Hedgehog-interacting protein orchestrates alveologenesis and protects against bronchopulmonary dysplasia and emphysema - PubMed (original) (raw)
Hedgehog-interacting protein orchestrates alveologenesis and protects against bronchopulmonary dysplasia and emphysema
Datian Ye et al. Sci Adv. 2025.
Abstract
Most of the lung's gas-exchange surface forms during alveologenesis and its disruption causes bronchopulmonary dysplasia (BPD) in infants, characterized by alveolar simplification and myofibroblast accumulation. BPD also increases the risk of adult emphysema, marked by alveolar loss. Despite this connection, mechanisms linking these conditions and effective treatments are still lacking. We identify hedgehog-interacting protein (HHIP), associated with both BPD and emphysema, as a critical regulator of alveologenesis. During this process, _Hhip_-expressing cells expanded, accompanied by hedgehog (Hh) signaling inhibition and myofibroblast transition. Stromal-specific Hhip deletion led to hyperactivation of Hh-IGF1 signaling axis, causing persistent SMA+ myofibroblasts and epithelial stem/progenitor cell senescence. Hyperactivation of this pathway was also observed in human BPD and hyperoxia-induced BPD models. Early Hhip deficiency resulted in adult emphysema with myofibroblast accumulation. We developed a therapeutic Fc-fused HHIP protein that mitigated BPD in neonatal mice and prevented adult emphysema. These findings establish HHIP as a critical regulator of alveologenesis and a therapeutic target for BPD and emphysema.
Figures
Fig. 1.. 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. 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.. Hhip deletion causes BPD that can be attenuated by inhibiting IGF1 signaling.
(A) H&E images of the lungs of _Hhip_-deleted and control mice. (B to D) Quantification of MLI, mean alveolus size, and alveolar density of _Hhip_-deleted and control lungs. (E) H&E images of the lungs of _Hhip_-deleted mice treated with vehicle or IGF1R inhibitor. (F to H) Quantification of MLI, mean alveolus size, and alveolar density of _Hhip_-deleted mice treated with vehicle or IGF1R inhibitor. (I) IF analysis of SPC and p21 in the lungs of _Hhip_-deleted mice treated with vehicle or IGF1R inhibitor. Arrow: p21+ SPC+ cells. (J and K) Quantification of the number of AT2s and percentage of p21+ AT2s in _Hhip_-deleted mice treated with vehicle or IGF1R inhibitor. Each data point represents one mouse [(B) to (D), (F) to (H), and (K)] 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.
Fig. 6.. HHIP-Fc protein alleviates hyperoxia-induced BPD phenotypes.
(A) Design strategy for HHIP-Fc recombinant protein and its analysis by Western blotting. (B) qPCR analysis of Gli1 expression in the lung stromal cells isolated from neonatal mice 3 and 5 days after one dose of HHIP or HHIP-Fc treatment. (C) H&E images of hyperoxia-exposed lungs, treated with HHIP-Fc or Fc fragment control. (D) MLI quantification of hyperoxia-exposed lungs, treated with HHIP-Fc or Fc. (E and F) IF analysis and quantification of myofibroblasts in hyperoxia-exposed lungs, treated with HHIP-Fc or Fc. (G to I) IF analysis and quantification of AT2 number and p21+ AT2 percentage in hyperoxia-exposed lungs, treated with HHIP-Fc or Fc. Arrow: p21+ SPC+ cells. (J) qPCR analysis of Igf1 expression in the lung stromal cells isolated from hyperoxia-exposed mice, treated with HHIP-Fc or Fc. Each data point represents one mouse [(B), (D), (F), and (H) to (J)] of an individual experiment. Data are expressed as mean ± SD. *P < 0.05, **P < 0.005, ***P < 0.0005, and ****P < 0.0001.
Fig. 7.. HHIP-Fc administration alleviates emphysema in adult mice with Hhip haploinsufficiency.
(A and B) H&E images and MLI quantification of the lungs from adult HhipLacZ/+ and WT mice. (C) IF analysis of PDGFRα and SMA in the lungs of adult HhipLacZ/+ and WT mice. Arrow: SMA+ PDGFRα+ cells. (D and E) Quantification of the number and percentage of myofibroblasts in adult HhipLacZ/+ and WT mice. (F and G) IF analysis of SPC and quantification of AT2s in the lungs of adult HhipLacZ/+ and WT mice. (H and I) H&E images and MLI quantification of the lungs from adult HhipLacZ/+ and WT mice treated with HHIP-Fc or Fc. (J and K) IF analysis and quantification of myofibroblasts in the lungs of adult HhipLacZ/+ and WT mice treated with HHIP-Fc or Fc. Arrow: SMA+ cells. (L and M) IF analysis and quantification of AT2s in the lungs of adult HhipLacZ/+ and WT mice treated with HHIP-Fc or Fc. Each data point represents one mouse [(B), (D), (E), (G), (I), (K), and (M)] of an individual experiment. Data are expressed as mean ± SD. *P < 0.05, **P < 0.005, ***P < 0.0005, and ****P < 0.0001.
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