Research ArticleExperimental Studies
Open Access

Inhibition of VEGFR1 TK Signaling in Peritoneal Macrophages Suppresses Endometriosis Development

AKIKO FURUE, MASAKO HONDA, KYOKO HATTORI, ERINA SATO, ATSUSHI YAMASHITA, MAYUKO OSADA, KANAKO HOSONO, MARIKO KAMATA, YOSHIYA ITO, MASABUMI SHIBUYA, KAZUYOSHI KATO and HIDEKI AMANO
In Vivo September 2025, 39 (5) 2584-2598; DOI: https://doi.org/10.21873/invivo.14059

Abstract

Background/Aim: Endometriosis is characterized by the accumulation of immune cells in endometrial lesions and the peritoneal cavity. Macrophages contribute to the growth and neovascularization of endometriotic lesions. Vascular endothelial growth factor receptor-1 (VEGFR1) is involved in neovascularization, while peritoneal macrophages (PMs) play a critical role in endometriosis development and establishment. We examined the role of VEGFR1 signaling in PMs during endometriosis development using a murine model of ectopic endometrial transplantation.

Materials and Methods: Endometrial fragments from female wild-type (WT) or VEGFR1 tyrosine kinase-deficient (TK−/−) donor mice were implanted into the peritoneal walls of recipient mice, either in a WT→WT or TK−/−→TK−/− combination. On day 14 after endometrial transplantation, the implant size, neovascular growth-promoting factors, macrophage accumulation in the implants and peritoneal cavity, and cytokine production were assessed. PMs from WT or TK−/− mice were transferred into the peritoneal cavity of WT→WT mice and their effects were assessed.

Results: Compared to WT→WT mice, TK−/−→TK−/− mice exhibited smaller implant sizes and reduced neovascularization, including angiogenesis and lymphangiogenesis. This was correlated with an increase in pro-inflammatory (M1) and a decrease in alternative (M2) large peritoneal macrophages (LPMs) within the peritoneal cavity. Transfer of TK−/−-PMs into the peritoneal cavity of WT→WT mice reduced endometriosis development and macrophage accumulation. This led to increased expression of M1 macrophage genes and decreased expression of M2 phenotype genes, compared to WT-PMs transfer. PMs from TK−/− mice exhibited increased M1-related and decreased M2-related gene expression.

Conclusion: Deletion of VEGFR1 TK signaling in PMs suppressed endometriosis progression and neovascularization by increasing M1 LPMs. Specific inactivation of VEGFR1 TK signaling may represent a potential therapeutic target for the management of endometriosis.

Keywords:

Introduction

Endometriosis is a persistent inflammatory disorder marked by the ectopic growth of tissue resembling the endometrium, typically manifesting as “lesions” within the peritoneal cavity (1). Although the etiology of endometriosis remains undefined, endometrial growth appears to be dependent on angiogenesis and lymphangiogenesis (2-4). In both human and murine endometrial lesions, there is an elevation of pro-angiogenic and pro-lymphangiogenic factors, including endothelial growth factor (VEGF)-A, as well as VEGF-C (5, 6). Additionally, anti-VEGF-A antibodies or VEGF receptor 3 (VEGFR3) kinase inhibitors attenuate angiogenesis and lymphangiogenesis, respectively, thereby suppressing endometrial growth in mice (3, 7).

VEGF-A exerts its activity through tyrosine kinase receptors, including VEGFR1 and VEGFR2. VEGFR1 is mainly expressed in macrophages, whereas VEGFR2 is primarily found on endothelial cells (8). Furthermore, macrophages expressing VEGFR1 are involved in angiogenesis during tumor growth (8), gastric ulcer healing (9), and blood flow recovery from ischemic limbs (10). Notably, VEGFR1-expressing macrophages also contribute to lymphangiogenesis in wound healing (11) by inducing key pro-lymphangiogenic cytokines, including VEGF-C and VEGF-D. These findings indicate that VEGFR1 in macrophages plays a role in inflammation-related angiogenesis and lymphangiogenesis.

In support of this view, endometriosis progression is associated with changes in the local immune microenvironment, particularly in the presence of macrophages. Endometriosis is characterized by the accumulation of immune cells in the endometrial lesions and the peritoneal cavity (12). In our study on VEGFR1 signaling in endometriosis, we discovered that endometriosis progression in mice is linked to both angiogenesis and lymphangiogenesis. This progression is facilitated by the secretion of neovascular growth-promoting cytokines from VEGFR1-expressing macrophages that accumulate in endometrial lesions (3, 7). These macrophages are mainly recruited from the bone marrow (BM) (7).

Furthermore, women with endometriosis exhibit a significant presence of macrophages in both their lesions and the peritoneal cavity (13). In patients with endometriosis, activated peritoneal macrophages (PMs) play a crucial role in modulating the clearance of cellular debris and enhancing cellular proliferation and angiogenesis. Deletion of PMs using clodronate liposomes suppresses endometrial lesion development (14). These observations indicate that PMs play a critical role in the development and establishment of endometriosis. Additionally, macrophages can be classified into two main phenotypes: M1 pro-inflammatory and M2 anti-inflammatory. In both patients and mouse models of endometriosis, M2-polarized macrophages are found in the peritoneal cavity (14, 15). The accumulation of M2 macrophages enhances angiogenesis and thereby plays a significant role in endometriosis development.

PMs are characterized by two distinct populations: large peritoneal macrophages (LPMs) and small peritoneal macrophages (SPMs) (16). LPMs represent most of the macrophage population in the peritoneum (17). MHC IIlow F4/80high LPMs are resident and long-lived with an embryonic origin and are suggested to self-renew independently from hematopoiesis (18). By contrast, MHC IIhigh F4/80low SPMs are short-lived and are recruited from BM-derived monocytes.

The aim of present study was to explore the involvement of PMs expressing VEGFR1 in the development of endometriosis. Therefore, we investigated whether VEGFR1 signaling in PMs contributes to the development of endometriosis.

Materials and Methods

Animals. Female C57BL/6 wild-type (WT) mice (8 weeks old) were obtained from CLEA Japan (Tokyo, Japan). Female VEGFR1 TK-deficient (TK−/−) mice were generated as described elsewhere (19). All mice were kept in a pathogen-free facility under controlled environmental conditions: humidity at 50±5%, temperature at 25±1°C, and a 12-h light-dark cycle. Animals were provided with unrestricted access to food and water. All experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of Kitasato University School of Medicine (approval no. 2024-019). All experimental protocols adhered to the institutional guidelines for animal research based on the Science Council of Japan’s Guidelines for Proper Conduct of Animal Experiments.

Experimental model of endometriosis. An endometrial transplantation model was established as previously described (20). Briefly, mice were anesthetized by intraperitoneal (i.p.) injection of a three-drug cocktail: 0.3 mg/kg of medetomidine hydrochloride (Nippon Zenyaku Kogyo, Fukushima, Japan), 4.0 mg/kg of midazolam (Astellas Pharma, Tokyo, Japan), and 5.0 mg/kg of butorphanol (Meiji Seika Pharma, Tokyo, Japan). To eliminate the effects of endogenous estrogen and the menstrual cycle, bilateral ovariectomy was performed through paravertebral incisions. Atipamezole was administered i.p. at 0.75 mg/kg to reverse the effects of medetomidine. All donor and recipient mice were subcutaneously administered estradiol valerate (100 mg/kg) (Pelanin Depot; Mochida Seiyaku, Osaka) weekly from the day of ovariectomy. Uterine tissues from donor mice were harvested 7 days after ovariectomy. A uterine fragment, circular and 3 mm in diameter, was implanted onto both sides of the peritoneal wall of recipient mice using 7–0 polypropylene sutures for attachment (B-Brown Ace Scrap, Inc., Tokyo). The recipient mice, either WT or TK−/−, were implanted with fragments from donor mice of the same type, designated as WT→WT and TK−/−→TK−/−, respectively. The day of implantation was designated as day 0. On day 14 after endometrial transplantation, the animals were put under anesthesia using isoflurane (Pfizer, Manhattan, NY, USA), and the implants from both sides were carefully excised. Subsequently, the animals were euthanized by cervical dislocation. The excised implants were digitally photographed, and the implant area (mm2) was determined using ImageJ (National Institutes of Health). Results are expressed as implant area in mm2. One implant sample was prepared for reverse transcription-quantitative PCR (RT-qPCR), while the other was prepared for histological analysis.

Cell preparation, adoptive transfer, and cell culture. Isolated PMs from WT or TK−/− mice were resuspended in Roswell Park Memorial Institute (RPMI) medium (Gibco, Thermo Scientific, Waltham, MA, USA), which was enriched with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin. These cells were seeded in 12-well plates (2×105 cells/well) and incubated at 37°C with 5% CO2 for 3 h. After incubation, cells were washed with phosphate-buffered saline (PBS) to remove non-adherent cells. Isolated PMs (1×106 cells/100 μl PBS) were adoptively transferred into the peritoneal cavity of WT→WT mice 1 day after endometrial transplantation

In a separate experimental setup, PMs (2×105 cells/well) were exposed to lipopolysaccharide (LPS) (10 ng/ml; Sigma-Aldrich, St. Louis, MO, USA) and recombinant murine interferon-gamma (IFN-γ) (20 ng/ml; BioLegend, San Diego, CA, USA) to facilitate polarization into M1 macrophages, or to recombinant murine interleukin (IL)-4 (20 ng/ml; BioLegend) to facilitate polarization into M2 macrophages. This process was conducted in RPMI 1640 medium over a period of 24 h. After culturing, PMs were collected and processed with TRIzol reagent (Thermo Fisher Scientific, Carlsbad, CA, USA) to evaluate mRNA levels using RT-qPCR.

Flow cytometric analyses. Following centrifugation of the peritoneal fluids, peritoneal exudate cells were collected. The peritoneal cells were incubated with anti-mouse CD16/32 antibody (TruStain FcX; BioLegend) to prevent primary monoclonal antibodies from binding non-specifically to the cell samples. The cells were stained with a set of specific reagents, including Brilliant Violet 450-conjugatedanti-CD45 (30-F11), APC-conjugated anti-Ly6G (1A8), PE/Cy7-conjugated anti-CD11b (M1/70), FITC-conjugated anti-F4/80 (BM8), APC/CY7-conjugated anti-MHC M5/114.15.2), BV421-conjugated anti-CD80 (16-10A1), and APC-conjugated anti-CD206 (C068C2). All antibodies were purchased from BioLegend. The analysis did not include cells that were positive for 7-aminoactinomycin D (BioLegend). The samples were analyzed using a FACS Verse cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The data were processed using Kaluza software version 2.1 (Beckman Coulter, Brea, CA, USA).

Cytokine determination. Peritoneal fluid was recovered by lavage with ice-cold PBS containing 2% FBS (5ml×3). Collected fluids were analyzed using a cytometric bead array (CBA) kit (BD Biosciences).

Immunofluorescence analysis. Excised endometrial tissue samples were fixed with periodate-lysine-paraformaldehyde at 4°C overnight. The samples were transferred to 30% sucrose in 0.1 M phosphate buffer. Tissue sections (8-μm-thick) were blocked with 1% bovine serum albumin in 0.5% Triton X-100 in PBS, followed by an overnight incubation at 4°C with the designated primary antibodies: rat anti-mouse F4/80 (1:100; Bio-Rad Laboratories, Hercules, CA, USA), rat anti-mouse CD31 monoclonal antibody (1:200; BD Biosciences), rabbit anti-mouse lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) (1:100; Abcam, Cambridge, UK), and rat anti-mouse VEGFR1 (1:100; R&D systems, Minneapolis, MN, USA). Sections were then incubated with the appropriate secondary antibodies: donkey anti-rabbit IgG Alexa Fluor 488, donkey anti-goat IgG Alexa Fluor 488, and donkey anti-goat IgG Alexa Fluor 594, all sourced from Molecular Probes (Eugene, OR, USA). DAPI staining was used to visualize the nuclei. Images were acquired using a fluorescence microscope (Biozero BZ-700 Series; KEYENCE, Osaka, Japan). The count of F4/80+ cells was conducted in five different fields of view (×400 magnification) within the endometrial tissue.

Determination of vessel density. Microvessel density (MVD) and lymphatic vessel density (LVD) within endometrial tissue implants have been used to assess angiogenesis and lymphangiogenesis, respectively (20). The number of CD31+ blood vessels and LYVE-1+ lymphatic vessels in four fields of view using a fluorescence microscope (Biozero BZ-700; Keyence Corporation) (×200 magnification) was counted. The results are presented as the number of CD31+ blood vessels or LYVE-1+ lymphatic vessels per square millimeter (MVD/mm2 or LVD/mm2). In addition, the area covered by blood and lymphatic vessels was determined using ImageJ and presented as the percentage of the total area observed [microvessel area (MVA)% or lymphatic vessel area (LVA)%].

Real-time quantitative PCR analysis. Total RNA was extracted from endometrial tissues using TRIzol reagent (Thermo Fisher Scientific, Carlsbad, CA, USA). To synthesize cDNA from 1 μg of extracted RNA, the ReverTra Ace qPCR RT Kit (TOYOBO Co., Ltd., Osaka, Japan) was utilized. The quantitative PCR amplification process was carried out using TB Green Premix Ex Taq II (Tli RNaseH Plus; Takara Bio, Inc. Shiga, Japan) under the following thermal cycling conditions: an initial step at 95°C for 10 s, followed by 40 cycles consisting of 95°C for 3 s and 60°C for 20 s. The mRNA expression levels were quantified using the comparative threshold cycle method and were normalized to the expression of glyceraldehyde-3-phosphate dehydrogenase in each sample. Details of the primer sequences are provided in Table I.

View this table:
Table I.

The primers used for reverse transcription and quantitative polymerase chain reaction (PCR) reactions.

Statistical analysis. The data are presented as the mean ± standard deviation (SD). Statistical analyses were conducted using GraphPad Prism version 8 (GraphPad Software, La Jolla, CA, USA). Data was compared between two groups using unpaired two-tailed Student’s t-tests and between multiple groups using one-way analysis of variance, followed by Tukey’s post hoc tests. Statistical significance was set at p<0.05.

Results

Deficient VEGFR1TK signaling attenuates endometrial growth and the formation of new blood and lymphatic vessels. We first investigated the role of VEGFR1 TK signaling in endometriosis development. After endometrial transplantation, the implant size in WT→WT mice increased on day 14. In the experiment where endometrial fragments from TK−/− mice were transplanted into TK−/− mice (TK−/−→TK−/− mice), the implant growth on day 14 was lower than that in WT→WT mice (Figure 1A). This result suggests that VEGFR1 TK signaling plays a role in endometriosis growth. The mRNA levels of VEGFR1 were higher in WT→WT mice than in TK−/−→TK−/− mice (Figure 1B). Additionally, VEGFR1 was co-localized with F4/80+ cells in WT→WT mice (Figure 1B). As we previously showed that endometriosis development is dependent on angiogenesis and lymphangiogenesis (3, 7), we examined pro-angiogenic and pro-lymphangiogenic factors. The gene expressions of angiogenic factors including VEGF-A and CD31 as well as lymphangiogenic factors including VEGF-C, VEGFR3, and LYVE-1 in WT→WT mice were higher than those in TK−/−→TK−/− mice (Figure 1C). Furthermore, we examined F4/80+ cells (macrophages) accumulation in the implants, because macrophages are a key player for angiogenesis and lymphangiogenesis in endometriotic lesions. Immunofluorescence analyses revealed that the number of F4/80+ cells in WT→WT mice was higher than that in TK−/−→TK−/− mice (Figure 1D). The mRNA levels of a chemokine, CCL2 and its receptor CCR2 in WT→WT mice were higher than those in TK−/−→TK−/− mice (Figure 1D). Collectively, these results indicate that increased endometrial growth was related to macrophage accumulation in the implants.

Figure 1.
Figure 1.

Deficiency of vascular endothelial growth factor receptor 1 (VEGFR1) tyrosine kinase (TK) signaling suppresses endometrial implant growth, angiogenesis, lymphangiogenesis, and macrophage accumulation. (A) Representative images of typical implant appearance in wild-type (WT)→WT and TK−/−→TK−/− mice and implant size (scale bar: 3 mm). (B) mRNA levels of VEGFR1 in WT→WT and TK−/−→TK−/− mice and representative microphotographs of double immunostaining of VEGFR1 (green) and F4/80 (red) in WT→WT mouse implants (scale bar: 50 μm). The arrowheads indicate double-positive cells. (C) mRNA levels of angiogenic factors (VEGF-A and CD31) and lymphangiogenic factors [VEGF-C, VEGFR3, and lymphatic vessel endothelial hyaluronan receptor (LYVE)-1] in WT→WT and TK−/−→TK−/− mouse implants. (D) Representative microphotographs of immunostaining of F4/80 (red) in the implants (scale bar: 50 μm). Cell nuclei were counterstained with DAPI (blue). The number of F4/80+ cells and mRNA level of C-C motif chemokine ligand 2 (CCL2) and C-C chemokine receptor type 2 (CCR2) in WT→WT and TK−/−→TK−/− mouse implants. Data are expressed as the mean±SD. *p<0.05, ***p<0.001.

Deficient VEGFR1 TK signaling increases pro-inflammatory LPMs and cytokines after implantation of endometrial tissues. As PMs, including LPMs and SPMs, participate in endometriosis development, we analyzed LPMs and SPMs in the peritoneal cavity from WT and TK−/− mice by flow cytometry. On day 14, gating was used to identify LPMs (F4/80highMHC-IIlow) and SPMs (F4/80lowMHC-IIhigh), and their numbers were subsequently determined (Figure 2A). The number of LPMs in TK−/−→TK−/− mice was higher than that in WT→WT mice. By contrast, there was no statistically significant difference in SPMs between the two groups. We also examined the phenotypes of LPMs on day 14, during the development of endometriosis. When compared to WT→WT mice, M1 macrophages (CD80 high/CD206 low) were more accumulated in the peritoneal cavity of TK−/−→TK−/− mice, while M2 macrophages (CD80low/CD206high) were less accumulated (Figure 2B). These results indicate that deficient VEGFR1 TK signaling increased M1-LPMs during the development of endometriosis. We further measured cytokine levels in the peritoneal fluids. The concentrations of pro-inflammatory cytokines, including TNFα and IL-6, were higher in TK−/−→TK−/− mice than in WT→WT mice (Figure 2C). Additionally, the concentration of the anti-inflammatory cytokine IL-10 was lower in TK−/−→TK−/− mice than in WT→WT mice (Figure 2C). These results suggest that increased M1-LPMs lacking VEGFR1 TK signaling are associated with inhibition of endometriosis progression as indicated by reduced implant sizes in WT→WT mice.

Figure 2.
Figure 2.

Accumulation of large peritoneal macrophages (LPMs) polarized towards M1 phenotype in the development of endometriosis. (A) Flow cytometry gating strategy of peritoneal macrophages (PMs) in wild-type (WT)→WT mice. F4/80high/MHC IIlow cells are defined as large peritoneal macrophages (LPMs) and F4/80low/MHC IIhigh cells are defined as small peritoneal macrophages (SPMs). The numbers of LPMs and SPMs were compared between WT→WT and tyrosine kinase (TK)−/−→TK−/− mice. (B) Representative dot plots of CD80/CD206 in LPMs from WT→WT and TK−/−→TK−/− mice and comparison of CD80+ or CD206+ LPMs between the two groups. (C) Levels of tumor necrosis factor alpha (TNFα), interleukin (IL)-6, and IL-10 cytokines in peritoneal fluids from WT→WT and TK−/−→TK−/− mice. Data are expressed as the mean±SD. *p<0.05, ***p<0.001.

Deficient VEGFR1 TK signaling in PMs attenuates endometrial growth and the formation of new blood and lymphatic vessels. To further confirm the involvement of VEGFR1 TK signaling in PMs in endometriosis progression, we adoptively transferred PMs isolated from WT or TK−/− mice to WT→WT mice. On day 14, the implant size in WT→WT mice transferred with WT-derived PMs was larger than that in WT→WT mice transferred with TK−/−-derived PMs (Figure 3A), suggesting that PMs from TK−/− mice reduced endometrial growth. The MVD, indicated by the number of CD31+ blood vessels in the implants, was larger in WT→WT mice transferred with WT-PMs than in those transferred with TK-deficient PMs (Figure 3B). The same was true for LVD, indicated by the number of LYVE-1+ lymphatic vessels in the implants. Furthermore, the mRNA levels of angiogenic factors, including VEGF-A and CD31 as well as lymphangiogenic factors, including VEGF-C, VEGFR3, and LYVE-1 in WT→WT mice transferred with TK−/−-derived PMs were lower than those in WT→WT mice transferred with WT-derived PMs (Figure 3C).

Figure 3.
Figure 3.

Transfer of vascular endothelial growth factor receptor 1 (VEGFR1) tyrosine kinase (TK)-deficient PMs suppresses endometrial growth and neovascularization in wild-type (WT)→WT mouse implants. (A) Representative images of typical implant appearance in WT→WT mice transferred with WT-PMs or VEGFR1 tyrosine kinase (TK) deficient (TK−/−)-PMs (scale bar: 3 mm) and comparison of implant size between the two groups. (B) Immunofluorescence staining for CD31 (red) and LYVE-1 (green) in the implants from WT→WT mice transferred with WT-PMs or TK−/−-PMs (scale bar: 100 μm). Microvessel density (MVD) and lymphatic vessel density (LVD) were compared between WT→WT mice transferred with WT-PMs or TK−/−-PMs. (C) mRNA levels of angiogenic factors including VEGF-A and CD31, and lymphangiogenic factors including VEGF-C, VEGFR3, and lymphatic vessel endothelial hyaluronan receptor (LYVE)-1 in WT→WT mice transferred with WT-PMs or TK−/−-PMs. Data are expressed as the mean±SD. *p<0.05, **p<0.01, ****p<0.0001.

Transfer of PMs deficient in VEGFR1 TK signaling reduced the accumulation of macrophages in the implants. We also examined macrophage infiltration in the implants as well as the peritoneal cavity after transfer of PMs to WT→WT mice. The number of F4/80+ cells in WT→WT mouse implants transferred with TK−/−-PMs was lower than the F4/80+ cell number in those transferred with WT-PMs. Double immunofluorescence analyses demonstrated that F4/80-expressing macrophages in WT→WT mouse implants transfer with WT-PMs were positive for VEGF-A and VEGF-C. The mRNA levels of M1 markers, including TNFα and IL-6, in WT→WT mice transferred with TK−/−-PMs were higher than those in WT→WT mice transferred with WT-PMs, while those of M2 markers, including MR and IL-10, were lower (Figure 4C). Regarding PMs in the peritoneal cavity, the number of LPMs in WT→WT mice that received TK−/−-PMs was greater than that in WT→WT mice that received WT-PMs. However, no statistically significant difference was observed in the number of SPMs between these two groups (Figure 4D). The peritoneal fluids of WT→WT mice that received TK−/−-PMs exhibited an increase in pro-inflammatory cytokine levels, including TNFα and IL-6, compared to the fluids of WT→WT mice that received WT-PMs (Figure 4E). No significant difference was noted in IL-10 levels between the two groups.

Figure 4.
Figure 4.
Figure 4.

Transfer of vascular endothelial growth factor receptor 1 (VEGFR1) tyrosine kinase (TK)-deficient PMs suppresses macrophage accumulation in wild-type (WT)→WT mouse implants and increases M1-LPMs in the peritoneal cavity of WT→WT mice. (A) Representative microphotographs of immunohistochemistry for F4/80 (red) in the implants of WT→WT mice transferred with WT-PMs or VEGFR1 TK-deficient (TK−/−) PMs (scale bar: 50 μm). Cell nuclei were counterstained with DAPI (blue). Quantified results are shown in the bar chart. (B) Immunofluorescence staining for F4/80 (red) and VEGF-A (green) or VEGF-C (green) in the implants from WT→WT mice transferred with WT-PMs or TK−/− -PMs. The arrowheads indicate double-positive cells (scale bar: 25 μm). (C) mRNA levels related to M1 macrophages including tumor necrosis factor alpha (TNFα) and interleukin (IL)-6 and M2 macrophages including mannose receptor (MR) and IL-10 in WT→WT mice transferred with WT-PMs or TK−/− -PMs. (D) Representative dot plots of LPMs and SPMs from WT→WT mice transferred with WT-PMs or TK−/− -PMs. Quantified results are shown in the bar chart. (E) Levels of TNFα, IL-6, and IL-10 in peritoneal fluids from WT→WT mice transferred with WT-PMs or TK−/− -PMs. Data are expressed as the mean±SD. *p<0.05, **p<0.01, ****p<0.0001.

Lack of VEGFR1 TK signaling in PMs induced M1 macrophage polarization in vitro. Finally, we examined whether the lack of VEGFR1 TK signaling plays a role in promoting PMs polarization in vitro. The stimulation of isolated PMs from TK−/− mice with LPS and IFN-γ increased the mRNA levels of M1 macrophages, including TNFα and IL-6, compared with PMs from WT mice (Figure 5A). Additionally, the stimulation of TK−/− PMs with IL-4 decreased the mRNA levels of M2 macrophages, including MR and IL-10, compared with WT-LPMs (Figure 5B).

Figure 5.
Figure 5.

M1 and M2 macrophages-related gene expressions in cultured PMs. (A) mRNA levels of tumor necrosis factor alpha (TNFα) and interleukin (IL)-6, cytokines related to M1 macrophages, in cultured PMs stimulated with LPS/IFN-γ or vehicle. (B) mRNA levels of the M2 macrophage-related mannose receptor (MR) and IL-10 in cultured PMs stimulated with IL-4 or vehicle. Data are expressed as the mean±SD. **p<0.01, ***p<0.001, ****p<0.0001.

Discussion

Macrophages contribute to endometriosis development; however, the mechanisms underlying their involvement in endometrial growth remain to be clarified. We previously showed that BM-derived macrophages expressing VEGFR1 in the endometrial implants promote angiogenesis and lymphangiogenesis by inducing neovascular-stimulating factors (3, 7). However, little is known about the involvement of PMs expressing VEGFR1. Our current study revealed that accumulation of VEGFR1 TK-deficient pro-inflammatory LPMs in the peritoneal cavity contributed to the suppression of endometriosis development and neovascularization. Furthermore, adoptive transfer of VEGFR1 TK-deficient PMs attenuated endometrial growth and neovascularization. These results suggest that inhibition of VEGFR1 TK signaling in PMs suppressed the promotion of endometriosis and neovascularization.

Macrophages are crucial components within the microenvironment of endometriosis. Specifically, PMs are thought to play a role in the advancement of endometriosis. Indeed, increased PMs are accompanied by endometrial growth (21). PMs represent a substantial component of the immune cell population in the pelvic cavity. In patients diagnosed with endometriosis, PMs not only increase in quantity but also undergo functional alterations (22, 23). Deletion of PMs with clodronate liposome reduces endometrial growth and angiogenesis (14), suggesting that PMs participate in the progression of endometriosis and angiogenesis in endometriotic lesions. Recent evidence indicates that elevation of PMs is related to increased biological activities related to angiogenesis and tissue adhesion in endometriotic lesions (24).

In the current study, mice with endometriosis showed higher numbers of LPMs than SPMs, indicating that mouse endometriosis development was associated with increases in LPMs, which is consistent with previous studies (25). Additionally, various molecules, including chemokines, mediate recruitment of PMs to the peritoneal cavity (26). As the levels of CCL2 are increased in the peritoneal cavity of women with endometriosis, CCL2 appears to contribute to the accumulation of PMs. However, the underlying mechanisms by which LPMs, as opposed to SPMs, are elevated during endometriosis progression remain to be elucidated. In contrast, using another endometriosis model where endometrial fragments obtained from estradiol-primed donor mice were injected into the peritoneal cavity of intact recipient mice, mice with endometriosis displayed lower numbers of LPMs and higher numbers of SPMs than PBS-treated control mice (27). The discrepant results may be due to differences in experimental designs, including induction of endometriosis and the animal models used.

Additionally, our data demonstrated that VEGFR1-deficient LPMs were related to suppression of endometriosis development and angiogenesis/lymphangiogenesis in the implants. These findings suggest that a lack of VEGFR1 TK signaling in LPMs contributes to inhibiting endometriosis development and neovascularization. This view was supported by experiments demonstrating that the adoptive transfer of VEGFR1 TK signaling-deficient PMs to the peritoneal cavity of WT mice transplanted with endometrial tissues reduced endometriosis development and neovascularization associated with blood/lymphatic vessel-stimulating factors.

The phenotypes of PMs involved in endometriosis have been appreciated. In patients with endometriosis, a significant presence of M2 macrophages is observed in the peritoneal cavity. Moreover, the administration of M2-polarized macrophages derived from BM into the peritoneal cavity of mice has been demonstrated to facilitate the formation of substantial ectopic lesions (14). In contrast, the administration of M1-polarized RAW264.7 macrophages into the peritoneal cavity of mice reduced endometrial growth (28). To understand the role of VEGFR1-TK signaling in LPMs in endometriosis development, we assessed the phenotypes of LPMs during endometrial growth. Our data revealed that M1 macrophages lacking VEGFR1-TK signaling contributed to the suppression of endometriosis development. Furthermore, these results suggest that VEGFR1 TK signaling deficiency polarized LPMs towards a pro-inflammatory phenotype. This speculation is supported by results showing that pro-inflammatory cytokines, including TNFα and IL-6, in the peritoneal fluids from TK−/−→TK−/− mice were increased compared with those in WT→WT mice. Additionally, our in vitro studies suggested that VEGFR1 TK signaling is involved in LPM polarization from a pro-inflammatory to an anti-inflammatory phenotype. However, further studies are required to elucidate the underlying mechanisms of peritoneal macrophage polarization by VEGFR1 TK signaling.

The present study demonstrated that the number of VEGFR1 TK-deficient macrophages in the peritoneal cavity increased; however, macrophage accumulation in the implants was suppressed. These findings may suggest that VEGFR1 TK-deficient LPMs failed to infiltrate into the implants. The critical issue is whether peritoneal macrophages promote endometrial growth and neo-vascularization by infiltrating to endometriotic lesions. Previous studies (29) have shown that LPMs expressing the transcriptional factor GATA6, a marker for LPMs, infiltrate endometriosis lesions; however, these infiltrated LPMs diminished the expression of GATA6 after entering the lesions, suggesting phenotypic changes after crossing into the lesion. Of interest, in the liver, PMs rapidly invade heat-induced injured lesions to repair damaged tissues without losing GATA6 expression (30). In contrast, no evidence of PM invasion into the hepatic sinusoids has been provided in several liver disease models (31). Thus, further studies are needed to examine whether PMs infiltrate endometriotic lesions and promote their growth, angiogenesis, and lymphangiogenesis by releasing growth factors.

There is an urgent need to identify new therapeutic targets for endometriosis that are both noninvasive and nonhormonal. PMs play a critical role in endometriosis pathology, and our data show that M1-polarized PMs by inhibiting VEGFR1 TK, suppress endometriosis progression and neovascularization. These findings suggest M1-polarized macrophages could prevent endometriosis development. Recent studies support this idea, showing that administering M1-polarized macrophages derived from RAW264.7 macrophages reduces endometriosis growth in murine models (28). Nanoparticle treatments reprogramming PMs to the M1 phenotype inhibit endometriosis progression in mice (32). Therefore, modulating the macrophage environment presents a promising treatment approach. However, it is important to recognize that macrophage modification has been based on the M1/M2 paradigm. A recent single-cell analysis in mice (33) has revealed more diverse macrophage subpopulations in endometriosis lesions and the peritoneal cavity than previously documented (29). Further studies are needed to determine which specific macrophage subpopulations are targeted and modified in the treatment of endometriosis.

Study limitations. First, the underlying regulatory mechanisms of VEGFR1 TK signaling in macrophage polarization remain unknown. Second, we employed an ectopic endometriosis transplantation model by suturing donor-derived endometrial tissue fragments to the peritoneal wall. This suturing model induces additional inflammation. In this regard, other endometriosis mouse models, such as those created by injection of endometrial fragments into the peritoneal cavity to mimic spontaneous lesion formation, should be considered. Third, we utilized an animal model to investigate the role of VEGFR1 signaling in PMs during endometriosis development; however, the current study does not include data from human samples. Further studies are necessary to validate the role of VEGFR1 signaling in PMs in human participants with endometriosis.

Conclusion

Deletion of VEGFR1 TK signaling increased pro-inflammatory LPMs, resulting in suppressed endometrial growth and neovascularization as indicated by decreased levels of angiogenic and lymphatic vessel markers and stimulating factors. In addition, deficiency of VEGFR1 TK signaling polarized PMs toward a pro-inflammatory phenotype. Specific inactivation of VEGFR1 TK signaling may represent a promising target for the therapeutic management of endometriosis progression.

Acknowledgements

The Authors thank Michiko Ogino for technical assistance.

Footnotes

  • Authors’ Contributions

    AF: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing - original draft; AY, MH, KH, ES, MO, KH, and MK: Data curation, Investigation, Methodology; YI: Formal analysis, Investigation, Writing - review & editing; MS: Methodology, Resources; KK and HA: Supervision, Validation, Writing - review & editing.

  • Conflicts of Interest

    The Authors declare that there are no conflicts of interest.

  • Funding

    This work was supported by a research grant from the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT) (24K19703). This study was also supported by the Parents’ Association Grant of the Kitasato University School of Medicine.

  • Artificial Intelligence (AI) Disclosure

    No artificial intelligence (AI) tools, including large language models or machine learning software, were used in the preparation, analysis, or presentation of this manuscript.

  • Received June 16, 2025.
  • Revision received July 8, 2025.
  • Accepted July 10, 2025.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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Inhibition of VEGFR1 TK Signaling in Peritoneal Macrophages Suppresses Endometriosis Development
AKIKO FURUE, MASAKO HONDA, KYOKO HATTORI, ERINA SATO, ATSUSHI YAMASHITA, MAYUKO OSADA, KANAKO HOSONO, MARIKO KAMATA, YOSHIYA ITO, MASABUMI SHIBUYA, KAZUYOSHI KATO, HIDEKI AMANO
In Vivo Sep 2025, 39 (5) 2584-2598; DOI: 10.21873/invivo.14059
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