Abstract
Abstract: :
Purpose: Opticin is a member of the small leucine–rich repeat protein/proteoglycan family. It is an extracellular matrix glycoprotein that is highly expressed in the eye throughout life and localises to the vitreous humour. The vitreous humour normally possesses anti–angiogenic properties to maintain its transparency. In this study we investigated whether opticin contributes to these properties. Methods: Recombinant bovine opticin was generated in 293–EBNA cells and purified from the conditioned media. The effects of opticin were tested on bovine aortic endothelial cells (BAEC) proliferation, migration and sprout formation stimulated by VEGF–A and FGF–2. Downstream signalling was analysed by semi–quantitative Western–blotting for phosphorylated ERK1/2. The effects of opticin on BAEC attachment, spreading and tubular formation were examined using a three dimensional type I collagen assay and VEGF–A, FGF–2 and bovine brain extract (BBE)–induced angiogenesis was performed to test the effects of opticin on in vivo chick choroiallantoic membrane (CAM) assay. Results: At 1 and 10 µg/ml, opticin had the ability to inhibit significantly both VEGF–A and FGF–2–stimulated proliferation, migration and sprout formation. The ERK1/2 phosphorylation downstream of VEGF–A and FGF–2 receptors was blocked by opticin. Opticin alone could disrupt attachment and spreading of BAEC on type I collagen matrix and blocked the BAEC attachment, spreading and tube–like structure formation stimulated by both VEGF–A and FGF–2. Opticin inhibited angiogenesis stimulated by VEGF–A, FGF–2 and BBE in the CAM assay. Conclusions: These results demonstrate that opticin has anti–angiogenic properties and inhibits the BAEC angiogenic responses to both VEGF–A and FGF–2 in in vitro cell culture systems. We also demonstrated that opticin inhibits angiogenesis in vivo. Opticin may play a key role in both the development of the eye (i.e. regression of the primary hyaloid vascular system) and in protection against vitreoretinal diseases such as proliferative diabetic retinopathy.
Keywords: extracellular matrix • vitreous • neovascularization