AGE induced macrophage-derived exosomes induce endothelial dysfunction in diabetes via miR-22-5p/FOXP1

Abstract

Background: Endothelial dysfunction is a pivotal contributor to cardiovascular complications in individuals with diabetes. However, the precise role of macrophages and their exosomes in the diabetic milieu remains elusive.

Methods: Exosomes (Exos) were isolated from the supernatants of macrophages treated with advanced glycation end products (AGE) or bovine serum albumin (BSA) using ultracentrifugation. Following coculture with AGE-Exos or BSA-Exos, human umbilical vein endothelial cells (HUVECs) were subjected to CCK-8, EdU, cell migration, monocyte adhesion, and tube formation assays. ELISA and Western blotting were employed to assess inflammatory cytokine release and protein expression levels in HUVECs. The miRNA expression profiles of AGE-Exos and BSA-Exos were analysed using miRNA arrays. Potential targets of miR-22-5p were predicted via miRNA databases and validated through RT‒qPCR, dual-luciferase reporter assays, and rescue experiments. Furthermore, a Rab27a knockout mouse model of type 2 diabetes mellitus (T2DM) was established by intraperitoneal injection of Streptozotocin. Aortic tissues were analysed via immunofluorescence for CD63 and CD31 expression, immunohistochemistry for VCAM-1 and ICAM-1 expression, and Western blotting for FOXP1 expression.

Results: AGE stimulation increased the secretion of exosomes from macrophages. Compared with BSA-Exos, AGE-Exos significantly impaired endothelial cell proliferation, migration, and tube formation capabilities while increasing monocyte adhesion and proinflammatory cytokine release without affecting cell viability. miR-22-5p was enriched in AGE-Exos, which were subsequently transferred to HUVECs, specifically targeting FOXP1, resulting in endothelial dysfunction. Overexpression of miR-22-5p in HUVECs using lentiviral vectors recapitulated the inflammatory effects observed with AGE-Exos, whereas anti-miR-22-5p conferred protective effects. Rab27a knockout significantly reduced exosome accumulation in T2DM model mouse aortic tissues, alleviating endothelial discontinuity, downregulating VCAM-1 and ICAM-1 expression, and upregulating FOXP1 expression.

Conclusions: AGE-induced release of macrophage-derived exosomes may partially depend on Rab27a transport, which delivers miR-22-5p to ECs. This miR-22-5p targets FOXP1 in ECs, leading to inflammation and resulting in endothelial dysfunction that accelerates the development of diabetic vascular lesions.

Keywords: Diabetes; Endotheliocytes; Exosomes; Inflammation; Macrophage; microRNAs.

Fig. 1

Identification of AGE-Exos and BSA-Exos. A TEM image of AGE-Exos(n = 3). Bar. 200 nm. B Reprmsentative images of the NTA of averge diameter of exosomes devived from BSA and AGE induced macrophages(n = 3). C. Western blotting of CD9, CD63, ALIX, Calnexin and GAPDH in macrophages, BSA-Exos and AGE-Exos. D NTA shows the concentration of the exosomes derived from macrophages under AGE or BSA treatments(n = 3). *P < 0.05, BSA vs. AGE

Fig. 2

AGE-Exos impair ECs function. A The uptake of PKH-26-labelled (red fluorescent dye) AGE-Exos by HUVECs. Bar. 100 μm. B CCK-8 shows the impact of AGE-Exos on HUVECs cell viability. C Proliferation of HUVECs treated with AGE-Exos and BSA-Exos for 24 h. Bar. 400 μm. D The migration of HUVECs treated with AGE-Exos and BSA-Exos for 24 h. Bar. 100 μm. E The tube formation ability of HUVEC indued by AGE-Exos and BSA-Exos. Bar. 100 μm. Mean ± SEM is represented as bar plot

Fig. 3

AGE-Exos enhance monocyte adhesion and increases adhesion molecule expression in ECs A Impact of AGE-Exos on THP-1 monocyte adhesion to HUVECs. Bar. 100 μm. B AGE-Exos decreased the expression of p-eNOs and increased the expression levels of VCAM-1, ICAM-1 and IL-6. All experiments were performed independently for at least three times. C AGE-Exos increased the release of sICAM-1, sVCAM-1 and IL-6 assayed through ELISA. n = 4. Mean ± SEM is represented as bar plot

Fig. 4

miR-22-5p is enriched in AGE-Exos and transferred to ECs. A, B Heatmap of RNA-sequencing analyses of AGE-Exos and BSA-Exos. C qPCR analyses on the miR-22-5p in macrophages treated with BSA or AGE, BSA-Exos and AGE-Exos. D Fluorescent imaging of HUVECs incubated with Cy3-labeled miR-22-5p AGE-Exos. E q-PCR analyses on the miR-22-5p in HUVECs treated with AGE-Exos and BSA-Exos. Mean ± SEM is represented as bar plot

Fig. 5

miR-22-5p mediates ECs dysfunction. A Fluorescent imaging of HUEVCs infected with lentivirus-GFP-miR-22-5p. qPCR analyses on miR-22-5p in HUVECs infected with lentivirus-GFP-miR-22-5p. B Effects of LV-miR-22-5p on the viability of HUVECs. C Western blotting data showing effects of LV-miR-22-5p on ICAM-1, VCAM-1, MCP-1, p-eNOS expression in HUVECs. D–F Effects of LV-miR-22-5p on proliferation(D), migration(E), and tube formation(F) of HUVECs. Bar: 200 μm in D and F, 100 μm in E. Mean ± SEM is represented as bar plot

Fig. 6

FOXP1 is a novel target of miR-22-5p. A The binding sequence of miR-22-5p in the 3’-UTR of FOXP1 transcript. B Dual-luciferase assay verified the binding relationship between miR-22-5p and the 3ʹ-UTR of FOXP1 transcripts (n = 3). C, D miR-22-5p regulates the mRNA level (C) and protein expression (D) of FOXP1. Mean ± SEM is represented as bar plot

Fig. 7

A miR-22-5p inhibitor partially mitigates the damage to ECs induced by AGE-Exos. A EdU staining showing the effects of miR-22-5p inhibitor on proliferation of HUVECs induced by AGE-Exos. Bar. 100 μm. B miR-22-5p inhibitor attenuated AGE-Exos effects on HUVECs in the transwell assay. Bar. 100 μm. C miR-22-5p inhibitor partially restored the expression levels of FOXP1, ICAM-1 and MCP-1 on HUVECs under AGE-Exos treatment. Mean ± SEM is represented as bar plot

Fig. 8

FOXP1 overexpression prevent miR-22-5p-induced ECs inflammation and dysfunction. A Western blotting data showing effects of LV-FOXP1 on expression of MCP-1, p-NF κB, ICAM-1, VCAM-1 and p-eNOS in mimic miR-22-5p stimulated HUVECs. B EdU staining showing the effects of LV-FOXP1 on proliferation of HUVECs induced by mimic miR-22-5p. Bar. 400 μm. C LV-FOXP1 alleviated the inhibitor role of mimic miR-22-5p on angiogenesis in HUVECs. Bar. 200 μm. Mean ± SEM is represented as bar plot

Fig. 9

Macrophage Rab27a inhibition mitigates the inflammation of ECs caused by AGE-stimulated macrophage exosomes. A Western blotting data showing effects of AGE on the expression of RAGE and Rab27a in THP-1. B Western blotting data showing effects of Si-Rab27a on the expression of Rab27a in THP-1. C Western blotting showing the expression of MCP-1, FOX-1, ICAM-1, VCAM-1 and p-eNOS in HUVECs induced by AGE-Exos derived from macrophage Rab27a inhibition. Mean ± SEM is represented as bar plot

Fig.10

Inhibition of exosome release reduces endothelial inflammation in T2DM model mice. A Blood glucose level of mouse from four groups. B Glucose tolerance test was performed by intraperitoneal injection of glucose. C Immunofluorescence of CD63 in the aorta of mouse from four groups. D Immunofluorescence of CD31 in the aorta of mouse from four groups. E Immunohistochemistry of ICAM-1 and VCAM-1 in the vascular endothelium. F Western blotting data showing the expression of FOXP1 and Rab27a in the aorta of mouse from four groups. Bar: 50 μm in D; 200 μm in F, 200 μm and 100 μm in F. Mean ± SEM is represented as bar plot

Fig. 11

Diagram of the signaling cascades involved in the effects of AGE-Exos on ECs. In the diabetic microenvironment, AGE stimulate the release of exosomes that are abundant in miR-22-5p, which via Rab27a-mediated pathway. These exosomes subsequently reach to endothelial cells, specifically targeting FOXP1, resulting in endothelial dysfunction and ultimately promoting diabetic vasculopathy. AGE, advanced glycosylation end products