Hedgehog-interacting protein orchestrates alveologenesis and protects against BPD and emphysema by modulating hedgehog-IGF1 signaling axis

A majority of the gas-exchange surface is generated during alveologenesis, and disruption of this process causes bronchopulmonary dysplasia (BPD) in preterm infants, characterized by alveolar simplification. BPD is a significant risk factor for adult emphysema, marked by alveolar loss. A comprehensive molecular understanding of alveologenesis and effective treatments for BPD and emphysema are still lacking. Here, we show that the cells expressing Hhip, a gene associated with both BPD and emphysema, expand within the alveoli during alveologenesis, followed by hedgehog (Hh) inhibition and myofibroblast transition. Stromal-specific deletion of Hhip leads to aberrant persistence of SMA+ alveolar myofibroblasts, mediated by a hyperactive Hh-IGF1 axis. Conditional knockout animal and 3D stroma-stem cell organoid models demonstrate that loss of Hhip induces alveolar stem/progenitor cell senescence in a non–cell-autonomous manner. Alveolar simplification and BPD phenotypes induced by Hhip deletion can be partially rescued by IGF1 signaling blockade. We also observe the downregulation of Hhip expression and hyperactivation of Hh-IGF1 signaling in hyperoxia-induced BPD animal models. In addition, adult mice deficient in Hhip expression develop emphysema phenotype with abnormal alveolar myofibroblasts. Finally, we develop a therapeutic Fc-fused HHIP recombinant protein that attenuates BPD phenotypes in postnatal mice and emphysema in adult mice. These findings underscore the critical role of HHIP in coordinating alveologenesis and preventing BPD and emphysema by restricting Hh-IGF1 signaling. For mouse scRNA-seq, all live mesenchymal and epithelial cells were sorted from two Gli1creERT/+: Hhipflox/flox 14 days post-birth. Mesenchymal cells were isolated based on forward and side scatter, DAPI, CD31, CD45 and CD326 exclusion. epithelial cells were isolated based on DAPI, CD31 and CD45 exclusion. One mouse was challenged with tamoxifen and one was treated with oil at the same timepoints as a control. The sorted mesenchymal and epithelial cells were resuspended in PBS containing 0.05% BSA in a 3:1 mixture. Cells were loaded onto a single lane per sample into the ChromiumTM Controller to produce gel bead-in emulsions (GEMs). GEMs underwent reverse transcription for RNA barcoding and cDNA amplification, with the library prepped using the Chromium Next GEM Single Cell 5' Kit v2. Each sample was sequenced in 1 lane of the HiSeq2500 (Illumina) in Rapid Run Mode.