Dysfunctional endothelial cells in patients with chronic thromboembolic pulmonary hypertension.

Dysfunctional endothelial cells in patients with chronic thromboembolic pulmonary hypertension. Rationale: 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. Mitochondrial disarrangements in endothelial cells might explain a hyperproliferative resistant phenotype that could explain vascular changes occurring in CTEPH. We aimed to develop an in vitro model of CTEPH using patient-derived cell lines and assess potential mitochondrial disturbances. Methods: Isolated cells from specimens obtained at PEA, were confirmed as being endothelial cells based on cobblestone morphology, endothelial phenotype (flow cytometry, RT-PCR, immunofluorescence) and functional analysis (tubule formation, proliferation and migration). We also measured: i) mitochondrial membrane potential (MMP), mitochondrial content and apoptosis/necrosis by flow cytometry and ii) mitochondrial dynamics (MD) by confocal microscopy. Results: Isolated cells maintained cobblestone morphology and stained positive for endothelial markers. 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). Functionally, they showed reduced capacity to form tubule structures (150±44 vs 96±21; p<0.03). CEPTH cells tended to show lower rates of depolarized MMP (49.91±14.70 vs. 59.87±8.41, p=NS), a decrease of mitochondrial content (148.94±69.96 vs. 295.57±178.60, PNS) and lower levels of necrosis/apoptosis (23.57±8.03 vs. 29.33±5.94, p=NS). Mitochondria from CTEPH patients tended to be smaller and to show higher circularity (0.45±0.009 vs. 0.43±0.012, p=NS), with less branching (2.77±0.14 vs. 2.93±0.06, p=NS) with respect to controls, both considered as pathologic markers. Conclusions: Endothelial cells obtained from PEA in CTEPH show a hyperproliferative phenotype, impaired function and mitochondrial material derangement, that may play a role in the pathogenesis of pulmonary hypertension after pulmonary embolism

Metabolic profile in endothelial cells of chronic thromboembolic pulmonary hypertension and pulmonary arterial hypertension.

Chronic thromboembolic pulmonary hypertension (CTEPH) and pulmonary arterial hypertension (PAH) are two forms of pulmonary hypertension (PH) characterized by obstructive vasculopathy. Endothelial dysfunction along with metabolic changes towards increased glycolysis are important in PAH pathophysiology. Less is known about such abnormalities in endothelial cells (ECs) from CTEPH patients. This study provides a systematic metabolic comparison of ECs derived from CTEPH and PAH patients. Metabolic gene expression was studied using qPCR in cultured CTEPH-EC and PAH-EC. Western blot analyses were done for HK2, LDHA, PDHA1, PDK and G6PD. Basal viability of CTEPH-EC and PAH-EC with the incubation with metabolic inhibitors was measured using colorimetric viability assays. Human pulmonary artery endothelial cells (HPAEC) were used as healthy controls. Whereas PAH-EC showed significant higher mRNA levels of GLUT1, HK2, LDHA, PDHA1 and GLUD1 metabolic enzymes compared to HPAEC, CTEPH-EC did not. Oxidative phosphorylation associated proteins had an increased expression in PAH-EC compared to CTEPH-EC and HPAEC. PAH-EC, CTEPH-EC and HPAEC presented similar HOXD macrovascular gene expression. Metabolic inhibitors showed a dose-dependent reduction in viability in all three groups, predominantly in PAH-EC. A different metabolic profile is present in CTEPH-EC compared to PAH-EC and suggests differences in molecular mechanisms important in the disease pathology and treatment