Strategies to improve hemorheology and clinical outcome of sickle cell anemia patients by red blood cell derived nitric oxide

近年来研究表明，红细胞一氧化氮合酶（RBC-NOS）产生的一氧化氮（NO）对维持和调节红细胞变形能力具有重要意义。部分RBC-NOS衍生的NO被进一步氧化为亚硝酸盐，作为NO储备发挥作用。NO的其他部分结合RBC蛋白，如α-血影蛋白和β-血影蛋白，从而促进RBC细胞骨架的稳定，这对RBC变形性有积极影响。镰状细胞性贫血（SCA）患者表现出受损的血液流变学，其特征在于RBC变形能力严重降低、高溶血率和红细胞增多。与Philippe Connes教授的研究小组合作，我们能够证明，与预期相反，SCA患者RBC中的NO产量与健康对照组相比增加。这是由Akt激酶途径的较高活化引起的，其因此增加RBC-NOS活化。所产生的NO与细胞骨架的蛋白质结合，最可能是α-血影蛋白和β-血影蛋白，但在这些患者中RBC变形性仍然显著降低。为了研究这一现象，并制定改善血液流变学和减少临床并发症的策略，我们确定了本项目建议书的以下目标：1。通过在体研究RBC-NOS和Akt信号通路阐明SCA患者的血液流变学变化：血液流变学、RBC-NOS谱与临床严重程度的关系。2.通过体外调节RBC-NOS依赖性和非依赖性NO产生，改善RBC变形性和细胞完整性，这是已知临床并发症的关键因素。红细胞一氧化氮合酶衍生的NO、氧化/亚硝化应激与红细胞变形性的关系3.通过施加剪切应力体外调节红细胞内NO含量改善红细胞变形性和细胞完整性：后续体内运动干预的预调查。4.通过体内运动干预改善红细胞中NO的形成，随后增加红细胞变形性。5.在基线条件下和血管闭塞事件期间，药理学增加对小鼠SCA模型全身NO含量的影响。

In recent years we have shown that red blood cell nitric oxide synthase (RBC-NOS) produced nitric oxide (NO) is of particular importance to maintain and regulate RBC deformability. Part of this RBC-NOS derived NO is further oxidized to nitrite to function as NO reserve. Other part of the NO binds to RBC proteins, such as a- und ß-spectrin, thus promoting the stabilization of the RBC cyto-skeleton, which positively affects RBC deformability. Sickle cell anemia (SCA) patients show im-paired blood rheology characterized by a severe reduction in RBC deformability, a high rate of hemolysis and enhanced eryptosis. In collaboration with the research group of Prof. Philippe Connes we were able to show that, against expectation, NO production in RBC of SCA patients is increased compared to healthy controls. This is caused by higher activation of the Akt kinase pathway, which consequently increases RBC-NOS activation. The produced NO binds to proteins of the cytoskeleton, most likely a- und ß- spectrin but RBC deformability is still significantly reduced in these patients. To investigate this phenomenon and to develop strategies to improve hemorheology and to reduce clinical complications, we defined the following objectives for this project proposal: 1. Elucidation of hemorheological changes in SCA patients by in vivo investigations of the RBC-NOS and Akt signaling pathway: Relationship between hemorheology, RBC-NOS profile and clinical se-verity. 2. Improvement of RBC deformability and cell integrity, key factors of the known clinical complica-tions, through in vitro modulation of RBC-NOS dependent and independent NO production. Rela-tionship between RBC-NOS derived NO, oxidative/nitrosative stress and RBC deformability. 3. Improvement of RBC deformability and cell integrity by in vitro modulation of intra-erythrocytic NO content by applied shear stress: Pre-investigation of subsequent in vivo exercise intervention. 4. Improvement of NO formation in RBC with subsequent increase in RBC deformability by in vivo exercise intervention. 5. Effects of pharmacological increase in systemic NO content in murine SCA models under base-line conditions and during vaso-occlusive events.