H2O2 and tumor necrosis factor-alpha induce differential binding of the redox-responsive transcription factors AP-1 and NF-kappaB to the interleukin-8 promoter in endothelial and epithelial cells.

We previously demonstrated that tumor necrosis factor-alpha (TNFalpha) and H2O2 differentially regulate interleukin-8 (IL-8) and intercellular adhesion molecule (ICAM-1) gene expression in endothelial and epithelial cells. H2O2 induced IL-8 expression in the A549 and BEAS-2B epithelial cell lines, but not in the human microvessel endothelial cell line, HMEC-1 or human umbilical vein endothelial cells. In contrast, H2O2 induced ICAM-1 only in endothelial cells. Unlike H2O2, the proinflammatory cytokine TNFalpha induced IL-8 and ICAM-1 in both cell types. In this study, we examine the role of the redox-responsive transcription factors AP-1 and nuclear factor-kappaB (NF-kappaB) in the differential expression of IL-8. DNA binding studies using nuclear protein extracts from HMEC-1 and A549 cells stimulated with H2O2 or TNFalpha demonstrated differential activation and promoter binding of AP-1 and NF-kappaB. H2O2 activated AP-1 but not NF-kappaB in A549, whereas TNFalpha activated AP-1 as well as NF-kappaB. In HMEC-1, TNFalpha activated NF-kappaB but not AP-1, while H2O2 did not activate either transcription factor. The differential activation of the factors was also reflected in their differential binding to the IL-8 promoter. Moreover, the H2O2 concentration dependent increase in epithelial IL-8 mRNA expression directly corresponded to the H2O2 concentration dependent binding of AP-1 to the IL-8 promoter. Supershift analysis revealed H2O2 as well as TNFalpha induced AP-1 complexes containing c-Fos and JunD. TNFalpha induced NF-kappaB complexes containing Rel A (p65). Immunohistochemical staining of HMEC-1 and A549 cells revealed TNFalpha stimulated nuclear localization of Rel A, whereas no translocation of Rel A was detected in either cell type stimulated by H2O2. These data indicate that the cell type-specific induction of IL-8 gene expression by H2O2 and TNFalpha in HMEC-1 and A549 cells can be explained by the differential binding of AP-1 and NF-kappaB to the IL-8 promoter.

Aminothiols protect endothelial cell proliferation against inhibition by lipopolysaccharide.

Lipopolysaccharide (LPS) is a primary agent of sepsis that damages the vascular endothelium. Endothelial cell proliferation is key to the repair of damaged endothelium, and drugs that counteract the antiproliferative impact of LPS on endothelial cells should be beneficial. Because LPS exerts much of its cytotoxicity by generating reactive oxygen and nitrogen intermediates, it would be helpful to know whether therapeutic antioxidant thiols maintain cell proliferation in injured endothelium. In this study, it was found that LPS inhibited bovine aortic endothelial cell proliferation by inducing apoptosis and by decreasing DNA synthesis. Because of its benefit to irradiated endothelial cells, we then treated the cells with a radio- and chemoprotective aminothiol, WR-1065 ([N-2-mecaptoethyl]-1-3-diaminopropane, the active form of Amifostine/Ethyol). WR-1065 attenuated the inhibition of DNA synthesis caused by LPS exposure. The disulfide of WR-1065, WR-33278, was tested and shown to both promote DNA synthesis and inhibit apoptosis. The effectiveness of the disulfide suggests that the reduction of cytotoxicity does not necessarily result from the scavenging of free radicals. These findings demonstrate a novel role for aminothiols in promoting DNA synthesis and lowering apoptosis in endothelium injured with LPS.

Aminothiol WR-1065 protects endothelial cell morphology against alterations induced by lipopolysaccharide.

In septic patients, lipopolysaccharide (LPS) damages the vascular endothelium, which manifests as tissue edema and impaired healing. This pathology occurs when LPS distorts endothelial cell morphology partly by generating free radicals. A radioprotector that scavenges free radicals, the aminothiol WR-1065 ([N-2-mercaptoethyl]-1-3-diaminopropane) was found in a prior study to normalize the morphology of irradiated endothelial cells (Mooteri SN, Podolski JL, Drab EA, et al: Radiat Res 145:217-224, 1996). The aim of this study was to determine whether WR-1065 also normalized endothelial cell morphology following exposure to LPS. For this aim, portions of bovine aortic endothelial cell cultures were denuded and exposed to LPS at 1 ng/mL. After 30 min, the apical membrane expressed increased integrin receptor to fibronectin, alpha5beta1. After 5 h, the morphology of the cells at the leading edge was distorted, and cell-cell contact was lessened. Also, filamentous actin-containing stress fibers were dissipated; however, filamentous actin content per cell was unchanged. Treatment with 2 mM WR-1065 for 2 h prior to LPS exposure attenuated the increased expression of alpha5beta1 and promoted cell-cell contact in the migrating endothelial cells. WR-1065 also promoted the retention of stress fibers and actin cytoskeletal shape in cells treated with LPS. Thus, LPS distorted endothelial cell morphology after increasing apical membrane expression of alpha5beta1 and dissipating stress fibers, effects prevented by WR-1065.