Dataset Information

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.

SUBMITTER: Sajja RK

PROVIDER: S-EPMC4373930 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:P-glycoprotein (P-gp) is expressed on brain microvessel endothelial cells of blood-brain barrier (BBB) and elevated after cerebral ischemia. In this study, we explored the influence and potential mechanisms of P-gp on BBB function in experimental ischemic stroke in vivo and in vitro. Middle cerebral artery occlusion/reperfusion (MCAO/R) was created in mice. Oxygen-glucose deprivation/reoxygenation (OGD/R) was performed in brain microvascular vessel-derived endothelial cells (bEnd.3) to mimic ischemia/reperfusion injury in vitro. P-gp-specific siRNA and pharmacological inhibitor cyclosporine A were used to inhibit P-gp, whereas pcDNA3.1 was utilized to overexpress P-gp. Twenty-four hours after reperfusion, acute ischemic stroke outcome, BBB integrity and permeability, autophagic proteins and relative signaling pathways were evaluated. P-gp levels were markedly elevated in mouse brain and endothelial cells following MCAO/R and OGD/R, respectively. P-gp siRNA silencing or pharmacologically inhibiting (cyclosporine A) reduced infarct volume and brain edema, attenuated brain pathology, and improved neurological behavior in association with attenuated accumulation of neutrophils and macrophages, reduced expression levels of inflammatory cytokines (TNF-α and IL-1β), matrix metalloproteinases (MMP-2 and MMP-9) and adhesion molecules (ICAM-1 and VCAM-1). P-gp silence also counteracted BBB leakage, restored the expressions of tight junction proteins (Claudin-5, Occludin and ZO-1), activated autophagic proteins (upregulated LC3-II/LC3-I and Beclin 1, and downregulated P62), and diminished Akt/mTOR signal activity in mice following MCAO/R. In the endothelial cell OGD/R assay, P-gp silence downregulated the expressions of inflammatory cytokines and adhesion molecules, inhibited leukocytes adhesion and migration, increased tight junction protein levels, and activated autophagy, all were reversible by forceful P-gp expression. Additionally, treatment with an autophagy inhibitor (3-methyladenine) abolished protections against ischemic stroke and tight junction proteins reduction followed by P-gp silence. In conclusion, increased P-gp expression after ischemic injury resulted in BBB dysfunction and hyperpermeability by suppressing Akt/mTOR-induced endothelial autophagy.

Dataset Information

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets