Abstract
Purpose :
The development of therapies that target blood-retinal barrier (BRB) disruption is hindered by a lack of reliable in vitro models that recapitulate the biology and pathophysiology of BRB-related diseases. We aimed to engineer a model of the human BRB and to validate the model by inducing changes in permeability and testing agents currently employed in the clinic.
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. The cocultures were treated with TNF-α (5 ng/mL) or IL1β (10 ng/mL) for 72 hrs then exposed to bevacizumab (0.25 ng/mL), aflibercept (500 ug/mL), prednisolone (100 nM), dexamethasone (100 nM) and/or VEGF-121 (100 ng/mL for 6 days. Expression of endothelial and pericyte markers was examined by immunofluorescence and barrier function was assessed by measuring transendothelial cell resistance (TEER).
Results :
The scaffolds supported the growth of the cocultures into a 3D structure. The endothelium expressed CD31, VE-cadherin, ZO1 and VWF whereas the PCs expressed NG2, PDGFR-β and NGFRP75. The TEER day 11 was 223.9±26.5 Ω*cm2 (N=9). Compared to monocultures, the constructs displayed enhanced expression of VE-cadherin (***P<.001, N=6), CD31 (**P<.005, N=6), PDGFR-β and NG2. Treatment with TNF-α or IL1β decreased ZO-1 and F-actin labeling and led to disrupted localization of VE-cadherin. Endothelial morphology changed from cobblestone to spindle-shaped, and F-actin was localized to the edges and tips of the cells, suggesting cell motility. Treatment with a combination of VEGF neutralizing agents and steroids rescued VE-cadherin expression and its localization at cell junctions, suggesting restoration of barrier integrity. VEGF neutralization alone led to more cytoplasmic localization of VE-cadherin.
Conclusions :
These results demonstrate the feasibility of creating a 3D model of the BRB with key physiological and biological characteristics that will be valuable for screening therapeutics as well as understanding the development and maintenance of the BRB.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.