Functional and metabolic characterization of endothelial cells in chronic thromboembolic pulmonary hypertension
- Autores: Valérie Smolders
- Directores de la Tesis: Marta Cascante Serratosa (dir. tes.), Paul H.a. Quax (codir. tes.), Olga Tura Ceide (codir. tes.)
- Lectura: En la Universitat de Barcelona ( España ) en 2020
- Idioma: inglés
- Tribunal Calificador de la Tesis: Josep Joan Centelles Serra (presid.), Ibrahim Halil Polat (secret.), Marta Casado Pinna (voc.)
- Programa de doctorado: Programa de Doctorado en Biotecnología por la Universidad de Barcelona
- Materias:
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- Resumen
Pulmonary Hypertension (PH) is a condition of increased blood pressure within the arteries of the lung (mPAP > 20mmHg) which affects approximately 1% of the global population. CTEPH, group 4 PH, is characterized by unresolved pulmonary emboli and pulmonary vascular remodeling of both occluded and non-occluded vessels. The gold standard treatment for CTEPH patients is pulmonary endarterectomy, the removal of occluding thromboembolic material from the pulmonary arteries, with a possible curative outcome. Although positive outcome associated with PEA surgery, 40% of CTEPH patients are not operable and up to one-third has persistent or recurrent PH pointing out the need for better understanding of CTEPH pathogenesis.
The general aim of this thesis was to improve the understanding of CTEPH pathophysiology by focusing on patient endothelial cell (EC) behaviour and function. For this purpose, we isolated ECs from vascular material collected at pulmonary endarterectomy in patients with CTEPH (referred to as CTEPH-EC) and validated them as an in vitro model for studying endothelial pathology in CTEPH.
In this thesis we described the isolation of EC from pulmonary endarterectomy specimen from patients with CTEPH. We validated these cells as an attractive in vitro model for endothelial pathology by assessing endothelial and mitochondrial function. In this work we showed alterations in oxidative/nitrative stress, antioxidant production, mitochondrial homeostasis and adhesion molecules that could be involved in the pathophysiology of CTEPH. In addition, we examined alterations in metabolism of CTEPH-EC that might be involved in EC dysfunction and subsequent vascular remodeling occurring in pulmonary arteries surrounding thrombi in CTEPH patients. We observed, in contrast to the increased glycolytic profile present in pulmonary arterial hypertension (PAH)-EC, a decrease in key and rate-limiting enzymes of the glycolytic pathway. In addition, we found a downregulation in pyruvate dehydrogenase kinase, the enzyme that prevents pyruvate entry into the mitochondria, together with a downregulation in glutamine metabolism. At last, pentose phosphate pathway- and fatty acid oxidation-associated key metabolic enzymes were not found different in CTEPH-EC compared to control ECs. Because of the metabolic differences in CTEPH-EC compared to what has been observed in PAH, we performed also a systemic metabolic comparison of EC isolated from CTEPH patients that underwent pulmonary endarterectomy and from PAH patients that underwent lung transplantation.
Interestingly, we showed a pronounced difference in gene expression of glycolytic and glutamine-related enzymes. No differences were observed in pentose phosphate pathway-related enzymes at both mRNA and protein level. PAH-EC seemed to have a higher basal viability compared to CTEPH-EC, which is in line with an increase in glycolysis in PAH-EC. Remarkably both CTEPH-EC and PAH-EC showed a drop in viability in response to metabolic inhibitors but no difference in viability between both groups was observed. At last, although clear metabolic differences between CTEPH-EC and PAH-EC are detectable, no difference in migration capacity could be observed between CTEPH-EC and PAH-EC. Finally, this thesis also focused on the inflammatory status of CTEPH-EC. We found that phospho-p65 was present in PECAM/CD31 positive vessels within pulmonary endarterectomy specimens from CTEPH patients. Furthermore we also showed increased presence of nuclear phospho-p65 in cultured CTEPH-EC. In line with these observations, several inflammatory factors downstream NF-κB signaling, such as IL-8, IL-1β, MCP-1, ICAM-1 and VCAM-1, were found to be upregulated in CTEPH-EC compared to control cells. When CTEPH-EC were incubated with a NF-κB inhibitor, we showed a significant decrease in mRNA levels of MCP-1 and VCAM-1. So, based on the results obtained in this study we can conclude that CTEPH-EC have a pro-inflammatory status under basal conditions as shown by the increased production of inflammatory cytokines IL-8, IL-1β, ICAM-1 and VCAM-1. More importantly, we showed that the increased inflammatory cytokines observed in CTEPH-EC are, at least partially, regulated through NF-κB signaling.
In conclusion, we identified several abnormalities in CTEPH-EC that could play a role in pathological mechanisms driving CTEPH-specific vascular changes. We described alterations in key processes such as angiogenesis and migration, oxidative stress, metabolism and inflammation. Each of these processes may represent targets for novel therapies or biomarkers
