Inhibition of VEGFR1 Tyrosine Kinase Signaling Attenuates Development of Lung Emphysema by Recruiting Monocyte-derived Macrophages

Abstract

Background/Aim

Macrophages are pivotal in the early stages of emphysema development within the lungs. Although vascular endothelial growth factor (VEGF) is implicated in emphysema formation, the contribution of signaling by VEGF receptor 1 (VEGFR1), which is highly expressed in monocytes and macrophages, to its progression remains uncertain. We explored whether VEGFR1 tyrosine kinase (TK) signaling in macrophages promoted emphysema progression and whether its inhibition mitigated disease by altering the phenotype of recruited macrophages.

Materials and Methods

Male Vegfr1 TK–deficient (TK^−/−) mice or wild-type (WT) mice were subjected to an elastase-induced lung emphysema model. We assessed airspace enlargement, inflammatory mediators, and macrophage accumulation.

Results

Compared with WT mice, TK^−/− mice showed attenuated emphysema development and lower levels of pro-inflammatory mediators, such as matrix metalloproteinase-12, while exhibiting increased anti-inflammatory mediators. Elastase induced a transient elevation of monocyte-derived macrophages (MDMs), peaking on day 3; this recruitment was greater in TK^−/− mice than in WT mice. MDMs and bone marrow-derived macrophages from TK^−/− mice demonstrated reduced pro-inflammatory and enhanced anti-inflammatory profiles. The transfer of WT-MDMs to TK^−/− mice aggravated emphysema and inflammation. Conversely, WT mice treated with a VEGFR1 inhibitor exhibited attenuation of elastase-induced lung emphysema, with reduced inflammation and increased MDMs.

Conclusion

These results suggest that inhibiting VEGFR1 TK signaling minimizes elastase-induced emphysema development by shifting recruited MDMs toward an anti-inflammatory profile and restraining matrix metalloproteinase-12-linked proteolytic inflammation, supporting VEGFR1 TK as a candidate therapeutic axis for early emphysema.

Introduction

Chronic obstructive pulmonary disease (COPD) is a common respiratory condition defined by a gradual reduction in lung function and airflow limitations (1). The primary pathogenic mechanism of COPD is an imbalance between excessive tissue destruction and inadequate repair, resulting in alveolar damage (2). Prolonged exposure to harmful substances markedly elevates the risk of emphysema, which is a principal characteristic of COPD. Emphysema arises from the degradation of parenchymal structures and disruption of epithelial regeneration (2). Emphysema substantially contributes to global morbidity and mortality (3). However, current therapeutic approaches focus mainly on relieving symptoms rather than on targeting the underlying causes of the disease (4).

Vascular endothelial growth factor (VEGF)-A is an essential growth factor crucial for preserving the functional and structural integrity of lung tissue (5). VEGFA exerts its biological effects through two tyrosine kinases (TKs): VEGF receptor-1 (VEGFR1) and VEGF receptor-2 (VEGFR2). VEGFR2 is predominantly expressed in vascular endothelial cells and mediates canonical VEGF functions, including the enhancement of vascular permeability, promotion of angiogenesis, and proliferation of endothelial cells. In contrast, VEGFR1 is highly expressed in monocytes and macrophages, where it regulates cell migration and chemotaxis (6). Both receptors share a common structural organization, consisting of seven immunoglobulin (Ig)-like domains in their extracellular region, a single transmembrane region, and an intracellular TK domain (6). Although the complete deletion of VEGFR1 results in embryonic lethality, mice lacking only the TK domain retain normal vascular development and viability (7). Furthermore, genetic ablation of the VEGFR1 TK domain suppresses VEGF-induced macrophage migration (7).

In models of smoking-induced emphysema and individuals with COPD, a significant reduction in the expression of VEGFA and its receptor in the airway epithelium is associated with airway damage and alveolar destruction (8). In contrast, elevated VEGFA levels in patients with COPD were shown to be linked to the progression of emphysema (9). These divergent findings suggest that VEGF signaling serves dual functions. Epithelial VEGFA/VEGFR2 signaling may offer protection, whereas VEGFR1 expressed by macrophages might promote inflammation independently of epithelial VEGF levels. The dual functions of VEGF signaling depend on receptor type and cellular context (5).

Regarding VEGFRs, blocking VEGF activity through VEGFR2 inhibition in rats resulted in alveolar apoptosis and the development of emphysema (10). Additionally, cigarette smoke adversely affects the VEGF–VEGFR2 pathway, leading to diminished levels of VEGF and its receptors in the pulmonary systems of rats and humans (11). Although VEGFR2 inhibition induces emphysema through endothelial apoptosis, the role of VEGFR1, predominantly expressed in macrophages, in emphysema progression remains unexplored. Our previous study demonstrated that VEGFR1 TK signaling facilitated compensatory lung growth post-pneumonectomy, primarily through the action of bone marrow (BM)-derived macrophages expressing VEGFR1 (12). Additionally, this signaling pathway was shown to contribute to the development of bleomycin-induced pulmonary fibrosis by promoting the accumulation of VEGFR1-expressing macrophages in fibrotic regions (13). VEGFR1 therefore appears to drive pro-fibrotic macrophage accumulation in bleomycin-induced fibrosis. However, it is unknown whether VEGFR1 signaling similarly promotes emphysema, which is a disease characterized by proteolytic, rather than fibrotic, remodeling. It is also unknown whether the distinct inflammatory milieu of emphysema alters VEGFR1-dependent macrophage phenotypes and pro-inflammatory mediator production. These observations raise the possibility that VEGF/VEGFR1 signaling contributes to COPD pathology by influencing the activity of recruited VEGFR1-expressing cells.

We propose that VEGFR1 TK signaling in macrophages facilitates the progression of emphysema through pro-inflammatory phenotypes, which is a role distinct from its pro-fibrotic function in bleomycin-induced lung injury. We therefore investigated whether inhibiting VEGFR1 TK would mitigate the development of emphysema by influencing macrophage recruitment and the production of inflammatory mediators.

Materials and Methods

Animals. Male VEGFR1 TK–deficient mice (TK^−/− mice) were produced as previously described (7). Male wild-type (WT) C57BL/6 mice were obtained from CLEA Japan (Tokyo, Japan). All mice were housed under specific pathogen-free conditions, with temperature consistently maintained at 25±1°C and humidity regulated at 50±5%. The mice were kept on a 12-h light–dark cycle and had continuous access to food and water. The Institutional Animal Care and Use Committee of Kitasato University School of Medicine approved all experimental protocols (approval no. 2025-151). The experiments were conducted according to the guidelines established by the Science Council of Japan for animal experiments.

Experimental protocols. Anesthesia was administered via intraperitoneal injection of a combination of anesthetics, including midazolam (4.0 mg/kg; Sandoz, Basel, Switzerland), medetomidine hydrochloride (0.75 mg/kg; Nippon Zenyaku Kogyo, Fukushima, Japan), and butorphanol (5.0 mg/kg; Meiji Seika Pharma, Tokyo, Japan). Porcine pancreatic elastase (E7885; Sigma-Aldrich, St. Louis, MO, USA) was dissolved in sterile phosphate-buffered saline (PBS) and delivered intratracheally to mice at a concentration of 50 μg/50 μl per mouse. This procedure was carried out using a 24G IV catheter (B. Braun Medical Inc., Bethlehem, PA, USA), as previously reported (14). After elastase administration, the effects of medetomidine were reversed by an intraperitoneal injection of atipamezole (0.75 mg/kg; Nippon Zenyaku Kogyo).

In separate experimental conditions, WT mice were initially treated with elastase (day 0). Subsequently, these mice were subjected to intraperitoneal administration of a monoclonal antibody specific to VEGFA (10 μg per mouse; 2G11-2A05; cat. no. BE0399; BioXcell, Lebanon, NH, USA) or a control IgG isotype (rat IgG2a, κ; BioLegend, San Diego, CA, USA) every other day (14). Lung tissues were then harvested for analysis on days 7 and 21 after elastase treatment.

In another set of experiments, TK^–/– mice were treated with elastase on day 0, followed by an intraperitoneal injection of C-C chemokine receptor type 2 (CCR2) inhibitor (INCB3344; Selleck, Houston, TX, USA) (10 mg/kg) (15) on days 0 and 2. The CCR2 inhibitor was dissolved in 4% dimethyl sulfoxide and diluted in 0.2 ml PBS. Lung tissues were collected for analysis on days 3 and 21 after elastase treatment.

In a separate experiment, VEGFR1 TK inhibitor ZM306416 (Selleck) (10 mg/kg) (16) was dissolved in 0.2 ml PBS containing 4% dimethyl sulfoxide. Following the administration of elastase to WT mice on day 0, these mice received intraperitoneal injections of either ZM306416 or vehicle every other day. Although ZM306416 does not inhibit VEGFR2, it suppresses the activity of epidermal growth factor receptor (EGFR) TK (17). Lung tissues were harvested for analysis on days 3 and 21 after elastase treatment.

Lung functional analysis. On day 21 post-elastase administration, the mice were prepared for analysis of lung function as previously described (12). In brief, mice were anesthetized using a combination of midazolam–medetomidine–butorphanol. The trachea was exposed and incised transversely. An intubation tube (20 G IV catheter; B. Braun Medical) was inserted into the trachea and ligated to form a sealed system. The tube was attached to a small animal ventilator which was managed by a computer system (FlexiVent; SCIREQ, Scientific Respiratory Equipment Inc., Montreal, QC, Canada). Mice were mechanically ventilated at 150 breaths/min with a tidal volume of 8 ml/kg and a positive end-expiratory pressure of 3 cm H₂O. Following a deep-inflation maneuver, respiratory system mechanics were assessed using the single-frequency forced oscillation maneuver (SnapShot-150) (18). Respiratory system compliance was calculated using flexiWare software (SCIREQ) and data were expressed as ml/cm H₂O.

Bronchoalveolar lavage. On days 3 and 21 post-elastase administration, mice were anesthetized using a mixture of midazolam, medetomidine, and butorphanol. The trachea was surgically exposed and cannulated with a 20 G IV catheter (B. Braun Medical). To collect bronchoalveolar lavage fluid (BALF), the trachea was perfused three times with 1 ml of sterile cold PBS containing 2 mM EDTA using a 1-ml syringe (14). Following centrifugation at 400 × g for 15 min at 4°C, the BALF supernatants were stored at −80°C for subsequent cytokine analysis. After BALF collection, the right lungs were harvested for RNA extraction; the animals were euthanized via cervical dislocation.

Flow cytometric analysis. Following centrifugation of the BALF, the cells were exposed to a red blood cell lysis buffer (BioLegend) for 2 min. Subsequently, the BAL cells were suspended in fluorescence-activated cell sorting buffer (BD Biosciences, Franklin Lakes, NJ, USA) and treated with an anti-mouse cluster of differentiation (CD)16/32 antibody (TruStain FcX; cat. no. 101320; BioLegend) for 10 min to inhibit non-specific binding. Cells were then stained with anti-CD45 (30-F11; cat. no. 103134), anti-lymphocyte antigen 6 complex locus G (Ly6G) (1A8; cat. no. 127614), anti-CD11b (M1/70; cat. no. 101216), and anti-sialic acid-binding immunoglobulin-like lectin F (SiglecF) (S17007L; cat. no. 155504) (all from BioLegend). Cells positive for 7-aminoactinomycin D (BioLegend) were excluded from the analyses. Data were acquired using a FACSVerse cytometer (BD Biosciences) and analyzed with Kaluza version 2.1 (Beckman Coulter, Brea, CA, USA). Cell numbers were quantified and expressed as the number of cells per milliliter.

Cell sorting. BALF samples were first passed through a cell strainer with a 70-μm mesh and then centrifuged at 400 × g for 10 min. Subsequently, the samples were treated with a staining solution containing anti-CD45 (30-F11; cat. no. 103134), anti-Ly6G (1A8; cat. no. 127614), anti-CD11b (M1/70; cat. no. 101216), and anti-SiglecF (S17007L; cat. no. 155504), all sourced from BioLegend. Immune cells were then sorted using an SH800S Cell Sorter (SONY, Tokyo, Japan).

Cell preparation, adoptive transfer of bone marrow (BM)-derived macrophages, and cell culture. BM-derived macrophages were produced by isolating BM cells from the femurs and tibias of 8-week-old male untreated WT and TK^−/− mice. These cells were then cultured in six-well plates, with each well containing 1.0×10⁶ cells, in RPMI 1640 medium supplemented with 10% fetal calf serum and 20 ng/ml macrophage colony-stimulating factor (BioLegend), following the method described in (14). On day 7 of culture, macrophages were collected; 3×10⁵ cells suspended in 50 μl PBS were then administered intratracheally to recipient mice 24 h after elastase treatment. Lung tissues were harvested for analysis on days 3 and 21 after elastase treatment.

In a separate experimental setup, on day 7 of culture, BM-derived macrophages (1.0×10⁶ cells/well) underwent 24-h treatment with lipopolysaccharide (LPS; 10 ng/ml; Sigma-Aldrich, St. Louis, MO, USA) and interferon-gamma (IFNγ; 20 ng/ml; BioLegend) or interleukin-4 (IL4; 20 ng/ml; BioLegend). After culturing, the BM-derived macrophages were harvested and homogenized using RNAiso Plus (Takara Bio, Shiga, Japan) to assess messenger RNA (mRNA) expression levels through real-time quantitative polymerase chain reaction (qPCR) analysis.

Cytokine quantification. To assess pro-matrix metalloproteinase-9 (MMP9) concentrations in isolated BM cells, an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA) was employed. MMP12 levels in the BALF were quantified using an ELISA kit (Abcam).

Histology and immunohistochemistry. In separate experiments, the lungs were infused with a 4% paraformaldehyde–phosphate buffer solution at a pressure of 20 cm H₂O and then isolated. The lung tissue samples were immediately preserved in 4% paraformaldehyde in PBS at 4°C for 24 h. Sections of paraffin-embedded tissues, each measuring 3.5 μm in thickness, were prepared and subsequently stained with hematoxylin and eosin. Images were acquired using a Biozero BZ-700 microscope (Keyence, Osaka, Japan). Hematoxylin and eosin-stained lung sections were examined, and 10 randomly selected microscopic fields were analyzed for each mouse, as previously described (19). The mean linear intercept, a key indicator of alveolar size, was calculated as the total length of the test lines divided by the number of intersections between the test lines and alveolar septa.

Immunofluorescence analysis. After excision, lung tissue samples were perfused with 4% paraformaldehyde and fixed for 24 h. They were then immersed in a mixture of 30% sucrose and 0.1 M phosphate buffer for 3 days at 4°C. Subsequently, the tissues were embedded in Tissue-Tek optimal cutting temperature (OCT) compound (Sakura Finetek, Torrance, CA, USA) and stored at −20°C. To prevent non-specific binding, 13-µm-thick frozen sections were initially incubated with a serum-free protein block (Dako) for 1 h at room temperature. Following this, sections were incubated overnight at 4°C with primary antibodies, including anti-mouse cluster of differentiation (CD68) (rat monoclonal antibody, FA-11; cat. no. MCA1957; 1:100; Bio-Rad Laboratories, Hercules, CA, USA), anti-mouse CD68 (rabbit polyclonal antibody, cat. no. ab125212; 1:100; Abcam, Cambridge, UK), VEGFA (goat polyclonal antibody, cat. no. AF1603; 1:100; R&D Systems, Minneapolis, MN, USA), anti-mouse VEGFR1 monoclonal antibody (rat polyclonal antibody, 141515; cat. no. MAB471; 1:100; R&D Systems), anti-mouse aquaporin 5 (rabbit polyclonal antibody, cat. no. ab78486; 1:100; Abcam), anti-mouse prosurfactant protein C (rabbit polyclonal antibody, cat. no. ab90716; 1:100; Abcam), and anti-mouse MMP12 (rabbit polyclonal antibody, cat. no. PA5-13181; 1:100; Invitrogen, Waltham, MA, USA). Following washing with PBS, the sections were incubated overnight at 4°C with appropriate secondary antibodies. These included donkey anti-rabbit IgG (cat. no. A21206) conjugated with Alexa Fluor 488, donkey anti-rat IgG (cat. no. A21208), and anti-goat IgG (cat. no. A11055), along with Alexa Fluor 594-conjugated donkey anti-rat IgG (cat. no. A21209) and anti-rabbit IgG (cat. no. A21207), all sourced from Molecular Probes (Eugene, OR, USA). Nuclei were counterstained using 4’,6-diamidino-2-phenylindole. Images were then acquired with a Keyence Biozero BZ-700 fluorescence microscope.

Real-time qPCR analysis. RNA was isolated from homogenized lung tissue using RNAiso Plus. The extracted mRNA was then converted into complementary DNA with a ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan). Quantitative PCR was conducted using TB Green Premix Ex Taq II (Tli RNase H Plus; Takara Bio) in reaction volumes of 20 μl, with 40 cycles of amplification. The expression levels of mRNA were normalized against those of glyceraldehyde-3-phosphate dehydrogenase (Gapdh). The primers employed are detailed in Table I.

Table I. The primers used for reverse transcription and quantitative polymerase chain reaction.

Statistical analysis. All data are presented as the mean±standard deviation. Data were analyzed using GraphPad Prism 9 (GraphPad Software, La Jolla, CA, USA). Comparisons between two groups were conducted using an unpaired two-tailed Student’s t-test. In contrast, comparisons among multiple groups were analyzed using one-way analysis of variance followed by Tukey’s post hoc test. A p-value of less than 0.05 was considered statistically significant.

Results

Deletion of VEGFR1 TK signaling suppressed elastase-induced pulmonary emphysema progression. To examine the role of VEGFR1 TK signaling in emphysema progression, mice were treated with elastase to induce pulmonary emphysema. Elastase treatment enlarged airspaces (Figure 1A), as indicated by a significant increase in mean linear intercept on days 14 and 21 (Figure 1B). Airspace enlargement in WT mice was significantly more pronounced than that in TK^−/− mice. Lung compliance in WT mice on day 21 was significantly greater than that in TK^−/− mice (Figure 1C). These results indicate that TK^−/− mice exhibited significantly reduced alveolar enlargement and lung compliance compared with WT mice after elastase administration.

Figure 1.

Vascular endothelial growth factor receptor 1 (VEGFR1) tyrosine kinase (TK) signaling deficiency ameliorates elastase-induced pulmonary emphysema progression. (A) Representative images of hematoxylin and eosin-stained lung sections from wild-type (WT) and TK-deficient (TK^−/−) mice on days 0, 7, 14, and 21. Scale bars=100 μm. (B) Alveolar diameters, as indicated by the mean linear intercept, in WT and TK^−/− mice after elastase administration. Data are expressed as the mean±standard deviation (SD) (n=4-6 per group). (C) Lung compliance levels on days 0 and 21. Data are expressed as the mean±SD (n=4-5 per group). (D) mRNA levels of Vegfa, Vegfr1, and Vegfr2 in lung tissues after elastase administration. Data are expressed as the mean±SD (n=4-6 per group). Representative images of immunofluorescence staining for (E) VEGFA (green) and CD68 (red), aquaporin 5 (AQP5) (red), or prosurfactant protein C (SFC) (red) on day 0 in lung tissues of WT mice, and for (F) VEGFR1 (green) and CD68 (red) on day 3 in lung tissues of WT mice. (E) Scale bars=100 μm, (F) Scale bar=50 μm. Arrowheads indicate double-stained cells.

To explore the involvement of VEGFA and its receptors, we determined their expression in lung tissue. The mRNA levels of Vegfa in both genotypes were transiently and significantly reduced on day 3 and then recovered, with no statistically significant differences found between them (Figure 1D). Vegfr1 levels showed trends similar to those of Vegfa; however, the levels in WT mice on days 7 and 21 were significantly higher than those in TK^−/− mice. The mRNA levels of Vegfr2, another receptor for VEGFA, were also significantly downregulated on day 3 but had recovered by day 21. No significant difference was observed in Vegfr2 levels between the two genotypes.

Immunofluorescence was conducted to identify cells expressing VEGFA. On day 0, VEGFA expression was observed to co-localize with cells expressing aquaporin 5, prosurfactant protein C, and CD68 (Figure 1E), corroborating our previous findings (14). The analysis of these results suggests that VEGFA is synthesized by pulmonary epithelial cells and macrophages. Furthermore, the immunofluorescence examination of lung tissues on day 3 revealed that VEGFR1 co-localized with CD68⁺ macrophages (Figure 1F).

Deletion of VEGFR1 TK signaling reduces pro-inflammatory mediators. To examine whether inflammation contributed to emphysema progression, the mRNA levels of pro-inflammatory mediators in lung tissues were assessed. The mRNA levels of Mmp9 and Mmp12, which contribute to emphysema formation, were significantly increased in WT mice on days 21 and 7, respectively, compared with those in TK^−/− mice (Figure 2A). MMP12 levels in the lungs on days 3 and 7 were significantly higher in WT mice than in TK^−/− mice (Figure 2B). MMP12 was expressed in CD68⁺ macrophages in WT mice on day 7 (Figure 2C). The mRNA levels of pro-inflammatory mediators tumor necrosis factor alpha (Tnfa), interleukin 1 beta (Il1b), and Il6, were significantly higher in WT mice than in TK^−/− mice (Figure 2D). In contrast, the mRNA levels of anti-inflammatory mediators, namely mannose receptor (Mr)and found in inflammatory zone 1 (Fizz1),were significantly higher in TK^−/− mice than in WT mice.

Figure 2.

mRNA levels of inflammatory mediators in lung tissues from wild-type (WT) and tyrosine kinase-deficient (TK^−/−) mice after elastase administration. (A) Pulmonary mRNA levels of matrix metalloproteinase-9 (Mmp9) and Mmp12 in WT and TK^−/− mice. Data are expressed as the mean±standard deviation (SD) (n=4-6 per group). (B) MMP12 levels in lung tissues on days 0, 3, and 7 in WT and TK^−/− mice. Data are expressed as the mean±SD (n=3-5 per group). (C) Representative images of immunofluorescence staining for MMP12 (green) and CD68 (red) on day 7. Arrowheads indicate double-stained cells. Scale bar=50 μm. (D) Pulmonary mRNA levels of pro-inflammatory mediators [tumor necrosis factor alpha (Tnfa), interleukin 1 beta (Il1b), and Il6], as well as anti-inflammatory mediators [mannose receptor (Mr) and found in inflammatory zone 1 (Fizz1)] in WT and TK^−/− mice. Data are expressed as the mean±SD (n=3-6 per group). Significantly different at: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Blockade of VEGFA mitigates emphysema progression and inflammation. To verify the role of VEGFA/VEGFR1 in pulmonary emphysema and inflammation, WT mice were treated with either a VEGFA antibody or control IgG. VEGFA antibody treatment reduced alveolar diameter on day 21 (Figure 3A). VEGFA antibody treatment did not affect Vegfa mRNA levels on days 7 and 21 in the two groups; however, it did significantly reduce the mRNA levels of Vegfr1, Vegfr1tk, and Vegfr2 (Figure 3B). The attenuated emphysema in VEGFA antibody–treated WT mice was associated with significantly reduced mRNA levels of pro-inflammatory mediators Tnfa, Il1b, Il6, Mmp9 and Mmp12, on days 7 and 21 compared with control mice (Figure 3C).

Figure 3.

Blockade of vascular endothelial growth factor-A (VEGFA) attenuates elastase-induced emphysema and inflammation. Following the administration of elastase to wild-type (WT) mice on day 0, these mice were subsequently treated with VEGFA antibody or immunoglobulin G (IgG) every other day. (A) Left: Representative images of hematoxylin and eosin-stained lung sections on day 21 from WT mice treated with VEGFA antibody or IgG. Scale bars=100 μm. Right: Alveolar diameter on days 7 and 21 in WT mice treated with VEGFA antibody or IgG. Data are expressed as the mean±standard deviation (SD) (n=4-6 per group). (B) mRNA levels of Vegfa, vascular endothelial growth factor receptor 1 (Vegfr1), VEGFR1 tyrosine kinase (Vegfr1tk), and Vegfr2 on days 7 and 21 in the lungs of WT mice treated with VEGFA antibody or IgG. Data are expressed as the mean±SD (n=3-5 per group). (C) mRNA levels of the pro-inflammatory mediators tumor necrosis factor alpha (Tnfa), interleukin 1 beta (Il1b), Il6, matrix metalloproteinase-9 (Mmp9), and Mmp12 on days 7 and 21 in the lungs of WT mice treated with VEGFA antibody or IgG. Data are expressed as the mean±SD (n=4-5 per group). Significantly different at: *p<0.05, **p<0.01, ****p<0.0001.

Involvement of macrophages during emphysema progression. Macrophages contribute to emphysema progression via inflammation (20). Therefore, we assessed changes in BALF macrophages during emphysema progression using flow cytometric analysis. The gating strategy revealed two main macrophage subtypes: alveolar macrophages (AMs) and monocyte-derived macrophages (MDMs) (Figure 4A), consistent with our previous report (14). Under basal conditions, most macrophages were AMs, with few MDMs observed (Figure 4B). On day 3, the number of AMs in WT and TK^−/− mice increased; however, no statistically significant difference was observed between the two groups. In contrast, there was a sharp increase in MDMs, which were higher in TK^−/− mice than in WT mice. The levels in both genotypes were markedly reduced on day 7 and remained low until day 21. We also determined the mRNA levels of chemokines. The mRNA levels of C-C motif chemokine ligand 2 (Ccl2) and its receptor Ccr2 in the lung tissues of TK^−/− mice on days 3 and 7 were significantly higher than those of WT mice (Figure 4C).

Figure 4.

Inflammatory cells during the progression of elastase-induced emphysema. (A) Flow cytometry gating strategy utilized to identify macrophages in lungs exposed to elastase. Following the gating of lymphocyte antigen 6 complex locus G (Ly6G)^high and CD11b^high cells (neutrophils), cells were segregated into two subsets based on the expression of sialic acid-binding immunoglobulin-like lectin F (SiglecF) and CD11b. SiglecF^high/CD11b^low cells were classified as alveolar macrophages (AMs), whereas SiglecF^low/CD11b^high cells were designated as monocyte-derived macrophages (MDMs). (B) Changes in AMs and MDMs in lungs exposed to elastase in wild-type (WT) and tyrosine kinase-deficient (TK^−/−) mice. Data are expressed as the mean±standard deviation (SD) (n=4-6 per group). (C) Pulmonary mRNA levels of C-C motif chemokine ligand 2 (Ccl2) and C-C chemokine receptor type 2 (Ccr2) in WT and TK^−/− mice. Data are expressed as the mean±SD (n=3-6 per group). (D) Left: The number of MDMs in the bone marrow of WT and TK^−/− mice on days 0 and 3. Data are expressed as the mean±SD (n=3-5 per group). Right: Pro-matrix metalloproteinase-9 (MMP9) levels in the bone marrow of WT and TK^−/− mice on days 0 and 3. Data are expressed as the mean±SD (n=4-6 per group). Significantly different at: *p<0.05, **p<0.01, ****p<0.0001, ****p<0.0001.

The above results suggest that the accumulation of MDMs in lung tissues contributes to emphysema progression. Therefore, we examined whether there were differences in the number of BM-derived MDMs between the two genotypes. The populations of MDMs in the BM of both genotypes on day 3 were significantly higher than those on day 0; those of TK^−/− mice on day 3 were higher than those of WT mice (Figure 4D). These results suggest that the increased number of MDMs in emphysematous lung tissues is derived from the BM. We previously reported that pro-MMP9 levels in the BM were associated with increased macrophage numbers in lung tissue (12). Based on these findings, we determined pro-MMP9 levels in the BM. The pro-MMP9 levels in the BM of TK^−/− mice on day 3 were significantly higher than those in WT mice (Figure 4D).

Furthermore, we determined the mRNA levels of inflammatory mediators in sorted day 3 MDM. The mRNA levels of pro-inflammatory mediators Il1b, Il6, and Mmp12, in MDMs from WT mice were significantly higher than those in TK^−/− mice (Figure 5A). In contrast, the mRNA levels of the anti-inflammatory mediator Fizz1 were significantly lower in WT mice than in TK^−/− mice. These results suggest that reduced pro-inflammatory and increased anti-inflammatory mediators in VEGFR1 TK–deficient MDMs contribute to suppressing emphysema progression.

Figure 5.

Pro- and anti-inflammatory mediators in monocyte-derived and bone marrow-derived macrophages. (A) mRNA levels on day 3 of proinflammatory mediators [tumor necrosis factor alpha (Tnfa), interleukin 1 beta (Il1b), Il6, matrix metalloproteinase-9 (Mmp9), and Mmp12], as well as anti-inflammatory mediators [mannose receptor (Mr) and found in inflammatory zone 1 (Fizz1)] in monocyte-derived macrophages from wild-type (WT) and tyrosine kinase-deficient (TK^−/−) mice. Data are expressed as the mean±standard deviation (SD) (n=3 per group). (B) mRNA levels of pro-inflammatory mediators (Tnfa, Il1b, Il6, Mmp9, Mmp12) in bone marrow-derived macrophages stimulated with vehicle or lipopolysaccharide (LPS)/interferon gamma (IFN-γ) and of anti-inflammatory mediators (Mr and Fizz1) in bone marrow-derived macrophages stimulated with vehicle or IL4. Data are expressed as the mean±SD (n=4-6 per group). Significantly different at: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

MDMs are derived from the BM (21). To examine whether VEGFR1 TK signaling was involved in the pro-inflammatory properties of MDMs, BM-derived macrophages from WT or TK^−/− mice were cultured and then stimulated with LPS/IFN-γ or IL4. In BM-derived macrophages stimulated with LPS/IFN-γ, the mRNA levels of pro-inflammatory mediators Tnfa, Il1b, Il6, Mmp9, and Mmp12, were significantly higher in WT mice than in TK^−/− mice (Figure 5B). Additionally, in BM-derived macrophages stimulated with IL4, the mRNA levels of anti-inflammatory mediators Mr and Fizz1, were significantly higher in TK^−/− mice than in WT mice. These results suggest that VEGFR1 TK signaling deficiency attenuates the pro-inflammatory properties of BM-derived macrophages and enhances their anti-inflammatory profile.

VEGFR1 TK signaling in BM-derived macrophages exacerbates pulmonary emphysema and inflammation. To verify the role of VEGFR1 TK signaling in MDMs in pulmonary emphysema, BM-derived macrophages from WT mice were adoptively transferred to TK^−/− mice. The adoptive transfer of WT-BM-derived macrophages to TK^−/− mice aggravated pulmonary emphysema, with increased alveolar diameters (Figure 6A) and mRNA levels of Vegfa, Vegfr1, and pro-inflammatory mediators Tnfa, Il1b, Il6, Mmp9, and Mmp12, compared with TK^−/− mice that received TK^−/−-BM-derived macrophages (Figure 6B).

Figure 6.

Adoptive transfer of bone marrow (BM)-derived macrophages during the progression of elastase-induced emphysema. On day 0, tyrosine kinase-deficient (TK^−/−) mice received an initial treatment with elastase. Subsequently, one day post-treatment, these TK^−/− mice were administered macrophages derived from either wild-type (WT) or TK^−/− BM-derived macrophages. (A) Left: Representative images of hematoxylin and eosin-stained lung sections from tyrosine kinase-deficient (TK^−/−) mice transferred with WT- or TK^−/−-BM-derived macrophages. Scale bars=100 μm. Right: Alveolar diameter on day 21. Data are expressed as the mean±standard deviation (n=6 per group). (B) mRNA levels of vascular endothelial growth factor A (Vegfa), vascular endothelial growth factor receptor 1 (Vegfr1), tumor necrosis factor alpha (Tnfa), interleukin 1 beta (Il1b), Il6, matrix metalloproteinase-9 (Mmp9), and Mmp12 on day 3 in the lungs of TK^−/− mice transferred with WT- or TK^−/−-BM-derived macrophages. Data are expressed as the mean±standard deviation (n=4-5 per group). Significantly different at: *p<0.05, **p<0.01, ****p<0.0001.

Inhibition of VEGFR1 TK-deficient MDMs increased emphysema progression and inflammation. To examine whether the accumulation of VEGFR1 TK-deficient MDMs in the lungs contributed to emphysema formation, TK^−/− mice were administered a CCR2 inhibitor. The CCR2 inhibitor significantly increased alveolar diameters on day 21 (Figure 7A) and mRNA levels of pro-inflammatory mediators Tnfa, Il1b, Il6, Mmp9 and Mmp12, on day 3 compared with vehicle-treated mice (Figure 7B). However, the CCR2 inhibitor significantly reduced the number of MDMs and AMs on day 3 (Figure 7C).

Figure 7.

Inhibition of C-C chemokine receptor type 2 (CCR2) aggravates elastase-induced emphysema and inflammation in tyrosine kinase-deficient (TK^−/−) mice. TK^−/− mice were initially treated with elastase on day 0, followed by an intraperitoneal injection of CCR2 inhibitor or vehicle on days 0 and 2. (A) Left: Representative images of hematoxylin and eosin-stained lung sections on day 21 from TK^−/− mice treated with CCR2 inhibitor or vehicle. Scale bars=100 μm. Right: Mean linear intercept on day 21 in TK^−/− mice treated with CCR2 inhibitor or vehicle. Data are expressed as the mean±standard deviation (SD) (n=4-5 per group). (B) mRNA levels of the pro-inflammatory mediators tumor necrosis factor alpha (Tnfa), interleukin 1 beta (Il1b), Il6, matrix metalloproteinase-9 (Mmp9), and Mmp12, on day 3 in the lungs of TK^−/− mice treated with CCR2 inhibitor or vehicle. Data are expressed as the mean±SD (n=4-5 per group). (C) The number of monocyte-derived macrophages (MDMs) and alveolar macrophages (AMs) on day 3 in the lungs of TK^−/− mice treated with CCR2 inhibitor or vehicle. Data are expressed as the mean±SD (n=4-5 per group). Significantly different at: *p<0.05, **p<0.01, ***p<0.001.

A VEGFR1 inhibitor suppressed the progression of pulmonary emphysema and inflammation. Finally, we examined whether VEGFR1 TK inhibition attenuated the progression of pulmonary emphysema. To this end, WT mice were treated with the VEGFR1 TK inhibitor ZM306416. The alveolar diameter of inhibitor-treated mice on day 21 was significantly lower than that of vehicle-treated mice (Figure 8A). Pulmonary mRNA levels of Vegfa and Vegfr1tk, but not Vegfr1 and Vegfr2, were significantly lower in inhibitor-treated mice than in vehicle-treated mice (Figure 8B). Additionally, inhibitor treatment reduced the mRNA levels of the pro-inflammatory mediators Tnfa, Il1b, Il6, Mmp9 and Mmp12 and significantly increased those of Mr and Fizz1 compared with vehicle treatment (Figure 8C). The VEGFR1 TK inhibitor also significantly increased the mRNA levels of Ccl2, and Ccr2 (Figure 8C). Moreover, the inhibitor increased the number of MDMs, but not AMs, compared with vehicle treatment (Figure 8D).

Figure 8.

Effects of vascular endothelial growth factor receptor 1 (VEGFR1) inhibitor on elastase-induced lung emphysema. Following the administration of elastase to WT mice on day 0, these mice received intraperitoneal injections of either VEGFR1 inhibitor ZM306416 or vehicle every other day. (A) Left: Representative images of hematoxylin and eosin-stained lung sections from wild-type (WT) mice treated with inhibitor or vehicle. Scale bars=100 μm. Right: Alveolar diameter on days 7 and 21 after elastase administration. Data are expressed as the mean±standard deviation (SD) (n=6 per group). (B) mRNA levels on days 3 and 21 of Vegfa, Vegfr1, Vegfr1 tyrosine kinase (Vegfr1tk), and Vegfr2 in the lungs of WT mice treated with inhibitor or vehicle. Data are expressed as the mean±SD (n=5-6 per group). (C) mRNA levels of the pro-inflammatory mediators tumor necrosis factor alpha (Tnfa), interleukin 1 beta (Il1b), Il6, matrix metalloproteinase-9 (Mmp9), and Mmp12; anti-inflammatory mediators mannose receptor (Mr) and found in inflammatory zone 1 (Fizz1); and C-C motif chemokine ligand 2 (Ccl2) and C-C chemokine receptor type 2 (Ccr2) on days 3 and 21 in the lungs of WT mice treated with inhibitor or vehicle. Data are expressed as the mean±SD (n=5-6 per group). (D) Left: Representative dot plots of monocyte-derived macrophages (MDMs) and alveolar macrophages (AMs) in the lungs of WT mice on day 3 treated with inhibitor or vehicle. Right: Quantified results expressed as the mean±SD (n=5 per group). Significantly different at: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Discussion

The present study showed that the deletion of VEGFR1 TK signaling suppressed the progression of elastase-induced pulmonary emphysema. This was associated with reduced pro-inflammatory mediators, including MMP12, and increased anti-inflammatory mediators. The absence of VEGFR1 TK signaling also led to the accumulation of VEGFR1 TK–deficient MDMs with reduced pro-inflammatory and elevated anti-inflammatory profiles in inflammatory airspaces. Our in vitro studies demonstrated that VEGFR1 TK signaling deficiency in MDMs reduced levels of inflammatory cytokine and MMPs while increasing anti-inflammatory profiles. Furthermore, the transplantation of BM-derived WT macrophages aggravated lung emphysema and inflammation in VEGFR1 TK-deficient mice. The inhibition of VEGFR1 TK mitigated the progression of lung emphysema, which was associated with decreased pro-inflammatory and enhanced anti-inflammatory responses in the lung. These results suggest that targeting VEGFR1 TK signaling in MDMs effectively attenuates elastase-induced pulmonary emphysema by modulating the balance between pro-inflammatory and anti-inflammatory mediators.

During the progression of elastase-induced pulmonary emphysema, lung macrophages, including AMs and MDMs, underwent considerable alterations. AMs, the resident lung macrophages in the alveolar space, were transiently elevated on day 3 and returned to their pre-injury values. In contrast, MDMs substantially increased on day 3 in response to elastase administration, diminishing on day 7. These results regarding the transient appearance of MDMs are consistent with those of other studies (22, 23); the increase in MDMs is associated with the development of emphysema (24).

Additionally, the deletion of VEGFR1 signaling further increased the number of MDMs. Our data showed that a transient increase in MDM levels in mice lacking VEGFR1 TK signaling was associated with suppression of progression of pulmonary emphysema and inflammation. Conversely, these results suggest that MDMs in VEGFR1 TK signaling promote pulmonary emphysema. To characterize the MDM profiles, we analyzed the gene expression profiles of pro-inflammatory macrophage phenotypes on day 3. The results indicated that MDMs from WT mice displayed an increased expression of genes related to pro-inflammatory profiles. Consistent with this, cultured BM-derived macrophages expressing VEGFR1 TK signaling exhibited pro-inflammatory properties. To understand the involvement of MDMs in VEGFR1 TK signaling in elastase-induced pulmonary emphysema, WT-BM-derived macrophages were transferred to VEGFR1 TK-deficient mice. This transfer increased emphysema, pro-inflammatory cytokines, and MMPs. Collectively, these findings suggest that the inhibition of VEGFR1 TK signaling attenuates pulmonary emphysema and inflammation by recruiting VEGFR1 TK-deficient MDMs to the lung tissue. Therefore, MDMs expressing VEGFR1 TK signaling are likely to participate in emphysema development and the enhancement of lung inflammation. Consistent with this, we previously reported that BM-derived macrophages expressing VEGFR1 TK signaling promoted bleomycin-induced fibrosis and inflammation (13). Similarly, adoptive transfer studies and monocytes demonstrated that the development of elastase-induced emphysema was dependent on monocyte recruitment (23).

MMPs have been implicated in emphysema formation through the enzymatic destruction of alveolar walls in smoke- or elastase-induced animal models of COPD and patients with COPD (25). Among these MMPs, MMP12 is essential for the development of emphysema induced by smoke and elastase; MMP12 levels in sputum are correlated with the extent of emphysema in patients with COPD (26). Mice with the deletion of MMP12 or administration of an MMP12 inhibitor did not develop emphysema (23). Consistent with this, our data demonstrated that increased MMP12 production in the alveolar space was associated with emphysema progression, whereas reduced MMP12 expression was associated with suppression of emphysema progression. Our in vitro study showed that Mmp12 upregulation in MDMs was dependent on VEGFR1 TK signaling. Thus, the present study suggests that elastase-induced emphysema can be partially attributed to MMP12 expression in VEGFR1-expressing MDMs.

In patients with emphysema, AMs produce an excess of MMPs, particularly MMP12, which contributes to structural changes in the lung (27). In mice, MMP12 released from AMs is involved in the development of elastase-induced emphysema (28). Our data showed that AMs were also increased after elastase administration; however, there were no significant differences in the number of AMs between the genotypes. These results suggest that VEGFR1 TK signaling in AMs does not play a role in the development of elastase-induced lung emphysema.

Persistent production of the chemokine CCL2 recruits CCR2-expressing macrophages, leading to sustained inflammation in the lungs and emphysema progression (22). Impaired recruitment of monocytes to emphysematous lungs by inhibition of the CCL2/CCR2 axis slows emphysema development (29, 30). Our data demonstrated that significant but transient MDM recruitment in TK^−/− mice during the early phase of elastase-induced lung emphysema formation was related to increased Ccl2 and Ccr2 expression. We also demonstrated that suppressing the recruitment of VEGFR1 TK-deficient MDMs using a CCR2 inhibitor aggravated pulmonary emphysema formation and inflammation. In contrast to previous reports, our findings suggest that the accumulation of MDMs lacking VEGFR1 TK attenuates emphysema progression and inflammation. However, our data revealed that the CCR2 inhibitor reduced the number of MDMs and AMs. Because both cell types were affected, we were unable to definitively attribute worsened emphysema solely to reduced MDM recruitment. The reduction in AMs, which have homeostatic and anti-inflammatory roles (31), may also contribute to disease progression. Therefore, a method specifically aimed at preventing the recruitment of MDMs to the lungs during the early phase of emphysema progression is necessary. We previously indicated that VEGFR1-expressing macrophages recruited in lung tissue were derived from the BM through the secretion of pro-MMP9 in the BM (11, 12). The present study also demonstrated that the number of MDMs in the BM of TK^−/− mice increased 3 days after elastase administration, which was associated with increased BM levels of pro-MMP9.

Study limitations. During the progression of elastase-induced emphysema, VEGFR1 expression was transiently reduced in WT mice, with levels higher than those in TK^−/− mice and increased on day 21. Considering that TK^−/− mice exhibited suppressed emphysema progression, VEGFR1 TK signaling contributed to emphysema progression. Consistent with this view, treatment with the VEGFR1 inhibitor ZM306416 exhibited inhibitory effects on elastase-induced lung emphysema progression, with reductions in inflammatory mediators, including MMP12. No specific inhibitor for VEGFR1 TK is available; thus, ZM306416 has been employed in experimental studies (32, 33). However, a critical limitation of this approach is that ZM306416 inhibits VEGFR1 and EGFR. As EGFR signaling in epithelial cells promotes repair and regeneration, we cannot definitively attribute the protective effects of ZM306416 to VEGFR1 inhibition alone. Future studies require either conditional Vegfr1 knockout in macrophages or superiorly selective VEGFR1 inhibitors to confirm target specificity.

Furthermore, VEGFA antibody suppressed progression of lung emphysema and inflammation despite reducing VEGFR1 and VEGFR2. VEGFR2 is involved in lung emphysema by inducing pulmonary endothelial and epithelial cell apoptosis (9). Therefore, it should be noted that the effects of VEGFA antibody were not solely due to VEGFR1 signaling.

We employed elastase-induced emphysema to isolate the role of VEGFR1 TK signaling in acute proteolytic injury and macrophage-driven MMP12 production, independent of chronic oxidative stress and adaptive immunity. However, it remains unproven whether VEGFR1-driven macrophage responses observed in acute elastase injury are mechanistically relevant to chronic cigarette smoke-induced COPD, which involves oxidative stress, adaptive immunity, and progressive remodeling (34). Additionally, some limitations were present in this study. Firstly, whilst the findings indicate that VEGFR1 TK signaling contributes to the development of elastase-induced lung emphysema by attracting pro-inflammatory MDMs through this signaling pathway, the specific molecular processes leading to the production of inflammatory mediators via VEGFR1 TK signaling remain undefined. Secondly, a global knockout model was used in this study. Conditional mouse knockout models are essential for understanding the role of VEGFR1 TK signaling in MDMs during the development of lung emphysema. Thirdly, we did not analyze VEGFR1 expression or VEGFR1-driven macrophage phenotypes in lung tissue or BAL samples from patients with COPD, limiting the clinical relevance of our conclusions.

Conclusion

The present study demonstrated that inhibiting VEGFR1 TK signaling suppressed the initiation of elastase-induced lung emphysema. This suppression appears to occur through the reduction of MMP12 production and recruitment of VEGFR1 TK-deficient MDMs, which exhibit diminished pro-inflammatory and enhanced anti-inflammatory profiles within the alveolar space. Consequently, the targeted inactivation of VEGFR1 TK signaling represents a promising therapeutic strategy for early-stage emphysema by modulating macrophage-driven inflammation. However, validation in cigarette smoke-induced models and human COPD samples is essential to establish clinical relevance and determine whether VEGFR1 inhibition can prevent or reverse established emphysema.

Conflicts of Interest

The Authors declare no conflicts of interest in relation to this study.

Authors’ Contributions

Conceptualization: SA, YI and HA. Data curation: SA, AY, TY and SH. Funding acquisition: SA and MO. Investigation: SA, KH and MK. Methodology: AY, MO and KH. Project administration: YA, TM and HA. Resources: MS. Supervision: YA, TM and HA. Validation: TM and HA. Writing – original draft: SA and YI. Writing – review and editing: TM and HA.

Acknowledgements

The Authors would like to thank Ms. Michiko Ogino and Ms. Kuniko Yoshimura for their support with our experiments.

Funding

This research was supported by a research grant (25K19885) from the Japanese Ministry of Education, Culture, Sports, Science, and Technology, and a grant from the Integrative Research Program of the Graduate School of Medical Science at Kitasato University.

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.