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Meredith Giblin, John S Penn; Cytokine-induced ECM alterations in DR pathogenesis. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2684.
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© ARVO (1962-2015); The Authors (2016-present)
An early structural abnormality of diabetic retinopathy (DR) is basement membrane (BM) thickening of retinal microvasculature. Recent studies suggest that this is a product of increased extracellular matrix (ECM) deposition and that it contributes to DR progression. The purpose of this study was to learn how diabetes-relevant stimuli affect BM constituent expression and how these diabetes-induced changes in ECM cause alterations in human retinal microvascular endothelial cells (HRMEC) that contribute to pathogenic leukostasis.
HRMEC and human retinal pericytes (HRP) were treated with diabetes-relevant stimuli, including inflammatory cytokines (TNFα or IL1β, 10ng/mL) and high glucose conditions (25mM D-glucose). Concentrations and treatment times were systematically optimized and expression of two primary BM components, collagen IV (COL4) and fibronectin (FN), was measured by qRT-PCR. To study how changes in ECM alter HRMEC behavior, HRMEC or HRP were treated with TNFα or IL-1β, respectively, for 48hrs before cultures were decellularized. Naïve HRMEC were plated on the decellularized matrices and analyzed 16hrs later for qRT-PCR of adhesion proteins or adhesion of peripheral blood mononuclear cells (PBMC).
High glucose-treated HRMEC or HRP showed no significant changes in COL4 or FN expression. In HRMEC, TNFα caused a 1.7-fold increase in COL4 (p<0.01) and .5-fold decrease in FN (p<0.01). In HRP, TNFα caused 2.7-fold (p<0.01) induction of COL4. IL-1β induced a 1.8-fold (p<0.01) increase in COL4 in HRMEC and HRP. ECM deposited by TNFα-treated HRP caused 349-, 6.5- and 3.3-fold inductions in SELE, ICAM and VCAM, respectively, in naïve HRMEC. ECM derived from IL-1β-treated HRMEC caused 4.2-(p<0.01), 2.2-(p<0.01) and 1.6-fold (p<0.01) inductions in SELE, ICAM and VCAM, respectively, in naïve HRMEC. HRMEC grown on IL-1β-conditioned ECM showed 2-fold (p<0.05) increase in PBMC adhesion.
Cytokines were shown to be more potent inducers of COL4 expression than high glucose conditions. Interestingly, TNFα caused contrasting expression changes in COL4 and FN in HRMEC, potentially compounding differences in the ratios of these two constituents in diabetic retinal BM. Additionally, decellularization experiments demonstrated that diabetes-relevant alterations in ECM composition alone change both adhesion molecule expression and PBMC adhesion to HRMEC, suggesting that BM thickening may drive other pathogenic behaviors in DR.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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