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Meredith J Giblin, John S Penn; Systematic Analysis of the Effects of Diabetes-Relevant Stimuli on Endothelial Expression of Extracellular Matrix Constituents. Invest. Ophthalmol. Vis. Sci. 2016;57(12):3235.
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© ARVO (1962-2015); The Authors (2016-present)
One of the earliest structural abnormalities of diabetic retinopathy (DR) is basement membrane (BM) thickening of the retinal microvasculature. Recent studies suggest that excessive extracellular matrix (ECM) deposition may cause alterations in BM composition and permeability; however much remains to be understood about how ECM deposition or BM thickening contributes to DR pathology. The purpose of this study was to develop an in vitro experimental platform to investigate the role of ECM deposition in DR.
Under normalized growth conditions, human retinal microvascular endothelial cells (HRMEC) were treated with a variety of diabetes-relevant stimuli. Included were inflammatory cytokines (10 ng/mL TNFα, 10 ng/mL IL-1β, 1 ng/mL IL-6, 1 ng/mL IL-8 or PBS vehicle); high glucose conditions (5 mM or 25 mM D-glucose, with L-glucose used as an osmotic control); and free fatty acids (50 μM oleic acid, 250 μM palmitic acid, 50 μM linoleic acid, or equimolar BSA). Concentrations, treatment times, and growth conditions were systematically altered to optimize induction of two primary BM components, collagen IV and fibronectin, which was measured by qRT-PCR.
HRMEC exposure to IL-1β caused a 3.3-fold (p=0.03) and 1.4-fold (p=0.007) increase in collagen IV and fibronectin, respectively. TNFα caused a 3.1-fold (p=0.01) increase in collagen IV. Exposure of HRMEC to high glucose showed a 1.5-fold (p=0.005) and 1.4-fold (p=0.03) increase in expression of collagen IV compared to normal glucose and L-glucose respectively. Treatment with IL-6, IL-8, and free fatty acids did not result in a significant increase in expression of fibronectin or collagen IV.
These results demonstrate the effect, or lack thereof, of a variety of diabetes-relevant stimuli on the expression of ECM proteins by HRMEC, providing a platform for the study of the underlying mechanisms of BM thickening. Inflammatory cytokines were shown to be more potent inducers of BM thickening than conditions designed to simulate hyperglycemia or hyperlipidemia. Future studies will employ this experimental platform to test cellular mechanisms underlying BM thickening.
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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