Figure 2.
Figure 2. Exposure of SSRBCs to NO reduces both SSRBC adhesion and the ability of SSRBCs to activate mononuclear leukocyte adhesion. (A) NO loading of SSRBCs inhibits adhesion to ECs in vitro. SSRBCs were sham loaded (deoxygenation under helium/reoxygenation at room air) or NO loaded as described in "Materials and methods," followed by exposure to vehicle alone or to epinephrine (Epi) for 1 minute. Adhesion of SSRBCs to HUVECs was tested in intermittent flow condition assays. Results are presented as percent adherent SSRBCs at a shear stress of 1 dyne/cm2. Error bars show SEMs of 3 different experiments using blood samples from 3 different patient donors with SCD. **P < .002 for vehicle-treated vs Epi-treated; *P < .01 for sham-loaded Epi-treated vs NO-loaded Epi-treated. (B) NO prevents SSRBCs from inducing mononuclear leukocyte adhesion to ECs in vitro. SSRBCs were loaded with NO before epinephrine treatment. Adhesion of PBMCs pre-incubated with washed Epi-treated SSRBCs or washed NO-loaded Epi-treated SSRBCs were then assayed. Results are presented as percent adherent PBMCs at a shear stress of 1 dyne/cm2. Error bars show SEMs of 3 different experiments using blood samples from 3 different patient donors with SCD. *P = .0207 for PBMCs + Epi-treated SS vs PBMCs + NO-loaded Epi-treated SS. (C-D) The effect of SSRBC NO supplementation on adhesion in vitro is independent of release of NO itself. Adhesion of SSRBCs and PBMCs to HUVECs was tested in intermittent flow condition assays, and results are presented as percent adherent cells at a shear stress of 1 dyne/cm2. Error bars show SEMs of 3 different experiments using blood samples from 3 different patients with SCD for panels C and D, and 3 different healthy donors for panel D. (C) SSRBCs were treated with Epi for 1 minute, NO loaded followed by stimulation with Epi, NO loaded followed by stimulation with Epi and then incubation with free HbA, or NO loaded followed by stimulation with Epi then incubation with albumin (alb). *P = .0003 for Epi-treated vs NO-loaded Epi-treated. There was no statistically significant difference in adhesion of NO-loaded Epi-treated SSRBCs vs SSRBCs that had NO-loaded and Epi-treated and then incubated with either free HbA or alb. (D) SSRBCs were Epi-treated or NO loaded then Epi-treated, before coincubation with PBMCs in the presence of free HbA or alb. ****P < .0001 for PBMCs + Epi-treated SSRBCs vs PBMCs alone and PBMCs + NO-loaded Epi-treated SSRBCs vs PBMCs + Epi-treated SSRBCs. There was no statistically significant difference measured for adhesion of PBMCs + NO-loaded Epi-treated SSRBCs vs PBMCs + NO-loaded Epi-treated SSRBCs in the presence of free HbA or alb. n.s., not significant.

Exposure of SSRBCs to NO reduces both SSRBC adhesion and the ability of SSRBCs to activate mononuclear leukocyte adhesion. (A) NO loading of SSRBCs inhibits adhesion to ECs in vitro. SSRBCs were sham loaded (deoxygenation under helium/reoxygenation at room air) or NO loaded as described in "Materials and methods," followed by exposure to vehicle alone or to epinephrine (Epi) for 1 minute. Adhesion of SSRBCs to HUVECs was tested in intermittent flow condition assays. Results are presented as percent adherent SSRBCs at a shear stress of 1 dyne/cm2. Error bars show SEMs of 3 different experiments using blood samples from 3 different patient donors with SCD. **P < .002 for vehicle-treated vs Epi-treated; *P < .01 for sham-loaded Epi-treated vs NO-loaded Epi-treated. (B) NO prevents SSRBCs from inducing mononuclear leukocyte adhesion to ECs in vitro. SSRBCs were loaded with NO before epinephrine treatment. Adhesion of PBMCs pre-incubated with washed Epi-treated SSRBCs or washed NO-loaded Epi-treated SSRBCs were then assayed. Results are presented as percent adherent PBMCs at a shear stress of 1 dyne/cm2. Error bars show SEMs of 3 different experiments using blood samples from 3 different patient donors with SCD. *P = .0207 for PBMCs + Epi-treated SS vs PBMCs + NO-loaded Epi-treated SS. (C-D) The effect of SSRBC NO supplementation on adhesion in vitro is independent of release of NO itself. Adhesion of SSRBCs and PBMCs to HUVECs was tested in intermittent flow condition assays, and results are presented as percent adherent cells at a shear stress of 1 dyne/cm2. Error bars show SEMs of 3 different experiments using blood samples from 3 different patients with SCD for panels C and D, and 3 different healthy donors for panel D. (C) SSRBCs were treated with Epi for 1 minute, NO loaded followed by stimulation with Epi, NO loaded followed by stimulation with Epi and then incubation with free HbA, or NO loaded followed by stimulation with Epi then incubation with albumin (alb). *P = .0003 for Epi-treated vs NO-loaded Epi-treated. There was no statistically significant difference in adhesion of NO-loaded Epi-treated SSRBCs vs SSRBCs that had NO-loaded and Epi-treated and then incubated with either free HbA or alb. (D) SSRBCs were Epi-treated or NO loaded then Epi-treated, before coincubation with PBMCs in the presence of free HbA or alb. ****P < .0001 for PBMCs + Epi-treated SSRBCs vs PBMCs alone and PBMCs + NO-loaded Epi-treated SSRBCs vs PBMCs + Epi-treated SSRBCs. There was no statistically significant difference measured for adhesion of PBMCs + NO-loaded Epi-treated SSRBCs vs PBMCs + NO-loaded Epi-treated SSRBCs in the presence of free HbA or alb. n.s., not significant.

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