Hypoxia-induced pulmonary hypertension drives mesenchymal remodeling and reveals a transitional fibroblast state contributing to neomuscularization

Rationale: Pulmonary hypertension (PH) is a fatal vascular disease characterized by distal and proximal vessel neomuscularization and right heart failure. While vascular smooth muscle cells (vSMCs) are traditionally considered the primary source of ACTA2+cells, the contribution of alternative mesenchymal lineages and transitional states remains unresolved. Objectives: To uncover the cellular origins and dynamic programs underlying ACTA2+ cell accumulation in hypoxia-induced PH and identify previously unrecognized intermediates contributing to vascular remodeling. Methods and Results: Using temporally controlled lineage tracing in Tg ( Acta2-CreERT2 ); tdTomato flox / flox mice, we distinguished pre-existing from newly induced ACTA2+ cells under normoxia and hypoxia. Hemodynamic and morphometric analyses confirmed vascular remodeling in both hypoxic groups; yet HOX–Tam lungs exhibited significantly more tdTom+ ACTA2+ cells, implicating non-vSMC-derived sources. Single-cell RNA sequencing revealed a complex mesenchymal landscape including alveolar fibroblasts, adventitial fibroblasts (AdvFs), and a previously undefined transitional adventitial fibroblast (TransAdV) population bridging fibroblastic and contractile states. TransAdVs co-expressed canonical smooth muscle markers such as Tagln and Myh11 with fibroblastic markers ( Pi16 , Scara5 and Dcn ) and were enriched for WNT signaling. RNA velocity estimation and dynamics mapped a trajectory from AdvFs toward vSMCs through TransAdVs, which expanded under hypoxia. Independent analysis of Gli1 CreERT2 ; tdTomato flox/flox mice confirmed emergence of TransAdV-like GLI1 + cells, indicating a conserved transitional state across lineages. Human PH lungs exhibited robust upregulation of GLI1 and PI16 transcripts within remodeled vessels. Complementary functional assays in cultured pulmonary arterial adventitial fibroblasts (PAAFs) demonstrated that WNT pathway activation (CHIR99021) and rhNGF treatment significantly increased ACTA2 expression under hypoxic conditions, underscoring the role of developmental and neurovascular signaling in driving mesenchymal plasticity. Conclusions: Our integrative approach using multi-tiered validation identifies TransAdVs as a novel fibroblast-derived transitional state that orchestrates vascular remodeling through developmental and stress-response pathways. These findings redefine the cellular hierarchy of PH and highlight new therapeutic targets to modulate mesenchymal plasticity in pulmonary vascular disease. Publication History Article published online: 26 February 2026 © 2026. Thieme. All rights reserved. Georg Thieme Verlag KG Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany