Macrophage derived VEGF regulates macrophage senescence to inhibit radiation-induced dermatitis

Abstract

Background: Macrophages are essential for maintaining tissue homeostasis and accelerating the repair processes; however, their functionality can be severely compromised in pathological conditions such as radiation-induced dermatitis. In this study we analyzed the role of macrophage derived Vascular Endothelial Growth Factor (VEGF) on regulation of macrophage senescence and its role on radiation-induced skin damage.

Methods: We used bone marrow-derived macrophages (BMMɸ) isolated from Csf1r-iCre; VEGF^fl/fl (VEGF-null) and wild-type (WT) mice. Macrophages were exposed to oxidative and genotoxic stress using H₂O₂, doxorubicin, and radiation exposure to evaluate senescence. Senescence was assessed via SA-β-Gal staining and expression of senescence-related genes. Additionally, VEGF receptor inhibition in WT macrophages was performed to determine the role of VEGF/VEGFR signaling in senescence regulation. Phagocytosis and migration assays were conducted to evaluate functional differences. For in vivo analysis, WT and Csf1r-iCre; VEGF^fl/fl mice were exposed to radiation, and skin toxicity, histological changes, and senescence markers in skin macrophages were assessed.

Results: VEGF-null macrophages showed increased sensitivity to senescence, with elevated SA-β-Gal staining and upregulated senescence-associated gene expression. WT macrophages treated with a VEGF receptor inhibitor displayed increased senescence-associated markers expression, highlighting the importance of VEGF/VEGF-R signaling in preventing macrophage senescence-like phenotypes. Additionally, VEGF-null macrophages have reduced phagocytic and migratory abilities. Our in vivo study using Csf1r-iCre; VEGF^fl/fl mice showed more severe radiation-induced dermatitis, including increased skin toxicity, hyperkeratosis, and elevated senescence-associated markers in skin macrophages compared to WT controls.

Conclusions: Absence of macrophage-derived VEGF leads to heightened macrophage dysfunction and exacerbates radiation-induced dermatitis. Targeting VEGF signaling may serve as a potential therapeutic strategy to mitigate radiation-related skin toxicity and improve patient outcomes during radiation therapy.

Keywords: Cell cycle arrest; Macrophage senescence; Phagocytosis; Radiation-induced dermatitis; Vascular endothelial growth factor.

Fig. 1

Absence of VEGF promotes increased expression of senescence-associated markers and decreased proliferation: (A-B) Representative images and quantification of SA-β-Gal staining of BMMɸ from WT and Csf1r-iCre; VEGF^fl/fl mice under basal conditions and after treatment with H₂O₂ (200 µM). Increased SA-β-Gal positive cells (blue staining) indicate higher senescence in VEGF-null macrophages compared to WT. (C) histogram of flow cytometry analysis of SA-β-Gal activity with 200 µM H₂O₂ showed higher mean fluorescence intensity in Csf1r-iCre; VEGF^fl/fl BMMɸ compared to WT BMMɸ (D) BrdU cell proliferation assay of BMMɸ from WT and Csf1r-iCre; VEGF^fl/fl mice treated with H₂O₂. Data show a significant reduction in proliferation of VEGF-null macrophages compared to WT. (E-G) qPCR analysis of p16, p21, p53 in BMMɸ from WT and Csf1r-iCre; VEGF^fl/fl mice treated with H₂O₂ (200 µM) and doxorubicin (0.1 µM). VEGF-null macrophages show increased expression of p16 and p21 under basal conditions, which is further upregulated upon treatment, indicating enhanced senescence. (H) Flow cytometry analysis of cell cycle distribution in BMMɸ from WT and Csf1r-iCre; VEGF^fl/fl mice using PI staining. VEGF-null macrophages exhibit a shorter S phase under basal conditions, which is further decreased after H₂O₂ or doxorubicin treatment, indicating impaired cell cycle progression compared to WT macrophages. Data presented as the mean ± SD. (Significant level, *: p < 0.05, **: p < 0.005, ***: p < 0.0005, ****: p < 0.00005, NS: not significant)

Fig. 2

WT BMMɸ conditioned media significantly ameliorates VEGF-null BMMɸ from senescence via VEGF-VEGFR interaction: (A) Schematic representation of WT and VEGF null BMMɸ culture followed by VEGF null BMMɸ treatment with WT BMMɸ conditioned media after H₂O₂ treatment (B) Representative images of SA-β-Gal staining in VEGF-null BMMɸ treated with WT BMMɸ conditioned media (CM). The WT CM-treated VEGF-null BMMɸ show reduced SA-β-Gal positivity compared to untreated VEGF-null BMMɸ, indicating a restoration to the WT phenotype. (C) Quantification of SA-β-Gal positive cells from the images shown in (B). bar graph, demonstrating significantly lower senescence in VEGF-null BMMɸ treated with WT CM. (D) Flow cytometry analysis of senescence in VEGF-null BMMɸ treated with WT CM. The histogram distribution pattern similar to WT BMMɸ, indicating conditioned media significantly ameliorates WT phenotype (MFI: Untreated BMMɸ: VEGF null 8523 vs. VEGF null + WT CM 5967) and (MFI: H₂O₂ treated BMMɸ: VEGF null 8911 vs. VEGF null + WT CM 5765). (E-F) qPCR analysis of p16, p21 and p53 in VEGF-null BMMɸ treated with WT CM. Gene expression patterns in these cells are similar to those of WT BMMɸ, confirming the restorative effect of WT CM. (G) schematic representation of BMMɸ treated with BAW2881, a VEGF receptor inhibitor or addition of recombinant VEGF (H-I) qPCR analysis of p16, p21 and p53 in WT BMMɸ treated with BAW2881, a VEGF receptor inhibitor or treated with BAW2881 and external addition of recombinant VEGF shows significantly higher expression of senescence markers compared to untreated WT BMMɸ, confirming the involvement of VEGF-VEGFR interaction in senescence inhibition. Data presented as the mean ± SD. (Significant level, *: p < 0.05, **: p < 0.005, ***: p < 0.0005, NS: not significant)

Fig. 3

Absence of VEGF increases sensitivity to radiation-induced expression of senescence-associated markers in macrophages: (A) Representative images of SA-β-Gal staining in VEGF-null and WT BMMɸ after exposure to 4 Gy and 6 Gy of radiation. VEGF-null BMMɸ show higher SA-β-Gal positivity compared to WT BMMɸ, indicating increased senescence. (B) Quantification of SA-β-Gal positive cells from the images shown in (A), demonstrating significantly higher senescence in VEGF-null BMMɸ at both 4 Gy and 6 Gy radiation doses. (C, D) histogram of flow-cytometry analysis of SA-β-Gal activity using 4 Gy and 6 Gy of radiation showing increased sensitivity of VEGF-null BMMɸ to radiation-induced senescence (4 Gy MFI: 15916 vs. 13070 and 6 Gy MFI: 8697 vs. 4066 respectively) compared to WT BMMɸ. (E-G) qPCR analysis of p16, p21 and p53 in untreated VEGF-null and WT BMMɸ and after exposure to 4 Gy and 6 Gy of radiation. VEGF-null BMMɸ show significantly higher expression of p16, p21 and p53 compared to WT BMMɸ, confirming increased senescence. (H) Phagocytosis assay using pHrodo Green E. coli BioParticles in VEGF-null and WT BMMɸ after treatment with 6 Gy of radiation. Senescent VEGF-null BMMɸ show significantly reduced phagocytosis (MFI: 16079 vs. 23206) compared to WT BMMɸ. (I) Migration assay using wound scratch method: (i and iv) Untreated VEGF-null BMMɸ exhibit slower migration rate, with 60% gap remaining compared to 30% in WT BMMɸ after 24 h. (ii and v) Senescent WT BMMɸ induced by 4 Gy radiation showed reduced migration compared to untreated WT BMMɸ but faster than VEGF-null BMMɸ. (iii and vi) Senescent WT BMMɸ induced by 6 Gy radiation showed reduced migration compared to untreated and 4 Gy treated WT BMMɸ but faster than VEGF-null BMMɸ. Data presented as the mean ± SD. (Significant level, **: p < 0.005, ****: p < 0.0005)

Fig. 4

Macrophage-specific deletion of VEGF increases sensitivity to radiation-induced dermatitis in mice. (A) Schematic representation of the experimental design for radiation induced dermatitis. Mice were irradiated with 8 Gy dose on alternate days, total 6 doses (1 dose/day). (B) photographs of mice showing the severity of radiation-induced dermatitis in Csf1r-iCre; VEGF^fl/fl mice compared to WT mice. Csf1r-iCre; VEGF^fl/fl mice developed more severe erythema and desquamation. (C) shows time-dependent increase in skin toxicity in Csf1r-iCre; VEGF^fl/fl compared to WT mice. Skin toxicity was assessed at multiple time points until the end of the study and provided a score as (No change: 0, redness on skin: 1, dry desquamation: 2, moist desquamation: 3, moist ulceration: 4) (D) H&E-stained dermatitis skin sections from Csf1r-iCre; VEGF^fl/fl and WT mice. Csf1r-iCre; VEGF^fl/fl mice exhibit increased inflammation, parakeratosis (PK), acanthosis (A), marked thickening of the sub-epidermal layer with spongiosis (S) and total epidermal thickening compared to WT mice. (E-G) Immunohistochemical staining for Ki-67 and cleaved Caspase 3 in skin sections from both the mice shows increased expression of Ki-67 antigen, and cleaved caspase 3 in Csf1r-iCre; VEGF^fl/fl mice compared to WT mice. The increased number of cleaved caspases 3 indicate increased numbers of apoptotic keratinocytes with high skin tissue damage compared to WT mice. Data presented as the mean ± SD. (Significant level, *: p < 0.005, ***: p < 0.0005)

Fig. 5

Increased senescence-associated markers expression in irradiated dermatitis skin of Csf1r.iCre; VEGF^fl/fl mice. (A, B) qPCR analysis of senescence-associated markers p16, p21, and p53 in macrophages sorted from control (non-irradiated) and irradiated dermatitis skin of WT and Csf1r.iCre; VEGF^fl/fl mice. Macrophages from Csf1r.iCre; VEGF^fl/fl mice show significantly increased expression of p16 and p53 compared to WT mice. No significant difference in p21 expression was observed between the two groups. (C, D) qPCR analysis of pro-inflammatory cytokine gene expression markers IL6, IL1α, and MMP9 in macrophages sorted from control (non-dermatitis) and irradiated dermatitis skin of WT and Csf1r.iCre; VEGF^fl/fl mice. Macrophages from Csf1r.iCre; VEGF^fl/fl mice show significantly increased expression of pro-inflammatory cytokine compared to WT mice skin sorted macrophages. (E-G) Immunofluorescent staining of dermatitis skin sections for senescence-associated markers p16 and p21 with macrophage marker (CD68). Csf1r.iCre; VEGF^fl/fl mice show higher expression of CD68 + p16 + and CD68 + p21 + in macrophages of dermatitis skin compared to WT mice. Data presented as the mean ± SD. (Significant level, **: p < 0.005; ***: p < 0.005, NS: not significant)