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Alireza Ziaei, Sharmila Masli; Differential Regulation of Endothelial VCAM-1 Expression in Ocular Inflammation via Modulation of Anti-oxidant Pathway. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1868.
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Thrombospondin-1 (TSP-1) is a critical molecule in regulation of an ocular inflammation. The Purpose of this study was to determine if TSP-1 utilizes Nrf2-driven antioxidant pathway in regulating the expression of adhesion molecules on vascular endothelial cells (ECs).
Flatmounts of retina harvested from C57BL/6 (WT), TSP-1null (6-8 wks.) and IRBP-immunized uveitic WT mice and vascular endothelial cells (2H-11) treated with either TNFα or IL-17 (10 ng/ml) or TSP-1 derived peptides (CD36 or CD47 binding) were stained with antibodies against ICAM-1, VCAM-1, Nrf2 and TSP-1 followed by fluorescence-conjugated secondary antibodies. Stained retinas and ECs were evaluated using fluorescence microscopy. Leukocyte adhesion assay was performed by using fluorescent dye-labeled WT lymph node cells and measuring fluorescence of cells adherent to ECs.
Both ICAM-1 and VCAM-1 immunostaining detected in TSP-1null retina resembled that seen in uveitic WT retina indicating a potential anti-inflammatory role of TSP-1. To evaluate possible TSP-1 dependent regulation of adhesion molecules on vascular endothelium during chronic inflammation we examined the expression of VCAM-1 and TSP-1 in 2H-11 ECs treated with IL-17. While VCAM-1 immunostaining and leukocyte adhesion was enhanced, TSP-1 immunostaining was reduced in ECs treated with IL-17 as compared to the untreated control cells. To elucidate if a specific TSP-1 receptor expressed on ECs is involved in regulating VCAM-1 expression, we used TSP-1 derived peptides that specifically bind TSP-1 receptors CD47 or CD36 on ECs. While CD47 ligation on ECs led to reduced VCAM-1 staining and leukocyte adhesion, CD36 ligation enhanced VCAM-1 staining compared to controls. This difference correlated with an enhanced staining of a transcription factor associated with anti-oxidant pathway, Nrf2, upon CD47 ligation and reduced staining of the same upon CD36 ligation on ECs. These results suggest a differential regulation of VCAM-1 expression on ECs by TSP-1 possibly via Nrf2-driven antioxidant pathway.
Our results suggest a TSP-1-mediated differential regulation of inflammation-induced expression of adhesion molecules in vascular endothelium via anti-oxidant pathway and further support a therapeutic potential of CD47 binding TSP-1 peptide during a chronic inflammatory condition.
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