We investigated the contributing role of the histone deacetylase 6 (HDAC6) to the early stages of diabetic retinopathy (DR). Furthermore, we examined the mechanism of action of HDAC6 in human retinal endothelial cells (HuREC) exposed to glucidic stress. Streptozotocin-induced diabetic rats (STZ-rats), a rat model of type 1 diabetes, were used as model of DR. HDAC6 expression and activity were increased in human diabetic postmortem donors and STZ-rat retinas and were augmented in HuREC exposed to glucidic stress (25 mM glucose). Administration of the HDAC6 specific inhibitor Tubastatin A (TS) (10 mg/kg) prevented retinal microvascular hyperpermeability and up-regulation of inflammatory markers. Furthermore, in STZ-rats, TS decreased the levels of senescence markers and rescued the expression and activity of the histone deacetylase sirtuin 1 (SIRT1), while downregulating the levels of free radicals and of the redox stress markers 4-hydroxynonenal (4-HNE) and nitrotyrosine (NT). The antioxidant effects of TS, consequent to HDAC6 inhibition, were associated with preservation of Nrf2-dependent gene expression and up-regulation of thioredoxin-1 activity. In vitro data, obtained from HuREC, exposed to glucidic stress, largely replicated the in vivo results further confirming the antioxidant effects of HDAC6 inhibition by TS in the diabetic rat retina. In summary, our data implicate HDAC6 activation in mediating hyperglycemia-induced retinal oxidative/nitrative stress leading to retinal microangiopathy and, potentially, DR.

Retinal ischemia contributes to visual impairment in ischemic retinopathies. A disintegrin and metalloproteinase ADAM17 is implicated in multiple vascular pathologies through its ability to regulate inflammatory signaling via ectodomain shedding. We investigated the role of endothelial ADAM17 in neuronal and vascular degeneration associated with retinal ischemia reperfusion (IR) injury using mice with conditional inactivation of ADAM17 in vascular endothelium. ADAM17Cre-flox and control ADAM17flox mice were subjected to 40 min of pressure-induced retinal ischemia, with the contralateral eye serving as control. Albumin extravasation and retinal leukostasis were evaluated 48 h after reperfusion. Retinal morphometric analysis was conducted 7 days after reperfusion. Degenerate capillaries were assessed by elastase digest and visual function was evaluated by optokinetic test 14 and 7 days following ischemia, respectively. Lack of ADAM17 decreased vascular leakage and reduced retinal thinning and ganglion cell loss in ADAM17Cre-flox mice. Further, ADAM17Cre-flox mice exhibited a remarkable reduction in capillary degeneration following IR. Decrease in neurovascular degeneration in ADAM17Cre-flox mice correlated with decreased activation of caspase-3 and was associated with reduction in oxidative stress and retinal leukostasis. In addition, knockdown of ADAM17 resulted in decreased cleavage of p75NTR, the process known to be associated with retinal cell apoptosis. A decline in visual acuity evidenced by decrease in spatial frequency threshold observed in ADAM17flox mice was partially restored in ADAM17-endothelial deficient mice. The obtained results provide evidence that endothelial ADAM17 is an important contributor to IR-induced neurovascular damage in the retina and suggest that interventions directed at regulating ADAM17 activity can be beneficial for alleviating the consequences of retinal ischemia.

Retinal microvascular dysfunction characterizes the pathophysiology of diabetic retinopathy (DR), a common complication of diabetes and a leading cause of blindness in the World. Oxidative stress and inflammation have been implicated as major pathogenic hubs for the induction and progression of DR. Reduced endogenous antioxidant activities within the retinal microvasculature can significantly contribute to oxidative stress in the diabetic retina. We have previously shown that diabetes inhibits the thioredoxin system (TrxS) and others have shown that the cytosolic histone deacetylase 6 (HDAC6) can promote oxidative stress in a not yet understood mechanism. Here we have investigated the role of HDAC6 in regulating the TrxS in the diabetic retinal microvasculature. HDAC6 expression and activity were assessed by immunoblotting and fluorimetric assay (respectively) in retinas of streptozotocin‐induced diabetic rats (STZ‐rats, 8 weeks of diabetes) and in primary culture of human retinal endothelial cells (HuREC) exposed to disease‐relevant glucose levels (HG=25mM D‐glucose) for 48 hours. Changes in TrxS were monitored by assessing the expression and activity of its functional unit, thioredoxin‐1 (Trx‐1), by immunoblotting and a commercially available fluorimetric assay, in STZ‐rats retinas and in HuREC exposed to HG. Some experiments in HuREC were conducted also in presence of 5μM of the specific HDAC6 inhibitor tubastatin A. CellROX assay and dot‐blot assessing 5‐nitrotyrosine (5‐NT) and 4 hydroxynonenal (4‐HNE) were used to measure oxidative stress in our experimental groups. HDAC6 expression and activity were found significantly elevated in retinas of STZ‐rats compared to normal age‐matched controls and in HuREC exposed to HG, but not to normal glucose (5mM D‐glucose) or the osmotic control L‐glucose (25mM). At the same time Trx‐1 expression was increased in diabetic retinas and in HuREC exposed to HG, whereas its activity was significantly decreased. Parallel to loss of Trx‐1 activity and increased HDAC6 expression and activity, we also found that oxidative stress parameters were also elevated. In HuREC, tubastatin A was able to block HG‐induced up‐regulation of oxidative stress parameters while up‐regulating Trx‐1 activity and normalizing its expression. In summary, our data confirm previous observations on the role of HDAC6 in promoting oxidative stress and disclose HDAC6 role in suppressing Trx‐1 activity. Therefore, our results, support HDAC6 pathogenic role in promoting oxidative stress in the diabetic retinal microvasculature and its implication in the pathogenesis of diabetic retinopathy.Support or Funding InformationEY022416This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.