Analysis of Metformin’s effect on Human Glaucomatous Lamina Cribrosa cells

Purpose: The Lamina Cribrosa (LC) is a key site of retinal ganglion cell axonal injury in primary open angle glaucoma (POAG). Metformin has been used in fibrotic disease and cancer models, as well as being associated with reduced POAG incidence in a large cross-sectional study. In this study, we assess metformin’s effect on glaucomatous LC cells by carrying out a systematic mitochondrial bioenergetic assessment and measuring markers of fibrosis and endoplasmic reticulum (ER) stress activity. In addition, we analyse a novel pathway involving MiRNA-26a and the oncogene HMGA1. Methods: Human LC cells from age matched normal (NLC) and glaucoma (GLC) donors were assessed using a Seahorse XFe96 Analyzer. GLC cells were treated with Metformin at different concentrations and a dose response curve was assessed. Three concentrations of metformin (0.1mM, 1.0mM and 5.0mM) and a MiRNA-26a mimic were then utilised to examine cell proliferation and apoptosis (BrdU staining), extracellular matrix (ECM) (Col1A1, a-SMA, and vitronectin), endoplasmic reticulum (ER) stress (CHOP) and HMGA1 gene (RT-PCR) and protein (western blotting) expression in NLC and GLC cells, Results: GLC cells had significantly lower maximal oxygen consumption rate, spare respiratory capacity and ATP production. Treatment with 0.1mM metformin significantly improved maximal OCR and spare respiratory capacity in GLC. GLC cells had significantly higher levels of HMGA1, MiRNA-26a, CHOP and ECM markers. All metformin concentrations and MiRNA-26a mimic significantly raised MiRNA-26a and decreased HMGA1 and ECM markers. Both treatments had no substantial effect on CHOP. Conclusion: Our results indicate the potential therapeutic benefit of metformin use in glaucoma. This may be achieved through improved mitochondrial functioning and ECM remodelling. Furthermore, we show evidence of a potential pathway of action for metformin, through up-regulated MiRNA-26a and subsequently downregulated HMGA1.