Impact of cGMP generated by NO-sensitive guanylyl cyclases on vascular smooth muscle remodelling

在血管系统中，NO/cGMP信号通路已经确立了调节平滑肌张力和抑制血小板聚集的功能，并预期NO/cGMP在导致血管重塑的平滑肌增殖中的作用。环状GMP是由形成cGMP的鸟苷酸环化酶(GC)在切应力激活的内皮一氧化氮合酶(NOS)激活的情况下产生的。NO敏感的GC存在两种亚型(NO-GC1和NO-GC2)，它们都存在于平滑肌细胞中，并介导血管松弛。我们产生了基因敲除(KO)小鼠，其中任何一种NO-GC亚型都被缺失。NO-Gc1异构体的缺失导致NO刺激的cGMP形成活性降低90%以上。然而，NO-GC1KO小鼠不会患上高血压。由于缺乏两种NO-GC亚型的完整KO显示出明显的血压升高，我们的结果表明，在生理条件下，只需要少量的cGMP来预防高血压。人们普遍认为，NO/cGMP途径的血管扩张作用被血管收缩因子血管紧张素II(Ang II)所抵消，血管紧张素II也可以诱导平滑肌细胞的增殖。在目前的项目中，将用血管紧张素转换酶抑制剂(Angii)治疗无GC1KO小鼠(2周和4周)，以了解cGMP形成能力的降低是否促进血管重构。血管紧张素转换酶抑制剂治疗后，血管重塑将通过血管结构的组织学检查(壁厚、管腔直径)和细胞过程的生化分析(细胞生长、死亡、迁移和细胞外基质的重组)进行评估。比较血管紧张素转换酶抑制剂诱导的WT和非GC1KO小鼠的变化，应该表明cGMP是否以及如何与促进增殖的网络相互作用。NO/cGMP在血管重塑中的作用也将在NO-GC1KO小鼠与低密度脂蛋白受体KO小鼠杂交并用高脂饮食挑战的动脉粥样硬化背景下进行评估。NO诱导的cGMP在血管重塑中的相关性将在进一步的小鼠模型中进行评估，即缺乏NO-GC异构体或对NO/cGMP信号的药物抑制。总之，这个项目将增加我们对cGMP在不同条件下对血管平滑肌细胞生长的作用的理解，并将在分子水平和(病理)生理背景下分析其与增殖相关过程的可能相互作用。

In the vascular system, the NO/cGMP signalling cascade has established functions in the regulation of smooth muscle tone and inhibition of platelet aggregation and a role of NO/cGMP in smooth muscle proliferation which results in vascular remodelling, is anticipated. Cyclic GMP is generated by the cGMP-forming guanylyl cyclases (GC) in response to NO formed by the endothelial NO synthase activated by shear stress. Two isoforms of the NO-sensitive GC exist (NO-GC1, NO-GC2) and both of them occur in smooth muscle cells and mediate vascular relaxation. We generated knockout (KO) mice in which either one of the NO-GC isoforms is deleted. The lack of the NO-GC1 isoform leads to a more than 90% reduction of NO-stimulated cGMP-forming activity. However, the NO-GC1 KO mice do not develop hypertension. As the complete KO lacking both NO-GC isoforms shows a pronounced increase of blood pressure, our results show that only a low amount of cGMP is required to prevent hypertension under physiological conditions. It is generally accepted that the vasodilatory effect of the NO/cGMP pathway is counter-balanced by the vasoconstrictor Angiotensin II (Ang II) that also induces proliferation of smooth muscle cells. In the current project, NO-GC1 KO mice will be treated with AngII (2 and 4 weeks) to find out whether the decreased cGMP-forming capacity promotes vascular remodelling. After AngII treatment, vascular remodelling will be assessed by histological inspection of vessel architecture (wall thickness, lumen diameter), and biochemical analysis of cellular processes (cell growth, death, migration and reorganization of extracellular matrix). The comparison of AngII-induced changes in WT and NO-GC1 KO mice should show whether and how cGMP interacts with networks promoting proliferation. The role of NO/cGMP in vascular remodelling will also be evaluated in the context of atherosclerosis in NO-GC1 KO mice crossbred with LDL receptor KO mice and challenged with a high fat diet. The relevance of NO-induced cGMP in vascular remodelling will be assessed in further mouse models, i.e. lack of both NO-GC isoforms or pharmacological inhibition of NO/cGMP signalling. In sum, this project will increase our understanding of cGMP's role in vascular smooth muscle cell growth under various conditions and will analyse its possible interplay with proliferation-related processes at the molecular level and in the (patho-)physiological context.