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Sudha Swamynathan, Shivalingappa K Swamynathan; Secreted Ly6/Urokinase-type Plasminogen activator Receptor-Related Protein-1 (SLURP1) Suppresses Angiogenic Inflammation through a Pathway that Involves Src, RhoA and NFκB. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1447. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Previously, we demonstrated that the Secreted Ly6/urokinase-type plasminogen activator receptor-related protein-1 (SLURP1) serves an immunomodulatory function in the ocular surface. Here, we examine the effect of SLURP1 on angiogenic inflammation, and the signaling pathways through which SLURP1 functions.
The effect of recombinant human SLURP1 (6XHis-tagged; expressed in E. coli and partially purified using Ni-ion columns) on angiogenesis was tested in vitro using human umbilical cord vascular endothelial cells (HUVEC) tube formation assay on matrigel-coated plates. HUVEC tube length, area and number of branch points were quantified by light and fluorescence microscopy, with the help of MetaMorph software. Human leukemia cell line HL60 was differentiated into a neutrophil-like state (HL60N) by incubating with 1.26% DMSO for six days. The effect of SLURP1 on (i) neutrophil chemotaxis was measured using HL60N cells and fMLP as a chemoattractant in Neuroprobe chemotaxis plates (5µm pore size), (ii) expression of IL-1a, IL-1b and MMP9 was measured by QPCR using total RNA from HL60N cells treated with SLURP1 or vehicle control, (iii) Src activity and nuclear localization of RhoA and NFκB was tested by immunofluorescent staining in control human corneal limbal epithelial (HCLE) cells or those stably transfected to express SLURP1 (HCLE-SLURP1), and (iv) actin polymerization in migrating HL60N and HCLE cells was visualized using fluorescently tagged phalloidin.
SLURP1 suppressed TNF-α-stimulated HUVEC tube formation without affecting their survival and/or proliferation. HUVEC tube length, area and number of branch points were significantly decreased in the presence of SLURP1. SLURP1 also suppressed (i) fMLP-stimulated HL60N chemotaxis, (ii) TNF-α-induced IL-1a, IL-1b and MMP9 production in HL60N cells, (iii) Actin polymerization in HL60N and HCLE migrating edges, (iv) Src activity in migrating edge of HCLE cells, (v) Nuclear localization of RhoA in HCLE cells, and (vi) TNF-α-induced nuclear translocation of NFκB in HCLE cells.
Taken together, these results suggest that SLURP1 suppresses TNF-α-stimulated angiogenic inflammation through a pathway that involves Src, RhoA and NFκB.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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