Abstract
Purpose :
The retinal pigment epithelium (RPE) is the major component of the blood-retinal barrier between the choroid and neurosensory retina. In Diabetic retinopathy (DR), RPE dysfunction is a consequence of hyperglycemia. Previous results obtained by our group show that Alpha-1 Antitrypsin (A1AT) acts as an anti-inflammatory agent that could play a role in DR treatment. Also, we elucidated A1AT mechanism of action that involves different signaling pathways. A1AT mitigates pathways related to inflammation, epithelial-mesenchymal transition, and oxidative stress. However, A1AT contribution to retinal protective mediators’ synthesis, as a complementary mechanism of action, has not been studied. Neuroprotectin D1 (NPD1) is involved in the adaptative response to the neurodegenerative and inflammatory process that occurs in early DR, protecting the retina. In this work, we explored the expression of different proteins involved in NPD1 synthesis, like cPLA2, using ARPE-19 cells exposed to high glycemia and A1AT treatment.
Methods :
ARPE-19 cells (ATCC® CRL-2302TM, Manassas, Virginia, USA) were maintained in DMEM/F12 (Invitrogen, Carlsbad, California, USA) containing 2 μM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% fetal bovine serum. ARPE-19 cells (passages 9 to 12) were incubated 16 hours with DMEM 5,5 mM glucose (Control), DMEM 5,5 mM glucose + 4.5 mg/ml A1AT (Control + A1AT), DMEM 30 mM glucose (High Glucose), DMEM 30 mM glucose + 4.5 mg/ml A1AT (High Glucose + A1AT). Cells were harvested with RIPA buffer for Western blot assay or Trizol for RT-qPCR. Immunoblot quantifications were performed with FIJI and relativized to Actin. Measurements were expressed as mean ± SD and statistically analyzed with ANOVA and Tukey multiple comparison test. A p-value <0.05 was considered to be statistically significant.
Results :
cPLA2 protein presented an increased expression with A1AT treatment: (0.29±0.04 Control vs. 0.57±0.12 Control + A1AT, p<0.01) and (0.61±0.19 High Glucose vs. 0.88±0.06 High Glucose + A1AT, p<0.01).
Conclusions :
These results might help to understand the molecular mechanisms behind A1AT. Further investigations on lipoxygenase-15 (LOX-15) and elongase-4 (ELOVL4) are being conducted to evaluate a possible synergistic effect of A1AT on NPD1 mediators to protect the retina.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.