The Leucine-Rich Repeat Domain of Opticin Confers Its Anti-Angiogenic Activity

Abstract

Purpose: : The glycoprotein opticin is found in the vitreous and is a member of the small leucine-rich repeat proteins (SLRP) family. Previously we have demonstrated that opticin inhibits preretinal neovascularisation using the mouse oxygen-induced retinopathy model and shown that it inhibits endothelial cell (EC) network formation in 3D matrices of collagen or Matrigel^TM. During angiogenesis matrix metalloproteinases (MMPs) including MMP2 and MMP9 are overexpressed, so we determined whether opticin is sensitive to MMP digestion and then investigated the anti-angiogenic properties of opticin fragments.

Methods: : Opticin was digested with MMP2 or MMP9 and the resultant fragments were analysed by electrophoresis, N-terminal sequencing and mass spectrometry. In vitro capillary networks were formed in 3D collagen gels using FGF2 stimulated ECs alone or in the presence of full-length opticin, a bacterially-expressed N-terminal fragment of opticin, or in co-culture with 293 cells expressing the leucine-rich repeat (LRR) domain of opticin.

Results: : Digestion of opticin with MMP2 or MMP9 cleaved opticin at the junction between its LRR domain and the N-terminal part of the molecule. Full-length opticin and the LRR domain of opticin inhibited capillary morphogenesis in the 3D assays. The N-terminal fragment of opticin did not inhibit capillary morphogenesis.

Conclusions: : MMP2 and MMP9 can cleave opticin. This cleavage produces an LRR domain that retains anti-angiogenic properties.

Keywords: vitreous • extracellular matrix • neovascularization