CXCR4 signaling at the ovine fetal-maternal interface regulates vascularization, CD34+ cell presence, and autophagy in the endometrium†

Abstract

Placenta development is characterized by extensive angiogenesis and vascularization but if these processes are compromised placental dysfunction occurs, which is the underlying cause of pregnancy complications such as preeclampsia and intrauterine growth restriction. Dysregulation of placental angiogenesis has emerged as one of the main pathophysiological features in the development of placental insufficiency and its clinical consequences. The signaling axis initiated by chemokine ligand 12 (CXCL12) and its receptor CXCR4 stimulates angiogenesis in other tissues, and may be central to placental vascularization. We hypothesized that CXCL12-CXCR4 signaling governs the pro-angiogenic placental microenvironment by coordinating production of central angiogenic factors and receptors and regulates endometrial cell survival essential for placental function and subsequent fetal longevity. The CXCR4 antagonist, AMD3100, was used to elucidate the role of CXCL12-CXCR4 signaling regarding uteroplacental vascular remodeling at the fetal-maternal interface. On day 12 postbreeding, osmotic pumps were surgically installed and delivered either AMD3100 or PBS into the uterine lumen ipsilateral to the corpus luteum. On day 20, endometrial tissues were collected, snap-frozen in liquid nitrogen, and uterine horn cross sections preserved for immunofluorescent analysis. In endometrium from ewes receiving AMD3100 infusion, the abundance of select angiogenic factors was diminished, while presence of CD34+ cells increased compared to control ewes. Ewes receiving AMD3100 infusion also exhibited less activation of Akt/mTOR signaling, and elevated LC3B-II, a marker of cellular autophagy in endometrium. This study suggests that CXCL12-CXCR4 signaling governs placental homeostasis by serving as a critical upstream mediator of vascularization and cell viability, thereby ensuring appropriate placental development.

Keywords: angiogenesis; chemokine; chemotaxis; conceptus; domestic animal reproduction; endometrium; female reproductive tract; implantation; placenta; placentation; ruminants; uterus.

Figure 1.

Pregnancy rates, as determined by presence of conceptus on day 20 post-breeding (100% AMD3100; 67% control).

Figure 2.

Protein abundance of (A) HIF1A, (B) FLT1, (C) VEGFA, and (D) CXCR4 in endometrial tissue of pregnant ewes, followed by (E) representative immunoblots. (F) Representative 20× hematoxylin and eosin staining of uterine cross sections. (G) Representative 40× magnification images of VEGFA (green) localization in superficial stroma (SS) of ovine uterine cross sections with DAPI (blue) as nuclear stain. Sections are from caruncle regions. Scale bars represent 20 μm. (H) Integrated density quantification of immunoreactive VEGFA in cross sections as shown in panel G. (I) Uterine cross sections were incubated with normal serum and secondary antibodies as negative controls; nuclei were counterstained with DAPI (blue). Data are represented by the mean ± SEM, and significance is denoted with an asterisk when P < 0.05 (*), or P < 0.01 (**).

Figure 3.

Protein abundance and representative immunoblots of VEGFA in ovine endometrial cells (oEP1) following treatment with (A) 50 ng/ml recombinant CXCL12 and (B) 300 ng/ml AMD3100. Data are represented by the mean ± SEM, and significance is denoted with an asterisk when P < 0.05 (*).

Figure 4.

Immunoreactive CD34 in ovine uterine cross sections. (A) Representative 40× magnification images of immunofluorescent staining for CD34 (yellow) with DAPI (blue) as nuclear stain. Scale bars represent 20 μm. (B) Integrated density quantification of immunoreactive CD34 in cross sections as shown in panel A. (C) Uterine cross sections were incubated with normal serum and secondary antibodies as negative controls; nuclei were counterstained with DAPI (blue). Data are represented by the mean ± SEM, and significance is denoted with an asterisk when P < 0.05 (*).

Figure 5.

Select pathways affected following inhibition of CXCR4 signaling at the fetal–maternal interface. Activation of (A) Akt and (B) mTOR, followed by (C) representative immunoblot. Protein abundance of cellular autophagy marker (D) LC3B-II and (E) representative immunoblot. Data are represented by the mean ± SEM, and significance is denoted with an asterisk when P < 0.05 (*), or P < 0.01 (**).