Abstract
Purpose: :
Opticin is a member of the small leucine–rich repeat protein/proteoglycan family. It is an extracellular matrix glycoprotein that was first identified associated with collagen fibrils in the vitreous and is highly expressed in the eye throughout life. The vitreous humour normally possesses anti–angiogenic properties to maintain its transparency but in pathological angiogenesis such as proliferative diabetic retinopathy, new blood vessels use the collagen network in the cortical vitreous as a "scaffold" for growth. In this study we investigated the effects of opticin on capillary morphogenesis in vitro and the role of integrin interactions.
Methods: :
Recombinant bovine opticin was generated in 293–EBNA cells and purified from the conditioned media. The effects of opticin on unstimulated or VEGF–, FGF–2–stimulated capillary morphogenesis were examined using 3D type I collagen assays. Cell spreading assays were used to study the interactions of opticin with endothelial cells (EC). In order to identify the integrins responsible for interactions, anti–functional antibodies raised against various α and ß chains were used as inhibitors during cell spreading assays. Direct binding studies between recombinant A–domain of the α2ß1 integrin and opticin were also performed using ELISA–based assays.
Results: :
The 3D collagen assays showed that opticin disrupted unstimulated and VEGF and FGF–2–stimulated capillary morphogenesis on type I collagen matrices. The cell spreading assays on opticin showed 60–70% of EC spreading when opticin was coated at 20 µg/ml. Inhibition assays revealed that the interaction between ECs and opticin could be blocked by anti–functional antibodies raised against α2 and ß1 chains. Direct binding studies revealed that opticin binds to the A–domain of the α2 chain with a Kd of 200 nM.
Conclusions: :
These results suggested that opticin may inhibit capillary morphogenesis on a collagen matrix by directly disrupting the interaction between collagen and its α2ß1 integrin receptor. Opticin may contribute to the anti–angiogenic properties of the vitreous by preventing EC interactions with collagen fibrils via the α2ß1 integrin and therefore, may play a role in protection against vitreoretinal diseases such as proliferative diabetic retinopathy.
Keywords: vitreous • neovascularization • protein structure/function