title: 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.
authors: Department of Pharmaceutical Sciences, Texas Tech University Health Sciences Center, Amarillo, Texas, 79106, United States of America.
authors: Ravi K
authors: RK
authors: Sajja
authors: Department of Chemistry, Texas Christian University, Fort Worth, Texas, 76129, United States of America.
authors: Kayla N
authors: KN
authors: Green
authors: Department of Pharmaceutical Sciences, Texas Tech University Health Sciences Center, Amarillo, Texas, 79106, United States of America.
authors: Luca
authors: L
authors: Cucullo
mesh_terms: D015703:Antigens, CD / Q000378:metabolism; D001812:Blood-Brain Barrier / Q000378:metabolism*; D015820:Cadherins / Q000378:metabolism; D002460:Cell Line; D062507:Claudin-5 / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism; D005260:Female; D006801:Humans; D007003:Hypoglycemia / Q000378:metabolism / Q000473:pathology; D007976:Leupeptins / Q000494:pharmacology; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000235:genetics / Q000378:metabolism; D051267:NF-E2-Related Factor 2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D010539:Permeability; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects; D062826:Zonula Occludens-1 Protein / Q000378:metabolism; D000098563:Seven in Absentia Proteins; D000099152:Cadherin 5
chemical_list: D015703:Antigens, CD; D015820:Cadherins; D062507:Claudin-5; D007976:Leupeptins; D016660:NAD(P)H Dehydrogenase (Quinone); D051267:NF-E2-Related Factor 2; D009687:Nuclear Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D044767:Ubiquitin-Protein Ligases; D062826:Zonula Occludens-1 Protein; D000098563:Seven in Absentia Proteins; D000099152:Cadherin 5; C447479:NQO1 protein, human; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde