Identifying a paracrine fibroblast-derived factor that stimulates fetal alveolar cells, thus potentially enhancing perinatal lung transition.

早产儿是最大的儿科患者群体，因此是一个巨大的社会挑战。这些婴儿的肺不成熟通常与呼吸窘迫相关，导致显著的发病率和死亡率。目前，对患有肺部并发症的婴儿的治疗主要是对症的，并且相当不特异，具有显著的不良副作用。这种情况突出了开发旨在刺激肺成熟的新治疗方法的迫切需要。肺间充质来源的旁分泌细胞信号刺激肺泡功能，如表面活性物质的合成，但其影响因素在很大程度上是未知的。在我们以前的工作中，我们确定了一个新的功能，肺成纤维细胞衍生的条件培养基（LF-CM），刺激上皮钠转运和ENaC基因表达在胎儿肺泡细胞。矢量钠转运，肺上皮细胞的主要功能，使肺液体吸收在出生时，从而围产期肺过渡，这是早产儿受损，并可能减轻LF-CM。因此，在拟议的项目中，我们的目标是确定负责刺激钠转运的因素。实验设置将从因子结合和使用4维分离方案的HPLC分离开始，然后是质谱因子鉴定和合成化合物确认。测试ENaC mRNA表达的响应，并用每个获得的LF-CM级分作为生物学读数进行确认。此外，计划建立更快、更稳健的读数，例如Corning Epic系统，以确认生物活性。第二种策略涉及RNA测序，比较应答细胞和非应答细胞，以鉴定LF-CM差异调节的途径。有了这些策略，我们希望确定新的内源性间充质源性因子能够刺激肺成熟，和/或通过确定额外的以前未知的肺上皮细胞的影响，扩大有关已知的因素的知识。 此外，我们希望描述所涉及的途径，以确定新的策略，以改善早产儿围产期肺转换。鉴定这些刺激间充质源性旁分泌成分将使我们小组和其他地方的临床科学家能够开发出急需的具有优化效果和减少副作用的新疗法，从而改善早产儿的长期健康。此外，这些结果还可能为成人肺衰竭开辟新的治疗方法，包括COVID-19肺炎，它也显示出肺泡液体清除的极端紊乱。新生儿研究小组和生物分析化学研究所与分子生物化学研究所和巴德瑙海姆的马克斯·普朗克心肺研究所合作，共同努力将确保在拟议的时间范围内成功执行该项目。

Preterm infants represent the largest paediatric patient group and thus a large societal challenge. Lung immaturity in these infants is frequently associated with respiratory distress leading to significant morbidity and mortality. Currently, the treatment of infants with pulmonary complications is mainly symptomatic and rather unspecific with salient unwanted side effects. This situation highlights the urgent need to develop new therapeutic approaches aiming at a stimulation of lung maturation. Lung mesenchymal-derived paracrine cell signals stimulate alveolar functions like surfactant synthesis, but the contributing factors are largely unknown. In our previous work we determined a novel function of lung fibroblast-derived conditioned medium (LF-CM), the stimulation of epithelial sodium transport and ENaC gene expression in fetal alveolar cells. Vectorial sodium transport, a major function of lung epithelial cells, enables lung fluid absorption at birth and thus perinatal lung transition, which is impaired in preterm infants and possibly alleviated by LF-CM. In the proposed project our aim therefore is to identify the factor(s) responsible for the stimulation of sodium transport. The experimental setup will start with factor binding and separation by HPLC using a 4-dimensional separation scheme, followed by mass spectrometric factor identification and confirmation with synthetic compounds. The response of ENaC mRNA expression is tested and confirmed with every obtained LF-CM fraction as biological readout. Furthermore, it is planned to establish a faster, more robust readout, e.g. the Corning Epic System, to confirm the biological activity. The second strategy involves RNA-Sequencing comparing responder and non-responder cells to identify differentially regulated pathways by LF-CM. With these strategies, we expect to identify novel endogenous mesenchymal-derived factors able to stimulate lung maturation, and/or expand the knowledge about already known factors by determining additional previously unknown effects on lung epithelia. Furthermore, we want to characterize the involved pathways to identify new strategies to improve perinatal lung transition in preterm infants. Identifying these stimulating mesenchymal-derived paracrine components will enable clinical scientists in our group and elsewhere to develop urgently needed novel therapies with optimized effects and reduced side effects, thus improving the long term health of premature infants. Furthermore, these results may also open new therapeutic approaches for adult lung failure, including COVID-19 pneumonia, which also shows extreme distubances of alveolar fluid clearance. The joint effort of the Neonatology research group and the Institute of Bioanalytical Chemistry in cooperation with the Institute of Molecular Biochemistry and the Max Planck Institute für Heart- and Lung Research in Bad Nauheim will ensure successful execution of the project within the proposed time frame.