Activation of NF-E2–Related Factor-2 Reverses Biochemical Dysfunction of Endothelial Cells Induced by Hyperglycemia Linked to Vascular Disease

Mingzhan Xue Qingwen Qian Antonysunil Adaikalakoteswari Naila Rabbani Roya Babaei-Jadidi Paul J. Thornalley OBJECTIVE-Sulforaphane is an activator of transcription factor NF-E2-related factor-2 (nrf2) that regulates gene expression through the promoter antioxidant response element (ARE). Nrf2 regulates the transcription of a battery of protective and metabolic enzymes. The aim of this study was to assess whether activation of nrf2 by sulforaphane in human microvascular endothelial cells prevents metabolic dysfunction in hyperglycemia. RESEARCH DESIGN AND METHODS-Human microvascular HMEC-1 endothelial cells were incubated in low and high glucose concentrations (5 and 30 mmol/l, respectively), and activation of nrf2 was assessed by nuclear translocation. The effects of sulforaphane on multiple pathways of biochemical dysfunction, increased reactive oxygen species (ROS) formation, hexosamine pathway, protein kinase C (PKC) pathway, and increased formation of methylglyoxal were assessed. RESULTS-Activation of nrf2 by sulforaphane induced nuclear translocation of nrf2 and increased ARE-linked gene expression, for example, three- to fivefold increased expression of transketolase and glutathione reductase. Hyperglycemia increased the formation of ROS-an effect linked to mitochondrial dysfunction and prevented by sulforaphane. ROS formation was increased further by knockdown of nrf2 and transketolase expression. This also abolished the counteracting effect of sulforaphane, suggesting mediation by nrf2 and related increase of transketolase expression. Sulforaphane also prevented hyperglycemia-induced activation of the hexosamine and PKC pathways and prevented increased cellular accumulation and excretion of the glycating agent methylglyoxal. CONCLUSIONS-We conclude that activation of nrf2 may prevent biochemical dysfunction and related functional responses of endothelial cells induced by hyperglycemia in which increased expression of transketolase has a pivotal role. Diabetes 57:2809-2817, 2008 - Tdiabetes that is a major cause of patient morbidhere is an increased risk of vascular disease in ity and mortality. This gives rise to a characteristic spectrum of diabetic microvascular disease (retinopathy, nephropathy, and neuropathy) and macrovascular disease (heart disease and stroke) (1 4). Vascular disease in diabetes is associated with dysfunction of endothelial cells in hyperglycemia. Activation of multiple pathways of biochemical dysfunction induced in vascular endothelial cells by high glucose concentration is thought to underlie the link of hyperglycemia in diabetes to the development of vascular disease (5,6). A common feature of endothelial cell dysfunction in hyperglycemia is increased formation of reactive oxygen species (ROS) by mitochondria, oxidative stress with inactivation of glyceraldehyde-3-phosphate dehydrogenase, and accumulation of triosephosphates and fructose-6-phosphate (79). There is an associated activation of protein kinase C (PKC), hexosamine pathway O-linked protein glycosylation, and increased glycation by methylglyoxal and other dicarbonyls forming advanced glycation end products (10 12). This appears to be driven mainly by the accumulation of glycolytic intermediates. Recent research has indicated that activation of the reduced pentosephosphate pathway by high-dose thiamine and related prodrug benfotiamine may counter this metabolic dysfunction (9,13,14), but little is known of the endogenous coordinated stress response to decrease triosephosphate accumulation and its link to increased ROS formation and oxidative stress in hyperglycemia. NF-E2related factor-2 (nrf2) is a member of the cap n collar subfamily of bZIP transcription factors. It is an essential transactivator of genes containing an antioxidant response element (ARE) in their promoter (rev. in 15,16). ARE-linked genes include a battery of protective and metabolic enzymes: -glutamylcysteine ligase, glutathione reductase (GSHRd), aldo-keto reductase (AKRd), glutathione transferases, quinone reductase (NQO1), nrf2 (17), and others (18). Nrf2-linked gene expression has a key role in the protection of cells against oxidative stress, carbonyl compounds, and electrophilic agents. Interestingly, the thiamine-dependent enzyme transketolase and transaldolase are also ARE-linked genes (18). Transketolase is considered to be the rate-controlling enzyme in the pentosephosphate pathway. Under basal conditions, nrf2 is complexed with Kelchlike ECH-associated protein 1 (Keap1), a BTB-Kelch protein. Keap1 is a substrate adaptor protein for a Cul 3 dependent E3 ubiquitin ligase complex, directing nrf2 for proteasomal degradation (19). Oxidative stress, electrophiles, and sulforaphane-like inducers disrupt the Keap1-nrf2 complex: nrf2 translocates to the nucleus and, combining with small maf protein (20), induces ARElinked gene expression (21). Sulforaphane releases nrf2 from Keap1 by modification of critical cysteine thiol residues (22). Keap1 has concurrent increased susceptibility to degradation but also has ARE-linked gene expression and may be induced by nrf2 activation, providing an Sulforaphane Nuclear membrane Nucleus Cell scaffolding (microfilaments) 5'-TMAnnRTGAYnnnGCR-3 ARE Increased expression of protective proteins (GST, NQO1, TK, TA, GCL, GSHRd, AKRd, nrf2, keap1 and others) Reductive pentose phosphate pathway Nrf2-mediated induction of transketolase expression Glucose MG DHAP G-3-P Mitochondrial dysfunction Pyruvate + NADH FIG. 1. Nrf2 activation and transketolase expression in human HMEC-1 endothelial cells in vitro. A: Schematic diagram showing activation of nrf2 and dynamic nuclear-cytoplasmic shuttling of nrf2 for ARE-linked expression. B: Multiple pathways of biochemical dysfunction induced by hyperglycemia in microvascular endothelial cells and effect of nrf2 activated, ARE-mediated induction of transketolase expression. Other mechanisms of biochemical dysfunction may be involved. autoregulatory feedback loop (23). Nrf2 also undergoes nuclear export, establishing cytoplasmic/nuclear dynamic shuttling (24). Recent research has suggested that the serine/threonine kinase CK2 has a role in nuclear import of nrf2 and that both CK2 and tyrosine kinase Fyn influence nuclear export and degradation of nrf2 (25,26) (Fig. 1A). There is an active nrf2Keap-1 system in vascular endothelial cells (27). The role, if any, of nrf2-linked gene expression in countering endothelial dysfunction in hyperglycemia has not been disclosed. Disposal of glyceraldehyde-3-phosphate and fructose-6-phosphate by the reductive pentosephosphate pathway induced by activation of nrf2 and increased expression of transketolase suggested a possible mechanism of intervention. In this report, we show that activation of nrf2 by the dietary activator sulforaphane, limited to concentration ranges found in plasma after consumption of broccoli (28) for relevance to future clinical dietary intervention, increased the (...truncated)