Reconstruction of a human blood-retina barrier and perfused vascular system to investigate the role of APOE variants in age-related macular degeneration - Project Summary/Abstract Age-related macular degeneration (AMD) is a leading cause of vision loss after 50 years of age. While genetic studies have identified various polymorphisms associated with AMD, there still is no clear picture of the mechanisms leading to disease initiation and progression. The main pathological hallmark of AMD is the accumulation of lipid-rich deposits, namely drusen, between the retinal pigmented epithelium (RPE) and the choroid, leading to retina degeneration. Several risk variants for AMD occur in lipid-related genes, including the apolipoprotein E (APOE) gene, but their role in AMD is unknown. We hypothesize that differences in lipid transport between APOE variants contribute to or inhibit AMD phenotypes. Unfortunately, there are currently no model systems for a rigorous assessment of the interplay between genetic factors and AMD pathology. Using induced pluripotent stem cell technology we developed an in vitro human outer blood-retina barrier (oBRB) that includes RPE and a choriocapillaris-like compartment and spontaneously forms sub-RPE deposits. Here, we propose to leverage these technologies and generate genetically modifiable retina tissue to investigate interactions between AMD phenotypes, lipid metabolism and APOE variants. In Aim 1, we will optimize the oBRB to study AMD phenotypes in a robust system that can also be assembled from cryopreserved cells. We will manipulate intrinsic and extrinsic factors to induce drusen in this system and correlate with human postmortem eyes. Next, we will generate isogenic oBRBs with different APOE variants to determine the mechanisms by which the APOE genotype influences AMD pathology by comparing their transcriptomic, lipidomic and functional profiles. Since the contribution of choriocapillaris hypoperfusion and function to the initiation of AMD is not well understood, in Aim 2, we will integrate microvascular perfusion into our system to investigate how genetically driven abnormalities affect AMD phenotypes. We will optimize a microfluidic approach with microvascular perfusion, generating a perfused oBRB (poBRB). Next, we will use a panel of live assays to investigate the contribution of APOE variants to poBRB vascular phenotypes and correlate that with the spatial distribution of drusen-like deposits. Collectively, we will unravel how APOE variants contribute to drusen formation, RPE and microvascular dysfunctional phenotypes and pinpoint cellular subtypes responsible for APOE effect. We will also determine the contribution of specific types of lipids to drusen formation. This work will establish a multimodal retina modeling approach to study AMD phenotypes. We anticipate that our work will reveal AMD therapeutic targets associated to lipid metabolism.
