Antiangiogenic effect of human apolipoprotein(a) kringle V on retinal neovascularization

Abstract

Purpose: : To investigate antiangiogenic effect of human apolipoprotein(a) kringle V (rhLK8) on retinal neovascularization

Methods: : Cell viability assay was performed to find out cellular toxicity of rhLK8. We injected rhLK8 intravitreally, and histologic analysis and TUNEL assay were performed to demonstrate retinal toxicity of rhLK8. Oxygen-induced retinopathy (OIR) was induced in mice, and to assess the antiangiogenic effect of rhLK8, we intravitreally injected rhLK8 at postnatal day 14. As in vitro angiogenesis assays, tube formation and wound migration assays were performed. We assessed the degree of cell adhesion and migration on different types of extracellular matrix proteins according to the treatment with rhLK8, antibodies to subtypes of integrins, or inhibitors of ERK, AKT, and JNK pathways. Protein microarray was performed to screen with which subunits of integrins rhLK8 exerted its action. Western blotting was used to investigate downstream signalling of interaction between rhLK8 and α3β1 integrin.

Results: : rhLK8 inhibited retinal neovascularization in the mouse model of OIR without definite toxicity on endothelial cells and retinal tissues at the therapeutic dosage. rhLK8 showed inhibitory effect on angiogenic processes in 2 in vitro angiogenesis assays: tube formation and wound migration assays. Interestingly, rhLK8 reduced particulary fibronectin-mediated migration of endothelial cells. Further experiments showed high binding affinity of rhLK8 to α3β1 integrin. Furthermore, rhLK8 inhibited phosphorylation of focal adhesion kinase, resulting in suppression of activation of JNK through action on p130CAS.

Conclusions: : Our data suggested the possible application of rhLK8 in the treatment of retinal neovascularization through its inhibitory action on fibronectin-mediated angiogenesis.

Keywords: diabetic retinopathy • retinal neovascularization