![Figure 4. RBC MPs mediate vascular endothelial damage in vitro and ex vivo. (A) MPs shed by wild-type (WT) and SAD RBC treated with 4N1-1 were purified and incubated on confluent murine SVEC-40 endothelial monolayers (25 MPs/μL). The cells were pretreated with inhibitors of NADPH oxydase (apocynin, 100μM; DPI, 10μM) or protein-C kinase (Gö-6976, 1μM; Ro-31-8220, 1μM) for 30 minutes. Alternatively, RBC MPs were pre-incubated with annexin-V (10 μg/mL) for 30 minutes. Fluorescent H2DFF-DA was added after 30 minutes and ROS production measured after 90 minutes. (*P < .05 vs WT control, none; #P < .05 vs WT MPs; $P < .05 vs SAD MPs+none). (B) Endothelial monolayers were exposed to RBC MPs for 30 minutes and washed off. RBCs were then left to adhere for 30 minutes. Nonadherent RBCs were eliminated and adherent RBCs counted (*P < .05 vs no MPs; $P < .05 vs WT MPs). (C) Representative micrographs of SAD RBC adhered to endothelium pre-treated with WT and SAD MPs (60×, bar is 5 μm). (D) Endothelial cells were treated with WT or SAD RBC MPs for 36 hours. Total DNA content was analyzed by FACS after propidium iodide coloration. Cells undergoing apoptosis (Sub-G1 phase) or proliferation (G2/M) were quantified. (E) Representative DNA profiles of endothelial cells incubated with WT or SAD RBC MPs for 16 hours (*P < .05 vs control, none). (F-G) Mesenteric resistances arteries were perfused with PSS alone, or supplemented with WT or SAD RBC MPs (200 MPs/μL) at 75 mmHg pressure and 20 μL/s flow. (F) Vasodilation in response to increasing ACH doses (10−4 to 10−7M) was quantified and expressed as percentage of passive diameter (*P < .05 vs control [solid line]; #P < .05 vs WT RBC MPs [dashes]). (G) Vessels were pretreated with ROS inhibitor Tempol (10μM; 30 minutes), or SAD MPs were saturated with annexin-V (1 μg/mL; 1 hour) before perfusion (*P < .05 vs control; #P < .05 vs SAD RBC MPs alone, +none).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/120/25/10.1182_blood-2012-02-413138/4/zh89991298680004.jpeg?Expires=1722712939&Signature=paWxZaO3XfJavDYNQ4kta~JxcH6HA55HcLYGrr3t7R7PYoH8O5sXtPRzW96g90SubTdvWit8EF7V0NbopJ4YTm~nLgJwebj-nu4BVz1FHUzGThwaLHJmzQoHaSA0~67Dkkuofqi5UFN7N1CejgipT~iKw14JZKm73JcJJ33H1jCO3FlKv9v6PI~miO2-4jr--EZRSW21h3Y2TTrmaTl2TBc0xpLuUSFanL1901uXfbhWFltn2zogjLI4SjrCQUgX2wh2WM0RSsjAfFv3nCp6mtJHyDLVVFrMJVtbjPpaMcNceqzoehGqcC1Dud-4N3mEjFPKvnMOkNJXPlVdbQYkzA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
RBC MPs mediate vascular endothelial damage in vitro and ex vivo. (A) MPs shed by wild-type (WT) and SAD RBC treated with 4N1-1 were purified and incubated on confluent murine SVEC-40 endothelial monolayers (25 MPs/μL). The cells were pretreated with inhibitors of NADPH oxydase (apocynin, 100μM; DPI, 10μM) or protein-C kinase (Gö-6976, 1μM; Ro-31-8220, 1μM) for 30 minutes. Alternatively, RBC MPs were pre-incubated with annexin-V (10 μg/mL) for 30 minutes. Fluorescent H2DFF-DA was added after 30 minutes and ROS production measured after 90 minutes. (*P < .05 vs WT control, none; #P < .05 vs WT MPs; $P < .05 vs SAD MPs+none). (B) Endothelial monolayers were exposed to RBC MPs for 30 minutes and washed off. RBCs were then left to adhere for 30 minutes. Nonadherent RBCs were eliminated and adherent RBCs counted (*P < .05 vs no MPs; $P < .05 vs WT MPs). (C) Representative micrographs of SAD RBC adhered to endothelium pre-treated with WT and SAD MPs (60×, bar is 5 μm). (D) Endothelial cells were treated with WT or SAD RBC MPs for 36 hours. Total DNA content was analyzed by FACS after propidium iodide coloration. Cells undergoing apoptosis (Sub-G1 phase) or proliferation (G2/M) were quantified. (E) Representative DNA profiles of endothelial cells incubated with WT or SAD RBC MPs for 16 hours (*P < .05 vs control, none). (F-G) Mesenteric resistances arteries were perfused with PSS alone, or supplemented with WT or SAD RBC MPs (200 MPs/μL) at 75 mmHg pressure and 20 μL/s flow. (F) Vasodilation in response to increasing ACH doses (10−4 to 10−7M) was quantified and expressed as percentage of passive diameter (*P < .05 vs control [solid line]; #P < .05 vs WT RBC MPs [dashes]). (G) Vessels were pretreated with ROS inhibitor Tempol (10μM; 30 minutes), or SAD MPs were saturated with annexin-V (1 μg/mL; 1 hour) before perfusion (*P < .05 vs control; #P < .05 vs SAD RBC MPs alone, +none).
