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Adropin Contributes to Anti-Atherosclerosis by Suppressing Monocyte-Endothelial Cell Adhesion and Smooth Muscle Cell Proliferation

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Adropin Contributes to Anti-Atherosclerosis by Suppressing Monocyte-Endothelial Cell Adhesion and Smooth Muscle Cell Proliferation

Kengo Sato et al. Int J Mol Sci.

Abstract

Adropin, a peptide hormone expressed in liver and brain, is known to improve insulin resistance and endothelial dysfunction. Serum levels of adropin are negatively associated with the severity of coronary artery disease. However, it remains unknown whether adropin could modulate atherogenesis. We assessed the effects of adropin on inflammatory molecule expression and human THP1 monocyte adhesion in human umbilical vein endothelial cells (HUVECs), foam cell formation in THP1 monocyte-derived macrophages, and the migration and proliferation of human aortic smooth muscle cells (HASMCs) in vitro and atherogenesis in Apoe-/- mice in vivo. Adropin was expressed in THP1 monocytes, their derived macrophages, HASMCs, and HUVECs. Adropin suppressed tumor necrosis factor α-induced THP1 monocyte adhesion to HUVECs, which was associated with vascular cell adhesion molecule 1 and intercellular adhesion molecule 1 downregulation in HUVECs. Adropin shifted the phenotype to anti-inflammatory M2 rather than pro-inflammatory M1 via peroxisome proliferator-activated receptor γ upregulation during monocyte differentiation into macrophages. Adropin had no significant effects on oxidized low-density lipoprotein-induced foam cell formation in macrophages. In HASMCs, adropin suppressed the migration and proliferation without inducing apoptosis via ERK1/2 and Bax downregulation and phosphoinositide 3-kinase/Akt/Bcl2 upregulation. Chronic administration of adropin to Apoe-/- mice attenuated the development of atherosclerotic lesions in the aorta, with reduced the intra-plaque monocyte/macrophage infiltration and smooth muscle cell content. Thus, adropin could serve as a novel therapeutic target in atherosclerosis and related diseases.

Keywords: atherosclerosis; endothelial cell; inflammation; macrophage; monocyte; smooth muscle cell.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Biosynthesis of adropin. Adropin precursor protein consisting of 76 amino acids produces the 43 amino acid adropin by the cleavage of a 33 amino acid signal peptide.
Figure 2
Figure 2
Expression of ENHO (adropin gene) and GPR19 in human vascular cells and the effect of adropin on foam cell formation and inflammatory phenotype in THP1 monocyte-derived macrophages. (A) mRNA expression levels of ENHO and GPR19 in THP1 monocytes, their derived macrophages, HASMCs, HUVECs, and HAECs were analyzed by RT-PCR. Glyceraldehyde-3-dehydrogenase (GAPDH) served as a loading control. Independent experiments were repeated twice to assure reproducibility. (B) THP1 monocytes were incubated for 6 days with the indicated concentrations of adropin, followed by a 19-h incubation with 50 μg/mL oxidized LDL in the presence of 100 μmol/L [3H]oleate. Foam cell formation was determined from reads of the intracellular radioactivity of cholesterol-[3H]oleate (n = 5). The results from 5 independent experiments are shown in different colors. (C) THP1 monocyte-derived macrophages cultured for 6 days were harvested before the addition of oxidized LDL for immunoblot for SRA, ACAT1, NCEH, ABCA1, and β-actin (n = 5–6). (D) THP1 monocytes were incubated for the indicated times with or without adropin (10 ng/mL). Cells were subjected to immunoblot for CD68 (a macrophage differentiation marker), MARCO (an M1 macrophage marker), arginase 1 (an M2 macrophage marker), p-NFκB, PPARγ, or β-actin (n = 3–4). The graph shows the expressions on day 6. * p < 0.05, p < 0.005 vs. 0 ng/mL of adropin.
Figure 3
Figure 3
Effects of adropin on inflammatory response and monocyte adhesion in HUVECs. (A) mRNA expression of ICAM1, VCAM1, and SELE (selectin E gene) was analyzed by RT-PCR. HUVECs were pre-treated with adropin (0, 10, 100 ng/mL) for 30 min and then incubated with adropin (0, 10, 100 ng/mL) + TNFα (0, 10 ng/mL) for 4 h. Representative images are shown; the graph on the right side indicates densitometry data following normalization relative to GAPDH (n = 3–4). (BD) HUVECs treated as described above were harvested and subjected to immunoblot to evaluate ICAM1, VCAM1, and selectin E protein expression (n = 4–5). Upper panels show representative immunoblots with densitometry data after normalization relative to β-actin shown beneath. (E) Confluent HUVECs were incubated in 0.5% fetal bovine serum (FBS)-EGM-2 for 16 h, and then pre-treated for 30 min with the indicated concentrations of adropin, followed by a 4-h incubation in the presence or absence of TNFα (10 ng/mL). Subsequently, calcein red-orange-labeled THP1 monocytes were plated on the HUVEC monolayer and incubated for 1 h. After washing, the adherent cells were observed by fluorescence microscopy (n = 4). Scale bar = 100 μm. Baseline (1 fold) = 66318.5 ± 4599.2 pixels. (BE) * p < 0.0001 vs. corresponding control of TNFα (−); p < 0.05, p < 0.001, § p < 0.005, || p < 0.0001 vs. corresponding control of TNFα (+).
Figure 4
Figure 4
Effects of adropin on migration, proliferation, apoptosis, and ECM expression in HASMCs. (A) Migration was determined in 10 HASMCs per plate using a BIOREVO BZ-9000 microscope in serum-free SmGM-2 with or without AngII (500 nmol/L) and adropin (0, 100, 1000 ng/mL). Four independent experimental replicates were performed. * p < 0.0001 vs. 0 ng/mL of adropin. p < 0.0005, p < 0.0001 vs. AngII. (B) The proliferation was determined by WST-8 assay following a 48-h incubation of HASMCs in 5% FBS-SmGM-2 with the indicated concentrations of adropin (n = 5). § p < 0.05 vs. 0 ng/mL of adropin. (C) HASMCs were stained as apoptotic cells (green) using the TUNEL method after a 48-h incubation in 5% FBS-SmGM-2 with the indicated concentrations of adropin. Nuclei were co-stained with 6-diamidino-2-phenylindole (blue). The graph indicates the percentage of apoptotic cells (n = 3). Scale bar = 100 μm. (D) HASMCs were incubated for 24 h in serum-free SmGM-2 with the indicated concentrations of adropin, and then harvested for immunoblot of collagen 1, collagen 3, fibronectin, elastin, MMP2, MMP9, and α-tubulin. Representative data showing protein expression (upper panels) with densitometry following normalization relative to α-tubulin (lower panels, n = 5–6). § p < 0.05 vs. 0 ng/mL of adropin.
Figure 5
Figure 5
Effects of adropin on intracellular signal transduction in HASMCs. HASMCs were incubated for 24 h in 5% FBS-SmGM-2 with the indicated concentrations of adropin. The effects of adropin on intracellular signals were assessed by immunoblots. Representative data showing protein expression or phosphorylation (upper panels) with densitometry following normalization relative to α-tubulin (lower panels, n = 3–4). * p < 0.05, p < 0.001, p < 0.005 vs. 0 ng/mL of adropin.
Figure 6
Figure 6
Effects of adropin on atherosclerotic lesion development in Apoe−/− mice. Of the 22 Apoe−/− mice at 17 weeks of age, six were sacrificed before infusion and six, five, and five were infused with adropin at doses of 0, 5, and 10 μg/kg/h using osmotic minipumps for 4 weeks. (A) The atherosclerotic lesions on the aortic internal surface were stained with Oil Red O (a–d). Cross-sections of the aortic sinus were stained with Oil Red O (e–h), with immunostains for monocyte-macrophage 2 (MOMA2; (i–l)) or α-smooth muscle actin (α-SMA; (m–p)). Scale bar = 200 μm. (BF) Statistical comparisons of atherosclerotic lesion area, atheromatous plaque size, monocyte-macrophage or VSMC contents, and the ratio of monocyte-macrophage contents/VSMC contents within atheromatous plaques among four groups. * p < 0.0001, p < 0.005, p < 0.0005, § p < 0.05. Bars indicate the mean values in the graphs.

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