Attachment of the conceptus trophoblast (Tr) to the uterine luminal epithelium (LE) is critical for successful implantation. This study determined whether alpha v (av) integrins (ITGAV) directly mediate porcine trophoblast cell adhesion to secreted phosphoprotein 1 (SPP1, also known as osteopontin (OPN)) and examined the temporal/spatial expression of ITGAV, beta 3 (b3, ITGB3) and beta 6 (b6, ITGB6) integrin subunits, and SPP1, at the uterine–placental interface of pigs. Knockdown of ITGAV in porcine Tr (pTr2) cells by siRNA reduced pTr2 attachment to SPP1. In situ hybridization confirmed the presence of ITGAV, ITGB3 and ITGB6 mRNAs in uterine LE and conceptus Tr between Days 9 and 60 of gestation, with no change in the magnitude of expression over the course of pregnancy. Exogenous E2 or P4 did not affect ITGAV, ITGB3 and ITGB6 mRNA expression in the uteri of ovariectomized gilts. Immunofluorescence identified ITGAV, ITGB3 and SPP1 proteins in large aggregates at the uterine LE-placental Tr/chorion interface on Day 25, but aggregates were no longer observed by Day 50 of gestation. These results are the first to directly demonstrate that pTr2 cells engage ITGAV-containing integrin receptors to adhere to SPP1 and suggest that mechanical forces generated by tethering elongating conceptuses to uterine LE leads to assembly of focal adhesions containing ITGAV and SPP1; however, as placentation progresses, subsequent folding/interdigitation at the uterine–placental interface disperses mechanical forces resulting in the loss of focal adhesions.
James W Frank, Heewon Seo, Robert C Burghardt, Kayla J Bayless and Greg A Johnson
Robert C Burghardt, James R Burghardt, James D Taylor II, Adele T Reeder, Bar T Nguen, Thomas E Spencer, Kayla J Bayless and Greg A Johnson
The integrity of the fetal–maternal interface is critical for proper fetal nourishment during pregnancy. Integrins are important adhesion molecules present at the interface during implantation; however, in vivo evidence for integrin activation and focal adhesion formation at the maternal–conceptus interface is limited. We hypothesized that focal adhesion assembly in uterine luminal epithelium (LE) and conceptus trophectoderm (Tr) results from integrin binding of extracellular matrix (ECM) at this interface to provide increased tensile forces and signaling to coordinate utero-placental development. An ovine model of unilateral pregnancy was used to evaluate mechanotransduction events leading to focal adhesion assembly at the maternal–conceptus interface and within the uterine wall. Animals were hysterectomized on days 40, 80, or 120 of pregnancy, and uteri immunostained for integrins (ITGAV, ITGA4, ITGA5, ITGB1, ITGB3, and ITGB5), ECM proteins (SPP1, LGALS15, fibronectin (FN), and vitronectin (VTN)), cytoskeletal molecules (ACTN and TLN1), and a signal generator (PTK2). Focal adhesion assembly in myometrium and stroma was also studied to provide a frame of reference for mechanical stretch of the uterine wall. Large focal adhesions containing aggregates of ITGAV, ITGA4, ITGA5, ITGB1, ITGB5, ACTN, and PTK2 were detected in interplacentomal uterine LE and Tr of gravid but not non-gravid uterine horns and increased during pregnancy. SPP1 and LGALS15, but not FN or VTN, were present along LE and Tr interfaces in both uterine horns. These data support the idea that focal adhesion assembly at the maternal–conceptus interface reflects adaptation to increasing forces caused by the growing fetus. Cooperative binding of multiple integrins to SPP1 deposited at the maternal–conceptus interface forms an adhesive mosaic to maintain a tight connection between uterine and placental surfaces along regions of epitheliochorial placentation in sheep.
Theodore T Wing, David W Erikson, Robert C Burghardt, Fuller W Bazer, Kayla J Bayless and Greg A Johnson
Angiogenesis is fundamental to the expansion of the placental vasculature during pregnancy. Integrins are associated with vascular formation; and osteopontin is a candidate ligand for integrins to promote angiogenesis. Endothelial progenitor cells (EPCs) are released from bone marrow into the blood and incorporate into newly vascularized tissue where they differentiate into mature endothelium. Results of studies in women suggest that EPCs may play an important role in maintaining placental vascular integrity during pregnancy, although little is known about how EPCs are recruited to these tissues. Our goal was to determine the αv integrin mediated effects of osteopontin on EPC adhesion and incorporation into angiogenic vascular networks. EPCs were isolated from 6 h old piglets. RT-PCR revealed that EPCs initially had a monocyte-like phenotype in culture that became more endothelial-like with cell passage. Immunofluorescence microscopy confirmed that the EPCs express platelet endothelial cell adhesion molecule, vascular endothelial cadherin, and von Willebrand factor. When EPCs were cultured on OPN-coated slides, the αv integrin subunit was observed in focal adhesions at the basal surface of EPCs. Silencing of αv integrin reduced EPC binding to OPN and focal adhesion assembly. In vitro siRNA knockdown in EPCs,demonstrated that OPN stimulates EPC incorporation into human umbilical vein endothelial cell (HUVEC) networks via αv-containing integrins. Finally, in situ hybridization and immunohistochemistry localized osteopontin near placental blood vessels. In summary, OPN binds the αv integrin subunit on EPCs to support EPC adhesion and increase EPC incorporation into angiogenic vascular networks.