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

在血管系统中，NO/cGMP信号级联在调节平滑肌张力和抑制血小板聚集中具有既定功能，并且预期NO/cGMP在平滑肌增殖中的作用导致血管重塑。环GMP由cGMP形成鸟苷酸环化酶（GC）响应于由剪切应力激活的内皮NO合酶形成的NO而产生。NO敏感GC有两种亚型（NO-GC 1，NO-GC 2），它们都存在于平滑肌细胞中并介导血管舒张。我们产生了敲除（KO）小鼠，其中任一种NO-GC亚型被删除。NO-GC 1亚型的缺乏导致NO刺激的cGMP形成活性降低90%以上。然而，NO-GC 1 KO小鼠不发生高血压。由于缺乏两种NO-GC亚型的完整KO显示血压显著升高，因此我们的结果表明，在生理条件下仅需要少量cGMP来预防高血压。一般认为，NO/cGMP途径的血管舒张作用被也诱导平滑肌细胞增殖的血管收缩剂血管紧张素II（Ang II）抵消。在目前的项目中，NO-GC 1 KO小鼠将接受AngII治疗（2周和4周），以确定cGMP形成能力的降低是否会促进血管重塑。AngII治疗后，将通过血管结构（壁厚、管腔直径）的组织学检查和细胞过程（细胞生长、死亡、迁移和细胞外基质重组）的生化分析评估血管重塑。WT和NO-GC 1 KO小鼠中AngII诱导的变化的比较应显示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与LDL受体KO小鼠杂交并用高脂肪饮食攻击的NO-GC 1 KO小鼠中的动脉粥样硬化的背景下进行评价。将在进一步的小鼠模型中评估NO诱导的cGMP在血管重塑中的相关性，即缺乏NO-GC同种型或NO/cGMP信号传导的药理学抑制。总之，该项目将增加我们对cGMP在各种条件下血管平滑肌细胞生长中的作用的理解，并将分析其在分子水平和（病理）生理背景下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.