Free Amino Acids Glycine and Glutamic Acid Inhibit Angiogenesis Induced by AGE in Bovine Retinal Endothelial Cells

Abstract

Previous studies from our lab have shown that amino acids act as antiglycating agents and can be beneficial in diabetes mellitus. Accumulation of advanced glycation end products (AGE) in uncontrolled diabetes mellitus can induce microvascular complications such as diabetic retinopathy that results in neovascularization in the retina. This study explored the effect of amino acids Glycine and Glutamic acid ( 0.5–2.5 mM) in mitigating the AGE (100 μg/ml) induced angiogenic effects in primary bovine retinal endothelial cells (BREC) cultured in vitro. Tube formation induced by AGE in the BREC cells were reduced by glycine and glutamic acid (p=0.05, p=0.008). Transwell migration assay revealed signficant inhibition of migration by glycine. Expression of actin cytoskelatal filaments that promotes migration was reduced predominantly by glycine than glutamic acid as seen by immuofluorescence. Leukocyte adhesion promoted by AGE treatment was reduced significantly by glycine (p=0.03) and glutamic acid (p=0.02). The mechanism was delineated in terms of AGE-RAGE/VEGF axis. The receptor for AGE (RAGE) and the VEGF expression was found to be decreased both at protein level and at mRNA level by glycine and glutamic acid treatment. mRNA expression of RAGE by qPCR revealed a maximal decrease of 60% by glycine at 2.5 mM but by glutamic acid at 0.5 mM and VEGF mRNA level showed maximal inhibition by glutamic acid at 2.5 mM. Western blot analysis showed that VEGF expression was predominantly reduced by glycine. This is the A¯Â¬Ârst study showing the anti angiogenic potential of the amino acids in an in vitro model of primary bovine retinal endothelial cell implying therapeutic potential in the management of diabetic retinopathy.