About the lab
Our research interests are in microvascular physiology. We study intrinsic properties of organ-specific endothelial cells and how they shape organ function and microenvironment, as well as their responses to external stimuli. We are also interested in understanding the role of the vasculature in cancer progression and dissemination, as well as organ pre-disposition for metastasis.
Featured research (6)
S ummary Microvascular endothelial cells (MVEC) are plastic, versatile and highly responsive cells, with morphological and functional aspects that uniquely match the tissues they supply. The response of these cells to oxygen oscillations is an essential aspect of tissue homeostasis, and is finely tuned to maintain organ function during physiological and metabolic challenges. Primary MVEC from two continuous capillary networks with distinct organ microenvironments, those of the lung and brain, were pre-conditioned at normal atmospheric (∼ 21 %) and physiological (5 and 10 %) O 2 levels, and subsequently used to compare organ-specific MVEC hypoxia response. Brain MVEC preferentially stabilise HIF-2α in response to hypoxia, whereas lung MVEC primarily accumulate HIF-1α; however, this does not result in significant differences at the level of transcriptional activation of hypoxia-induced genes. Glycolytic activity is comparable between brain and lung endothelial cells, and is affected by oxygen pre-conditioning, while glucose uptake is not changed by oxygen pre-conditioning and is observed to be consistently higher in brain MVEC. Conversely, MVEC mitochondrial activity is organ-specific; brain MVEC maintain a higher relative mitochondrial spare capacity at 5% O 2 , but not following hyperoxic priming. If maintained at supra-physiological O 2 levels, both MVEC fail to respond to hypoxia, and have severely compromised and delayed induction of the glycolytic shifts required for survival, an effect which is particularly pronounced in brain MVEC. Oxygen preconditioning also differentially shapes the composition of the mitochondrial electron transport chain (ETC) in the two MVEC populations. Lung MVEC primed at physioxia have lower levels of all ETC complexes compared to hyperoxia, an effect exacerbated by hypoxia. Conversely, brain MVEC expanded in physioxia display increased complex II (SDH) activity, which is further augmented during hypoxia. SDH activity in brain MVEC primed at 21 % O 2 is ablated; upon hypoxia, this results in the accumulation of near-toxic levels of succinate in these cells. Our data suggests that, even though MVEC are primarily glycolytic, mitochondrial integrity in brain MVEC is essential for metabolic responses to hypoxia; these responses are compromised when cells are exposed to supra-physiological levels of oxygen. This work demonstrates that the study of MVEC in normal cell culture environments do not adequately represent physiological parameters found in situ , and show that the unique metabolism and function of organ-specific MVEC can be reprogrammed by external oxygen, significantly affecting the timing and degree of downstream responses. Graphical Abstract In brief Hypoxia sensing by microvascular endothelial cells (MVEC) is organ-specific, and efficacy of response is affected by external oxygen. While glycolytic capacity is mostly maintained in MVEC regardless of organ or origin, mitochondrial function is required for adequate sensing and timely metabolic shift to glycolysis. Hyperoxygenation of MVEC compromises mitochondrial function, glycolytic shift and survival to hypoxia. Highlights Environmental O 2 influences MVEC hypoxia response in an organ-specific fashion Brain MVEC are unable to respond and survive to hypoxia if hyperoxygenated prior to stress MVEC glycolytic capacity is not affected by O 2 , but the increase in glucose uptake and shift to glycolytic metabolism stifled and delayed in hyperoxidized MVEC High O 2 ablates activity of mitochondria complex II in brain MVEC, significantly disturbing succinate levels Disruption of mitochondrial integrity compromises hypoxia sensing irrespective of glycolytic capacity
Anthracycline-based chemotherapy is a common treatment for cancer patients. Because it is delivered intravenously, endothelial cells are exposed first and to the highest concentrations, prior to diffusion to target cells. Not surprisingly, vascular dysfunction is a consequence of anthracycline therapy. While chemotherapy-induced endothelial damage at administration sites has been investigated, the effects of lower doses encountered by distant microvascular networks has not. The aim of this study was to investigate the impact of epirubicin, a widely used anthracycline, on healthy endothelial cells to elucidate its effects on microvascular physiology. Here, endothelial cells were briefly exposed to low doses of epirubicin to recapitulate levels in circulation following dilution in the blood and compound half-life in circulation. Both immediate and prolonged responses to treatment were assessed to determine changes in endothelial function. Epirubicin caused a decrease in proliferation and viability in hUVEC, with lower doses resulting in a senescent phenotype in a large proportion of cells, accompanied by a significant increase in pro-inflammatory cytokines and a significant decrease in metabolic activity. Epirubicin exposure also impaired endothelial function with delayed wound closure, reduced angiogenic potential and increased monolayer permeability downstream of VE-cadherin internalization. Primary lung endothelial cells obtained from epirubicin-treated mice similarly demonstrated reduced viability and functional impairment. In vivo , epirubicin treatment resulted in persistent reduction in lung vascular density and significantly increased infiltration of myeloid cells. Modulation of endothelial status and inflammatory tissue microenvironment observed in response to low doses of epirubicin may predict risk for long-term secondary pathologies associated with chemotherapy.
Animals require an immediate response to oxygen availability to allow rapid shifts between oxidative and glycolytic metabolism. These metabolic shifts are highly regulated by the HIF transcription factor. The factor inhibiting HIF (FIH) is an asparaginyl hydroxylase that controls HIF transcriptional activity in an oxygen-dependent manner. We show here that FIH loss increases oxidative metabolism, while also increasing glycolytic capacity, and that this gives rise to an increase in oxygen consumption. We further show that the loss of FIH acts to accelerate the cellular metabolic response to hypoxia. Skeletal muscle expresses 50-fold higher levels of FIH than other tissues: we analyzed skeletal muscle FIH mutants and found a decreased metabolic efficiency, correlated with an increased oxidative rate and an increased rate of hypoxic response. We find that FIH, through its regulation of oxidation, acts in concert with the PHD/vHL pathway to accelerate HIF-mediated metabolic responses to hypoxia.
Thrombosis can cause localized ischemia and tissue hypoxia, and both of these are linked to cancer metastasis. Vascular micro-occlusion can occur as a result of arrest of circulating tumour cells in small capillaries, giving rise to microthrombotic events that affect flow, creating localized hypoxic regions. To better understand the association between metastasis and thrombotic events, we generated an experimental strategy whereby we modelled the effect of microvascular occlusion in metastatic efficiency by using inert microbeads to obstruct lung microvasculature before, during and after intravenous tumour cell injection. We found that controlled induction of a specific number of these microthrombotic insults in the lungs caused an increase in expression of the hypoxia-inducible transcription factors (HIFs), a pro-angiogenic and pro-tumorigenic environment, as well as an increase in myeloid cell infiltration. Induction of pulmonary microthrombosis prior to introduction of tumour cells to the lungs had no effect on tumorigenic success, but thrombosis at the time of tumour cell seeding increased number and size of tumours in the lung, and this effect was strikingly more pronounced when the micro-occlusion occurred on the day following introduction of tumour cells. The tumorigenic effect of microbead treatment was seen even when thrombosis was induced five days after tumour cell injection. We also found positive correlations between thrombotic factors and expression of HIF2$\alpha$ in human tumours. The model system described here demonstrates the importance of thrombotic insult in metastatic success and can be used to improve understanding of thrombosis-associated tumorigenesis and its treatment.
In this issue of Cancer Cell, Cantelmo et al. describe how reduction of PFKFB3 enzyme activity can promote vascular normalization. The authors show in turn how this affects vascular permeability and can ultimately improve the efficacy of chemotherapeutic agents.
- Patrick G Johnston Centre for Cancer Research
About Cristina Branco
- Studying endothelial cell metabolism and physiology in health and disease, and impact of the microvasculature on organ function and cancer metastasis. We use primary endothelial cells and in vivo models, state of the art equipment and technology to assess baseline and challenge responses in real time. Currently working on role of HIF transcription factors in EC organ remodelling and organ specific microvascular properties.