Abstract
Purpose :
To determine if the intracellular uptake of Catalase in sodium hyaluronate-albumin-chitosan microparticles inhibits choroidal neovascularization and subsequent permeation of retinal epithelial cells ARPE-19 via Bruch’s membrane and Human Microvascular Endothelial Cells HMECs-(a model for the choroidal microvasculature) caused by exposure to bacterial lipopolysaccharide LPS. Wet Age-Related Macular Degeneration AMD exposes the choroidal blood vessels to high-levels of reactive oxygen species ROS due to oxidative stress LPS and inflamatory mediators such as nitrates. This may change the characteristics of the vasculature - resulting in perhaps increased invasive neovascularization and scarring through the retinal epithelium and permeability to blood components such as albumin. TNF and other pro-inflammatory cytokines, have also been shown to be released in response to endotoxin. This can also further cause changes in the nature of vascular endothelial cells.
Methods :
ARPE-19 retinal epithelial and HMEC endothelial cells were cultured separately to confluency onto trans-well plates, pore size 3µm allowing passage of albumin and other plasma components. Monolayer integrity of retinal epithelial and endothelial cells was confirmed by Trans-epithelial electrical resistance TEER measurement. Fluorescent FITC tagged albumin was added to the upper chamber of the transwell cells. The effect of pretreatment with: 1) 5mg/ml Blank microspheres, 2) 5mg/ml Catalase microparticles and 3) 10 mg/ml Catalase microparticles on increasing LPS( 0, 0.5, 1.0 and 10 µg/ml), was determined by TEER and basolateral FITC tagged albumin. Cells with 0 conc of endotoxin served as the control group. Alamar Blue assay was used to determine biocompatibility.
Results :
Dose response in TEER measurement and FITC-BSA permeability to LPS was prevalent. 5mg/mL and 10mg/mL Catalase microparticles showed approximately 35% and 80% inhibition of endotoxin LPS compared to control respectively. Cell viability of approximately 90% was determined in both cell lines.
Conclusions :
Microencapsulated Catalase inhibited the oxidative stress and may mitigate the angiogenic effects of LPS in retinal epithelial and microvascular endothelial cells. Biocompatibility of the Catalase microparticle formulation with the ex-vivo models was demonstrated via Alamar Blue assay.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.