Derivation and Characterisation of Endothelial Cells from Patients with Chronic Thromboembolic Pulmonary Hypertension.

Blanco

et al. 2022

Sci Rep

Self Cite

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.

Section: Methodsmentioning

confidence: 99%

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

Blanco

et al. 2022

Sci Rep

Self Cite

“…ECs isolated from endarterectomy specimens, referred to as CTEPH-ECs, were cultured as previously described [28]. In short, ECs were plated onto 0.1% gelatin-coated wells and grown in an endothelium cell medium (ScienceCell Research Laboratories) supplemented with endothelial cell growth supplement, 5% fetal bovine serum (FBS), and Penicillin/Streptomycin solution (ScienceCell Research Laboratories).…”

Section: Pulmonary Endothelial Cell Isolation and Culturementioning

confidence: 99%

“…In short, ECs were plated onto 0.1% gelatin-coated wells and grown in an endothelium cell medium (ScienceCell Research Laboratories) supplemented with endothelial cell growth supplement, 5% fetal bovine serum (FBS), and Penicillin/Streptomycin solution (ScienceCell Research Laboratories). The cell phenotype was characterized by staining the cells with antibodies against a panel of endothelial and smooth muscle cell-specific markers, including endothelial nitric oxide synthase (eNOS) and alpha smooth muscle Actin (αSMA) [28]. The patient characteristics are presented in Table 1.…”

Section: Pulmonary Endothelial Cell Isolation and Culturementioning

confidence: 99%

The Inflammatory Profile of CTEPH-Derived Endothelial Cells Is a Possible Driver of Disease Progression

Lodder

et al. 2021

Cells

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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.

“…ECs isolated from endarterectomy specimens of CTEPH patients, referred to as CTEPH-EC, were isolated and cultured as previously described 18 . EC phenotype was characterized by positive staining of endothelial and smooth muscle cell-speci c markers, including endothelial nitric oxide synthase (eNOS) and alpha-smooth muscle actin (α-SMA) as previously described [21]. ECs from two heritable and four idiopathic PAH patients (referred to as PAH-EC) were isolated from lung tissues and artery rings of endstage PAH patients obtained from lung transplantations and cultured as previously described [22,23].…”

Section: Pulmonary Endothelial Cell Isolation and Culturementioning

confidence: 99%

Metabolic Profile in Endothelial Cells of Chronic Thromboembolic Pulmonary Hypertension and Pulmonary Arterial Hypertension.

Blanco

et al. 2021

Preprint

Self Cite

Background 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. Methods 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. Results 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 HPAE. 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. Conclusions 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.