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Sumbul Bushra, Nasser Rizk, Isha Sharma; ASSOCIATION BETWEEN INSULIN AND NITRIC OXIDE IN HUMAN RETINAL MICROVASCULAR ENDOTHELIAL CELLS IN VITRO. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5426.
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© ARVO (1962-2015); The Authors (2016-present)
NO involved in regulation of vascular homeostasis is reported to be released by insulin dependent PI3K/ Akt signaling pathway. Endothelial dysfunction impairs ocular hemodynamics by reducing the bioavailability of NO and increasing the production of reactive oxygen species (ROS) and may be responsible for the pathogenesis of vascular dysfunction in retinopathy.The aim of this study was to examine the effect of insulin on NO production in human retinal microvascular endothelial cells cultured in hyperglcemic conditions.
In the current study HRMECs cells were cultured in high glucose (30mM) and normal (5mM) glucose for 24 hours. Subsequently, the cells were treated with 100nM insulin for 10 minutes, 1, 2, and 4 hours. The various parameters of PI3K/ Akt signaling pathway were analyzed. IRS-1, IRS-2, PI3K, Akt, eNOS, VEFGA, NFkB were analyzed for gene expression. Adhesion molecules P-selectin and ICAM-1 assessed by flow cytometry. ROS and NO production measured by immunofluorescence and fluorometry respectively. The cell viability, cell cycle, apoptosis and total oxidative stress were evaluated by imaging flowcytometery.
This study demonstrated that hyperglycemia causes an increase in ROS/oxidative stress and apoptosis, while insulin promotes significant reduction in ROS and apoptosis. eNOS mediated NO production increases with hyperglycemia but remarkably reduced with insulin treatment after 1hour, 2 hours and 4 hours. The current study demonstrated that gene expression of PI3K, Akt and eNOS were significantly elevated in high glucose. Insulin treatment up regulated IRS-1 and IRS-2 genes after 1 hour however, PI3K, Akt and eNOS were significantly reduced. Although hyperglycemia induced upregulation of angiogenic marker VEGFA, the current study showed a significant downregulation upon 100nM insulin treatment. Consistent with the VEGFA gene expression HG significantly increased the cell migration and angiogenesis while insulin treatment improved barrier function. This study also demonstrated that hyperglycemia significantly increases adhesion protein P-selectin with significant reduction at 4hrs insulin treatment.
This may suggest that insulin could counteract the hyperglycemic effect on AKT/PI3 kinase which mediates NO production and VEGF-A, with decreased adhesion molecules such as p-selectin that is involved in barrier disorder of retinal endothelial cells.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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