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Elizabeth A Berger, Eliisa Strand, Laura A Young, Haoshen Shi, David J Kracht, Thomas W Carion; Innovative Flow-Based Co-Culture System to Investigate REC:PMN Interactions Under High Glucose Conditions. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3562.
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© ARVO (1962-2015); The Authors (2016-present)
Microvascular pathology is well associated with the development and progression of diabetic retinopathy (DR). Leukostasis is also implicated in the pathogenesis of this disease. Given the close interaction between retinal microvascular endothelial cells (REC) and infiltrating inflammatory cells in the retinal vasculature during the progression of DR, it is imperative to generate in vitro conditions that closely mimic what is observed in vivo in order to appropriately examine this interrelationship. As such, the current study uses the innovative flow-based hollow-fiber modeling system, which allows for co-culture of human (H) REC with mouse PMN to better understand how this interaction and resultant cellular responses contribute to DR pathogenesis.
HREC were grown as a monolayer in the FiberCell Systems Polysulfone Plus cartridge by seeding primary cells (15 × 106) and allowing them to adhere to the activated hollow fibers for 12h. After removal of non-adherent cells, the system was circulated with normal glucose (NG, 5nM) or high glucose (HG, 25nM) media for up to 28 days. PMN derived from B6 mice were then added directly to the cartridge system and circulated with the media. Samples were obtained from the ‘extravascular space’ via a sampling port throughout the entire study. After 28 days of HREC:PMN co-culture, cell lysates were processed for protein analyses by ELISA and Western blot.
When cultured alone, HREC exposed to HG displayed increased VEGF levels versus NG; however, no differences were observed in key inflammatory mediators, such as IL-1β. Significant increases in VEGF, IL-1β, IL-8/MIP-2, COX-2, 5-LOX and 15-LOX were detected in HG compared to NG upon the addition of B6-derived PMN. In addition, adhesion molecules ICAM-1 and VCAM-1 were significantly upregulated with HG exposure of HREC co-cultured with PMN.
Overall, these data provide evidence for an important interaction between PMN and REC under high glucose conditions, which may be important in the inflammatory response that is observed in DR. Further examination of this system should provide physiologically relevant data that cannot be studied under static culture conditions for the effects of REC:PMN interactions regarding adhesion, leukostasis and the role of each cell type in chronic inflammation associated with the pathogenesis of DR.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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