A novel growth-friendly system alleviates pulmonary dysplasia in early-onset scoliosis combined with thoracic insufficiency syndrome: Radiological, pathological, and transcriptomic assessments

Abstract

Background: The posterior procedure utilizing growth-friendly techniques is the golden standard for patients with early-onset scoliosis combined with thoracic insufficiency syndrome (EOS + TIS). Pulmonary hypoplasia is the main cause of dying prematurely in the EOS + TIS. This study assessed the therapeutic impact of a novel growth-friendly system on the pulmonary development of piglet's EOS + TIS model.

Methods: The animal procedure period lasts 12 weeks, of which the construction of the EOS + TIS was performed at 0-8 weeks, and implantation of a novel growth-friendly system was applied at 8-12 weeks. During the animal procedure, X-rays and CT were performed to observe scoliosis, thorax, and lungs. After 12 weeks, pathological changes in lung tissue were assessed using HE and IHC staining. RNA-seq characterized novel growth-friendly system-associated differentially expressed genes (DEGs) and validated using RT-qPCR, western blotting, and IHC.

Results: Implantation of the novel growth-friendly system increased body weight, body length, and total lung volume, as well as decreased the coronal and sagittal Cobb angles for the EOS + TIS model. It also ameliorated EOS + TIS-induced thickening of the alveolar wall, increased alveolar spaces, and decreased alveolar number and diameter. In lung tissue, a total of 790 novel growth-friendly system-associated DEGs were identified, and they were mainly involved in the regulation of immune, inflammatory, calcium transport, and vascular development. Among these DEGs, BDKRB1, THBS1, DUSP1, IDO1, and SPINK5 were hub genes, and their differential expression was consistent with RNA-seq results in lung tissues.

Conclusion: The novel growth-friendly system has mitigated scoliosis and pulmonary hypoplasia in the EOS + TIS model. We further elucidate the molecular mechanisms underlying the amelioration of pulmonary hypoplasia.

Keywords: Early-onset scoliosis; Growing rod; Pathology; Pulmonary hypoplasia; Radiology; Thoracic insufficiency syndrome; Transcriptomics.

Fig. 1

Effect of novel growth-friendly system on scoliosis in the EOS + TIS. A: Flowchart of the animal procedures in this study. B: Overall image of the spine-tethering device and the novel growth-friendly system. B1: Gland screw; B2: Transversal connector; B3: Eye screw. Statistical analysis of piglet weights at 0th, 8th, 10th, and 12th weeks for Sham, Model, and Treatment groups (N = 5). D: Statistical analysis of changes in body length of piglets (N = 5). E: Representative X-ray images of coronal and sagittal planes and statistical analysis of changes in coronal Cobb angle (θs) and sagittal Cobb angle (θk) of piglets at the 0th, 8th, and 12th weeks, respectively (N = 5). Markers are θs and θk for each group of piglets. Compared to the Sham group, *P < 0.05; **P < 0.01; ***P < 0.001. Compared to the Model group, ^&P < 0.05; ^&&P < 0.01.

Fig. 2

Effect of novel growth-friendly system on total lung volume in the EOS + TIS. A: (Upper) Representative images of 3D CT reconstruction of the lungs and spine in the Sham, Model, and Treatment groups; (Bottom) Representative pictures of 3D CT reconstruction of the thorax and lungs at the T7-T10 level. B: Statistical analysis of total TLVs and TLVs at the T7-T10 level in all groups of piglets (N = 5). Compared to the Sham group, *P < 0.05; **P < 0.01; ***P < 0.001. Compared to the Model group, ^&P < 0.05; ^&&P < 0.01.

Fig. 3

Effect of novel growth-friendly system on lung histopathology in the EOS + TIS. A: HE staining was applied to observe the pathological changes of lung tissues in the Sham, Model, and Treatment groups. Scale bar: 500 μm. B: IHC staining was performed to detect the effect of the novel growth-friendly system on the expression of M1-type macrophage markers (CD68 and iNOS), M2-type macrophage markers (CD206), and angiogenesis-related proteins (VEGFA and VEGFR2) in lung tissues of the EOS + TIS model. Scale bar: 50 μm. C: Statistical analysis of changes in mean optical density values of CD68, iNOS, CD206, VEGFA, and VEGFR2 for lung tissues in each group (N = 5). Compared to the Sham group, *P < 0.05; **P < 0.01; ***P < 0.001. Compared to the Model group, ^&P < 0.05; ^&&P < 0.01.

Fig. 4

Identification of novel growth-friendly system-associated DEGs. A: Violin plots display the FPKM values after the normalization of mRNA expression profiles for each sample in the Model and Treatment groups. B: Cluster analysis characterizes the distance between the mRNA expression profiles of each sample. Darker colors mean shorter distances. C: The mRNA expression profiles of each sample were dimensionalized by principal component analysis (PCA) to characterize the variability. D: The volcano plot visualizes the expression of all genes in the Treatment group compared to the Model group. DEGs satisfy both |log₂FC|>1 and q < 0.05. E: Heatmap presents the clustering of mRNA expression profiles and the expression of DEGs for each sample. F: Radar graph visualizes the FC of DEGs with |log₂FC| TOP30. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

GO/KEGG enrichment analysis of novel growth-friendly system-associated DEGs. A: Bar plot visualizes the GO terms of the -log₁₀p-value TOP30 obtained by enrichment of the novel growth-friendly system-associated DEGs. B: The chord diagram exhibits the DEGs involved in the GO term for the -log10p-value TOP10. C: Bubble plot visualizes KEGG terms with enrichment score TOP20 obtained by enrichment of the novel growth-friendly system-associated DEGs. D: Enrichment of DEGs in KEGG terms with enrichment score TOP10.

Fig. 6

PPI networks for Novel growth-friendly system-related DEGs. A: Venn diagram visualizes the intersection of DEGs in Model vs Sham and Treatment vs Model. B: A PPI network of the novel growth-friendly system-related DEGs constructed based on the String website, Cytoscape software, and Network Analysis Tool. A larger circle means a larger Indegree and a redder color means a larger clustering coefficient. C: Indegree and clustering coefficient within the TOP30 DEGs in the PPI network of the novel growth-friendly system-related DEGs. D: Cytoscape software and MCODE APP were applied to cluster modules in the PPI network. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

Expression validation of Novel growth-friendly system-associated DEGs. A: RT-qPCR was conducted to detect the expression differences of DEGs in lung tissues of Sham, Model, and Treatment groups (N = 5). B: Representative images of gel blot for BDKRB1 and THBS1 proteins and statistical analysis in lung tissues of each group (N = 3). C: IHC staining was performed to analyze the mean optical density values of BDKRB1 and THBS1 proteins in lung tissues (N = 5). Scale bar: 50 μm. D: (Upper) Diagram of the calcium pathway involved in BDKRB1; (Bottom) Diagram of the TGF-β pathway involved in THBS1. E: Representative IHC images of calcium pathway-associated proteins (CaMKIIα/δ) and TGF-β pathway-associated proteins (TGF-β and p-Smad3) in lung tissues and their statistical analyses (N = 5). Scale bar: 50 μm. F: Effect of the novel growth-friendly system on the expression of CaMKIIα/δ, TGF-β, and p-Smad3 proteins in lung tissues of the EOS + TIS model was detected by western blotting (N = 3). The supplementary material provided the original images of gels and blots. *, **, *** represent P < 0.05, P < 0.01, and P < 0.001, respectively; ns represents no significant difference.