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.
