VEGFR-1 blockade disrupts peri-implantation decidual angiogenesis and macrophage recruitment

Background Angiogenesis and macrophage recruitment to the uterus are key features of uterine decidualization; the progesterone-mediated uterine changes that allow for embryo implantation and initiation of pregnancy. In the current study, we characterized the expression of vascular endothelial growth factor receptor-1 (VEGFR-1) in macrophages and endothelial cells of the peri-implantation uterus and determined if VEGFR-1 function is required for decidual angiogenesis, macrophage recruitment, and/or the establishment of pregnancy. Methods Expression of VEGFR-1 in uterine endothelial cells and macrophages was determined with immunohistochemistry. To assess the effect of continuous VEGFR-1 blockade, adult female mice were given VEGFR-1 blocking antibody, MF-1, every 3 days for 18 days. After 6 doses, females were mated and a final dose of MF-1 was given on embryonic day 3.5. Endothelial cells and macrophages were quantified on embryonic day 7.5. Pregnancy was analyzed on embryonic days 7.5 and 10.5. Results F4/80+ macrophages are observed throughout the stroma and are abundant adjacent to the endometrial lumen and glands prior to embryo implantation and scatter throughout the decidua post implantation. VEGFR-1 expression is restricted to the uterine endothelial cells. F4/80+ macrophages were often found adjacent to VEGFR-1+ endothelial cells in the primary decidual zone. Continuous VEGFR-1 blockade correlates with a significant reduction in decidual vascular and macrophage density, but does not affect embryo implantation or maintenance of pregnancy up to embryonic day 10.5. Conclusions We found that VEGFR-1 functions in both decidual angiogenesis and macrophage recruitment to the implantation site during pregnancy. VEGFR-1 is expressed by endothelial cells, however blocking VEGFR-1 function in endothelial cells results in reduced macrophage recruitment to the uterus. VEGFR-1 blockade did not compromise the establishment and/or maintenance of pregnancy.


Introduction
Implantation of the fertilized oocyte induces uterine decidualization, the rapid proliferation and differentiation of stromal fibroblasts into glycogen and lipid rich decidual cells. Endothelial cells within the decidua proliferate to form a rudimentary vascular plexus necessary to maintain pregnancy prior to placenta development [1][2][3]. Cells of the innate immune system, including natural killer cells and macrophages, infiltrate the decidua and function to establish and maintain maternal-fetal tolerance [4,5]. These events are coordinated by ovarian estrogen and progesterone. Angiogenesis during decidualization is required for a successful pregnancy, however the signaling pathways that regulate this process have yet to be fully characterized.
Embryo implantation and trophoblast invasion create a pro-inflammatory environment leading to the recruitment of immune cells that mediate maternal tolerance to the semi-allogeneic embryo [5,10]. Decidual macrophages are the second most abundant immune cell population at the implantation site, comprising 20-30% of immune cells in the uterine decidua [4,11,12]. Dysregulated macrophage activation in the decidua has been implicated in recurrent miscarriages [13]. Decidual macrophage numbers, as well as the proportion of pro-apoptotic Fas ligand expressing decidual macrophages, are increased in spontaneous miscarriages [14]. Thus, understanding the signaling pathways that regulate decidual macrophages may elucidate causes of early pregnancy failures.
Macrophages are closely associated with endothelial cells during physiologic angiogenesis during ovarian corpus luteum development and postnatal retinal development, [15,16]. Activation of macrophage VEGFR-1 has a role in macrophage recruitment to sites of active angiogenesis [17,18]. Macrophages have been implicated in promoting angiogenesis by releasing pro-angiogenic factors such as VEGF-A and angiopoietin [15] and in anastomosis, the formation of a bridge between two angiogenic tip cells followed by vascular sprout fusion [15,19]. Ablation of ovarian macrophages leads to significant endothelial cell depletion and hemorrhage [16,20]. Macrophages have been implicated in the pregnant uterus, including acting in the coordination of the maternal immune response and promoting angiogenesis. However, the relationship between decidual macrophages, endothelial cells and VEGFR-1 has not been investigated.
In this study, we determined the expression pattern of VEGFR-1 with respect to endothelial cells and macrophages in the pre-and post-implantation uterus. VEGFR-1 neutralizing antibodies were administered prior to and during implantation. Our goal was to determine if inhibition of VEGFR-1 affects decidual angiogenesis, macrophage recruitment, and/or the establishment and maintenance of pregnancy in mice. Using two different VEGFR-1 blocking antibodies, MF-1 and R&D Systems AF471 that prevent ligand binding to VEGFR-1, we found that both vascular density and macrophage recruitment to the pregnant uterus were significantly reduced. Despite this, embryos implanted in the uterus and pregnancy progressed after VEGFR-1 blockade. We conclude that VEGFR-1 activity is required for proper decidual angiogenesis and macrophage recruitment to the implantation site during pregnancy.

Animal model
The Columbia University Institutional Animal Care and Use Committee approved animal studies. Adult wildtype CD1 female mice and CD1 male mice of proven fertility were used. When mice were bred, noon on the day a mating plug was observed was designated embryonic day (E) 0.5. Uteri of non-pregnant female mice and pregnant females at E3.5 and E6.5 were embedded in Tissue-Tek® O.C.T.™ Compound (Sakura Fine Technical Co, Ltd, Tokyo, Japan), snap-frozen on dry ice in ethanol and stored at −80°C.
To determine the effect of continuous VEGFR-1 blockade, females were intraperitoneally (i.p.) injected with VEGFR-1 blocking monoclonal antibody (MF-1, 132 mg/kg animal, ImClone Systems, Inc.; n = 5 mice), anti-mouse VEGFR-1 antibody (AF471, 2 mg/kg animal, R&D systems; n = 1 mice), or saline (n = 5 mice) every 3 days for 18 days. MF-1 has an elimination half-life of 3 days and reaches maximal plasma concentrations 6 hours after dosing [27]. After 6 doses, females were mated and a final dose of antibody or saline was given on E3.5. Pregnant females were sacrificed on E7.5. Implantation sites were counted, uterine weight measured, and uteri freshly frozen in O.C.T.™ compound for sectioning. Pregnant females were sacrificed on E10.5 to determine the number of implantation sites and uterine weight.

Histological staining
For uterine analyses, 5 μm transverse sections through the non-pregnant and E3.5 uteri were generated. For implantation site analyses, frontal sections through uterus/implantation site were generated for E6.5 at 7 μm and for E7.5 with VEGFR-1 blockade at 12 μm. Implantation was confirmed by H&E staining every 5th section. Specific staining was performed at least 3 times and 5 different uterine sections or implantation sites were analyzed.
Imaging IHC and H&E staining images were captured with a Nikon Eclipse E800 microscope and Nikon DXM 1200 digital camera and NIS-Elements D3.10 software or ImagePro Plus v.4.01 software. Fluorescent images were captured using a Nikon A1 scanning confocal microscope on an Eclipse Ti microscope stand (Nikon Instruments, Melville, NY). Standard lasers and filters were used to image DAPI, AlexaFluor 488, and TRITC. Images were taken using the 10×/0.4 and 60×/1.49 objectives. Representative Z-stacked maximum intensity images are shown.

Quantitation of decidual macrophage and vascular density
The percentage of decidua occupied by blood vessels or macrophages was calculated from E7.5 decidua frontal uterine sections by dividing the total area of CD31 or F4/80 staining by the area of the decidua multiplied by 100. Images were captured using a Nikon Eclipse E800 microscope and Nikon DXM 1200 digital camera, and data were processed using ImagePro Plus Version 4.01 (Media Cybernetics) [28].

Serum progesterone levels
Blood was obtained from all animals by cardiopuncture. Serum progesterone levels were measured using a competitive chemiluminescent immunoassay (Diagnostic Products Corp./Siemens) [9].

Statistical analysis
Data are presented as mean ± standard error of the mean (sem). Unpaired Student t-test was used to compare sample means. P < 0.05 was considered a statistically significant difference. Statistical analyses were performed using the Statistical Package for Social Science version 15.0 (SPSS, Inc., Chicago, Il).
At E6.5, the embryo is readily detected in the postimplantation uterus ( Figure 1A). VEGFR-1 + cells are most abundant in the decidua directly surrounding the implanted embryo or the primary decidual zone ( Figure 1B-F). CD31 + and endomucin + ECs are observed throughout the decidua ( Figure 1B, C), while VE-cadherin + ECs are most abundant in the anti-mesometrial decidua ( Figure 1D). The positive VE-cadherin staining directly adjacent to the implanted embryo does not have a vascular pattern and does not represent ECs ( Figure 1D). VEGFR-1 is expressed in decidual ECs that express CD31 ( Figure 1B), endomucin ( Figure 1C) or VE-cadherin ( Figure 1D). CD11b + monocytes and F4/80 + macrophages are scattered throughout the decidua, but their distribution patterns differ ( Figure 1E, F). VEGFR-1 expression is not detected in CD11b + or F4/80 + decidual monocytes/ macrophages ( Figure 1E, F), in contrast to VEGFR-1 expression in decidual ECs. VEGFR-1 is expressed in cells that are directly adjacent to decidual monocytes/macrophages. Since VEGFR-1 expression closely overlaps with CD31 expression, CD31 was used as the EC marker for all additional experiments.

VEGFR-1 is expressed in endothelial cells of the non-pregnant uterus
In the non-pregnant uterus, CD31 + ECs and F4/80 + macrophages are distributed throughout the surrounding stroma and myometrium, but excluded from the uterine lumen and tubular endometrial glands that are lined by a single layer of columnar epithelium (Figure 2A-C). VEGFR-1 expression is restricted to a subset of stromal ECs ( Figure 2D). VEGFR-1 is not expressed by F4/80 + macrophages, but is expressed on cells adjacent to stromal macrophages ( Figure 2E, F).

VEGFR-1 is expressed in endothelial cells of the pre-implantation uterus
By E3.5, progesterone secretion by ovarian corpora lutea has been initiated to prepare the endometrium for embryo implantation. The gross appearance of the E3.5 pre-implantation uterus is similar to the non-pregnant uterus (Figures 2A and 3A). As in the non-pregnant state, CD31 + ECs ( Figure 3B) and F4/80 + macrophages ( Figure 3C) are distributed throughout the stroma and myometrium. Similar to the non-pregnant state, CD31 + ECs and F4/80 + macrophages ( Figure 3C, F) are abundant adjacent to the lumen and glands at E3.5. VEGFR-1 + cells are observed throughout the stroma ( Figure 3D). VEGFR-1 expression was cell associated consistent with the antibody detecting full-length VEGFR-1 on the cell surface ( Figure 3E, inset). Co-staining with CD31 reveals that most VEGFR-1 + cells are ECs ( Figure 3E). As in the non-pregnant state, VEGFR-1 + cells are found adjacent to F4/80 + macrophages ( Figure 3F) and CD11b + monocytes ( Figure 3G).

VEGFR-1 is expressed in endothelial cells of the post-implantation uterus
With embryo-uterine contact during implantation, stromal cells are transformed into large, polygonal, glycogen and lipid rich decidual cells. By E6.5, the primary and secondary decidual zones are defined by morphologic differences in decidual cells, as well as differential expression of tight junction proteins and VEGF receptors [9,29,38,39] (Figure 4A). CD31 + ECs are abundant throughout the decidua, the endometrium surrounding the implanted embryo, and myometrium ( Figure 4B, F, and G). F4/80 + macrophages are scattered throughout the decidua and most abundant in the myometrium and stroma directly adjacent to the myometrium ( Figure 4C, I, J). Within the decidua, the secondary decidual zone (SDZ) contains the highest concentration of F4/80 + macrophages ( Figure 4C). VEGFR-1 + cells are most abundant in the primary decidual zone (PDZ) ( Figure 4D, E, H). Most VEGFR-1 + cells are ECs ( Figure 4E) and within the PDZ, VEGFR-1 + cells are found adjacent to F4/80 + macrophages ( Figure 4H). As in the pre-implantation uterus, VEGFR-1 expression in decidual ECs is consistent with full-length VEGFR-1 ( Figure 4E, H, insets). The distribution pattern of VEGFR-1 + ECs and macrophages suggests that VEGFR-1 + EC and F4/80 + macrophages are in direct contact in both the non-pregnant, peri-implantation and post-implantation uterus.    Previous studies demonstrated that a single dose of VEGFR-1 blocking antibody MF-1, administered on E3.75, had no effect on decidual angiogenesis or pregnancy [9]. In this study, we administered 6 doses of MF-1 prior to mating and a single dose on E3.5, one day prior to implantation, to determine the effect of continuous VEGFR-1 blockade on pregnancy. Administration of VEGFR-1 blocking antibody MF-1 had no effect on the number of implantation sites or uterine weights at E7.5 and E10.5 (Table 1). Histological analysis of uterine tissues sections confirmed normal embryonic development at E7.5 and E10.5 (data not shown). However, differences in macrophage and vascular density were observed. At E7.5, F4/80 + macrophage and CD31 + EC densities were both significantly decreased in MF-1 treated uteri compared to controls ( Figure 5 and Table 1). VEGFR-1 blockade with R&D Systems AF471 antibody also resulted in decreased macrophage numbers and vascular density at E7.5 ( Figure 5B).
To confirm that blocking VEGFR-1 did not affect ovarian function, we measured serum progesterone levels at E7.5. The mean serum progesterone level was similar in MF-1 treated mice and controls (23.4 ± 2.3 vs. 27.9 ± 1.1 ng/mL, Table 1), indicating that VEGFR-1 blockage did not affect corpora lutea function in this model. Thus, the observed uterine defects can be attributed to the direct affects of VEGFR-1 blockade on the uterus.

Discussion
We have previously shown that peri-implantation inhibition of VEGFR-2 significantly reduces vascular density and causes pregnancy loss by E10.5. We showed that only a subset of endothelial cells express VEGFR-1 and peri-implantation inhibition with a single dose of VEGFR-1 blocking antibody at E3.75 does not affect vascular density or pregnancy [9]. Our present evaluation of VEGFR-1 expression with respect to endothelial cells and macrophages shows that VEGFR-1 expression is mainly restricted to uterine and decidual endothelial cells, while VEGFR-1 is not expressed in monocytes and macrophages. Endothelial cells expressing VEGFR-1 were often in direct contact with CD11b + monocytes and F4/80 + macrophages. Finally repeated pre-implantation injections of a VEGFR-1 blocking antibody, significantly decrease both the number of macrophages and vascular density in the decidua of E7.5 pregnant mice. Taken together, our results suggest that the decreased vascularity observed at E7.5 is a direct result of VEGFR-1 blockade, whereas the reduction in macrophages may be an indirect result of the decrease in VEGFR-1 function in the endothelial cells.
Treatment with MF-1, which blocks binding of VEGF-A and PlGF to VEGFR-1, can have pro-or anti-angiogenic effects, depending on the context of VEGFR-1 function [26,[40][41][42]. PlGF signaling, via membrane-localization of full length VEGFR-1, plays a pro-angiogenic role in pathological angiogenesis [26,42]. In murine decidual angiogenesis, VEGFR-1 appears to be membrane associated consistent with full-length receptor expression (Figures 1  and 3E, H). Thus, the reduced angiogenesis we observed is likely due to blocking ligand-dependent VEGFR-1 proangiogenic signaling. However, it has been previously shown that the VEGFR-1 TK domain is not necessary for murine decidual angiogenesis [29]. Thus the proangiogenic role for VEGFR-1, we observed may be independent of VEGFR-1 TK signaling pathway.
We have shown that a single dose of MF-1 administered within 24 hours of implantation does not affect vascular density [9], but continuous VEGFR-1 blockade results in a 48% reduction in vascular density ( Figure 5D). A greater than 50% reduction in decidual vascular density with VEGFR-2 blockade results in pregnancy loss, likely due to loss of angiogenesis in both the primary and secondary decidual zones [9]. In contrast, inhibition of VEGFR-3 results in a less than 50% decrease in primary decidual zone angiogenesis, but does not lead to pregnancy loss [9]. Combined, these data suggest that implantation and early pregnancy can tolerate a less than 50% reduction of vascular density in the primary decidual zone.
Macrophages are prominent in the adult ovary and uterus, two organs with physiological cyclical angiogenesis regulated by the VEGF/VEGFR-2 signaling pathway [9,28,43]. The number and phenotype of macrophages in the non-pregnant uterus changes with the ovarian cycle and additional macrophages are recruited into the endometrium during the peri-implantation period [20,44]. It has been shown that pre-implantation macrophage ablation prevents embryo implantation and results in infertility [20]. However, fertility is restored when macrophagedepleted mice are supplemented with exogenous progesterone suggesting a defect in corpora lutea function. In (See figure on previous page.) Figure 5 VEGFR-1 blockade reduces peri-implantation macrophage and vascular density at E7.5. (A, B) Double staining for F4/80 (green) and CD31 (red) in E7.5 uterine frontal sections from mice treated with VEGFR-1 blocking antibodies (MF-1 or AF471) or from control mice. Reduced expression of F4/80 and CD31 is observed in treatment groups. Scale bars = 50 μm. (C, D) F4/80 + or CD31 + signal was determined and normalized by total decidual area. F4/80 + macrophage and CD31 + EC density is significantly reduced in uteri of mice that received MF-1 compared to controls. fact, macrophages have been shown to regulate corpus lutea development and function, specifically the synthesis of progesterone that is required for embryo implantation and establishing pregnancy [20,45]. Thus, uterine macrophages may not be necessary for embryo implantation. We found that pregnancy can tolerate a reduction of macrophages by 37% of normal. Taken together, the data suggest that macrophages do not have an obligatory role in the uterine decidualization and implantation of the embryo. However the role of macrophages after embryo implantation still remains to be elucidated.