Magali M. Le Goff

Identification in Vitreous and Molecular Cloning of Opticin, a Novel Member of the Family of Leucine-rich Repeat Proteins of the Extracellular Matrix

A prominent 45-kDa component was identified by protein staining following SDS-polyacrylamide gel electrophoresis of a 4 m guanidine hydrochloride extract from bovine vitreous collagen fibrils. Peptide sequences obtained from this component were used as a basis for the cloning (from human retinal cDNA) and sequencing of a novel member of the leucine-rich repeat extracellular matrix protein family that we have named opticin. Opticin mRNA was found by reverse transcription polymerase chain reaction in ligament and skin as well as in retina. An open reading frame containing 332 amino acids was identified, the first 19 amino acids representing a signal peptide. The deduced amino acid sequence of the mature protein encodes a 35-kDa protein with a calculated isoelectric point of 5.4. The central domain of this protein consists of six B-type leucine-rich repeats. This domain is flanked by cysteine clusters including a C-terminal two-cysteine cluster containing an additional leucine-rich repeat. The N-terminal region contains a cluster of potential O-glycosylation sites, and analysis of bovine vitreous opticin demonstrated the presence of sialylatedO-linked oligosaccharides substituting the core protein. Opticin shows highest protein sequence identity to epiphycan (42%) and osteoglycin (35%) and belongs to Class III of the leucine-rich repeat extracellular matrix protein family.

Opticin Exerts Its Anti-angiogenic Activity by Regulating Extracellular Matrix Adhesiveness

Background: Recently, we demonstrated that the glycoprotein opticin is anti-angiogenic. Here, the underpining mechanism is explored. Results: By binding to collagen, opticin competitively inhibits integrin-mediated endothelial cell adhesion. Conclusion: Opticin inhibits angiogenesis by weakening endothelial cell adhesion to the surrounding extracellular matrix. Significance: Elucidating the regulatory mechanisms of angiogenesis is important for understanding pathology and drug discovery. Opticin is an extracellular matrix glycoprotein that we identified associated with the collagen network of the vitreous humor of the eye. Recently, we discovered that opticin possesses anti-angiogenic activity using a murine oxygen-induced retinopathy model: here, we investigate the underlying mechanism. Using an ex vivo chick chorioallantoic membrane assay, we show that opticin inhibits angiogenesis when stimulated by a range of growth factors. We show that it suppresses capillary morphogenesis, inhibits endothelial invasion, and promotes capillary network regression in three-dimensional matrices of collagen and MatrigelTM. We then show that opticin binds to collagen and thereby competitively inhibits endothelial cell interactions with collagen via α1β1 and α2β1 integrins, thereby preventing the strong adhesion that is required for proangiogenic signaling via these integrins.

The Vitreous Glycoprotein Opticin Inhibits Preretinal Neovascularization

PURPOSE Opticin is an extracellular matrix glycoprotein that the authors discovered in the vitreous humor of the eye. It is synthesized by the nonpigmented ciliary epithelium and secreted into the vitreous cavity and, unusually for an extracellular matrix molecule, high-level synthesis is maintained into adult life. Here the authors investigated the hypothesis that opticin influences vascular development in the posterior segment of the eye and pathologic angiogenesis into the normally avascular, mature (secondary) vitreous. METHODS Opticin was localized in murine eyes by immunohistochemistry. An opticin knockout mouse was established and vascular development was compared between knockout and wild-type mice. Wild-type and opticin null mice were compared in the oxygen-induced retinopathy model, a model of pathologic angiogenesis, and this model was also used to assess the effects of intravitreal injection of recombinant opticin into eyes of wild-type mice. RESULTS Opticin colocalizes with the collagen type II-rich fibrillar network of the vitreous, the inner limiting lamina, the lens capsule, the trabecular meshwork, and the iris. Analyses of the hyaloid and retinal vasculature showed that opticin has no effect on hyaloid vascular regression or developmental retinal vascularization. However, using the oxygen-induced retinopathy model, the authors demonstrated that opticin knockout mice produce significantly more preretinal neovascularization than wild-type mice, and the intravitreal delivery of excess opticin inhibited the formation of neovessels in wild-type mice. CONCLUSIONS A lack of opticin does not influence vascular development, but opticin is antiangiogenic and inhibits preretinal neovascularization.