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Jinling Yang, Magali Saint-Geniez, Yin Shan Eric Ng, Marsha A. Moses, Patricia A D'Amore; Shedding of Endomucin by Endothelial Cells under Inflammatory Conditions: Involvement of Matrix Metalloproteases. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3104. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Interactions between inflammatory cells and endothelial cells (ECs) are critical to vascular inflammation, which has been heavily implicated in many ocular diseases. We have previously shown that endomucin (EMCN), an apically localized, EC-specific transmembrane mucin, blocks adhesion of inflammatory cells to ECs under non-inflammatory conditions and is downregulated by TNF-α. Furthermore, EMCN overexpression blocks TNF-α-induced inflammatory cell adhesion to EC in vitro and inflammatory cell infiltration in vivo. Activation of matrix metalloproteases (MMPs) that occurs during inflammation mediates protein cleavage. This study investigated the role of MMPs in the regulation of EMCN under inflammatory conditions.
Human umbilical vein ECs (HUVECs) or human retinal ECs (HRECs) were treated with TNF-α, IL-1β and IFN-γ for 24 hrs or pervanadate (50 mM, 30 min), a strong oxidant that mimics inflammatory conditions. The role of MMPs was examined by pretreatment of HUVECs with MMP inhibitors (BB94 5 mM or GM6001 10 mM). Total and cell surface EMCN protein levels were evaluated by immunoblotting alone or following biotinylation. EMCN localization was revealed by immunocytochemistry.
TNF-α, IL-1β or IFN-γ downregulated total EMCN protein in HUVECs in a dose-dependent manner; at 1 ng/ml, total EMCN protein was reduced to 33.2±1.1% (P<0.001), 27.8±5.4% (P<0.01), 59.0±2.6% (P<0.01) of controls, respectively. In HREC, 1 ng/ml TNF-α also reduced total EMCN protein to 37.9±0.8% (P<0.001). TNF-α-induced reduction in total EMCN protein in HUVECs was partially blocked by BB94 (59.5±4.2% P<0.001) or GM6001 (70.7±14.2% P<0.05). BB94 led to a significant preservation of cell surface EMCN (31.2±10.3% vs. 65.6±5.4%, P<0.01) in TNF-α-treated HUVECs. Immunocytochemical localization confirmed that BB94 partially prevented EMCN loss from the cell surface and cell margins. Pervanadate treatment reduced total EMCN protein to 59.3±4.3% of controls (P<0.001) and this was nearly totally normalized (92.0±12.9% P<0.01) by BB94. A C-terminal EMCN antibody reacted with a low molecular weight fragment (~15kD) in lysates from pervanadate-treated cells, which was absent in BB94- or vehicle-treated groups.
These results indicate a role for MMPs in the shedding of EMCN under inflammatory conditions and suggest a novel therapeutic target for inflammatory ocular diseases.
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