Systemic application of 3-methyladenine markedly inhibited atherosclerotic lesion in ApoE-/- mice by modulating autophagy, foam cell formation and immune-negative molecules

Abstract

A growing body of evidence demonstrates that autophagy, an evolutionarily conserved intracellular degradation process, is involved in the pathogenesis of atherosclerosis and has become a potential therapeutic target. Here we tested the effect of two inhibitors of phosphatidylinositol 3-kinase, 3-methyladenine (3-MA) and 2-(4-morpholinyl)-8-phenyl-chromone (LY294002), commonly used as inhibitors of autophagy, in atherosclerosis in apolipoprotein E^-/- mice. Systemic application of 3-MA but not LY294002 markedly reduced the size of atherosclerotic plaque and increased the stability of lesions in high-fat diet-fed mice as compared with controls. Furthermore, 3-MA had multiple atheroprotective effects, including modulating macrophage autophagy and foam cell formation and altering the immune microenvironment. Long-term treatment with 3-MA promoted oxidized low-density lipoprotein (oxLDL)-induced macrophage autophagy and suppressed foam cell formation and cell viability in vitro. Furthermore, systemic application of 3-MA promoted lipid droplet breakdown and decreased apoptosis, most likely associated with autophagy. 3-MA treatment strikingly enhanced the expression of immune-negative molecules such as interleukin 10 (IL-10), transforming growth factor β and IL-35, as well as forkhead box P3 (Foxp3), the specific transcriptional factor for regulatory T cells, but did not affect the level of proinflammatory cytokines in the arterial wall. We provide strong evidence for the potential therapeutic benefit of 3-MA in inhibiting atherosclerosis development and improving plaque stability.

Figure 1

3-MA markedly inhibited the development of atherosclerotic lesion in ApoE^−/− mice fed with HFD. (a) Representative images of Oil Red O-stained plaque burden (red) in aortas from control mice (6 mice from 14 control mice) and 3-MA-treated mice (6 mice from the 16 3-MA-treated mice). Data are percentage ORO-stained plaque areas in the entire aorta and regions (aortic arch, thoracic aorta and abdominal aorta). (b and c) Representative hematoxylin and eosin (H&E) staining and Oil Red O staining of cross-sections of aortic root in control mice (n=14) and 3-MA-treated mice (n=16). Scale bar, 200 μm. Data are mean±S.D. *P<0.05, **P<0.01, ***P<0.001, unpaired two-tailed Student's t-test

Figure 2

3-MA application improved the stability of plaque. (a–d and f) Cross-sections of aortic root from control mice (n=14) and 3-MA-treated mice (n=16) stained for T cells (CD3), macrophages (MOMA-2) and smooth muscle cells (αSMC-Actin). Sirius red staining was used to detect collagen fibers. TUNEL staining was used to detect apoptotic cells. Scale bar, 100 μm. (e) Analysis of vulnerability index of control mice (n=14) and 3-MA-treated mice (n=16). Data are mean±S.D. **P<0.01, ***P<0.001, unpaired two-tailed Student's t-test

Figure 3

3-MA affected autophagy both in vitro and in plaque in vivo. (a) Cultured peritoneal macrophages were preincubated with 3-MA for 30 min, then stimulated with 25 μg/ml oxLDL. Western blot analysis of autophagy activity at different times after oxidized low-density lipoprotein (oxLDL) stimulation. The asterisk indicates cross-reactive band of p62 antibody. Data are mean±S.D. **P<0.01, ***P<0.001, two-way ANOVA. (b) Western blot analysis of LC3 and p62 level in whole-aorta lysates from control mice (4 mice from 14 control mice) and 3-MA-treated mice (4 mice from 16 3-MA–treated mice). Data are mean±S.D. from three independent experiments. *P<0.05, unpaired two-tailed Student's t-test. (c) Immunofluorescence staining of LC3 co-stained with MOMA-2 in aortic root sections from control mice and 3-MA-treated mice. Right panels of LC3 staining (white arrows; scale bar, 25 μm) are the enlargements of boxed areas in left panels (scale bar, 25 μm)

Figure 4

3-MA decreased foam cell macrophage formation in vitro and accumulation of lipid droplets within atherosclerotic plaque. (a) Representative images of BODIPY (green) and LC3 (red) in aortic plaque from control mice and 3-MA-treated mice. Red boxes highlight LC3-positive area and yellow boxes LC3-negative areas. Scale bar, 50 μm. (b) Cells were preincubated with 3-MA for 30 min, then stimulated with 25 μg/ml oxLDL for foam cell induction. Oil Red O staining was performed at different times after oxLDL stimulation. The proportion of Oil Red O-positive cells from three independent experiments was calculated and data are mean±S.D. **P<0.01, ***P<0.001, unpaired two-tailed Student's t-test

Figure 5

3-MA inhibited viability of oxLDL-stimulated macrophages in vitro. Cells were preincubated with 3-MA or rapamycin for 30 min, and then stimulated with 25 μg/ml oxLDL. CCK-8 assay was performed at different times after oxLDL stimulation to measure the viability of microphages. Data from three independent experiments are mean±S.D. (n=3 replicates). **P<0.01, ***P<0.001, unpaired two-tailed Student's t-test

Figure 6

3-MA improved anti-inflammatory microenvironment in atherosclerotic plaque. Quantitative RT-PCR analysis of messenger RNA (mRNA) expression of IL-6, IL-17, interferon-gamma (IFN-γ), IL-10, TGF-β, Ebi3, IL-12α, p28, IL-12β and Foxp3 in the aorta of control mice (4 mice from 14 control mice) and 3-MA-treated mice (6 mice from 16 3-MA-treated mice). Data from three replicates in three independent experiments are mean±S.D. *P<0.05, **P<0.01, unpaired two-tailed Student's t-test