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. 2013 Sep 19;4(9):e806.
doi: 10.1038/cddis.2013.317.

Targeting annexin A7 by a small molecule suppressed the activity of phosphatidylcholine-specific phospholipase C in vascular endothelial cells and inhibited atherosclerosis in apolipoprotein E⁻/⁻mice

H Li  1 , S Huang, S Wang, J Zhao, L Su, B Zhao, Y Zhang, S Zhang, J Miao
Affiliations

Targeting annexin A7 by a small molecule suppressed the activity of phosphatidylcholine-specific phospholipase C in vascular endothelial cells and inhibited atherosclerosis in apolipoprotein E⁻/⁻mice

H Li et al. Cell Death Dis. .

Abstract

Phosphatidylcholine-specific phospholipase C (PC-PLC) is a key factor in apoptosis and autophagy of vascular endothelial cells (VECs), and involved in atherosclerosis in apolipoprotein E⁻/⁻ (apoE⁻/⁻) mice. But the endogenous regulators of PC-PLC are not known. We recently found a small chemical molecule (6-amino-2, 3-dihydro-3-hydroxymethyl-1, 4-benzoxazine, ABO) that could inhibit oxidized low-density lipoprotein (oxLDL)-induced apoptosis and promote autophagy in VECs, and further identified ABO as an inhibitor of annexin A7 (ANXA7) GTPase. Based on these findings, we hypothesize that ANXA7 is an endogenous regulator of PC-PLC, and targeting ANXA7 by ABO may inhibit atherosclerosis in apoE⁻/⁻ mice. In this study, we tested our hypothesis. The results showed that ABO suppressed oxLDL-induced increase of PC-PLC level and activity and promoted the co-localization of ANXA7 and PC-PLC in VECs. The experiments of ANXA7 knockdown and overexpression demonstrated that the action of ABO was ANXA7-dependent in cultured VECs. To investigate the relation of ANXA7 with PC-PLC in atherosclerosis, apoE⁻/⁻ mice fed with a western diet were treated with 50 or 100 mg/kg/day ABO. The results showed that ABO decreased PC-PLC levels in the mouse aortic endothelium and PC-PLC activity in serum, and enhanced the protein levels of ANXA7 in the mouse aortic endothelium. Furthermore, both dosages of ABO significantly enhanced autophagy and reduced apoptosis in the mouse aortic endothelium. As a result, ABO significantly reduced atherosclerotic plaque area and effectively preserved a stable plaques phenotype, including reduced lipid deposition and pro-inflammatory macrophages, increased anti-inflammatory macrophages, collagen content and smooth muscle cells, and less cell death in the plaques. In conclusion, ANXA7 was an endogenous regulator of PC-PLC, and targeting ANXA7 by ABO inhibited atherosclerosis in apoE⁻/⁻ mice.

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Figures

Figure 1
Figure 1
ABO treatment inhibited the increased level and activity of PC-PLC induced by oxLDL in endothelial cells via promoting co-localization of ANXA7 and PC-PLC in an ANXA7-dependent manner. (a) Effects of ABO on the protein levels of PC-PLC in HUVECs and MS1 cells under oxLDL treatment detected by western blot analysis. ABO (50 μM) and oxLDL (50 μg/ml for HUVECs and 100 μg/ml for MS1 cells) were used. (b) Quantification of PC-PLC protein levels shown in A. Protein levels were normalized to GAPDH. Values are mean±S.E.M. *P<0.05, versus control, #P<0.05, versus oxLDL, n=3. (c) Double immunohistochemical staining showed that ANXA7 (red) and PC-PLC (green) exhibited agglomeration and co-localization after ABO treatment in HUVECs upon oxLDL stimulus. Overlays of the red and green signal with a yellow color indicated co-localization. Scale bars, 16 μm. (d and e) Overexpression (pCMV-ANXA7) and knockdown (siANXA7) of ANXA7 were performed. (d) Analysis of co-localization between ANXA7 and PC-PLC in HUVECs. Co-localization in confocal microscopy is defined by the presence of three or more ANXA7 and PC-PLC residing at the same physical location in a specimen. The bar chart shows quantification of co-localization of ANXA7 and PC-PLC. Data are mean±S.E.M. *P<0.05, **P<0.01; n=3. (e) Quantification of PC-PLC activity. Data are mean±S.E.M. *P<0.05, **P<0.01, ***P<0.001, #P<0.05, n=3
Figure 2
Figure 2
ABO treatment inhibited the increased level and activity of PC-PLC in apoE−/− mice. (a) Double-stained images of co-localization (yellow) of PC-PLC and CD31-positive VECs. Scale bars, 60 μm. (b) Quantification of PC-PLC level in endothelium of baseline, control and ABO-treated groups. Data are mean±S.E.M. *P<0.05 versus baseline, #P<0.05 versus control, n=6. (c) Relative PC-PLC activity normalized to baseline activity (equal to 1). Data are mean±S.E.M. *P<0.05 versus baseline, #P<0.05 versus control, n=6
Figure 3
Figure 3
ABO increased ANXA7 level in the endothelium of apoE−/− mice. (a) Double-stained images of co-localization (yellow) of ANXA7 with CD31-positive VECs. Scale bars, 60 μm. (b) The relative fluorescent intensity of ANXA7 in the endothelium of apoE−/− mice. Data are mean±S.E.M. #P<0.05 versus control, n=6
Figure 4
Figure 4
ABO induced autophagy in cultured endothelial cells under oxLDL treatment and in the endothelium of apoE−/− mice. (a) Effects of ABO on the protein level of LC3-II and LC3-I conversion in HUVECs and MS1 cells under oxLDL treatment detected by western blot analysis. ABO (50 μM) and oxLDL (50 μg/ml for HUVECs and 100 μg/ml for MS1 cells) were used. (b) Quantification of LC3-II protein levels and the ratio of LC3-II/LC3-I shown in a. Protein levels were normalized to GAPDH. Values are mean±S.E.M. *P<0.05, **P<0.01 versus control, #P<0.05, ##P<0.01 versus oxLDL, n=3. (c) TEM of autophagosome formation in the endothelium of ABO-treated groups (N, nucleus; arrow, autophagosomes and autolysosomes). (d) Autophagic vesicles (AVs), including autophagosomes and autolysosomes, were counted from 8 μm2 sampling regions per cell. Data are mean±S.E.M. #P<0.05 versus control. (e) Dual immunofluorescence for CD31 and LC3 of en face aortic arch. Confocal images of en face aortic arch labeled with DAPI (blue), rat-anti-CD31 (red) and rabbit-anti-LC3 (green). Scale bars, 60 μm. (f) The bar chart separately showed quantification of LC3 dots per cell in the endothelium. Data are mean±S.E.M. *P<0.05 versus baseline, #P<0.05, ##P<0.01 versus control, n=6
Figure 5
Figure 5
ABO decreased p62 level in cultured endothelial cells under oxLDL treatment and in the endothelium of apoE−/− mice. (a) Effects of ABO on the protein level of p62 in HUVECs and MS1 cells under oxLDL treatment detected by western blot analysis. ABO (50 μM) and oxLDL (50 μg/ml for HUVECs and 100 μg/ml for MS1 cells) were used. (b) Quantification of p62 protein levels shown in a. Protein levels were normalized to GAPDH. Values are mean±S.E.M. *P<0.05, versus control, #P<0.05, versus oxLDL, n=3. (c) Double-stained images of co-localization (yellow) of p62 with CD31-positive VECs. Scale bars, 60 μm. (d) The relative fluorescent intensity of p62 in the endothelium of apoE−/− mice. Data are mean±S.E.M. *P<0.05 versus baseline, #P<0.05 versus control, n=6
Figure 6
Figure 6
Images and quantifications of cell death in the aortic root lesions of apoE−/− mice. (a) Left, sections from the aortic root were labeled by TUNEL to detect apoptotic cells and counterstained with PI to detect nuclei; right, images of H&E-stained aortic root lesions. Red lines show the boundary of the developing necrotic core. Scale bars, 60 μm. (b) From the top to bottom panel, mean percent of TUNEL-positive cells in the lesions, TUNEL-positive cells in the endothelium and necrotic areas in the lesions. Values are mean±S.E.M. *P<0.05 versus baseline, #P<0.05 versus control, n=6
Figure 7
Figure 7
Effects of ABO on the size and phenotype of aortic atherosclerotic plaque in apoE−/− mice. (a) Representative oil-red O–stained aortas from apoE−/− mice (Scale bars, 3 mm, n=5). (b) H&E staining of brachiocephalic artery (scale bars, 200 μm, n=6) and (c) aortic root lesions (scale bars, 500 μm, n=6). Bar graphs show en face plaque area and ratio of intima to lumen (data are mean±S.E.M. *P<0.05 versus baseline, #P<0.05 versus control). (d) From the top to bottom panel, oil-red O staining of atherosclerotic lesions, masson trichrome staining of collagen (in blue), immunostaining for mouse α-smooth muscle actin, double-stained images of co-localization (yellow) of CD68 (red) and CD11C (green)-positive areas, double-stained images of co-localization (yellow) of CD68(red) and CD206 (green) -positive areas and in situ zymography detecting MMP-2/9 activity (n=4–5 sections per tissue, at least three sites of analysis per slide). Scale bars for oil-red O staining, 500 μm and others, 80 μm. (e) Quantification of lipid, collagen, actin area in atherosclerotic lesion, weighted co-localization coefficients for CD11C and CD206-positive areas, and MMP-2/9 activity in baseline, control and ABO-treated groups. Data are mean±S.E.M. *P<0.05 versus baseline, #P<0.05 versus control, n=6
Figure 8
Figure 8
Effects of ABO on body weight, organ coefficients and serum lipid levels of apoE−/− mice and oxLDL-induced IL-6 and IL-8 secretion in HUVECs. (a) Body weight was measured every week. Data are mean±S.E.M.; n=6. (b) Blood was collected at the end of the experiment for measuring lipid levels. Data are mean±S.E.M. *P<0.05 versus baseline, n=6. (c) ABO decreased the secretion of IL-6 and IL-8 from oxLDL-treated HUVECs. ABO (50 μM) and oxLDL (50 μg/ml) were used. IL-6 and IL-8 secretions from HUVECs were measured by enzyme-linked immunosorbent assay. Data are mean±S.E.M. **P<0.01 versus control, ##P<0.01 versus oxLDL, n=3
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