Importance of erythrocyte arginase-1 for vascular homeostasis and disease

实验证据和第一批临床数据表明，红细胞积极参与血管功能调控。然而，红细胞直接作用于血管壁细胞并参与心血管疾病过程的概念直到最近才出现，其红分泌活动所涉及的介质和信号通路在很大程度上尚不清楚。在这个项目的第一个资助期，我们已经证明了来自溶解红细胞膜的一氧化氮促进了平滑肌细胞矿化和动脉粥样硬化病变的钙化。基于这些发现，我们产生了红细胞中缺乏精氨酸酶-1的高胆固醇血症小鼠，以使红细胞中的精氨酸酶-eNOS平衡转向NO生成增加。正在进行的对这一新的小鼠品系的鉴定可以证实，在体外和体内，红细胞释放的NO增强促进了血管钙化。此外，我们观察到NO生物利用度的增加对血管内稳态有全身性影响，这表明红细胞在这些过程中发挥了重要作用。因此，在第二个资助期，我们计划更详细地研究红细胞对血管动态平衡和疾病过程的贡献。具体地说，我们将在C57BL6/J和高胆固醇血症载脂蛋白E缺乏的背景下，对红细胞特异性精氨酸酶-1缺失和红细胞源NO释放增加的小鼠进行表型，包括血管功能调节(WP1)、新内膜形成(WP2)和动脉粥样硬化斑块进展和稳定性(WP3)。为了确定红细胞精氨酸酶-1对全身NO生物利用度的贡献，研究结果将与内皮细胞特异性精氨酸酶缺失的小鼠进行比较。根据NO可能通过诱导血管细胞合成其他气体递质的观察结果和自己的初步数据，我们将检测血管内皮细胞和平滑肌细胞在药物抑制或RNAi介导的硫化氢产生酶遗传功能丧失后对小鼠和人红细胞的反应。为了将我们在小鼠和细胞中的发现转化为人类，涉及NO生物利用度和血管系统信号转导的因素的表达和活性的差异将在高危患者或已确诊的心血管疾病患者的红细胞中进行检测。

Experimental evidence and first clinical data suggest that erythrocytes actively participate in vascular function control. However, the concept that erythrocytes directly act on cells of the vessel wall and contribute in cardiovascular disease processes has only recently emerged, and the mediators and signalling pathways involved in their erythrocrine activities are largely unknown. During the first funding period of this project we have shown that nitric oxide from lysed erythrocyte membranes promotes smooth muscle cell mineralisation and atherosclerotic lesion calcification. Based on these findings, we generated hypercholesterolaemic mice lacking arginase-1 specifically in red blood cells to shift the arginase-eNOS balance in erythrocytes towards increased NO generation. The ongoing characterisation of this new mouse line could confirm that the enhanced release of NO from erythrocytes promotes vascular calcification in vitro and in vivo. In addition, we observed that the increased NO bioavailability has systemic effects on vascular homeostasis suggesting a major role of erythrocytes in these processes. In the second funding period, we therefore plan to examine the contribution of erythrocytes to vascular homeostasis and disease processes in more detail. Specifically, we will phenotype mice with erythrocyte-specific arginase-1 deletion and increased erythrocyte-derived NO release, both on the C57BL6/J and the hypercholesterolaemic apolipoprotein E-deficient background, including vascular function regulation (WP1), neointima formation (WP2) and atherosclerotic plaque progression and stability (WP3). To determine the contribution of red blood cell arginase-1 to systemic NO bioavailability, findings will be compared to mice with endothelial cell-specific arginase deletion. Based on observations that NO may act via inducing the synthesis of other gasotransmitters in vascular cells and own preliminary data, we will examine the response of endothelial and smooth muscle cells after pharmacological inhibition or RNAi-mediated genetic loss-of-function of H2S generating enzymes to murine and human erythrocytes. To translate our findings in mice and cells to humans, differences in the expression and activity of factors involved in the bioavailability of NO and signal transduction in the vascular system will be examined in RBCs from patients at risk or with established cardiovascular disease.