Fig 2: Hhip deletion induces the persistence of myofibroblasts.(A) IF analysis of SMA in the alveoli of Hhip-deleted (HHIP CKO) and control mice at P14. (B) Number of myofibroblasts per unit alveolar area of Hhip-deleted and control mice. (C and D) UMAP showing cell clusters in the lung fibroblasts of Hhip-deleted and control mice at P14. (E) Violin plots showing the expression of Pdgfra, Acta2, Hhip, and Cdh4 in alveolar myofibroblasts (ALMF), ductal myofibroblasts (DMF), peribronchial fibroblasts (Perib), adventitial fibroblasts (Adv), and alveolar fibroblasts (Alv). (F) Expression of Acta2, Myh11, Tagln, and Igf1 in ALMFs and DMFs of Hhip-deleted and control mice. (G) qPCR analysis of Igf1 expression in the lung stromal cells isolated from Hhip-deleted and control mice. (H) Analysis of Igf1 (RNA in situ) and SMA expression in the alveoli. (I) Number of SMA+ Igf1+ cells per unit alveolar area of Hhip-deleted and control mice at P14. (J) Percentage of SMA+ Igf1+ cells in total Igf1+ cells. (K) Top 10 activated pathways in Hhip-deleted, relative to control myofibroblasts, analyzed with IPA. (L) qPCR analysis of Gli1, Igf1, and Acta2 expression in the lung stromal cells treated with PBS, SHH, and SHH plus HHIP. (M) qPCR analysis of Acta2 expression in SHH-stimulated lung stromal cells treated with vehicle or IGF1R inhibitor. (N) IF analysis of SMA expression in the alveoli of Hhip-deleted mice administered with vehicle or IGF1R inhibitor. (O) Number of myofibroblasts per unit alveolar area of Hhip-deleted mice administered with vehicle or IGF1R inhibitor. All in vitro experiments have been repeated at least one time with consistent results for validation. Each data point represents one mouse [(B), (G), (I), (J), and (O)] of an individual experiment. Data are expressed as mean ± SD. *P < 0.05, **P < 0.005, ***P < 0.0005, and ****P < 0.0001.

Fig 3: Deletion of Hhip induces cell senescence.(A) Violin plots showing the expression of Cdkn1a in ALMFs and DMFs of Hhip-deleted and control mice. (B) Senescence β-galactosidase staining of Hhip-deleted and control lungs. (C) IF analysis of SMA, p21, and CDH4 in Hhip-deleted and control lungs. Arrow: p21+ DMFs; arrowhead: p21+ ALMFs. (D and E) Number of p21+ ALMFs (D) and DMFs (E) per unit alveolar area of Hhip-deleted and control mice at P14. (F) IF analysis of SPC and p21 in Hhip-deleted and control lungs. Arrow: p21+ SPC+ cells. (G) Percentage of p21+ cells in AT2s. (H) Activation of senescence pathways in the AT2s of Hhip-deleted mice, relative to control AT2s, and analyzed with IPA. (I) Top 5 upstream regulators in the AT2s of Hhip-deleted mice, relative to control AT2s, analyzed with IPA. (J) AT2 organoids cocultured with lung stromal cells (R26RSmoM2/+) pre-infected with adenovirus-empty and adenovirus-Cre, treated with anti-IGF1 antibody and IgG. (K and L) Quantification of colony-forming efficiency (CFE) and organoid size. (M) IF analysis of SPC and p21 in AT2 organoids. Arrow: p21+ SPC+ cells. (N) Percentage of p21+ cells in AT2s in the organoid assay. All in vitro experiments have been repeated at least one time with consistent results for validation. Each data point represents one mouse [(D), (E), and (G)] of an individual experiment. Data are expressed as Mean ± SD. *P < 0.05, **P < 0.005, ***P < 0.0005, and ****P < 0.0001.

Fig 4: Expansion of HHIP+ cells, Hh inhibition, and myofibroblast transition during alveologenesis.(A) Quantification of SMA+ and HHIP+ cells in the alveoli during alveologenesis. (B) Immunofluorescence (IF) analysis of SMA-lineage cells (staining tdT) and SMA in the alveoli of Acta2DreER/+:R26RRSR-tdT/+ mice at P7 and P14. (C) Histology quantification of the percentage of SMA+ myofibroblasts in total SMA-lineage cells. (D) IF analysis of HHIP, PDGFRα, and SMA expression in the alveoli. (E) Histology quantification of the percentage of myofibroblasts in HHIP+ cells. (F) X-gal staining of Gli1lacZ/+ reporter demonstrates Gli1 expression in the alveoli of P7 and P14 mice. (G) qPCR analysis of Hhip and Gli1 expression in the lung stromal cells of P7 and P14 mice. (H) IF analysis of GLI1-lineage cells (staining tdT) and SMA in the alveoli of Gli1CreER/+:R26RtdT/+mice. (I) Histology quantification of the percentage of myofibroblasts in total GLI1-lineage cells. Each data point represents one mouse [(A), (C), (E), (G), and (I)] of an individual experiment. Data are expressed as mean ± SD. *P < 0.05, **P < 0.005, and ****P < 0.0001.

Fig 5: Overactivation of Hh-IGF1 signaling in BPD.(A) snRNA-seq analysis of GLI1, PATCH1, IGF1, and ACTA2 expression in ALMFs and DMFs of human BPD. (B) H&E images of the lungs of hyperoxia-treated and control mice. (C) Quantification of MLI of hyperoxia-treated and control lungs. (D) qPCR analysis of Hhip, Gli1, and Igf1 expression in the lung stromal cells isolated from hyperoxia-treated and control mice. (E) IF analysis of SMA in the lungs of hyperoxia-treated and control mice. (F) Histology quantification of the number of myofibroblasts of hyperoxia-treated and control mice. (G) qPCR analysis of Acta2 expression in the lung stromal cells isolated from hyperoxia-treated and control mice. (H) qPCR analysis of Cdkn1a expression in the lung epithelial cells isolated from hyperoxia-treated and control mice. (I) IF analysis of p21 and SPC in the lungs of hyperoxia-treated and control mice. Arrow: p21+ SPC+ cells. (J and K) Quantification of the percentage of p21+ AT2s and number of AT2s in hyperoxia-treated and control mice. Each data point represents one mouse [(C), (D), (F) to (H), (J), and (K)] of an individual experiment. Data are expressed as mean ± SD. *P < 0.05, **P < 0.005, and ****P < 0.0001.