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Meredith Giblin, John S Penn; Cytokine-induced ECM alterations in DR pathogenesis. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3567. doi: https://doi.org/.
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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 pathogenic retinal cell behaviors. Yet, to date, studies regarding BM alterations and DR pathology remain inconclusive. The purpose of this study was to learn how diabetes-relevant stimuli affect expression of BM components and how these diabetes-induced changes affect human retinal microvascular endothelial cells’ (HRMEC) expression of adhesion proteins that are known contributors 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 deposited ECM alter HRMEC behavior, HRMEC or HRP were treated with TNFα or IL-1β, respectively, for 48hrs before cultures were decellularized. Naïve HRMEC were then plated on the decellularized matrices and collected 16hrs later for qRT-PCR.
High glucose treatment of HRMEC or HRP produced no significant changes in COL4 or FN expression. In HRMEC, TNFα caused a 1.7-fold increase in COL4 (p<0.01) and a .5-fold decrease in FN (p<0.01). In HRP, TNFα caused a 2.7-fold (p<0.01) induction of COL4. IL-1β induced a 1.8-fold (p<0.01) induction of COL4 in both HRMEC and HRP. TNFα-conditioned HRP ECM caused 349-, 6.5- and 3.3-fold inductions in SELE, ICAM and VCAM, respectively. IL-1β-conditioned HRMEC ECM caused 2.8-fold induction in SELE.
Cytokines were shown to be more potent inducers of COL4 expression than conditions designed to simulate hyperglycemia. Interestingly, TNFα caused contrasting expression changes in HRMEC indicating that ratios of BM constituents may be significantly altered in the diabetic retinal BM. HRP demonstrated higher levels of COL4 induction than HRMEC, arguing that HRMEC should not be the only focus in understanding BM thickening. Additionally, decellularization experiments suggest that diabetes-relevant alterations in ECM composition alone can alter adhesion molecule expression by HRMEC, indicating that BM thickening may drive other pathogenic behaviors in DR.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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