Molecular insights using spatial transcriptomics of the distal lung in congenital diaphragmatic hernia

Abstract

Abnormal pulmonary vascular development and function in congenital diaphragmatic hernia (CDH) is a significant factor leading to pulmonary hypertension. The lung is a very heterogenous organ and has marked cellular diversity that is differentially responsive to injury and therapeutic agents. Spatial transcriptomics provides the unmatched capability of discerning the differences in the transcriptional signature of these distinct cell subpopulations in the lung with regional specificity. We hypothesized that the distal lung parenchyma (selected as a region of interest) would show a distinct transcriptomic profile in the CDH lung compared with control (normal lung). We subjected lung sections obtained from male and female CDH and control neonates to spatial transcriptomics using the Nanostring GeoMx platform. Spatial transcriptomic analysis of the human CDH and control lung revealed key differences in the gene expression signature. Increased expression of alveolar epithelial-related genes (SFTPA1 and SFTPC) and angiogenesis-related genes (EPAS1 and FHL1) was seen in control lungs compared with CDH lungs. Response to vitamin A was enriched in the control lungs as opposed to abnormality of the coagulation cascade and TNF-alpha signaling via NF-kappa B in the CDH lung parenchyma. In male patients with CDH, higher expression of COL1A1 (ECM remodeling) and CD163 was seen. Increased type 2 alveolar epithelial cells (AT-2) and arterial and lung capillary endothelial cells were seen in control lung samples compared with CDH lung samples. To the best of our knowledge, this is the first use of spatial transcriptomics in patients with CDH that identifies the contribution of different lung cellular subpopulations in CDH pathophysiology and highlights sex-specific differences.NEW & NOTEWORTHY This is the first use of spatial transcriptomics in patients with congenital diaphragmatic hernia (CDH) that identifies the contribution of different lung cellular subpopulations in CDH pathophysiology and highlights sex-specific differences.

Keywords: congenital diaphragmatic hernia; pulmonary hypertension; spatial transcriptomics.

Graphical abstract

Figure 1.

Histopathological characterization of the CDH lungs: representative H&E stained lung tissue sections from CDH (A) and control lungs (B). A: lung section from patient with CDH showing severe pulmonary hypoplasia with airways (*) seen approaching pleural surface and markedly decreased radial alveolar count of 1–2 (double-headed arrows, H&E, ×100). B: lung section from control patient showing appropriately developed pulmonary parenchyma with radial alveolar count of 4–5 (double arrowheads; H&E, ×40). Representative low (C)- and high (D)-magnification lung section stained with a nuclear marker (SYTO 13), PanCK (Pancytokeratin; epithelial marker), and CD45 (immune cell marker) with demarcation of the region of interest for the selection of the distal lung parenchyma. CDH, congenital diaphragmatic hernia; H&E, hematoxylin-eosin.

Figure 2.

Spatial transcriptomic analysis of the human CDH and control lung reveals key differences in the gene expression signature. Spatial transcriptomic analysis of lung sections obtained from patients with CDH (n = 4/sex/group) were compared with age- and sex-matched controls. A: volcano plots showing differentially expressed genes (DEGs) in control vs. patients with CDH. DEGs upregulated in controls are shown on the left (negative) and genes upregulated in CDH are on the right (positive). Gray dot: NS, orange: P < 0.05, green: FDR < 0.05. B: violin plots showing normalized expression values (counts) of selected genes; epithelial (SFTPA1, SFTPA2, SFTPC, and CD74) and endothelial (EPAS1 and CLDN5) in CDH and control lungs. C: enriched biological pathways in the CDH vs. control lung. CDH, congenital diaphragmatic hernia; FDR, false discovery rate; NS, not significant.

Figure 3.

Marked sex-specific differences in differentially expressed genes and enriched biological pathways in male and female CDH in lung parenchymal regions analyzed by spatial transcriptomics. Spatial transcriptomic analysis of lung sections obtained from male and female patients with CDH (n = 4/sex/group) were compared with age- and sex-matched controls. Volcano plots showing differentially expressed genes (DEGs) in male control vs. patients with CDH (A) and female control vs. patients with CDH (B). DEGs upregulated in controls are shown on the left (negative) and genes upregulated in CDH are on the right (positive). Gray dot: NS, orange: P < 0.05, green: FDR < 0.05. C: violin plots showing normalized expression values (counts) of selected genes; in male and female CDH and control lungs. D: four-way Venn diagram showing minimal overlap between males and females in differentially expressed genes. E: enriched biological pathways in the male and female CDH lung compared with sex-matched control lungs. F: four-way Venn diagram showing very little overlap between males and females in enriched biological pathways. CDH, congenital diaphragmatic hernia; FDR, false discovery rate.

Figure 4.

Quantitative differences in cell type abundance using spatial deconvolution in the human CDH lung reveals involvement of alveolar epithelial and pulmonary capillary endothelial cells. Spatial deconvolution analysis of lung sections obtained from patients with CDH (n = 4/sex/group) were compared with age- and sex-matched controls. Violin plots showing cell type abundance in control vs. patients with CDH and male control vs. patients with CDH. CDH, congenital diaphragmatic hernia.