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

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)

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

Endothelial cells and organ function: applications and implications of understanding unique and reciprocal remodelling

The microvasculature is a heterogeneous, dynamic and versatile component of the systemic circulation, with a unique ability to locally self-regulate and to respond to organ demand and environmental stimuli. Endothelial cells from different organs display considerable variation but it is currently unclear to what extent functional properties of organ-specific endothelial cells are intrinsic, acquired and/or reprogrammable. Vascular function is a fundamental pillar of homeostasis, and dysfunction results in systemic consequences for the organism. Additionally, vascular failure can occur downstream of organ disease or environmental stress, often driving an exacerbation of symptoms and pathologies originally independent of the local circulation. The understanding of the molecular mechanisms underlying endothelial physiology and metabolism holds the promise to inform and improve diagnosis, prognosis and treatment options for a myriad of conditions as unrelated as cancer, neurodegeneration or pulmonary hypertension, and likely everything in between, if we consider that also treatments for such conditions are primarily distributed via the blood stream. However, studying endothelial function has its challenges: the origin, isolation, culture conditions and pre-conditioning stimuli make this an extremely variable cell type to study, and difficult to source. Animal models exist but are neither trivial to generate nor necessarily adequately translatable to human disease. In this article, we aim to illustrate the breath of microvascular functions in different environments, highlighting current and pioneering studies that have advanced our insight into the importance of the integrity of this tissue, as well as the limitations posed by its heterogeneity and plasticity.</p

Argus Vision: A Tracking Tool for Exhibition Designers

Abstract 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.</jats:p

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

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

Organ-specific heterogeneity in endothelial cell hypoxia response

The microvasculature is a heterogeneous, dynamic and versatile component of the systemic circulation, with a unique ability to locally self-regulate, to respond to organ demand and systemic stimuli, with deep effects on pathophysiological responses. In spite of the variation found among EC from different organs, it is not clear if that is intrinsically determined or a result of microenvironmental cues. We compared the hypoxia response of primary murine lung microvascular EC (lMVEC), which we have largely explored in the context of metastatic disease and pulmonary hypertension, to those of the brain (bMVEC), a network of very distinct environment and demands. Unexpectedly, our results show that bMVECs lose viability earlier and more dramatically when exposed to 1% O2 than lMVEC. This correlates with increased expression of the autophagic protein BNIP3. Hypoxic bMVECs also show metabolic dormancy, and are unresponsive to mitochondrial toxins, and only modest increases in glycolytic rate, compared to lMVECs, in keeping with mild induction of hypoxia-induced transcription factor HIF-1훼 (regulator of the switch to glycolysis). This was further seen in glucose uptake rates (glycolytic input) and extracellular acidification (glycolytic output); Overall bMVEC have reduced and attenuated hypoxia response compared to lMVEC. We investigated if these differences could be a result of environmental reprogramming, and subsequently cultured these cells at physiological oxygen levels. Cells cultured at 10% O2 (average O2 in lung) and 5% O2 (physiological O2 in brain), we found that the metabolic response to hypoxia (1% O2) was strikingly different, both between the two EC types and within the same cells cultured at different atmospheres. In all cases, higher O2 exposure elevates baseline OXPHOS and stifles the capacity to trigger glycolysis. At 10% O2 the response to hypoxia in bMVEC is still impaired compared to lMVEC, but when cultured at 5% O2, this is reversed, and bMVEC show a quick and pronounced increase in glycolytic rate when transferred to 1% O2, whereas lMVECs display no further increase in glycolysis upon 1%, suggesting 5% O2 is already hypoxic for a lMVEC. These results show that microvascular plasticity and responses are intrinsic, but to a large extent reprogrammable by environmental priming, and this knowledge is central in understanding and treating a myriad of insults to the microvasculature (surgery, wounding, any kind of circulating therapeutics, or other systemic signals)

Object Semantic Segmentation in Point Clouds—Comparison of a Deep Learning and a Knowledge-Based Method

Through the power of new sensing technologies, we are increasingly digitizing the real world. However, instruments produce unstructured data, mainly in the form of point clouds for 3D data and images for 2D data. Nevertheless, many applications (such as navigation, survey, infrastructure analysis) need structured data containing objects and their geometry. Various computer vision approaches have thus been developed to structure the data and identify objects therein. They can be separated into model-driven, data-driven, and knowledge-based approaches. Model-driven approaches mainly use the information on the objects contained in the data and are thus limited to objects and context. Among data-driven approaches, we increasingly find deep learning strategies because of their autonomy in detecting objects. They identify reliable patterns in the data and connect these to the object of interest. Deep learning approaches have to learn these patterns in a training stage. Knowledge-based approaches use characteristic knowledge from different domains allowing the detection and classification of objects. The knowledge must be formalized and substitutes the training for deep learning. Semantic web technologies allow the management of such human knowledge. Deep learning and knowledge-based approaches have already shown good results for semantic segmentation in various examples. The common goal but the different strategies of the two approaches engaged our interest in doing a comparison to get an idea of their strengths and weaknesses. To fill this knowledge gap, we applied two implementations of such approaches to a mobile mapping point cloud. The detected object categories are car, bush, tree, ground, streetlight and building. The deep learning approach uses a convolutional neural network, whereas the knowledge-based approach uses standard semantic web technologies such as SPARQL and OWL2to guide the data processing and the subsequent classification as well. The LiDAR point cloud used was acquired by a mobile mapping system in an urban environment and presents various complex scenes, allowing us to show the advantages and disadvantages of these two types of approaches. The deep learning and knowledge-based approaches produce a semantic segmentation with an average F1 score of 0.66 and 0.78, respectively. Further details are given by analyzing individual object categories allowing us to characterize specific properties of both types of approaches.</jats:p