Characterization of endothelial cell metabolism under different microenvironmental challenges: exploring therapeutic implications of targeting glycogen degradation

Abstract

[eng] Angiogenesis is the formation of blood vessels from the pre-existing ones and is required for distributing nutrients and oxygen to the tissues through the blood flow. This process is important for tumour tissues as well, which recruit the neighbouring blood vessels for their survival and propagation, the process termed as tumour angiogenesis. Endothelial cells are those that line the blood vessels and are the key players in the process of angiogenesis. Many anti-angiogenic therapies based on targeting the pro-angiogenic factors have not shown to be completely effective against the tumour vessels and pose adverse side-effects. Since metabolism is the downstream effect of any type of cellular induction, exploring metabolic reprogramming of endothelial cells during angiogenic induction could provide alternative therapeutic strategies for targeting tumour angiogenesis. VEGF is identified to be one of the most versatile and potent pro-angiogenic factors for its roles in multiple steps of blood vessel formation. Hypoxia has also been found to be a key regulator of angiogenesis and has been identified to induce a higher production of VEGF by the tumour and stromal cells. Thus in this thesis we present our observations upon exploring the metabolic reprogramming of endothelial cells in the presence of different stimulating factors to understand their effects in the central carbon metabolic pathways. 1. In the first part of the study, methodologies like the tracer-based metabolomics and fluxomics have been used to explore the effects of external factors like hypoxia and VEGF on endothelial cell metabolism. The results revealed a major metabolic adaptation induced by hypoxic condition compared to normal oxygen condition and a subtle, but disctint changes induced by VEGF under both normoxia and hypoxia in endothelial cells. VEGF also showed a major metabolic effect in glycogen metabolism of endothelial cells which has not been explored in detail in the past. 2. In the next part of the study the direct effect on metabolic pathways upon growing endothelial cells with tumour cells has been explored for the first time. This preliminary observation was made by growing endothelial cells with tumour cells that possessed distinct metastatic abilities of low and high metastasis. It was observed that endothelial cells grown with low metastatic tumours showed changes in metabolic pathways similar to those changes by VEGF, and the high metastatic tumour showed a completely distinct metabolic change reiterating the metastatic characteristics, compared to the other. This pioneering study has revealed that even the same types of tumours, when distinct only by their invasive and metastatic abilities, can cause different metabolic adaptations in their neighbouring cells, especially revealing the changes in endothelial cells. 3. In addition to the above observations we found that glycogen metabolism is active in endothelial cells and targeting the glycogen degradation reduced endothelial cell viability and functions related to angiogenesis in the in vitro and in vivo studies, which showed that targeting glycogen metabolism can be an alternative strategy against tumour angiogenesis. 4. A general characterization of glycogen metabolism in endothelial cells was performed to understand this pathway in detail. This part of the study has revealed the characteristics of the key enzymes related to glycogen metabolism and the changes in their activities following environmental perturbations like differing glucose and oxygen contents. The glycogen metabolism in endothelial cells has not been explored before and this study emphasizes the importance of regulating the environmental factors when this metabolic pathway is considered as a target for anti-angiogenesis. Thus these observations provide us clues on exploring metabolic targets as alternative anti-cancer therapies by affecting endothelial cell viability and function, which in turn will affect the angiogenic process.