Abstract

P 149

Soluble VEGF receptor 1 is a major anti-angiogenic factor of human amniotic membrane

Mikk Pauklin, Henning  Thomasen, Klaus-Peter Steuhl, Daniel Meller
Zentrum für Augenheilkunde, Universitätsklinikum Essen, Essen

Objective
Human amniotic membrane (AM) has been used for several years in ophthalmic surgery because of its anti-angiogenic, anti-inflammatory, and wound healing promoting properties. The major angiogenic factor, vascular endothelial growth factor (VEGF) is in several tissues neutralized by a soluble splice variant of its receptor 1 (sFLT-1). Our aim was to analyze if sFLT-1 is expressed in AM and could be responsible for its anti-angiogenic properties.
Methods
Total RNA and protein were isolated from six samples of AM, human central cornea, corneal epithelium and human limbal epithelial cells (HLEC) cultivated on cryo-preserved AM. The expression of sFLT-1 was analyzed using Real-Time PCR and Western blotting. AM samples were stored in cryo-medium at -80°C for at least three months. After thawing, the cryo-preserved AM was incubated in culture medium (0.5ml/cm2) at 37°C to produce AM conditioned medium (CM) and the medium was changed after 1, 2, 3, 5, 7, 9, 12 and 14 days, or after 3 days. The amount of sFLT-1 released from AM during the mentioned time-course was  measured using ELISA. Medium conditioned by each of the AM samples for 3 days, was incubated with either a neutralizing anti-sFLT-1 or isotype control antibody. To detect the effect of CM and sFLT-1 on angiogenesis, human umbilical vein endothelial cells (HUVECs) were cultivated for 3 days in 20, 40, 60, 80, or 100% of the above-mentioned AM-CM and the cell proliferation was analyzed by MTT assay.
Results
The mean expression of sFLT-1 was in AM 5-fold higher than in cornea, 170-fold higher than in corneal epithelium and more than 1000-fold higher than in HLECs (p<0.05). 0.3 ng/cm2 sFLT-1 was released from AM during the first day, 0.04 ng/cm2 during the second day, and no detectable amounts of sFLT-1 were released after that. AM-CM had a concentration-dependent inhibitory effect on HUVEC proliferation, reaching 33% with undiluted AM-CM. Most of the inhibitory effect could be reversed by a prior neutralization of sFLT-1 in CM.
Conclusions
The expression of sFLT-1 is in AM significantly higher than in cornea, corneal epithelium or HLECs. sFLT-1 is released from the AM during the first days after use and is probably one of its key ant-angiogenic factors.

Copyright: 2009 Porstmann Kongresse GmbH. All rights reserved. Impressum, Disclaimer.