Figure 1.
Figure 1. FVIIa downregulates TNF-α-induced inflammatory gene expression in endothelial cells. HUVECs were serum-starved for 12 hours and stabilized for 1 hour in the serum-free medium. Cells were exposed to FVIIa or APC (100 nM) for 1 hour in serum-free medium and then treated with TNF-α (5 ng/mL) for 6 hours. (A-C) Total RNA was extracted from cells, and levels of mRNA were analyzed by quantitative RT-PCR for ICAM-1 (A), VCAM-1 (B), and IL-6 (C). mRNA levels present in nonstimulated (NS) cells treated with a control vehicle (Con) were taken as 1, and values in the experimental treatments were expressed relative to this value (RE). (D-E) FVIIa suppression of TNF-α-induced ICAM-1, VCAM-1, and IL-6 protein expression. The experimental conditions were the same as described above. ICAM-1 and VCAM-1 protein expression in cell lysates was analyzed by western blot analysis, and the signals were quantified by densitometric analysis using ImageJ software (D); IL-6 protein levels in the supernatants were measured by an ELISA (E). (F) HUVECs grown on the glass coverslip were treated with FVIIa and TNF-α (5 ng/ml) for 4 hours as described above. THP-1 cells labeled with a green fluorescent cell linker (PKH67) were added to the endothelial cell monolayer and allowed to adhere for 30 minutes in serum-rich RPMI medium. The nonadherent THP-1 cells were washed with serum-free medium, and the cells were fixed with 4% paraformaldehyde. The images of the adherent cells were captured by a fluorescent microscope at 100× magnification. UN, unstimulated cells. (G). Dose-dependent effect of FVIIa and APC on attenuation of TNF-α-induced VCAM-1 expression. HUVEC were treated with varying concentrations of FVIIa or APC (0 to 100 nM) for 1 hour and then stimulated with TNF-α (5 ng/ml) for 6 hours. Cell extracts were subjected to immunoblot analysis to determine VCAM-1 protein expression and actin (as a loading control). Band intensities were quantitated by densitometric analysis and normalized to the loading control. VCAM-1 expression levels in cells treated with TNFα alone was taken as 100%. Data shown in the bar graphs represent the mean ± SEM of 3 to 6 independent experiments. *P < .05; **P < .01; ***P < .001; ns, not statistically significant.

FVIIa downregulates TNF-α-induced inflammatory gene expression in endothelial cells. HUVECs were serum-starved for 12 hours and stabilized for 1 hour in the serum-free medium. Cells were exposed to FVIIa or APC (100 nM) for 1 hour in serum-free medium and then treated with TNF-α (5 ng/mL) for 6 hours. (A-C) Total RNA was extracted from cells, and levels of mRNA were analyzed by quantitative RT-PCR for ICAM-1 (A), VCAM-1 (B), and IL-6 (C). mRNA levels present in nonstimulated (NS) cells treated with a control vehicle (Con) were taken as 1, and values in the experimental treatments were expressed relative to this value (RE). (D-E) FVIIa suppression of TNF-α-induced ICAM-1, VCAM-1, and IL-6 protein expression. The experimental conditions were the same as described above. ICAM-1 and VCAM-1 protein expression in cell lysates was analyzed by western blot analysis, and the signals were quantified by densitometric analysis using ImageJ software (D); IL-6 protein levels in the supernatants were measured by an ELISA (E). (F) HUVECs grown on the glass coverslip were treated with FVIIa and TNF-α (5 ng/ml) for 4 hours as described above. THP-1 cells labeled with a green fluorescent cell linker (PKH67) were added to the endothelial cell monolayer and allowed to adhere for 30 minutes in serum-rich RPMI medium. The nonadherent THP-1 cells were washed with serum-free medium, and the cells were fixed with 4% paraformaldehyde. The images of the adherent cells were captured by a fluorescent microscope at 100× magnification. UN, unstimulated cells. (G). Dose-dependent effect of FVIIa and APC on attenuation of TNF-α-induced VCAM-1 expression. HUVEC were treated with varying concentrations of FVIIa or APC (0 to 100 nM) for 1 hour and then stimulated with TNF-α (5 ng/ml) for 6 hours. Cell extracts were subjected to immunoblot analysis to determine VCAM-1 protein expression and actin (as a loading control). Band intensities were quantitated by densitometric analysis and normalized to the loading control. VCAM-1 expression levels in cells treated with TNFα alone was taken as 100%. Data shown in the bar graphs represent the mean ± SEM of 3 to 6 independent experiments. *P < .05; **P < .01; ***P < .001; ns, not statistically significant.

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