Recombinant alpha2(IV)NC1 domain of type IV collagen is an effective regulator of retinal capillary endothelial cell proliferation and inhibits pre-retinal neovascularisation.
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.


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).


CONCLUSIONS
alpha2(IV)NC1 inhibits angiogenesis in the retinal microvasculature. This recombinant protein has potential for the treatment of neovascularisation in proliferative retinopathies.
Coleman
Gary
G
Centre for Vision Science, School of Biomedical Science, Queen's University Belfast, Royal Victoria Hospital, Grosvenor Road, Belfast, BT12 6BA, Northern Ireland, UK.
Gardiner
Tom A
TA
Boutaud
Ariel
A
Stitt
Alan W
AW
D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects; D002196:Capillaries; D002417:Cattle; D002448:Cell Adhesion / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D024141:Collagen Type IV / Q000494:pharmacology*; D004261:DNA Replication; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000187:drug effects*; D004797:Enzyme-Linked Immunosorbent Assay; D051379:Mice; D008810:Mice, Inbred C57BL; D010100:Oxygen / Q000633:toxicity; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000494:pharmacology; D015861:Retinal Neovascularization / Q000473:pathology / Q000517:prevention & control*; D012171:Retinal Vessels / Q000166:cytology*
D024141:Collagen Type IV; D011994:Recombinant Proteins; D010100:Oxygen