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Meredith Giblin, John S Penn; Cytokine-induced ECM alterations in DR pathogenesis. Invest. Ophthalmol. Vis. Sci. 2020;61(7):1766.
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© ARVO (1962-2015); The Authors (2016-present)
Capillary basement membrane (BM) thickening is an early structural abnormality of diabetic retinopathy (DR). The exact mechanisms involved in this event and its contribution to other DR events are unclear. Our aim was to utilize quantitative mass spectrometry (qMS) to identify changes in extracellular matrix (ECM) deposition by human retinal endothelial cells (hRMEC) under diabetes-relevant inflammatory conditions and to understand how ECM changes affect hRMEC behaviors that contribute to pathogenic leukostasis.
hRMEC were treated with IL1β (10ng/mL) for 48hrs before cultures were decellularized and underlying ECM was solubilized for qMS. MaxQuant software was used for quantification of ECM (n=3). Lamininβ3 (LAMB3) and nidogen-2 (NID2) were chosen for further study based on differential deposition under IL1β. To study how ECM affects hRMEC behavior, hRMEC were treated with IL1β or 24hr treatment with LAMB3 or NID2 siRNA. Cultures were then decellularized and naïve hRMEC were plated on IL1β- or siRNA-conditioned ECM. Naïve hRMEC were analyzed 16hrs later for adhesion protein expression by qRT-PCR or for adhesion of peripheral blood mononuclear cells (PBMC).
IL1β caused a 1.4-fold increase (p<0.05) in total deposited ECM protein. ECM constituency was significantly altered, including 7.2- and 3.2-fold increases (p<0.01) in LAMB3 and NID2 deposition, respectively. When grown on IL1β-conditioned ECM, naïve hRMEC showed 4.2- and 2.2-fold increases (p<0.01) in SELE and ICAM, respectively. IL1β-conditioned ECM also caused a 2-fold (p<0.05) increase in PBMC adhesion to hRMEC. Naïve hRMEC grown on LAMB3 siRNA-conditioned ECM showed 3.5- and 2.5-fold increases in SELE and VCAM, respectively. NID2 siRNA-conditioned ECM caused 5.6- and 4.3-fold inductions in SELE and VCAM, respectively.
Conditioned ECM studies demonstrated that diabetes-relevant ECM alterations alone increase both adhesion molecule expression and PBMC adhesion. LAMB3 and NID2 were identified as two constituents with significantly increased deposition under IL1β treatment. When LAMB3 or NID2 deposition was inhibited, adhesion molecule expression was also increased, suggesting a biphasic response to the presence and concentration of these constituents. These results indicate that alterations of BM composition in DR may exert pathologic influences through hRMEC expression of adhesion proteins and identify two BM constituents as potential therapeutic targets.
This is a 2020 ARVO Annual Meeting abstract.
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