Altered Nrf2 Signaling Mediates Hypoglycemia-Induced Blood–Brain Barrier Endothelial Dysfunction In Vitro

Abstract

Hypoglycemia impairs blood-brain barrier (BBB) endothelial function; a major hallmark in the pathogenesis of various CNS disorders. Previously, we have demonstrated that prolonged hypoglycemic exposure down-regulated BBB endothelial NF-E2 related factor-2 (Nrf2) expression; a redox-sensitive transcriptional factor that regulates endothelial function. Here, we sought to determine the functional role of Nrf2 in preserving BBB integrity and molecular mechanisms underlying hypoglycemia-induced Nrf2 down-regulation in vitro using human cerebral microvascular endothelial cell line (hCMEC/D3). Cell monolayers were exposed to normal or hypoglycemic (5.5 or 2.2mM D-glucose) media for 3-24h. Pharmacological or gene manipulation (by silencing RNA) approaches were used to investigate specific molecular pathways implicated in hypoglycemia-induced Nrf2 degradation. BBB integrity was assessed by paracellular permeability to labeled dextrans of increasing molecular sizes (4-70kDa). Silencing Nrf2 expression in hCMEC/D3 cells abrogated the expression of claudin-5 and VE-cadherin, while ZO-1 was up-regulated. These effects were paralleled by a decrease in electrical resistance of hCMEC/D3 monolayers and potential increase in permeability to all labeled dextrans. Hypoglycemic exposure (3-24h) led to progressive and sustained down-regulation of Nrf2 (without affecting mRNA) and its target, NQO-1, with a concomitant increase in the cytosolic pool of E3 ubiquitin ligase, Siah2 (but not Keap1). Pretreatment with protease inhibitor MG132, or selective knock-down of Siah2 (but not Keap1) significantly attenuated hypoglycemia-induced Nrf2 destabilization. While hypoglycemic exposure triggered a significant increase in BBB permeability to dextrans, silencing Siah2 gene abrogated the effects of hypoglycemia and restored BBB integrity. In summary, our data indicate a potential role for Nrf2 signaling in regulating tight junction integrity and maintaining BBB function. Nrf2 suppression by increased Siah2-driven proteasomal degradation mediates hypoglycemia-evoked endothelial dysfunction and loss of BBB integrity. Overall, this study suggests that sustained activation of endothelial Nrf2 signaling could have therapeutic potential to prevent hypoglycemia-induced cerebrovascular dysfunction.

Rutin Prevents High Glucose-Induced Renal Glomerular Endothelial Hyperpermeability by Inhibiting the ROS/Rhoa/ROCK Signaling Pathway

“…The endothelial tight junction (TJ) regulates the permeability of endothelial cells, and exposure of monolayers of cultured renal endothelial cells to HG can significantly increase the permeability [20,21]. In this study, HRGECs were treated with 12.5, 25, or 50 µM rutin and/or HG for 24 h. We detected HG-induced endothelial hyperpermeability by measuring the passage of FITC-dextran across the endothelium.…”

Novel Function of E3 Ubiquitin Ligase Siah2 to Regulate ROS Metabolism

“…Recent work has demonstrated that Siah2 protein is a promising therapeutic target in pathological conditions accompanied by a low level of Nrf2 expression [7,40] . The inhibition of Siah2 expression restores the decreased expression of Nrf2-inducible genes and subsequently improves symptoms.…”

“…Knockdown of Siah2 rescues hypoxia-induced reduction of both Nrf2 mutants mimicking phosphorylation at Ser40 and lacking this phosphorylation site, suggesting that Siah2 contributes to the degradation of Nrf2 irrespective of its phosphorylation status [7] ( Figure 1). In addition, the knockdown of Siah2, but not Keap1, was shown to significantly attenuate the hypoglycemia-mediated reduction of Nrf2 expression [40] . Mitochondria are critical for cell survival, and their morphology is associated with susceptibility to cell death signals [41] .…”

“…Recent studies have also suggested that NF-E2-related factor (Nrf2), which plays an important role in ROS metabolism, is negatively regulated by Siah2 under stressful conditions [7,40] . Under basal conditions, Nrf2 is a well-known substrate for Kelchlike ECH-associated protein 1 (Keap1) [6] ; however, Siah2, but not Keap1, is involved in the degradation of Nrf2 under particular conditions.…”

“…Interfering with Nrf2 down-regulation through Siah inhibition is effective to induce the expression of cytoprotective genes [7,40] , as it may have an impact on the cellular adaptive responses to oxidative stress more broadly than the inhibition of Keap1 alone.…”