title: Impact of cigarette smoke extract and hyperglycemic conditions on blood-brain barrier endothelial cells.
abstract: BACKGROUND
  Diabetes and tobacco smoking are significant public health concerns which have been shown to independently impact the blood-brain barrier (BBB). Since smoking is a risk factor for diabetes and shares some of the common pathological pathways leading to metabolic abnormalities, it is hypothesized that their combination would produce additive or synergistic BBB dysfunction. Therefore, the objective of this study was to assess this hypothesis and evaluate the magnitude of these effects in vitro using hCMEC/D3 cells; a well-established human BBB endothelial cell line.
  
  
  METHODS
  Monolayers of hCMEC/D3 cells were exposed to hyperglycemic conditions (HG; 35 mM) or 5% soluble cigarette smoke extracts (CSE, model of mainstream smoke exposure) for 12-24 h. Cells were then harvested for subsequent biochemical analyses. Transendothelial electrical resistance (TEER) and paracellular permeability to florescent dextrans were used to assess monolayer integrity. Analysis of released factors and cytokines was carried out by ELISA. Western blot (WB) analysis/immunofluorescence of relevant molecular targets was carried out. P-gp efflux activity was measured using rhodamine 123.
  
  
  RESULTS
  Immunofluorescence and WB data showed a significant ZO-1 down-regulation by HG and/or CSE over 24 h exposure. CSE in presence of HG produced a synergistic increase in release of vascular endothelial growth factor that was accompanied by decreased TEER and augmented permeability to labeled dextrans in a size-dependent manner. Moreover, CSE increased the expression of GLUT-1 and SGLT-1 in isolated membrane fractions of hCMEC/D3 cells. The effect was amplified by HG. Both, HG and CSE elicited the membrane upregulation of P-glycoprotein (P-gp) expression which however, was not paralleled by a comparable efflux activity. Interestingly, concomitant exposure to HG and CSE evoked a marked upregulation of PECAM-1 and other pro-inflammatory markers including IL-6 and -8, when compared to each condition alone. Moreover, exposure to all tested conditions amplified (to a different degree) cellular oxidative stress response denoted by increased Nrf2 nuclear translocation.
  
  
  CONCLUSIONS
  Overall, our results have clearly shown an additive pattern in the release of angiogenic and inflammatory factors following concomitant exposure to HG and CSE. This suggests the involvement of common key modulators in BBB impairment by both CS and HG possibly through the activation of oxidative stress responses.
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, 1300 S. Coulter Street, Amarillo, TX, 79106, USA. prasad.shikha@ttuhsc.edu.
authors.forename: Shikha
authors.initials: S
authors.lastname: Prasad
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, 1300 S. Coulter Street, Amarillo, TX, 79106, USA. ravi.sajja@ttuhsc.edu.
authors.forename: Ravi K
authors.initials: RK
authors.lastname: Sajja
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, 1300 S. Coulter Street, Amarillo, TX, 79106, USA. JH.park@ttuhsc.edu.
authors.forename: Jee Hyun
authors.initials: JH
authors.lastname: Park
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, 1300 S. Coulter Street, Amarillo, TX, 79106, USA. pooja.naik@ttuhsc.edu.
authors.forename: Pooja
authors.initials: P
authors.lastname: Naik
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, 1300 S. Coulter Street, Amarillo, TX, 79106, USA. md.a.kaisar@ttuhsc.edu.
authors.forename: Mohammad Abul
authors.initials: MA
authors.lastname: Kaisar
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, 1300 S. Coulter Street, Amarillo, TX, 79106, USA. luca.cucullo@ttuhsc.edu.|Center for Blood Brain Barrier Research, Texas Tech University Health Sciences Center, Amarillo, TX, 79106, USA. luca.cucullo@ttuhsc.edu.
authors.forename: Luca
authors.initials: L
authors.lastname: Cucullo
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D001812:Blood-Brain Barrier / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism*; D051246:Glucose Transport Proteins, Facilitative / Q000378:metabolism; D006801:Humans; D006943:Hyperglycemia / Q000378:metabolism*; D007249:Inflammation / Q000378:metabolism; D062789:Tobacco Products / Q000633:toxicity*; D062826:Zonula Occludens-1 Protein / Q000378:metabolism
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D051246:Glucose Transport Proteins, Facilitative; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein