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Role of peroxisomal fatty acid β-oxidation in vessel sprouting

Periodic Reporting for period 1 - PerMet (Role of peroxisomal fatty acid β-oxidation in vessel sprouting)

Reporting period: 2018-04-01 to 2020-03-31

Endothelial cells (ECs) form the inner lining of blood vessels and are involved in the development of devastating diseases like cancer or the eye disease age-related macular degeneration (AMD). ECs are traditionally divided into three subtypes: While being quiescent 'phalanx' cells in established blood vessels, they differentiate into actively proliferating 'stalk' cells and migrating 'tip' cells during the formation of new vessel branches (vessel sprouting or ‘angiogenesis’). However, ECs might be far more heterogeneous than this simple taxonomy suggest, considering the distinctly different functions they serve across tissues. Blood vessel formation from existing quiescent blood vessels in cancer and AMD is promoted by inducing excessive EC proliferation and migration. In cancer, this leads to the formation of highly disordered vessels which enable the survival of the tumor. Thus, anti-angiogenic therapies aimed to reduce tumor vessel development constitute an important approach for cancer therapeutics. The host lab recently showed that angiogenesis is controlled by metabolic processes in ECs, like for instance mitochondrial fatty acid oxidation (FAO). The manipulation of EC metabolism could thus be therapeutically used to normalize blood vessel formation in cancer and other diseases.
Peroxisomes are small metabolic organelles and are present in virtually every human cell. Genetic defects causing the loss of peroxisomes can have severe consequences for patients. Although our knowledge about peroxisomes is very limited, this indicates that these organelles are vital for human health. Among others, they are crucial for oxidizing specific fatty acids (e.g. very long chain fatty acids, VLCAs) by peroxisomal FAO (pFAO). Though peroxisomes are numerously present in ECs, their role in the formation of blood vessels is entirely unknown. Investigating their role could offer valuable insights into the metabolic workings of ECs and thus provide us with potential therapeutic approaches for diseases characterized by abnormal angiogenesis.
The objective of this project was to study the function of peroxisomes in ECs by investigating the effects of peroxisomal gene silencing on EC function and vessel sprouting in cultured ECs and in an EC-specific knock-out mouse model (Aim 1). Additionally, we planned to characterize the (metabolic) gene signature of different EC subtypes (beyond quiescent, tip and stalk cells), and investigate the impact of peroxisomal gene loss on the distribution of different subtypes using single-cell RNA-sequencing (scRNAseq, Aim 2). Furthermore, we aimed to characterize the metabolic fate of peroxisomally oxidized VLCFAs (Aim 3).
Aim 1 – The role of peroxisomes in angiogenesis
To investigate the role of peroxisomes and pFAO in angiogenesis, we silenced specific genes crucial for pFAO or peroxisome biogenesis in endothelial cells (ECs). First, we performed a battery of in vitro assays in ECs, in which we used lentiviral vectors to knock down the expression of peroxisomal genes (“KD-ECs”): We validated that this knockdown in ECs indeed impaired peroxisomal function and caused for instance disturbed levels of specific peroxisomal metabolites. We assessed the proliferation (a characteristic of dividing stalk cells during angiogenesis) of KD-ECs by measuring the incorporation of radioactively labelled thymidine into newly built DNA. Using wound scratch assays, we measured the migration of KD-ECs, a characteristic of tip cells. Finally, we assessed the development of sprouts from KD-EC spheroids as an 3D in vitro model of vessel sprouting. These experiments revealed decreased sprouting and proliferation in KD-ECs, and thus indicated an important role of peroxisomes and pFAO for in vitro EC vessel sprouting. Using gene expression (transcriptomic) analyses as well as a multitude of metabolic assays, we observed that the loss of peroxisomes additionally affects multiple other cellular functions and metabolic pathways, underlining the importance of peroxisomes for EC function.
To study the role of peroxisomes in the formation of blood vessels in vivo, we successfully generated mice with an EC-specific loss of peroxisomal genes and applied various angiogenesis models. These experiments confirmed the important role of peroxisomes in the function of ECs and angiogenesis in vivo.

Aim 2 – scRNAseq of ECs
To investigate which kinds of different EC subtypes exist in vessels across tissues in vivo, we established a single-cell transcriptome atlas of murine endothelial cells. We isolated ECs from multiple organs from healthy adult mice and performed scRNAseq, revealing that ECs are much more heterogeneous than previously assumed, and can be divided in over 75 subtypes, depending on organ, tissue bed and activation state. This data will serve as an invaluable resource for future studies of ECs. In addition to the proposed experiments, we used scRNAseq of ECs to investigate the endothelial metabolic plasticity in pathological angiogenesis in AMD and cancer. This approach identified multiple novel metabolic pathways in ECs, which are crucial for pathological angiogenesis and which could thus serve as potential targets to manipulate angiogenesis for therapeutic purposes. All this data was published in top tier scientific journals, reaching a high number of scientists from various fields.
To investigate the effect of the loss of peroxisomes on the mRNA expression pattern in ECs, we performed RNA-sequencing of ECs upon silencing of peroxisomal genes in vitro (see Aim 1).

Aim 3 – VLCFA isotope tracing
To investigate the metabolic fate of VLCFAs in ECs, we have started optimizing the preparation of isotope-labelled versions of these highly hydrophobic molecules (because their solubilization proved to be very challenging), as well as developing protocols for VLCFA uptake by ECs.
Aim 1: The study of loss of functional peroxisomes has confirmed our hypothesis that peroxisomes in ECs are important during angiogenesis. This discovery can help establishing novel anti-angiogenic therapies based on manipulating the metabolism of ECs. Our findings regarding the vital role of peroxisomes in ECs can not only give us a deeper insight into the metabolic framework of angiogenesis but could additionally offer valuable insights into the function of peroxisomes in the context of peroxisomal diseases, and can in this way contribute to finding therapeutic approaches to these incurable diseases.

Aim 2: The atlases of murine EC subtypes in health and disease, which we have published, present a powerful discovery and resource tool for future research that can be used for instance to identify promising targets or investigate molecular mechanisms in ECs and blood vessels. Moreover, the RNA-sequencing of ECs upon silencing of peroxisomal genes will help us to investigate the consequences of the loss of peroxisomes in ECs.

Aim 3: The investigation of the metabolic fate of peroxisomally metabolized VLCFAs and other peroxisomal metabolites will provide the first comprehensive insight into which metabolic pathways oxidized VLCFA and other metabolites are utilized, helping us to fill the gaps in our knowledge of peroxisomal and EC metabolism. This data can also provide valuable clues about previously unknown metabolite alterations in patients with peroxisomal diseases, that might contribute to the disease-specific defects and could potentially be targeted therapeutically.
PerMet Graphical Abstract