Construction of neonatal pulmonary hypoperfusion model
We created a neonatal pulmonary hypoperfusion model by performing PAB surgery on P1 rats, as reported previously [9, 21, 22]. The color Doppler ultrasound showed an irregular colorful blood flow signal accompanied with a significant narrowing in the PAB group (Fig. 1A, arrow), indicating a high-speed turbulent flow at the banding site due to stenosis. Quantification data showed that PA-VTI, peak velocity, and PPG in the PAB group increased by 4.9-, 2.5-, and 6.9-folds compared to those in the sham group, respectively (Fig. 1A–D). These results confirmed the successful creation of PA stenosis.
Verification of neonatal pulmonary hypoperfusion model
We calculated the pulmonary perfusion through the left ventricular outflow tract (Fig. 2A), and found that the pulmonary blood flow was significantly lower in the PAB group than in the sham group (Fig. 2B) at P7. These results suggested that the neonatal pulmonary hypoperfusion model was successfully created.
Pulmonary hypoperfusion caused alveolar dysplasia
At P21, smaller lungs and lung volume were observed in the PAB group (Fig. 3A, B). At the tissue level, there was a significant decrease in the number of alveoli and a significant increase in Lm in the PAB compared to the sham groups (Fig. 3C–E), suggesting impaired alveolarization due to PAB. Furthermore, a lower blood vessel density was also observed in the PAB than in the sham groups (Fig. 3F, G), indicating impaired vascularization due to PAB. These results confirmed that pulmonary hypoperfusion leads to alveolar dysplasia [9].
Transcriptomics of postnatal alveolar development because of pulmonary hypoperfusion
To investigate how pulmonary hypoperfusion affects the gene expression during the AVL stage, we obtained lung tissues at P7 and P14 to perform RNA-seq analysis (n = 5, except for the P7 group, which contained four rats because one died before harvesting). As shown in the volcano map in Fig. 4A, during normal postnatal alveolar development, there were 7721 DEGs between the Sham_14 and Sham_7 groups, among which 3879 were upregulated and 3842 were downregulated. Under the influence of pulmonary hypoperfusion, this number decreased to 6011, of which 2991 were upregulated and 3020 were downregulated (Fig. 4B). The heat map revealed a high similarity between samples within the same group but apparent difference between samples from different groups (Fig. 4C), suggesting a high biological reproducibility of PAB surgery. The principal component analysis (PCA) plot further confirmed the high reproducibility of PAB surgery, and showed a different postnatal developmental track between normal and pulmonary hypoperfusion-influenced lungs (Fig. 4D). The differences between the PAB and sham groups were more significant at P7 than at P14 (Fig. 4D). These results demonstrated that the postnatal alveolar developmental track is changed by pulmonary hypoperfusion.
Biological processes (BP), cellular components (CC), and molecular functions (MF) of postnatal alveolar development changed by pulmonary hypoperfusion as indicated by GO analysis
We performed GO analysis on the DEGs to identify the changes in BP, CC, and MF during postnatal alveolar development due to pulmonary hypoperfusion.
During normal postnatal alveolar development, the top 10 most enriched GO terms of BP were regulation of response to stimulus, regulation of signal transduction, regulation of cell communication, regulation of signaling, small GTPase-mediated signal transduction, Rho protein signal transduction, regulation of Rho protein signal transduction, regulation of small GTPase-mediated signal transduction, DNA replication, and ras protein signal transduction (Fig. 5A, B). The top 10 most enriched GO terms of CC were kinetochore, chromosomal region, chromosome, centromeric region, intrinsic component of plasma membrane, bounding membrane of organelle, organelle subcompartment, myosin complex, nucleus, intrinsic component of organelle membrane, and integral component of organelle membrane (Fig. 5A, B). The top 10 most enriched GO terms of MF were Rho GTPase binding, GTPase binding, Ras GTPase binding, small GTPase binding, enzyme binding, guanyl-nucleotide exchange factor activity, Ras guanyl-nucleotide exchange factor activity, Rho guanyl-nucleotide exchange factor activity, extracellular matrix structural constituent, and growth factor binding (Fig. 5A, B). These results highlight the critical role of cell–cell communication and signal transduction during normal postnatal alveolar development, which is consistent with a previous publication [20].
Under the influence of pulmonary hypoperfusion, the top 10 most enriched GO terms of BP were DNA metabolic process, DNA replication, chromosome organization, DNA-dependent DNA replication, intracellular signal transduction, DNA replication initiation, DNA repair, organelle organization, response to stress, and cellular response to DNA damage stimulus (Fig. 5C, D). The top 10 most enriched GO terms of CC were nucleus, chromosome and centromeric region, chromosomal region, kinetochore, chromosome, nuclear part, chromosomal part, membrane-enclosed lumen, organelle lumen, and intracellular organelle lumen (Fig. 5C, D). The top 10 most enriched GO terms of MF were DNA-dependent ATPase activity, catalytic activity acting on DNA, DNA helicase activity, cysteine-type endopeptidase inhibitor activity, cytoskeletal protein binding, GTPase binding, coenzyme binding, hydrolase activity, acting on acid anhydrides, monooxygenase activity, and NADP binding (Fig. 5C, D). These results suggest a significant increase in cell cycle activity during pulmonary hypoperfusion-influenced postnatal alveolar development.
The aforementioned results suggest that cell–cell communication and signaling transduction during normal postnatal alveolar development were partly switched to cell cycle activity because of pulmonary hypoperfusion.
Pathways of postnatal alveolar developmental trajectory were changed by pulmonary hypoperfusion
KEGG pathway analysis was used to identify the pathways regulating alveolar development that were affected by pulmonary hypoperfusion. The results indicated that the top 20 enriched pathways during normal alveolar development were cell cycle, microRNAs in cancer, axon guidance, AGE-RAGE signaling pathway in diabetic complications, rap1 signaling pathway, proteoglycans in cancer, bacterial invasion of epithelial cells, leukocyte transendothelial migration, other types of O-glycan biosynthesis, platelet activation, p53 signaling pathway, focal adhesion, progesterone-mediated oocyte maturation, small cell lung cancer, MAPK signaling pathway, glycerolipid metabolism, valine, leucine and isoleucine degradation, amoebiasis, PI3K-Akt signaling pathway, and aminoacyl-tRNA biosynthesis (Fig. 6A, B). These results suggest that cell cycle and axon guidance are the primary pathways that regulate normal postnatal alveolar development, consistent with a previous publication [15].
Under the influence of pulmonary hypoperfusion, the top 20 enriched pathways were cell cycle, DNA replication, RNA transport, microRNAs in cancer, PI3K-Akt signaling pathway, cellular senescence, focal adhesion, progesterone-mediated oocyte maturation, mismatch repair, insulin resistance, ECM-receptor interaction, spliceosome, oocyte meiosis, human papillomavirus infection, Fanconi anemia pathway, pyrimidine metabolism, AGE-RAGE signaling pathway in diabetic complications, one carbon pool by folate, other types of O-glycan biosynthesis, and platelet activation (Fig. 6C, D). Notably, the − log10 (padj) of cell cycle was 7.27 and 3.59 in the PAB and sham groups, respectively, and axon guidance was not included in the list of enriched terms (Fig. 6A, C). These results suggest that pulmonary hypoperfusion led to overactivation of the cell cycle and absent axon guidance during postnatal alveolar development.
Verification of RNA-seq results by examination of cell cycle and axon guidance markers
To confirm the RNA-seq results, 10 randomly selected cell cycle and axon guidance associated genes were verified by qRT-PCR (Fig. 7A, B). The results showed that the expression levels of cell cycle-associated genes were significantly higher (Fig. 7A), while the expression levels of axon guidance-associated genes were significantly lower (Fig. 7B), in the PAB group compared to the sham group. These results suggest that cell cycle was promoted and axon guidance was inhibited during postnatal alveolar development due to pulmonary hypoperfusion.
Ki67, a cell cycle marker, was selected to confirm the RNA-seq results. As shown in Fig. 8A and B, the proportion of Ki67-positve cells was significantly increased in PAB group than in the sham group, indicating over-activated cell cycle because of pulmonary hypoperfusion. In contrast, the axon guidance markers SEMA3A and Nrp1 were lower in the PAB group than in the sham group (Fig. 8C–E), indicating inhibition of axon guidance caused by pulmonary hypoperfusion. To visualize semaphorin distribution and axon trajectories, we performed SEMA7a immunofluorescence staining, which showed that SEMA7a was located at bronchioles and downregulated under pulmonary hypoperfusion conditions (Fig. 8F, G). To assess cell–cell communication, we determined the expression of AT1 cells, a hub of cell–cell communication for postnatal alveolar development [19]. The results showed downregulation of AT1 cells under pulmonary hypoperfusion conditions (Fig. 8H, I).
Furthermore, we observed a significantly higher percentage of TUNEL-positive cells in the PAB group than in the sham group (Fig. 9A, B), suggesting higher apoptosis in the PAB group than the sham group because of pulmonary hypoperfusion.