Gene Silencing of the Mitochondrial Adaptor p66 ^Shc Suppresses Vascular Hyperglycemic Memory in Diabetes

Abstract

Rationale: Hyperglycemic memory may explain why intensive glucose control has failed to improve cardiovascular outcomes in patients with diabetes. Indeed, hyperglycemia promotes vascular dysfunction even after glucose normalization. However, the molecular mechanisms of this phenomenon remain to be elucidated.Objective: The present study investigated the role of mitochondrial adaptor p66 Shc in this setting. Methods and Results:In human aortic endothelial cells (HAECs) exposed to high glucose and aortas of diabetic mice, activation of p66Shc by protein kinase C ␤II (PKC␤II) persisted after returning to normoglycemia. Persistent p66Shc upregulation and mitochondrial translocation were associated with continued reactive oxygen species (ROS) production, reduced nitric oxide bioavailability, and apoptosis. We show that p66Shc gene overexpression was epigenetically regulated by promoter CpG hypomethylation and general control nonderepressible 5-induced histone 3 acetylation. Furthermore, p66Shc -derived ROS production maintained PKC␤II upregulation and PKC␤II-dependent inhibitory phosphorylation of endothelial nitric oxide synthase at Thr-495, leading to a detrimental vicious cycle despite restoration of normoglycemia. Moreover, p66Shc activation accounted for the persistent elevation of the advanced glycated end product precursor methylglyoxal. In vitro and in vivo gene silencing of p66Shc , performed at the time of glucose normalization, blunted ROS production, restored endothelium-dependent vasorelaxation, and attenuated apoptosis by limiting cytochrome c release, caspase 3 activity, and cleavage of poly (ADP-ribose) polymerase. Conclusions: p66Shc is the key effector driving vascular hyperglycemic memory in diabetes. Our study provides molecular insights for the progression of diabetic vascular complications despite glycemic control and may help to define novel therapeutic targets. (Circ Res. 2012;111:278-289.) Key Words: vascular disease Ⅲ diabetes mellitus Ⅲ free radicals Ⅲ endothelium T he prevalence of diabetes has dramatically increased worldwide, with a further rise anticipated in the next decades. 1,2 Morbidity and mortality from cardiovascular disease is 2-to 8-fold higher in subjects with than in those without diabetes. 3 Editorial, see p 262Recent prospective clinical trials have failed to confirm unequivocal benefits from normalization of glycemia on cardiovascular outcomes. 4 -8 In these trials, intensive glucoselowering therapy was started after a median duration of diabetes ranging from 8 to 11 years. 4 -8 By contrast, early treatment of hyperglycemia reduces the risk of myocardial infarction, diabetes-related deaths, and all-cause mortality. 9 -11 These observations support the concept that hyperglycemic environment may be remembered in the vasculature. 12 Reactive oxygen species (ROS) are probably involved in this phenomenon defined "hyperglycemic memory," but the underlying molecular mechanisms remain unknown. [13][14][15] Overproduction of ROS by mitochondria is considered as a causal link betwe...

Activated protein C ameliorates diabetic nephropathy by epigenetically inhibiting the redox enzyme p66 Shc

“…2A). However, glucose failed to induce p66 Shc expression in primary mouse glomerular endothelial cells (GENCs), whereas, in agreement with previous work (15), glucose induced p66 Shc expression in various non-GENCs (Fig. 2 A and B).…”

“…3B). Glucose-induced H3 hyperacetylation is mediated-at least in par-by the H3 acetyltransferase GCN5 (15). Consistently, glucose-dependent induction of GCN5 was observed in podocytes, and this induction was prevented by aPC (Fig.…”

“…Separate promoter regions control expression of these structurally and functionally distinct Shc splicing variants. Interestingly, the p66 Shc promoter is subject to epigenetic modifications (14), and glucose-induced hypomethylation and hyperacetylation of the p66 Shc promoter results in high levels of p66 Shc expression and ROS generation in endothelial cells (15). In agreement with these data, p66 Shc -deficient mice are protected against diabetic nephropathy (16,17).…”

“…These observations, together with the current findings, imply the existence of a self-regulatory feed-forward mechanism, which may be disrupted in diseases like diabetes mellitus. Glucose-induced hypomethylation and hyperacetylation of the p66 Shc promoter, resulting in increased p66 Shc expression (15), may disrupt this protective feed-forward system, inhibiting TM expression and initiating a self-propagating cycle of diabetic vascular complications. Fortunately, the observed effects of exogenously administered aPC indicate that this protective feed-forward pathway may be restored by therapeutic interventions.…”

“…3A). Hypomethylation of the p66 Shc promoter has been shown to be associated with histone 3 (H3) hyperacetylation (15). Indeed, glucose was also found to induce H3 acetylation, an effect that was prevented by aPC (Fig.…”