Organ-specific heterogeneity in the endothelial cell hypoxia response

Change log

The aim of this thesis is to study the hypoxia response pathways within microvascular endothelial cells. In the first results chapter I investigated the differential role of the two hypoxia inducible transcription factors HIF-1α and HIF-2α in the murine lung endothelium during cancer metastasis, and how the hypoxia response of the vascular endothelium remodels the lung pre-metastatic niche. Previous work had shown that lung endothelial-specific knockouts of HIF-1α and HIF-2α respectively inhibit and promote metastatic success. Thus, hypoxic stimuli of varying lengths were used to preferentially stabilise either HIF isoform prior to tumour cell injection. Acute hypoxia resulted in stabilisation of HIF-1α, endothelial cell death, and increased vascular permeability, facilitating tumour cell extravasation. This was potentiated by the recruitment and retention of specific myeloid cells that further supported a pro-metastatic environment. Chronic hypoxia on the other hand reduced metastatic success, in conjunction with HIF-2α-mediated increases in endothelial cell viability and vascular integrity. These effects were reversed by the endothelial-specific deletion of each isoform. Having investigated the role of the HIF pathway lung metastasis, the second results chapter aimed to broaden the scope to the vasculature of a different metastatic target of breast cancer, the brain. To achieve this, I cultured microvascular endothelial cells (MVECs) of both organs in vitro and exposed them to hypoxia (1% O₂). My initial observations suggested that brain MVECs fared much more poorly in hypoxia than their lung counterparts. This was puzzling, given that the brain microvasculature is exposed to much lower oxygen levels in vivo and should thus presumably be better equipped to deal with hypoxia. Crucially however, both tissues contain much less oxygen than the 18.5% O₂ found in standard tissue culture incubators, and thus the cellular responses seen in these conditions are unlikely to relevantly represent the adaptations seen in physiological conditions. To investigate this possibility, the subsequent experimental set-up included culturing each cell population also in physiological O₂ tensions. Indeed, this resulted in a much more pronounced hypoxia response, both in terms of viability and expression of key genes. Still, there were pronounced differences between the two cell types, most notably a differential stabilisation of HIF isoforms. Brain MVECs relied much more heavily on HIF-2α whereas lung MVECs contained considerably higher levels of HIF-1α. The third results chapter further explored the effect of hyperoxia versus physioxia on brain and lung MVECs, with a specific focus on their metabolic response. I performed metabolic stress tests to measure the cells’ mitochondrial and glycolytic capacities if cultured at normoxia or physioxia, both at baseline and after subsequent exposure to hypoxia. Furthermore, I assessed the metabolic adaptation of each cell type to hypoxia in real time. Similar to what was observed in chapter 2, both cell types but particularly brain MVECs grown at ambient oxygen levels were less able to increase glycolysis in response to hypoxia. Furthermore, brain MVECs also displayed severely impaired mitochondrial metabolism, as evidenced by reduced maximal respiration and expression of respiratory complex proteins. Overall, this thesis highlights the importance of hypoxia response pathways in endothelial cells in pathology, such as during cancer metastasis, as well as during normal cell physiology, shown by the detrimental effects of non-physiological oxygen levels.

Branco, Cristina
Endothelial cell, Metabolism, Hypoxia, HIF, Metastasis
Doctor of Philosophy (PhD)
Awarding Institution
University of Cambridge