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

The Columbia University Institutional Animal Care and Use Committee approved animal studies. Adult wild-type 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.

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.

Sections were stained as previously described [27]. Primary antibodies included anti-mouse VEGFR-1 (R&D Systems, AF471), anti-mouse F4/80 (eBioscience, 14–4801), anti-mouse CD31 (BD Biosciences, 553370), anti-mouse endomucin (Santa Cruz, sc-65495), anti-mouse VE-cadherin (BD Biosciences, 550548), and anti-mouse CD11b (Abcam ab8878). For IHC staining, biotin rabbit anti-goat IgG (Vector, BA-5000), biotin goat anti-rat IgG (BD Biosciences, 559286), the avidin/biotin blocking kit (Vector, SP-2001), the Vectastain ABC kit and DAB substrate kit (Vector, SK-4100) were used. Sections were counterstained with hematoxylin. For IF experiments, the following secondary antibodies were used: donkey anti goat-IgG Alexa-Fluor 594 (Invitrogen, A11058) and donkey anti rat-IgG Alexa-Fluor 488 (Invitrogen, A2108). Slides were covered with Vectashield containing with 4′, 6-diamidino-2-phenylindole (Vector, H-1200) for nuclear visualization.

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.

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].

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

We determined the expression of VEGFR-1 in uterine endothelial cells (ECs) and macrophages in the post-implantation murine decidua at E6.5 (Figure 1). To determine EC expression, VEGFR-1 was co-stained with three endothelial cell markers, CD31 [9, 28, 29], endomucin [30] and VE-cadherin [31, 32]. To determine macrophage VEGFR-1 expression, uterine sections were stained for VEGFR-1 and either the monocyte marker, CD11b [33, 34] or the macrophage marker, F4/80 [35‐37].

At E6.5, the embryo is readily detected in the post-implantation 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.

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).

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).

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.