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Investigating the role of HIFs in myeloid cells during experimental choroidal neovascularisation

Final Report Summary - AMD_CNV_HIF (Investigating the role of HIFs in myeloid cells during experimental choroidal neovascularisation)

Retinal neovascularisation is when new, fragile blood vessels grow, uncontrolled at the back of the eye. These vessels can leak blood, which damages the light-sensing cells of the retina causing sight loss. Neovascularisation is a feature of several conditions including diabetes and macular degeneration. It also occurs in some cases where premature babies have required treatment with high doses of oxygen to support the development of their immature lungs. The retinas of these children are also immature, and the high oxygen levels can inhibit proper blood vessel growth in the eyes. On return of these babies to normal air, blood vessel growth in the eyes starts again, but in an uncontrolled and defective manner. The best-known activator of neovascularisation is a molecule called Vascular Endothelial Growth Factor (VEGF). There are drugs that can stop VEGF working, but these only slow the progression of sight loss, which means there are other factors which must also influence neovascularisation. The Hypoxia Inducible Factors (HIFs) family of molecules regulate VEGF in many different cell types in the body. HIFs of the immune system are thought to be another mechanism for driving neovascularisation. We have been investigating the role of HIFs in neovascularisation to determine if by stopping/increasing their action we can further slow or even stop this process. To do this, we have created mice which lack the genes to create the HIF molecules (Hif1α, Hif2α and Von Hippel-Lindau tumor suppressor (Vhl)) in their myeloid cells, the type of immune cells that are involved in neovascularisation. These mice have normal HIF genes in the rest of the cells of their bodies, allowing us to investigate just the role of HIFs in myeloid cells and specifically their contribution to neovascularisation individually and in combination with one another. To induce neovascularization in these mice, we use a model called Oxygen-induced Retinopathy (OIR). This model mimics what happens in premature babies who required high levels of oxygen after birth. Mice pups are placed in an oxygen chamber with high oxygen levels (75% of oxygen) for five days after which they are returned to normal air (21% of oxygen). When they come out of the chamber, there is a big area on the surface of their retina without blood vessels, because of the halted growth. Five days later, uncontrolled vessels have grown and filled in part of this empty area, forming what we call neovascular tufts (Figure 1A). When we place mice lacking Hif1α, Hif2α or both (from their myeloid cells) in the chamber, we don’t find any difference in the empty area or the formation of neovascular tufts when compared with normal mice (Figure 1B), meaning that Hif1α and Hif2α in myeloid cells are not essential for neovascularisation. However, when we look at the same responses in mice lacking Vhl in myeloid cells, we see the same neovascular tufts as in control mice, but the empty area in the middle of the retina is smaller (Figure 1b). Vhl inactivates Hif1α and Hif2α, meaning that it turns off their activity. Therefore, in mice without Vhl, HIFs are over-activated and their effects increased. These data suggest that increased activity of HIFs in myeloid cells helps blood vessels to grow faster but in a more controlled fashion to cover the empty areas of the retina. We are still investigating why and how this happens and if it could be useful when designing new therapies for neovascularisation in the future. In parallel to this central part of the project, we have further optimised the OIR model. We have tested an alternative version of the model that reduces the time the mice pups spend in the oxygen chamber from 5 to 3 days. We have found that this shorter version of the model is equal to the original model both for safety to the mice and in the stimulation of neovascularisation. The shorter model may provide researchers with a faster yet equally reliable way to study OIR which may also improve the wellbeing of the animals used.

Figure 1. A) Schematic representation of OIR protocol. Mice are place for five days in a chamber with 75% of oxygen, what stops the growing of the vessels of the retina and a central area “empty” area can be measured right after oxygen exposure (depicted in white). Five days later, uncontrolled vessels form neovascular tufts (depicted in yellow) that invade part of the “empty” area. B) Mice lacking Hif1α, Hif2α or both specifically in myeloid cells showed the same neovascular responses than control mice, after OIR protocol (from Liyanage et al. ATVB 2016). C) Mice lacking Vhl in myeloid cells, which have then increased activity of HIFs, showed the same neovascularisation than control mice but the remaining empty area was significantly smaller, after OIR.