OPN binds alpha V integrin to promote endothelial progenitor cell incorporation into vasculature.

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.

Identification of appropriate reference genes for qPCR analyses of placental expression of SLC7A3 and induction of SLC5A1 in porcine endometrium.

INTRODUCTION: Endometria and placentae undergo developmental changes that affect the stability of genes used as references for normalization of qPCR data. We identified genes that are stable within the porcine endometrium and placenta throughout pregnancy, and elucidated the temporal/spatial mRNA localization of the glucose and arginine transporters, solute carrier family (SLC) 5A1 and SLC7A3, respectively. MATERIALS AND METHODS: qPCR was performed for 10 genes within porcine endometria from Days 5, 11, and 15 of the estrous cycle and 11, 15, 25, 40, 60, and 85 of pregnancy; and chorioallantois from Days 30, 35, 45, 50, 60, and 85. Gene stability was analyzed using GeNorm and NormFinder algorithms. qPCR and in situ hybridization determined temporal/spatial localization of SLC5A1 and SLC7A3 at the uterine-placental interface. RESULTS: The geometric mean of TATA-binding protein (TBP), hypoxanthine phosphoribosyl transferase 1 (HPRT1), and tubulin alpha 1B (TUBA1B) provides acceptable reference values for porcine placenta. The geometric mean of TBP, beta actin (ACTB), and succinate dehydrogenase complex subunit A flavoprotein (SDHA) is acceptable for endometria. SLC5A1 is induced by estrogen in endometrial luminal epithelium (LE) on Days 12 and 13 of pregnancy. SLC7A3 is expressed in the chorion. DISCUSSION AND CONCLUSION: Using appropriate reference genes resulted in complementary results between qPCR and in situ hybridization techniques for SLC5A1 and SLC7A3 mRNAs. SLC5A1 is induced in uterine LE by estrogen of trophectoderm origin when the blastocyst is free-floating and dependent on glucose from the endometrium, and SLC7A3 is expressed by the established placenta to support fetal growth.