M2 macrophage-derived extracellular vesicles protect against abdominal aortic aneurysm by modulating macrophage polarization through miR221-5p

Abstract

Background: Extracellular vesicles (EVs) derived from M2 macrophages (M2-EVs) play a protective role in the pathogenesis of acute lung injury. However, their roles and mechanisms in abdominal aortic aneurysm (AAA) are unknown.

Methods: The effects of M2-EVs in AAA were examined in ApoE^-/- mice with angiotensin II infusion. After M2 macrophages were stimulated with antisense oligonucleotides of miR221-5p (miR221-5p-ASOs), EVs were extracted and administered to mice via the tail vein. In vitro, the primary bone marrow-derived monocytes (BMDMs) were isolated and co-cultured with human aortic endothelial cells (HAECs) in Transwell chambers.

Results: M2-EVs significantly reduced AAA incidence and maximal aortic diameters, improved fiber continuity, increased α-SMA, and reduced macrophage infiltration in AAA mice. RNA sequencing revealed that miR221-5p was upregulated in M2-EVs and downregulated in AAA. miR221-5p-ASOs reduced the protection of M2-EVs in AAA mice. M2-EVs induced M2 macrophage polarization, while miR221-5p-ASOs had no effect. Moreover, M2-EVs alleviated oxidative stress and inflammatory responses in HAECs. Mechanistically, miR221-5p bound to poly(ADP-ribose) polymerase 1 (PARP-1) mRNA and reduced PARP-1 expression; PARP-1 was bound to protein phosphatase 1ɑ (PP-1ɑ) and negatively regulated its expression. In vitro experiments showed miR221-5p modulated macrophage polarization through the PARP-1/PP-1ɑ/JNK/c-Jun pathway. Macrophage deletion of PARP-1 inhibited AAA formation and phosphorylation of JNK/c-Jun in mice.

Conclusions: miR221-5p in M2-EVs plays a critical role in AAA pathophysiology by modulating macrophage polarization through PARP-1/PP-1ɑ/JNK/c-Jun signaling. M2-EVs and miR221-5p represent promising therapeutic options for AAA.

Keywords: Abdominal aortic aneurysm; Extracellular vesicles; M2 macrophages; Macrophage polarization; microRNA 221-5p.

Fig. 1

M2-EVs protected against AAA formation in mice. A The morphological characteristics of M2-EVs were examined by TEM (scale bar = 200 nm). B NTA was used to determine the average size of M2-EVs. C ARF6, CD81, and Alix protein levels were analyzed by western blot analysis in cells and M2-EVs. D Survival rates were assessed in the control, Ang II, Ang II + M0-EV, and Ang II + M2-EV groups. E M2-EVs reduced the incidence of AAA in mice. F Morphology of the aorta was analyzed in mice (scale bar = 0.5 cm). G H&E staining of the abdominal aortas in mice (scale bar = 200 µm). H The maximal abdominal aortic diameters were analyzed in mice. I Ultrasonography of the abdominal aorta. AAA, abdominal aortic aneurysm; M0-EVs, extracellular vesicles derived from M0 macrophages; M2-EVs, extracellular vesicles derived from M2 macrophages; TEM, transmission electron microscopy; NTA, nanoparticle tracking analyzer; Ang II, angiotensin II; *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 2

M2-EVs improved AAA remodeling in mice. A Masson staining of abdominal aortas in mice. B EVG staining of the abdominal aortas in mice was performed to assess the elastic fibers. C M2-EVs increased α-SMA expression in Ang II-infused mice. D M2-EVs reduced Ang II-induced macrophage infiltration in AAA lesions. E Quantification of α-SMA expression in abdominal aortas. F Quantification of CD68 expression in abdominal aortas. Scale bar = 200 µm. AAA, abdominal aortic aneurysm; M2-EVs, extracellular vesicles derived from M2 macrophages; EVG, Elastica van Gieson; Ang II, angiotensin II; ns, nonsignificant; ***P < 0.001

Fig. 3

M2-EVs inhibited the inflammatory response in AAA mice. A IL-1β expression was analyzed by IHC in abdominal aortas (scale bar = 200 µm). B Quantification of IL-1β expression in abdominal aortas. C TNF-α, ICAM-1, and iNOS mRNA expression levels in mouse abdominal aortas were analyzed by RT-PCR. D TNF-α, ICAM-1, and iNOS expression levels were assessed by western blot analysis. E Quantification of TNF-α protein expression in abdominal aortas. F Quantification of ICAM-1 protein expression in abdominal aortas. G Quantification of iNOS protein expression in abdominal aortas. AAA, abdominal aortic aneurysm; M2-EVs, extracellular vesicles derived from M2 macrophages; IHC, immunohistochemistry; Ang II, angiotensin II; TNF-α, tumor necrosis factor α; ICAM-1, intercellular adhesion molecule 1; iNOS, inducible nitric oxide synthase; IL-1β, interleukin 1β; ns, nonsignificant; **P < 0.01, ***P < 0.001

Fig. 4

miR221-5p-ASOs reduced the protection of M2-EVs in AAA mice. A A heatmap of RNA sequencing results revealed increased expression of miR221-5p in M2-EVs. B miR221-5p expression was analyzed in M0 and M2 macrophages. C miR221-5p expression was analyzed in M0-EVs and M2-EVs. D miR221-5p expression was analyzed in normal control and AAA mice. E The survival rate of mice was assessed in the control, Ang II, miR221-5p-ASOs, and Ang II + M2-EVs groups. F miR221-5p-ASOs increased AAA incidence in mice. G Morphology of the aorta was analyzed in mice (scale bar = 1 cm). H AAA was analyzed by H&E, Masson, EVG, and IHC of α-SMA and CD68 in mice. I Quantification of α-SMA expression in abdominal aortas. J Quantification of CD68 expression in the abdominal aorta. K Ultrasonography was performed to evaluate the abdominal aorta. L Maximal abdominal aortic diameters were analyzed in mice. AAA, abdominal aortic aneurysm; M2-EVs, extracellular vesicles derived from M2 macrophages; Ang II, angiotensin II; miR221-5p-ASOs, antisense oligonucleotides of miR221-5p; EVG, Elastica van Gieson; IHC, immunohistochemistry; *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

M2-EVs modulated macrophage polarization in vitro. A After BMDMs were stimulated by LPS (10 ng/ml) and IFN-γ (10 ng/ml) to induce M1 polarization, M2-EVs or miR221-5p-ASOs were added for 24 h, and IL-1β expression was analyzed by RT-PCR. B iNOS expression was analyzed using RT-PCR. C IL-1β and iNOS expressions were assessed by western blot analysis. D Quantification of IL-1β protein expression in BMDMs. E Quantification of iNOS protein expression in BMDMs. F M1 macrophage polarization was analyzed by flow cytometry. G After BMDMs were stimulated with IL-4 (20 ng/ml) to induce M2 polarization, M2-EVs or miR221-5p-ASOs were added for 24 h, and IL-10 expression was analyzed by RT-PCR. H Arg-1 mRNA expression was analyzed by RT-PCR. I IL-10 and Arg-1 expression levels were assessed by western blot analysis. J Quantification of IL-10 protein expression in BMDMs. K Quantification of Arg-1 protein expression in BMDMs. L M2 macrophage polarization was analyzed by flow cytometry. BMDMs, primary bone marrow-derived monocytes; LPS, lipopolysaccharide; M2-EVs, extracellular vesicles derived from M2 macrophages; miR221-5p-ASOs, antisense oligonucleotides of miR221-5p; IL-4, interleukin 4; IL-1β, interleukin 1β; iNOS, inducible nitric oxide synthase; IL-10, interleukin 10; Arg-1, arginase 1; ns, nonsignificant; *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6

miR-221-5p inhibited oxidative stress and inflammation in HAECs. A Illustration of the co-culture of HAECs with BMDMs stimulated by LPS, M2-EVs, or M2-EVs-miR221-5p-ASOs. B M2-EVs were ingested by BMDMs. Blue, DAPI; red, cytoplasm; green, M2-EVs. C DHE staining was performed to assess the oxidative stress in HAECs (scale bar = 50 µm). D Quantification of DHE fluorescence in HAECs. E ICAM-1 expression in HAECs was analyzed by immunofluorescence (scale bar = 50 µm). F Quantification of ICAM-1 in HAECs. G Adhesion of THP-1 cells to HAECs was evaluated using immunofluorescence (scale bar = 100 µm). H Quantification of THP-1 cell adhesion to HAECs. BMDMs, primary bone marrow-derived monocytes; HAECs, human aortic endothelial cells; LPS, lipopolysaccharide; M2-EVs, extracellular vesicles derived from M2 macrophages; miR221-5p-ASOs, antisense oligonucleotides of miR221-5p; DHE, dihydroethidium; ICAM-1, intercellular adhesion molecule 1; ***P < 0.001

Fig. 7

PARP-1 was increased in AAA and reduced by miR221-5p. A The binding sites of PARP-1 and miR221-5p were predicted using bioinformatics analysis. B Luciferase reporter assays demonstrating the ability of miR221-5p to bind to the PARP-1 3′UTR. C, D miR221-5p was transfected into BMDMs, and the mRNA and protein expression levels of PARP-1 were analyzed in vitro. E, F PARP-1 mRNA and protein expression in human AAA lesions was evaluated using RT-PCR and western blot analysis. G PARP-1 activity was analyzed in serum of patients with AAA, as assessed by ELISA (n = 15). H, I Human aortas were analyzed by H&E and Masson staining (scale bar = 200 μm). PAR expression was assessed in human AAA lesions and normal tissues by immunohistochemistry (scale bar = 50 µm). J, K Mice were stimulated with Ang II and treated with M2-EVs, then PARP-1 mRNA and protein expression in the aortas was evaluated by RT-PCR and western blot analysis. AAA, abdominal aortic aneurysm; Ang II, angiotensin II; ELISA, enzyme-linked immunosorbent assay; PARP-1, poly(ADP-ribose) polymerase 1; PAR, poly(ADP-ribose); M2-EVs, extracellular vesicles derived from M2 macrophages; BMDMs, primary bone marrow-derived monocytes; NC, negative control; ns, nonsignificant; ***P < 0.001

Fig. 8

PARP-1 binds to PP-1α to modulate JNK/c-Jun phosphorylation. A BMDMs were stimulated by LPS; immunofluorescence was performed and revealed that PARP-1 was co-expressed with protein phosphatase 1ɑ (PP-1ɑ) in the nucleus. Red, PP-1ɑ; green, PARP-1; blue, nucleus. B IP analysis revealed that PARP-1 binds to PP-1α. C Heatmap of the differentially expressed genes in BMDMs from control and PARP-1^f/f/lyzM^Cre mice after LPS stimulation (10 ng/ml). D KEGG pathway enrichment analysis of genes involved in cell signaling pathways. E Phosphorylation of JNK was analyzed by western blot analysis. F Quantification of phosphorylated JNK expression. G Phosphorylated JNK and c-Jun were analyzed by western blot analysis in BMDMs from PARP-1^f/f/lyzM^Cre mice and BMDMs transfected or treated with PP-1α plasmid, SP600125 (a JNK inhibitor), or miR221-5p-ASOs. H Quantification of phosphorylated JNK and c-Jun protein expression. IP, immunoprecipitation; PARP-1, poly(ADP-ribose) polymerase 1; MAPK, mitogen-activated protein kinase; LPS: lipopolysaccharide; PP-1α, protein phosphatase 1α; BMDM, primary bone marrow-derived monocyte; SP600125, a JNK inhibitor; ns, nonsignificant; ***P < 0.001

Fig. 9

miR221-5p modulates M1 macrophage polarization through the PARP-1/PP-1α/JNK/AP-1 pathway. The study findings indicate that miR221-5p inhibits M1 macrophage polarization through the PARP-1/PP-1α/JNK/c-Jun pathway. AAA, abdominal aortic aneurysm; PP-1α, protein phosphatase 1α; PARP-1, poly(ADP-ribose) polymerase 1; AP-1, activator protein 1; IL-1β, interleukin 1β; TNF-α, tumor necrosis factor α; IL-6, interleukin 6; EV, extracellular vesicle