Guenat, O. T. | Search bioRxiv

bioRxiv2025

LOSS OF ROR2 TYROSINE KINASE RECEPTOR IS ASSOCIATED WITH ENDOTHELIAL DYSFUNCTION IN PAH VIA INAPPROPRIATE INTEGRIN BETA 1 ACTIVATION

RationaleEndothelial dysfunction is a key feature of pulmonary arterial hypertension (PAH). We previously identified Wnt7a, a ligand of the Wnt planar cell polarity (PCP) pathway, as essential for pulmonary angiogenesis, with its loss linked to PAH. Given the importance of Wnt/PCP to lung endothelial function and angiogenesis, our goal is to elucidate how Wnt/PCP regulates angiogenic responses in pulmonary microvascular endothelial cells (PMVECs). ROR2, a tyrosine kinase receptor specific to Wnt/PCP, is crucial for cardiovascular development, but its role in PAH is unclear. We hypothesized that ROR2 supports endothelial homeostasis, and its loss would impair angiogenesis, contributing to PAH. MethodsEndothelial-specific ROR2 knockout (ROR2 ECKO) and wild-type (WT) mice were studied under normoxia and chronic hypoxia using echocardiography, hemodynamics, and lung morphometry. PMVECs from healthy and PAH lungs were transfected with ROR2 siRNA/constructs for functional and molecular studies. Focal adhesion (FA) activation and force generation were assessed via FRET-based methods. Bulk and single-cell transcriptomic analyses were performed on siROR2 PMVECs and ROR2 ECKO lungs. ResultsROR2 ECKO mice exhibited worsened pulmonary hypertension, right ventricular remodeling, microvascular loss, and muscularization in hypoxia. Single-cell RNA sequencing of lung endothelial cells showed dysregulation of pathways involved in barrier formation and angiogenesis. Evans blue dye extravasation confirmed reduced endothelial barrier integrity in ROR2 ECKO mice. ROR2-deficient PAH PMVECs displayed increased adhesion, permeability, and FA numbers, with reduced VE-cadherin at cell junctions. Confocal imaging revealed ROR2 localization in FAs, interacting with integrin {beta}1 (ITGB1). FRET analysis showed that ITGB1 remained in an active, adhesion-promoting state in ROR2-deficient cells. Restoring ROR2 in PAH PMVECs normalized adhesion, barrier function, and FA abundance. Transcriptomic analysis identified Rab12 as a key mediator of ROR2-ITGB1 crosstalk, with Rab12 knockdown mimicking ROR2 deficiency in PMVECs. ConclusionsROR2 regulates pulmonary angiogenesis by maintaining endothelial barrier integrity and facilitating integrin recycling. Restoring ROR2 signaling could be a potential therapeutic approach for PAH.

cell biology↗

bioRxiv2019

Second-generation lung-on-a-chip array with a stretchable biological membrane

The complex architecture of the lung parenchyma and the air-blood barrier is difficult to mimic in-vitro. Recently reported lung-on-a-chips used a thin, porous and stretchable PDMS membrane, to mimic the air-blood barrier and the rhythmic breathing motions. However, the nature, the properties and the size of this PDMS membrane differ from the extracellular matrix of the distal airways. Here, we present a second-generation lung-on-a-chip with an array of in vivo-like sized alveoli and a stretchable biological membrane. This nearly absorption free membrane allows mimicking in vivo functionality of the lung parenchyma at an unprecedented level. The air-blood barrier is constituted by human primary lung alveolar epithelial cells from several patients and co-cultured with primary lung endothelial cells. Typical markers of lung alveolar epithelial cells could be observed in the model, while barrier properties were preserved for up to three weeks. This advanced lung alveolar model reproduces some key features of the lung alveolar environment in terms of composition, alveolar size, mechanical forces and biological functions, which makes this model a more analogous tool for drug discovery, diseases modeling and precision medicine applications.

bioengineering↗