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Organ function relies on microvascular networks to maintain homeostatic equilibrium, which varies widely in different organs and during different physiological challenges. The endothelium role in this critical process can only be evaluated in physiologically relevant contexts. Comparing the responses to oxygen flux in primary murine microvascular EC (MVEC) obtained from brain and lung tissue reveals that supra-physiological oxygen tensions can compromise MVEC viability. Brain MVEC lose mitochondrial activity and undergo significant alterations in electron transport chain (ETC) composition when cultured under standard, non-physiological atmospheric oxygen levels. While glycolytic capacity of both lung and brain MVEC are unchanged by environmental oxygen, the ability to trigger a metabolic shift when oxygen levels drop is greatly compromised following exposure to hyperoxia. This is particularly striking in MVEC from the brain. This work demonstrates that the unique metabolism and function of organ-specific MVEC (1) can be reprogrammed by external oxygen, (2) that this reprogramming can compromise MVEC survival and, importantly, (3) that ex vivo modelling of endothelial function is significantly affected by culture conditions. It further demonstrates that physiological, metabolic and functional studies performed in non-physiological environments do not represent cell function in situ, and this has serious implications in the interpretation of cell-based pre-clinical models.

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.

Growth of a fetus is dependent upon the functional capacity of its placenta, but how the latter is matched to fetal demands is currently unknown. Critically, there is continuous expansion of the feto-placental microvasculature throughout pregnancy, along with morphogenic modifications in the overlying trophoblast epithelium. Here we demonstrate, through fetal and trophoblast specific genetic manipulations in the mouse, that signalling by IGF2 from the feto-placental endothelium and endocrine actions of circulating fetal IGF2 are required. We provide evidence that endothelial and fetal-derived IGF2 plays an important role in trophoblast morphogenesis, acting through Gcm1 and Synb. The effects on placental microvasculature expansion are mediated through IGF2R and angiopoietin-Tie2/TEK signalling. Thus, our study reveals a direct role for IGF2-IGF2R axis on matching fetal demand to placental supply and establishes the principle that hormone-like signals from the fetus play important roles in the control of placental vascularization and trophoblast morphogenesis, findings that have potential clinical implications.

COVID-19 has proven to be a metabolic disease resulting in adverse outcomes in individuals with diabetes or obesity. Patients infected with SARS-CoV-2 and hyperglycemia suffer from longer hospital stays, higher risk of developing acute respiratory distress syndrome (ARDS), and increased mortality compared to those who do not develop hyperglycemia. Nevertheless, the pathophysiological mechanism(s) of hyperglycemia in COVID-19 remains poorly characterized. Here we show that insulin resistance rather than pancreatic beta cell failure is the prevalent cause of hyperglycemia in COVID-19 patients with ARDS, independent of glucocorticoid treatment. A screen of protein hormones that regulate glucose homeostasis reveals that the insulin sensitizing adipokine adiponectin is reduced in hyperglycemic COVID-19 patients. Hamsters infected with SARS-CoV-2 also have diminished expression of adiponectin. Together these data suggest that adipose tissue dysfunction may be a driver of insulin resistance and adverse outcomes in acute COVID-19., Competing Interests: Conflict of interest R.E.S. is on the scientific advisory board of Miromatrix Inc. R.E.S. is a speaker and consultant for Alnylam Inc. The other authors have no conflict of interest.