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Sphingosine‐1‐phosphate and 8‐pCPT‐2′‐O‐Me‐cAMP Can Restore Alcohol‐induced Junctional Disorganization and Barrier Dysfunction of Human Brain Microvascular Endothelial Cells
Author(s) -
Alves Natascha Guimaraes,
Adderley Shaquria,
Yuan Sarah,
Breslin Jerome
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.950.8
Subject(s) - blood–brain barrier , tight junction , barrier function , paracellular transport , sphingosine 1 phosphate , chemistry , endothelial stem cell , microbiology and biotechnology , homeostasis , sphingosine , endocrinology , medicine , central nervous system , permeability (electromagnetism) , biology , biochemistry , in vitro , membrane , receptor
The blood‐brain‐barrier (BBB) is a highly selective endothelial barrier that protects the brain from blood‐born cytotoxins and ensures proper homeostasis of the central nervous system. Alcohol has previously been shown to compromise the BBB, however the mechanisms involved are incompletely understood. We hypothesized that alcohol increases BBB permeability by activating the p38 MAP kinase, disrupting paracellular junction proteins, such as VE‐Cadherin and Claudin‐5, and that microvascular barrier stabilizers such as sphingosine‐1‐phosphate (S1P) and 8‐pCPT‐2′‐O‐Me‐cAMP (8‐CPT) can resolve alcohol‐induced BBB dysfunction. Cultured primary human brain microvascular endothelial cell (HBMEC) monolayers were used to model BBB function so that endothelial‐specific mechanisms could be studied. Trans‐endothelial electrical resistance (TER) was used as an index of barrier function. Localization of VE‐cadherin and Claudin‐5 was determined by immunofluorescence microscopy. S1P (1 μM) or 8‐CPT (100 μM) were applied following treatment with alcohol to test their efficacies to rescue barrier function. The results show that treatment of HBMEC with alcohol (50–100 mM) causes a biphasic hyperpermeability response, characterized by an initial transient decrease in TER (10 min) that recovers, followed by a prolonged decrease in TER that lasts several hours. Surprisingly, we observed that pretreatment with the selective p38 MAPK inhibitor SB208530 (20 μM) does not block the alcohol‐induced decrease in HBMEC monolayer TER. Predictably, treatment of HBMEC monolayers with either S1P or 8‐CPT significantly enhances barrier function. However, pretreatment with S1P or 8‐CPT does not prevent the disruption caused by 75 mM alcohol, and S1P pretreatment can even exacerbate the alcohol‐induced decrease in TER. In contrast, application of S1P or 8‐CPT, after alcohol has already evoked a decrease in TER, causes a hastened recovery to normal HBMEC monolayer barrier function. Our Immunocytochemistry studies demonstrate colocalization of VE‐Cadherin and Claudin‐5 at intercellular junctions, and alcohol disrupts this organization. Importantly, treatment with S1P (after alcohol) can restore junctional integrity. Taken together, our results show that alcohol causes a biphasic decrease in HBMEC monolayer barrier function that does not require p38 MAP kinase activation, but does involve reorganization of junctional protein complexes. In addition, S1P and 8‐CPT can enhance barrier function and may be useful for restoring BBB function in the presence of alcohol intoxication. Support or Funding Information Supported by a grant from the USF MCOM Office of Research.