Hypoxia is thought to be one of the main causes of tissue remodeling and endothelial cell (EC) dysfunction in CTEPH. Studying cells under different oxygen (O₂) culture conditions can be delicate. The aim is to evaluate the effect on ECs derived from patients with CTEPH (EC-CTEPH) under different O₂ conditions compared to healthy controls. The specific objectives are validation of a novel system for continuous hypoxia; and study of the effect hypoxia in EC-CTEPH. A new technology has been optimized based on the development of newly designed biochips. Fiber optic sensor measures, supernatant (SN) analysis and qRT-PCR were used to validate the technique with Healthy human pulmonary artery endothelial (HPAE) cells (n=5). To assess the role of hypoxia in EC dysfunction RT-PCR, SN analysis and wound healing assay were performed using EC-CTEPH (n=5). HPAE cells were used as control group (n=5). Our results show that O₂ arrives faster to the cells and hypoxic conditions are maintained throughout time. Expression of hypoxia-induced genes, such as VEGF and HIF-1 is more accentuated in ECs cultured within the new system. The chips have been validated and compared with conventional hypoxia incubator. The new system induces a more stable expression of hypoxic genes. Hypoxic conditions are reached faster than in a conventional hypoxia incubator. These results lead the way to a more profound study to determine the role of hypoxia in EC-CTEPH and to understand the effects of different O₂ conditions in ECs dysfunction. Further studies are needed to assess the role of hypoxia in EC-CTEPH compared to HPAE. Supported by grants SOCAP, SEPAR, ISCIII (CP17/00114, PI18/00960), FCHP, CIBERES.

The mechanisms underlying the development and severity of pulmonary hypertension (PH) in chronic obstructive pulmonary disease (COPD) and interstitial lung disease (ILD) remain poorly understood. To investigate the contribution of genetic factors, we performed targeted sequencing of PH-related genes in 63 patients with COPD, 51 with ILD, and 38 with idiopathic pulmonary arterial hypertension (IPAH), stratifying COPD and ILD patients according to PH severity, assessed by right heart catheterisation. Patients with ILD-associated PH harboured fewer genetic variants than those without PH, whereas an opposite trend was observed in COPD-associated PH. Variants of uncertain significance (VUS) in PAH-associated genes (BMPR2, BMPR1B) were identified in two out of 28 patients with severe COPD-PH and in one with nonsevere ILD-PH (SMAD9). VUS in extracellular matrix-related genes (THBS1, LOXL2) were also identified in four patients with severe COPD-PH, and in inflammatory-related genes (TNFAIP6) in two patients with ILD-PH. Pathogenic/likely pathogenic variants in PAH-associated genes were exclusive to IPAH. We conclude that there is limited genetic overlap between COPD-PH and ILD-PH, with a stronger genetic contribution to PH in COPD, particularly in its severe form, where a subset of patients carries VUS in PAH-associated genes or genes involved in vascular remodelling.

Pulmonary endarterectomy (PEA) specimens provide a unique source of endothelial cells (ECs) to model chronic thromboembolic pulmonary hypertension (CTEPH) in vitro. This study investigates the impact of chronic hypoxia on PEA-derived ECs, focusing on mechanisms of endothelial dysfunction and vascular remodeling. ECs from PEA specimens (EC-CTEPH) and controls were exposed to normoxia, hypoxia, and reoxygenation. Cell morphology, proliferation, migration, and expression of angiogenic and hypoxia-responsive genes were assessed. Pharmacological HIF stabilization with dimethyloxalylglycine (DMOG) was compared with hypoxia. Oxidative stress responses were evaluated using hydrogen peroxide. EC-CTEPH showed impaired adaptation to hypoxia, with reduced induction of glycolytic and angiogenic genes, altered morphology, delayed wound closure, and persistent oxidative stress after reoxygenation, consistent with defective hypoxia sensing. DMOG partially restored metabolic gene expression, indicating improved adaptation through HIF stabilization. Despite elevated basal ROS levels, oxidative challenge did not trigger adaptive glycolytic or angiogenic responses and induced distinct transcriptional profiles compared with controls. CTEPH endothelial cells display an altered response to hypoxia and oxidative stress, consistent with impaired hypoxia sensing and stress adaptation. This model highlights maladaptive endothelial features and provides a framework for future studies exploring HIF-targeted approaches in CTEPH.