This study sought to determine the distribution of opticin, an extracellular matrix small leucine-rich repeat protein secreted by the non-pigmented ciliary body epithelium (CBE), in pathological eye tissues including posterior hyaloid membranes (PHM) and epiretinal membranes (ERM) from subjects with proliferative diabetic retinopathy (PDR), central retinal vein occlusion (CRVO) and proliferative vitreoretinopathy (PVR). Eight enucleated eyes and eleven surgically excised PHMs/ERMs from patients with PDR, CRVO or PVR were analysed by immunohistochemistry for the presence and distribution of opticin, vitreous (delineated by a type II collagen antibody) and blood vessels (using CD31 and CD34 antibodies as endothelial markers). Opticin was present at the basal surface of the non-pigmented CBE and, in a patchy distribution, within CBE cells in all 8 enucleated globes. It also co-localised with the type II collagen of vitreous, where present, in these eyes. Opticin was present in 16 of the 19 PHMs/ERMs, where it was arranged in layers (10 membranes), diffusely (4 membranes) or in foci (2 membranes). Where in a layered pattern, opticin co-localised with vitreous type II collagen incorporated into the membrane, whereas the other two patterns did not co-localise with type II collagen labelling. We concluded that even in advanced proliferative retinal disease, the CBE continues to express and secrete opticin. Opticin was co-distributed with vitreous type II collagen and was also present in the pre-retinal membranes of proliferative retinopathies, where it could play a role in their development.

Purpose Preretinal neovascularisation is a form of angiogenesis i.e. growth of new blood vessels (into the vitreous) from the pre-existing vessels (the retinal vasculature). The vitreous contains pro- and anti-angiogenic molecules and normally the balance favours an anti-angiogenic state. However in diseases such as proliferative diabetic retinopathy, excess pro-angiogenic molecules, e.g. VEGF, stimulate preretinal neovascularisation. Anti-angiogenic molecules identified in the vitreous to date include PEDF, thrombospondin-1 and endostatin, but their relative importance in inhibiting preretinal neovascularisation remains unclear. Here we show that an endogenous glycoprotein of the vitreous called opticin has potent anti-angiogenic properties, and provide evidence that it has a pre-eminent role in inhibiting pre-retinal neovascularisation. Methods Opticin null mice were generated and bred onto a C57/BL6 background. The opticin null mice and wild-type mice +/- intravitreal injection with recombinant opticin (at P14) were investigated using the oxygen-induced retinopathy model (75% oxygen from P7-12, and analysis at P17 by serial histological sectioning). Results The opticin null mice demonstrated increased preretinal neovascular nuclei per cross section (109 +/- 6 S.E) compared to wild-type controls (73 +/- 3) (P < 0.0001). The opticin injected wild-type eyes showed a decrease in preretinal neovascular nuclei per cross-section (38 +/- 3) compared to eyes injected with PBS alone (76 +/- 4) (P < 0.0001). Conclusion Opticin inhibits preretinal neovascularisation in a dose-dependent manner. This is the first time that a knockout mouse has demonstated increased neovascularisation using this model. Opticin, or molecules derived from opticin, represent potential therapeutics.

Angiogenesis plays a key role in ocular development and disease. Normally following regression of the hyaloid vascular system the vitreous humour is avascular and anti-angiogenic. However, in diseases such as proliferative diabetic retinopathy, new blood vessels grow from the retina into the vitreous humour potentially leading to blindness. Opticin is a glycoprotein that was first isolated from vitreous humour of the eye and is a member of the extracellular matrix small leucine-rich repeat proteoglycan/protein family. As it is highly expressed in the eye throughout life we hypothesised that it contributes towards the anti-angiogenic properties of the vitreous humour. Purified recombinant opticin was generated as previously described (Le Goff et al. JBC 2003, 278:45280). Using the recombinant opticin we show that opticin potently inhibits VEGF and FGF-2-induced human retinal endothelial cell and bovine aortic endothelial cell proliferation, migration, formation of tube-like structures in matrigel and collagen, and invasion. In vivo studies showed that opticin significantly decreased FGF-2 and VEGF induced angiogenesis in chick chorioallantoic membrane assays. We also demonstrate that opticin decreases signalling through ERK1/2. These observations suggest that opticin is a key regulator of angiogenesis in the eye and further studies will elucidate its mechanisms of action.

Opticin is a class III member of the extracellular matrix small leucine-rich repeat protein (SLRP) family that was initially identified in the eye in association with the collagen fibrils of the vitreous humor. Recombinant and tissue-extracted forms of bovine opticin were subjected to biochemical and biophysical characterization. Following SDS-PAGE the predominant component produced by both forms was a broad band between 45–52 kDa. There was evidence for two-stage processing and, additionally, a proteolytic cleavage product of ∼25 kDa. Deconvolution of circular dichroism spectra revealed β-sheet (41%), β-turn (21%), and α-helix (10%), and thermal denaturation experiments showed a transition with a midpoint of 47 °C. Weight-averaged molecular mass measurements using both light scattering and analytical ultracentrifugation demonstrated that opticin exists in solution as a stable dimer of ∼90 kDa, which can be dissociated into a monomer by denaturation with 2.5 m guanidine hydrochloride or during SDS-polyacrylamide electrophoresis. Opticin remains a dimer after removal of the amino-terminal region by O-sialoglycoprotein endopeptidase digestion, suggesting that dimer formation is mediated by the leucine-rich repeats. Dimerization could have a number of functional consequences, including divalent ligand interactions.

Recently, several groups have published new information regarding the origins and structure of the vitreous humour, and the inner limiting lamina (ILL) of the retina. This short article provides an overview of this new information. It is proposed that vitreous proteins are derived from several different cell types with the posterior half of the non-pigmented ciliary epithelium being prominent in the expression of several connective tissue macromolecules. In addition, some basement membrane macromolecules are also expressed by the ciliary body and may subsequently be assembled on the surface of the Müller cells to form the ILL. New data suggest that the posterior half of the non-pigmented ciliary epithelium has substantial secretory activity and is likely to play a pivotal role in eye development.

PURPOSE:To determine the structure, location, and tissue-specific expression of the mouse opticin gene (Optc) and to compare expression in the eye with that of Prelp, collagen II, and collagen IX.METHODS:Expressed sequence tags (ESTs) to mouse opticin were identified and the full-length sequence obtained after PCR reactions using a 15-day-postconception (dpc) whole-mouse embryo cDNA library. The mouse chromosomal localization of Optc was determined by radiation hybrid mapping and its genomic structure determined using an Optc-containing BAC clone. Tissue-specific expression of opticin, PRELP, collagen II, and collagen IX mRNAs was investigated by in situ hybridization and by dot blot hybridization for opticin.RESULTS:The Optc gene was localized to mouse chromosome 1 at 74.3 cM and consisted of seven exons spanning 10 kb. The Optc gene was less than 4 kb from the Prelp gene. In situ hybridization localized opticin mRNA exclusively to the presumptive ciliary body during development and to the nonpigmented ciliary epithelium of the adult mouse eye. Expression of Prelp was also detected in the nonpigmented ciliary epithelium of the adult eye. However, expression of collagen types II and IX was detected largely in the developing mouse eye, with type IX expression confined primarily to the presumptive ciliary body.CONCLUSIONS:The Optc, Prelp, and fibromodulin (Fmod) genes form a cluster on mouse chromosome 1. Opticin may represent a marker for ciliary body differentiation. Continued expression of opticin in the adult mouse eye suggests functions other than that of putative regulator of vitreous collagen fibrillogenesis.