Identification of antiangiogenic and antiinflammatory proteins in human amniotic membrane

Purpose. To identify the potential antiangiogenic and antiinflammatory proteins expressed in human amniotic membrane tissue. Methods. Human amniotic epithelial 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. Antiangiogenic and antiinflammatory proteins were detected by the reverse transcriptase-polymerase chain reaction (RT-PCR) technique and further confirmed by DNA sequencing of PCR-amplified transcripts. The distribution of tissue inhibitors of metalloproteinase (TIMPs) were studied further by immunohistochemistry performed on paraffin-embedded amniotic membrane tissue. Results, RT-PCR results showed that both human amniotic epithelial and mesenchymal cells express interleukin-1 receptor antagonist, all four TIMPs, collagen XVIII, and interleukin-10. Thrombospondin-1 was expressed in all of the epithelial cell specimens and in one out of five mesenchymal cell specimens. Furthermore, immunohistochemistry studies performed on freshly prepared amniotic membrane confirmed that all members of the TIMP family were present in epithelial and mesenchymal cells as well as in the compact layer of the amniotic stroma. In cryopreserved ed amniotic membranes, positive staining was seen in residual amniotic cells and stroma. Conclusions. Human amniotic membrane epithelial and mesenchymal cells express various antiangiogenic and antiinflammatory proteins. Some of those proteins also were found in amniotic membrane stroma. These findings map explain in part the antiangiogenic and antiinflammatory effects of amniotic membrane transplantation.