Background: A recombinant form of the alpha2(IV)NC1 domain of type IV collagen has been shown to have potent anti-angiogenic activity although this peptide has not been studied in the context of proliferative retinopathies. In the current investigation we examined the potential for alpha2(IV)NC1 to regulate retinal microvascular endothelial cell function using a range of in vitro and in vivo assay systems.
Materials And Methods: alpha2(IV)NC1 at concentrations between 0.1 and 1 mug/ml was added to retinal microvascular endothelial cells (RMECs) followed by assessment of cell attachment, proliferation and survival. This agent was also tested within a novel in vitro three-dimensional retinal angiogenesis assay and the number of angiogenic sprouts quantified. alpha2(IV)NC1 was also delivered intra-vitreally to mice with oxygen-induced proliferative retinopathy (OIR) and neovascularisation evaluated in comparison with vehicle-treated controls.
Results: RMECs treated with alpha2(IV)NC1 (0.1, 0.5 and 1 microg/ml) showed delayed attachment at 3 h post-seeding, although this deficit had been restored at the 6-h time point. BrdU assay of DNA replication revealed that confluent RMECs treated with alpha2(IV)NC1 showed no measurable response in comparison with vehicle-treated controls. By contrast, proliferation of sub-confluent RMECs was significantly reduced by alpha2(IV)NC1 at 0.5 microg/ml (P<0.01). alpha2(IV)NC1 also induced apoptosis in RMECs and inhibited angiogenesis of pre-existing retinal vascular networks in vitro (P<0.001). Intra-vitreal injection of alpha2(IV)NC1 in the OIR model significantly inhibited pre-retinal neovascularisation compared with vehicle-treated controls (P<0.001).
Conclusion: alpha2(IV)NC1 inhibits angiogenesis in the retinal microvasculature. This recombinant protein has potential for the treatment of neovascularisation in proliferative retinopathies.
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