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TJ Hollingsworth, Mei Du, Krysten M Farjo; RBP4 Induces Inflammation in Human Retinal Capillary Endothelial Cells by a TLR4-Dependent Mechanism. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1026.
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Serum retinol-binding protein (RBP4) is the transport protein for Vitamin A (retinol) in the blood, but RBP4 also has retinol-independent activity that contributes to the development of insulin resistance in type 2 diabetes. Moreover, serum RBP4 levels are significantly increased in patients with proliferative diabetic retinopathy compared to diabetic patients with mild or no retinopathy. We have previously shown that elevation of RBP4 induces pro-inflammatory gene expression in human retinal capillary endothelial cells (HRCEC) through a retinol-independent mechanism that is dependent on NADPH oxidase and NF-κB, and thus may contribute to the formation of vascular lesions in diabetic retinopathy. However, the cell membrane receptor that mediates RBP4-induced endothelial inflammation is unclear, since HRCEC do not express the RBP4 receptor for retinol uptake, STRA6. The current study focuses on identifying the receptor and signal transduction pathways activated by RBP4 to induce inflammation in HRCEC.
Since previous studies have shown that RBP4 stimulates pro-inflammatory activity in macrophages via TLR4-MAPK signaling and inhibits insulin signaling in adipocytes via STRA6-STAT5 signaling, we tested if RBP4 stimulated either of these pathways in HRCEC. HRCEC were treated with purified recombinant human RBP4 in the presence or absence of inhibitors for TLR4, JNK, ERK, p38, STATs, NADPH oxidase, and NF-κB. MAPK and STAT pathway activation was assessed by western blotting for phospho-JNK,-ERK, -p38, -STAT1, -STAT3, and -STAT5. Pro-inflammatory gene expression was analyzed by western blotting and ELISA. In vitro leukostasis assays were performed to measure the net effect of inhibitors on RBP4-induced inflammation.
Both a chemical TLR4 inhibitor (TAK-242) and TLR4 neutralizing antibodies significantly blocked RBP4-induced expression of pro-inflammatory genes, including VCAM-1, ICAM-1, IL-6, MCP-1, and E-selectin. We observed phospho-activation of MAPK signaling, but not STAT signaling in response to RBP4 treatment. Similarly, MAPK inhibitors significantly reduced RBP4-mediated inflammation, whereas STAT inhibition did not have a significant effect.
RBP4 induces inflammation in HRCEC via TLR4 and MAPK signaling. This suggests RBP4 and TLR4 may be therapeutic targets in diabetic retinopathy.
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