Friedmacher Florian, Hofmann Alejandro Daniel, Takahashi Hiromizu, Takahashi Toshiaki, Gosemann Jan-Hendrik, Puri Prem
National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin 12, Ireland.
Pediatr Surg Int. 2014 Feb;30(2):129-35. doi: 10.1007/s00383-013-3444-z.
Pulmonary hypoplasia (PH), characterized by alveolar immaturity, remains the main cause of neonatal mortality and long-term morbidity in infants with congenital diaphragmatic hernia (CDH). Lipid-containing interstitial fibroblasts (LIFs) are critically important for normal alveolar development. Thymocyte antigen 1 (Thy-1) is a highly expressed cell-surface protein in this specific subset of lung fibroblasts, which plays a key role in fetal alveolarization by coordinating the differentiation and lipid homeostasis of alveolar LIFs. Thy-1 increases the lipid content of LIFs by upregulation of adipocyte differentiation-related protein (ADRP), a lipogenic molecular marker characterizing pulmonary LIFs. Thy-1 (-/-) mice further show impaired alveolar development with reduced proliferation of pulmonary LIFs, resulting in a PH-similar phenotype. We hypothesized that pulmonary Thy-1 signaling is disrupted in experimentally induced CDH, which may has an adverse effect on the lipid content of alveolar LIFs.
Timed-pregnant Sprague-Dawley rats were treated with either 100 mg nitrofen or vehicle on embryonic day 9.5 (E9.5). Fetuses were killed on E21.5, and lungs were divided into controls (n = 14) and CDH-associated PH (n = 14). Pulmonary gene expression levels of Thy-1 and ADRP were assessed by quantitative real-time PCR. ADRP immunohistochemistry and oil-red-O staining were used to localize alveolar LIF expression and lipid droplets. Immunofluorescence double staining for Thy-1 and oil-red-O was performed to evaluate Thy-1 expression and lipid content in alveolar LIFs.
Radial alveolar count was significantly reduced in CDH-associated PH with significant downregulation of pulmonary Thy-1 and ADRP mRNA expression compared to controls. ADRP immunoreactivity and lipid droplets were markedly diminished in alveolar interstitial cells, which coincided with decreased alveolar LIF expression in CDH-associated PH compared to controls. Confocal laser scanning microscopy confirmed markedly decreased Thy-1 expression and lipid content in alveolar LIFs of CDH-associated PH compared to controls.
Our study provides strong evidence that disruption of pulmonary Thy-1 signaling results in reduced lipid droplets in alveolar LIFs and may thus contribute to PH in the nitrofen-induced CDH model. Treatment modalities aimed at increasing lipid content in alveolar LIFs may therefore have a therapeutic potential in attenuating CDH-associated PH.
以肺泡发育不成熟为特征的肺发育不全（PH）仍是先天性膈疝（CDH）患儿新生儿死亡和长期发病的主要原因。含脂质的间质成纤维细胞（LIFs）对正常肺泡发育至关重要。胸腺细胞抗原1（Thy-1）是肺成纤维细胞这一特定亚群中高表达的细胞表面蛋白，通过协调肺泡LIFs的分化和脂质稳态在胎儿肺泡化过程中起关键作用。Thy-1通过上调脂肪细胞分化相关蛋白（ADRP）增加LIFs的脂质含量，ADRP是一种表征肺LIFs的生脂分子标志物。Thy-1（-/-）小鼠进一步表现出肺泡发育受损，肺LIFs增殖减少，导致类似PH的表型。我们推测，在实验性诱导的CDH中肺Thy-1信号通路被破坏，这可能对肺泡LIFs的脂质含量产生不利影响。
在胚胎第9.5天（E9.5）给定时怀孕的Sprague-Dawley大鼠注射100mg硝基芬或赋形剂。在E21.5处死胎儿，将肺分为对照组（n = 14）和与CDH相关的PH组（n = 14）。通过定量实时PCR评估Thy-1和ADRP的肺基因表达水平。采用ADRP免疫组织化学和油红O染色定位肺泡LIFs表达和脂滴。进行Thy-1和油红O免疫荧光双重染色以评估肺泡LIFs中Thy-1表达和脂质含量。
与对照组相比，与CDH相关的PH组的放射状肺泡计数显著降低，肺Thy-1和ADRP mRNA表达显著下调。与对照组相比，肺泡间质细胞中ADRP免疫反应性和脂滴明显减少，这与CDH相关的PH组肺泡LIFs表达降低一致。共聚焦激光扫描显微镜证实，与对照组相比，CDH相关的PH组肺泡LIFs中Thy-1表达和脂质含量明显降低。
我们的研究提供了强有力的证据，表明肺Thy-1信号通路的破坏导致肺泡LIFs中脂滴减少，因此可能导致硝基芬诱导的CDH模型中的PH。因此，旨在增加肺泡LIFs脂质含量的治疗方式可能对减轻CDH相关的PH具有治疗潜力。
