Studies on the Healthy and Diseased Blood-Brain Barrier

Brain vasculature is supplying the brain with oxygen and nutrients, which is important for brain function. Brain capillaries, the main interface for exchange of these nutrients and oxygen span the whole brain with a network of approximately 640 km of vessels. Since the brain has only very limited energy storage capacity, it is highly dependent on continuous blood flow and efficient exchange of components from blood to brain parenchyma. Changes in blood flow are directly influencing the brain in physiological conditions e.g. during neuronal activity or in pathological conditions e.g. stroke. However, uncontrolled exchange of blood borne molecules is detrimental for the brain, since the blood contains a plethora of molecules, which can act as neurotoxins (e.g. hemoglobin, free ion) when entering the brain. These substances have to be kept out the brain. Therefore, a structure called the blood-brain barrier is lining the brain`s entire vasculature, sheltering the brain from peripheral influence while at the same time allowing controlled exchange of nutrients and gas into brain parenchyma and waste export into the blood stream. The first part of this thesis is focusing on differences and similarities of healthy, adult brain vasculature of genetically different mouse lines. To confirm that brain vasculature of these different lines present with intact and functional endothelial cells and blood-brain barrier, morphological analysis and analysis of extravasation of exogenous tracer were performed. After verifying that no major differences were found in morphology and BBB permeability between vasculature of different backgrounds, the transcriptomic analysis of the healthy endothelium was performed. I investigated the transcriptome of endothelial cells – the cell type lining blood vessels – of different regions of male and female mice of different background. In line with previous knowledge, male and female endothelium is expressing sex specific genes, e.g. Xist, Tsix, Lars2 or Uty. Furthermore, animals of long inbred strains compared to recently wild derived strains showed differences in gene expression of brain endothelium mostly involved in immune response, oxygen level response and transmembrane ion transport. 15 The second part of this thesis contains analysis of brain vasculature in healthy and tumor condition. Within this part, the transcriptome of endothelial cell enriched samples from glioblastoma, brain metastases, and epilepsy and autopsy controls were analyzed focusing on blood-brain barrier genes. Glioblastoma and brain metastases endothelial cells showed differences in genes encoding for regulation of cell proliferation, cell migration, angiogenesis, and extracellular matrix composition when compared to controls. Furthermore, we found evidence for the presence of two different subpopulations of fibroblasts differentially expressed at glioblastoma vasculature pointing towards a less recognized role of vessel-associated fibroblast-like cells in glioblastoma. The third part of this thesis is focusing on the development of a protocol to investigate the diversity of brain tumor endothelial cells using single cell RNA sequencing (SmartSeq2). A protocol for isolation of endothelial cells from fresh glioblastoma and control tissue was developed. Endothelial cells were sorted from the tumor and healthy tissue using two different endothelial surface markers. The sorted endothelial cells were subjected to single cell RNA sequencing using the Smartseq2 protocol. Analysis of RNAseq data pointed to a high variability in endothelial cells genetic profiles with only few cells expressing typical endothelial gene signatures