Inhibitory effect of engineered exosomes loaded with A485 on hypoxia-induced pathological neovascularization by targeted inhibition of lactylation

Authors:Fan Yuanyuan,Luo Yufan, Xie Ping, Hu Zizhong

Corresponding author: Hu Zizhong, Email: huzizhong@njmu.edu.cn

Published:2026-09-10

DOI:10.3760/cma.j.cn115989-20260427-00191

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ABSTRACT 

Objective To construct engineered exosomes loaded with the p300 inhibitor A485 (A485@EV) and to investigate their effects and mechanisms in inhibiting hypoxia-induced pathological neovascularization by targeting inhibition of lactylation.

Methods A485@EV was prepared by sonication, and its markers, morphology and physical and chemical properties were identified. The encapsulation efficiency, loading efficiency and release profile of A485 were determined. Human umbilical vein endothelial cells (HUVECs) and human microglial cell line 3 (HMC3) were divided into control group, free A485 group, and A485@EV group, and cultured under 1% O ₂ for 24 hours to establish a hypoxia model. Then equal volumes of phosphate buffered saline (PBS), free A485 or A485@EV were added to the cell culture system correspondingly, so that the final concentration of free A485 and A485@EV was 20 μmol/L and 20 μg/ml, respectively. EdU proliferation assay, Transwell migration assay, and tube formation assay were used to evaluate the proliferation, migration, and tube formation of HUVECs. EdU proliferation assay, Transwell migration assay, wound healing assay, and Western blot were used to evaluate the proliferation, migration, and wound healing of HMC3 as well as the cyclooxygenase-2 (COX-2) protein expression. Eighteen healthy 7-day-old male C57BL/6J mice were selected to establish an oxygen-induced retinopathy (OIR) model. The OIR mice were randomly divided into control group, free A485 group, and A485@EV group using the random number table method, with 6 mice in each group and mice received an intravitreal injection of 1 μl of PBS, 1 mmol A485, or 1 μg A485@EV, respectively. Avascular area, neovascular area, and the lactylation levels of retinal endothelial cells and microglia were detected by immunofluorescent imaging. The study protocol was approved by the Animal Ethics Committee of Nanjing Medical University (No. IACUC-2202032-1).

Results The successfully constructed A485@EV exhibited a typical cup-shaped morphology, with a mean particle size of (167.0±70.6)nm, a zeta potential of -35.63 mV, an encapsulation efficiency of (40.91±2.30)%, a loading efficiency of (20.45±1.15)%, and a 24-hour cumulative release rate of approximately 73.1%. Compared with the control group and free A485 group, the EdU positive rate, the number of migrating cells and the tube formation ability of HUVECs in A485@EV group were significantly reduced (all P<0.05). Compared with the control group and free A485 group, the EdU positive rate, the number of migrating cells, scratch healing rate and relative expression of COX-2 protein in HMC3 cells were also significantly reduced (all P<0.05). In the OIR mouse model, avascular and neovascular areas were both lower in the A485@EV group than in the control and free A485 groups, with statistically significant differences (all P<0.05). IB4-positive endothelial cells and IBA1-positive microglia showed markedly weaker fluorescence of lactylation in the A485@EV group than in the control and free A485 groups.

Conclusions Engineered exosome loaded with A485 may attenuate hypoxia-induced endothelial cell tube formation and microglial activation by targeting inhibition of the lactylation effect, thereby inhibiting pathological neovascularization.

KEYWORDS：

Pathological neovascularization;Engineered exosomes;A485;Lactylation

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Authors Info & Affiliations 

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Figures & Tables

Figure 1 Lactylation levels in mice retinal endothelial cells and microglia in each group (Scale bar=20 μm)　A: Co-localization of endothelial cells (IB4, green) and pan-lactylation (red)　B: Co-localization of microglia (IBA1, green) and pan-lactylation (red)　OIR: oxygen-induced retinopathy

Figure 2 Identification of exosomes and construction and characterization of engineered exosomes (A485@EV) 　A: Electrophoretogram of markers expression of exosomes and A485@EV CD63 and CD9 proteins were highly expressed and the endoplasmic reticulum marker Calnexin was not obviously expressed　B: Size distribution and TEM images of exosomes and A485@EV (Scale bar=100 nm)　B1: exosomes　B2: A485@EV　C: Average zeta potential of exosomes and A485@EV　D: Encapsulation efficiency and loading efficiency of A485@EV　E: Release profile of A485@EV　HUMSCs: human umbilical cord mesenchymal stem cells; TEM: transmission electron microscopy

Figure 3 Proliferation, migration, and tube formation of different HUVECs group 　A: Staining images of EdU proliferation assay (Scale bar=100 µm)　B: Representative images of Transwell migration assay (crystal violet, Scale bar=100 µm)　C: Representative images of tube formation assay (Scale bar=100 µm)　HUVECs: human umbilical vein endothelial cells

Table 1 Comparison of proliferation, migration, and tube formation of different HUVECs groups (mean±SD) Note: Compared with control group, ^a P<0.05; compared with free A485 group, ^b P<0.05 (One-way ANOVA, Tukey test)　HUVECs: human umbilical vein endothelial cells

Figure 4 Proliferation, migration, wound healing, and COX-2 expression in different HMC3 groups 　A: Staining images of EdU proliferation assay (Scale bar=100 µm)　B: Representative images of Transwell migration assay (crystal violet, Scale bar=100 µm)　C: Representative images of wound healing assay (Scale bar=100 µm)　D: Electrophoretogram of COX-2 protein expression　HMC3: human microglial cell line 3; COX-2: cyclooxygenase-2

Table 2 Comparison of proliferation, migration, wound healing of HMC3 cells and COX-2 protein expression among different groups (mean±SD) Note: Compared with control group, ^a P<0.05; compared with free A485 group, ^b P<0.05 (One-way ANOVA, Tukey test)　HMC3: human microglial cell line 3; COX-2: cyclooxygenase-2

Figure 5 Comparison of the degree of lactylation and the areas of non-vascular and neovascular regions in OIR mice retina among different groups 　A: Co-localization of endothelial cells (IB4, green) and pan-lactylation (red) (Scale bar=50 µm)　B: Co-localization of microglia (IBA1, green) and pan-lactylation (red) (Scale bar=50 µm)　C: Avascular areas (outlined in yellow lines) and neovascular areas (covered in white shadow) (Scale bar=1 mm)　D: Comparison of retinal avascular area　 F=21.98, P<0.05. Compared with control group, ^aP<0.05; compared with free A485 group, ^bP<0.05 (One-way ANOVA, Tukey test; n=6)　E: Comparison of retinal neovascular area F=59.59, P<0.05. Compared with control group, ^aP<0.05; compared with free A485 group, ^bP<0.05 (One-way ANOVA, Tukey test; n=6)　OIR: oxygen-induced retinopathy

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