Chronic thromboembolic pulmonary hypertension (CTEPH) is a form of pulmonary hypertension characterized by the presence of fibrotic intraluminal thrombi and causing obliteration of the pulmonary arteries. Although both endothelial cell (EC) dysfunction and inflammation are linked to CTEPH pathogenesis, regulation of the basal inflammatory response of ECs in CTEPH is not fully understood. Therefore, in the present study, we investigated the role of the nuclear factor (NF)-κB pro-inflammatory signaling pathway in ECs in CTEPH under basal conditions. Basal mRNA levels of interleukin (IL)-8, IL-1β, monocyte chemoattractant protein-1 (MCP-1), C-C motif chemokine ligand 5 (CCL5), and vascular cell adhesion molecule-1 (VCAM-1) were upregulated in CTEPH-ECs compared to the control cells. To assess the involvement of NF-κB signaling in basal inflammatory activation, CTEPH-ECs were incubated with the NF-κB inhibitor Bay 11-7085. The increase in pro-inflammatory cytokines was abolished when cells were incubated with the NF-κB inhibitor. To determine if NF-κB was indeed activated, we stained pulmonary endarterectomy (PEA) specimens from CTEPH patients and ECs isolated from PEA specimens for phospho-NF-κB-P65 and found that especially the vessels within the thrombus and CTEPH-ECs are positive for phospho-NF-κB-P65. In summary, we show that CTEPH-ECs have a pro-inflammatory status under basal conditions, and blocking NF-κB signaling reduces the production of inflammatory factors in CTEPH-ECs. Therefore, our results show that the increased basal pro-inflammatory status of CTEPH-ECs is, at least partially, regulated through activation of NF-κB signaling and potentially contributes to the pathophysiology and progression of CTEPH.

Skeletal muscle repair relies on heterogeneous populations of satellite cells (SCs). The mechanisms that regulate SC homeostasis and state transition during activation are currently unknown. Here, we investigated the emerging role of non-genetic micro-heterogeneity, i.e., intrinsic cell-to-cell variability of a population, in this process. We demonstrate that micro-heterogeneity of the membrane protein CRIPTO in mouse-activated SCs (ASCs) identifies metastable cell states that allow a rapid response of the population to environmental changes. Mechanistically, CRIPTO micro-heterogeneity is generated and maintained through a process of intracellular trafficking coupled with active shedding of CRIPTO from the plasma membrane. Irreversible perturbation of CRIPTO micro-heterogeneity affects the balance of proliferation, self-renewal, and myogenic commitment in ASCs, resulting in increased self-renewal in vivo. Our findings demonstrate that CRIPTO micro-heterogeneity regulates the adaptative response of ASCs to microenvironmental changes, providing insights into the role of intrinsic heterogeneity in preserving stem cell population diversity during tissue repair.

Background: Chronic thromboembolic pulmonary hypertension (CTEPH) and pulmonary arterial hypertension (PAH) are two subtypes of pulmonary hypertension (PH). Endothelial dysfunction is believed the main cause of the hyperproliferative and antiapoptotic phenotype in PH characterized by increased glycolysis in PAH. It is unknown whether PAH and CTEPH share the same metabolic abnormalities. The aim of our study is to perform a systematic metabolic comparison of endothelial cells (ECs) derived from PAH and CTEPH patients. Methods: ECs from human pulmonary artery endothelial cells HPAECs (n=3), CTEPH (n=7) and PAH (n=6) were expanded until the same passage. Metabolic targets were analysed by qRT-PCR and Western Blot. Viability and migration were studied using MTT and wound healing assay. Supernatants were assessed by BGEM Test Cards. All values are shown as mean±SD. The analysis was performed using one-way ANOVA (p<0.05, statistically significant). Results: Gene and protein expression of key glycolytic enzymes were significantly upregulated in PAH-ECs compared to CTEPH-ECs and HPAECs. Mitochondrial enzymes, pentose phosphate pathway and glutamine metabolism-related enzymes were upregulated in PAH-ECs compared to the CTEPH-ECs and HPAECs. Both PAH and CTEPH-ECs showed significant higher viability compared to HPAECs. Acidification of the supernatant was detected in both patients’ lines. Statistical higher glucose and lower lactate concentration in the supernatant were shown in HPAECs compared to patient cell lines. Conclusion: PAH-ECs were found to be metabolically different than CTEPH-ECs indicating distinct pathogenic mechanisms in the development of the two PH subtypes. This creates the opportunity to specifically target the distinct metabolic abnormalities in both diseases.

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.