome pathway [30].

The potential role of HDAC6 in regulating cellular redox homeostasis has been previously implicated in the pathogenesis of several neurodegenerative diseases [31,32]. In addition, similar findings have been obtained in studies showing beneficial antioxidant effects of HDAC6 inhibitors in models of retinal neurodegenerative diseases [17,18]. Thus, the results here described are in agreemen<sup>t</sup> with these previous findings by demonstrating a role for HDAC6 in DR and retinal microangiopathy through pro-oxidant effects.
