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Induction of decay-accelerating factor by thrombin through a protease-activated receptor 1 and protein kinase C–dependent pathway protects vascular endothelial cells from complement-mediated injury

Elaine A. Lidington, Dorian O. Haskard and Justin C. Mason

Article Figures & Data

Figures

  • Fig. 1.

    Up-regulation of DAF expression on HUVECs following stimulation with thrombin.

    The expression of DAF on resting and thrombin-stimulated HUVECs was assessed by flow cytometry using MoAb 1H4. The figure shows background fluorescence (FITC-labeled rabbit antimouse antibody alone) (shaded histograms) and DAF expression (open histograms) on unstimulated HUVECs (A) and HUVECs stimulated with thrombin (10 U/mL) for 6 hours (B), 12 hours (C), 24 hours (D), 48 hours (E), and 72 hours (F). The RFI for DAF expression at each time point is shown in brackets. The RFI represents the mean MFI with the test MoAb divided by the MFI using an isotype-matched irrelevant MoAb. The results are representative of 3 experiments on separate EC cultures.

  • Fig. 2.

    Dose response for thrombin-induced DAF and ICAM-1 expression.

    HUVEC monolayers were incubated for 24 hours in the presence or absence of increasing concentrations of thrombin before harvesting and analysis by flow cytometry. DAF and ICAM-1 were detected using MoAbs 1H4 and 6.5B5, respectively. The results are expressed as mean ± SEM for RFI DAF (A) and ICAM-1 (B). The results are representative of 3 separate experiments performed in triplicate wells using different EC lines.

  • Fig. 3.

    Effect of hirudin on thrombin-induced DAF expression and comparison of TRAP-6 to thrombin for induction of DAF.

    (A) HUVEC monolayers were treated with hirudin (30 U/mL) or plain medium alone for 30 minutes prior to the addition of thrombin (10 U/mL) and cultured for a further 24 hours. Following harvesting, ECs were analyzed by flow cytometry for the expression of DAF using MoAb 1H4. The data are expressed as a percentage of the DAF expression (RFI) on thrombin-stimulated ECs and are presented as the mean ± SEM. (B) HUVEC monolayers were treated with increasing concentrations of TRAP-6 or vehicle alone for 24 hours and then harvested, stained with MoAb to DAF (1H4), and analyzed by flow cytometry. The data are expressed as a percentage of the DAF expression on thrombin-treated cells and are presented as the mean ± SEM of 3 experiments performed on separate HUVEC cultures.

  • Fig. 4.

    Effect of pharmacologic inhibitors of PKC, p38 and p42/44 MAPK, and NF-κB on thrombin-induced DAF.

    HUVEC monolayers were preincubated with (A) the PKC inhibitor RO31-8220 (1 μmol/L), (B) the p38 inhibitor SB202190 (25 μmol/L), (C) the MEK-1 inhibitor of p42/44 MAPK phosphorylation PD98059 (50 μmol/L), or (D) the NF-κB inhibitor PSI (10 μmol/L) for 1 hour prior to the addition of 10 U/mL thrombin for 24 hours. The ECs were then harvested and stained with MoAb to DAF (1H4) for analysis by flow cytometry. The data are expressed as a percentage of the DAF expression on thrombin-treated cells and are presented as the mean ± SEM of 3 experiments performed on separate HUVEC cultures.

  • Fig. 5.

    Thrombin-induced surface expression of DAF requires de novo protein synthesis.

    HUVECS were plated at confluence (5 × 105 cells/well) in 6-well dishes and cultured overnight at 37°C. They were then pretreated with CHX (1 μg/mL) for 30 minutes prior to addition of thrombin (10 U/mL) for a further 24 hours. Following harvesting, DAF expression was measured by flow cytometry using MoAb 1H4. CHX at these concentrations was not toxic to ECs, as assessed by examination of the monolayers prior to staining using phase-contrast microscopy, cell counting, and estimation of trypan blue exclusion. The data are expressed as the RFI ± SEM from 2 similar experiments performed on separate HUVEC cultures.

  • Fig. 6.

    Thrombin-induced DAF gene transcription is independent of de novo protein synthesis.

    (A) HUVECs in 75-cm2 flasks were pretreated with CHX (1 μg/mL) or plain medium alone for 30 minutes prior to the addition of thrombin (10 U/mL) and culture for up to 24 hours. Total RNA was isolated, and Northern blots were prepared. The figure shows unstimulated HUVEC (lane 1) thrombin treatment for 2 hours (lane 2), 4 hours (lane 3), 6 hours (lane 4), and 24 hours (lane 5); CHX alone for 4 hours (lane 6); and CHX and thrombin for 2 hours (lane 7) and 4 hours (lane 8). The ethidium bromide–stained gels confirmed equal loading of RNA in each lane. (B) Quantification of mRNA levels in resting and thrombin-stimulated ECs using densitometric scanning. (C) HUVECs in 75-cm2 flasks were pretreated with CHX (1 μg/mL) or plain medium alone for 30 minutes prior to the addition of VEGF (25 ng/mL) and culture for up to 24 hours. Total RNA was isolated, and Northern blots were prepared. The figure shows unstimulated HUVECs (lane 1); VEGF treatment for 3 hours (lane 2), 6 hours (lane 3), and 9 hours (lane 4); CHX alone for 6 hours (lane 5); CHX and VEGF for 6 hours (lane 6); and CHX and VEGF for 9 hours (lane 7). The ethidium bromide–stained gels confirmed equal loading of RNA in each lane. (D) Quantification of mRNA levels in resting and VEGF-stimulated ECs using densitometric scanning.

  • Fig. 7.

    Thrombin-induced DAF expression protects ECs against complement-mediated injury.

    HUVECs were plated at confluence (5 × 105 cells/well) in 6-well dishes and cultured overnight at 37°C prior to stimulation with 10 U/mL thrombin or plain medium alone (unstimulated) for 24 hours. Following harvesting, ECs were incubated with the antiendoglin MoAb RMAC8 or plain medium alone for 30 minutes at 4°C. The ECs were then washed in HBSS/1% BSA prior to addition of up to 5% NHS for 3 hours at 37°C. Binding of C3 was detected by flow cytometry using FITC-conjugated rabbit antihuman C3. (A) Percent C3 binding ± SD (n = 3) to unstimulated (black bars) and thrombin-stimulated HUVECs (hatched bars), with binding to unstimulated ECs (RFI ± SD = 8.1 ± 2.7) shown as 100%. The negative control represents C3 binding in the presence of HIHS but without preincubation with MoAb RMAC8. (B) C3 binding (RFI ± SD, n = 3) on unstimulated (black bars) and thrombin-stimulated HUVECs (hatched bars) in the presence of MoAbs 1H4 (anti-DAF) and EN4 (anti-CD31), both at 50 μg/mL. (C) HUVECs plated at confluence (1.5 × 105 cells/well) in 24-well plates were cultured overnight prior to stimulation with 10 U/mL thrombin or plain medium alone for 24 hours. Following loading with calcein acetoxymethyl ester, ECs were incubated with MoAb RMAC8 for 30 minutes at 37°C. The ECs were then washed in HBSS/1% BSA prior to the addition of baby rabbit complement or HIHS for 45 minutes at 37°C. Calcein release was measured and percent cell lysis calculated as outlined in “Materials and methods.” The data are presented as percent cell lysis ± SD, n = 3. The figure is representative of 3 similar experiments performed on different EC cultures. * P < .05, ** P < .001.