Inhibition of Semicarbazide-sensitive Amine Oxidase Reduces Atherosclerosis in Cholesterol-fed New Zealand White Rabbits

Abstract

Inflammation, oxidative stress, and the formation of advanced glycated end-products (AGEs) are important components of atherosclerosis. Vascular adhesion protein-1 (VAP-1) participates in inflammation. Its enzymatic activity, semicarbazide-sensitive amine oxidase (SSAO), can catalyze oxidative deamination reactions to produce hydrogen peroxide and aldehydes, leading to the subsequent generation of AGEs. This study aimed to investigate the effect of VAP-1/SSAO inhibition on atherosclerosis. In our study, immunohistochemical staining showed that atherosclerotic plaques displayed higher VAP-1 expression than normal arterial walls in apolipoprotein E-deficient mice, cholesterol-fed New Zealand White rabbits and humans. In cholesterol-fed rabbits, VAP-1 was expressed on endothelial cells and smooth muscle cells in the thickened intima of the aorta. Treatment with PXS-4728A, a selective VAP-1/SSAO inhibitor, in cholesterol-fed rabbits significantly decreased SSAO-specific hydrogen peroxide generation in the aorta and reduced atherosclerotic plaques. VAP-1/SSAO inhibition also lowered blood low-density lipoprotein cholesterol, reduced the expression of adhesion molecules and inflammatory cytokines, suppressed recruitment and activation of macrophages, and decreased migration and proliferation of SMC. In conclusion, VAP-1/SSAO inhibition reduces atherosclerosis and may act through suppression of several important mechanisms for atherosclerosis.

Figure 1

The distribution of vascular adhesion protein-1 (VAP-1) in the aorta. (A) Stronger VAP-1 staining (black arrows) is seen in atherosclerotic arterial walls than in normal parts in apolipoprotein-E deficient mice, rabbits, and humans. (B) Strong VAP-1 staining (white arrows) is seen in the markedly thickened intima of cholesterol-fed rabbits and is closely co-localized with endothelial cells (EC) and smooth muscle cells (SMC). The scale bar = 100 μm.

Figure 2

Effect of PXS-4728A on semicarbazide-sensitive amine oxidase (SSAO) activity in (A) thoracic aorta, (B) lung, and (C) epididymal fat. SSAO activity is expressed as the SSAO-specific production rate of H₂O₂. The values are the mean ± SEM (N = 5–6 in each group). *p < 0.05 compared to the cholesterol-fed group.

Figure 3

Effects of semicarbazide-sensitive amine oxidase (SSAO) inhibition by PXS-4728A on atherosclerotic lesions. (A) Representative photographs showing oil red O-stained atherosclerotic plaques of the aorta in different groups. (B) Quantification of oil red O-stained atherosclerotic plaques in different groups. (C) Representative photographs showing Hematoxylin and Eosin staining of atherosclerotic plaques of the aorta in different groups. (D) Quantification of the ratio of intima/media area in different groups. (E) Representative photographs showing atherosclerotic plaques of the aorta by micro-computed tomography (micro-CT) in different groups. (F) Quantification of atherosclerotic plaques by micro-CT in different groups. In B, D and F, the data are expressed as a ratio and calculated as the mean ± SEM (n = 4–6 in each group); *p < 0.05 compared to the control group (CTRL). ^†p < 0.05 compared to the cholesterol-fed group.

Figure 4

Effects of semicarbazide-sensitive amine oxidase (SSAO) inhibition by PXS-4728A on the adhesion, inflammation, and macrophages activation in atherosclerotic plaques of thoracic aorta in rabbits in different groups. (A) Expression of adhesion molecules in the atherosclerotic plaques in different groups. Immunohistochemical staining for intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin expression are shown. (B) Expression of inflammatory cytokines including cyclooxygenase-2 (COX-2), monocyte chemoattractant protein-1 (MCP-1), and interleukin-6 (IL-6) by immunohistochemical staining in different groups. (C) Expression of markers for macrophage activation and recruitment in different groups. Immunohistochemical staining for Toll-like receptor -4 (TLR-4), CD36, lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), receptor for advanced glycation end-product (RAGE), macrophage (Mϕ) and smooth muscle cells (SMC) expression are shown. Strong TLR-4, CD36, LOX-1, and RAGE staining (white arrowheads) is seen in the markedly thickened intima of cholesterol-fed rabbits and is closely co-localized with macrophages (Mϕ) and SMC. Expression of adhesion molecules, cytokines, and macrophage activation markers are higher in cholesterol-fed group than that in control (CTRL) group and cholesterol-fed/PXS-4728A group. The internal elastic lamina is indicated by the arrows. The scale bar = 100 μm. Quantification of IHC staining-positive areas in atherosclerotic plaques of thoracic aorta were shown on the lower panel of indicated antibodies (N = 3–4 in each group). Histograms of their integrated optical density (IOD) value were measured by Image-Pro Plus software. The values are expressed as mean ± SEM. (*P < 0.05). (D) Expression levels of the markers of adhesion, inflammation, and macrophages activation in rabbit aorta are detected by Western blot analysis in different groups. The cropped blots are used in the figure, and the full-length blots are presented in Supplementary Figure S5.

Figure 5

The immunohistochemical analysis for the matrix metalloproteinase-9 (MMP-9) expressions, smooth muscle cells (SMC), proliferative cell nuclear antigen (PCNA) (brown color) in aortas of rabbits in different groups. (A) Compared to rabbits with normal diet (CTRL), cholesterol-fed rabbits had markedly thickened intima with increased MMP-9, SMC, and PCNA staining. The immunopositive staining of MMP-9, SMC, PCNA, and co-localization between PCNA and SMC expressions in cholesterol-fed rabbits with PXS-4728A treatment (Cholesterol-fed/PXS-4728A) were weaker and lower than that in cholesterol-fed rabbits. The internal elastic lamina is indicated by the arrows. The scale bar = 100 μm. (B) The integral optical density (IOD) of MMP-9 immunostaining intensity quantified by Image-Pro Plus software (N = 3–4 in each group). (C) Percentage of proliferating cells (PCNA-positive) determined by dividing the number of PCNA-positive cells by the number of total cells per section (N = 3–4 in each group). The values are expressed as mean ± SEM. *p < 0.05 compared to control group (CTRL). ^†p < 0.05 compared to Cholesterol-fed group.