Pulmonary endothelial heterogeneity shapes divergent molecular vulnerabilities under SMAD4 deficiency

Abstract

Aim: Pulmonary vascular endothelial cells (ECs) comprise functionally distinct subpopulations, including arterial, venous, general capillary (gCap), and aerocyte capillary (aCap) ECs, that are essential for the maintenance of lung function. SMAD family member 4 (SMAD4) signaling is a critical contributor to vascular homeostasis. This study aims to elucidate how SMAD4 affects individual endothelial subpopulations in the lung.

Methods: We curated a previously published single-cell RNA sequencing (scRNA-seq) dataset from Smad4-deficient (Smad4^fl/fl; Rosa26CreER^T2) and control (Smad4^fl/fl) mouse lungs, using a unified bioinformatic pipeline. Integrated analyses of cell clustering, differential gene expression, high-dimensional weighted gene co-expression network analysis (hdWGCNA), and chromatin immunoprecipitation sequencing were applied to resolve subpopulation-specific transcriptomic shifts and regulatory modules in the pulmonary endothelial compartment.

Results: SMAD4 deficiency induced markedly asymmetric transcriptomic changes across pulmonary vascular endothelial subpopulations. Upon Smad4 loss, arterial ECs largely preserved their core transcriptomic identity, whereas venous and gCap ECs transitioned into a vulnerable state characterized by aberrant proteostasis and reduced survival. Smad4-null gCap ECs also exhibited disrupted vascular homeostatic and morphogenic processes. Smad4-null aCap ECs displayed a distinct transcriptomic shift, manifested as gas-exchange dysregulation and immune activation, alongside shared defects in translation/protein folding and vascular morphogenic capacity. Regulatory modules and putative hub genes associated with venous/gCap EC vulnerability and aCap-specific transcriptomic shift upon SMAD4 deficiency were further identified.

Conclusion: These findings define a lineage-resolved framework for SMAD4 signaling in the pulmonary endothelium and uncover molecular vulnerabilities that may inform EC subpopulation-targeted vascular interventions.

Keywords

Pulmonary endothelial subpopulation / SMAD4 / molecular heterogeneity / single-cell transcriptomics

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