Identification of antiangiogenic and antiinflammatory proteins in human amniotic membrane

Purpose. To identify the potential antiangiogenic and antiinflammatory prot eins expressed in human amniotic membrane tissue. Methods. Human amniotic e pithelial and mesenchymal cells were isolated from human amniotic membranes by sequential trypsin and collagenase digestion. Total RNAs were harvested from freshly obtained human amniotic epithelial and mesenchymal cells. Ant iangiogenic and antiinflammatory proteins were detected by the reverse tran scriptase-polymerase chain reaction (RT-PCR) technique and further confirme d by DNA sequencing of PCR-amplified transcripts. The distribution of tissu e inhibitors of metalloproteinase (TIMPs) were studied further by immunohis tochemistry performed on paraffin-embedded amniotic membrane tissue. Result s, RT-PCR results showed that both human amniotic epithelial and mesenchyma l cells express interleukin-1 receptor antagonist, all four TIMPs, collagen XVIII, and interleukin-10. Thrombospondin-1 was expressed in all of the ep ithelial cell specimens and in one out of five mesenchymal cell specimens. Furthermore, immunohistochemistry studies performed on freshly prepared amn iotic membrane confirmed that all members of the TIMP family were present i n epithelial and mesenchymal cells as well as in the compact layer of the a mniotic stroma. In cryopreserved ed amniotic membranes, positive staining w as seen in residual amniotic cells and stroma. Conclusions. Human amniotic membrane epithelial and mesenchymal cells express various antiangiogenic an d antiinflammatory proteins. Some of those proteins also were found in amni otic membrane stroma. These findings map explain in part the antiangiogenic and antiinflammatory effects of amniotic membrane transplantation.