Vascular leakage
Vascular leakage is the results of hyperpermeability which leads to extravasation of fluids and proteins, resulting in interstitial oedema. Vascular leakage is a common feature of many diseases ranging from cancer and inflammation to metabolic diseases such as diabetes, and in the eye it is a hallmark feature of diabetic retinopathy and neovascular age-related macular degeneration. VEGF is a major leakage-inducing factor and many therapies have been developed to contrast its action. However, these therapies only work in a limited percentage of patients.
At the UCL Institute of Ophthalmology I studied the role of VEGF in retinal vascular leakage. We used explants of rodent retinae to study acute neurovascular permeability and signal transduction and the role of AMP-activated protein kinase (AMPK). Here we observed that VEGF leads to AMPK activation with consequent phosphorylation of endothelial nitric oxide synthase (eNOS) and VE-cadherin. In parallel, AMPK also mediates phosphorylation of p38 MAP kinase and HSP27, indicating that it regulates paracellular junctions and cellular contractility, both previously associated with endothelial permeability (Dragoni et al., J Cell Sci, 2021).
.png)
In the ex-vivo retina model, the carotid artery of the animal is cannulated and perfused with heparin to avoid blood clothing, cardioplegic solution to stabilise the vasculature and Evan's blue to colour the vessels.
Next, the eye is removed and enucleated and the retina is isolated together with the attached sclera. The retina is flattened and mounted into a silicon pad with a metal ring and pins to keep it still. A glass needle is made with a puller and sharpened with a grinder. Sulforhodamine-B is sucked into the needle and injected into the retinal vasculature. Sulforhodamine-B is fluorescent as well as the whole retinal vasculature after the injection. A vessel is chosen and a baseline is recorded for about 30 seconds, before the agonist is added to the top of the retina, whilst recoding.
Permeability is then measured as loss of fluorescence over time.
To expand my knowledge on VEGF-induced permeability, I am now defining the role of its co-receptor, the transmembrane protein neuropilin 1 (NRP1), in retinal endothelial cells. Firstly, my work seeks to determine how NRP1 promotes VEGF signalling along several distinct downstream signalling pathways. The outcomes will be immediately relevant for our understanding of VEGF-induced vascular permeability mechanisms in the eye. Secondly, my work seeks to determine why a previously developed NRP1 inhibitor, designed to impair pathological blood vessel growth, activates rather than inhibits vascular leakage.