Organ-specific heterogeneity in the endothelial cell hypoxia response

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

Acute and chronic hypoxia differentially predispose lungs for metastases

Abstract: Oscillations in oxygen levels affect malignant cell growth, survival, and metastasis, but also somatic cell behaviour. In this work, we studied the effect of the differential expression of the two primary hypoxia inducible transcription factor isoforms, HIF-1α and HIF-2α, and pulmonary hypoxia to investigate how the hypoxia response of the vascular endothelium remodels the lung pre-metastatic niche. Molecular responses to acute versus chronic tissue hypoxia have been proposed to involve dynamic HIF stabilization, but the downstream consequences and the extent to which differential lengths of exposure to hypoxia can affect HIF-isoform activation and secondary organ pre-disposition for metastasis is unknown. We used primary pulmonary endothelial cells and mouse models with pulmonary endothelium-specific deletion of HIF-1α or HIF-2α, to characterise their roles in vascular integrity, inflammation and metastatic take after acute and chronic hypoxia. We found that acute hypoxic response results in increased lung metastatic tumours, caused by HIF-1α-dependent endothelial cell death and increased microvascular permeability, in turn facilitating extravasation. This is potentiated by the recruitment and retention of specific myeloid cells that further support a pro-metastatic environment. We also found that chronic hypoxia delays tumour growth to levels similar to those seen in normoxia, and in a HIF-2α-specific fashion, correlating with increased endothelial cell viability and vascular integrity. Deletion of endothelial HIF-2α rendered the lung environment more vulnerable to tumour cell seeding and growth. These results demonstrate that the nature of the hypoxic challenge strongly influences the nature of the endothelial cell response, and affects critical parameters of the pulmonary microenvironment, significantly impacting metastatic burden. Additionally, this work establishes endothelial cells as important players in lung remodelling and metastatic progression

Author Correction: Acute and chronic hypoxia differentially predispose lungs for metastases.

An amendment to this paper has been published and can be accessed via a link at the top of the paper

Argus Vision : A Tracking Tool for Exhibition Designers

Contemporary exhibitions are increasingly staged using extensive and often interactive media. To create such exhibitions, exhibition design companies employ professionals from a wide range of different disciplines. The support of interdisciplinary exhibition designers in the design process is one goal of research in Human-Computer Interaction. This includes the deployment of Do-It-Yourself (DIY) Tools that enable professionals from all disciplines involved to design and create interactive media themselves. In this paper, we will present Argus Vision, a DIY Tool, which allows exhibition designers the use of camera-tracking to rapidly prototype and develop immersive exhibitions and interactive installations. We successfully used Argus Vision in two real-world case studies both in the prototyping and in the deployment of two installations in exhibitions. Additionally, we conducted expert interviews with exhibition designers, investigating the tool’s usefulness for them.publishe

Argus Vision : A Tracking Tool for Exhibition Designers

Contemporary exhibitions are increasingly staged using extensive and often interactive media. To create such exhibitions, exhibition design companies employ professionals from a wide range of different disciplines. The support of interdisciplinary exhibition designers in the design process is one goal of research in Human-Computer Interaction. This includes the deployment of Do-It-Yourself (DIY) Tools that enable professionals from all disciplines involved to design and create interactive media themselves. In this paper, we will present Argus Vision, a DIY Tool, which allows exhibition designers the use of camera-tracking to rapidly prototype and develop immersive exhibitions and interactive installations. We successfully used Argus Vision in an exhibition as well as in a proof-of-concept study in our lab. Additionally, we conducted expert interviews with exhibition designers, investigating its usefulness for them.publishe

Acute and chronic hypoxia differentially predispose lungs for metastases

Abstract: Oscillations in oxygen levels affect malignant cell growth, survival, and metastasis, but also somatic cell behaviour. In this work, we studied the effect of the differential expression of the two primary hypoxia inducible transcription factor isoforms, HIF-1α and HIF-2α, and pulmonary hypoxia to investigate how the hypoxia response of the vascular endothelium remodels the lung pre-metastatic niche. Molecular responses to acute versus chronic tissue hypoxia have been proposed to involve dynamic HIF stabilization, but the downstream consequences and the extent to which differential lengths of exposure to hypoxia can affect HIF-isoform activation and secondary organ pre-disposition for metastasis is unknown. We used primary pulmonary endothelial cells and mouse models with pulmonary endothelium-specific deletion of HIF-1α or HIF-2α, to characterise their roles in vascular integrity, inflammation and metastatic take after acute and chronic hypoxia. We found that acute hypoxic response results in increased lung metastatic tumours, caused by HIF-1α-dependent endothelial cell death and increased microvascular permeability, in turn facilitating extravasation. This is potentiated by the recruitment and retention of specific myeloid cells that further support a pro-metastatic environment. We also found that chronic hypoxia delays tumour growth to levels similar to those seen in normoxia, and in a HIF-2α-specific fashion, correlating with increased endothelial cell viability and vascular integrity. Deletion of endothelial HIF-2α rendered the lung environment more vulnerable to tumour cell seeding and growth. These results demonstrate that the nature of the hypoxic challenge strongly influences the nature of the endothelial cell response, and affects critical parameters of the pulmonary microenvironment, significantly impacting metastatic burden. Additionally, this work establishes endothelial cells as important players in lung remodelling and metastatic progression

Protocol to isolate and culture primary mouse feto-placental endothelial cells.

In the mouse, feto-placental endothelial cells (FPEC) line the inner surface of the feto-placental blood vessels located within placental labyrinthine zone and play critical roles in placental development and function. Here, we present a detailed protocol for isolation and culture of primary mouse FPEC, as well as two complementary methods (immunohistochemistry staining and flow cytometry analysis) to assess their purity. These cells are suitable for downstream ex vivo studies to investigate their functional properties, both in normal and pathological contexts. For complete details on the use and execution of this protocol, please refer to Sandovici et al. (2022).This work was supported by the Biotechnology and Biological Sciences Research Council (grant BB/H003312/1 to M.C.) and the Medical Research Council (MRC_MC_UU_12012/4 to M.C.)

Hyperoxia Reprogrammes Microvascular Endothelial Cell Response to Hypoxia in an Organ-Specific Manner.

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

Blended Museum : The Interactive Exhibition "Rebuild Palmyra?"

"Rebuild Palmyra?" is a multimedia exhibition about the ancient city of Palmyra, its destruction by Daesh, and the question of whether it should be rebuilt. As such, it tackles today's pressing question of how humanity should deal with the destruction of cultural heritage. In the design of the exhibition, we pursued the Blended Museum approach, in which we strive to seamlessly integrate interactive media into exhibition design to increase the overall visitor experience. In this work, we present the exhibition, which consists of four rooms. We focus on three interactive installations in which the topic of reconstructing Palmyra is mediated using new technologies such as 3D Printing, Augmented Reality, and Virtual Reality. The installations helped visitors in developing their own point of view on the question of rebuilding Palmyra. Lastly, we provide insights into the technical implementation of the installations and discuss the results of quantitative and qualitative evaluations.publishe