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 the mechanisms underlying the pathophysiology of TBX4-related lung disease remain poorly understood. We developed a lung mesenchyme-specific Tbx4 loss of function ( Tbx4cKO ) mouse model that bypasses embryonic lethality to investigate TBX4-related lung disease. Echocardiography of adult Tbx4cKO mice demonstrated significant hemodynamic changes consistent with PH. Three-dimensional whole-mount analysis of embryonic day 18.5 lungs revealed reduced lobe volumes and decreased distance between pleural edges and muscularized vessels. In adult Tbx4cKO lungs, high-resolution spatial quantitation identified extensive vascular remodeling characterized by significant medial thickening, distal muscularization of small diameter arteries, and extension of muscularized vessels into normally non-muscularized subpleural zones. Contrary to previous reports suggesting vascular simplification with Tbx4 loss, our comprehensive three-dimensional approach demonstrated an elaborated arterial tree with pathologic muscularization. Additional heterozygous loss of Tbx5 ( Tbx4cKO;Tbx5he t) exacerbated vascular phenotypes without worsening hemodynamic parameters. We also documented dysregulated airway smooth muscle patterning and prominent subpleural smooth muscle bands that share molecular features with myofibroblasts and airway smooth muscle cells, echoing pathologic findings in human TBX4 syndrome lung tissue. Collectively, our findings identify TBX4 as a critical suppressor of smooth muscle differentiation across multiple pulmonary compartments. This model recapitulates key features of human TBX4 syndrome and reveals mild developmental underpinnings with subsequent progressive postnatal smooth muscle dysregulation, highlighting a postnatal window during which therapeutic regulation of mesenchymal differentiation may be beneficial.