Molecular Heterogeneity of VEGF–A in the Vitreous of Patients with Proliferative Diabetic Retinopathy

Abstract

Introduction: : There is compelling evidence that vascular endothelial growth factor–A (VEGF–A) is a key mediator of intraocular neovascularization associated with ischemic retinal diseases and wet age–related macular degeneration. VEGF–A exists as one of several isoforms, of which VEGF–A₁₆₅ is the most abundant. However, VEGF–A₁₆₅ may be cleaved at the COOH terminus by various proteases (e.g., plasmin and MMP3) to generate bioactive proteolytic fragments lacking the heparin–binding domain. Previous studies (Aiello et al, NEJM 331:1480–7,1994) demonstrated that the levels of VEGF–A are elevated in the eye fluids of patients with active proliferative retinopathies. However, the assay employed in these early studies had a format that measured intact VEGF–A₁₆₅ but not VEGF–A₁₂₁ or proteolytic fragments of VEGF–A₁₆₅ such as VEGF–A₁₁₀, thus potentially underestimating the VEGF levels.

Methods: : To test this hypothesis, we employed a novel multi–site format that measures total VEGF–A, which was used in parallel with the conventional assay to test a panel of vitreous samples.

Results: : The levels of "total" VEGF–A were significantly higher than the levels of VEGF–A₁₆₅, suggesting that a significant portion of VEGF–A is either alternatively spliced VEGF–A₁₂₁ or – perhaps more likely – proteolytically cleaved VEGF–A. Consistent with this hypothesis, levels of MMP3 and plasminogen in the vitreous of patients with proliferative diabetic retinopathy were significantly higher than control vitreous samples. Furthermore, ion exchange chromatography documented the presence in such vitreous samples of molecular forms of VEGF consistent with VEGFA₁₂₁ or proteolytic fragments of VEGF–A₁₆₅.

Conclusions: : These findings suggest that non–heparin–binding VEGF–A forms generated by alternative mRNA splicing and/or proteolysis potentially play an important role in pathological neovascularization. Thus, neutralization of such forms of VEGF–A, in addition to VEGF–A₁₆₅, may be important to achieve optimal clinical benefit.

Keywords: diabetic retinopathy • vitreous • growth factors/growth factor receptors