Publicação

Metabolic remodeling of endothelial cells upon hypoxia and β-blockers as anti-angiogenic drugs

Detalhes bibliográficos
Resumo:	Angiogenesis occurs when new blood vessels sprout from pre-existing ones through proliferation of endothelial cells (ECs). This process plays a major role in several physiological and pathophysiological processes, including atherosclerosis, chronic inflammation and cancer. The tumor associated neovasculature is responsible for the supply of nutrients and oxygen, promoting tumor growth and metastasis, even though these blood vessels are structurally and functionally abnormal. During tumor progression, rapid and uncontrolled proliferation of tumor cells occurs, leading to a hypoxic microenvironment with insufficient blood supply. Therefore, tumor cells start to release pro-angiogenic factors, such as VEGF (vascular endothelial growth factor), and hypoxia leads to the stabilization of HIF-1α (hypoxia-inducible factor 1 α), inducing the expression of genes that will stimulate the formation of a new vessel. The first objective of this thesis was to unravel the hypoxia effect in metabolic remodeling during the endothelial cell activation. An NMR (nuclear magnetic resonance) analysis of HUVECs (human umbilical vein endothelial cells) exposed to CoCl2-mimicked hypoxia showed decreased lactate and 2-hydroxyisobutyrate extracellular levels in hypoxia. In addition, an increase of intracellular formate levels was detected in hypoxia, which suggests a proliferating phenotype due to formate involvement in purine synthesis. On the other hand, there was a decrease in the intracellular levels of glucose, lactate and acetate upon exposure to hypoxia, precursors of pyruvate. Concerning endothelial activation upon hypoxia, an in vitro tube forming assay was performed and indicated a promotion of vessel-like structures formation by hypoxia. However, in an ex vivo rat aortic ring assay the exposition to CoCl2 resulted in sprouting inhibition. Besides, a wound healing assay demonstrated a migration inhibition on cells exposed to CoCl2. This makes us wonder if the concentration of CoCl2 used is actually mimicking hypoxia. Cysteine (Cys) is an important ROS (reactive oxygen species) scavenger, showed to have an involvement in cancer cell remodeling. Since ROS act as pro-angiogenic molecules, we had the intent to investigate the effect of Cys in endothelial activation. An aortic ring assay showed a tendency for a sprouting increase when exposed to Cys and higher branch points density when exposed to H2O2. However, these levels decreased when exposed to Cys and H2O2, what suggests that ROS may play a role in inducing angiogenesis, being Cys able to somehow revert this process. Many anti-angiogenic strategies were developed over the years, but they have failed mostly because the mechanisms involved in neoangiogenesis are not fully understood. Propranolol (a nonselective β-blocker) has emerged as an effective treatment for infantile hemangiomas, leading to its regression. Hence, our second objective was to determine the effect of propranolol in neo-angiogenesis. A tube forming assay was performed and showed that propranolol inhibited the formation and destroyed the already formed vessel-like structures. Its inhibitory effects extended to migration, without affecting cell viability. Besides, an immunofluorescence assay showed that propranolol affected the expression of some EC markers, particularly when the exposure was shorter. The effect was also accessed ex vivo, in which propranolol inhibited the sprout of endothelial cells from the aortic ring, corroborating the in vitro results. The metabolic remodeling involved during aortic ring sprouting was also accessed by NMR spectroscopy, showing the differential expression of metabolites from different pathways, glycolysis, aminoacyl-tRNA biosynthesis, valine, leucine and isoleucine biosynthesis and degradation. This study gives some insights into the metabolic remodeling induced by hypoxia in endothelial cells, opening new cues on the metabolic adaptation underlying angiogenesis which, despite being a process involving non-cancerous cells it accounts for cancer progression. The results on the effect of propranolol in angiogenesis reinforces its use as a therapeutic alternative to target angiogenesis and treat cancer not only vascular tumors, being a starting point to further investigations on the mechanisms underlying its action.
Autores principais:	Martins, Filipa Alexandra Dias
Assunto:	Células endoteliais Angiogenese Microambiente tumoral Hipoxia Propranolol Teses de mestrado - 2019
Ano:	2019
País:	Portugal
Tipo de documento:	dissertação de mestrado
Tipo de acesso:	acesso aberto
Instituição associada:	Universidade de Lisboa
Idioma:	inglês
Origem:	Repositório da Universidade de Lisboa