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This Article Full Text Full Text (PDF) All Versions of this Article: 297/4/C945    most recent 00606.2008v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Sarai, K. Articles by Makino, H. PubMed PubMed Citation Articles by Sarai, K. Articles by Makino, H.

EXTRACELLULAR MATRIX, CELL INTERACTIONS

Endothelial barrier protection by FTY720 under hyperglycemic condition: involvement of focal adhesion kinase, small GTPases, and adherens junction proteins

¹Department of Medicine and Clinical Science, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, ²Faculty of Health Sciences, Okayama University Medical School, ³Department of Medical Engineering, Kawasaki Medical School, Okayama, Japan

Submitted 24 November 2008 ; accepted in final form 4 August 2009

Recently, sphingosine 1-phosphate (S1P) has been highlighted as an endothelial barrier-stabilizing mediator. FTY720 is a S1P analog originally developed as a novel immunosuppressant. The phosphorylated form of FTY720 binds to S1P receptors to exert S1P-like biological effects, suggesting endothelial barrier promotion by FTY720. To elucidate whether FTY720 induces signaling events related to endothelial barrier enhancement under hyperglycemic conditions, human microvascular endothelial cells (HMVECs) preincubated with hyperglycemic (30 mM) medium were treated with 100 nM FTY720 for 3 h. Immunofluorescent microscopy and coprecipitation study revealed FTY720-induced focal adhesion kinase (FAK)-associated adherens junction (AJ) assembly at cell-cell contacts coincident with formation of a prominent cortical actin ring. FTY720 also induced transmonolayer electrical resistance (TER) augmentation in HMVEC monolayers in both normoglycemic and hyperglycemic conditions, implying endothelial barrier enhancement. Similar to S1P, site-specific FAK tyrosine phosphorylation analysis revealed FTY720-induced FAK [Y⁵⁷⁶] phosphorylation without phosphorylation of FAK [Y³⁹⁷/Y⁹²⁵]. Furthermore, FTY720 conditioned the phosphorylation profile of FAK [Y³⁹⁷/Y⁵⁷⁶/Y⁹²⁵] in hyperglycemic medium to the same pattern observed in normoglycemic medium. FTY720 challenge resulted in small GTPase Rac activation under hyperglycemic conditions, whereas increased Rho activity in hyperglycemic medium was restored to the basal level. Rac protein depletion by small interfering RNA (siRNA) technique completely abolished FTY720-induced FAK [Y⁵⁷⁶] phosphorylation. These findings strongly suggest the barrier protective effect of FTY720 on HMVEC monolayers in hyperglycemic medium via S1P signaling, further implying the possibility of FTY720 as a therapeutic agent of diabetic vascular disorder.

endothelial barrier function; human microvascular endothelial cell; diabetes mellitus; transmonolayer electrical resistance; cytoskeleton