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，婴儿肺部并发症的治疗主要是对症治疗，而且不具有特异性，而且有明显的不良副作用。这种情况突出了迫切需要开发新的治疗方法，旨在刺激肺成熟。肺间质来源的旁分泌细胞信号刺激肺泡功能，如表面活性剂的合成，但影响因素在很大程度上是未知的。在我们之前的工作中，我们确定了肺成纤维细胞衍生条件培养基（LF-CM）的一种新功能，即刺激上皮钠转运和胎儿肺泡细胞中ENaC基因表达。载体钠转运是肺上皮细胞的一项主要功能，可促进出生时肺液的吸收，从而实现围产期肺转运，这在早产儿中受损，LF-CM可能会减轻这一功能。因此，在提议的项目中，我们的目标是确定刺激钠运输的因素。实验设置将从使用四维分离方案的高效液相色谱进行因子结合和分离开始，然后进行质谱因子鉴定和合成化合物的确认。以每个获得的LF-CM片段作为生物学读数，检测并确认ENaC mRNA表达的响应。此外，计划建立一个更快，更可靠的读数，例如康宁Epic系统，以确认生物活性。第二种策略涉及rna测序，比较反应细胞和非反应细胞，以确定LF-CM的差异调节途径。通过这些策略，我们期望发现能够刺激肺成熟的新型内源性间充质衍生因子，并/或通过确定其他先前未知的对肺上皮的影响来扩展对已知因子的了解。此外，我们希望表征相关途径，以确定改善早产儿围产期肺过渡的新策略。确定这些刺激间充质衍生的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迫切需要的新疗法，优化效果，减少副作用，从而改善早产儿的长期健康。此外，这些结果也可能为成人肺衰竭开辟新的治疗方法，包括COVID-19肺炎，这也显示了肺泡液清除的极端距离。新生儿研究小组和生物分析化学研究所的共同努力，与分子生物化学研究所和马克斯普朗克研究所<s:1>心肺研究所在巴德瑙海姆合作，将确保项目在拟议的时间框架内成功执行。

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.