Excessive postnatal smooth muscle differentiation in a lung-specific model of TBX4-related pulmonary hypertension
Lea C. Steffes, Kaylie A. Chiles, Sehar R. Masud, Aleen Rahman, Madeline Dawson, Csaba Galambos, Maya E. Kumar, Ripla Arora
Lea C. Steffes, Kaylie A. Chiles, Sehar R. Masud, Aleen Rahman, Madeline Dawson, Csaba Galambos, Maya E. Kumar, Ripla Arora
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Research Article Cardiology Pulmonology

Excessive postnatal smooth muscle differentiation in a lung-specific model of TBX4-related pulmonary hypertension

Abstract

Heterozygous TBX4 variants are the second most common genetic cause of pediatric pulmonary hypertension (PH), yet mechanisms underlying TBX4-related lung disease remain poorly understood. This study developed a lung-mesenchyme-specific Tbx4 loss-of-function (Tbx4cKO) mouse model that bypasses embryonic lethality to investigate this condition. Adult Tbx4cKO mice demonstrated significantly impaired pulmonary flow acceleration consistent with PH. Three-dimensional analysis of embryonic lungs revealed reduced lobe volumes and decreased distance between pleural edges and muscularized vessels. In adult Tbx4cKO lungs, we identified extensive vascular remodeling characterized by medial thickening and the extension of muscularized arteries into normally non-muscularized subpleural parenchymal zones. Contrary to previous reports suggesting vascular simplification, 3-dimensional analysis demonstrated an elaborated pulmonary artery tree in addition to pathologic wall muscularization. Depletion of a single Tbx5 allele in addition to both Tbx4 alleles exacerbated histologic phenotypes, with worsened right ventricular dilation. This model also demonstrated dysregulated airway smooth muscle patterning and prominent subpleural smooth muscle bands, similar to those in human TBX4 syndrome. We identify TBX4 as a critical regulator of smooth muscle differentiation and patterning across multiple lung compartments. Our model recapitulates key features of human TBX4 syndrome and identifies dysregulated smooth muscle differentiation as a potential future therapeutic target.

Authors

Lea C. Steffes, Kaylie A. Chiles, Sehar R. Masud, Aleen Rahman, Madeline Dawson, Csaba Galambos, Maya E. Kumar, Ripla Arora

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Figure 6

Loss of T-box genes leads to expansion of ectopic subpleural smooth muscle bands.

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Loss of T-box genes leads to expansion of ectopic subpleural smooth musc...
(A–C) Ventral views of right caudal lobes from control, Tbx4cKO, and Tbx4cKO;Tbx5het, stained in whole mount to identify ACTA2+ cells (white). Small bands of subpleural smooth muscle (arrowheads) are occasionally found in control specimens, with the size and extent of the subpleural smooth muscle banding increasing in Tbx4cKO, and with Tbx4cKO;Tbx5het animals showing extensive banding wrapping all faces of the lobe. (D and E) Subpleural accumulation of ACTA2+ smooth muscle (asterisks) is a feature of TBX4 syndrome but absent in control (see Supplemental Figure 3). (F) Confocal micrograph of immunostained cryosection shows that mouse ACTA2+ bands lie directly beneath the pleura and are composed of bundles of tightly aligned cells that lack apparent contact with internal lung structures. Molecular characterization of ectopic subpleural bands in a Tbx4cKO;Tbx5het mouse by fluorescent in situ hybridization shows ACTA2+ bands express high levels of Cnn1 (G and H) and Lgr6 (I), minimal levels of Hhip (J), while Notch3 and Pdgfrb are undetectable, not fully consistent with any canonical lung smooth muscle identity, but intermediate between airway smooth muscle and myofibroblast. Pleura are marked with a white dotted line in F–J; subpleural band cells are marked with a yellow dotted line in I and J. Scale bars: 500 μm (A–C), 100 μm (D–F), and 50 μm (G–J).