In vitro anti-angiogenic effects of cryo-preserved amniotic membrane and the role of TIMP2 and thrombospondin

Abstract

Abstract Background Amniotic membrane (AM) has been used as a biological substrate in ophthalmology and other fields for several years. It has a valuable role in ocular surface burns and non-healing ulcers. It is known that AM is non-immunogenic and has anti-inflammatory and anti-fibrotic properties and supports epithelial cell proliferation and migration. These are supposedly mediated through a host of growth factors and cytokines. Despite its clinically accepted role in reducing corneal vascularisation when applied on the eye, there have been contradicting reports on the effect of AM on angiogenesis. Methods The effect of soluble factors released from clinically prepared cryo-preserved AM on cultured human umbilical vein endothelial cell (HUVEC) proliferation was quantified and angiogenesis assessed on matrigel. Antibody blockage of soluble tissue inhibitors of metalloproteinase (TIMP)2 and Thrombospondin, identified on Searchlight protein arrays, was used to evaluate their role in the effect of cryo-preserved AM. Results AM conditioned medium (AMCM) was found to reduce HUVEC proliferation and angiogenesis. Angiogenesis was reduced by direct application of AM, a gradient effect was observed, where HUVEC closer to the AM failed completely in forming any tubules. In addition, HUVEC failed to survive directly on the AM. Analysis of the soluble factors released by the amnion included high levels of anti-angiogenic factors, thrombospondin and TIMP 1 and 2. Antibody blockage of TIMP2 and thrombospondin prevented the anti-angiogenic effect of AMCM and AM. Conclusion AM in vitro has potent anti-angiogenic properties dependent on secreted TIMP2 and thrombospondin, validating some of the effects observed clinically.