Decreased Glycolysis as Metabolic Fingerprint of Endothelial Cells in Chronic Thromboembolic Pulmonary Hypertension

Chronic thromboembolic pulmonary hypertension (CTEPH) is characterized by thrombofibrotic obstruction of proximal pulmonary arteries. The cellular and molecular mechanisms underlying the pathogenesis remain incompletely understood, although we recently evidenced the potential involvement of the inflammatory marker C-reactive protein (CRP). We aimed to investigate the intracellular mechanisms induced by CRP in proximal pulmonary arterial endothelial cells (PAEC). PAEC were isolated from vascular material obtained during pulmonary endarterectomy. RNA was extracted from CRP-stimulated PAEC, and first-stand cDNA was generated. A RT2 profiler PCR Array was used to evaluate the expression of 84 key genes related to NF-κB-mediated signal transduction. CRP-induced NF-κB activation was studied. The effects of pyrrolidine-dithio-carbamate ammonium (PDTC), an inhibitor of the NF-κB pathway, were investigated on CRP-induced adhesion of monocytes to PAEC, adhesion molecule expression, endothelin-1 (ET-1), interleukin-6 (IL-6), and von Willebrand factor (vWF) secretion. Compared with nonstimulated PAEC, serotonin receptor 2B was downregulated by 25%, inhibitor of NF-κB kinase subunit epsilon (IKBKE) by 30%, and toll-like receptor-4 and -6 by 18 and 39%, respectively, in CRP-stimulated PAEC. The transcription factor FOS was threefold upregulated. CRP induced RelA/NF-κBp65 phosphorylation. PDTC dose dependently inhibited the adhesion of monocytes to CRP-stimulated PAEC. PDTC also inhibited the CRP-induced expression of ICAM-1 at the surface of PAEC. PDTC impaired the secretion of ET-1 by 18% and tended to inhibit the secretion of IL-6 by CRP-stimulated PAEC by 46%. PDTC did not inhibit the CRP-induced secretion of vWF. These results suggest an involvement of the NF-κB pathway in mediating different effects of CRP on proximal CTEPH-PAEC.

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

Scientific Reports ◽

10.1038/s41598-021-98320-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Olga Tura-Ceide ◽

Valérie F. E. D. Smolders ◽

Núria Aventin ◽

Constanza Morén ◽

Mariona Guitart-Mampel ◽

...

Keyword(s):

Endothelial Cells ◽

Pulmonary Hypertension ◽

Cell Model ◽

Mitochondrial Dynamics ◽

Chronic Thromboembolic Pulmonary Hypertension ◽

Molecular Signature ◽

Thromboembolic Pulmonary Hypertension ◽

Novel Targets

AbstractPulmonary endarterectomy (PEA) resected material offers a unique opportunity to develop an in vitro endothelial cell model of chronic thromboembolic pulmonary hypertension (CTEPH). We aimed to comprehensively analyze the endothelial function, molecular signature, and mitochondrial profile of CTEPH-derived endothelial cells to better understand the pathophysiological mechanisms of endothelial dysfunction behind CTEPH, and to identify potential novel targets for the prevention and treatment of the disease. Isolated cells from specimens obtained at PEA (CTEPH-EC), were characterized based on morphology, phenotype, and functional analyses (in vitro and in vivo tubule formation, proliferation, apoptosis, and migration). Mitochondrial content, morphology, and dynamics, as well as high-resolution respirometry and oxidative stress, were also studied. CTEPH-EC displayed a hyperproliferative phenotype with an increase expression of adhesion molecules and a decreased apoptosis, eNOS activity, migration capacity and reduced angiogenic capacity in vitro and in vivo compared to healthy endothelial cells. CTEPH-EC presented altered mitochondrial dynamics, increased mitochondrial respiration and an unbalanced production of reactive oxygen species and antioxidants. Our study is the foremost comprehensive investigation of CTEPH-EC. Modulation of redox, mitochondrial homeostasis and adhesion molecule overexpression arise as novel targets and biomarkers in CTEPH.

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Shear stress-exposed pulmonary artery endothelial cells fail to upregulate HSP70 in chronic thromboembolic pulmonary hypertension

PLoS ONE ◽

10.1371/journal.pone.0242960 ◽

2020 ◽

Vol 15 (12) ◽

pp. e0242960

Author(s):

William Salibe-Filho ◽

Thaís L. S. Araujo ◽

Everton G. Melo ◽

Luiza B. C. T. Coimbra ◽

Monica S. Lapa ◽

...

Keyword(s):

Endothelial Cells ◽

Pulmonary Hypertension ◽

Shear Stress ◽

Molecular Chaperones ◽

Chronic Thromboembolic Pulmonary Hypertension ◽

Annexin A2 ◽

Hsp70 Expression ◽

Potential Biomarker ◽

Thromboembolic Pulmonary Hypertension ◽

Static Conditions

The pathophysiological mechanisms underlying chronic thromboembolic pulmonary hypertension (CTEPH) are still unclear. Endothelial cell (EC) remodeling is believed to contribute to this pulmonary disease triggered by thrombus and hemodynamic forces disbalance. Recently, we showed that HSP70 levels decrease by proatherogenic shear stress. Molecular chaperones play a major role in proteostasis in neurological, cancer and inflammatory/ infectious diseases. To shed light on microvascular responses in CTEPH, we characterized the expression of molecular chaperones and annexin A2, a component of the fibrinolytic system. There is no animal model that reproduces microvascular changes in CTEPH, and this fact led us to isolated endothelial cells from patients with CTEPH undergoing pulmonary endarterectomy (PEA). We exposed CTEPH-EC and control human pulmonary endothelial cells (HPAEC) to high- (15 dynes/cm2) or low- (5 dynes/cm2) shear stress. After high-magnitude shear stress HPAEC upregulated heat shock protein 70kDa (HSP70) and the HSP ER paralogs 78 and 94kDa glucose-regulated protein (GRP78 and 94), whereas CTEPH-ECs failed to exhibit this response. At static conditions, both HSP70 and HSP90 families in CTEPH-EC are decreased. Importantly, immunohistochemistry analysis showed that HSP70 expression was downregulated in vivo, and annexin A2 was upregulated. Interestingly, wound healing and angiogenesis assays revealed that HSP70 inhibition with VER-155008 further impaired CTEPH-EC migratory responses. These results implicate HSP70 as a novel master regulator of endothelial dysfunction in type 4 PH. Overall, we first show that global failure of HSP upregulation is a hallmark of CTEPH pathogenesis and propose HSP70 as a potential biomarker of this condition.

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LSC - 2020 - Comparison of metabolic profile in endothelial cells of chronic thromboembolic pulmonary hypertension and pulmonary arterial hypertension

10.1183/13993003.congress-2020.1549 ◽

2020 ◽

Keyword(s):

Endothelial Cells ◽

Pulmonary Hypertension ◽

Pulmonary Arterial Hypertension ◽

Arterial Hypertension ◽

Metabolic Profile ◽

Chronic Thromboembolic Pulmonary Hypertension ◽

Thromboembolic Pulmonary Hypertension ◽

Pulmonary Arterial

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Protein network analyses of pulmonary endothelial cells in chronic thromboembolic pulmonary hypertension

Scientific Reports ◽

10.1038/s41598-021-85004-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Sarath Babu Nukala ◽

Olga Tura-Ceide ◽

Giancarlo Aldini ◽

Valérie F. E. D. Smolders ◽

Isabel Blanco ◽

...

Keyword(s):

Oxidative Stress ◽

Endothelial Cells ◽

Pulmonary Hypertension ◽

Endothelial Dysfunction ◽

Chronic Thromboembolic Pulmonary Hypertension ◽

Protein Carbonyl ◽

Protein Network ◽

Protein Carbonyl Content ◽

Network Analyses ◽

Thromboembolic Pulmonary Hypertension

AbstractChronic thromboembolic pulmonary hypertension (CTEPH) is a vascular disease characterized by the presence of organized thromboembolic material in pulmonary arteries leading to increased vascular resistance, heart failure and death. Dysfunction of endothelial cells is involved in CTEPH. The present study describes for the first time the molecular processes underlying endothelial dysfunction in the development of the CTEPH. The advanced analytical approach and the protein network analyses of patient derived CTEPH endothelial cells allowed the quantitation of 3258 proteins. The 673 differentially regulated proteins were associated with functional and disease protein network modules. The protein network analyses resulted in the characterization of dysregulated pathways associated with endothelial dysfunction, such as mitochondrial dysfunction, oxidative phosphorylation, sirtuin signaling, inflammatory response, oxidative stress and fatty acid metabolism related pathways. In addition, the quantification of advanced oxidation protein products, total protein carbonyl content, and intracellular reactive oxygen species resulted increased attesting the dysregulation of oxidative stress response. In conclusion this is the first quantitative study to highlight the involvement of endothelial dysfunction in CTEPH using patient samples and by network medicine approach.

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