Role of cGMP signalling in pericytes during lung fibrosis

研究NO/cGMP介导的细胞收缩、生长和存活等多种功能的NO/cGMP信号转导机制，已被证明是研究NO/cGMP在多种细胞功能中的良好模型。在过去三年工作的基础上，我们现在将集中讨论肺生理学和病理生理学，特别是纤维化。虽然NO-GC首先从大鼠和牛肺中分离出来，但到目前为止还没有鉴定出表达高水平该酶的细胞类型。在过去的资助期间，我们发现NO-GC在肺周细胞中高表达。周细胞是在毛细血管前小动脉、毛细血管和毛细血管后小静脉中发现的壁细胞。周细胞中NO-GC的鉴定在临床上很有趣，因为这些细胞几乎在所有的器官和组织中都能找到。周细胞的确切功能仍在调查中。基于周细胞收缩和松弛的能力，已假定周细胞调节毛细血管直径，因此可能对血压调节起作用。周细胞也被认为参与了肺纤维化的发展。有趣的是，最近在我们的全球KO小鼠身上证实了NO-GC在纤维化过程中的作用。我们已经获得了一株在Foxd1启动子控制下在周细胞前体细胞中表达Cre重组酶的小鼠品系。在谱系追踪实验中，Foxd1表达的细胞被证明发育成肺周细胞，在博莱霉素诱导的肺纤维化过程中，细胞外基质沉积和瘢痕形成具有重要意义。Foxd1-Cre小鼠目前正在与我们的无GC小鼠杂交。利用这些动物，我们将阐明NO-GC在博莱霉素诱导的肺纤维化发展中的作用。这将包括1)分离和培养周细胞，以鉴定NO/cGMP级联的成员以及收缩/松弛研究；2)博莱霉素在我们的细胞特异性KO菌株中诱导肺纤维化，包括作为预防/缓解纤维化过程的新的NO-GC激活剂和刺激剂的临床相关性评估；以及3)NO-GC对香烟烟雾暴露动物与肺纤维化的作用。此外，我们将试图剖析哪种NO-GC亚型(NO-GC1或NO-GC2)在疾病中具有主要影响。在与本研究单位其他小组的合作下，这些数据将使用缺乏cGKI和BK通道的小鼠进行补充。此外，我们将使用在Foxd1来源的周细胞中表达cGMP生物传感器的转基因小鼠来监测疾病发展过程中培养和全肺中cGMP的动力学。总之，我们的项目将提供关于cGMP在一种迄今被忽视的细胞类型--周细胞中的功能的有价值的信息，并将阐明NO-GC在肺纤维化中的作用。

Mice lacking NO-sensitive guanylyl cyclase (NO-GC) globally or in specific cell types have been proven to be excellent models to study NO/cGMP-mediated signalling in many cellular functions including contraction, growth and survival. Based on our work in the previous three years, we will now concentrate on lung physiology and pathophysiology with a special focus on fibrosis. Although NO-GC has been first isolated from rat and bovine lung, the cell type that expresses high levels of the enzyme has not been identified to date. In the last funding period, we have found NO-GC to be highly expressed in lung pericytes. Pericytes are mural cells found in precapillary arterioles, capillaries and postcapillary venules. The identification of NO-GC in pericytes is clinically intriguing as these cells are found in virtually all organs and tissues. The exact function of pericytes is still under investigation. Based on their ability to contract and relax, pericytes have been postulated to regulate capillary diameter and thus possibly contribute to blood pressure regulation. Pericytes are also thought to be involved in the development of lung fibrosis. Interestingly, a role of NO-GC in fibrotic processes has been demonstrated recently using our global KO mice. We have acquired a mouse strain that expresses Cre recombinase in pericyte precursor cells under the control of the Foxd1 promotor. In lineage tracing experiments, Foxd1-expressing cells have been shown to develop into lung pericytes which contribute significantly to extracellular matrix deposition and scarring during bleomycin-induced lung fibrosis. Foxd1-Cre mice are currently being crossed with our floxed NO-GC mice. Using these animals, we will elucidate the role of NO-GC in the development of bleomycin-induced lung fibrosis. This will include 1) the isolation and culture of pericytes for identification of members of the NO/cGMP cascade as well as contraction/relaxation studies, 2) bleomycin induction of lung fibrosis in our cell-specific KO strains, including the clinically relevant evaluation of novel NO-GC activators and stimulators as agents to prevent/relieve fibrotic processes and 3) the effect of NO-GC on cigarette smoke-exposed animals in conjunction with lung fibrosis. In addition, we will try to dissect which of the NO-GC isoforms (NO-GC1 or NO-GC2) has the major impact in the disease. In collaboration with other groups of this research unit, these data will be complemented using mice lacking cGKI and BK channel. Furthermore, we will use transgenic mice expressing a cGMP biosensor in Foxd1-derived pericytes to monitor cGMP kinetics in culture and in whole lung during disease development. In sum, our project will provide valuable information on the function of cGMP in a so far neglected cell type, the pericyte, and will shed light on the role of NO-GC during lung fibrosis.