Role of cGMP signalling in pericytes during lung fibrosis

小鼠缺乏NO-敏感鸟苷酸环化酶（NO-GC）的全球或在特定的细胞类型已被证明是很好的模型，研究NO/cGMP介导的信号转导在许多细胞功能，包括收缩，生长和生存。基于我们过去三年的工作，我们现在将集中于肺生理学和病理生理学，特别关注纤维化。虽然NO-GC已首先从大鼠和牛肺中分离出来，但迄今为止尚未鉴定出表达高水平酶的细胞类型。在上一个资助期间，我们发现NO-GC在肺周细胞中高度表达。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的鉴定在临床上是有趣的，因为这些细胞几乎在所有器官和组织中发现。周细胞的确切功能仍在研究中。基于其收缩和舒张的能力，周细胞被假定为调节毛细血管直径，从而可能有助于血压调节。周细胞也被认为参与肺纤维化的发展。有趣的是，NO-GC在纤维化过程中的作用最近已经使用我们的全球KO小鼠得到证实。我们已经获得了一个小鼠品系，表达Cre重组酶在周细胞前体细胞的Foxd 1启动子的控制下。在谱系追踪实验中，已显示Foxd 1表达细胞发育成肺周细胞，其在博来霉素诱导的肺纤维化期间显著促进细胞外基质沉积和瘢痕形成。Foxd 1-Cre小鼠目前正在与我们的floxed NO-GC小鼠杂交。利用这些动物，我们将阐明NO-GC在博来霉素诱导的肺纤维化发展中的作用。这将包括1）用于鉴定NO/cGMP级联的成员的周细胞的分离和培养以及收缩/舒张研究，2）在我们的细胞特异性KO菌株中肺纤维化的博来霉素诱导，包括新的NO-GC活化剂和刺激剂作为预防/减轻纤维化过程的药剂的临床相关评价和3）NO-GC对香烟烟雾的作用-暴露的动物与肺纤维化有关。此外，我们将尝试剖析NO-GC亚型（NO-GC 1或NO-GC 2）中的哪一种在疾病中具有主要影响。与该研究单位的其他小组合作，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Foxd 1衍生的周细胞中表达cGMP生物传感器的转基因小鼠来监测疾病发展过程中培养物和整个肺中的cGMP动力学。总之，我们的项目将提供有价值的信息cGMP的功能，在一个迄今为止被忽视的细胞类型，周细胞，并将阐明在肺纤维化过程中的作用NO-GC。

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.