Abstract
Purpose :
Pathological angiogenesis is the hallmark of proliferative diabetic retinopathy (DR) at the advanced stage of this ocular disease. In response to prolonged tissue injury, human retinal microvascular endothelial (HRMEC) cells form new blood vessels to ensure the supply of oxygen and nutrients to the eye; however, progressive fibrovascular proliferation can eventually lead to retinal detachment and blindness. Caspase-8, the responsible protease for apoptosis has also been shown to modulate pathological and developmental angiogenesis but whether it exerts this role in a hyperglycemic environment has not yet been explored.
Methods :
HRMEC were propagated using Complete Classic Medium with 1% Antibiotic-Antimycotic and 10% FBS. Conditioned medium was harvested from cells seeded into 24 well plates at a density of 800K and stored at -20°. HRMECs were treated with 5.5mM or 30mM of glucose, and the Casp-8 Glo assay was performed 24hrs following treatment. Cells were transfected with siRNA at 80% confluent. With a final concentration of siRNA solution 600pmol, samples were probed with p38 MAPK Antibody and Phospho-p38 MAPK. After transfected cells were confluent, they were starved in 2% FBS DMEM medium overnight. A wound was induced by scratching the cell monolayer with a P200 pipette tip. Cell migration was observed under the light microscope 24hrs after treatment.
Results :
After 24 hours of glucose treatment, there were 232.5K viable cells in 5.5mM compared to 119K in 30mM glucose. Additionally, high glucose also increased VEGF secretion after 24hrs of glucose treatment (5.5mM of glucose 16.30pg VEGF/mL, 5.5 mM vs 22.41pg/mL 30mM). Hyperglycemic conditions also resulted in increased Pro-Caspase 8 expression, as the concentration of Pro-Caspase 8 expression from 1.25 fold change at 5.5mM to 2.5 fold (compared to 18S RNA). Western blots showed near-complete blocking of p38 phosphorylation in HRMEC with Caspase 8 KD.
Conclusions :
In conclusion, caspase 8 activity was confirmed by Caspase-Glo assay in HRMEC when treated at both 5.5mM and 30mM of glucose, specifically at a luminescence of 1.75 RLU and 1.90 RLU, respectively. In addition, Caspase-8 KD resulted in a significant reduction of protein expression from 2.1-fold change to 1.1 along with an inhibition of cell migration.Taken together, our data strongly suggest that Caspase-8 promotes HRMEC migration via MAPK pathway involving p38 phosphorylation.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.