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 4

Whole-mount analysis reveals increased vascular smooth muscle coverage of peripheral pulmonary artery networks in Tbx4cKO and Tbx4cKO;Tbx5het.

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Whole-mount analysis reveals increased vascular smooth muscle coverage o...
(A–C) Intact right caudal lobes stained to highlight smooth muscle α-actin (ACTA2) protein (white), cleared, and visualized in 3 dimensions from control, Tbx4cKO, and Tbx4cKO;Tbx5het animals allows visualization of both airway, arterial, and venous smooth muscle networks. The same branch (dorsal view of RCd.L3) is shown for each genotype with distal-most airway bifurcation marked with asterisk to aid comparison. Images in A–C are multifocal projections of stereoscope z-stacks. Scale bars: 500 μm. (D–F) Schematic drawings of arterial networks for each genotype. The gray dotted line in E at 160 μm demarcates the subpleural nonmuscular zone (SNZ) where muscularized pulmonary arteries are absent in control lungs. (G) Highly muscularized arteries (H&E stain of paraffin section) are found close to the pleural surface in TBX4 syndrome lungs but not in control (see Supplemental Figure 3). (H) In both Tbx4cKO and Tbx4cKO;Tbx5het, muscularized arteries extend significantly closer to the pleura (yellow dotted lines in A–C; black dotted lines in D–F). (I) Consistent with abnormal smooth muscle coverage of slender distal pulmonary arteries, the diameter of the distal-most fully muscularized arteries drops significantly in both Tbx4cKO and Tbx4cKO;Tbx5het animals. Control n = 7, Tbx4cKO n = 5, Tbx4cKO;Tbx5het n = 3. Welch’s ANOVA (assuming unequal variances) was performed to assess differences across genotypes, followed by Games-Howell post hoc tests for pairwise comparisons between groups.