Preconditioning of primary human endothelial cells with inflammatory mediators alters the "set point" of the cell.
Endothelial cells are highly sensitive to changes in the extracellular milieu. Sepsis results in activation of inflammatory and coagulation pathways. We hypothesized that sepsis-associated mediators may alter the response capacity (so-called "set point") of endothelial cells. Human umbilical vein endothelial cells (HUVEC) were preincubated in the presence or absence of tumor necrosis factor (TNF)-alpha, lipopolysaccharide (LPS), hypoxia, hyperthermia, and/or high glucose; treated with or without thrombin for 4 h; and then processed for RNase protection assays of selected activation markers. Priming with TNF-alpha and LPS significantly inhibited thrombin-mediated induction of vascular cell adhesion molecule-1, intercellular adhesion molecule-1, tissue factor, and E-selectin, but not platelet-derived growth factor-A or CD44. In electrophoretic mobility shift assays, thrombin-treated HUVEC demonstrated inducible binding of p65 NF-kappaB, an effect that was significantly blunted by pretreatment of cells with TNF-alpha and LPS. Consistent with these results, TNF-alpha and LPS attenuated the effect of thrombin on IkappaB phosphorylation, total cytoplasmic IkappaB, and nuclear translocation of p65 NF-kappaB. The inhibitory effect of TNF-alpha on thrombin signaling persisted for up to 24 h following removal of the cytokine. Taken together, these data suggest that inflammatory mediators prime endothelial cells to modulate subsequent thrombin response.
Wada
Youichiro
Y
The Center for Vascular Biology Research and Division of Molecular and Vascular Medicine, Beth Israel Deaconess Medical Center, Boston MA 02215, USA.
Otu
Hasan
H
Wu
Shengqian
S
Abid
Md Ruhul
MR
Okada
Hitomi
H
Libermann
Towia
T
Kodama
Tatsuhiko
T
Shih
Shu-Ching
SC
Minami
Takashi
T
Aird
William C
WC
D021581:Active Transport, Cell Nucleus; D002478:Cells, Cultured; D003470:Culture Media / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D017931:DNA Primers; D004305:Dose-Response Relationship, Drug; D019040:E-Selectin / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology*; D004730:Endothelium, Vascular / Q000166:cytology* / Q000473:pathology; D005334:Fever / Q000378:metabolism; D006801:Humans; D018960:Hyaluronan Receptors / Q000096:biosynthesis; D000860:Hypoxia / Q000378:metabolism; D020672:I-kappa B Proteins / Q000378:metabolism; D007249:Inflammation; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism; D008954:Models, Biological; D019001:Nitric Oxide Synthase / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000378:metabolism; D021381:Protein Transport; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012260:Ribonucleases / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction; D013917:Thrombin / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors; D051996:Transcription Factor RelA / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D014471:Umbilical Veins / Q000166:cytology*; D019010:Vascular Cell Adhesion Molecule-1 / Q000378:metabolism
D003470:Culture Media; D017931:DNA Primers; D019040:E-Selectin; D018960:Hyaluronan Receptors; D020672:I-kappa B Proteins; D008070:Lipopolysaccharides; D010982:Platelet-Derived Growth Factor; C498143:RELA protein, human; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D019010:Vascular Cell Adhesion Molecule-1; C075278:platelet-derived growth factor A; D018799:Intercellular Adhesion Molecule-1; D019001:Nitric Oxide Synthase; D012260:Ribonucleases; D013917:Thrombin