Figure 4
Figure 4. Endothelial TLR4 signaling is required for WPB activation and vascular stasis induced by heme. (A) HUVECs were incubated with vehicle (DMSO), heme (10 µM), or heme plus the TLR4-signaling inhibitor TAK-242 (400 nM). After a 15-minute incubation, cells were fixed and stained for surface P-selectin (green) and VWF (red). Nuclei were counterstained with DAPI (blue). Data are representative of at least 3 experiments. (B) mPVECs were isolated and cultured from TLR4+/+ and TLR4−/− mice. mPVECs were incubated with heme (10 µM), Hb (10 µM heme), or LPS (10 ng/ml) for 15 minutes (n = 6 wells per treatment). Cells were fixed and surface VWF was measured by enzyme immunoassay (*P < .05 for TLR4+/+ vs TLR4−/−). (C) Percent stasis was measured in the subcutaneous venules of NY1DD and C57BL/6 mice with DSFCs as described in Figure 1. Mice were given a bolus infusion (0.012 mL/g) of the indicated treatments at the specified heme doses: vehicle (DMSO/saline, 1:9 vol/vol) + heme (3.2 µmol/kg), TAK-242 (2 mg/kg, IP 30 minutes before heme) + heme, vehicle + LPS (1 mg/kg), TAK-242 + LPS, isotype control IgG (30 µg/mouse) + LPS, anti-LPS IgG (30 µg per mouse) + LPS, isotype control IgG + heme, anti-LPS IgG + heme, vehicle + H/R, TAK-242 + H/R, or LPS alone. The numbers of mice (n) in each treatment group are indicated. Bars are mean % stasis + SD with mean stasis values written above the bars. *P < .01 vs control; #P < .001 C57 + LPS vs NY1DD vehicle + LPS. (D) Chimeric NY1DD βS/TLR4−/− and NY1DD βS/TLR4+/+ mice were generated by transplanting BM from NY1DD βS sickle mice into TLR4−/− or TLR4+/+ mice (n = 3 recipients per group). C57BL/6 mice were used as TLR4+/+ mice. Negative control chimeric C57 βMurine/TLR4−/− mice were generated by transplanting BM from C57BL/6 mice into TLR4−/− mice. The presence of human βS was confirmed by isoelectric focusing (IEF) at 3 months posttransplant. Representative IEF bands for each group are shown (bottom). Stasis was measured in transplanted mice 1 to 2 weeks after IEF determination after IV infusion of Hb (0.32 µmol heme/kg) (*P < .01 NY1DD βS/TLR4−/− vs NY1DD βS/TLR4+/+). Nontransplanted NY1DD βS mice (n = 2) served as positive controls. All stasis values are mean percent stasis + SD. (E) Leukocyte rolling flux was measured in venules of NY1DD mice with DSFCs before (baseline) and 1 hour after infusion of heme (3.2 µmol/kg). Half of the mice (n = 4) were treated with TAK-242 after baseline (+TAK-242, 2 mg/kg, IP, 30 minutes before heme). Control mice (n = 4) were untreated with TAK-242 (−TAK-242). Values are mean number of rolling cells per minute + SD. (F) Leukocyte adhesion was measured in the same venules as described in panel C. Values are mean number of adherent cells per 100 µm + SD.

Endothelial TLR4 signaling is required for WPB activation and vascular stasis induced by heme. (A) HUVECs were incubated with vehicle (DMSO), heme (10 µM), or heme plus the TLR4-signaling inhibitor TAK-242 (400 nM). After a 15-minute incubation, cells were fixed and stained for surface P-selectin (green) and VWF (red). Nuclei were counterstained with DAPI (blue). Data are representative of at least 3 experiments. (B) mPVECs were isolated and cultured from TLR4+/+ and TLR4−/− mice. mPVECs were incubated with heme (10 µM), Hb (10 µM heme), or LPS (10 ng/ml) for 15 minutes (n = 6 wells per treatment). Cells were fixed and surface VWF was measured by enzyme immunoassay (*P < .05 for TLR4+/+ vs TLR4−/−). (C) Percent stasis was measured in the subcutaneous venules of NY1DD and C57BL/6 mice with DSFCs as described in Figure 1. Mice were given a bolus infusion (0.012 mL/g) of the indicated treatments at the specified heme doses: vehicle (DMSO/saline, 1:9 vol/vol) + heme (3.2 µmol/kg), TAK-242 (2 mg/kg, IP 30 minutes before heme) + heme, vehicle + LPS (1 mg/kg), TAK-242 + LPS, isotype control IgG (30 µg/mouse) + LPS, anti-LPS IgG (30 µg per mouse) + LPS, isotype control IgG + heme, anti-LPS IgG + heme, vehicle + H/R, TAK-242 + H/R, or LPS alone. The numbers of mice (n) in each treatment group are indicated. Bars are mean % stasis + SD with mean stasis values written above the bars. *P < .01 vs control; #P < .001 C57 + LPS vs NY1DD vehicle + LPS. (D) Chimeric NY1DD βS/TLR4−/− and NY1DD βS/TLR4+/+ mice were generated by transplanting BM from NY1DD βS sickle mice into TLR4−/− or TLR4+/+ mice (n = 3 recipients per group). C57BL/6 mice were used as TLR4+/+ mice. Negative control chimeric C57 βMurine/TLR4−/− mice were generated by transplanting BM from C57BL/6 mice into TLR4−/− mice. The presence of human βS was confirmed by isoelectric focusing (IEF) at 3 months posttransplant. Representative IEF bands for each group are shown (bottom). Stasis was measured in transplanted mice 1 to 2 weeks after IEF determination after IV infusion of Hb (0.32 µmol heme/kg) (*P < .01 NY1DD βS/TLR4−/− vs NY1DD βS/TLR4+/+). Nontransplanted NY1DD βS mice (n = 2) served as positive controls. All stasis values are mean percent stasis + SD. (E) Leukocyte rolling flux was measured in venules of NY1DD mice with DSFCs before (baseline) and 1 hour after infusion of heme (3.2 µmol/kg). Half of the mice (n = 4) were treated with TAK-242 after baseline (+TAK-242, 2 mg/kg, IP, 30 minutes before heme). Control mice (n = 4) were untreated with TAK-242 (−TAK-242). Values are mean number of rolling cells per minute + SD. (F) Leukocyte adhesion was measured in the same venules as described in panel C. Values are mean number of adherent cells per 100 µm + SD.

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