Division of Cardiology, Children's Hospital of Los Angeles, USA.
Division of Hematology, Children's Hospital of Los Angeles, USA.
Free Radic Biol Med. 2021 Aug 1;171:143-155. doi: 10.1016/j.freeradbiomed.2021.05.007. Epub 2021 May 8.
Sickle cell anemia (SCA) is characterized by decreased red blood cell (RBC) deformability due to polymerization of deoxygenated hemoglobin, leading to abnormal mechanical properties of RBC, increased cellular adhesion, and microcirculatory obstruction. Prior work has demonstrated that NO• influences RBC hydration and deformability and is produced at a basal rate that increases under shear stress in normal RBC. Nevertheless, the origin and physiological relevance of nitric oxide (NO•) production and scavenging in RBC remains unclear. We aimed to assess the basal and shear-mediated production of NO• in RBC from SCA patients and control (CTRL) subjects. RBCs loaded with a fluorescent NO• detector, DAF-FM (4-Amino-5-methylamino- 2',7'-difluorofluorescein diacetate), were imaged in microflow channels over 30-min without shear stress, followed by a 30-min period under 0.5Pa shear stress. We utilized non-specific nitric oxide synthase (NOS) blockade and carbon monoxide (CO) saturation of hemoglobin to assess the contribution of NOS and hemoglobin, respectively, to NO• production. Quantification of DAF-FM fluorescence intensity in individual RBC showed an increase in NO• in SCA RBC at the start of the basal period; however, both SCA and CTRL RBC increased NO• by a similar quantity under shear. A subpopulation of sickle-shaped RBC exhibited lower basal NO• production compared to discoid RBC from SCA group, and under shear became more circular in the direction of shear when compared to discoid RBC from SCA and CTRL, which elongated. Both CO and NOS inhibition caused a decrease in basal NO• production. Shear-mediated NO• production was decreased by CO in all RBC, but was decreased by NOS blockade only in SCA. In conclusion, total NO• production is increased and shear-mediated NO• production is preserved in SCA RBC in a NOS-dependent manner. Sickle shaped RBC with inclusions have higher NO• production and they become more circular rather than elongated with shear.
镰状细胞贫血 (SCA) 的特征是脱氧血红蛋白聚合导致红细胞 (RBC) 变形能力下降，从而导致 RBC 机械性能异常、细胞黏附增加和微循环阻塞。先前的工作表明，NO•会影响 RBC 的水合和变形能力，并以基础速率产生，在正常 RBC 受到切应力时会增加。然而，RBC 中一氧化氮 (NO•) 的产生和清除的起源和生理相关性仍不清楚。我们旨在评估 SCA 患者和对照 (CTRL) 受试者 RBC 的基础和切变介导的 NO•产生。将负载荧光 NO•探测器 DAF-FM（4-氨基-5-甲基氨基-2'，7'-二氟荧光素二乙酸酯）的 RBC 成像在微流通道中，在无切应力下进行 30 分钟，然后在 0.5Pa 切应力下进行 30 分钟。我们利用非特异性一氧化氮合酶 (NOS) 阻断和血红蛋白的一氧化碳 (CO) 饱和来分别评估 NOS 和血红蛋白对 NO•产生的贡献。个体 RBC 中 DAF-FM 荧光强度的定量显示，在基础期开始时 SCA RBC 中的 NO•增加；然而，在切应力下，SCA 和 CTRL RBC 都以相似的数量增加了 NO•。与 SCA 组盘形 RBC 相比，镰状 RBC 的基础 NO•产生量较低的亚群，与 SCA 和 CTRL 组的盘形 RBC 相比，在切应力下变得更圆，呈椭圆形。CO 和 NOS 抑制均导致基础 NO•产生减少。所有 RBC 中的 CO 均降低了切变介导的 NO•产生，但仅在 SCA 中 NOS 阻断降低了切变介导的 NO•产生。总之，SCA RBC 中的总 NO•产生增加，并且以 NOS 依赖的方式保存了切变介导的 NO•产生。含包涵体的镰状 RBC 具有更高的 NO•产生，并且它们在切应力下变得更圆而不是拉长。
