Derivation and characterisation of endothelial cells from patients with chronic thromboembolic pulmonary hypertension

The material obtained from pulmonary endarterectomy (PEA) offers the unique opportunity to study the pathophysiological mechanisms of chronic thromboembolic pulmonary hypertension (CTEPH) at disease site. Metabolic changes and mitochondrial disarrangements in endothelial cells might elucidate a hyperproliferative apoptosis-resistant phenotype that could explain vascular changes occurring in CTEPH. The objective of our study was to develop an in vitro model of CTEPH using patient derived cell lines and assess endothelial cell metabolism and potential mitochondrial disturbances. Isolated cells from patients obtained after PEA, were confirmed as being endothelial cells based on cobblestone morphology, endothelial phenotype (flow cytometry, RT-PCR and immunofluorescence) and functional analysis (tubule formation, proliferation and migration). We also measured: i) hypoxia-inducible factor 1-alpha (HIF-1a) expression by RT-PCR, ii) glycolytic enzymes and metabolites by colorimetric assays (such as lactate dehydrogenase (LDH) and lactate). Isolated cells maintained cobblestone morphology and stained positive for endothelial markers (such as CD31, VE-cadherin, eNOS and VWF). They showed a hyperproliferative phenotype when compared with control human pulmonary artery endothelial cell lines (HPAE): number of Ki67+cells (50.33±13.4 vs 32.5±9.5; p<0.05), and fold expansion (1.56±0.08 vs 0.8±0.05; p<0.002). HIF-1α was higher expressed in patient derived cells compared with HPAE (3.21±1.38 vs 1.54±0.87; p<0.001). Functionally, they showed reduced capacity to form tubule structures (150±44 vs 96±21; p