Abstract
Purpose :
Diabetic retinopathy (DR) is a leading cause of blindness in working-age population and is regarded as a microvascular complication due to the breakdown of the endothelial barrier. The retinal pigment epithelium (RPE) is the major component of the blood-retinal barrier between the choroid and neurosensory retina. RPE is essential to prevent the passage of molecules and ions, and maintain cell polarity allowing the correct functioning of the retina and maintain retinal immune privilege. One of the early events in DR is the dysfunction of RPE, affecting the retina. In DR, RPE dysfunction is consequence of hyperglycemia, leading to a dysregulation on different protein expression, which, in turn, contributes to oxidative stress and, eventually, angiogenesis. Previous results obtained by our group show that Alpha-1 Antitrypsin (A1AT) acts like an anti-inflammatory agent that could play a role on DR treatment. However, it is important to know signaling pathways involved in its mechanism of action. In this work, we explore the expression of different genes and proteins from NFkB pathway, Wnt signaling pathway and mTOR on 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 16h with DMEM 5,5 mM glucose (Control), DMEM 5,5 mM glucose + 4.5 mg/ml A1AT (Control + A1AT), DMEM 30 mM glucose (Diabetic), DMEM 30 mM glucose + 4.5 mg/ml A1AT (Diabetic + A1AT). Cells were harvested with RIPA buffer for Western blot assay or Trizol for RT-qPCR.
Results :
NFkB p65, TNF-a,TG2, AKT, pAKT and HIF1α proteins were diminished with A1AT treatment. Also, CTNNB1 and mTOR genes presented a lower expression with A1AT while, PTEN and GSK3B were augmented.
Conclusions :
These results help to understand molecular mechanisms behind pathophysiologic processes in diabetic retinopathy and how A1AT works. Also shows that A1AT acts in a wide range of signaling pathways and cellular activities, making it as an appropriate molecule in diabetic retinopathy treatment.
This is a 2020 ARVO Annual Meeting abstract.