Altered Nrf2 signaling mediates hypoglycemia-induced blood-brain barrier endothelial dysfunction in vitro.

Ravi K. Sajja,Luca Cucullo,Kayla N. Green

doi:10.1371/journal.pone.0122358

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.

Highlights

Hypoglycemia is a potential clinical challenge in the management of type 1 and advanced type 2 diabetes mellitus and aggravates the pathophysiology of long-term microvascular complications associated with diabetes [1,2,3]
Fluorescein isothiocyanate (FITC) and Rhodamine B isothiocyanate (RITC)-dextrans were purchased from Sigma-Aldrich, while Cascade Blue-dextran, anti-claudin 5 (#35–2500) and occludin were obtained from Invitrogen (Eugene, OR, USA)
To determine the specific role of NF-E2 related factor-2 (Nrf2) in maintenance of blood-brain barrier (BBB) integrity, we selectively knocked down Nrf2 expression by siRNA-based gene silencing

Summary

Introduction

Hypoglycemia is a potential clinical challenge in the management of type 1 and advanced type 2 diabetes mellitus and aggravates the pathophysiology of long-term microvascular complications associated with diabetes [1,2,3]. Clinical and preclinical studies suggest that iatrogenic or hypoxia-associated hypoglycemic episodes initiate progressive neuronal injury and widespread brain damage resulting in cognitive dysfunction and learning deficits [4,5,6]. In this line, emerging evidence indicates a detrimental impact of hypoglycemia on cerebrovasculature at the level of blood-brain barrier (BBB), a physical, transport and metabolic interface that guards and maintains the homeostasis of brain microenvironment [7,8,9]. The molecular mechanisms underlying hypoglycemia-elicited oxidative stress and BBB endothelial dysfunction are not well understood and require critical investigation for the development of effective therapeutic strategies to prevent cerebrovascular damage [15]

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