63 Inhibition of CXCR4 at the fetal-maternal interface during placentation results in altered production of vascular endothelial growth factor receptors in the placenta on Day 90 of pregnancy

Abstract

Placental development is characterised by extensive angiogenesis and vascularization; if these processes are compromised, placental dysfunction occurs, which is the underlying cause of complications such as preeclampsia and intrauterine growth restriction. The signalling axis initiated by chemokine ligand 12 (CXCL12) and its receptor CXCR4 stimulate angiogenesis critical to placental vascularization. Our laboratory and others demonstrated stimulation of vascular endothelial growth factor (VEGF) synthesis by CXCL12/CXCR4 signalling, and recently, we reported less production of the VEGF receptor, FLT-1, on Day 20 in pregnant sheep following interference of intrauterine CXCL12-dependent signalling. While no animal model fully recapitulates human placentation, the sheep is arguably the most applicable animal model to study fetal-maternal interactions and placentation. Based on our studies, we hypothesised that inhibiting CXCR4 at the fetal-maternal interface during initial placentation alters placental production of VEGF receptors, FLT-1 and KDR, at mid-gestation. To test this hypothesis, AMD3100, a CXCR4 antagonist, was used to elucidate the role of CXCL12/CXCR4 signalling at the ovine fetal-maternal interface. On Day 12 post-breeding, osmotic pumps were surgically installed and delivered either AMD3100 or phosphate-buffered saline (PBS) into the uterine lumen ipsilateral to the corpus luteum for either 7 days (n = 7 PBS and n = 8 AMD3100) or 14 days (n = 7 PBS and n = 8 AMD3100). The objectives were to determine whether disruption of the CXCL12/CXCR4 axis during placentation affects fetal survival and alters VEGF receptor synthesis and whether duration of CXCR4 inhibition affects placental vascular remodelling. On Day 90 of pregnancy, ewes were anaesthetised; reproductive tracts were removed; and maternal caruncle (CAR) and fetal cotyledon (COT) components were separated, snap frozen in liquid nitrogen, and stored at −80°C until protein isolation. Pregnancy success was not affected by treatment or duration of treatment (71% PBS vs. 62% AMD3100 for 7 days; 85% PBS vs. 62% AMD3100 for 14 days). In addition, fetal weight on Day 90 (530.8 ± 28.2 g PBS vs. 540.5 ± 20.3 g AMD3100 for 7 days; 494.3 ± 23.9 g PBS vs. 532.7 ± 11.8 g AMD3100 for 14 days) was not affected by treatment. Immunoblotting was used to detect protein abundance, and an unpaired two-tailed Student's t-test was used to determine significant changes. Greater FLT-1 (P < 0.05) was evident in CAR and COT tissue on Day 90 for both the 7-day treatment (0.92 ± 0.16 CAR PBS vs. 1.48 ± 0.18 CAR AMD3100; 0.12 ± 0.16 COT PBS vs. 0.62 ± 0.16 COT AMD3100) and the 14-day treatment (0.18 ± 0.05 CAR PBS vs. 0.43 ± 0.001 CAR AMD3100; 0.04 ± 0.005 COT PBS vs. 0.11 ± 0.02 COT AMD3100) of CXCR4 inhibition compared with controls, whereas KDR levels did not change (P > 0.05). Interestingly, elevated FLT-1, but not KDR, is a marker of preeclampsia in women, and because of its role as a VEGF scavenger, overexpression of FLT-1 often leads to an anti-angiogenic state. We suggest that CXCL12/CXCR4 signalling during initial placental development serves as an upstream regulator of placental vascularization, thereby ensuring appropriate placental development.