Wing, Theodore (2013-11). Two Novel Mechanistic Paradigms in Pig Pregnancy: I. Osteopontin Binds to the ITGAV (the ?v Integrin Subunit) to Promote Porcine Endothelial Progenitor Cell Incorporation into Developing Vasculature. II. Expression and Regulation of Genes for Glucose and Arginine Transporters in Pig Uteri, Conceptuses and Placentae Increases during Pregnancy.. Master's Thesis.
Thesis
During pregnancy, uterine and placental blood vessels develop to facilitate maximal transfer of nutrients to the fetus. These studies focus on the incorporation of endothelial progenitor cells (EPCs) into the growing placental and uterine vasculatures (Study 1), and on the expression of the sodium-dependent glucose transporter (SLC5A1) and amino acid transporter (SLC7A3) in uterine and placental tissues (Study 2). For Study 1, RT-PCR indicated that purified monocytes isolated from newborn piglets differentiated into an endothelial-like phenotype in culture. While human umbilical vein endothelial cells (HUVECs) alone invaded a 3D collagen model to form multicellular tubes with lumens and branching structures, EPCs alone did not invade the matrices. However, when cultured in the presence of HUVECs, EPCs incorporated into vascular structures. Osteopontin (OPN) is a matricellular molecule highly expressed in regions of angiogenesis within the pig placenta. When HUVECs and EPCs were cultured in the presence of OPN, the number of EPCs that incorporated into newly-forming HUVEC sprouts increased significantly. EPCs express integrins, which act as transmembrane receptors for OPN. Affinity chromatography followed by immunoprecipitation studies suggested that the ITGAV subunit on EPCs directly bound OPN. Knocking down the ITGAV subunit using siRNA significantly decreased the ability of EPCs to adhere to OPN-coated culture wells in adhesion assays. For Study 2, in situ hybridization and qPCR revealed that SLC5A1 mRNA expression in uterine luminal (LE) and peaked on Day 12-13 of pregnancy. in situ hybridization analysis confirmed that SLC5A1 expression increased specifically in LE of pseudopregnant model and qPCR revealed that estrogen increased SLC5A1 mRNA (P<0.05) in vivo and in vitro. SLC7A3 was induced in the chorion between Days 25 and 30. qPCR confirmed a significant increase in SLC7A3 mRNA in Day 60 as compared to Day 30 placentae. In conclusion, we hypothesize: (1) that OPN binds to ITGAV-containing integrins on EPCs as an essential initial step in EPC incorporation into the growing vasculature to maximize placental vascularization; and (2) that SLC5A1 is increased in uterine LE of pigs by estrogens to mediate glucose transport, while SLC7A3 may mediate arginine transport across the placenta to support developing fetuses in pigs.
