Myeloid-derived growth factor inhibits inflammation and alleviates endothelial injury and atherosclerosis in mice

Abstract

Whether bone marrow modulates systemic metabolism remains unknown. Here, we found that (i) myeloid cell-specific myeloid-derived growth factor (MYDGF) deficiency exacerbated vascular inflammation, adhesion responses, endothelial injury, and atherosclerosis in vivo. (ii) Myeloid cell-specific MYDGF restoration attenuated vascular inflammation, adhesion responses and leukocyte homing and alleviated endothelial injury and atherosclerosis in vivo. (iii) MYDGF attenuated endothelial inflammation, apoptosis, permeability, and adhesion responses induced by palmitic acid in vitro. (iv) MYDGF alleviated endothelial injury and atherosclerosis through mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4)/nuclear factor κB (NF-κB) signaling. Therefore, we concluded that MYDGF inhibits endothelial inflammation and adhesion responses, blunts leukocyte homing, protects against endothelial injury and atherosclerosis in a manner involving MAP4K4/NF-κB signaling, and serves as a cross-talk factor between bone marrow and arteries to regulate the pathophysiology of arteries. Bone marrow functions as an endocrine organ and serves as a potential therapeutic target for metabolic disorders.

Fig. 1. Myeloid cell–specific MYDGF deficiency is associated with endothelial injury and inflammation in mice.

KO and WT mice aged 4 to 6 weeks were divided into four groups (NCD-WT, NCD-KO, WD-WT, and WD-KO mice) and were fed their respective diets for 12 weeks (10 mice in each group). (A and B) The vasodilation responses to (A) acetylcholine (Ach) and (B) sodium nitroprusside (SNP) (n = 6). (C) Representative images of terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining in sections of thoracic aortas. TUNEL (apoptotic cells, red), anti-CD31 (endothelial cells, green), and 4′,6-diamidino-2-phenylindole (DAPI) (nuclei, blue). Arrows indicate CD31/TUNEL colocalization. Scale bars, 200 μm. (D) The percentage of apoptotic endothelial cells (n = 6). (E) Representative electron microscopy images of endothelium. The arrows show endothelial cell (EC); IEL, internal elastic lamina. Scale bars, 50 μm. (F) The mRNA levels of inflammation (TNF-α, IL-1β, and IL-6) in MAECs of mice (n = 8). The data are presented as the means ± SEM. *P < 0.05 versus NCD-WT, **P < 0.01 versus NCD-WT, ***P < 0.001 versus NCD-WT; †P < 0.05 versus WD-WT, †† P < 0.01 versus WD-WT, †††P < 0.001 versus WD-WT

Fig. 2. Myeloid cell–specific MYDGF deficiency is associated with atherosclerotic plaque formation in AKO mice.

AKO and DKO mice aged 4 to 6 weeks were fed a WD for 12 weeks (10 mice in each group). (A and B) The vasodilatation reaction induced by Ach (A) and SNP (B) (n = 10). (C) Representative images of en face atherosclerotic lesions. (D) Quantitative analysis of (C) (n = 5). (E) Representative images of the cross-sectional area of the aortic root (n = 8). Scale bars, 500 μm. (F) Quantitative analysis of (E). (G) Representative immunohistochemical staining images of VSMCs [α–smooth muscle actin (α-SMA)], collagen (Masson), macrophages (anti-CD68), and T lymphocytes (anti-CD3) in aortic plaques. Scale bar, 100 μm. (H) Quantitative analysis of (G) (n = 8). (I and J) The mRNA levels of adhesion molecules (VCAM-1, ICAM-1, and E-selectin) (I) and inflammation (TNF-α, IL-1β, and IL-6) (J) in MAECs of mice (n = 5). The data are presented as the means ± SEM. *P < 0.05 and **P < 0.001.

Fig. 3. BMT alleviated endothelial injury and atherosclerosis in mice.

As shown in fig. S4C, BMT was performed, and atherosclerosis was assessed after WD feeding for 12 weeks (10 mice in each group). (A) The aortic vasodilatation induced by Ach in KO mice (n = 10). (B) Representative images of TUNEL staining in sections of thoracic aortas. Scale bars, 200 μm. (C) The percentage of apoptotic endothelial cells (n = 5). (D) Representative electron microscopy images of endothelium in KO mice (n = 5). Scale bars, 50 μm. (E) Representative images of en face atherosclerotic lesion areas in AKO and DKO mice. (F) Quantitative analysis of (E) (n = 5). (G) Representative images of the cross-sectional area of the aortic root in AKO and DKO mice. Scale bars, 500 μm. (H) Quantitative analysis of (G) (n = 8). (I) Representative immunohistochemical staining images of VSMCs, collagen, macrophages, and T lymphocytes in aortic plaques. Scale bar, 100 μm. (J) Quantitative analysis of (I) (n = 5). The data are presented as the means ± SEM. *P < 0.05 versus WT → WT and **P < 0.01 versus WT → WT; #P < 0.05 versus WT → KO and ##P < 0.001 versus WT → KO; †P < 0.01 versus WT → AKO; ‡P < 0.001 versus WT → DKO.

Fig. 4. The MYDGF overexpression of bone marrow in situ alleviated atherosclerosis.

In situ MYDGF overexpression in bone marrow was performed in KO, AKO, and DKO mice aged 4 to 6 weeks. Then, the mice were fed a WD for 12 weeks, and atherosclerosis was assessed at the end of the experiment (10 mice in each group). (A) The aortic vasodilatation induced by Ach in KO mice (n = 10). (B) Representative images of TUNEL staining in sections of thoracic aortas. Scale bars, 200 μm. (C) The percentage of apoptotic endothelial cells (n = 9). (D) Representative electron microscopy images of endothelium. Scale bars, 50 μm. (E) Representative images of en face atherosclerotic lesions. (F) Quantitative analysis of (E) (n = 5). (G) Representative images of the cross-sectional area of the aortic root. Scale bars, 500 μm. (H) Quantitative analysis of (G) (n = 9). (I) Representative immunohistochemical staining images of VSMCs, collagen, macrophages, and T lymphocytes in aortic plaques. Scale bar, 100 μm. (J) Quantitative analysis of (I) (n = 5). The data are shown as the means ± SEM. *P < 0.05 versus KO-GFP and **P < 0.001 versus KO-GFP; †P < 0.001 versus AKO-GFP; ‡P < 0.001 versus DKO-GFP.

Fig. 5. The MYDGF overexpression of bone marrow in situ decreased the leukocytes homing within aortic plaques from DKO mice.

MYDGF overexpression of bone marrow in situ was performed in DKO mice aged 4 weeks, and leukocyte homing was analyzed in DKO-GFP or DKO-MYDGF mice that had been fed a WD for 12 weeks. (A) The mRNA expression of the macrophage markers F4/80 and CD68 in aortas. (B) The mRNA expression of the chemokines in aortas. (C and D) The homing of GFP leukocytes to atherosclerotic plaques 48 hours after intravenous injection into DKO-GFP and DKO-MYDGF mice that were fed a WD for 12 weeks. (C) Fluorescence micrograph of aortic root plaques. The dashed line indicates the plaque border. Inset, magnification of GFP leukocytes. Left, DAPI; middle, GFP; right, merge. Scale bars, 150 μm. (D) Quantification of GFP leukocytes per square millimeter of plaque (n = 5). The data represent the means ± SEM; *P < 0.001.

Fig. 6. Endothelial-specific MAP4K4 KD vitiates MYDGF-mediated protection of endothelial injury in vivo.

MAP4K4 KD/KO and control/KO mice were fed a WD for 12 weeks, and endothelial injury and NF-κB signaling were investigated. (A) Schematic of the transgenic construct used to generate MAP4K4 KD animals. (B and C) MAECs were derived from MAP4K4 KD and control mice. (B) MAP4K4 and VEGFR2 (vascular endothelial growth factor receptor 2) protein expression in MAECs and unselected cell lysates (n = 3). (C) mRNA levels of MAP4K4 in immune-selected or unselected cells (n = 3). (U6, promoter; WRE, woodchuck hepatitis virus posttranscriptional regulatory element). (D) The expression levels of NF-κB signaling in MAECs of MAP4K4 KD/KO and control/KO mice (n = 6). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (E) The aortic vasodilatation induced by Ach in MAP4K4 KD/KO and control/KO mice (n = 10). (F) Representative images of TUNEL staining in sections of thoracic aortas. (G) The percentage of apoptotic endothelial cells (n = 6). (H) Representative electron microscopy images of endothelium. Scale bars, 50 μm. (I) The mRNA levels of adhesion molecules and (J) inflammation in MAECs of aortas (n = 10). The data are shown as the means ± SEM. *P < 0.001.

Fig. 7. Schematic showing that MYDGF plays a protective role in atherosclerosis via MAP4K4/NF-κB signaling pathway.