In‐vitro anti angiogenic effects of cryo‐preserved amniotic membrane

Abstract

PurposeAmniotic membrane (AM) has long been used as a biological substrate in Ophthalmology. It has a valuable role in ocular surface burns, ulcers, and nonhealing epithelial defects. AM is non‐immunogenic and has anti‐inflammatory and anti‐fibrotic properties. AM is also supports epithelial cells proliferation and migration.These effects are 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 its in‐vitro anti angiogenic effect. The purpose of this study was to establish the effect of clinically prepared AM on angiogenesis and to identify the factors responsible for this role.MethodsThe effect of clinically prepared cryo‐preserved AM on human umbilical cord endothelial cells (HUVEC) proliferation was quantified using. The amniotic membrane effect on angiogenesis was quantified on matrigel. Responsible anti‐angiogenic factors were identified using searchlight protein analysis and immun‐blocking experiments.ResultsAM had a profound and significant direct effect on angiogenesis. A gradient effect was observed where HUVEC closer to the AM failed completely to migrate and form any tubules. HUVEC also failed to survive directly on the AM. proliferation was reduced significantly when exposed to AM conditioned medium. Analysis of the soluble factors released by the AM included high levels of anti‐angiogenic factors,Thrombospondin and tissue inhibitors of metalloproteinase 1 and 2.ConclusionsThe AM in‐vitro has significant anti‐angiogenic properties. This effect is attributable to an array of soluble anti angiogenic factors. This can opens a new field of using AM conditioned solutions for ocular surface vascularisation if the physical presence of the AM over prolonged period of time is not desired.