Abstract
Purpose :
Chronic hyperglycemia and hypoxia results in disrupted vascular permeability. Hypoxia is a critical player in the development of diabetic retinopathy. The development of therapies that target hypoxia at the blood-retinal barrier (BRB) is hindered by a lack of reliable human in vitro models that recapitulate hypoxia-induced vascular destabilization. We engineered a 3D model of the human BRB under hypoxic conditions and validated changes in key markers associated with barrier breakdown.
Methods :
Scaffolds coated with hyaluronan-based hydrogel were seeded with primary human retinal microvascular endothelial cells (hRMVECs) and allowed to grow for 7 days. Scaffolds were inverted, primary human pericytes (PCs) were added, and were grown for an additional 6 days until confluent. The cocultures were placed in a normoxic (20% oxygen) incubator and hypoxic chamber (1% oxygen) over time ranging from 8 hours to 9 days. Expression of VE-Cadherin, ZO-1, and F-actin were evaluated by immunocytochemistry. Expression levels of VEGF, ANG2, and ANG1, and HIF1a in the supernatant were measured using ELISA.
Results :
The bioengineered 3D model depicts intact endothelial cell-cell junctions under normoxic (20% oxygen) conditions and disrupted endothelial cell-cell junctions and elevated secreted VEGF and ANG2/ANG1 ratio levels under hypoxic conditions (1% oxygen) that are typically observed during vascular destabilization. ANG2/ANG1 levels progressively increased over time with hypoxia treatment depicting progression of disease. Changes in cytoskeletal (F-actin) morphology of endothelial cells were observed indicating cell migration. These results corroborate our previous protocol of diseasing the bioengineered BRB tissue using TNFα and IL1β.
Conclusions :
These results demonstrate the feasibility of creating a 3D hypoxic model of the BRB with key physiological and biological characteristics depicting vascular destabilization observed during diabetic retinopathy.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.