Efficacy of epidermal growth factor in suppressing inflammation and proliferation in pterygial fibroblasts through interactions with microenvironmental M1 macrophages

Abstract

The protein epidermal growth factor (EGF), which plays a crucial role in promoting cell proliferation and survival, has recently demonstrated potential in reducing inflammation. In this study, we examined the impact of EGF on the anti-inflammatory and anti-proliferative properties of pterygium, a prevalent hypervascular proliferative disease affecting the ocular surface. In surgically excised tissues, markers for fibrotic and inflammatory signals, including VIM, ACTA2, FAP, MMP2, VCAM1, ICAM1, CD86, IL6, and IL1B were upregulated in the pterygium body stroma compared to the normal conjunctival stroma. EGF exerted anti-inflammatory and anti-vasculogenic effects on pterygial fibroblasts when co-cultured with M1 macrophages. Moreover, exosomes derived from EGF-preconditioned M1 macrophages suppressed the heightened inflammatory and vasculogenic signals in pterygial fibroblasts induced by exosomes from M1 macrophages. Paradoxically, the proliferation of pterygial fibroblasts was inhibited by EGF in the in vitro microenvironment with M1 macrophages, despite EGF being known as a growth factor. EGF-preconditioning of M1 macrophages rescued the increased proliferation of pterygial fibroblasts induced by exosomes from M1 macrophages. In conclusion, our findings demonstrate that EGF effectively mitigates inflammation and proliferation in pterygial fibroblasts within a microenvironment containing M1 macrophages.

Keywords: EGF; Exosome; Inflammation; Proliferation; Pterygial fibroblast; Pterygium.

Fig. 1

Activation of fibrosis-related signals in pterygium body stroma compared to normal conjunctival stroma. (A-D) Real-time RT-PCR analysis of VIM, ACTA2, FAP, and MMP2 in conjunctival stromal (CJ) and pterygium body stromal (PT) tissues. Wilcoxon signed-rank test (A and C) or paired t-test (B and D). N = 8 to 11. *p0.05 and **p0.01.

Fig. 2

Activation of gene expressions for adhesion molecules and M1 macrophage in pterygium body stroma compared to normal conjunctival stroma. (A-F) Real-time RT-PCR analysis of adhesion molecules including VCAM1 and ICAM1, M1 macrophage marker CD86 and its secreting cytokine IL6 and IL1B, and M2 macrophage marker MRC1 in conjunctival stromal (CJ) and pterygium body stromal (PT) tissues. Wilcoxon signed-rank test. N = 14 to 16. *p0.05, **p0.01, ***p0.001 and ****p0.0001.

Fig. 3

EGF-induced attenuation of inflammatory, fibrotic, and vasculogenic signals in pterygial fibroblasts during co-culture with M1 macrophages. (A) Scheme of co-culture of M1 macrophages and pterygium fibroblasts using transwell system with EGF treatment (10 ng/mL for 24 h). (B-H) Real-time RT-PCR analysis for IL6, IL1B, MMP2, VCAM1, ICAM1, VEGFA and VEGFC expressions in pterygial fibroblast with and without EGF (10 ng/mL) during co-culture with M1 macrophages (24 h). Analysis of variance followed by Tuckey’s post-hoc test. N = 4. *p0.05, **p0.01, ***p0.001, ****p0.0001, and ns: not significant. EGF, epidermal growth factor; PF, pterygial fibroblast.

Fig. 4

The relative attenuation of inflammatory, fibrotic, and vasculogenic signals in pterygial fibroblasts by the delivery of EGF-preconditioned M1 macrophage-derived exosomes. (A) The low- and high-magnified images of the delivered PKH26 dye-labeled M1 macrophage-derived exosomes delivered and penetrated into the cultured pterygial fibroblasts. Scale bars: 200 μm (white) and 20 μm (black). (B-H) Real-time RT-PCR analysis for IL6, IL1B, MMP2, VCAM1, ICAM1, VEGFA, and VEGFC expressions in pterygial fibroblasts treated with M1 macrophage-derived exosome (M1 Exo, 20 µg/mL for 24 h) or EGF-preconditioned M1 macrophage-derived exosome (EGF-M1 Exo, 20 µg/mL for 24 h). Analysis of variance followed by Tuckey’s post-hoc test. N = 4. *p0.05, **p0.01, ***p0.001, ****p0.0001, and ns: not significant. EGF, epidermal growth factor; PF, pterygial fibroblast.

Fig. 5

Effect of EGF on the anti-proliferation of cultured pterygial fibroblasts during during co-culture with M1 macrophages. (A) Representative images of Ki-67 staining of cultured pterygial fibroblasts with or without EGF treatment (10 ng/mL) during co-culture with M1 macrophages. Scale bars: 200 μm. (B) Comparison of proportion of Ki-67⁺ cells with or without EGF treatment (10 ng/mL) during co-culture with M1 macrophages. Analysis of variance followed by Tuckey’s post-hoc test. N = 3. **p0.01, ***p0.001, and ns: not significant. EGF, epidermal growth factor; PF, pterygial fibroblast.

Fig. 6

Suppression of pterygial fibroblast proliferation induced by M1 macrophage-derived exosomes using preconditioning of M1 macrophages with EGF. Growth curves of pterygial fibroblasts in the absence or presence of M1-derived exosomes (M1 Exo) or EGF (10 ng/mL)-preconditioned M1-derived exosomes (EGF-M1 Exo) over 72 h. Analysis of variance followed by Tuckey’s post-hoc test. N = 4. *p0.05 (Vehicle vs. M1 Exo) and ^#p0.05 (EGF-M1 Exo vs. M1 Exo). EGF, epidermal growth factor.