Abstract
Purpose:
In our previous clinic study, we have found that metformin use is associated with lower incidence of severe diabetic retinopathy. Subsequent in vitro study revealed that metformin has anti-angiogenic and anti-inflammatory effects on human retinal microvascular endothelial cells (hRVECs). This study investigates whether metformin protects hRVECs from direct high glucose or advanced glycation end products (AGEs) -induced insults.
Methods:
Early passages of primary human RVECs (ACBRI 181) were exposed to either high glucose (30 mM) or AGEs (100 µg/ml) with or without metformin co-treatment. Transwell permeability of hRVECs monolayer to FITC-dextran was assayed. The production of monocyte chemotactic protein-1 (MCP-1) and interleukin-8 (IL-8) were determined by ELISA. The expression levels of occludin, claudin 5 and phosphorylated AMP-activated protein kinase (AMPK) were assessed by Western blot.
Results:
AGEs induced a marked increase of hRVEC monolayer permeability to FITC-dextran by about 2 fold, which was completely blocked by 5 mM metformin at (p < 0.05). High glucose exposure led to a significant (p < 0.05) downregulation of adhesion molecules, claudin 5 and occludin by about 30%. Such effect was reversed partially by metformin at 2.5 mM and completely at 5 mM (p < 0.05). AGEs significantly increased secretion of inflammatory chemokines, MCP-1 and IL-8 in hRVECs by 2 fold and 1.7 fold, respectively (p < 0.001), and it decreased the level of phosphorylated AMPK in hRVEC. Metformin completely blocked AGEs-induced upsurge secretion of MCP-1 to below its baseline level, and IL-8 to basal level, respectively (p < 0.05). Meanwhile, metformin also restored phosphorylated AMPK (p < 0.05).
Conclusions:
Metformin protects hRVECs from high glucose and/or AGEs-induced increase of permeability, reduction of adhesion molecules, and upregulation of inflammatory chemokines. These effects are at least partially mediated by restoring the activity of metabolic regulator AMPK. These results provide further support that metformin directly protect the integrity and normal function of retinal vascular endothelial cells from diabetic insults.