Investigating mechanisms which control blood vessel formation and function: How does GPCR signalling control vascular permeability within the Blood-Br

To ensure tissue homeostasis, the central nervous system must be protected from hormones, neurotransmitters or pathogens circulating in the blood, while still allowing vital nutrients to reach the brain. To achieve this, blood vessels which vascularize the central nervous system (CNS) display unique properties, termed the blood-brain barrier (BBB). The BBB heavily restricts vessel permeability and thus protects the brain from injury and disease. Loss of barrier properties during diseases including stroke, diabetes and vascular dementia contribute to underlying pathology and disease progression. Conversely, the restrictive permeability of the BBB poses challenges for drug delivery to the CNS. The genetic mechanisms which regulate permeability of the BBB are poorly understood but are potential therapeutic targets where abnormal vascular barrier function contributes to disease. We have identified a G-Protein Coupled Receptor (GPCR) essential for normal BBB permeability, but how it achieves this function remains unknown.

We use zebrafish to study how vascular permeability is controlled because zebrafish embryos are optically translucent and develop outside of the parent. This allows us to label blood vessels fluorescently and directly observe leaky blood vessels in zebrafish embryos using a microscope. In zebrafish, the BBB is quickly established between 2 and 3 days post fertilisation and importantly, mechanisms which regulate blood vessel formation and function in zebrafish are highly conserved with humans.

Using CRISPR/Cas9 genome editing, we have generated novel zebrafish mutants in a GPCR complex. Our zebrafish mutants possess a leaky BBB. By employing transgenic and mutant zebrafish embryos with fluorescently labelled blood vessels, this project will examine the role of signalling via this GPCR in regulating vessel permeability and BBB function.

Zebrafish GPCR mutants display hyperpermeability within the developing brain vasculature at stages where these vessels are normally intact, and the BBB is normally functional. Interestingly, extravasation of large numbers of microvesicles in our mutants suggests vascular hyperpermeability is a transcellular process. We hypothesise that our GPCR mutants display an activation of transcellular permeability pathways leading to increased permeability of the BBB.

To test this hypothesis, this project will utilise RNA sequencing to identify the transcriptional mechanisms underlying GPCR -mediated vascular permeability. To test candidate genes, we will employ cutting edge CRISPR/Cas9 and CRISPR interference technologies developed within our group. To determine how the GPCR complex controls BBB permeability, we will perform live imaging of blood vessel formation and function within zebrafish embryos using confocal and lightsheet fluorescence microscopy. This project will identify novel molecular mechanisms which control vessel permeability and which may be candidates for therapeutic manipulation during disease.