Department of Pediatrics, University of Rochester Medical Center, Rochester, New York, United States.
Department of Laboratory Medicine and Pathology, University of Washington, University of Washington, Seattle, Washington, United States.
Am J Physiol Lung Cell Mol Physiol. 2023 Oct 1;325(4):L419-L433. doi: 10.1152/ajplung.00130.2023. Epub 2023 Jul 25.
Bronchopulmonary dysplasia (BPD) is a disease of prematurity related to the arrest of normal lung development. The objective of this study was to better understand how proteome modulation and cell-type shifts are noted in BPD pathology. Pediatric human donors aged 1-3 yr were classified based on history of prematurity and histopathology consistent with "healed" BPD (hBPD, = 3) and "established" BPD (eBPD, = 3) compared with respective full-term born ( = 6) age-matched term controls. Proteins were quantified by tandem mass spectroscopy with selected Western blot validations. Multiplexed immunofluorescence (MxIF) microscopy was performed on lung sections to enumerate cell types. Protein abundances and MxIF cell frequencies were compared among groups using ANOVA. Cell type and ontology enrichment were performed using an in-house tool and/or EnrichR. Proteomics detected 5,746 unique proteins, 186 upregulated and 534 downregulated, in eBPD versus control with fewer proteins differentially abundant in hBPD as compared with age-matched term controls. Cell-type enrichment suggested a loss of alveolar type I, alveolar type II, endothelial/capillary, and lymphatics, and an increase in smooth muscle and fibroblasts consistent with MxIF. Histochemistry and Western analysis also supported predictions of upregulated ferroptosis in eBPD versus control. Finally, several extracellular matrix components mapping to angiogenesis signaling pathways were altered in eBPD. Despite clear parsing by protein abundance, comparative MxIF analysis confirms phenotypic variability in BPD. This work provides the first demonstration of tandem mass spectrometry and multiplexed molecular analysis of human lung tissue for critical elucidation of BPD trajectory-defining factors into early childhood. We provide new insights into the natural history of bronchopulmonary dysplasia in donor human lungs after the neonatal intensive care unit hospitalization. This study provides new insights into how the proteome and histopathology of BPD changes in early childhood, uncovering novel pathways for future study.
支气管肺发育不良（BPD）是一种与正常肺发育停滞有关的早产儿疾病。本研究旨在更好地了解在 BPD 病理中如何注意到蛋白质组调节和细胞类型转移。根据早产儿病史和组织病理学表现，将 1-3 岁的儿科人类供体分为“愈合”BPD（hBPD，n = 3）和“已建立”BPD（eBPD，n = 3），并与相应的足月出生（n = 6）年龄匹配的足月对照进行比较。通过串联质谱法对蛋白质进行定量，并进行了Western blot 验证。对肺切片进行多重免疫荧光（MxIF）显微镜检查，以计数细胞类型。使用 ANOVA 比较组间蛋白质丰度和 MxIF 细胞频率。使用内部工具和/或 EnrichR 进行细胞类型和本体富集。蛋白质组学检测到 eBPD 与对照相比有 5746 种独特的蛋白质上调，186 种下调，534 种下调，而 hBPD 与年龄匹配的足月对照相比，差异表达的蛋白质较少。细胞类型富集表明肺泡 I 型、肺泡 II 型、内皮/毛细血管和淋巴管减少，平滑肌和成纤维细胞增加，与 MxIF 一致。组织化学和 Western 分析也支持 eBPD 中与对照相比铁死亡上调的预测。最后，eBPD 中几个映射到血管生成信号通路的细胞外基质成分发生改变。尽管蛋白质丰度的比较明显，但比较 MxIF 分析证实了 BPD 中的表型变异性。这项工作首次展示了人类肺组织的串联质谱和多重分子分析，用于深入阐明 BPD 轨迹定义因素进入儿童早期。我们提供了关于新生儿重症监护病房住院后供体人类肺部支气管肺发育不良自然史的新见解。这项研究为进一步研究提供了新的见解，了解 BPD 的蛋白质组和组织病理学如何在儿童早期发生变化。
