Kontrollmechanismen der endothelialen NO-Synthase-Expression

内皮细胞中一氧化氮（NO）的产生不足或过度降解与动脉粥样硬化的发病机制有关。因此，在编码内皮细胞NO合酶（NOS-3）的基因的启动子中的单核苷酸多态性（SNP）与心血管疾病相关，该单核苷酸多态性明显导致酶表达的下降。根据我们的准备工作，-786位的胸苷被胞嘧啶纯合取代导致一种尚未鉴定的蛋白质（称为因子X）与nos-3基因的启动子紧密结合，这不仅阻止了STAT-3的邻近结合，而且阻止了一种假定的剪切应力敏感性转录因子的邻近结合。虽然STAT-3介导抗炎细胞因子白细胞介素-10对NOS-3表达的刺激作用，但层流剪切应力是维持酶在体内表达的生理学上最重要的刺激。与-786 C/T-SNP纯合子个体（高达高加索人群的15%）内皮细胞NO合成能力降低一致，我们可以进一步证明该SNP与发生冠状动脉疾病以及类风湿性关节炎的风险显著增加相关。 基于这些发现，本研究计划有三个目标：（i）通过亲和层析和质谱分析相结合的方法鉴定X因子;（ii）通过利用nos-3基因的剪切应力敏感性是通过启动子-786C/T SNP附近的序列介导的这一事实，来表征迄今为止难以捉摸的剪切应力敏感性转录因子及其结合基序;和（iii）建立动物实验概念模型（心脏移植血管病）的验证，以证明开发用于阻断因子X或促进NOS-3表达的基于核酸的药物对-786C/T SNP的功能校正的功效。由于这种SNP似乎不存在于其他哺乳动物中，因此将为此目的产生两种含有人C型启动子和用于比较的T型启动子的敲入小鼠品系。

An insufficient production or excessive degradation of nitric oxide (NO) in endothelial cells has been linked to the pathogenesis of atherosclerosis. Consequently, a single nucleotide polymorphism (SNP) in the promoter of the gene encoding endothelial cell NO synthase (NOS-3) that apparently causes a decline in enzyme expression has been associated with cardiovascular disease. According to our preparatory work, homozygous replacement of the thymidine at position -786 by a cytosine results in the tight binding of an as yet unidentified protein, termed factor X, to the promoter of the nos-3 gene which prevents not only the nearby binding of STAT-3 but also that of a putative shear stress-sensitive transcription factor. While STAT-3 mediates the stimulatory effect of the anti-inflammatory cytokine interleukin-10 on NOS-3 expression, laminar shear stress is the physiologically most important stimulus for maintaining expression of the enzyme in vivo. Consistent with the decreased NO-synthesizing capacity of endothelial cells in individuals homozygous for the -786C/T-SNP (up to 15% of the Caucasian population), we could further demonstrate that this SNP is associated with a significantly increased risk to develop coronary artery disease as well as rheumatoid arthritis. Based on these findings, the proposed project has three objectives: (i) To identify factor X by using a combination of affinity chromatography and mass spectrometry analysis; (ii) to characterize the hitherto elusive shear stress-sensitive transcription factor and its binding motif by exploiting the fact that shear stress sensitivity of the nos-3 gene is mediated through the promoter sequence around the -786C/T SNP; and (iii) to set-up an animal experimental proof of concept model (cardiac transplant vasculopathy) to demonstrate efficacy of nucleic acid-based drugs, developed to block factor X or boost NOS-3 expression, for functional correction of the -786C/T SNP. Since this SNP does not seem to occur in other mammals, two knock in mouse strains harboring the human C-type and, for comparison, T-type promoter will be generated for this purpose.