Endostatin Up–Regulates Occludin and Tie–2 Expression via a Distinct Intra–Cellular Signalling Cascade, Whilst Decreasing VEGF165 Mediated Blood Retinal Barrier Permeability in vivo

Abstract

Abstract: : Endostatin is a 20kDa fragment of collagen XVIII, derived by selective proteolytic degradation of vascular basement membranes. This peptide can prevent tumour angiogenesis and has also been shown to be efficacious in the eye by preventing retinal and choroidal neovascularisation. The Purpose: of this study was to evaluate the effects of endostatin on the levels of expression and phosphorylation of the tight junction protein occludin and the receptor tyrosine kinsase Tie–2, whose ligand angiopoietin–1 is a known anti–permeability factor. Moreover, it was hypothesised that changes in levels of expression and phosphorylation of these proteins are dependent on a cascade of intra–cellular signalling events involving MAPK. The Methods:involved pre–treatment of retinal microvascular endothelial cells (RMEC’s) for 1 hour with a specific inhibitor of p44/p42 MAPK (PD98059) (0µM, 0.1 µM, 1.0 µM and 10 µM), and subsequent treatment for 24 hours with 1µg/ml endostatin, followed by western blot analysis to determine Occludin and Tie–2 expression. The levels of expression of Occludin and Tie–2 were shown to be significantly decreased when compared to RMEC’s treated with 1µg/ml endostatin alone. RMEC’s treated with 1µg/ml endostatin also showed a significant decrease in levels of expression of the stress response kinase JNK–1.Results:showed 1µg/ml endostatin treatment caused a 4–fold increase in occludin levels and increased phosphorylation of occludin on serine and threonine residues in retinal microvascular endothelial cells. Whilst not activating Tie–2 by causing tyrosine phosphoylation, 1µg/ml endostatin treatment for 24 hours caused levels of expression of the receptor to be significantly up–regulated (P ≤ 0.05). In conjunction with these results, we also show that 60ng endostatin can decrease evan’s blue albumin permeation at the inner retina of C57/BL6 mice administered with an intra–ocular injection of 30ng VEGF. This is the first report of the effects endostatin exerts on the tight junction protein occludin expression and its effects on levels of expression of the receptor tyrosine kinase receptor Tie–2. Our Conclusions: are that endostatin mediated up–regulation of occludin and Tie–2 involves a distinct cascade of events involving p44/p42 MAPK, and this may play an important role in mediating its anti–angiogenic effect at the iBRB.

Keywords: retina • cell adhesions/cell junctions • protein structure/function